MAPPING THE TAU PROTEIN INTERACTOME USING THE BIOID2 IN SITU LABELLING APPROACH By Ahmed Atwa A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Neuroscience—Master of Science 2022 ABSTRACT MAPPING THE TAU PROTEIN INTERACTOME USING THE BIOID2 IN SITU LABELLING APPROACH By Ahmed Atwa Pathological inclusions composed of tau protein are hallmarks of neurodegenerative diseases collectively known as tauopathies, of which the most common is Alzheimer’s Disease (AD). Tau is most well-known as a microtubule-associated protein involved in regulating microtubule dynamics, but accumulating evidence suggests tau is involved in many biological functions. Deciphering the tau protein interactome is critical for better understating the physiological and pathological roles of tau. This work aimed to identify tau interacting partners using the in situ protein labelling BioID2 method by creating fusion proteins between full-length human tau and either BioID2 on the N-terminus (BioID2-Tau) or C-terminus (Tau-BioID2). A total of 372 proteins were identified, of which 269 interacted with Tau-BioID2, 169 with BioID2-Tau, and 66 proteins overlapped between both tau proteins. Gene Ontology (GO) cellular component analysis mapped protein interactions in the mitochondria, cytoskeleton, dendrites, nucleus, synaptic vesicles, and the ribonucleoprotein complex. While GO molecular function pathways identified proteins involved in RNA binding, translation regulation, ubiquitin ligase activity, kinase binding, mitochondrial oxidoreductase, and peroxidase activity. KEGG pathway analysis identified proteins associated with neurodegenerative diseases, including AD, Parkinson’s disease, Huntington’s disease, and Amyotrophic lateral sclerosis. Thus, this approach can identify members of the tau interactome via in situ labeling, that may help shed light on tau’s functional roles and provide novel therapeutic strategies for neurodegenerative diseases. This thesis is dedicated to Mom, Dad, my Wife, and my Baby Boy. Thank you for always believing in me. iii ACKNOWLEDGEMENTS I would like to start this by expressing my utmost gratitude to my mentor, Dr. Nicholas Kanaan. Since day one, Dr. Kanaan has been extremely supportive on both the academic and the personal levels. Dr. Kanaan introduced me to this project and guided me through the experimental planning, he always excelled at putting me on the right track. Dr. Kanaan encouraged me to present my work at conferences and supported my application to receive a travel fellowship award. I am extremely grateful for the opportunities he gave me; he is a true mentor and I am very much looking forward to my next years in his lab working on my PhD. I would like to thank all members of the Kanaan lab. Tessa is a great lab manager, she always made my life a lot easier. Dr. Benjamin Combs, Dr. Mattew Benskey, Mohammed, and Rebecca helped me a lot with the experiments. All members of the lab are extremely supportive, friendly, and this work couldn’t have been finished on time without their contribution. I would like to thank my committee members Dr. Irving Vega, Dr.Scott Counts, Dr. Min-Hao Kuo, and Dr. Nicholas Kanaan. They guided me during this thesis work. Our discussions always challenged me and certainly made me a better critical thinker. Finally, I would like to thank my family and friends. They showed all kinds of emotional support during those past two years of a pandemic. My wife Shahd and my baby Jayd are my greatest support in life, I was very lucky to have such a wonderful supportive wife. This couldn’t have been done without you. Thank you for everything. iv TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………….……......viii LIST OF FIGURES……………………………………………………………………..…….... ix . KEY TO ABBREVIATIONS……………………………………………………………….…….x INTRODUCTION…………………………………………………………………………………1 . Tau protein expression and structure………………………………………………….1 . Physiological functions of the tau protein………………………………………………3 Tau protein pathology in neurodegenerative diseases…………………….…….….5 Tau protein-protein interactions………………………………………………………10 Enzyme-mediated proximity labeling: A novel approach to study protein-protein interactions…………………………………………………………………………...... 14 . THESIS OBJECTIVE…………………………………………………………………………..18 Aim Statement…………………………………………………………………………. 19 . Hypothesis……………………………………………………………………………... 19 . MATERIALS AND METHODS………………………………………………………………..20 Creating Tau-BioID2 expression plasmids using Restriction Cloning…………… 20 . Site-directed mutagenesis…………………………………………………….20 Preparation of LB/agar plates and LB broth medium………………………22 Transformation of XL10-Gold ultracompetent cells..…………...…………..22 Transformation of Stbl3 competent E.coli……………………………….......23 Plasmid DNA Miniprep………………………..……………………………….23 Plasmid DNA Maxiprep………………………………...……………………...24 Restriction Digestion…………………………………………………………...24 DNA Gel Electrophoresis……………………………………………………...24 Gel purification and DNA ligation……………………………………………..25 Human embryonic kidney (HEK293T) cell culture……………………...…..26 Transfection of HEK293T cells………………………………………………. 26 . HEK293T protein lysate collection…………………………………………... 27 . SDS-PAGE and Western blotting……………...……………………………..27 Generation of Lentiviruses that express the BioID2 proteins……………………...28 Lentiviruses production in HEK293T cells………………………………….. 28 . Immunocytochemistry (ICC)…………………………………………………..29 Lentiviral functional titer determination using ICC…………………………. 30 . Embryonic day 18 mouse primary cortical neuron culture preparation......31 Immunocytofluorescence (ICF)……………………………………………….32 Lentivirus transduction efficiency……………………………………………..32 Lentiviral transduction in neurons…………………………………………….33 Endogenous tau vs lentiviral tau expression levels………………………...33 v Mass Spectrometry identification of biotinylated proteins………………………….34 Optimization of Biotin dose and incubation time……………………………34 . Primary neurons protein lysate collection…………………………………... 35 . Biotin-streptavidin affinity pulldown………………...………………………...35 Liquid Chromatography-MS/MS……………………………………………...36 Mass spectrometry protein identification…………………………...………..38 Label-free quantification of identified proteins…………………………… …38 . Functional protein association networks……………………………………. 39 . Gene Ontology enrichment analysis………………………………………… 40 . Validation of tau protein interacting partners………………………………..40 Antibodies……………………………………………………………………… 41 . Data representation and statistical analysis………………………………...42 RESULTS……………………………………………………………………………………….43 Generation and validation of Tau-BioID2 expression plasmids and lentiviruses..43 Mutagenesis of the BioID2 expression plasmids and creating the Tau-BioID2 fusion proteins…………………………………………………………………………. 44 . Creating BioID2 lentiviral transfer plasmids……………………………………...….48 Lentiviruses generation and determination of the functional titer………………....52 Optimizing lentiviral transduction of primary cortical neurons……………………..55 Determining optimal titer for primary neuron transduction………………………...55 Lentiviruses express the Tau-BioID2 proteins at near physiological levels……...59 Optimization of biotin dose and incubation time…………………………………….61 Affinity pulldown and mass spectrometry identification of biotinylated tau interacting proteins……………………………………………………………………..64 Optimization of the biotin-streptavidin affinity pulldown using HEK293T cell lysates…………………………………………………………………………………...65 Optimization of the biotin-streptavidin affinity pulldown using primary cortical neurons lysates………………………………………………………………………...68 Identification of the tau protein interactome using mass spectrometry…………..71 . Gene ontology enrichment analysis of tau interactome.…………………………...74 GO enrichment analysis reveals distinct cellular components for Tau-BioID2 and BioID2-Tau interacting partners……………………………………………………… 77 . Validation of the identified tau interacting partners………………………………… 80 . DISCUSSION………………………………………………………………………………….. 82 . Tau interactome mapping……………………………………………………………..82 Distinct and overlapping interactions between BioID2-Tau and Tau-BioID2…….83 Tau protein interactions in various cellular localizations…………………………...86 Technical development of the approach……………………………………………. 89 . Cloning of BioID2 and Tau fusion proteins…………………………………. 90 . Lentiviruses production……………………………………………………….. 91 . Lentiviral-mediated transduction of primary cortical mouse neurons……. 91 . Mass spectrometry identification of tau interacting proteins……………… 93 . Validation of identified tau interacting partners……………………………..94 . vi Limitations of the approach……………………………………….………………….. 95 . Future directions………………………………………………………………………..97 APPENDICES……………………………………………………………………………….....99 APPENDIX A: Table 3: List of the identified tau protein interacting partners…..100 APPENDIX B: Table 4: List of tau interacting partners identified in ClueGo cellular components ……………………….…………………………………………116 APPENDIX C: Table 5: List of tau interacting partners identified in ClueGo molecular function pathways…………………..…………………………………….125 REFERENCES………………………………………………………………………………..130 vii LIST OF TABLES Table 1: Cycling parameters for mutagenesis reactions……………………………………22 Table 2: KEGG pathway analysis associated proteins with neurological disorders..…...76 Table 3: List of the identified tau protein interacting partners…………………………… 100 . Table 4: List of tau interacting partners identified in ClueGo cellular components…… 116 . Table 5: List of tau interacting partners identified in ClueGo molecular function pathways……………………………………………………………………………………… 125 . viii LIST OF FIGURES Figure 1: Design of the BioID2 constructs……………………………………………….…. 43 . Figure 2: Restriction cloning of the BioID2 fusion proteins…………………………….…. 46 . Figure 3: Validation the lentiviral transfer plasmids expressing the BioID2 constructs...50 . Figure 4: Lentiviruses functional titer and validation………………………………………. 53 . Figure 5: Expression of the BioID2 proteins in primary cortical neurons…………….…..57 Figure 6: Lentiviral expression of Tau-BioID2 compared to WT Tau expression……….60 . Figure 7: Optimization of biotin dose and incubation time………………………………....62 Figure 8: Schematic representation of the experimental paradigm selected for primary neurons affinity purification and mass spectrometry experiments………………………..63 Figure 9: Affinity capture of biotinylated proteins using streptavidin Dynabeads………. 64 . Figure 10: Optimization of affinity pulldown in HEK293T cell lysates…………………….66 Figure 11: Optimization of affinity pulldown in primary neurons lysates………………… 69 . Figure 12: Venn diagrams of the identified preferential interacting proteins…………….72 . Figure 13: STRING analysis for the identified total tau interactome…………………..….73 Figure 14: ClueGo gene ontology enrichment analysis for the total tau interactome…..75 Figure 15: GO enrichment cellular component analysis……………………………….…..78 Figure 16: Validation of Tau interacting partners by western blotting…………………….81 ix KEY TO ABBREVIATIONS ACN acetonitrile AD Alzheimer’s disease ALS amyotrophic lateral sclerosis CBD corticobasal degeneration CMF calcium-and magnesium-free Co-IP co-immunoprecipitation CTE chronic traumatic encephalopathy ddH2O double-distilled water DIV day in vitro DMEM Dulbecco's Modified Eagle Medium DPBS Dulbecco's phosphate-buffered saline E.coli Escherichia coli FBS fetal bovine serum FTDP frontotemporal dementia with parkinsonism GO gene ontology GS goat serum HD Huntington’s disease HEK human embryonic kidney ICC immunocytochemistry ICF immunocytofluorescence MAP microtubule-associated protein x MCL Markov clustering MOI multiplicity of infection MS mass spectrometry MTBR microtubule binding region NBM neurobasal medium NFTs neurofibrillary tangles PART primary age-related tauopathy PD Parkinson’s disease PL proximity labeling PPI protein-protein interactions PSP progressive supranuclear palsy PTMs post-translational modifications SDS sodium dodecyl sulfate STRING search tool for the retrieval of interacting genes/proteins TKO tau knockout WT wild type xi INTRODUCTION Tau protein expression and structure The human tau protein is encoded by the microtubule-associated protein tau (MAPT) gene on chromosome 17q21 which contains 16 exons (Neve et al., 1986). Exons E4A, E6, and E8 are transcribed preferentially in the peripheral tissue (Caillet-Boudin et al., 2015). In the human brain, the 5’- untranslated region is encoded by exons E0 and E1. while the 3’- untranslated region is encoded by exon 14. Constitutive exons in the MAPT gene are E1, E4, E5, E7, E9, E11, E12, and E13 while exons E2, E3, and E10 are subject to alternative splicing in the adult brain (Himmlert, 1989). Exon E3 expression is dependent on the presence of exon E2 while exon E2 can be expressed independently of exon E3 (Andreadis et al., 1992). Thus, alternative splicing of exons E2, E3, and E10 yields the six MAPT mRNAs found in the adult human brain (Goedert et al., 1989). Splicing of exons E2 and E3 give rise to tau transcripts that are different in the amino- terminus (N-terminus) by the absence or presence of one or two N-terminus inserts (0N, 1N or 2N). Exon 10 encodes for the repeat domain R2, alternative splicing of exon E10 yields transcripts that either have three or four repeat domains (3R or 4R). Accordingly, the six tau isoforms found in the human brain are 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R ranging from 352 to 441 amino acids (Goedert & Jakes, 1990). The expression of the human tau protein isoforms is developmentally regulated. In the fetal brain, only the shortest tau isoform which lacks exons E2, E3, and E10 is present (0N3R, 352 amino acids)(Lee et al., 2001b). In the adult brain, equal levels of the 3R and 4R isoforms are expressed while the expression levels of the 0N, 1N, and 2N isoforms are ~37%, ~54%, and ~9% respectively (Y. Wang & Mandelkow, 2016). In the adult 1 mouse brain, only the 4R tau isoforms are present. In addition, the longest isoform of the human tau protein contains an N-terminus segment (amino acids 17-28) that is not found in the murine tau protein which might play a role in tau protein-protein interactions (PPIs) (Hernández et al., 2020a). The tau protein has four major domains: the N-terminus projection domain, the proline rich region, the microtubule binding region (MTBR), and the C-terminus domain. The longest isoform of the human tau protein (2N4R, hTau40) contains the two highly acidic 29-amino-acid inserts (exons E2-E3) followed by the basic proline rich region (amino acids Isoleucine 151- glutamine 244). The N-terminus domain (amino acids methionine 1 – tyrosine 197) does not bind to the microtubules and is followed by the MTBR which contains the four repeat domains encoded by exons E9-E12 (R1-R4; amino acids Glutamine 244 – Asparagine 368). The C-terminus domain contains amino acids Lysine 369 – Leucine 441 (Mandelkow et al., 1995; Sergeant et al., 2005; Y. Wang & Mandelkow, 2016). Under physiological condition, tau is a highly soluble protein that is structurally dynamic and lacks a stable globular conformation as is characteristic of members of the so-called intrinsically disordered protein family. Tau has an asymmetric distribution of charges due to the presence of 56 negative aspartic acid/glutamic acid residues and 58 positive lysine/arginine residues. The electrostatic interaction between the opposite charges is consistent with the proposed paperclip-like confirmation in which the C- terminus domain folds over the MTBR and the N-terminus projection domain folds back and on the C-terminus (Jeganathan et al., 2006). The high degree of flexibility tau displays 2 is thought to play a role in its folding as well as its functional interactions with other proteins. Physiological functions of the tau protein Tau protein was first isolated in 1975 from porcine brain as a heat-stable protein that associates with tubulin and is required for the assembly and stabilization of the cerebral microtubules (Weingarten et al., 1975). However, accumulating evidence suggests that tau exhibits several biological functions in various neuronal subcellular compartments including axons, dendrites, and the nucleus. Tau is a microtubule-associated protein (MAP) that stabilizes and promotes the assembly of microtubules in vitro by binding to the interface between α- and β- tubulin heterodimers at its MTBR evolutionary conserved residues 224–237, 245–253, 275–284, and 300–317, while the intervening unbound residues are flexible supporting the dynamic nature of the tau protein binding to microtubules within the context of in vitro assays (Kadavath et al., 2015). In another study using neurons, tau was instead shown to regulate the dynamics of the microtubules rather than stabilizing them (Qiang et al., 2018). Tau was more abundant on the labile region of the microtubules rather than the stable region. Depletion of tau in cultured rat neurons reduced the labile domain of the microtubules (marked by the presence of tyrosinated tubulin) and increased the mass of the stable regions of the microtubules (an observation attributed to the increased expression of MAP6 upon tau depletion). Thus, tau can be considered as a multi-functional MAP that might have functional connections with other MAPs (e.g. MAP6) and is likely to play a role regulating the dynamics of the microtubules rather than their stability in neurons. 3 Within the axon, several studies have identified roles for tau in regulating both anterograde and retrograde axonal transport. Early studies suggested tau might compete with kinesin and dynein motor proteins for binding to microtubules affecting both types of active axonal transport of cytoplasmic organelles (Dixit et al., 2008). In the same study, the authors reported that kinesin motors tended to detach from the microtubules in regions of bound tau (shortest and longest tau isoforms) while the dynein motors did not detach from the microtubules but rather tended to reverse direction. However, other studies have challenged the notion that tau physically interferes with motor proteins (Morfini et al., 2007) by showing that exogenous perfused tau in isolated squid axoplasm had no effect on axonal transport. Tau can also regulate kinesin-mediated anterograde axonal transport via its phosphatase-activating domain (Kanaan et al., 2011a). The phosphatase activating domain is an N-terminus region comprised of amino acids 2-18 that was found to activate PP1 which in turn dephosphorylates and activates GSK3β leading to phosphorylation of kinesin motors causing cargo release. Thus, tau has important functions in regulating kinesin- and dynein- mediated axonal transport within the neurons which might be modulated by its protein-protein interactions. Tau also localizes to the somatodendritic compartment (Papasozomenos & Binder, 1987) and the nucleus (Loomis et al., 1990). However, the physiological functions in these compartments are not well characterized. Tau was required for the dendritic and synaptic maturation of new-born hippocampal granule neurons as well as the formation of dendritic spines and postsynaptic densities (Pallas‐Bazarra et al., 2016). In vitro and in vivo studies showed that physiological tau localizes to the nucleus and binds double- stranded and single-stranded DNA as well as the histone-DNA complex. Tau binding 4 maintains the integrity of genomic DNA (Camero et al., 2014) and protects DNA against reactive oxygen species-induced heat stress (Sultan et al., 2011). Moreover, tau deficient mice showed altered integrity of genomic DNA, cytoplasmic, and nuclear RNA suggesting a role for physiological tau in maintaining the integrity of the nucleic acids (Violet et al., 2014). Studies in tau knockout (TKO) mice suggested a function of physiological tau in neurogenesis and synaptic plasticity. TKO mice showed impaired hippocampal neurogenesis (Hong et al., 2010) as well as selective deficit in hippocampal long-term depression but not long-term potentiation (Kimura et al., 2014). In contrast, Ahmed and colleagues reported that tau deletion impaired long-term potentiation but not long-term depression (Ahmed et al., 2014). Tau was also found to interact with neural plasma membrane through its N-terminus projection domain (Brandt et al., 1995), mediated by Annexins A2 and A6 (Gauthier-Kemper et al., 2018a). Several of the interacting partners and functional roles of tau are currently being elucidated and likely will contribute to tau’s participation in the pathogenesis of human neurodegenerative diseases. Tau protein pathology in neurodegenerative diseases Intracellular tau accumulation is a hallmark of a heterogenous group of neurodegenerative disorders collectively known as tauopathies, of which the most common form is Alzheimer’s disease (AD). Mutations in the MAPT gene as well as post- translational modifications (PTMs) of the tau protein might reduce the affinity of tau binding to microtubules and mediate the tendency for intracellular aggregation of abnormal tau species (reviewed in (Alquezar et al., 2021; Pîrşcoveanu et al., 2017).To date, 112 MAPT mutations were identified in both the intronic and exonic sequences. Several of the mutations were experimentally linked to tau neuropathological alterations, 5 while the functions of other mutations remain understudied (ALZFORUM, 2022). Tau PTMs and the mutations of the MAPT gene might alter the protein interactome of tau which in turn may impact its functional role in disease pathogenesis. The switch from soluble physiological -to- insoluble aggregated tau is suggested to follow a sequence of events that is not entirely understood. Insoluble filamentous tau aggregates including straight filaments (SF) and paired helical filaments (PHF) are predominant in tauopathies and comprise the various hallmark inclusions of each tauopathy. This includes neurofibrillary tangles (NFTs), globose tangles, neuropil threads, neuritic plaques, Pick bodies, astrocytic plaques, tufted astrocytes, and coiled bodies (Chung et al., 2021). Aggregation is thought to evolve from the successive multimerization of soluble tau species. Tau monomers self-associate into dimers which contribute to the formation of smaller order soluble tau oligomers that can either be on-pathway or off- pathway for filament formation. On-pathway oligomeric tau can then form filamentous aggregates that coalesce into the inclusions described above. Many lines of current research are focused on understanding the mechanistic regulators involved in tau seeding and aggregation under pathological conditions (reviewed in (Cowan & Mudher, 2013; Mamun et al., 2020)). Tauopathies can be classified into a) 3R-tauopathies such as Pick’s disease (PiD) (Pickering-Brown et al., 2004), b) 4R-tauopathies such as corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Huntington’s disease (HD) (Baba et al., 2007), c) tauopathies involving both 3R and 4R Tau isoforms including AD, Lewy body dementia, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART), and tangle-only dementia; and d) inherited frontotemporal dementia with 6 parkinsonism linked to chromosome 17 (FTDP-17) characterized by autosomal dominant mutations of the MAPT gene on chromosome 17q21 that can present with 3R, 4R or mixed isoform pathologies (Goedert & Jakes, 2005; Wszolek et al., 2006). Tauopathies also are categorized as primary or secondary tauopathies depending upon whether tau is the primary defining pathology or another form of pathology is present as a defining feature, respectively. Alzheimer’s disease is a secondary tauopathy first described in 1906 by Alois Alzheimer (Alzheimer, 1911). The neuropathological hallmarks of AD as described by the National Institute on Aging – Alzheimer’s Association (NIA-AA) include the deposition of the extracellular amyloid-β plaques, intracellular aggregation of the tau protein, and neurodegeneration (Jack et al., 2018). Tau pathology progresses from the entorhinal and transentorhinal cortices (Braak stages І and ІІ), to the hippocampal formation (stages ІІІ and ІV) and later spreads to the cerebral cortices (stages V and VІ) (Braak et al., 1995, 2006; Braak & Braak, 1991). Abnormal phosphorylation of all six isoforms of tau was reported in AD suggesting that some forms of phosphorylated tau may play a role in the development of tau pathologies (Goedert et al., 1992; Grundke-Iqbal et al., 1987; IHARA et al., 1986). The longest tau isoform (2N4R) has 85 potential phosphorylation sites (serine, threonine, and tyrosine residues) making phosphorylation the most prominent PTM of the tau protein. Phosphorylation at some sites within tau can reduce microtubule binding and may increase the aggregation propensity of tau (Alonso et al., 2001; Bramblett et al., 1993; Lindwall & Cole, 1984). More recent work, also suggest specific tau modifications may precipitate pathogenic changes in tau (e.g. exposure of the N- terminal phosphatase activating domain) that facilitate mechanisms of tau-mediated 7 toxicity (Combs et al., 2019; Mueller et al., 2021). Other tau PTMs reported in AD include acetylation, methylation, ubiquitination, SUMOylation, O-GlcNAcylation, N-glycosylation, and truncation (reviewed in (Alquezar et al., 2021; Carroll et al., 2021)). Further studies are required to better understand the crosstalk between the various roles tau PTMs play in regulating tau pathological progression in AD. Other secondary tauopathies include HD, synucleinopathies, and CTE, among several others. HD is an autosomal dominant disorder characterized by the aggregation of huntingtin protein (Labbadia & Morimoto, 2013). Increased levels of rod-like tau deposits of the 4R isoform, tau oligomers as well as abnormally phosphorylated tau (colocalized with mutant huntingtin) were reported in post-mortem HD brains (Fernández- Nogales et al., 2014; Vuono et al., 2015). Synucleinopathies are neurodegenerative disorders characterized by the presence of α-synuclein aggregates (Lewy bodies). Both NFTs and increased levels of phosphorylated tau were reported in Lewy body dementia, suggesting a synergistic interaction between tau and α-synuclein in synucleinopathies (Chin et al., 2020; Irwin et al., 2013; Joachim et al., 1987). CTE is another secondary tauopathy involving both 3R and 4R tau isoforms. The main driver of CTE is believed to be various forms of brain injury (e.g. concussive and/or repetitive subconcussive injuries) and is characterized by elevated levels of phosphorylated tau in neuronal and glial inclusions, axonal injury, and progressive neurodegeneration (Katsumoto et al., 2019). To date, more than 13 neurological disorders have been identified as primary tauopathies in which tau is considered the major contributor of the pathological processes. Those include FTDP-17, PiD, PSP, CBD, and PART, among others (Josephs, 2017). FTDP-17 is characterized by several mutations of the MAPT gene (Strang et al., 8 2019a). FTDP-17 mutations were suggested to alter tau polymerization kinetics, morphology of tau aggregates, propensity to aggregate, and microtubule binding (Combs & Gamblin, 2012). The P301L and the P301S missense mutations in exon E10 induced tau phosphorylation and aggregation (4R isoforms), NFT formation, synaptic dysfunction, and neuronal loss in several brain regions of transgenic mutant tau mouse models (Götz & Ittner, 2008; Lewis et al., 2000; Sperfeld et al., 1999). Accordingly, tau transgenic mouse models expressing P301L and P301S mutations are extensively used to study tau aggregation, axonal degeneration, and impaired memory function. Other FTDP-17 mutations (K257T, G272V, AK280, V337M, G389R, and R406W) were reported to reduce tau binding to microtubules, induce microtubule instability, and promote abnormal tau phosphorylation (reviewed in (Lee et al., 2001a; Strang et al., 2019b)). Moreover, FTDP- 17 tau mutant carriers present the diverse clinical and neuropathological presentations that resemble other primary sporadic tauopathies such as PiD, PSP, and CBD. PiD is characterized by severe atrophy in the hippocampus, frontal cortex and temporal cortex with neuronal tau inclusions of the 3R isoforms known as Pick bodies. Pathological phosphorylated tau was reported in the cortical and subcortical regions of post-mortem brains of subjects with PiD (Delacourte et al., 1996). PSP is characterized by tau inclusions composed primarily of the 4R isoform (Richter-Landsberg, 2016). NFTs, tau-positive tufted astrocytes and oligodendroglia coiled bodies were reported in PSP patients (Dickson et al., 2007). CBD is another 4R tauopathy characterized by astrocytic plaques, coiled bodies, and dense glial threads (Komori, 1999; Komori et al., 1998). The term PART was first proposed in 2014 to describe the progression of NFTs in cognitively normal aged individuals as well as in demented patients previously referred to as tangle- 9 only dementia, tangle-predominant senile dementia, and preferential development of NFT without senile plaques. PART is characterized by the presence of NFT with spatiotemporal progression similar to that seen in AD and without or with few amyloid-β plaques (NFT+/ amyloid-β −) (Crary et al., 2014). Taken together, there is a heterogeneity in the pathognomonic tau inclusions in both primary and secondary tauopathies that could be regulated in part by tau PTMs, MAPT mutations, as well as differences in tau protein- protein interactions. Tau protein-protein interactions Tau participates in various microtubule-dependent and -independent processes at the physiological and pathological level (reviewed in (Guo et al., 2017; Y. Wang & Mandelkow, 2016)). Although pathological tau dysfunction is the hallmark of the heterogenous group of neurodegenerative tauopathies, the varying disease-related mechanisms involving tau mutations, PTMs, and aggregation await further elucidation (Arendt et al., 2016; M. Morris et al., 2011). One way to better understand the various biological roles of tau in health and disease is by studying the tau protein interactome. The physiological and pathological functions of tau in the various cellular compartments are regulated by its multifaceted direct and indirect protein-protein interactions (Limorenko & Lashuel, 2022; Stancu et al., 2019). For example, tau not only acts as a substrate for other proteins such as kinases and phosphatases (reviewed in (Mietelska- Porowska et al., 2014)), but also act as a scaffolding protein that may regulate several kinase and phosphatase-mediated signaling pathways (Mueller et al., 2021). Several studies have utilized crosslinking, co-immunoprecipitation (Co-IP) and mass spectrometry (MS) to identify tau protein interacting partners. In a human 10 neuroblastoma SH-SY5Y cell model, the authors transfected SH-SY5Y cells with enhanced green fluorescent proteins fused to the C-terminus of a) Full-length tau (amino acids 1-441), b) tau (amino acids 1-255), c) tau (amino acids 256-441), and d) tau amino acids 1-441 with the FTDP P301L mutation (Geeth Gunawardana et al., 2015). Transfected cells were crosslinked, lysed and the tau proteins interactors were affinity captured using GFP-trap beads, and then quantified by MS analysis. Proteins were identified as interacting partners based on an enrichment ratio >1.2 compared to the enhanced green fluorescent proteins-only transfected cell lysates. Utilizing this approach, the authors identified 190 tau interacting proteins which included RNA binding proteins, translation initiation and elongation factors, ribosomal proteins, histones, cytoskeletal, and proteasomal proteins. Histones, RNA binding, ribosomal, and translation proteins were preferentially interacting with the N-terminus of tau (amino acids 1-255), while members of the 14-3-3 protein family, actin binding, heat shock, and proteasomal proteins were preferentially interacting with the C-terminus of tau (amino acids 256-441). Moreover, the P301L mutation impaired tau binding to heat shock proteins, and proteasomal proteins, while it enhanced binding to ribosomal proteins. In another study, in vivo crosslinking was used in 7-month-old wild type (WT) and transgenic rats expressing tau amino acids 151-391 of the 4R isoform (Sinsky et al., 2020). The protein lysates were extracted from the brain stem, separated by affinity chromatography using tau antibodies DC18 (amino acids 168 -181), DC25 (amino acids 347 -353), and DC190 (amino acids 368 - 376). Tau cross-linked proteins were then identified by mass spectrometry. Utilizing this approach, the authors identified 175 11 proteins as tau interactors, of which 39 were associated with AD, 10 with Parkinson’s disease, and 22 with both. Alternative splicing of the MAPT gene at exons E2 and E3 generates the 0N,1N, and 2N tau isoforms that are different by the expressing either 0,1, or 2 N-terminal inserts. The mouse MAPT gene generates only the 4R tau isoforms 0N4R, 1N4R, and 2N4R (Hernández et al., 2020b) . To identify the interacting partners with each of the three isoforms of mouse tau, whole brain lysates from WT mouse were co-immunoprecipitated by antibodies specific to either 0N, 1N, or 2N isoforms (C. Liu et al., 2016). MS analysis identified a total of 101 proteins in all three tau isoforms lysates that were at least 10% enriched compared to lysates from TKO mice. The identified proteins were localized to the cytoplasm, cytoskeleton, and membrane-bound organelles including mitochondria, plasma membrane, and vesicles. Moreover, the authors reported preferential interactions with specific tau isoforms. Other tau interactome mapping studies utilized the lysis/co-IP approach coupled to MS analysis to identify tau PPIs, followed by a more targeted approach to confirm and study the biological relevance of some of the novel tau interactors. In one study, WT mouse brain lysates were co-immunoprecipitated using three tau antibodies (Tau5, Tau21, and Tau24). Wang and colleagues identified 204 proteins as tau interactors and this included members of the dynactin complex, heat shock proteins, and members of the ubiquitin-proteasome system (P. Wang et al., 2017). Otub1 was identified as a novel tau deubiquitinase that significantly promoted phosphorylation and oligomerization of tau P301S in vivo and in vitro in transgenic models expressing TauP301S, an effect that was dependent on the catalytic domain of Otub1. 12 In another study, tau protein interacting partners were immunoprecipitated (Tau13 antibody) from frontal cortex lysate of 2.5-month-old uninduced (control) and induced rTg4510 tauopathy mouse model expressing the 0N4R tau isoform with the P301L mutation (Maziuk et al., 2018a). Doxycycline induction increases the expression level of the mutant tau protein. Interacting partners belonging to nucleotide binding, heat shock, and phosphoproteins were upregulated in the induced rTg4510 mice while proteins associated with the ribonucleoprotein complex, RNA binding, and mitochondria proteins were decreased in P301L expressing mice. Furthermore, nucleotide binding proteins including hnRNPA0, EWSR1, RPL7, and PABP colocalized with phosphorylated tau (pS202 tau, CP13 antibody) primarily in the neuronal soma and formed insoluble aggregates in the frontal cortex of 8 months old rTg4510 mice. Pathological deposits of RNA binding proteins (hnRNPA0, DDX60) were also observed adjacent (but not colocalized) to NFTs in the temporal cortex of human AD patients (Braak stage V and VІ). Phosphorylated tau accumulation is a key characteristic for tauopathies. Tau phosphorylation alters tau structure and function. A recent study used two phospho-tau antibodies, the AT8 and PHF1, to identify interacting partners via traditional IP approaches (Drummond et al., 2020). The hippocampus and entorhinal cortex of AD patients was used to purify AT8-positive tau and then used MS analysis to identify co- IPed proteins. The 542 proteins identified included APOE, GAPDH, CDK5, and ubiquitin. Samples from the frontal cortex of AD patients were immunoprecipitated with the PHF1 antibody and co-IPed proteins were identified by MS analysis. Utilizing this approach, 125 proteins were identified as potential interactors with phosphorylated tau. Among these, 75 proteins were also identified in AT8-purified samples, including ubiquitin, 14-3-3 13 protein family members, nucleotide binding proteins (e.g. HNRNPK, HNRNPA2B1, and HNRNPA1), and members of phagosome maturation protein family (e.g ATP6V0D1, NSF, PRDX5, PRDX6, and VAMP2). Enzyme-mediated proximity labeling: A novel approach to study protein-protein interactions Traditional methods for studying protein-protein molecular interactions relied mainly on antibody-based affinity purification coupled to MS analysis. These approaches allow for the identification of interacting partners that form a relatively stable complex with the protein of interest. However, the main limitations of this approach are the requirement of the non-physiological cell lysis which is susceptible to losing transient and low-affinity PPIs, the potential to artificially induce interactions, lysis and IP buffer-mediated effects, and a critical dependence on the availability of a high-quality antibodies. Other methods used to identify PPIs include the yeast two-hybrid system and coupling cross-linking to antibody affinity purification, however, they are prone to increased rate of false positives and yeast may not effectively recapitulate physiological interactions in mammalian cells (reviewed in (Berggård et al., 2007; Hayes et al., 2016)). Over the past decade, proximity labeling (PL) techniques have emerged as a complementary approach that overcome several of the limitations of the traditional techniques. PL approaches involve the fusion of a protein of interest to an engineered enzyme which allow in situ biotin labeling of the proximal proteins (proximity-dependent biotinylation). The biotinylated proteins interacting with or in close proximity to the protein of interest are affinity captured using streptavidin-coated beads (leveraging the highly specific and strong biotin/streptavidin interaction) and identified using mass spectrometry. 14 To date, the engineered enzymes are either biotin ligases such as BioID, BioID2, BASU, TurboID, and miniTurbo, or peroxidases such as APEX, APEX2, and HRP. Biotin ligases catalyze the reaction between biotin and ATP to create the activated easter biotinyl-5’- AMP intermediate, which binds to lysine residues on the proximal proteins. Peroxidases oxidize biotin-phenol substrates in the presence of hydrogen peroxide to generate biotin- phenoxyl radical which reacts with tyrosine residues in the proximal proteins. The biotinylation radius of the enzymes (~10 nm in living cells) as well as the short half-life of the intermediates favors the labeling of the proximal proteins over the distant proteins that do not interact with the protein of interest (comprehensively reviewed in (Gingras et al., 2019; Qin et al., 2021; Zhou & Zou, 2021)). The BioID biotin ligase (named for biotin identification) utilizes the Escherichia coli (E. coli) biotin ligase BirA. BioID is a mutated form of BirA which includes the R118G missense mutation. The mutated BioID overcomes the biotinylation specificity of BirA for the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase and allows BioID to promiscuously biotinylate all proximal lysine resides (Roux et al., 2012, 2013b). BioID2 was identified as an improved smaller form of BioID developed from the Aquifex aeolicus biotin ligase. BioID2 has the R40G mutation for promiscuous biotinylation of proximal lysines in interacting proteins in live cells. For efficient biotin labeling of proteins, BioID2 requires biotin supplementation and incubation for several hours (~16 -18 hours) or days as the protein labeling accumulates over time in living cells (Kim et al., 2016). APEX is a cytosolic plant ascorbate peroxidase (hence APEX) that was first engineered as a tag for electron microscopy (Martell et al., 2012) and was first used for proteome labeling in living cells to identify mitochondrial matrix proteins (Rhee et al., 15 2013). In 2014, APEX2 was developed from APEX to include the A134P mutation which improved the kinetics, stability, and cellular activity of the enzyme (Lam et al., 2014). The main difference between APEX2 and BioID2 is the significantly shorter time (~ 1 min) utilized for protein labeling by APEX2 in live cells, which allows for a shortened cross- sectional snapshot of the interactome in live cells. Also, APEX2 is a peroxidase which requires the addition of toxic hydrogen peroxide to the cells which limits its in vivo and long-term application (Qin et al., 2021; Samavarchi-Tehrani et al., 2020). In a recent study, the APEX2 approach and an antibody-based immunoprecipitation approach were used to study the tau protein interactome in human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) (Tracy et al., 2022). The i3Neurons were modified to express one of the following proteins a) APEX2-Tau, b) Tau-APEX2, c) APEX2- α Tubulin, d) WT-Tau-Flag, e) TauP301L-Flag, and f) TauV337M-Flag. The APEX2-based PL experiments was used to identify tau PPIs by fusing APEX2 to either the N-terminus or the C-terminus of the 2N4R human tau protein. An APEX2-α-tubulin was used as the control fusion protein. This PL approach identified a total of 246 tau interacting partners, of which 136 were identified in APEX2- Tau and Tau-APEX2, 68 were unique to APEX2-Tau, and 42 were identified only with Tau-APEX2. This interactome snapshot identified proteins localized to the cytoskeleton, synapse, ribonucleoprotein complex, somatodendritic compartment, autophagosome, ubiquitin proteasome system, and synaptic vesicles. The antibody-based affinity purification experiment was used to study the effects of the FTD mutations P301L and V337M on the tau protein interactome. Protein lysates were immunoprecipitated using the Flag tag antibody and identified by mass spectrometry. Compared with WT-Tau, 16 TauP301L exhibited increased interaction with 14 proteins and decreased interaction with 108 proteins. While TauV337M showed increased interaction with 69 proteins and decreased interaction with 184 proteins. The enriched proteins with TauP301L were involved in nucleoside metabolism while those enriched with TauV337M were members of the synaptic vesicle membrane and spliceosome complex. Interestingly, the decreased interactions with both TauP301L and TauV337M included ribosomal and mitochondrial proteins which were presumably disrupted by the two FTD mutations. 17 THESIS OBJECTIVE This thesis aims to develop a method to identify the protein interactome of tau. The microtubule-dependent and -independent functions of the tau protein are mediated by its interacting partners under physiological and pathological conditions. The identified tau protein interactome provides a comprehensive mapping of tau’s growing cellular functions in neurons which could help to develop novel tau-targeted therapeutic strategies for the tau-mediated neurodegenerative processes. I utilized the BioID2 biotin ligase approach which allows for the in situ biotin labeling in living neurons. The biotinylated proteins can then be identified by biotin-targeted pulldown with streptavidin and identified using mass spectrometry. I created fusion proteins between BioID2 and tau. BioID2 was fused to either the N-terminus of the 2N4R human tau protein (BioID2-Tau) or the C-terminus (Tau-BioID2). Both constructs featured a 13x-linker between BioID2 and tau which adds flexibility between the two proteins and minimizes the undesired interactions between them. Two control constructs were created, the Myc-BioID2 was used as a control for BioID2-Tau while the BioID2-HA was used as a control for Tau-BioID2. Lentiviruses expressing the BioID2 proteins were generated and used to transduce E18 TKO primary cortical mouse neurons. The long-term goal of this project is to provide an approach that will facilitate the detection of bona fide tau interactors that may be transient/weak interactors and/or difficult to detect using more traditional approaches. 18 Aim Statement Develop a novel Tau-BioID2 assay to map tau protein-protein interactions which mediate the cellular functions of tau under normal physiological conditions. Hypothesis Tagging BioID2 to either the N-terminus or C-terminus of full-length 2N4R human tau protein will reveal known as well as novel tau interacting partners in primary cortical neurons. 19 MATERIALS AND METHODS Creating Tau-BioID2 expression plasmids using Restriction Cloning The below methods were used to create plasmids that express the Tau-BioID2 fusion proteins. The BioID2-expressing plasmids MCS-13X Linker-BioID2-HA (BioID2-HA, Addgene, #80899) and Myc-BioID2-13X Linker-MCS (Myc-BioID2, Addgene, #92308) were a kind gift from the Kyle Roux lab (Kim et al., 2016). MCS-13X Linker-BioID2-HA plasmid was used to fuse BioID2 to the C-terminus of the tau protein (Tau-BioID2) while the mycBioID2-13X Linker-MCS plasmid was used to fuse BioID2 to the N-terminus of the tau protein (BioID2-Tau). Both plasmids feature a 13x-linker which is a serine-glycine repeat sequence that acts as a flexible spacer sequence which could increase the biotinylation range (Kim et al., 2016). The BioID2 constructs were cloned into the pFIN vector (Addgene, #44352) to generate lentiviruses for primary neuron transductions. The longest tau isoform, hT40 or the 2N4R isoform (Tau, 441 amino acids) was used for all experiments. All plasmid constructs were validated by restriction digestion, Sanger sequencing, and western blotting. Site-directed mutagenesis Single-site and multi-site mutagenesis reactions were performed using QuikChange Lightning site-directed mutagenesis Kit (Agilent Technologies, #210518 and #210516). The BioID2-HA plasmid was mutated to include a KpnI restriction site upstream of the BioID2 sequence and then again to include an EcoRV site and a Kozak sequence 20 uprstream of the KpnI site. Then a NotI restriction site was inserted downstream of the BioID2 sequence. The following primers were used: • KpnI site insertion: 5’- cggattcgaattcggatccGGTACCttttcggaattcggatccg-3’ • EcoRV site and Kozak sequence insertion: 5’-cggtcgtacgtctccgGATATCAAGCCACCATGggtaccttttcg-3’ • NotI site insertion: 5’-gatgtaccggattacgcatagGCGGCCGCcgctgatcagcctc-3’ The Myc-BioID2 plasmid was mutated to insert an EcoRV restriction site upstream of the BioID2 sequence and an NdeI restriction site downstream of the BioID2 sequence, and to delete the endogenous NdeI resitriction site upstream of the BioID2 sequence. The following primers were used: • EcoRV site insertion: 5’- cactatagggagacccaagcGATATCgccaccatggaac-3’ • NdeI site insertion: 5’- gtggatcggcgcgccgtCATATGaacctcgagc-3’ • NdeI site deletion: 5’- gcagtacatcaagtgtatcCGCGgccaagtacgccccctattgacg-3’ The following mutagenesis reaction protocol was used: 19.25 µl nuclease-free H2O, 2.5 µl QCL buffer, 0.75 µl Quiksolution reagent, 0.5 µl dNTP mix, 0.5 µl (10 µM) primers, 25 ng DNA template, and 0.5 µl QCL enzyme. The mutagenesis reaction was transferred into a thermal cycler (Applied Biosystems, #4375786) and the cycling parameters in Table 1 were used. After mutagenesis, 2 µl of Dpn I restriction enzyme was added to the reaction and incubated for 10 minutes at 37 °C. Dpn I enzyme digests the template double stranded DNA (i.e. methylated) in the reaction. 21 Table 1: Cycling parameters for mutagenesis reactions Step Number of Cycles Temperature Time 1 1 95 °C 2 minutes 2 30 95 °C 30 seconds 55 °C 30 seconds 65 °C 30 seconds/kilobase of plasmid length 3 1 65 °C 5 minutes 4 1 4 °C Infinity Preparation of LB/agar plates and LB broth medium The LB/agar was prepared by dissolving 10 grams of LB broth base (Sigma-Aldrich, #L3522), 7.5 grams of Agar (Sigma-Aldrich, #A1296) in 500 ml double-distilled water (ddH2O) and autoclave using a liquid cycle. LB/agar was cooled at room temperature, 50 mg ampicillin (Sigma-Aldrich, #A9518) were added while still warm, and approximately 20 ml of LB/agar were poured into petri dishes and left at room temperature to solidify. LB/agar plates were used directly or stored at 4°C. LB broth base was prepared by dissolving 10 grams of LB in 500 ml ddH2O. Transformation of XL10-Gold ultracompetent cells XL10-Gold ultracompetent cells (provided with QuikChange Lightning kit) were thawed on ice and then 40 µl of cells and 4 µl of DNA were transferred into a microcentrifuge tube and incubated on ice for 10 minutes. Cells were heat-shocked for 30 seconds at 42 °C and left on ice for 2 minutes. Pre-heated LB broth (360 µl) was added to the tube and cells were incubated for 1 hour at 37 °C with shaking at 300 rpm. The entire culture was 22 poured on one LB/agar plate, streaked to cover the plate, and incubated at 37 °C for 12- 16 hours. Transformation of Stbl3 competent E.coli For each transformation reaction, one vial of one shot Stbl3 cells (ThermoScientific, #C7373-03) was thawed on ice. 5 μl DNA were added to the cells and left on ice for 30 minutes. Stbl3 cells were heat-shocked at 42 °C for 45 seconds and placed on ice for 2 minutes. S.O.C medium (250 µl) was added to the Stbl3 cells, and the transformation reaction was incubated for 1 hour at 37 °C with shaking at 225 rpm. The entire culture was streaked to cover an LB/agar plate, and the plate was incubated at 37 °C for 12-16 hours. Plasmid DNA Miniprep Single colonies (5-10 colonies/construct) from the streaked LB/agar plates were picked using a micropipette tip and inoculated into a culture of 5 ml LB containing ampicillin (100 μg/ml) in a sterile glass test tube and incubated for 12-16 hours at 37 °C with shaking at 225 rpm. Bacterial cells were centrifuged at 8000 x g for 3 minutes at room temperature and supernatant was discarded. Plasmid DNA extraction was performed using QIAprep spin miniprep kit (Qiagen, #27104) following the manufacturer’s instructions. Plasmid DNA concentration was measured using NanoDrop spectrophotometer (ThermoScientific, #ND-2000). Validation of plasmid DNA was done by both restriction digestion and Sanger sequencing (Genewiz). 23 Plasmid DNA Maxiprep A single colony was picked from LB/agar plate, inoculated into a 5 ml LB sterile glass test tube containing ampicillin (100 μg/ml), and incubated overnight at 37 °C with shaking at 225 rpm. The 5ml bacterial culture was inoculated into 250 ml LB containing ampicillin (100 μg/ml) in a flask. The bacterial culture was left to grow overnight at 37 °C with shaking at 225 rpm. The bacterial cells were harvested by centrifugation at 6,000 x g for 15 minutes at 4 °C, and the plasmid DNA maxiprep was performed using the Qiagen plasmid maxi kit (Qiagen, #12162) following the manufacture’s protocol. Restriction Digestion Digestion reactions were prepared by adding 500 ng plasmid DNA samples, 1 μl of each restriction enzyme, 2 μl fast digest green buffer and nuclease-free H2O to a total volume of 20 μl. Digestion reaction was incubated for 1 hour at 37°C. Restriction enzymes used for the plasmids created were fast digest EcoRV (ThermoScientific, #FD0303), KpnI (ThermoScientific, #FD0524), NdeI (ThermoScientific, #FD0584), NotI (ThermoScientific, #FD0596), and XhoI (ThermoScientific, #FD0694). DNA Gel Electrophoresis One liter of 50X TAE buffer stock solution was prepared by adding 242.28 g Tris-base (Sigma-Aldrich, #10708976001), 57.2 ml glacial acetic acid, 100 ml of 500 mM EDTA buffer (pH 8.0) and adjusting the volume to one liter by adding ddH2O. One liter 0.5X TAE 24 buffer working solution was prepared by adding 10 ml 50X TAE buffer to 990 ml ddH 2O. Agarose gel (1%) was prepared by dissolving 2 grams of agarose (Bio-Rad, #1613101) in 200 ml 0.5X TAE buffer and microwaving for 2-4 minutes until fully dissolved, and adding 20 μl ethidium bromide (Bio-Rad, #1610433). The agarose gel was poured into a gel electrophoresis system (ThermoScientifc, #09-528-110B), and left to polymerize for 30-40 minutes at room temperature. 20 μl of 1kilobase DNA ladder (NEB, #N3232S) was added to the first well then 20 μl digestion reaction was added. The gel was run at 150 constant volts for 45-60 minutes and the DNA bands were visualized under a UV transilluminator (Accuris, #E3100). Gel purification and DNA ligation The DNA gels were placed on a UV transilluminator and the desired DNA band was cut out using a razor blade and placed it into a microcentrifuge tube. DNA gel extraction was performed using QIAquick Gel Extraction Kit (Qiagen, #28706) following manufacturer's instructions. Then, the ligation reaction was prepared by adding 2 μl of open plasmid backbone, 8 μl of the desired insert (with compatible overhangs), 1 μl of 10X T4 buffer, 1 μl of T4 DNA ligase enzyme (ThermoScientific, #EL0011), adjusting the final volume to 20 μl with ddH2O, and incubating overnight at 16 °C. The ligation reaction was used to transform a bacterial strain, purified using QIAprep spin miniprep kit (Qiagen, #27104), and validated by restriction digestion and Sanger sequencing (Genewiz). 25 Human embryonic kidney (HEK293T) cell culture Complete Dulbecco's Modified Eagle Medium (DMEM) (500 ml Gibco, DMEM #11995073, 5% fetal bovine serum (FBS), and 1% penicillin-streptomycin Sigma-Aldrich, #P0781) was prepared. Complete DMEM was filtered through a disposable vacuum 0.22 µm filter (Corning, #097611) and stored at 4 °C for up to one month. HEK293T cells were retrieved by thawing 2 x 10^6 cells in 37°C bead bath, centrifuged at 200 x g for 2 minutes, supernatant was discarded, and the cell pellet was resuspended in 5 ml complete DMEM. Cells were transferred to a T25 flask (ThermoScientific, #156340), and maintained for 4 days at 37 °C, 5% CO2 in a humidified incubator. For routine maintenance of cells, old medium was discarded, and monolayers of 80-90% confluent cells were detached by adding 1 ml 0.5% trypsin (Gibco, #15400054) and incubating for 5 minutes at 37 °C in the humidified incubator. Detached cells were collected in a 15 ml conical tube (Fisher scientific, #22010075), centrifuged at 200 x g for 2 minutes. Cell pellet was resuspended in 10 ml complete DMEM, split 1:10 in a new T75 flask (ThermoScientific, #156472), and maintained at 37°C, 5% CO2 in a humidified incubator. Transfection of HEK293T cells HEK293T cells (4.8 x 10^6) were plated in a poly-d-lysine-coated 12-well plate (Corning, #354470) and left overnight in a humidified incubator. Transfection reactions for each well in a 12-well plate were prepared by adding 1 μg plasmid DNA (expressing BioID2-only controls or Tau-BioID2 fusion proteins), 10 μl polyethylenimine, 200 μl of 150 mM NaCl, and was kept at room temperature for 20 minutes. HEK293T Cells were transected at 26 ~80% confluency by adding drops of the transfection mixture and were maintained for 24 hours in a humidified incubator. Finally, 100 μM biotin (Sigma-Aldrich, #B4501) was supplemented in complete DMEM and cells were incubated for 24 hours before collecting cell lysates. HEK293T protein lysate collection Cell culture media was removed, and cells were washed twice with 1x Dulbecco's phosphate-buffered saline (DPBS) (Gibco, #14200075). Cell lysates were collected in lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.4% SDS, 1% NP-40, 1 mM EGTA, and 1.5 mM MgCl2 supplemented with 1x protease inhibitors added immediately before use (pepstatin A, leupeptin, bestatin, aprotinin, and PMSF all purchased from ThermoScientific). Cell lysates were sonicated for 3 x 10 seconds (Misonix XL-2000 series) and centrifuged at 12,000 x g for 10 minutes at 4°C. Protein concentration was measured using Pierce rapid gold BCA protein assay kit (ThermoScientifc, #A53225) following manufacturer's instructions and protein lysates were stored at -80 °C until use. SDS-PAGE and Western blotting Lysates were prepared in 1x sample buffer (containing 2% Sodium dodecyl sulfate (SDS), 5% 2-mecaptoethanol, 10% glycerol, 0.002% bromophenol blue, 20 mM Tris-HCl pH 6.8) and heated to 95 °C for 5 minutes. Precast SDS-PAGE gels (Bio-Rad, #4561095) were used to separate proteins via electrophoresis in 1x running buffer (25 mM Tris base pH 8.3, 190 mM glycine, 0.1% SDS). Protein lysates (20 µg/lane) and protein standard 27 markers (1.5 µl/lane; Bio-Rad #1610373) were run for 32 minutes at constant 250 volts. Gels and nitrocellulose transfer membrane (Bio-Rad, #1620213) were soaked for 5 minutes in 1x transfer buffer (25 mM Tris pH 8.3, 192 mM glycine, and 20% methanol). Proteins were transferred from the gel onto a nitrocellulose membrane by running the transfer cassette for 50 minutes at constant 400 milliampere. Membranes were blocked in 2% non-fat milk for 1 hour at room temperature. Membranes were incubated in primary antibodies diluted in the blocking buffer overnight at 4 °C. Membranes were washed three times in 1x TBS-T (50 mM Tris, 150 mM NaCl, pH 7.4, 1% Tween-20) and then incubated in secondary antibodies IRDye 680LT goat anti-mouse IgG and IRDye 800CW goat anti- rabbit IgG, diluted in the blocking buffer (1:20,000). Membranes were washed three times in 1x TBST and imaged using LI-COR Odyssey® infrared system. Generation of Lentiviruses that express the BioID2 proteins Lentiviruses production in HEK293T cells For each lentiviral preparation, 4 x 150 mm cell culture dishes (Corning, #430599) were plated at density 1 x 10^6 cells/dish in 25 ml complete DMEM. Culture dishes were incubated overnight in a humidified incubator. Two hours prior to transfection, DMEM was removed, and fresh DMEM was added. The following transfection reactions were then prepared for each 150 mm culture dish: 45 μg plasmid DNA (22.5 μg of the BioID2 plasmid DNA, 15 μg of pNHP packaging vector (Addgene, #22500), 7.5 μg of pHEF- VSVG envelope vector (Addgene, #22501), 300 μl polyethylenimine and 6 ml of 150 mM NaCl. The transfection reagent was incubated for 20 minutes at room temperature, slowly 28 added to each culture dish, then transfected HEK293T cells were maintained overnight in a humidified incubator. Complete DMEM was substituted by freshly prepared viral medium (98% DMEM, 1% FBS, and 1% penicillin-streptomycin) and transfected HEK293T cells were maintained for 48 hours in a humidified incubator. Medium containing lentiviruses was transferred to a 50 ml conical tube, centrifuged at 675 x g for 5 minutes and the supernatant containing lentiviruses was filtered through a 0.45 μm filter. Lentiviruses were harvested by layering on 20% sucrose solution, and ultracentrifugation at 82,700 x g for 2 hours at 4°C using Sorvall™ WX+ ultracentrifuge (ThermoScientific, #75000100). The lentiviral pellets were resuspended in 500 μl sterile PBS, aliquoted, snap-frozen in crushed dry ice, and stored at -80°C until use. Immunocytochemistry (ICC) HEK293T cells were plated in a 24-well plate at a density of 75,000 cells/well and incubated overnight. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, #15714) prepared in 1x cytoskeleton buffer (10 mM MES, 138 mM KCl, 3 mM MgCl2, 4 mM EGTA pH 6.1, all from Sigma-Aldrich). Cells were rinsed four times by 1x TBS and incubated for 1 hour at room temperature in blocking buffer containing 5% goat serum (GS, VWR#10152-212),1% bovine serum albumin (Gemini Bio Labs#700-101P), 0.2% Triton-X100 (Bio-Rad, #161-0407) prepared in 1x TBS. Cells were incubated in primary antibodies diluted in 2% GS-1x TBS and maintained overnight at 4 °C. Cells were rinsed four times by 1x TBS and were incubated for one hour in biotinylated secondary antibodies diluted 1:500 in 2% GS. Avidin-biotinylated horseradish peroxidase (HRP) complex (ABC, ThermoScientific, #32020) was prepared by adding 1 drop of each 29 reagent A and B to 10 ml 1x TBS and incubating for 30 minutes at 4 °C. Cells were rinsed four times in 1x TBS and were incubated in ABC solution or 1 hour at room temperature. The peroxidase substrate was prepared by adding 2.5 mg DAB (Sigma-Aldrich, #281751) to 5 ml 1x TBS and then adding 0.5 μl 30% H2O2. Cells were rinsed four times in 1x TBS and the peroxidase substrate was added for 5 minutes at room temperature. The assay development was stopped by removing the substrate and rinsing four times in 1x TBS. Cells were visualized under inverted light microscope Nikon eclipse TE2000-U. Lentiviral functional titer determination using ICC HEK293T cells were transduced by lentiviruses expressing either the fusion proteins Tau- BioID2, BioID2-Tau or the respective controls BioID2-HA, Myc-BioID2. Cells were transduced by lentiviruses at 10-fold dilutions from 1:10^2 to 1:10^9 and maintained for 72 hours in a humidified incubator. Cells were fixed in 4% paraformaldehyde and the ICC protocol was followed as described above. HA-tag and Myc-tag primary antibodies were used at dilutions 1:4,000 in 2% GS. Biotinylated goat anti-mouse IgG and goat anti-rabbit IgG secondary antibodies were diluted 1:500 in 2% GS. The lentiviral functional titer was calculated by multiplying the number of transduced cells at the highest viral dilution by the dilution factor. Lentivirus functional titer (transducing unit/μl) = # transduced cells in the highest viral dilution x the dilution factor 30 Embryonic day 18 mouse primary cortical neuron culture preparation Timed-pregnant female TKO mice (JAX, 007251) were euthanized by intraperitoneal injection of 50-100 mg sodium pentobarbital diluted in saline per kg. Mouse pups were collected on embryonic day 18 (E18) and kept in ice-cold 0.9% saline. Pups were decapitated and heads were isolated and placed on a 60 mm dish containing ice-cold saline and placed under a dissecting microscope. Skin and skull were removed, and the brain tissue was extracted. The cerebellum was discarded, the two hemispheres were separated, and the cerebral cortices were dissected. Cortices from all pups were cut into small pieces and collected in a tube containing ice-cold calcium- and magnesium-free solution (CMF; contains 1x DPBS, 1x amphotericin B (Gibco, #15290018), 1x gentamicin (Gibco, #15750060), and 10% glucose (Sigma-Aldrich, #G8270)). Where indicated, E18 WT C57/BL6 (BrainBits LLC, C57ECX) cortical tissue was used where indicated. The E18 cortical tissue pieces were washed four times in CMF and incubated in 0.25% trypsin solution (Gibco, #15090046) for 15 minutes at 37 °C. Trypsin was removed and cortices were washed two times in CMF. Trypsin inactivation solution (3 ml) was added containing 2.1 ml Hank’s Balanced Salt Solution (Gibco TM#24020117), 0.6 ml newborn calf serum (GibcoTM#16010167), and 0.3 ml DNase solution (Worthington# LS002006). A homogenous cell suspension was obtained by gentle trituration of the tissue (30 x 14-gauge needle, 30 x 15-gauge needle, 20 x 16-gauge needle, 20 x 18- gauge needle, and 15 x 21-gauge needle). Cell suspensions were layered onto 5 ml sterile-filtered FBS and centrifuged at 200 x g for 5 minutes. Primary neuron cell pellets were resuspended in 1ml complete neurobasal medium containing 96% neurobasal media (NBM; Gibco, #21103049), 1% L-glutamine (Gibco, #25030081), 1% amphotericin 31 B (Gibco, #15290018), 1% B-27 Supplement (Gibco, #17504044), and 1% gentamicin (Gibco, #15750060). Cell counts were determined using Countess™ 3 automated cell counter (Invitrogen, #AMQAX2000), primary neurons were plated at density 3.6 x 10^6 cells per plate and maintained in a humidified incubator by adding 10% NBM twice a week. Immunocytofluorescence (ICF) Primary neurons were fixed in 4% paraformaldehyde for 20 minutes, washed three times in 1x TBS, and blocked for one hour in blocking buffer (5% GS, 1% bovine serum albumin, 0.2% Triton-X100 diluted in 1xTBS). Cells were then incubated overnight in primary antibodies diluted in 2% GS. Cells were washed three times in 1xTBS then incubated in secondary antibodies diluted 1:500 in 2%GS for one hour at room temperature. Cells were washed three times in 1xTBS and counterstained using DAPI counterstain diluted 1:10,000 in 1xTBS. Cells were visualized under inverted microscope Nikon eclipse Ti2. Lentivirus transduction efficiency E18 WT primary cortical mouse neurons were transduced on 4th day in vitro (DIV) by lentiviruses expressing the BioID2 proteins (Tau-BioID2, BioID2-Tau, Myc-BioID2, and BioID2-HA). For each lentiviral construct, primary neurons were transduced at multiplicity of infection (MOI) of 50,100, and 200. On DIV9, primary neurons were fixed and ICF was performed as described above. Primary antibodies used were HA-tag or Myc-tag antibodies (diluted 1:800 in 2%GS), and Tau13 antibody diluted (1:5,000 in 2%GS). 32 Secondary antibodies used were Alexa Fluor 568 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG (diluted 1:500 in 2%GS). Transduction efficiency was calculated by dividing the number of transduced cells by the total number of cells (identified by the DAPI nuclear counterstain). 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑟𝑎𝑛𝑠𝑑𝑢𝑐𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 Transduction efficiency = 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 Lentiviral transduction in neurons On DIV4, primary neurons were treated with lentiviruses expressing the fusion proteins BioID2-Tau, Tau-BioID2 or the respective controls Myc-BioID2, and BioID2-HA. Lentiviruses were diluted in complete NBM, and primary neurons were transduced at MOI 200. Every other day, 10% NBM was supplemented, and the primary neuronal culture was checked for the toxicity of the lentiviral vectors expressing the BioID2 proteins. The neuronal culture was maintained for four days before supplementing exogenous biotin. This allows sufficient time for the lentiviral genome reverse transcription, integration into the host cell DNA and expression of the proteins of interest using the host cell transcription and translation machinery. Endogenous tau vs lentiviral tau expression levels E18 WT and TKO primary cortical neurons were plated at density 150,000 cells/well in a 24-well plate (n=3 technical replicates). On DIV4, TKO neurons were transduced by lentivirus expressing Tau-BioID2. Both neuronal cultures were maintained by adding 10% 33 NBM every other day. On DIV12, primary neurons were fixed and ICF was performed as described above with Tau5 primary antibody (diluted 1:5,000 in 2%GS) and Alexa Fluor 568 goat anti-mouse IgG secondary antibody (diluted 1:500 in 2%GS). To quantify tau protein expression level, fluorescent signals were imaged at 20x magnification (0.9 mm field of view). Images were randomly selected within the well ( 5 images/well and 3 wells/condition) using identical image acquisition settings (1 second exposure, all other parameters at the default). Image analysis was preformed using FiJi software (Schindelin et al., 2012) by setting the minimum intensity threshold at 12,000 and the maximum intensity threshold at 60,000. Signal intensity was assessed within each image using the analyze particles function and taking the average pixel intensity values for comparison between WT cells and Tau-BioID2 transduced TKO neurons. Mass Spectrometry identification of biotinylated proteins Optimization of Biotin dose and incubation time E18 TKO primary cortical neurons were transduced on DIV4 by lentivirus expressing BioID2-Tau. On DIV8, exogenous biotin prepared in neurobasal medium was supplemented in the following doses 0, 50, or 100 μM. Cells were incubated for either 24, 48 or 96 hours in a humidified incubator. Three biological replicates were performed for each biotin dose and incubation time condition. Protein lysates were collected and 10 μg was used for western blotting. Membranes were blocked in 2% non-fat milk for 1 hour at room temperature and incubated in anti-biotin primary antibody diluted 1:1,000 overnight at 4°C. Membranes were washed three times in 1x TBS-T and incubated in Li-cor IRDye 34 800CW goat anti-rabbit secondary antibody diluted 1:20,000. The blots were imaged using LI-COR Odyssey® infrared system. The biotin signal was quantified using LI-COR Image StudioTM lite version 5.2. Primary neurons protein lysate collection E18 TKO primary cortical neurons were plated at density 3.6 x 10^6 cells per plate. On DIV4, each plate was transduced by lentivirus at MOI 200 (n=3 independent experimental replicates for each lentiviral transduction). On DIV8, 100 μM biotin was supplemented. On DIV12, cells were washed twice in 1x DPBS and protein lysates were collected in 1ml lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.4% SDS, 1% NP-40, 1 mM EGTA, and 1.5 mM MgCl2 supplemented with 1x protease inhibitors immediately before use (pepstatin A, leupeptin, bestatin, aprotinin, and PMSF). Lysates were sonicated 3 x 10 seconds and centrifuged at 12,000 x g for 10 minutes at 4°C. Protein concentration was measured using Pierce™ rapid gold BCA protein assay kit. Biotin-streptavidin affinity pulldown Ethanol-clean low retention microcentrifuge tubes (ThermoScientific, #3453) were placed in a magnetic separation stand. Streptavidin magnetic Dynabeads C1 (400 μl; Invitrogen, #65002) were used per condition. The beads were washed twice in 1ml lysis buffer and then 1 mg of protein lysate was added to the beads and rotated overnight at room temperature. The following day unbound supernatant proteins were collected as the post- pulldown sample and stored at 4°C until further analysis. The beads containing bound 35 biotinylated proteins were washed twice in lysis buffer, then resuspended in 1ml 50 mM TrisCl, pH 7.4. 900 μl was transferred to a new low retention tube for mass spectrometry analysis while the remaining 100 μl was transferred to another tube for western blotting validation. The tube containing the 100 μl for western blotting was placed on a magnetic separation stand and the supernatant was discarded. The beads were resuspended in elution buffer containing 25 mM biotin prepared in lysis buffer. Efficient elution of biotinylated proteins was performed by competition with free biotin in the elution buffer and heating to 95°C for 15 minutes. The tube was placed on a magnetic separation stand and the supernatant containing biotinylated proteins was transferred to a new tube and western blotting validation was performed as described above using anti-biotin antibody. Liquid Chromatography-MS/MS The tube containing the 900 μl was placed on magnetic separation stand and the supernatant was discarded. The beads were washed six times in 25 mM ammonium bicarbonate (pH 8) and then resuspended in 150 μl of 25 mM ammonium bicarbonate - 50% acetonitrile (ACN). On-bead protein digestion was performed by adding 100 ng of rlys-C enzyme (Promega, #V1671) and incubating for 90 minutes at 37°C then adding 3 μg trypsin (Promega, #V5280) and incubating for 16-18 hours at 37°C. The tubes were placed on magnetic separation band and the digestion solution was collected in a new low retention tube. The samples were dried to completion in a speed vacuum centrifuge 36 at 30 °C and the samples were resuspended in 50 μl of 25 mM ammonium bicarbonate /5% ACN. NanoLC-MS/MS separations were performed with a Thermo Scientific™ Ultimate™ 3000 RSLCnano System. Peptides were desalted in-line using a 3 μm diameter bead, C18 Acclaim™ PepMap™ trap column (75 μm × 20 mm) with 2% ACN, 0.1% formic acid (FA) for 5 min with a flow rate of 5 μl/min at 40°C. The trap column was then brought in line with a 2 μm diameter bead, C18 EASY-Spray™ column (75 μm × 250 mm) for analytical separation over 60 min with a flow rate of 350 nl/min at 40°C. The mobile phase consisted of 0.1% FA (buffer A) and 0.1% FA in ACN (buffer B). The separation gradient was as follows: 5 min desalting, 40 min 4–40% B, 2 min 40–65% B, 2 min 65–95% B, 7 min 95% B, 1 min 95–4% B, 3 min 4% B. One microliter of each sample was injected. Top 20 data-dependent mass spectrometric analysis was performed with a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer. MS1 resolution was 60K at 200 m/z with a maximum injection time of 45 ms, AGC target of 3e6, and scan range of 300–1500 m/z. MS2 resolution was 30K at 200 m/z, with a maximum injection time of 54 milliseconds, AGC target of 1e5, and isolation range of 1.3 m/z. HCD normalized collision energy was 28. Only ions with charge states from +2 to +6 were selected for fragmentation, and dynamic exclusion was set to 30 s. The electrospray voltage was 1.9 kV at a 2.0 mm tip to inlet distance. The ion capillary temperature was 280°C and the RF level was 55.0. All other parameters were set as default. 37 Mass spectrometry protein identification Raw mass spectrometry data were searched with Sequest HT against the reviewed Mus musculus Uniprot proteome database (UP000000589, 25285 unique sequences) with Thermo Scientific Proteome Discoverer software (version 2.5). Enzyme specificity was set to trypsin with an MS1 tolerance of 10 ppm and a fragment tolerance of 0.02 Da. Oxidation (M), biotinylation (K), acetylation (protein N-term), methionine loss (protein N- term), and biotinylation (protein N-term) were set as dynamic modifications. Peptides and proteins were filtered by 1% false discovery rate with threshold determined via decoy search using the Percolator algorithm. At least two peptide identifications were required to identify a protein. All other parameters were set at the defaults. Label-free quantification of identified proteins Label-free quantification was performed using Proteome Discoverer software (version 2.5). Peptide abundance was calculated based on the peak intensity of the peptide spectral matches. Peptide abundance was normalized to the sample group with highest peptide abundance. The protein abundance ratio was calculated using the pairwise ratio approach by measuring the abundance ratios for each peptide observed in both the tau sample and its respective control and selecting the median ratio as the protein abundance ratio. The following criteria were used to define proteins as Tau-BioID2 or BioID2-Tau interactors: 1) being identified in at least two of the independent replicates, and 2) being detected at ≥1.5-fold increase compared to the respective BioID2 control. Proteins identified in only one independent replicate as well as background proteins including 38 keratins, and endogenously biotinylated carboxylases were removed from further analysis. Functional protein association networks Functional protein-protein interaction networks were visualized by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis. STRING V11.5 was used to visualize the protein interactome network including both functional associations retrieved from published databases, text mining, computational prediction methods and physical interactions retrieved from experimental data (genetic, biochemical, and biophysical techniques) with a minimum required confidence interaction score set at 0.7 and above (high confidence interaction) (Szklarczyk et al., 2015). The network map was then imported to the stringApp (Doncheva et al., 2019) (a built-in app in Cytoscape V.3.9.1) (Cline et al., 2007; Paul Shannon et al., 1971), the Cytoscape app ClusterMaker Cluster Network (J. H. Morris et al., 2011) was used to perform Markov Clustering (MCL) (Brohée & van Helden, 2006) with inflation value of 3.0 to reduce the cluster size. The string enrichment app (Cytoscape) was used to retrieve the functional enrichment analysis visualized on the protein network map with false discovery rate set at 1%, for simplicity. GO cellular component was added to the map to visualize the intracellular localization of the mapped proteins (Doncheva et al., 2019). 39 Gene Ontology enrichment analysis GO enrichment analyses were performed using ClueGo (V.2.5.8) (Bindea et al., 2009) app plugged in Cytoscape V.3.9.1. I performed ClueGO cellular component, molecular function pathways and KEGG pathways with GO interval minimum level of 3 and maximum level of 9 showing only pathways with p-value ≤0.05. For KEGG pathway analysis, only diseases with more than 26 identified proteins were included. All other parameters were set at the defaults. Validation of tau protein interacting partners TKO primary cortical neurons (E18) were cultured at density 150,000 cells/well in a 24- well plate. Primary neurons were transduced by lentiviruses at MOI 200 on DIV4. Transduced neurons were maintained in a 37°C, 5% CO2 humidified incubator. Protein lysates were collected on DIV12 in the high-concentration detergent lysis buffer and protein concentration was measured using Pierce rapid gold BCA protein assay kit (ThermoScientifc, #A53225). For each neuronal lysate, 250 μg proteins were rotated overnight at room temperature with 200 μl streptavidin magnetic Dynabeads C1 (Invitrogen, #65002). Validation of captured biotinylated proteins was done by probing for potential tau interacting partners in the elution samples using standard SDS-PAGE and western blotting. The protein lysate collection, biotin-streptavidin affinity pulldown, and western blotting were performed as described above. 40 Antibodies Primary antibodies used were anti-biotin (Cell Signaling Technology Cat# 5597, RRID: AB_10828011), anti-BioID2 (Abcam, #ab232733), HA-tag antibody (Cell Signaling Technology Cat#3724, RRID: AB_1549585), Myc-tag antibody (Cell Signaling Technology Cat#2276, RRID:AB_331783), and GAPDH (Cell Signaling Technology Cat#5174, RRID:AB_10622025). Tau antibodies used were produced in-house, including Tau13 (Combs et al., 2016; García-Sierra et al., 2003) (amino acids 8-9, 13-21; in-house, RRID: AB_2721193), and Tau5 (Carmel et al., 1996; Kanaan & Grabinski, 2021; LoPresti et al., 1995; Porzig et al., 2007) (amino acids 218-225; in-house, RRID: AB_2721194). For validation experiments of identified proteins, antibodies used were MAP2 (Cell Signaling Technology Cat#8707, RRID: AB_2722660), and MAP6 (Cell Signaling Technology Cat#4265, RRID: AB_2140993). Secondary antibodies used were IRDye 680LT goat anti-mouse IgG (LI-COR Biosciences Cat#926-68020, RRID:AB_10706161), IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences Cat#926-32211, RRID:AB_621843), Alexa Fluor 568 goat anti-mouse IgG (Thermo Fisher Scientific Cat#A-11031, RRID:AB_144696), Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific Cat#A-11034, RRID:AB_2576217), biotinylated goat anti-mouse IgG (Jackson ImmunoResearch Labs Cat#115-065-166, RRID:AB_2338569), and biotinylated goat anti-rabbit (Vector Laboratories Cat#BA-1000, RRID:AB_2313606). For western blotting, Tau13 and Tau5 were used at dilution 1:100,000 while all commercial antibodies were used at dilution 1:1000. For ICC, HA-tag, and Myc-tag antibodies were diluted 1:800. For ICF, Tau13 and Tau5 were diluted 1:5,000 while HA-tag and Myc-tag antibodies were diluted 1:800. 41 Data representation and statistical analysis Statistical tests and bar graphs were done using GraphPad Prism (V.8.0.1), GraphPad Software, La Jolla California USA, www.graphpad.com. Fluorescent microscopy images were taken using NIS-Elements BR (V.5.02.00, 64-bit) software, image analysis and quantification was preformed using FiJi (Schindelin et al., 2012). Western blot quantification was performed using LI-COR Image StudioTM lite version 5.2. Mass spectrometry label free quantification was performed using Thermo Scientific Proteome Discoverer software (PD; V 2.5). Lists of identified proteins were curated and filtered according to the defined criteria using RStudio (V.1.4.1717) (RStudio Team, 2021). Schematic figures were created with BioRender.com. 42 RESULTS Generation and validation of Tau-BioID2 expression plasmids and lentiviruses The BioID2-HA plasmid was used to create the Tau-BioID2 construct with BioID2 fused to the C-terminus of the longest human tau isoform (Tau, 2N4R or hT40 isoform with 441 amino acids) and served as the control protein. The Myc-BioID2 plasmid was used to create the BioID2-Tau construct with BioID2 fused to the N-terminus of Tau and served as the control protein (Figure 1). All plasmid DNA sequences were validated by restriction digestion and Sanger sequencing (GENEWIZ). Figure 1: Design of the BioID2 constructs. Two fusion and two BioID2-only control constructs were created. Fusion constructs are BioID2 tagged to either the N-terminus or the C-terminus of Tau sequence to create BioID2-Tau or Tau-BioID2, respectively. The control constructs were designed to express only the BioID2 protein. The Myc-BioID2 construct was used as a control for BioID2-Tau and the BioID2-HA construct was used as a control for Tau-BioID2. All constructs featured a 13xlinker between tau and BioID2. BioID2-Tau and Myc-BioID2 had a Myc tag on the N-terminus. Tau-BioID2 and BioID2- HA had an HA tag on the C-terminus. 43 Mutagenesis of the BioID2 expression plasmids and creating the Tau-BioID2 fusion proteins The BioID2-HA plasmid was mutated to insert EcoRV, Kozak sequence, and KpnI restriction sites upstream of the 13xLinker-BioID2 sequence and NotI restriction site downstream of the BioID2-HA sequence as shown in Figure 2A. Mutagenesis reactions were validated by restriction digestion to confirm the presence of newly inserted sites (Figure 2A). Plasmid DNA from two bacterial colonies were screened first for KpnI and NotI restriction sites insertion, one plasmid DNA had only the NotI site insertion while the other plasmid DNA had both restriction sites inserted (Figure 2A, red box). Then, the plasmid DNA was screened for EcoRV restriction site insertion. DNA gel shows successful insertion of all restriction sites (Figure 2A). Single cut plasmid DNA shows the linearized plasmid compared to the uncut supercoiled plasmid while the double cut plasmid shows the 13xLinker-BioID2 region of interest at 973 base pairs (Figure 2A, bottom panel red box). The final BioID2-HA plasmid that passed restriction digestion analysis was further confirmed by Sanger sequencing. To create the Tau-BioID2 fusion protein, both pCMV-Tau and the mutated BioID2- HA plasmids were cut by KpnI and NotI (Figure 2B). DNA bands of the open pCMV plasmid and the BioID2-HA insert were extracted from the gel, ligated, and transformed into XL10-Gold ultracompetent cells. Plasmid DNA from two colonies were extracted and screened using EcoRV and NotI restriction digestion, one plasmid DNA showed successful ligation of the Tau-BioID2 sequence at 2,293 base pairs (Figure 2B, red box). The final Tau-BioID2 plasmid that passed restriction digestion analysis was further confirmed by Sanger sequencing. 44 The Myc-BioID2 plasmid was mutated to insert an EcoRV restriction site upstream of the BioID2-13xLinker sequence and an NdeI restriction site downstream of the BioID2- 13xLinker sequence, then the endogenous NdeI restriction site upstream of the BioID2- 13xLinker sequence was deleted as shown in Figure 2C. Plasmid DNA was screened first for EcoRV and NdeI restriction sites insertion. The DNA gel EcoRV/NdeI single cut shows two bands for the open plasmid and the region between the inserted EcoRV/NdeI sites and the endogenous NdeI site (Figure 2C). The DNA plasmid was then validated for the deletion of the endogenous NdeI restriction site. NdeI single cut shows only the linearized plasmid which indicates the deletion of the endogenous NdeI site, while the EcoRV and NdeI double cut shows the BioID2-13xLinker region of interest (Figure 2C, red box). The final Myc-BioID2 plasmid that passed restriction digestion analysis was further confirmed by Sanger sequencing. To create the BioID2-Tau fusion protein, the pT7c-Tau and the mutated Myc- BioID2 plasmids were cut with NdeI and XhoI (Figure 2D). The tau band (from the pT7c plasmid) and the open Myc-BioID2 plasmid were extracted from the gel, ligated, and transformed into XL10-Gold ultracompetent cells. Plasmid DNA was extracted and screened using EcoRV and NotI restriction digestion. Plasmid DNA showed successful ligation of the BioID2-13xLinker-Tau sequence at 2,320 base pairs (Figure 2D, red box). The final BioID2-Tau plasmid that passed restriction digestion analysis was further confirmed by Sanger sequencing. 45 Figure 2: Restriction cloning of the BioID2 fusion proteins. A) Map illustrations for the mutated BioID2-HA plasmid show the insertion of the EcoRV, Kozak and KpnI sites. Restriction digestion validation gels show a purified plasmid that does not contain both restriction sites (lanes 2-5) and the plasmid used for further construct generation that correctly included both KpnI and NotI restriction sites (lanes 5-8). Lane 1 is the DNA 46 Figure 2 (cont’d) size ladder. The red box shows the expected digestion product with both sites inserted. The other validation gel shows the insertion of the EcoRV restriction, and the final expected digestion product is shown in the red box. B) Cloning of the Tau-BioID2 construct. The fusion protein was created by replacing the Flag tag in the pCMV-Tau plasmid by the BioID2-HA sequence. The pCMV-Tau and the BioID2-HA plasmids were cut by KpnI and NotI, the insert and open plasmid were extracted and ligated. Restriction digestion validation gel shows the purified ligated plasmid containing the Tau-BioID2 fusion protein (red box). C) Map illustrations for the mutated Myc-BioID2 plasmids show the insertion of EcoRV and NdeI restriction sites, and deletion of endogenous NdeI site. The red box shows the final expected digestion product with insertion of both EcoRV and NdeI restriction sites and deletion of the endogenous NdeI restriction site. D) Cloning of the BioID2-Tau construct. The fusion protein was created by inserting the tau sequence from the pT7c-Tau plasmid into the mutated Myc-BioID2 plasmid. Both plasmids were cut by NdeI and XhoI, the tau insert and the Myc-BioID2 open plasmid were ligated. Restriction digestion validation gel shows the purified ligated plasmid containing the BioID2-Tau fusion protein (red box). All plasmid DNA sequences were further validated by Sanger sequencing (GENEWIZ). 47 Creating BioID2 lentiviral transfer plasmids The Tau-BioID2 fusion proteins and the respective BioID2-only controls were cloned into the pFIN vector to generate lentiviruses. The pFIN vector is the lentiviral transfer plasmid which encodes the protein of interest as well as the human immunodeficiency virus-1 Gag, RRE, and WPRE sequences flanked by the 5’- and 3’- long terminal repeats. The pFIN-CBA-Tau plasmid was cut by EcoRV and NotI to open the pFIN-CBA plasmid backbone and remove the tau sequence, allowing ligation of the the Tau-BioID2 and BioID2-Tau inserts as well as the BioID2-HA and Myc-BioID2 control inserts. To create the pFIN-BioID2-HA and the pFIN-Tau-BioID2, the BioID2-HA and Tau- BioID2 sequences were cut by EcoRV and NotI from the mutated BioID2-HA plasmid and the pCMV-Tau-BIoID2 plasmid respectively (Figure 2 A and B). The DNA bands of the pFIN open plasmid, the BioID2-HA and the Tau-BioID2 inserts were extracted from the gel, ligated, and transformed into Stbl3 competent E. coli which reduce the homologous recombination events of the long terminal repeats in the pFIN vector. The ligated plasmid DNA was extracted and screened by EcoRV and NotI restriction digestion. Plasmid DNA showed successful ligation of the BioID2-HA and Tau-BioID2 into the pFIN plasmid backbone (Figure 3A, red box). The final pFIN-BioID2-HA and pFIN-Tau-BioID2 plasmids that passed restriction digestion analysis were further confirmed by Sanger sequencing. To create the pFIN-Myc-BioID2 and the pFIN-BioID2-Tau, the Myc-BioID2 and BioID2-Tau sequences were cut by EcoRV and NotI from the mutated Myc-BioID2 plasmid and the Myc-BioID2-Tau plasmid respectively (Figure 2 C and D). The DNA bands of the pFIN open plasmid, the Myc-BioID2 and the BioID2-Tau inserts were extracted from the gel, ligated, and transformed into Stbl3 competent E. coli. The ligated 48 plasmid DNA was extracted and screened by EcoRV and NotI restriction digestion. Plasmid DNA showed successful ligation of the Myc-BioID2 and BioID2-Tau into the pFIN plasmid backbone (Figure 3B, red box). The final pFIN-Myc-BioID2 and pFIN-BioID2-Tau plasmids that passed restriction digestion analysis were further confirmed by Sanger sequencing. Prior to packaging into lentiviruses, the pFIN plasmids were further validated for the mammalian cell expression of the BioID2 constructs. HEK293T cells were transfected with either pFIN-BioID2-HA, pFIN-Tau-BioID2, pFIN-Myc-BioID2 or pFIN-BioID2-Tau plasmids and were maintained overnight. The next day, biotin (100 μM) was supplemented and the HEK293T cells were maintained overnight before collecting the protein lysates. Expression and functionality of the BioID2 proteins were validated by western blotting and probing the membranes with BioID2 and biotin antibodies. As expected, BioID2 antibody showed reactive bands were for Myc-BioID2, BioID2-Tau, BioID2-HA and Tau-BioID2 at ~37, ~90, ~37, and ~90 kiloDaltons, respectively (Figure 3C). In addition, biotin antibody showed that the expressed BioID2 proteins were functional (Figure 3C). 49 Figure 3: Validation the lentiviral transfer plasmids expressing the BioID2 constructs. A) Cloning of the BioID2-HA and the Tau-BioID2 sequences into the pFIN plasmid backbone. pFIN-BioID2-HA and pFIN-Tau-BioID2 plasmids were created by replacing tau sequence in the pFIN-Tau plasmid with the BioID2-HA and the Tau-BioID2 sequences. pFIN-Tau, mutated BioID2-HA, and pCMV-Tau-BioID2 50 Figure 3 (cont’d) plasmids were cut by EcoRV and NotI. The pFIN open plasmid, the BioID2-HA insert, and the Tau-BioID2 insert DNA bands were extracted from the gel and ligated. EcoRV and NotI restriction digestion validation gel shows the purified ligated plasmids containing the BioID2-HA and the Tau-BioID2 fusion protein (red boxes). B) Cloning of the Myc-BioID2 and the BioID2-Tau sequences into the pFIN plasmid backbone. pFIN-Myc-BioID2 and pFIN-BioID2-Tau plasmids were created by replacing tau sequence in the pFIN-Tau plasmid by the Myc-BioID2 and the BioID2-Tau sequences. pFIN-Tau, mutated Myc- BioID2, and Myc- BioID2-Tau plasmids were cut by EcoRV and NotI. The pFIN open plasmid, the Myc-BioID2 insert, and the BioID2-Tau insert DNA bands were extracted from the gel and ligated. EcoRV and NotI restriction digestion validation gel shows the purified ligated plasmids containing the Myc-BioID2 and the BioID2-Tau fusion protein (red boxes). C) HEK293T cells were transfected by pFIN-Myc-BioID2, pFIN-BioID2-Tau, pFIN-BioID2-HA, and pFIN-Tau-BioID2 plasmids. Exogenous biotin was supplemented to validate the functionality of the BioID2 proteins. The expression and functionality of the BioID2 proteins was confirmed by western blotting. Antibodies used were anti-BioID2 (green) and anti-biotin (red) primary antibodies.……………………………………………… 51 Lentiviruses generation and determination of the functional titer To generate lentiviruses (LV) expressing the BioID2 proteins, HEK293T cells were transfected with the lentiviral transfer plasmids (encodes BioID2 proteins), the pNHP packaging vector (encodes for Gag, Pol, Rev, and Tat), and the pHEF-VSVG envelope vector (encodes VSV-G envelope protein). Lentiviruses were harvested by centrifugation and the lentiviral functional titer was determined by ICC of transduced HEK293T cells. The functional titer for each lentivirus was as follows: a) the Tau-BioID2 LV was 3.70x107 TU/μl, b) BioID2-HA LV was 3.45x107 TU/μl, c) BioID2-Tau LV was 2.85x107 TU/μl, and d) Myc-BioID2 LV was 1.42x107 TU/μl (Figure 4A-B). To further validate the expression of the BioID2 proteins, HEK293T cells were transduced at MOI of 50 by Myc-BioID2, BioID2-HA, BioID2-Tau, or Tau-BioID2 lentiviruses. Lysates were analyzed using western blotting with Myc tag and HA tag antibodies and the protein bands for Myc-BioID2, BioID2- HA, BioID2-Tau, and Tau-BioID2 were found at the predicted molecular weights ~37, ~37, ~90, and ~90 kiloDaltons, respectively (Figure 4C). Moreover, to validate the expression of tau protein, HEK293T cell lysates were analyzed using western blotting with Tau13 antibody. The protein bands for BioID2-Tau and Tau-BioID2 show that lentiviruses express tau protein at similar levels (Figure 4D). Collectively, these data suggest that the molecular cloning of the BioID2 lentiviral transfer plasmids (pFIN vector) was successful as validated by restriction digestion, Sanger sequencing, and western blotting. Also, the lentiviral production and purification was successful in generating viable viral particles capable of transducing and expressing the BioID2 proteins in HEK293T cells. 52 Figure 4: Lentiviruses functional titer and validation. A) For each lentiviral construct, HEK293T cells were transfected by lentiviral plasmids (pFIN transfer plasmids expressing 53 Figure 4 (cont’d) the BioID2 protein, pNHP packaging plasmid, and pHEF-VSVG envelope plasmid). Functional titer of the generated lentiviruses was assessed by immunocytochemistry (ICC). The functional titer was calculated by multiplying the number of transduced cells at the highest viral dilution by the respective dilution factor. B) Representative image for the ICC of the lentiviral transduction of HEK293T cells at the lowest viral concentration that was used to calculate the functional titer. C) HEK293T cells were transduced by lentiviruses at MOI 50 to validate the viability of the created lentiviruses. Lysates were analyzed by western blot which shows the expression of all four BioID2 proteins at the expected molecular weights. Antibodies used were Myc tag and HA tag primary antibodies. Note the BioID2-Tau and Tau-BioID2 bands are indicated by the arrow, the Myc-BioID2 and BioID2-HA control bands by an arrowhead, and a non-specific band identified with the HA antibody with an asterisk. D) HEK293T cell lysates were validated for the expression of tau protein. Western blotting with Tau13 antibody shows the expression of BioID2-Tau and Tau-BioID2 proteins (arrow) at the expected molecular weight and at a similar expression level. MOI; multiplicity of infection, TU; transducing unit 54 Optimizing lentiviral transduction of primary cortical neurons Three important optimizations were performed for lentivirus-mediated expression of the BioID2 proteins in E18 WT and TKO primary cortical neurons. First, a range of multiplicity of infections were used to optimize the amount of mouse E18 cortical neurons transduced. Second, the level of exogenous tau expression was measured to determine whether near-physiological or supraphysiological levels were expressed. Third, a dose response for exogenous biotin supplemented into the medium was tested. These optimization steps were critical to ensure sufficient transduction efficiency in primary neurons, establish whether overexpression artifacts may cloud interpretation, and determine the functionality of the BioID2 enzymes. Determining optimal titer for primary neuron transduction The E18 WT primary cortical neurons were transduced by the BioID2 lentiviruses on DIV 4 at MOI of 50,100, and 200. Primary neurons were maintained five days post- transduction (DIV 9) and the transduction efficiency was determined by ICF. Quantification of the ICF images showed increased lentiviral transduction efficiency at higher MOI achieving ~50% transduction efficiency at MOI 200 (Figure 5A). Accordingly, MOI of 200 was used for the lentiviral transduction of primary neurons in all the downstream experiments. Representative images show successful expression of the BioID2 proteins in WT primary cortical neurons probed by Myc tag, HA tag, and Tau13 antibodies (Figure 5B). The BioID2-Tau and Tau-BioID2 proteins were probed by the Tau13 antibody, which binds to an epitope in the N-terminus of the human tau protein (amino acids 8-9, 13-21) not present in rodent tau (i.e. Tau13 is human tau-specific) (Combs et al., 2016; García-Sierra et al., 2003). The Tau13 antibody will only bind to the 55 human expressed by lentiviral transduction (Figure 5B). For the downstream experiments, TKO primary cortical neurons were used to avoid the competition with the endogenous mouse tau. 56 Figure 5: Expression of the BioID2 proteins in primary cortical neurons. A) E18 WT primary cortical neurons were transduced by lentiviruses expressing Myc-BioID2, BioID2- Tau, BioID2-HA, and Tau-BioID2 proteins. Lentiviral transduction was done at a range of 57 Figure 5 (cont’d) multiplicity of infections (MOI 50, MOI 100, and MOI 200). For each lentiviral transduction, ICF was performed, and the transduction efficiency was calculated at each MOI by dividing the number of transduced neurons by the total number of neurons. Myc-BioID2 positive cells were analyzed by Myc tag antibody, BioID2-HA positive cells were analyzed by HA tag antibody while BioID2-Tau and Tau-BioID2 positive cells were analyzed by Tau13 antibody which does not bind mouse tau. The bar graph shows the quantification transduction efficiency for each lentivirus and at each MOI (5 images/well used for analysis). Primary neurons transduced at MOI 200 showed the highest transduction efficiency for all lentiviruses (~50%). Values are represented as mean ± SEM. B) Representative images showing the increased expression of the BioID2 proteins at increased lentiviral MOI. Antibodies used were Myc tag, HA tag, and Tau13. E18; embryonic day 18, DIV; days in vitro, MOI; multiplicity of infection, ICF; immunocytofluorescence. Scale bar, 50 μm (applies to all panels). 58 Lentiviruses express the Tau-BioID2 proteins at near physiological levels Viral vector-mediated expression of exogenous proteins can lead to overexpression and subsequent artifacts. Thus, the level of lentivirus-mediated expression of the Tau-BioID2 protein in TKO mouse cortical neurons was assessed and compared to the expression levels of endogenous Tau in WT mouse cortical neurons. On DIV 4, TKO neurons were transduced by lentivirus expressing the Tau-BioID2 protein at MOI 200 and ICF was performed at DIV 12 along with WT neurons (untransduced) of the same age. Tau signal intensity was determined by densitometry measurements of Tau5 ICF. The Tau5 antibody binds to both the human and mouse tau protein (Kanaan & Grabinski, 2021). Quantification of the average pixel intensity of tau signal shows similar expression levels between the lentiviral expressed Tau-BioID2 and the WT Tau (Figure 6A-B). 59 Figure 6: Lentiviral expression of Tau-BioID2 compared to WT Tau expression. A) E18 TKO cortical neurons were transduced by Tau-BioID2 lentivirus on DIV 4. Transduced TKO neurons and WT cortical neurons were maintained until DIV 12. Cortical neurons were fixed and ICF was performed at DIV 12 using Tau5 antibody. Densitometry quantification of the average pixel intensity for tau signal shows similar expression between the Tau-BioID2 protein expressed in TKO primary cortical neurons and the WT mouse tau (n=3 wells/condition and 5 images/well). Values are represented as mean ± SEM. B) Representative images of the physiological tau in WT cortical neurons and the Tau-BioID2 expression (used as a representative lenti-tau construct) in transduced TKO cortical neurons of the same age. Scale bar, 50 μm. TKO; tau knockout, WT: wild type, E18; embryonic day 18, DIV; days in vitro, MOI; multiplicity of infection, ICF; immunocytofluorescence 60 Optimization of biotin dose and incubation time E18 TKO primary cortical neurons were transduced on DIV 4 by the BioID2-Tau lentivirus (MOI 200) and on DIV 8 exogenous biotin was supplemented at 0, 50, or 100 μM. Cells were analyzd 24, 48, or 96 hours after biotin supplementation. Quantification of the western blotting for biotinylated proteins showed a time- and dose-dependent increase in the biotinylation signal detected, with a maximum signal achieved at 100 μM biotin for 96 hours (Figure 7A). The biotinylation signal observed at 0 μM supplemented biotin dose was due to the presence of biotin in the B-27 supplement added to the neurobasal medium. Taken together, the lentiviruses were effective at transducing ~50% of the primary cortical neurons at MOI 200. The expression level of the exogenous Tau-BioID2 protein was comparable to the expression level of endogenous WT mouse tau. Finally, optimal biotin supplementation was at 100 μM followed by a four-day post-supplementation incubation period. Based upon these optimization experiments, an experimental paradigm was selected for all downstream studies (Figure 8). 61 Figure 7: Optimization of biotin dose and incubation time. E18 TKO primary cortical neurons were transduced by lentivirus expressing the BioID2-Tau protein at MOI 200 on DIV 4. Lysates were collected on DIV 12 and western blotting was performed using biotin antibody. A) Quantification of the biotinylation signal detected by western blotting, values are represented as mean ± SEM (n=3 independent experiments). B) Representative image of the western blotting. TKO; tau knockout, E18; embryonic day 18, DIV; days in vitro, MOI; multiplicity of infection. 62 Figure 8: Schematic representation of the experimental paradigm selected for primary neurons affinity purification and mass spectrometry experiments. E18 TKO primary cortical neurons were transduced on DIV 4 (n=3 for each lentiviral transduction). Exogenous biotin (100 μM) was supplemented on DIV 8 and protein lysates were collected on DIV 12. TKO; tau knockout, E; embryonic day, DIV; day in vitro. 63 Affinity pulldown and mass spectrometry identification of biotinylated tau interacting proteins Protein lysates containing in situ biotin-labeled interacting proteins from TKO cortical neurons transduced with either BioID2-HA, Tau-BioID2, Myc-BioID2 or BioID2-Tau were collected on DIV 12 (Figure 8). The biotinylated proteins were captured by streptavidin- coated magnetic beads. The purified biotinylated protein samples were split into a) 10% for western blotting to confirm biotinylation and validate identified interacting proteins, and b) 90% for MS protein identification (Figure 9). Figure 9: Affinity capture of biotinylated proteins using streptavidin Dynabeads. Streptavidin-coated beads capture the biotinylated labeled proteins. Then 10% of the sample was used to validate effective pulldown of biotinylated proteins and the expression of exogenous proteins (BioID2-HA, Tau-BioID2, Myc-BioID2 and BioID2-Tau). The other 90% of the captured protein sample was used for MS identification of tau interacting proteins. MS; Mass spectrometry 64 Optimization of the biotin-streptavidin affinity pulldown using HEK293T cell lysates The first set of affinity pulldown optimizations was performed using HEK293T cell lysates. HEK293T cells were transfected by pFIN-Myc-BioID2, pFIN-BioID2-Tau, and pFIN-Tau- BioID2 plasmids. First, the protein lysates containing the biotinylated proteins were collected in 1x co-immunoprecipitation (Co-IP) lysis buffer containing 20 mM Tris pH 7.5, 0.5 mM dithiothreitol, 150 mM NaCl, and 0.5% Triton X-100. The biotinylated proteins were eluted in 1x sample buffer. Capturing and eluting the biotinylated proteins was unsuccessful (Figure 10A). Next, a method adapted from (Roux et al., 2013a) was used, which includes a lysis buffer containing 50 mM Tris pH 7.4, 500 mM NaCl, 0.2% SDS, and 1 mM dithiothreitol and elution in 1x sample buffer. Again, capturing and eluting the biotinylated proteins was unsuccessful (Figure 10B). Finally, the method described in (Cheah & Yamada, 2017) was tested. A high concentration detergent lysis buffer was used that contained 50 mM Tris pH 7.4, 150 mM NaCl, 0.4% SDS, 1% NP-40, 1 mM EGTA, and 1.5 mM MgCl2. Elution of the biotinylated proteins was performed by competition with high concentration of free biotin (25 mM) prepared in lysis buffer and heating the sample to 95ᵒC for 15 minutes. This method showed efficient capturing and elution of the biotinylated proteins and the BioID2 proteins (Figure 10C). For these optimization experiments, the sample-bead incubation was set to four hours at room temperature. Based on these results, the high concentration detergent and biotin competition/heat elution method was used. 65 Figure 10: Optimization of affinity pulldown in HEK293T cell lysates. HEK293T cells were transfected by pFIN-Myc-BioDI2, pFIN-Tau-BioID2 plasmids, or pFIN-BioID2-Tau plasmids. Exogenous biotin (100 μM) was supplemented, and cells were maintained for 48 hours. Protein lysates were collected, and biotinylated proteins were captured by streptavidin-coated magnetic beads. Captured proteins were eluted and western blotting was performed. Three lysis buffers and three elution methods were 66 Figure 10 (cont’d) performed to validate the optimal pulldown conditions. A) Co-IP lysis buffer was used, and biotinylated proteins were eluted in 1x sample buffer. No proteins were detected in the elution. B) Low concentration detergent lysis buffer was used, and elution was done in 1x sample buffer. No proteins were detected in the elution. C) High concentration detergent lysis buffer was used, and elution was done in 25 mM biotin with sample heating to 95ᵒC showing successful capturing and elution of biotinylated proteins (elution lanes in western blots). Note the effective capture and elution of biotinylated proteins. Biotin and BioID2 antibodies were used for the western blotting. Pre- refers to the input lysate sample, Post- refers to the flow through after capturing, and Elution refers to the eluted sample from the beads. Co-IP; co-immunoprecipitation. 67 Optimization of the biotin-streptavidin affinity pulldown using primary cortical neurons lysates The optimized affinity pulldown protocol was then tested with primary neurons lysates. Primary cortical neurons (E18) from TKO mice were transduced by lentiviruses to express BioID2-HA, BioID2-Tau, Myc-BioID2, or Tau-BioID2. Protein lysates containing the biotinylated proteins were collected on DIV 12 in the high-detergent concentration lysis buffer and elution was done in 25 mM biotin at 95 ᵒC. In this set of experiments, the sample-bead incubation time and temperature were optimized. Capturing and eluting biotinylated proteins was unsuccessful when samples were incubated with pulldown beads for four hours at room temperature (Figure 11A). Next, samples were incubated with beads overnight at 4ᵒC, but this also was unsuccessful (Figure 11B). Finally, incubating the sample-bead mixture overnight at room temperature showed successful capturing and elution of the biotinylated proteins (Figure 11C). Taken together, these optimization experiments demonstrated that the ideal pulldown and elution conditions for neuron lysates includes incubating samples with beads overnight at room temperature followed by elution in a high concentration detergent buffer, competition with free biotin and heat. These parameters were used in subsequent experiments described below. 68 Figure 11: Optimization of affinity pulldown in primary neurons lysates. E18 TKO primary cortical neurons were transduced by BioID2-HA, BioID2-Tau, Myc-BioID2, or Tau-BioID2 lentiviruses on DIV4. Exogenous biotin (100 μM) was supplemented on DIV8, and cells lysates were collected on DIV12. Biotinylated proteins were captured by streptavidin-coated magnetic beads and western blotting was performed to validate the 69 Figure 11 (cont’d) successful capturing and elution of tau interacting proteins. Three conditions were optimized for the successful capturing by the streptavidin-coated magnetic beads. A) Sample-bead incubation was done for four hours at room temperature, biotinylated proteins were not captured by the beads, as indicated by similar levels in both the input lysate (pre-) and the flow through (post-). B) Sample-bead incubation was done overnight at 4ᵒC, biotinylated proteins were not captured by the beads, as indicated by similar levels in both the input lysate (pre-) and the flow through (post-). C) Sample-bead incubation was done overnight at room temperature. This method successfully captured biotinylated proteins and provided effective elution of biotinylated proteins (elution lanes in western blots). Biotin antibody was used. Pre- refers to the input lysate sample, Post- refers to the flow through after capturing, and Elution refers to the eluted sample from the beads 70 Identification of the tau protein interactome using mass spectrometry To identify the interacting partners with Tau-BioID2 and BioID2-Tau proteins, label-free quantification analysis was performed on the mass spectrometry raw data files. Potential interactors with either Tau-BioID2 or BioID2-Tau were defined as follows: a) identified in at least two of the experimental replicates, and b) was ≥1.5-fold increased compared to the respective control. Following these criteria, 269 proteins were identified as potential interactors with the Tau-BioID2 protein (≥1.5-fold increase versus BioID2-HA control) and 219 showed < 1.5-fold change and were not considered potential interactors (Figure 12A). For the BioID2-Tau interactors, 169 proteins showed ≥1.5-fold increase compared to the Myc-BioID2 control, and 140 proteins showed < 1.5-fold change (Figure 12B). Out of the 269 preferential interactors with Tau-BioID2 and the 169 preferential interactors with the BioID2-Tau: 1) 66 proteins were identified as interactors with both proteins, 2) 203 proteins were identified only as Tau-BioID2 interactors, and 3) 103 proteins were identified only as BioID2-Tau interactors (Figure 12C). Taken together, a total of 372 proteins were identified as potential tau interacting partners. STRING analysis was performed to map the functional and physical associations network of the identified tau protein interactome with MCL clustering inflation value of 3.0. Major cellular component categories of the identified interacting partners included mitochondrial proteins, cytoskeletal proteins, synaptic proteins, nuclear proteins and ribonucleoproteins ranked by the number of identified proteins in the cellular component (Figure 13). The full list of the identified proteins is shown in Appendix A. 71 Figure 12: Venn diagrams of the identified preferential interacting proteins. A) Tau- BioID2 and BioID2-HA shows 269 proteins considered as preferential Tau-BioID2 interactors (>1.5-fold change compared to BioID2-HA). B) BioID2-Tau and Myc-BioID2 shows 169 proteins considered as BioID2-Tau interactors (>1.5-fold change compared to Myc-BioID2), C) Tau-BioID2 and BioID2-Tau interactors collectively showing 372 proteins considered potential members of the tau interactome. 72 Figure 13: STRING analysis for the identified tau interactome. The network included both functional and physical associations with high confidence interaction score (>0.7). MCL Clustering was then performed using the String app with an inflation parameter of 3.0. Clustering was performed to reduce the complexity in the network to visualize the individual nodes. Major cellular component categories of the identified proteins are grouped by color. 73 Gene ontology enrichment analysis of tau interactome ClueGO cellular component and molecular function pathways were analyzed for the list of tau interacting partners (Figure 14). Cellular component analysis mapped synaptic proteins localized to axonal growth cone, dendritic spine, pre-synapse, post-synapse and synaptic vesicle membrane. Cytoskeletal proteins belonging to the microtubule and actin cytoskeleton were identified. Mitochondrial proteins belonging to the mitochondrial respirasome, inner mitochondrial membrane, and the mitochondrial matrix were identified. The identified proteins were mapped to both the cytosolic and the mitochondrial ribosomal proteins, while proteins also were mapped to the peroxisome and the ribonucleoprotein granule (Figure 14A). ClueGo molecular function pathway enrichment identified RNA binding proteins (dsRNA, mRNA, rRNA binding proteins), ribonucleoprotein complex binding proteins, cytoskeletal binding proteins (tau, microtubule, and actin binding proteins), kinase binding and ubiquitin ligase binding proteins. Proteins regulating translation initiation and nucleotide binding were also identified. Mitochondrial pathways included NADH dehydrogenase, oxidoreductase, peroxidase, FAD binding, fatty-acyl-coA binding, and electron transfer pathways were identified (Figure 14B). The full list of the identified proteins in cellular components and molecular function pathways are shown in Appendices B and C. Interestingly, the GO KEGG pathways analysis revealed proteins involved in neurological diseases. Those included AD (26 proteins), Parkinson’s disease (PD; 28 proteins), Amyotrophic lateral sclerosis (ALS; 31 proteins), HD (24 proteins), and prion diseases (25 proteins). The list of the associated proteins is shown in Table 2. 74 Figure 14: ClueGo gene ontology enrichment analysis for the identified tau interactome. A) Cellular component enrichment analysis, B) Molecular function pathways analysis. Only pathways with p-value ≤0.05 were included. 75 Table 2: KEGG pathway analysis associated proteins with neurological disorders % of Disease # of Proteins Associated Genes Genes [Atp5b, Atp5h, Atp5o, COX2, Casp3, Cox4i1, Cox5a, Dvl2, Gapdh, Gsk3b, Alzheimer’s Kif5b, Ndufa10, Ndufa9, Ndufs1, Ndufs2, 26 7.05 Disease Ndufv1, Psmc2, Sdhb, Snca, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5] [Atp5b, Atp5h, Atp5o, COX2, Camk2b, Casp3, Cox4i1, Cox5a, Kif5b, Ndufa10, Parkinson’s Ndufa9, Ndufs1, Ndufs2, Ndufv1, Psmc2, 28 11.34 Disease Sdhb, Septin5, Snca, Trap1, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Uba1, Uchl1] [Actb, Atp5b, Atp5h, Atp5o, COX2, Casp3, Cox4i1, Cox5a, Dctn1, Fus, Hnrnpa3, Kif5b, Ndufa10, Ndufa9, Amyotrophic 31 8.40 Ndufs1, Ndufs2, Ndufv1, Pfn2, Psmc2, Lateral Sclerosis Rac1, Sdhb, Srsf3, Srsf7, Tardbp, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5] [Atp5b, Atp5h, Atp5o, COX2, Casp3, Cltc, Cox4i1, Cox5a, Dctn1, Kif5b, Huntington Ndufa10, Ndufa9, Ndufs1, Ndufs2, 24 7.95 Disease Ndufv1, Psmc2, Sdhb, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5] [Atp5b, Atp5h, Atp5o, COX2, Casp3, Cox4i1, Cox5a, Gsk3b, Hspa8, Kif5b, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Prion Disease 25 9.33 Ndufv1, Psmc2, Rac1, Sdhb, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5] 76 GO enrichment analysis reveals distinct cellular components for Tau-BioID2 and BioID2-Tau interacting partners ClueGO cellular component analysis was performed separately on: a) proteins identified in both Tau-BioID2 and BioID2-Tau (66 shared proteins), b) proteins identified in Tau- BioID2 but not BioID2-Tau (203 proteins), and c) proteins identified in BioID2-Tau but not Tau-BioID2 (103 proteins). The 66 shared proteins were localized to mitochondria, ribosomes, axonal growth cone, dendrites, and cytoskeleton (Figure 15A). The 203 proteins identified with only Tau-BioID2 were localized to the cytoskeleton, ribonucleoprotein granule, cytoplasmic stress granule, ribosomes, axonal growth cone, dendritic shaft, synapse, and synaptic vesicles (Figure 15B). The 103 proteins identified with only BioID2-Tau were primarily localized to mitochondria and ribosomal subunits (Figure 15C). Taken together, the identified tau protein interactome mapped synaptic, nuclear mitochondrial, RNA binding, ribosomal, and cytoskeletal proteins. GO cellular component analysis revealed a preferential interaction of synaptic and RNA binding proteins with the C-terminus tagged tau protein (Tau-BioID2), while the mitochondrial proteins showed preferential interaction with the N-terminus tagged Tau protein (BioID2-Tau). 77 Figure 15: GO enrichment cellular component analysis. A) Proteins identified as interacting partners with both Tau-BioID2 and BioID2-Tau (66 proteins). B) Proteins 78 Figure 15 (cont’d) identified as Tau-BioID2 only interacting partners (203 proteins). C) Proteins identified as BioID2-Tau only interacting partners (103 proteins). Only pathways with p-value ≤0.05 were included 79 Validation of the identified tau interacting partners Primary E18 cortical neurons from TKO mice were transduced by lentiviruses expressing the BioID2 proteins and candidate tau interacting partners identified by mass spectrometry were validated by western blotting. Proteins identified by mass spectrometry as potential interactors with both Tau-BioID2 and BioID2-Tau proteins were chosen for validation experiments as these likely represent robust tau interactors. First, MAP2 was identified by mass spectrometry as an enriched protein in both Tau-BioID2 and BioID2- Tau when compared to their respective controls BioID2-HA and Myc-BioID2 (i.e. 3-fold increase in Tau-BioID2 compared to BioID2-HA and 2-fold increase in BioID2-Tau compared to Myc-BioID2). Immunoblotting confirmed that MAP2 shows an increased interaction with Tau-BioID2 and BioID2-Tau compared to the control samples (Figure 16A). Second, MAP6 was identified by mass spectrometry as a specific interactor with both BioID2-Tau and Tau-BioID2 proteins and was not detected in either Myc-BioID2 or BioID2-HA control proteins. Immunoblotting confirms that MAP6 was uniquely found with both tau proteins and was undetectable in the controls (Figure 16B). 80 Figure 16: Validation of candidate Tau interacting partners by western blotting. TKO primary cortical neurons (E18) were transduced by BioID2-HA, Tau-BioID2, Myc- BioID2, and BioID2-Tau lentiviruses. Biotinylated Tau interacting partners were captured by streptavidin-coated magnetic beads. Western blotting was performed to validate the mass spectrometry results by probing with potential tau interacting partners that were enriched in either Tau-BioID2 or BioID2-Tau samples compared to their respective controls BioID2-HA and Myc-BioID2. Western blots were probed for A) MAP2, and B) MAP6. MAP2 and MAP6 antibodies were used. 81 DISCUSSION Accumulating evidence suggests there are physiological microtubule-dependent and - independent functions of the tau protein. Understanding the diverse biological processes in which the tau protein participates is critical to advancing our understanding of normal tau physiology and potentially tau’s role in pathological mechanisms of tauopathies. The work presented in this thesis provides a novel in situ labeling approach to study tau protein-protein interactions in living neurons. Tau interactome mapping Utilizing the BioID2 in situ labelling approach, 372 proteins were identified that are candidate members of the tau protein interactome. The cellular localization of the identified tau protein interactions was consistent with previous findings and included proteins mapped to the mitochondria, cytoskeleton, synaptic vesicles, ribosomes, the ribonucleoprotein complex as well as heat shock proteins and regulators of the ubiquitin- proteasome system (Geeth Gunawardana et al., 2015; C. Liu et al., 2016; Tracy et al., 2022; P. Wang et al., 2017). Moreover, the BioID2 approach identified tau protein interactions previously reported in the literature including PP1α, GSK3β (Combs et al., 2021; Kanaan et al., 2011b), EFhd2 (Ferrer‐Acosta et al., 2013), Annexins A2, and A6 (Gauthier-Kemper et al., 2018b), and α-synuclein (Esposito et al., 2007). These results provide supportive evidence of the various physiological functions of the tau protein and demonstrate the capability of the BioID2 approach in mapping the tau protein interactome. Interestingly, the identified tau interactome revealed proteins associated with neurodegenerative diseases including AD, PD, ALS, HD, and prion disease. Among 82 which; a) GSK3β, Dvl2, α-synuclein and GAPDH associated with AD, b) α-synuclein , Camk2b, Trap1, Uba1, Uchl1, and Septin5 are associated with PD, c) Actb, Dctn1, Hnrnpa3, Fus, Srsf3, Srsf7, and Tardbp are associated with ALS, d) Hspa8, and Rac1 are associated with prion disease, and e) Atp5b, Atp5h, Atp5o, Cox2, Cox4i1, Cox5a, Casp3, Kif5b, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1, Psmc2, Sdhb, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, and Tubb5 are associated with all five diseases. These results align with a previous study showing that a tau protein consisting of amino acids 151-391 interacted with 39 proteins associated with AD, 10 associated with PD, and 22 associated with both diseases (Sinsky et al., 2020). However, the associated interactions detected here were different. One possible explanation for the difference in specific proteins identified is the experimental strategy used. Sinsky et al., utilized a rat brainstem crosslinking approach coupled to mass spectrometry to identify the interactome of a truncated version of tau expressing amino acids 151-391, while here the in situ labeling BioID2 approach with full-length tau was used. Distinct and overlapping interactions between BioID2-Tau and Tau-BioID2 I reported distinct interactions between the N- versus C- terminus tagged tau proteins, BioID2-Tau and Tau-BioID2. The preferential interactors with BioID2-Tau (103 proteins) were mapped to the mitochondria and ribosomal subunits while the preferential interactors with Tau-BioID2 (203 proteins) were mapped to cytoplasmic stress granule, ribosomes, cytoskeleton, axonal growth cone, dendritic shaft, synapse, and synaptic vesicles. This is possibly due to where the interactors bind tau as BioID2 labeling will be influenced by the physical distance and/or orientation of binding partners. The labeling radius of the BioID2 protein is estimated at ~10 nm (Kim et al., 2014). Adding a 25 nm 83 13xlinker between BioID2 and the protein of interest was suggested to increase the biotinylating range of the BioID2 protein (Kim et al., 2016). The length of the tau protein was estimated to be ~63-70 nm with the longest average length of human tau estimated at 96.2 nm (Hagestedt et al., 1989; Ruben et al., 1991; Tracy et al., 2022). Thus, the placement of the BioID2 may facilitate or restrict access to specific sets of interacting partners. Tracy et al., used APEX2 in situ labeling fused to either the N-terminus (APEX- Tau) or C-terminus (Tau-APEX2) of full-length human tau in human neurons derived from a human induced pluripotent stem cell line (WTC11) (Tracy et al., 2022). The APEX2 is a proximity labeling enzyme similar in principle to BioID2, both proteins involve biotin labeling in a ~10-20 nm labeling radius. A major difference is that APEX2 is peroxidase which requires adding toxic hydrogen peroxide and accordingly biotinylates proteins in a ~1 min time frame identifying the interactome as a cross-sectional snapshot that can be utilized to study tau dynamics (i.e. tau secretion during activity-dependent neuron excitability). Similar to our results, they found enrichment of different interactors with each protein. They reported synaptic, dendritic, mRNA binding, and proteasomal proteins interacting preferentially with APEX2-Tau, while Tau-APEX2 preferentially interacted with proteins involved in heat shock response, vesicle mediated transport, and transport along microtubules. Out of the 372 proteins identified with the BioID2 approach, only 62 proteins were also identified in the APEX2 approach and were mapped to the cytoskeleton, synapse, somatodendritic compartment and mitochondria. Importantly, out of the 62 overlapping proteins between both APEX2 and BioID2 approaches, 10 proteins (α- 84 synuclein, Rac1, Pfn2, Cltc, Hspa8, Psmc2, Tubb2a, Tubb2b, Tubb3, and Tubb4b) were annotated to neurodegenerative diseases including AD, PD, HD, ALS, and prion disease. In addition, BioID2 identified 66 overlapping proteins between both BioID2-Tau and Tau-BioID2 and were mapped to mitochondria, and mitochondrial ribosomes (e.g. Timm44, Tbrg4, Pdhx, Ak4, Mut, Cox4i1, Fdxr, Mrpl19, Mrpl37, and Mrpl38), somatodendritic compartment, and axonal growth cone (Dcx, β-synuclein, α-synuclein, Pclo, and MAP6), and the microtubule cytoskeleton (e.g. Map2, Map6, Stmn1, Map1b, Kif2a, Map4, Camsap3, and Tbcb). While Tracy et al., identified 136 overlapping proteins between APEX2-Tau and Tau-APEX2 and included tubulin binding, actin binding, nucleic acid binding, as well as members of the SNARE complexes (Tracy et al., 2022). Again, differences in specific proteins identified between Tracy et al. and this work might be due to the experimental approaches and cellular models utilized. Neither study can assign the differences to where in the tau protein the interactors bind, but they indicate that in situ labeling may require tagging both ends of a protein to effectively label the full spectrum of interactors. The use of truncated portions of the full-length tau protein are more likely to facilitate the identification of tau domain-specific interactors. Geeth Gunawardana et al., utilized a crosslinking approach to identify tau interactors with an N-terminal tau construct (amino acids 1-255) and a C-terminal tau construct (amino acids 256-441) in the human neuroblastoma SH-SY5Y cell model. The Tau1-255 protein showed interactions with histones, RNA binding, and translation proteins, while the Tau256-441 protein interacted with heat shock proteins, actin binding proteins, and members of the proteasomal system 85 (Geeth Gunawardana et al., 2015). A natural extension of this work is to utilize the BioID2 approach to identify tau domain-specific interactors in neurons. Tau protein interactions in various cellular localizations GO cellular component analysis revealed tau protein interactions in the mitochondria, cytoskeleton, synaptic vesicles, ribosomes, and the ribonucleoprotein complex. Perhaps not surprisingly, several tubulin cytoskeleton-related proteins were identified as interactors with tau using the BioID2 approach, but additional and potentially important other interactions were identified. Tau protein was traditionally described as a microtubule stabilizing protein. This finding was later challenged, and tau was reported to regulate microtubule dynamics rather than stabilizing them (Qiang et al., 2018). Tau, as well as other MAPs, were suggested to crosslink the actin and microtubules cytoskeletons (reviewed in (Mohan & John, 2015)). This aligns with our finding that tau interacts with proteins involved in tubulin, and actin binding, as well as the interaction with other MAPs including MAP2, MAP1b, MAP4, MAP6, EML4, MAPRE1, and JPT2. I provided additional support that the tau-cytoskeletal protein interactions exist, which might modulate the various functions of tau in regulating cytoskeletal dynamics as well as tau’s role as a scaffolding protein that may localize binding partners to cytoskeleton. The interaction between tau and mitochondria is extensively reported in literature. Early studies examined the effect of tau binding to the microtubules on the mitochondria kinesin-mediated axonal transport (Ebneth et al., 1998), a process potentially regulated by GSK3β (Llorens-Martin et al., 2011; Tatebayashi et al., 2004). More recent studies reported the effect of mitochondria dysfunction on tau pathology (Du et al., 2022; Terada et al., 2021), while others correlated tau mutations and accumulation to mitochondria 86 reduction and/or dysfunction (Cummins et al., 2019; Li et al., 2016; Rodríguez-Martín et al., 2016; Tracy et al., 2022). However, it’s not well characterized whether tau accumulation leads to mitochondrial dysfunction, or whether mitochondrial dysfunction is a major contributor to tau pathology. Much less is known about a potential physiological role for tau in mitochondrial function, but it is notable that 164 proteins involved in mitochondria function were found in this work. Those included proteins involved in mitochondria respirasome, mitochondrial large and small ribosomal subunits, mitochondrial oxidoreductase activity, electron transfer, NADH dehydrogenase activity, and FAD binding. Future studies may investigate the physiological tau-mitochondria interaction which will further our understanding of the pathological mechanisms that involve tau accumulation and mitochondrial dysfunction. Tau was also reported to bind RNA-binding proteins in the dendrites which regulates the formation of stress granules as well as tau pathological misfolding (Apicco et al., 2018; Jiang et al., 2021; Maziuk et al., 2018b; Vanderweyde et al., 2016). Tau interaction with RNA binding proteins and members of the ribonucleoprotein complex was observed in this work. The identified proteins included HNRNPA3, HNRNPM, HNRNPQ, TARDBP, FARSB, FUS, FXR1, among others. Oligomeric tau was reported to inhibit translation by increasing the tendency of RNA stress granules formation, a process mediated by tau binding to HNRNPA2/B1(Jiang et al., 2021). Other RNA binding proteins (i.e. TIA1) were also found to mediate tau toxicity and increase the tendency of tau aggregation and neurodegeneration. The role of physiological tau in binding to RNA binding proteins and localization to the dendrites is a relatively new area of investigation and not well-understood (Maziuk et al., 2018a; Vanderweyde et al., 2016). Studying the 87 molecular mechanisms involved in tau localization to the dendrites and binding to RNA binding proteins may elucidate the physiological roles and the pathological mechanisms involved in stress granules formation and pathologic tau accumulation in neurodegenerative diseases. The BioID2 approach identified small and large ribosomal proteins (RPL11, RPL13, RPL14, RPL3, RPL4, RPS15a, RPS3, RPS9, RPSa), translation initiation factors (EIF4b, EIF4a2, EIF5a, among others), and proteins localized to the dendrites (DCX, PP1r9b, PCLO, SNAP47, NSF, NAP1l4, among others). These findings provide additional evidence of microtubule-independent functions of the tau proteins which require further investigation to understand the molecular mechanisms mediated by tau protein-protein interactions. Tau also localizes to the synapses in both physiological and pathological states. Tau is suggested to play a role in synaptic plasticity. For instance, tau knockout mice show impairment in long-term potentiation (Ahmed et al., 2014) and long-term depression (Kimura et al., 2014). Physiological tau undergoes activity-dependent translocation to the post-synaptic compartment where it acts as a scaffolding protein by binding to Fyn kinase and PSD-95 localizing Fyn kinase to phosphorylate and stabilize NMDA receptors, a process critical to long-term potentiation (Ittner et al., 2010; Mueller et al., 2021). In AD, pathological tau accumulates in the synaptic terminals causing synaptic loss and impairment of synaptic plasticity (Wu et al., 2021; Yoshiyama et al., 2007). Pathological tau can induce presynaptic dysfunction by binding synaptogrin-3 on synaptic vesicles which leads to accumulation of synaptic vesicles and blocking exocytosis in the presynaptic terminals (McInnes et al., 2018) . The localization of tau to the synapses is consistent with the findings presented in this thesis. Tau interactions were reported in the 88 presynaptic terminals (e.g. Bin1, PCLO, FXR1, α-synuclein, β-synuclein, Srcin1, Tnik, among others), post synapses (e.g. Neurabin-2, srGAP2, DCLK1, DNM2, ARHGEF2, CFL1, FUS, PRKAR2B, PSMC2, Ank2, among others), and synaptic vesicles membrane (e.g. ATP2b1, CLTC, DNM1L, Rab14, Rab2a, Rab7, Snap47, and α-synuclein). Understanding the signaling mechanisms of synaptic tau protein-protein interactions will help us better identify and study various roles of synaptic tau in both health and disease states. Taken together, the BioID2 proximity labeling approach provides an effective route to map the tau interactome highlighting the broad physiological functions mediated by the tau protein. Understanding the molecular signaling pathways mediated by the physiological tau interactome may help elucidate the pathological mechanisms involved in the switch from physiologic-to-insoluble aggregated pathologic tau in the neurodegenerative tauopathies. Technical development of the approach The proposed experimental framework involved four main phases; a) molecular cloning to create fusion proteins between the BioID2 biotin ligase and tau followed by the production of the fusion proteins in lentiviruses, b) culturing TKO primary cortical neurons and expression of the BioID2 proteins via lentiviral-mediated transduction, c) affinity capturing and mass spectrometry identification of the biotinylated tau-interacting proteins, and d) validation of the identified proteins by affinity capturing and immunoblotting. 89 Cloning of BioID2 and Tau fusion proteins To map the total tau protein interactome, I tagged BioID2 to the N-terminus (BioID2-Tau) or the C-terminus (Tau-BioID2) of the 2N4R human tau isoform. The two fusion proteins were cloned from the Myc-BioID2-13xlinker and the 13xlinker-BioID2-HA plasmids respectively. The two plasmids are different in the place of the tag and the 13xlinker sequence. Accordingly, the Myc-BioID2-13xlinker was used as a control protein for BioID2-Tau and the 13xlinker-BioID2-HA was used as a control for Tau-BioID2. This step is critical for proper elimination of background proteins (proteins identified due to non- specific interactions or direct interactions with BioID2 and/or the 13x linker), endogenously biotinylated proteins (e.g.carboxylases), and contaminant proteins (e.g. streptavidin, trypsin, keratins). Fusion proteins Tau-BioID2, BioID2-Tau and the respective controls BioID2-HA and Myc-BioID2 were cloned into a pFIN vector backbone which encodes for essential elements for the lentiviral production including Gag, RRE, and WPRE sequences. The pFIN plasmids are prone to homologous recombination events between the 5’- and 3’- long terminal repeats which makes it challenging to produce the constructs. To avoid this, I used Stbl3 competent E. coli to produce the DNA constructs. Stbl3 cells are known to reduce the homologous recombination events. I also noted that incubating transformed Stbl3 cells for the least amount of time (~12 hours) increases the chances of successful viral plasmid preparations. To ensure proper cloning of the DNA construct, the created pFIN plasmids were validated by restriction digestion and Sanger sequencing for the correct insertion of the BioID2 protein sequences. These steps are important to the generation of high-quality lentiviral preparations. 90 Lentiviruses production The BioID2 proteins were produced in lentiviruses by transfection of HEK293T cells by pFIN transfer plasmids carrying the BioID2 sequences, pNHP packaging vector, and pHEF-VSVG envelope vector. Lentiviruses were harvested by centrifugation and the functional titer was determined by immucnocytochemistry in HEK293T cells. The functional titer protocol is based on the ability of the functional viruses in infecting the cells and expressing the protein of interest. I determined the lentiviral functional titer in HEK293T cells which is time and cost effective. HEK293T cells were then transduced at a similar MOI of 50 by BioID2-Tau and Tau-BioID2 lentiviruses to establish whether the functional titers were accurate. The results showed that the lentiviruses expressed the fusion proteins at nearly similar levels validating the functional titer protocol. Ensuring that the lentiviruses achieved similar expression levels in mammalian cells facilitates a more robust interpretation of the identified Tau-BioID2 and BioID2-Tau protein interactomes by mitigating expression level differences as a potential driver of disparate interactors identified between each tau protein. Lentiviral-mediated transduction of primary cortical mouse neurons The second phase of the project involved a series of optimizations to define an experimental paradigm before affinity capturing and MS identification of biotinylated proteins. First, I identified the lentiviral transduction efficiency by transuding E18 TKO primary cortical neurons at a varying range of multiplicity of infections (50, 100, and 200). I found that all four lentiviruses expressing Tau-BioID2, BioID2-Tau, Myc-BioID2, and 91 BioID2-HA are capable of infecting ~50% of primary neurons at MOI 200. Although increasing the titer further may have produced higher efficiency, this may also increase the chance or extent of non-physiological overexpression leading to unwanted artifacts. Next, the level of lentiviral-mediated tau expression was measured to determine whether near-physiological or supraphysiological levels of expression occurred. Similar expression levels of the physiological WT tau and lentiviral-expressed Tau-BioID2 were determined. This optimization step was important to ensure that the identified tau protein interactions represented the physiological state and were not identified due to the high levels of expression of the Tau-BioID2 proteins by lentiviruses. Although gene therapy- based approaches are effective at delivering exogenous expression systems to neurons, the potential artifacts induced by overexpression can be difficult to address. Fortunately, densitometry analyses suggest that the level of lentiviral-mediated tau expression was similar to WT neuron tau levels. These results suggested the approach used here was unlikely to be affected significantly by overexpression artifacts. Finally, I showed that the highest biotinylation signal was achieved by supplementing 100 μM biotin and incubating for 96 hours. This allows sufficient time for the BioID2 in situ biotin labeling of the proximal proteins. Our experimental paradigm involved the lentiviral transduction (MOI 200) of 3.6 x 10^6 E18 TKO primary cortical neurons on DIV4, supplementing biotin (100 μM) on DIV8 and collecting the lysates of biotinylated proteins on DIV 12. Notably, I could have revisited these optimization steps if I were not able to detect biotinylated proteins by mass spectrometry. This could have included, a) increasing the number of TKO primary cortical neurons per lentiviral 92 transduction, b) transducing TKO neurons at a higher MOI (i.e. 300), and/or c) supplementing a higher biotin dose and/or increasing the incubation time. Mass spectrometry identification of tau interacting proteins The third phase involved affinity capturing of the biotinylated proteins using streptavidin conjugated magnetic beads and identification of the labelled proteins by mass spectrometry. For efficient capturing of biotinylated proteins, I performed two rounds of optimizations using lysates from HEK293T cells and primary cortical neurons. Although the interaction between biotin and streptavidin is one of the strongest known biological interactions (Haugland & You, 2008), the most efficient capturing was with a high- concentration detergent lysis buffer. One possible explanation for this might be that denaturing the secondary structures of the proteins by a high concentration of detergents (i.e. SDS and NP-40) was required to expose the biotinylated residues and allow binding to the streptavidin magnetic beads. Importantly, the in situ labeling of interactors prior to cell lysis affords harsher conditions that traditional IP-type approaches would not allow. Due to the high affinity interaction between biotin and streptavidin, elution of biotinylated proteins bound to the magnetic beads required competition with a high concentration of free biotin (25 mM) and heat. Furthermore, when I used lysates from primary neurons, the sample-bead incubation time (overnight) and temperature (room temp) were critical for the efficient capturing of the biotinylated proteins. This might be due to the lower lentiviral-mediated expression of the BioID2 proteins (hence lower biotinylation levels) compared to the expression in HEK293T cells mediated by pFIN plasmids transfection and similar approaches others have used (Kim et al., 2016; Roux et al., 2013b). 93 For the mass spectrometry identification, I performed label free quantification to determine the abundances of the identified proteins in the Tau-BioID2 and BioID2-Tau samples compared to the respective controls BioID2-HA and Myc-BioID2. I set the following criteria for defining proteins as tau interactors: 1) Proteins must be identified in at least two out of the three independent replicates, and 2) proteins should be detected at ≥1.5-fold increase compared to the respective BioID2 control. These criteria were chosen to balance minimizing false positives and false negatives. I chose the abundance ratio based on the previously reported enrichment ratios in the antibody-based affinity purification experiments such as ≥1.2 in (Geeth Gunawardana et al., 2015), ≥ 1.1 in (C. Liu et al., 2016), and ≥1.5 in (Tracy et al., 2022). I identified 372 tau-interacting partners by MS using these criteria, however, MS identification ultimately requires validation to confirm whether these proteins are bona fide tau interactors. Validation of identified tau interacting partners Finally, I chose some of the identified interactors for proof-of-concept validation by western blotting. I successfully validated MAP2 and MAP6 as tau interactors using this approach. Notably, the validation of interacting proteins by western blotting is limited to the amount of biotinylation on the protein, and subsequently the amount that will be captured by the beads. Mass spectrometry is more sensitive at identifying low levels of biotinylated proteins compared to western blotting. Future experiments are planned to validate additional novel tau interactors that were identified using the BioID2 approach using tau antibody-based affinity purification coupled with the use of brain lysates from either human tau knock-in mouse brains and/or post-mortem human brain tissue. 94 Reversal of the co-immunoprecipitation using antibodies against the identified tau interacting partners will help to strengthen the confirmation as a bona fide interactor. Finally, the use of in situ labeling immunostaining techniques, such as proximity ligation assays, are viable means to further validate not only the interaction but where in neurons the proteins interact. Limitations of the approach Enzyme-mediated proximity labeling approaches, including the BioID2 approach, provide a complementary framework for studying protein-protein interactions. The proximity labeling approaches overcome several of the limitations of the traditional lysis and antibody-based techniques. However, some limitations remain that should be considered when planning experiments and interpreting results. First, proximity labeling techniques are based on creating relatively bulky fusion proteins which might directly interfere with protein interactions. Fusion proteins can alter the functionality and/or folding of the target protein. However, our group has worked with Tau-GFP fusion proteins and found that microtubule binding and in vitro aggregation are unaffected (Kanaan et al., 2020). Also, biotinylation of the fused target protein (i.e. tau) may affect its interactions. Moreover, labeling is dependent on the biotinylating radius of the enzyme (~10 nm) as well as the surface-exposed lysine residues available for biotinylation by the BioID2 enzyme. Thus, the orientation and/or accessibility of an interactor may impact whether it is effectively biotinylated. Indeed, we and others (Tracy et al., 2022) noted differences in the set of interactors identified in the BioID2 or APEX2 fusions are on the N- or C-terminus of full- length tau. Another limitation is that the BioID2 enzyme not only biotinylates the stable and transient/weak interactions but could also label proteins that are in close proximity 95 either in a complex (i.e. indirectly interacting) or not interacting (i.e. non-specific detection) with the target protein. Nonetheless, the in situ labeling of interactors provided by BioID2 approach is advantageous and provides a robust addition to the toolkit available for studying tau protein-protein interactions, but these caveats require consideration and identified interactors require validation. Identifying protein interactions using this approach is also limited by the tau isoform used, solubility of the proteins in the lysis buffer, and the neuronal model. In the adult human brain, six tau isoforms are expressed due to the alternative splicing of exons E2, E3, and E10 of the MAPT gene. In this study, I tagged BioID2 to the longest isoform of the tau protein (2N4R isoform). Thus, the identified tau interacting partners are limited to potential interactors with the 2N4R isoform. Liu and colleagues previously reported preferential interactions to specific mouse tau isoforms (C. Liu et al., 2016). To better represent candidates of the physiological tau protein interactome, BioID2 could be tagged to all six tau isoforms. Solubility of the proteins is another limiting factor. I used lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.4% SDS, 1% NP-40, 1 mM EGTA, and 1.5 mM MgCl2. The identified proteins are those soluble in SDS and NP-40 detergents, non-soluble pelleted proteins were not identified using this approach as the pellet was not analyzed. A more comprehensive assessment to try capturing the full set of interacting partners would include analysis of biotinylated proteins in the lysate pellet. Tau knockout primary cortical neurons were transduced on the 4th day in vitro. TKO mice display a compensatory upregulation of other MAPS (MAP1A, MAP1B and MAP2), but whether this occurs in TKO primary neurons is unknown. (Harada et al., 1994; G. Liu 96 et al., 2019; Ma et al., 2014). The increased expression levels of MAPs in TKO neurons might interfere or compete with the physiological interacting partners of the DIV 4 expressed lenti-tau. However, this requires further investigation to study the crosstalk between lenti-tau and compensatory mechanisms such as changes in other MAPs. Finally, the model system used here is a developing neuron in culture, which may influence the tau interactome. Indeed, developmental-dependent differences in tau expression are well known (i.e. 0N3R is expressed in early development and later additional isoforms are expressed) (Y. Wang & Mandelkow, 2016). The influence of an actively developing neuron on tau interactions is an area of high interest as it has not been well-studied. Confirming the identified tau interactors in adult CNS tissue sources (as discussed above) could help clarify whether the interactors identified here are relevant to the adult nervous system. Taken together, I identified candidate members of the tau interactome using the BioID2 approach and several of the identified proteins overlapped with prior work and some of the proteins were further confirmed by immunoblotting. However, due to the limitations discussed abover, there might be other physiological tau interacting partners that were not effectively detected using this approach or were due to specific technical aspects and the model system used. Future directions I demonstrated that the BioID2 in situ labeling approach facilitates the detection of tau interactors. Future studies can adapt this technique to address several questions regarding tau proteins interactions at both the physiological and pathological states. I plan on dissecting the role of each tau domain in protein interactions by creating BioID2 fusion proteins with the following tau domains: a) 1-224 amino acids (Nterm-BioID2), b) 225-380 97 amino acids (MTBR-BioID2), c) 381-441 amino acids (Cterm-BioID2), d) 1-380 amino acids (Nterm-MTBR-BioID2), and e) 225-441 amino acids (MTBR-Cterm-BioID2). This approach will help further our understanding of the various physiological functions of the tau protein mediated by the protein-protein interactions of its different domains. Moreover, this technique can be adapted to identify tau protein interactions at the level of the disease-related modifications including pathogenic post-translational modifications and mutations of the MAPT gene involved in the neurodegenerative tauopathies. The BioID2 approach was successfully adapted by others to identify protein interactions in vivo (Feng et al., 2020; Pronobis et al., 2021). Thus, the presented approach can be utilized to study in vivo tau protein-protein interactions in tauopathy mouse models. This could help us better understand the spatiotemporal development of the disease-causing mechanisms mediated by tau protein interactions. If adapted to a normal physiological model, perhaps by fusing to normal tau in mice, the "physiological” tau interactome during neurodevelopment and/or in specific brain regions or cell types may be elucidated. The approach adapted in this thesis is a discovery-based approach in which I was interested in identifying candidates of the physiological tau interactome. Future studies may couple the discovery-based approach to a more targeted approach to study the downstream signaling pathways of the novel tau protein interactions identified. This could help reveal novel regulatory mechanisms of the various biological functions of the tau protein which may reveal novel therapeutic targets to counteract tau- mediated neurodegenerative processes. 98 APPENDICES 99 APPENDIX A Table 3: List of the identified tau protein interacting partners Accession Gene Protein Description Tau- BioID2- ID BioID2 Tau P19783 Cox4i1 Cytochrome c oxidase subunit 4 isoform 1 Yes Yes P28740-1 Kif2a Isoform 1 of Kinesin-like protein KIF2A Yes Yes P31324 Prkar2b cAMP-dependent protein kinase type II- Yes Yes beta regulatory subunit P46460 Nsf Vesicle-fusing ATPase Yes Yes Q07417 Acads Short-chain specific acyl-CoA Yes Yes dehydrogenase Q3UHX2 Pdap1 28 kDa heat- and acid-stable Yes Yes phosphoprotein Q61191 Hcfc1 Host cell factor 1 Yes Yes Q7TSJ2 Map6 Microtubule-associated protein 6 Yes Yes Q80Y14 Glrx5 Glutaredoxin-related protein 5 Yes Yes Q8BJZ4 Mrps35 28S ribosomal protein S35 Yes Yes Q91YT0 Ndufv1 NADH dehydrogenase [ubiquinone] Yes Yes flavoprotein 1 Q9WUR9 Ak4 Adenylate kinase 4 Yes Yes O88712 Ctbp1 C-terminal-binding protein 1 Yes Yes P35293 Rab18 Ras-related protein Rab-18 Yes Yes P85094 Isoc2a Isochorismatase domain-containing Yes Yes protein 2A Q60875 Arhgef2 Rho guanine nucleotide exchange factor Yes Yes 2 Q6NS60 Fbxo41 F-box only protein 41 Yes Yes Q80VC9 Camsap3 Calmodulin-regulated spectrin- Yes Yes associated protein 3 100 Table 3 (cont’d) Q8BK64 Ahsa1 Activator of 90 kDa heat shock protein Yes Yes ATPase homolog 1 Q8K0D5 Gfm1 Elongation factor G Yes Yes Q91YM4 Tbrg4 Transforming growth factor beta Yes Yes regulated gene 4; Protein TBRG4; May play a role in cell cycle progression; Belongs to the FAST kinase family Q921S7 Mrpl37 39S ribosomal protein L37 Yes Yes Q99LB2 Dhrs4 Dehydrogenase/reductase SDR family Yes Yes member 4 Q9D1E6 Tbcb Tubulin-folding cofactor B Yes Yes Q9D338 Mrpl19 39S ribosomal protein L19 Yes Yes Q9DCX2 Atp5h ATP synthase subunit d Yes Yes Q80TK0 Kiaa1107 AP2-interacting clathrin-endocytosis Yes Yes protein P27546-4 Map4-4 Isoform 4 of Microtubule-associated Yes Yes protein 4 Q9ER88 Dap3 28S ribosomal protein S29 Yes Yes P20357 Map2 Microtubule-associated protein 2 Yes Yes Q6A065 Cep170 Centrosomal protein of 170 kDa Yes Yes P54227 Stmn1 Stathmin Yes Yes O88809 Dcx Neuronal migration protein doublecortin Yes Yes P27546 Map4 Microtubule-associated protein 4 Yes Yes Q9ERD7 Tubb3 Tubulin beta-3 chain Yes Yes Q61578 Fdxr NADPH:adrenodoxin oxidoreductase Yes Yes Q9CPY7 Lap3 Cytosol aminopeptidase Yes Yes Q8BH04 Pck2 Phosphoenolpyruvate carboxykinase Yes Yes Q91ZZ3 Sncb Beta-synuclein Yes Yes P99024 Tubb5 Tubulin beta-5 chain Yes Yes Q8BJH1 Zc2hc1a Zinc finger C2HC domain-containing Yes Yes protein 1A P14873 Map1b Microtubule-associated protein 1B Yes Yes 101 Table 3 (cont’d) P16332 Mut Methylmalonyl-CoA mutase Yes Yes Q9CQR4 Acot13 Acyl-coenzyme A thioesterase 13 Yes Yes O35857 Timm44 Mitochondrial import inner membrane Yes Yes translocase subunit TIM44 Q9QYX7 Pclo Protein piccolo Yes Yes Q9JLM8 Dclk1 Serine/threonine-protein kinase DCLK1 Yes Yes Q8K2M0 Mrpl38 39S ribosomal protein L38 Yes Yes Q8JZN5 Acad9 Complex I assembly factor ACAD9 Yes Yes Q99KE1 Me2 NAD-dependent malic enzyme Yes Yes Q8CFA2 Amt Aminomethyltransferase Yes Yes Q8BIJ6 Iars2 Isoleucine--tRNA ligase Yes Yes Q8JZQ2 Afg3l2 AFG3-like protein 2 Yes Yes Q9JJV2 Pfn2 Profilin-2 Yes Yes Q8JZU2 Slc25a1 Tricarboxylate transport protein Yes Yes Q91WD5 Ndufs2 NADH dehydrogenase [ubiquinone] iron- Yes Yes sulfur protein 2 Q9R0U0 Srsf10 Serine/arginine-rich splicing factor 10 Yes Yes Q7TSQ8 Pdpr Pyruvate dehydrogenase phosphatase Yes Yes regulatory subunit Q9Z2I8 Suclg2 Succinate--CoA ligase [GDP-forming] Yes Yes subunit beta Q8BKZ9 Pdhx Pyruvate dehydrogenase protein X Yes Yes component O55042 Snca Alpha-synuclein Yes Yes Q9CZU6 Cs Citrate synthase Yes Yes Q91V41 Rab14 Ras-related protein Rab-14 Yes Yes Q8BH95 Echs1 Enoyl-CoA hydratase Yes Yes Q8K411 Pitrm1 Presequence protease Yes Yes 102 Table 3 (cont’d) Q9JJL8 Sars2 Serine--tRNA ligase Yes Yes O08788 Dctn1 Dynactin subunit 1 Yes No O09012 Pex5 Peroxisomal targeting signal 1 receptor Yes No P10630 Eif4a2 Eukaryotic initiation factor 4A-II Yes No P35235-1 Ptpn11 Isoform 2 of Tyrosine-protein Yes No phosphatase non-receptor type 11 P47857 Pfkm ATP-dependent 6-phosphofructokinase Yes No P49443 Ppm1a Protein phosphatase 1A Yes No P62245 Rps15a 40S ribosomal protein S15a Yes No P62774 Mtpn Myotrophin Yes No P83510 Tnik Traf2 and NCK-interacting protein kinase Yes No P97447 Fhl1 Four and a half LIM domains protein 1 Yes No Q0KL02 Trio Triple functional domain protein Yes No Q3UMY5 Eml4 Echinoderm microtubule-associated Yes No protein-like 4 Q61990 Pcbp2 Poly(rC)-binding protein 2 Yes No Q6PIU9 Uncharacterized protein FLJ45252 Yes No homolog Q80U49 Cep170b Centrosomal protein of 170 kDa protein B Yes No Q80X90 Flnb Filamin-B Yes No Q8BHG2 Czib CXXC motif containing zinc binding Yes No protein Q8BL97 Srsf7 Serine/arginine-rich splicing factor 7 Yes No Q8CIB5 Fermt2 Fermitin family homolog 2 Yes No Q8CIN4 Pak2 Serine/threonine-protein kinase PAK 2 Yes No Q8R570 Snap47 Synaptosomal-associated protein 47 Yes No Q8VBT9 Aspscr1 Tether containing UBX domain for GLUT4 Yes No 103 Table 3 (cont’d) Q99KC8 Vwa5a von Willebrand factor A domain- Yes No containing protein 5A Q99N95 Mrpl3 39S ribosomal protein L3 Yes No Q99N96 Mrpl1 39S ribosomal protein L1 Yes No Q9CQI6 Cotl1 Coactosin-like protein Yes No Q9D0E1 Hnrnpm Heterogeneous nuclear ribonucleoprotein Yes No M Q9DCT8 Crip2 Cysteine-rich protein 2 Yes No Q9QXZ0 Macf1 Microtubule-actin cross-linking factor 1 Yes No A2AAY5 Sh3pxd2b SH3 and PX domain-containing protein Yes No 2B O08709 Prdx6 Peroxiredoxin-6 Yes No O35098 Dpysl4 Dihydropyrimidinase-related protein 4 Yes No O35685 Nudc Nuclear migration protein nudC Yes No O54734 Ddost Dolichyl-diphosphooligosaccharide-- Yes No protein glycosyltransferase 48 kDa subunit O88342 Wdr1 WD repeat-containing protein 1 Yes No P00405 mt-Co2 Cytochrome c oxidase subunit 2 Yes No P06151 Ldha L-lactate dehydrogenase A chain Yes No P14094 Atp1b1 Sodium/potassium-transporting ATPase Yes No subunit beta-1 P14148 Rpl7 60S ribosomal protein L7 Yes No P14824 Anxa6 Annexin A6 Yes No P16460 Ass1 Argininosuccinate synthase Yes No P39054 Dnm2 Dynamin-2 Yes No P40124 Cap1 Adenylyl cyclase-associated protein 1 Yes No P45376 Akr1b3 Aldo-keto reductase family 1 member B1 Yes No P46471 Psmc2 26S proteasome regulatory subunit 7 Yes No P47941 Crkl Crk-like protein Yes No P50247 Ahcy Adenosylhomocysteinase Yes No 104 Table 3 (cont’d) P50516 Atp6v1a V-type proton ATPase catalytic subunit A Yes No P56959 Fus RNA-binding protein FUS Yes No P58404 Strn4 Striatin-4 Yes No P61082 Ube2m NEDD8-conjugating enzyme Ubc12 Yes No P62814 Atp6v1b2 V-type proton ATPase subunit B Yes No P63001 Rac1 Ras-related C3 botulinum toxin substrate Yes No 1 P63028 Tpt1 Translationally-controlled tumor protein Yes No P63242 Eif5a Eukaryotic translation initiation factor 5A- Yes No 1 P67984 Rpl22 60S ribosomal protein L22 Yes No P70271 Pdlim4 PDZ and LIM domain protein 4 Yes No P70677 Casp3 Caspase-3 Yes No P97930 Dtymk Thymidylate kinase Yes No Q2NL51 Gsk3a Glycogen synthase kinase-3 alpha Yes No Q3UU96 Cdc42bpa Serine/threonine-protein kinase MRCK Yes No alpha Q52KI8 Srrm1 Serine/arginine repetitive matrix protein 1 Yes No Q61316 Hspa4 Heat shock 70 kDa protein 4 Yes No Q61584 Fxr1 Fragile X mental retardation syndrome- Yes No related protein 1 Q68FF0 Kiaa1841 Uncharacterized protein KIAA1841 Yes No Q6NZJ6 Eif4g1 Eukaryotic translation initiation factor 4 Yes No gamma 1 Q6P1F6 Ppp2r2a Serine/threonine-protein phosphatase 2A Yes No 55 kDa regulatory subunit B alpha isoform Q8K1M6 Dnm1l Dynamin-1-like protein Yes No Q8K2K6 Agfg1 Arf-GAP domain and FG repeat- Yes No containing protein 1 Q8R1B4 Eif3c Eukaryotic translation initiation factor 3 Yes No subunit C 105 Table 3 (cont’d) Q921F2 Tardbp TAR DNA-binding protein 43 Yes No Q9CYZ2 Tpd52l2 Tumor protein D54 Yes No Q9CZM2 Rpl15 60S ribosomal protein L15 Yes No Q9D0G0 Mrps30 28S ribosomal protein S30 Yes No Q9D2R0 Aacs Acetoacetyl-CoA synthetase Yes No Q9EQI8 Mrpl46 39S ribosomal protein L46 Yes No Q9ESX4 Zcchc17 Nucleolar protein of 40 kDa Yes No Q9WTX2 Prkra Interferon-inducible double-stranded Yes No RNA-dependent protein kinase activator A Q9WV60 Gsk3b Glycogen synthase kinase-3 beta Yes No Q9WV92 Epb4.1l3 Band 4.1-like protein 3 Yes No Q9CX00 Ist1 IST1 homolog Yes No Q9EQF6 Dpysl5 Dihydropyrimidinase-related protein 5 Yes No P52196 Tst Thiosulfate sulfurtransferase Yes No Q61166 Mapre1 Microtubule-associated protein RP/EB Yes No family member 1 Q8C8R3 Ank2 Ankyrin-2 Yes No Q9QXL1 Kif21b Kinesin-like protein KIF21B Yes No Q61792 Lasp1 LIM and SH3 domain protein 1 Yes No Q9CY58 Serbp1 Plasminogen activator inhibitor 1 RNA- Yes No binding protein A2AGT5 Ckap5 Cytoskeleton-associated protein 5 Yes No Q8BRT1 Clasp2 CLIP-associating protein 2 Yes No Q8BGT8 Phyhipl Phytanoyl-CoA hydroxylase-interacting Yes No protein-like P97855 G3bp1 Ras GTPase-activating protein-binding Yes No protein 1 Q62418 Dbnl Drebrin-like protein Yes No P52480-2 Pkm-2 Isoform M1 of Pyruvate kinase PKM Yes No 106 Table 3 (cont’d) Q6ZPJ3 Ube2o (E3-independent) E2 ubiquitin- Yes No conjugating enzyme UBE2O Q8VDJ3 Hdlbp Vigilin Yes No Q8CI94 Pygb Glycogen phosphorylase Yes No Q62448 Eif4g2 Eukaryotic translation initiation factor 4 Yes No gamma 2 Q8VE88- Fam114a2 Isoform 2 of Protein FAM114A2 Yes No 2 Q9JHI5 Ivd Isovaleryl-CoA dehydrogenase Yes No Q9DAW9 Cnn3 Calponin-3 Yes No P07356 Anxa2 Annexin A2 Yes No Q62188 Dpysl3 Dihydropyrimidinase-related protein 3 Yes No Q9R0Q6 Arpc1a Actin-related protein 2/3 complex subunit Yes No 1A Q9DBT5 Ampd2 AMP deaminase 2 Yes No Q9WUM4 Coro1c Coronin-1C Yes No P63017 Hspa8 Heat shock cognate 71 kDa protein Yes No Q8BMG7 Rab3gap2 Rab3 GTPase-activating protein non- Yes No catalytic subunit Q60598 Cttn Src substrate cortactin Yes No Q8CDG3 Vcpip1 Deubiquitinating protein VCIP135 Yes No Q8VDQ8 Sirt2 NAD-dependent protein deacetylase Yes No sirtuin-2 P68369 Tuba1a Tubulin alpha-1A chain Yes No Q8CI51 Pdlim5 PDZ and LIM domain protein 5 Yes No Q6PGC1 Dhx29 ATP-dependent RNA helicase DHX29 Yes No P70296 Pebp1 Phosphatidylethanolamine-binding Yes No protein 1 P05064 Aldoa Fructose-bisphosphate aldolase A Yes No A2AHC3 Camsap1 Calmodulin-regulated spectrin-associated Yes No protein 1 Q9D8Y0 Efhd2 EF-hand domain-containing protein D2 Yes No 107 Table 3 (cont’d) B1AZI6 Thoc2 THO complex subunit 2 Yes No Q8CH77 Nav1 Neuron navigator 1 Yes No Q62417 Sorbs1 Sorbin and SH3 domain-containing Yes No protein 1 Q78ZA7 Nap1l4 Nucleosome assembly protein 1-like 4 Yes No P17742 Ppia Peptidyl-prolyl cis-trans isomerase A Yes No P05213 Tuba1b Tubulin alpha-1B chain Yes No Q8BTM8 Flna Filamin-A Yes No Q80Y56 Zfyve20 Rabenosyn-5 Yes No P68372 Tubb4b Tubulin beta-4B chain Yes No P62908 Rps3 40S ribosomal protein S3 Yes No Q6R891 Ppp1r9b Neurabin-2 Yes No Q61171 Prdx2 Peroxiredoxin-2 Yes No P18872 Gnao1 Guanine nucleotide-binding protein G(o) Yes No subunit alpha Q6PB44 Ptpn23 Tyrosine-protein phosphatase non- Yes No receptor type 23 P10649 Gstm1 Glutathione S-transferase Mu 1 Yes No O55131 7-Sep Septin-7 Yes No G5E829 Atp2b1 Plasma membrane calcium-transporting Yes No ATPase 1 Q922W5 Pycr1 Pyrroline-5-carboxylate reductase 1 Yes No Q8BGC4 Zadh2 Prostaglandin reductase-3 Yes No Q9CWF2 Tubb2b Tubulin beta-2B chain Yes No Q9CQW2 Arl8b ADP-ribosylation factor-like protein 8B Yes No P19157 Gstp1 Glutathione S-transferase P 1 Yes No Q62167 Ddx3x ATP-dependent RNA helicase DDX3X Yes No P11031 Sub1 Activated RNA polymerase II Yes No transcriptional coactivator p15 P58252 Eef2 Elongation factor 2 Yes No Q8C8R3- Ank2-2 Isoform 2 of Ankyrin-2 Yes No 2 108 Table 3 (cont’d) Q80X50 Ubap2l Ubiquitin-associated protein 2-like Yes No Q6PGH2 Hn1l Jupiter microtubule associated homolog 2 Yes No Q8BG05 Hnrnpa3 Heterogeneous nuclear ribonucleoprotein Yes No A3 Q91W50 Csde1 Cold shock domain-containing protein E1 Yes No P97427 Crmp1 Dihydropyrimidinase-related protein 1 Yes No P53994 Rab2a Ras-related protein Rab-2A Yes No O08553 Dpysl2 Dihydropyrimidinase-related protein 2 Yes No O08539 Bin1 Myc box-dependent-interacting protein 1 Yes No P18760 Cfl1 Cofilin-1 Yes No P60843 Eif4a1 Eukaryotic initiation factor 4A-I Yes No P07901 Hsp90aa1 Heat shock protein HSP 90-alpha Yes No Q6P549 Inppl1 Phosphatidylinositol 3 Yes No Q9R0P9 Uchl1 Ubiquitin carboxyl-terminal hydrolase Yes No isozyme L1 Q61768 Kif5b Kinesin-1 heavy chain Yes No E9QAT4 Sec16a Protein transport protein Sec16A Yes No Q9D8E6 Rpl4 60S ribosomal protein L4 Yes No P35700 Prdx1 Peroxiredoxin-1 Yes No Q60838 Dvl2 Segment polarity protein dishevelled Yes No homolog DVL-2 P23198 Cbx3 Chromobox protein homolog 3 Yes No P29341 Pabpc1 Polyadenylate-binding protein 1 Yes No Q9WUA2 Farsb Phenylalanine--tRNA ligase beta subunit Yes No P60710 Actb Actin Yes No P70349 Hint1 Histidine triad nucleotide-binding protein Yes No 1 Q7TMM9 Tubb2a Tubulin beta-2A chain Yes No P51880 Fabp7 Fatty acid-binding protein Yes No 109 Table 3 (cont’d) Q02053 Uba1 Ubiquitin-like modifier-activating enzyme Yes No 1 O70318 Epb4.1l2 Band 4.1-like protein 2 Yes No Q7TMK9 Syncrip Heterogeneous nuclear ribonucleoprotein Yes No Q P11499 Hsp90ab1 Heat shock protein HSP 90-beta Yes No Q6PAJ1 Bcr Breakpoint cluster region protein Yes No Q8VDD5 Myh9 Myosin-9 Yes No Q8CHT0 Aldh4a1 Delta-1-pyrroline-5-carboxylate Yes No dehydrogenase Q9CR57 Rpl14 60S ribosomal protein L14 Yes No Q9D8N0 Eef1g Elongation factor 1-gamma Yes No Q80UG5 9-Sep Septin-9 Yes No Q61753 Phgdh D-3-phosphoglycerate dehydrogenase Yes No P12367 Prkar2a cAMP-dependent protein kinase type II- Yes No alpha regulatory subunit P84244 H3f3b Histone H3.3 Yes No P62259 Ywhae 14-3-3 protein epsilon Yes No Q9QZQ1 Mllt4 Afadin Yes No Q8BGD9 Eif4b Eukaryotic translation initiation factor 4B Yes No P57776 Eef1d Elongation factor 1-delta Yes No Q7TSC1 Prrc2a Protein PRRC2A Yes No Q9Z1N5 Ddx39b Spliceosome RNA helicase Ddx39b Yes No P62827 Ran GTP-binding nuclear protein Ran Yes No Q99PT1 Arhgdia Rho GDP-dissociation inhibitor 1 Yes No P60335 Pcbp1 Poly(rC)-binding protein 1 Yes No Q9Z2Q6 5-Sep Septin-5 Yes No P28652 Camk2b Calcium/calmodulin-dependent protein Yes No kinase type II subunit beta 110 Table 3 (cont’d) P54923 Adprh [Protein ADP-ribosylarginine] hydrolase Yes No P62869 Tceb2 Elongin-B Yes No Q68FD5 Cltc Clathrin heavy chain 1 Yes No P20152 Vim Vimentin Yes No P27659 Rpl3 60S ribosomal protein L3 Yes No P16858 Gapdh Glyceraldehyde-3-phosphate Yes No dehydrogenase Q61553 Fscn1 Fascin Yes No Q9CXW4 Rpl11 60S ribosomal protein L11 Yes No Q8VEH3 Arl8a ADP-ribosylation factor-like protein 8A Yes No P50136 Bckdha 2-oxoisovalerate dehydrogenase subunit Yes No alpha Q64521 Gpd2 Glycerol-3-phosphate dehydrogenase No Yes Q8BG05- Hnrnpa3-2 Isoform 2 of Heterogeneous nuclear No Yes 2 ribonucleoprotein A3 Q8CC88 Vwa8 von Willebrand factor A domain- No Yes containing protein 8 Q8QZS1 Hibch 3-hydroxyisobutyryl-CoA hydrolase No Yes Q99N93 Mrpl16 39S ribosomal protein L16 No Yes Q9CXI0 Coq5 2-methoxy-6-polyprenyl-1 No Yes Q9DCM0 Ethe1 Persulfide dioxygenase ETHE1 No Yes Q9JKF7 Mrpl39 39S ribosomal protein L39 No Yes P20108 Prdx3 Thioredoxin-dependent peroxide No Yes reductase P47934 Crat Carnitine O-acetyltransferase No Yes P52825 Cpt2 Carnitine O-palmitoyltransferase 2 No Yes Q14C51 Ptcd3 Pentatricopeptide repeat domain- No Yes containing protein 3 Q3TBW2 Mrpl10 39S ribosomal protein L10 No Yes Q3UQ84 Tars2 Threonine--tRNA ligase No Yes Q501J6 Ddx17 Probable ATP-dependent RNA helicase No Yes 111 Table 3 (cont’d) Q5SUF2 Luc7l3 Luc7-like protein 3 No Yes Q60759 Gcdh Glutaryl-CoA dehydrogenase No Yes Q61425 Hadh Hydroxyacyl-coenzyme A dehydrogenase No Yes Q80X85 Mrps7 28S ribosomal protein S7 No Yes Q91Z67 Srgap2 SLIT-ROBO Rho GTPase-activating No Yes protein 2 Q99N94 Mrpl9 39S ribosomal protein L9 No Yes Q9D0Q7 Mrpl45 39S ribosomal protein L45 No Yes Q9D6M3 Slc25a22 Mitochondrial glutamate carrier 1 No Yes Q9DC69 Ndufa9 NADH dehydrogenase [ubiquinone] 1 No Yes alpha subcomplex subunit 9 Q9JLJ2 Aldh9a1 4-trimethylaminobutyraldehyde No Yes dehydrogenase O88935- Syn1 Isoform Ib of Synapsin-1 No Yes 1 P12787 Cox5a Cytochrome c oxidase subunit 5A No Yes Q9R020 Zranb2 Zinc finger Ran-binding domain- No Yes containing protein 2 P42125 Eci1 Enoyl-CoA delta isomerase 1 No Yes P84104 Srsf3 Serine/arginine-rich splicing factor 3 No Yes Q7TNC4 Luc7l2 Putative RNA-binding protein Luc7-like 2 No Yes O08749 Dld Dihydrolipoyl dehydrogenase No Yes Q99JR1 Sfxn1 Sideroflexin-1 No Yes Q8VEM8 Slc25a3 Phosphate carrier protein No Yes Q99J99 Mpst 3-mercaptopyruvate sulfurtransferase No Yes Q8BK72 Mrps27 28S ribosomal protein S27 No Yes Q8BWT1 Acaa2 3-ketoacyl-CoA thiolase No Yes P14206 Rpsa 40S ribosomal protein SA No Yes P51174 Acadl Long-chain specific acyl-CoA No Yes dehydrogenase 112 Table 3 (cont’d) Q9JLZ3 Auh Methylglutaconyl-CoA hydratase No Yes Q99NB1 Acss1 Acetyl-coenzyme A synthetase 2-like No Yes Q9D7B6 Acad8 Isobutyryl-CoA dehydrogenase No Yes Q8VE22 Mrps23 28S ribosomal protein S23 No Yes Q9WTP7 Ak3 GTP:AMP phosphotransferase AK3 No Yes Q9JHS4 Clpx ATP-dependent Clp protease ATP- No Yes binding subunit clpX-like Q9QWI6 Srcin1 SRC kinase signaling inhibitor 1 No Yes Q9DBL1 Acadsb Short/branched chain specific acyl-CoA No Yes dehydrogenase Q99LC3 Ndufa10 NADH dehydrogenase [ubiquinone] 1 No Yes alpha subcomplex subunit 10 P29758 Oat Ornithine aminotransferase No Yes Q8C5H8 Nadk2 NAD kinase 2 No Yes Q9EQ20 Aldh6a1 Methylmalonate-semialdehyde No Yes dehydrogenase [acylating] P47738 Aldh2 Aldehyde dehydrogenase No Yes P70404 Idh3g Isocitrate dehydrogenase [NAD] subunit No Yes gamma 1 Q9CZN7 Shmt2 Serine hydroxymethyltransferase No Yes Q9CZS1 Aldh1b1 Aldehyde dehydrogenase X No Yes Q6ZWN5 Rps9 40S ribosomal protein S9 OS No Yes P50171 H2-Ke6 Estradiol 17-beta-dehydrogenase 8 No Yes P99029 Prdx5 Peroxiredoxin-5 No Yes Q925I1 Atad3a ATPase family AAA domain-containing No Yes protein 3 Q9CQ62 Decr1 2,4-dienoyl-CoA reductase No Yes Q9CQN1 Trap1 Heat shock protein 75 kDa No Yes Q5M8N0 Cnrip1 CB1 cannabinoid receptor-interacting No Yes protein 1 113 Table 3 (cont’d) Q5FWK3 Arhgap1 Rho GTPase-activating protein 1 No Yes Q04750 Top1 DNA topoisomerase 1 No Yes Q9D6R2 Idh3a Isocitrate dehydrogenase [NAD] subunit No Yes alpha Q6PB66 Lrpprc Leucine-rich PPR motif-containing protein No Yes Q8VH51- Rbm39 Isoform 2 of RNA-binding protein 39 No Yes 2 Q91VD9 Ndufs1 NADH-ubiquinone oxidoreductase 75 kDa No Yes subunit Q6P3A8 Bckdhb 2-oxoisovalerate dehydrogenase subunit No Yes beta P61922 Abat 4-aminobutyrate aminotransferase No Yes Q9Z110 Aldh18a1 Delta-1-pyrroline-5-carboxylate synthase No Yes O88696 Clpp ATP-dependent Clp protease proteolytic No Yes subunit P47963 Rpl13 60S ribosomal protein L13 No Yes Q9CQA3 Sdhb Succinate dehydrogenase [ubiquinone] No Yes iron-sulfur subunit P35486 Pdha1 Pyruvate dehydrogenase E1 component No Yes subunit alpha Q99JY0 Hadhb Trifunctional enzyme subunit beta No Yes Q9Z2Z6 Slc25a20 Mitochondrial carnitine/acylcarnitine No Yes carrier protein Q8R164 Bphl Valacyclovir hydrolase No Yes Q8BMF4 Dlat Dihydrolipoyllysine-residue No Yes acetyltransferase component of pyruvate dehydrogenase complex Q8CGK3 Lonp1 Lon protease homolog No Yes Q8BIW1 Prune Exopolyphosphatase PRUNE1 No Yes P54071 Idh2 Isocitrate dehydrogenase [NADP] No Yes Q9DCW4 Etfb Electron transfer flavoprotein subunit beta No Yes 114 Table 3 (cont’d) P97807 Fh1 Fumarate hydratase No Yes Q8BMS1 Hadha Trifunctional enzyme subunit alpha No Yes O55125 Nipsnap1 Protein NipSnap homolog 1 No Yes Q99KI0 Aco2 Aconitate hydratase No Yes Q3UNH4 Gprin1 G protein-regulated inducer of neurite No Yes outgrowth 1 P08249 Mdh2 Malate dehydrogenase No Yes Q9WUR2 Eci2 Enoyl-CoA delta isomerase 2 No Yes Q9Z1J3 Nfs1 Cysteine desulfurase No Yes P45952 Acadm Medium-chain specific acyl-CoA No Yes dehydrogenase Q8QZT1 Acat1 Acetyl-CoA acetyltransferase No Yes P56480 Atp5b ATP synthase subunit beta No Yes P01942 Hba-a2 Hemoglobin subunit alpha No Yes Q8VDC0 Lars2 Probable leucine--tRNA ligase No Yes Q9DB20 Atp5o ATP synthase subunit O No Yes P26443 Glud1 Glutamate dehydrogenase 1 No Yes Q80XN0 Bdh1 D-beta-hydroxybutyrate dehydrogenase No Yes Q99LC5 Etfa Electron transfer flavoprotein subunit No Yes alpha P50544 Acadvl Very long-chain specific acyl-CoA No Yes dehydrogenase Q91VA6 Poldip2 Polymerase delta-interacting protein 2 No Yes P51150 Rab7 Ras-related protein Rab-7a No Yes 115 APPENDIX B Table 4: List of tau interacting partners identified in ClueGo cellular components GO term Number of % Associated Associated genes found genes genes mitochondrion 164 8.627038002 Abat, Acaa2, Acad8, Acad9, Acadl, Acadm, Acads, Acadsb, Acadvl, Acat1, Aco2, Acot13, Acss1, Afg3l2, Ak3, Ak4, Aldh18a1, Aldh1b1, Aldh2, Aldh4a1, Aldh6a1, Aldh9a1, Aldoa, Amt, Ank2, Anxa6, Ass1, Atad3a, Atp5b, Atp5h, Atp5o, Atp6v1a, Auh, Bckdha, Bckdhb, Bdh1, Bphl, COX2, Cfl1, Clpp, Clpx, Cltc, Coq5, Cox4i1, Cox5a, Cpt2, Crat, Cs, Csde1, Dap3, Decr1, Dhrs4, Dhx29, Dlat, Dld, Dnm1l, Dnm2, Dpysl2, Dtymk, Echs1, Eci1, Eci2, Etfa, Etfb, Ethe1, Fdxr, Fh1, Gapdh, Gcdh, Gfm1, Glrx5, Glud1, Gpd2, Gsk3a, Gsk3b, Gstp1, H2-Ke6, Hadh, Hadha, Hadhb, Hibch, Hsp90aa1, Hsp90ab1, Hspa4, Iars2, Idh2, Idh3a, Idh3g, Isoc2a, Ivd, Lap3, Lars2, Ldha, Lonp1, Lrpprc, Mdh2, Me2, Mmut, Mpst, Mrpl1, Mrpl10, Mrpl16, Mrpl19, Mrpl3, Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps23, Mrps27, Mrps30, Mrps35, Mrps7, Nadk2, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1, Nfs1, Nipsnap1, Oat, Pck2, Pdha1, Pdhx, Pdpr, Pebp1, Pex5, Phyhipl, Pitrm1, Poldip2, Prdx1, Prdx2, Prdx3, Prdx5, Prdx6, Ptcd3, Pycr1, Rps15a, Rps3, Sars2, Sdhb, Sfxn1, Shmt2, Sirt2, Slc25a1, Slc25a20, Slc25a22, Slc25a3, Snca, Sncb, Srgap2, Suclg2, Tars2, Tbrg4, Timm44, Trap1, Tst, Uba1, Vwa8, Ywhae, Zadh2 116 Table 4 (cont’d) mitochondrial 83 23.78223419 Abat, Acaa2, Acadl, Acadm, Acads, matrix Acadsb, Acadvl, Acat1, Acss1, Ak3, Ak4, Aldh1b1, Aldh2, Aldh4a1, Atad3a, Atp5b, Bckdha, Bckdhb, Bdh1, Clpp, Clpx, Coq5, Cs, Dap3, Dlat, Dld, Dtymk, Echs1, Eci1, Etfa, Etfb, Ethe1, Gcdh, Glrx5, Glud1, H2-Ke6, Hadh, Hadha, Hadhb, Iars2, Ivd, Lars2, Lonp1, Lrpprc, Mdh2, Me2, Mmut, Mrpl1, Mrpl10, Mrpl16, Mrpl19, Mrpl3, Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps23, Mrps27, Mrps30, Mrps35, Mrps7, Ndufa10, Ndufa9, Nfs1, Oat, Pdha1, Pdhx, Pdpr, Pitrm1, Poldip2, Prdx1, Rps3, Sars2, Shmt2, Snca, Suclg2, Tars2, Tbrg4, Timm44, Trap1, Tst mitochondrial 59 8.452721596 Acaa2, Acad9, Acadl, Acadm, Acads, membrane Acadvl, Acat1, Afg3l2, Aldh18a1, Ass1, Atad3a, Atp5b, Atp5h, Atp5o, Bckdhb, Bdh1, COX2, Cfl1, Clpx, Coq5, Cox4i1, Cox5a, Cpt2, Crat, Csde1, Dnm1l, Dnm2, Eci1, Fdxr, Gcdh, Glud1, Gpd2, Hadh, Hadha, Hadhb, Idh2, Ivd, Mdh2, Mpst, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1, Nipsnap1, Pebp1, Rps3, Sdhb, Sfxn1, Shmt2, Slc25a1, Slc25a20, Slc25a22, Slc25a3, Snca, Srgap2, Timm44, Trap1, Tst mitochondrial 63 8.333333015 Acaa2, Acad9, Acadl, Acadm, Acads, envelope Acadvl, Acat1, Afg3l2, Ak3, Ak4, Aldh18a1, Ass1, Atad3a, Atp5b, Atp5h, Atp5o, Bckdhb, Bdh1, COX2, Cfl1, Clpx, Coq5, Cox4i1, Cox5a, Cpt2, Crat, Csde1, Dnm1l, Dnm2, Dtymk, Eci1, Fdxr, Gcdh, Glud1, Gpd2, H2- Ke6, Hadh, Hadha, Hadhb, Idh2, Ivd, Mdh2, Mpst, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1, Nipsnap1, Pebp1, Rps3, Sdhb, Sfxn1, Shmt2, Slc25a1, Slc25a20, Slc25a22, Slc25a3, Snca, Srgap2, Timm44, Trap1, Tst 117 Table 4 (cont’d) mitochondrial 38 14.61538506 Afg3l2, Atp5b, Atp5h, Atp5o, Bckdha, protein- Bckdhb, Clpx, Cox4i1, Cox5a, Dap3, containing Dlat, Etfa, Etfb, Hadha, Hadhb, Mrpl1, complex Mrpl10, Mrpl16, Mrpl19, Mrpl3, Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps23, Mrps27, Mrps30, Mrps35, Mrps7, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1, Sdhb, Suclg2 cytoskeleton 96 4.125904083 Acot13, Actb, Aldoa, Ank2, Arhgef2, Arl8a, Arl8b, Arpc1a, Bin1, Camk2b, Camsap1, Camsap3, Cap1, Cbx3, Cdc42bpa, Cep170, Cep170b, Cfl1, Ckap5, Clasp2, Cltc, Cnn3, Coro1c, Cotl1, Crmp1, Cttn, Dbnl, Dctn1, Dcx, Ddx3x, Dnm1l, Dnm2, Dpysl2, Dpysl3, Dvl2, Eml4, Epb41l2, Epb41l3, Fermt2, Flna, Flnb, Fscn1, Gapdh, Gsk3a, Gsk3b, Hspa8, Inppl1, Ist1, Kif21b, Kif5b, Lasp1, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Mtpn, Myh9, Nav1, Nudc, Pclo, Pdlim4, Pdlim5, Pfn2, Ppp1r9b, Prkar2a, Prkar2b, Ptpn23, Rac1, Ran, Rps3, Septin5, Septin7, Septin9, Sh3pxd2b, Shmt2, Sirt2, Snca, Sorbs1, Srcin1, Stmn1, Tbcb, Tnik, Tpt1, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Vim, Wdr1, Ywhae neuron 84 4.929577351 Aak1, Abat, Acad9, Acadm, Actb, Afdn, projection Ahcy, Ank2, Arhgef2, Arl8a, Arl8b, Ass1, Atp2b1, Bcr, Bin1, Camk2b, Cfl1, Clasp2, Cnn3, Crip2, Crmp1, Cttn, Dbnl, Dctn1, Dcx, Dnm2, Dpysl2, Dpysl3, Dpysl5, Eif4b, Eif4g2, Eif5a, Epb41l3, Flna, Flnb, Fscn1, Fus, Fxr1, Glrx5, Gnao1, Gprin1, Gsk3a, Gsk3b, Hcfc1, Hnrnpa3, Hsp90aa1, Hsp90ab1, Hspa8, Kif21b, Kif5b, Map1b, Map2, Map4, Map6, Mpst, Mtpn, Nap1l4, Nav1, Nsf, Pabpc1, Pclo, Pdlim4, Pebp1, Ppm1a, Ppp1r9b, Prkar2b, Psmc2, Pygb, Rac1, Rps3, Septin5, Septin7, Sirt2, Snap47, Snca, Sncb, Srcin1, Srgap2, Stmn1, Strn4, Tubb3, Uchl1, Vim, Ywhae 118 Table 4 (cont’d) microtubule 62 4.751299381 Arhgef2, Arl8a, Arl8b, Bin1, Camk2b, cytoskeleton Camsap1, Camsap3, Cbx3, Cep170, Cep170b, Ckap5, Clasp2, Cltc, Crmp1, Cttn, Dctn1, Dcx, Ddx3x, Dnm1l, Dnm2, Dpysl2, Eml4, Gapdh, Gsk3a, Gsk3b, Hspa8, Ist1, Kif21b, Kif5b, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Myh9, Nav1, Nudc, Prkar2a, Prkar2b, Ptpn23, Rac1, Ran, Rps3, Septin5, Septin7, Septin9, Shmt2, Sirt2, Sorbs1, Stmn1, Tbcb, Tpt1, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Ywhae microtubule 41 9.213482857 Arhgef2, Bin1, Camsap1, Camsap3, Cep170, Cep170b, Ckap5, Clasp2, Cltc, Dctn1, Dcx, Dnm1l, Dnm2, Dpysl2, Eml4, Gsk3a, Gsk3b, Hspa8, Kif21b, Kif5b, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Nav1, Nudc, Septin9, Sirt2, Stmn1, Tbcb, Tpt1, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5 organelle inner 47 9.845560074 Acaa2, Aldh18a1, Atad3a, Atp5b, membrane Atp5h, Atp5o, Bckdhb, Bdh1, COX2, Clpx, Coq5, Cox4i1, Cox5a, Cpt2, Crat, Csde1, Eci1, Fdxr, Gcdh, Glud1, Gpd2, Hadh, Hadha, Hadhb, Idh2, Lrpprc, Mdh2, Mpst, Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1, Nipsnap1, Rps3, Sdhb, Sfxn1, Shmt2, Slc25a1, Slc25a20, Slc25a22, Slc25a3, Snca, Srgap2, Timm44, Trap1, Tst supramolecular 61 5.888031006 Actb, Aldoa, Ank2, Arhgef2, Bin1, fiber Camsap1, Camsap3, Cep170, Cep170b, Ckap5, Clasp2, Cltc, Cotl1, Cttn, Dbnl, Dctn1, Dcx, Dnm1l, Dnm2, Dpysl2, Dpysl3, Eml4, Fermt2, Flna, Flnb, Fxr1, Gsk3a, Gsk3b, Hspa8, Kif21b, Kif5b, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Myh9, Nav1, Nudc, Pdlim4, Pdlim5, Rac1, Rpl15, Rpl4, Rpl7, Septin9, Sirt2, Snca, Stmn1, Tbcb, Tpt1, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Vim 119 Table 4 (cont’d) axon 52 6.110458374 Aak1, Acadm, Actb, Afdn, Arl8a, Arl8b, Bcr, Bin1, Cfl1, Clasp2, Crip2, Crmp1, Cttn, Dctn1, Dcx, Dnm2, Dpysl2, Dpysl3, Eif4g2, Epb41l3, Flna, Fscn1, Fxr1, Gprin1, Gsk3a, Gsk3b, Hcfc1, Hsp90aa1, Hsp90ab1, Hspa8, Kif21b, Kif5b, Map1b, Map2, Map4, Map6, Mtpn, Nav1, Pclo, Pebp1, Ppp1r9b, Pygb, Septin5, Septin7, Sirt2, Snca, Sncb, Srcin1, Tubb3, Uchl1, Vim, Ywhae postsynapse 50 6.180469513 Actb, Ank2, Arhgef2, Atp2b1, Bcr, Camk2b, Cfl1, Ckap5, Cnn3, Crkl, Cttn, Dbnl, Dclk1, Dnm2, Eif4b, Eif4g1, Eif4g2, Epb41l3, Flna, Fus, Fxr1, Gapdh, Gsk3b, Hnrnpa3, Hspa8, Macf1, Map1b, Map2, Map4, Nsf, Pak2, Pcbp2, Pclo, Pdlim4, Pdlim5, Pfn2, Ppp1r9b, Prkar2b, Psmc2, Rac1, Rpl14, Rpl4, Rpl7, Rps3, Snap47, Snca, Srcin1, Srgap2, Strn4, Tnik dendrite 48 5.73476696 Acad9, Arhgef2, Atp2b1, Bcr, Bin1, Camk2b, Cfl1, Cnn3, Crmp1, Cttn, Dbnl, Dcx, Dnm2, Dpysl2, Dpysl5, Eif4b, Eif5a, Flna, Fscn1, Fus, Fxr1, Glrx5, Gnao1, Gsk3b, Hcfc1, Hsp90aa1, Hsp90ab1, Hspa8, Kif21b, Kif5b, Map1b, Map2, Map6, Nap1l4, Nsf, Pabpc1, Pclo, Pdlim4, Ppp1r9b, Prkar2b, Psmc2, Rac1, Rps3, Snap47, Srcin1, Srgap2, Strn4, Tubb3 distal axon 34 7.852193832 Aak1, Actb, Bin1, Cfl1, Clasp2, Crip2, Crmp1, Cttn, Dcx, Dnm2, Dpysl2, Dpysl3, Flna, Fscn1, Fxr1, Gprin1, Gsk3b, Hsp90aa1, Hsp90ab1, Hspa8, Kif21b, Kif5b, Map1b, Map2, Pclo, Pebp1, Ppp1r9b, Septin5, Septin7, Sirt2, Snca, Sncb, Tubb3, Ywhae postsynaptic 34 7.744874477 Actb, Ank2, Arhgef2, Bcr, Camk2b, density Cnn3, Dbnl, Dclk1, Dnm2, Eif4g2, Epb41l3, Fxr1, Gapdh, Gsk3b, Hnrnpa3, Hspa8, Macf1, Map1b, Map2, Map4, Nsf, Pak2, Pcbp2, Pclo, Pdlim5, Ppp1r9b, Rpl14, Rpl4, Rpl7, Rps3, Snap47, Srcin1, Srgap2, Tnik 120 Table 4 (cont’d) polymeric 51 6.488549709 Actb, Arhgef2, Bin1, Camsap1, cytoskeletal Camsap3, Cep170, Cep170b, Ckap5, fiber Clasp2, Cltc, Cotl1, Cttn, Dbnl, Dctn1, Dcx, Dnm1l, Dnm2, Dpysl2, Dpysl3, Eml4, Flna, Gsk3a, Gsk3b, Hspa8, Kif21b, Kif5b, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Nav1, Nudc, Pdlim4, Pdlim5, Rac1, Septin9, Sirt2, Stmn1, Tbcb, Tpt1, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Vim actin 36 6.923077106 Actb, Aldoa, Arhgef2, Arpc1a, Cap1, cytoskeleton Cdc42bpa, Cfl1, Cnn3, Coro1c, Cotl1, Crmp1, Cttn, Dbnl, Dpysl3, Epb41l2, Fermt2, Flna, Flnb, Fscn1, Lasp1, Macf1, Map2, Mtpn, Myh9, Pdlim4, Pdlim5, Ppp1r9b, Rac1, Septin5, Septin7, Septin9, Sh3pxd2b, Snca, Sorbs1, Srcin1, Wdr1 ribosome 36 14.87603283 Dap3, Ddx3x, Dhx29, Eef2, Hba-a1, Mrpl1, Mrpl10, Mrpl16, Mrpl19, Mrpl3, Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps23, Mrps27, Mrps30, Mrps35, Mrps7, Ptcd3, Rpl11, Rpl13, Rpl14, Rpl15, Rpl22, Rpl3, Rpl4, Rpl7, Rps15a, Rps3, Rps9, Rpsa, Snca, Zcchc17 ribosomal 33 15.86538506 Dap3, Ddx3x, Dhx29, Hba-a1, Mrpl1, subunit Mrpl10, Mrpl16, Mrpl19, Mrpl3, Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps23, Mrps27, Mrps30, Mrps35, Mrps7, Rpl11, Rpl13, Rpl14, Rpl15, Rpl22, Rpl3, Rpl4, Rpl7, Rps15a, Rps3, Rps9, Rpsa, Zcchc17 large ribosomal 21 15.67164135 Mrpl1, Mrpl10, Mrpl16, Mrpl19, Mrpl3, subunit Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps30, Rpl11, Rpl13, Rpl14, Rpl15, Rpl22, Rpl3, Rpl4, Rpl7, Zcchc17 cytosolic 17 13.7096777 Ddx3x, Dhx29, Hba-a1, Mrpl1, Rpl11, ribosome Rpl13, Rpl14, Rpl15, Rpl22, Rpl3, Rpl4, Rpl7, Rps15a, Rps3, Rps9, Rpsa, Zcchc17 121 Table 4 (cont’d) mitochondrial 17 18.88888931 Dap3, Mrpl1, Mrpl10, Mrpl16, Mrpl19, ribosome Mrpl3, Mrpl37, Mrpl38, Mrpl39, Mrpl45, Mrpl46, Mrpl9, Mrps23, Mrps27, Mrps30, Mrps35, Mrps7 cell cortex 30 8.40336132 Actb, Anxa2, Cap1, Cfl1, Clasp2, Coro1c, Cotl1, Crip2, Ctbp1, Cttn, Dbnl, Dctn1, Dvl2, Epb41l2, Fermt2, Flna, Flnb, Fscn1, Lasp1, Macf1, Mapre1, Myh9, Pclo, Ppp1r9b, Rac1, Septin5, Septin7, Septin9, Snca, Wdr1 growth cone 27 10.93117428 Cfl1, Clasp2, Crip2, Crmp1, Cttn, Dcx, Dnm2, Dpysl2, Dpysl3, Flna, Fscn1, Fxr1, Gprin1, Gsk3b, Hsp90aa1, Hsp90ab1, Kif21b, Kif5b, Map1b, Map2, Pclo, Ppp1r9b, Sirt2, Snca, Sncb, Tubb3, Ywhae presynapse 27 4.02985096 Aak1, Actb, Atp2b1, Bin1, Btbd8, Cltc, Ctbp1, Dnm1l, Dnm2, Dpysl2, Fxr1, Hspa8, Pclo, Pdlim5, Pebp1, Pfn2, Rab14, Rab2a, Rab7, Rpl22, Septin5, Septin7, Snap47, Snca, Sncb, Srcin1, Tnik dendritic spine 18 7.594936848 Atp2b1, Bcr, Cfl1, Cnn3, Cttn, Dnm2, Fus, Fxr1, Gsk3b, Hspa8, Map1b, Pdlim4, Ppp1r9b, Prkar2b, Psmc2, Rac1, Srgap2, Strn4 ribonucleoprotein 15 5.836575985 Actb, Csde1, Ddx3x, Eif4g1, Fxr1, granule G3bp1, Hnrnpa3, Pabpc1, Pcbp1, Psmc2, Rac1, Tardbp, Top1, Tuba1a, Tubb5 cortical 14 11.19999981 Actb, Cap1, Cfl1, Clasp2, Cotl1, Cttn, cytoskeleton Dbnl, Flna, Lasp1, Mapre1, Myh9, Pclo, Ppp1r9b, Wdr1 actin filament 13 13.26530647 Actb, Cfl1, Fermt2, Flna, Flnb, Fscn1, bundle Myh9, Pdlim4, Pdlim5, Septin5, Septin7, Septin9, Sorbs1 melanosome 13 12.0370369 Ahcy, Anxa2, Anxa6, Atp6v1b2, Cltc, Hsp90aa1, Hsp90ab1, Hspa8, Rab2a, Rab7, Rac1, Ran, Ywhae actomyosin 12 12.12121201 Actb, Cdc42bpa, Fermt2, Flnb, Fscn1, Myh9, Pdlim4, Pdlim5, Septin5, Septin7, Septin9, Sorbs1 122 Table 4 (cont’d) inner 12 9.677419662 Afg3l2, Atp5b, Atp5h, Atp5o, Cox4i1, mitochondrial Cox5a, Ndufa10, Ndufa9, Ndufs1, membrane Ndufs2, Ndufv1, Sdhb protein complex mitochondrial 12 20.6896553 Mrpl1, Mrpl10, Mrpl16, Mrpl19, Mrpl3, large ribosomal Mrpl37, Mrpl38, Mrpl39, Mrpl45, subunit Mrpl46, Mrpl9, Mrps30 neuron 12 5.381165981 Aak1, Actb, Bin1, Dpysl2, Hspa8, Pclo, projection Pebp1, Septin5, Septin7, Snca, Sncb, terminus Uchl1 peroxisome 12 7.547169685 Crat, Dhrs4, Dnm1l, Eci2, Idh2, Pex5, Prdx1, Prdx5, Prdx6, Vim, Vwa8, Zadh2 small ribosomal 12 14.28571415 Dap3, Ddx3x, Dhx29, Hba-a1, Mrps23, subunit Mrps27, Mrps35, Mrps7, Rps15a, Rps3, Rps9, Rpsa cytosolic large 10 13.33333302 Mrpl1, Rpl11, Rpl13, Rpl14, Rpl15, ribosomal Rpl22, Rpl3, Rpl4, Rpl7, Zcchc17 subunit mitochondrial 10 20.83333397 Acadvl, Atad3a, Atp5b, Clpx, Hadha, nucleoid Hadhb, Lonp1, Lrpprc, Poldip2, Shmt2 actin filament 9 6.617647171 Actb, Cotl1, Cttn, Dbnl, Dpysl3, Flna, Pdlim4, Pdlim5, Rac1 cortical actin 9 10.11236 Cap1, Cfl1, Cotl1, Cttn, Dbnl, Lasp1, cytoskeleton Myh9, Ppp1r9b, Wdr1 respiratory chain 9 12.16216183 COX2, Cox4i1, Cox5a, Ndufa10, complex Ndufa9, Ndufs1, Ndufs2, Ndufv1, Sdhb synaptic vesicle 9 6.766917229 Atp2b1, Bin1, Cltc, Dnm1l, Rab14, membrane Rab2a, Rab7, Snap47, Snca mitochondrial 8 11.4285717 Cox4i1, Cox5a, Ndufa10, Ndufa9, respirasome Ndufs1, Ndufs2, Ndufv1, Sdhb cytosolic small 7 14 Ddx3x, Dhx29, Hba-a1, Rps15a, Rps3, ribosomal Rps9, Rpsa subunit dendritic shaft 7 10.14492798 Arhgef2, Flna, Gsk3b, Hspa8, Map2, Nsf, Prkar2b 123 Table 4 (cont’d) microtubule end 6 17.1428566 Camsap1, Camsap3, Ckap5, Clasp2, Dctn1, Mapre1 mitochondrial 5 16.66666603 Dap3, Mrps23, Mrps27, Mrps35, Mrps7 small ribosomal subunit peroxisomal 5 17.8571434 Eci2, Pex5, Prdx1, Prdx5, Prdx6 matrix postsynaptic 5 15.15151501 Arhgef2, Bcr, Dnm2, Gapdh, Tnik density, intracellular component 124 APPENDIX C Table 5: List of tau interacting partners identified in ClueGo molecular function pathways GO term Number % Associated Associated genes found of genes genes nucleotide 124 5.218446732 Acadm, Acads, Acadsb, Acadvl, Acat1, binding Acss1, Actb, Afg3l2, Ahcy, Ak3, Ak4, Aldh18a1, Aldh2, Aldh4a1, Aldh9a1, Anxa6, Arhgdia, Arhgef2, Arl8a, Arl8b, Ass1, Atad3a, Atp2b1, Atp5b, Atp6v1a, Atp6v1b2, Bcr, Camk2b, Cdc42bpa, Clpx, Ctbp1, Dclk1, Ddx17, Ddx39b, Ddx3x, Decr1, Dhx29, Dld, Dnm1l, Dnm2, Dtymk, Eci2, Eef1d, Eef2, Eif4a1, Eif4a2, Eif4b, Eif4g1, Etfa, Etfb, Farsb, Fdxr, G3bp1, Gapdh, Gcdh, Gfm1, Glud1, Gnao1, Gsk3a, Gsk3b, H2-Ke6, Hadh, Hadha, Hint1, Hsp90aa1, Hsp90ab1, Hspa4, Hspa8, Iars2, Idh2, Idh3a, Idh3g, Ivd, Kif21b, Kif5b, Lars2, Ldha, Lonp1, Me2, Mrpl39, Myh9, Nadk2, Ndufs2, Ndufv1, Nsf, Pak2, Pck2, Pebp1, Pfkm, Phgdh, Prkar2a, Prkar2b, Psmc2, Rab14, Rab18, Rab2a, Rab3gap2, Rab7, Rac1, Ran, Sars2, Septin5, Septin7, Septin9, Sirt2, Stmn1, Suclg2, Tars2, Timm44, Tnik, Top1, Trap1, Trio, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Uba1, Ube2m, Ube2o, Vwa8 RNA 71 6.027164459 Auh, Cltc, Csde1, Ddx17, Ddx39b, Ddx3x, binding Dhx29, Eef1d, Eef1g, Eef2, Eif3c, Eif4a1, Eif4a2, Eif4b, Eif4g1, Eif4g2, Eif5a, Farsb, Fus, Fxr1, G3bp1, Gfm1, Hdlbp, Hnrnpa3, Hnrnpm, Hsp90aa1, Hsp90ab1, Hspa8, Iars2, Lonp1, Lrpprc, Luc7l2, Luc7l3, Mrpl1, Mrpl16, Mrps27, Mrps7, Nfs1, Pabpc1, Pcbp1, Pcbp2, Prkra, Ptcd3, Ran, Rpl11, Rpl13, Rpl14, Rpl15, Rpl22, Rpl3, Rpl4, Rpl7, Rps3, Rps9, Sars2, Serbp1, Shmt2, Srrm1, Srsf10, Srsf3, Srsf7, Syncrip, Tardbp, Thoc2, Trap1, Tst, Tuba1b, Tubb4b, Vim, Zcchc17, Zranb2 125 Table 5 (cont’d) purine 104 4.693140984 Aacs, Aak1, Acad9, Acadl, Acadm, Acads, nucleotide Acadvl, Acat1, Acss1, Actb, Afg3l2, Ahcy, binding Ak3, Ak4, Aldh18a1, Anxa6, Arhgdia, Arhgef2, Arl8a, Arl8b, Ass1, Atad3a, Atp2b1, Atp5b, Atp6v1a, Atp6v1b2, Bcr, Camk2b, Cdc42bpa, Clpx, Dclk1, Ddx17, Ddx39b, Ddx3x, Dhx29, Dnm1l, Dnm2, Dtymk, Eci2, Eef1d, Eef2, Eif4a1, Eif4a2, Eif4b, Eif4g1, Farsb, G3bp1, Gcdh, Gfm1, Glud1, Gnao1, Gsk3a, Gsk3b, Hadha, Hsp90aa1, Hsp90ab1, Hspa4, Hspa8, Iars2, Idh3g, Kif21b, Kif5b, Lars2, Lonp1, Myh9, Nadk2, Nsf, Pak2, Pck2, Pebp1, Pfkm, Prkar2a, Prkar2b, Psmc2, Rab14, Rab18, Rab2a, Rab3gap2, Rab7, Rac1, Ran, Sars2, Septin5, Septin7, Septin9, Stmn1, Suclg2, Tars2, Timm44, Tnik, Top1, Trap1, Trio, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Uba1, Ube2m, Ube2o, Vwa8 ribonucleotide 104 4.684684753 Aacs, Aak1, Acad9, Acadl, Acadm, Acads, binding Acadvl, Acat1, Acss1, Actb, Afg3l2, Ak3, Ak4, Aldh18a1, Anxa6, Arhgdia, Arhgef2, Arl8a, Arl8b, Ass1, Atad3a, Atp2b1, Atp5b, Atp6v1a, Atp6v1b2, Bcr, Camk2b, Cdc42bpa, Clpx, Dclk1, Ddx17, Ddx39b, Ddx3x, Dhx29, Dnm1l, Dnm2, Dtymk, Eci2, Eef1d, Eef2, Eif4a1, Eif4a2, Eif4b, Eif4g1, Farsb, G3bp1, Gcdh, Gfm1, Glud1, Gnao1, Gsk3a, Gsk3b, Hadha, Hsp90aa1, Hsp90ab1, Hspa4, Hspa8, Iars2, Idh3g, Kif21b, Kif5b, Lars2, Lonp1, Myh9, Nadk2, Ndufv1, Nsf, Pak2, Pck2, Pebp1, Pfkm, Prkar2a, Prkar2b, Psmc2, Rab14, Rab18, Rab2a, Rab3gap2, Rab7, Rac1, Ran, Sars2, Septin5, Septin7, Septin9, Stmn1, Suclg2, Tars2, Timm44, Tnik, Top1, Trap1, Trio, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb3, Tubb4b, Tubb5, Uba1, Ube2m, Ube2o, Vwa8 126 Table 5 (cont’d) cytoskeletal 69 6.66023159 Actb, Afdn, Aldoa, Ank2, Anxa2, Anxa6, protein Arhgef2, Arl8b, Arpc1a, Bin1, Camsap1, binding Camsap3, Cap1, Cfl1, Ckap5, Clasp2, Cltc, Cnn3, Coro1c, Cotl1, Crmp1, Cttn, Dbnl, Dctn1, Dcx, Ddx3x, Dnm1l, Dnm2, Dpysl2, Dpysl3, Dpysl4, Dpysl5, Eef2, Eml4, Epb41l2, Epb41l3, Fermt2, Flna, Flnb, Fscn1, Fus, Gapdh, Gsk3b, Hsp90aa1, Hsp90ab1, Inppl1, Kif21b, Kif5b, Lasp1, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Myh9, Pdlim4, Pdlim5, Pfn2, Ppp1r9b, Ppp2r2a, Prune1, Rab14, Rps3, Snca, Sncb, Stmn1, Wdr1 ATP binding 62 4.060248852 Aacs, Aak1, Acss1, Actb, Afg3l2, Ak3, Ak4, Aldh18a1, Ass1, Atad3a, Atp2b1, Atp5b, Atp6v1a, Atp6v1b2, Bcr, Camk2b, Cdc42bpa, Clpx, Dclk1, Ddx17, Ddx39b, Ddx3x, Dhx29, Dtymk, Eif4a1, Eif4a2, Eif4b, Eif4g1, Farsb, G3bp1, Glud1, Gsk3a, Gsk3b, Hsp90aa1, Hsp90ab1, Hspa4, Hspa8, Iars2, Idh3g, Kif21b, Kif5b, Lars2, Lonp1, Myh9, Nadk2, Nsf, Pak2, Pebp1, Pfkm, Psmc2, Sars2, Suclg2, Tars2, Timm44, Tnik, Top1, Trap1, Trio, Uba1, Ube2m, Ube2o, Vwa8 kinase 42 4.833141327 Actb, Ank2, Arhgdia, Atp1b1, Camk2b, binding Clasp2, Cltc, Dctn1, Dcx, Dnm2, Dpysl2, Dvl2, Eef2, Fermt2, Flna, Gsk3a, Gsk3b, Gstm1, Gstp1, Hsp90aa1, Hsp90ab1, Kif5b, Ldha, Map2, Mapre1, Nsf, Pak2, Pdlim5, Pebp1, Pfkm, Ppp1r9b, Prdx3, Prkar2a, Prkar2b, Prkra, Ptpn23, Rac1, Rps3, Sorbs1, Srcin1, Trap1, Vim actin binding 34 7.439825058 Afdn, Anxa6, Arpc1a, Bin1, Camsap3, Cap1, Cfl1, Clasp2, Cnn3, Coro1c, Cotl1, Crmp1, Cttn, Dbnl, Eef2, Epb41l2, Epb41l3, Fermt2, Flna, Flnb, Fscn1, Inppl1, Lasp1, Lrpprc, Macf1, Map1b, Map2, Myh9, Pdlim4, Pdlim5, Pfn2, Ppp1r9b, Snca, Wdr1 127 Table 5 (cont’d) tubulin binding 30 7.556674957 Arhgef2, Arl8b, Camsap1, Camsap3, Ckap5, Clasp2, Cnn3, Dctn1, Dcx, Ddx3x, Dnm1l, Dnm2, Dpysl2, Dpysl5, Eml4, Gapdh, Kif21b, Kif5b, Lrpprc, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Prune1, Rps3, Snca, Sncb, Stmn1 mRNA binding 26 8.30670929 Auh, Ddx3x, Eif3c, Eif4g1, Eif4g2, Fus, Fxr1, G3bp1, Hdlbp, Hnrnpa3, Hnrnpm, Hsp90aa1, Luc7l2, Luc7l3, Mrps7, Pabpc1, Pcbp1, Pcbp2, Rpl7, Rps3, Serbp1, Shmt2, Srsf3, Syncrip, Tardbp, Thoc2 microtubule 24 8.450704575 Arhgef2, Camsap1, Camsap3, Ckap5, binding Clasp2, Cnn3, Dctn1, Dcx, Dnm1l, Dnm2, Dpysl2, Dpysl5, Eml4, Gapdh, Kif21b, Kif5b, Macf1, Map1b, Map2, Map4, Map6, Mapre1, Rps3, Snca actin filament 22 10 Afdn, Anxa6, Arpc1a, Bin1, Camsap3, binding Cfl1, Clasp2, Coro1c, Cotl1, Crmp1, Cttn, Dbnl, Eef2, Fermt2, Flna, Fscn1, Lasp1, Lrpprc, Macf1, Myh9, Ppp1r9b, Wdr1 GTPase 19 5.9375 Afdn, Anxa2, Arhgap1, Arhgdia, Arhgef2, binding Bin1, Coro1c, Dnm1l, Dvl2, Flna, Hsp90aa1, Nsf, Pak2, Pex5, Rab3gap2, Rab7, Rac1, Rbsn, Srgap2 ubiquitin 19 5.9375 Dnm1l, Elob, Gsk3b, Hsp90aa1, protein ligase Hsp90ab1, Hspa8, Lrpprc, Ndufs2, Pcbp2, binding Prdx5, Prdx6, Prkar2a, Prkar2b, Rpl11, Sorbs1, Tuba1b, Tubb5, Uchl1, Ywhae small GTPase 17 6.007067204 Afdn, Anxa2, Arhgap1, Arhgdia, Arhgef2, binding Coro1c, Dnm1l, Dvl2, Flna, Nsf, Pak2, Pex5, Rab3gap2, Rab7, Rac1, Rbsn, Srgap2 translation 14 13.5922327 Dhx29, Eef1d, Eef1g, Eef2, Eif3c, Eif4a1, regulator Eif4a2, Eif4b, Eif4g1, Eif4g2, Eif5a, Gfm1, activity, nucleic Pcbp1, Shmt2 acid binding oxidoreductase 13 20.3125 Acad8, Acad9, Acadl, Acadm, Acads, activity, acting Acadsb, Acadvl, Crat, Decr1, Gcdh, Ivd, on the CH-CH Sdhb, Zadh2 group of donors rRNA binding 12 15 Eef2, Mrpl16, Mrps27, Mrps7, Ptcd3, Rpl11, Rpl3, Rpl4, Rpl7, Rps3, Rps9, Tst 128 Table 5 (cont’d) translation factor 12 14.28571415 [Dhx29, Eef1d, Eef1g, Eef2, Eif3c, Eif4a1, activity, RNA Eif4a2, Eif4b, Eif4g1, Eif4g2, Eif5a, Gfm1] binding ribonucleoprotein 12 7.272727489 [Ckap5, Ddx3x, Dhx29, Eef2, Eif3c, Eif4b, complex binding Eif4g1, Eif5a, Mrps27, Ptcd3, Rpsa, Serbp1] electron transfer 11 12.94117641 [Acadsb, COX2, Cox4i1, Cox5a, Etfa, Etfb, activity Ndufa10, Ndufs1, Ndufs2, Ndufv1, Sdhb] flavin adenine 11 12.22222233 [Acad8, Acad9, Acadl, Acadm, Acads, dinucleotide Acadsb, Acadvl, Dld, Etfa, Gcdh, Ivd] binding double-stranded 10 11.49425316 [Cltc, Ddx3x, Eif4a1, Eif4b, Fxr1, RNA binding Hsp90ab1, Prkra, Tuba1b, Tubb4b, Vim] acyl-CoA 9 81.8181839 [Acad8, Acad9, Acadl, Acadm, Acads, dehydrogenase Acadsb, Acadvl, Gcdh, Ivd] activity fatty-acyl-CoA 8 29.62962914 [Acad9, Acadl, Acadm, Acads, Acadvl, binding Eci2, Gcdh, Hadha] peroxidase 8 12.5 [Gstp1, Hba-a1, Prdx1, Prdx2, Prdx3, activity Prdx5, Prdx6, Snca] 5S rRNA binding 6 46.15384674 [Eef2, Rpl11, Rpl3, Rpl4, Rpl7, Tst] tau protein 6 25 [Bin1, Gsk3b, Hsp90aa1, Hsp90ab1, binding Ppp2r2a, Snca] thioredoxin 5 71.42857361 [Prdx1, Prdx2, Prdx3, Prdx5, Prdx6] peroxidase activity translation 5 25 [Eef1d, Eef1g, Eef2, Eif5a, Gfm1] elongation factor activity NADH 5 21.73913002 [Ndufa10, Ndufa9, Ndufs1, Ndufs2, Ndufv1] dehydrogenase activity negative 5 15.15151501 [Gsk3b, Gstp1, Prdx5, Prdx6, Snca] regulation of oxidoreductase activity NADH 4 22.22222137 [Ndufa10, Ndufs1, Ndufs2, Ndufv1] dehydrogenase (ubiquinone) activity ribosomal small 4 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