Tau is enriched in the axonal compartment in healthy neurons but is mislocalized to the somatodendritic compartment in disease, playing an important role in tauopathy pathogenesis. The localization of tau in axons involves the axon initial segment (AIS), which is a specialized region of the proximal axon that acts as a retrograde diffusion barrier for tau. Here, we examined the timing of AIS development alongside the differential distribution of tau in axons of hippocampal neurons in culture. We also examined AIS morphology and levels of axonal and somatic tau in aging process of Fisher 344 rats. Using tau domains, pseudophosphorylation, and a familial tau mutation combined with a photoconvertible fluorescent construct, we analyzed the diffusion behavior of tau in living hippocampal neurons. We discovered that the microtubule binding region is necessary and sufficient to prevent diffusion from the axon to the soma, and that disease-related changes in tau such as phosphorylation and familial mutation of tau show enhanced axosomatic tau diffusion. Analysis of aged rat brains showed that the AIS-associated protein, Ankyrin G, remains properly localized and abundant during aging, while the axosomatic distribution of tau does not change in hippocampal neuron populations. To further elucidate the mechanisms by which the AIS inhibits retrograde tau mislocalization we used shRNAs to knockdown the expression AIS proteins: Ankyrin G (AnkG) and tripartite motif containing protein 46 (TRIM46). We show that TRIM46 plays a critical role in maintenance of the diffusion barrier in cultured hippocampal neurons. Knockdown of TRIM46 is sufficient to allow somatic diffusion of axonal tau into the somata of neurons and reduce the relative axonal enrichment of tau. Knockdown of AnkG does not change tau localization or axo-somatic distribution. Immunoprecipitation and mass spectrometry was used to characterize this connection between tau and TRIM46, but we conclude that tau and TRIM46 do not interact directly. Instead, we identified TRIM46 interactions with several microtubule-associated and actin-associated proteins supporting an integral role in maintenance of the AIS cytoarchitecture. We propose that the tight regulation of microtubule organization in the AIS by TRIM46 prevents somatic diffusion of tau, and tau modifications that disrupt its interactions with microtubules contributes to axosomatic mislocalization.
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