Alzheimer’s disease (AD) is a devastating neurodegenerative disease for which there is no cure. There is a critical need for disease-modifying therapies that can help halt or even reverse the disease course. Gaining a better understanding of the mechanisms underlying the neuronal dysfunction and neurodegenerative processes observed in AD can guide the development of effective therapies. The progressive accumulation of pathological tau protein is a defining feature of AD. Tau pathology accumulates throughout the brain in a stereotypical pattern that correlates with disease severity, making it a promising target. Whether the origins of tau pathology is from cell-to-cell seeding (a prominent hypothesis in the field) or through cell autonomous routes, the subsequent functional consequences of pathogenic forms of tau require further definition. Indeed, tau is capable of seeding aggregation in vitro and in vivo, and here I leveraged this phenomenon to induce the formation of pathogenic tau in a neuron culture model of the tauopathy associated with sporadic AD. Specifically, I seeded primary neurons derived from human tau knock-in (MAPT-KI) mice with insoluble tau purified from human AD brains (AD-tau) to address critical gaps in our knowledge of the intracellular consequences of pathogenic tau. First, I performed a rigorous characterization of the model and of the seeded tau inclusions. Then, I investigated the consequences of the seeded inclusions on axon integrity and on synaptic density and function. I show that treating MAPT-KI primary neurons with AD-tau results in the progressive formation of intracellular tau pathology. I used immunological methods to show that the seeded inclusions have AD-associated modifications including phosphorylation at the PHF1, AT8, and pS422 epitopes, and conformation changes including oligomerization and exposure of an N-terminal domain (phosphatase activating domain; PAD) that is linked to axonal transport deficits. PAD activates protein phosphatase 1 (PP1), which in turn activates glycogen synthase kinase 3β (GSK3β). GSK3β phosphorylates kinesin light chains, causing these molecular motors to release their cargo. Therefore, aberrant PAD-exposure in disease may disrupt axonal transport through overactivation of this signaling pathway. I observed co-localization of active GSK3β and cargo proteins [i.e. synaptophysin and amyloid precursor protein (APP)] with PAD-exposed tau inclusions. Furthermore, I observed PAD-exposed tau and APP colocalized in swollen processes reminiscent of axonal swellings or spheroid bodies observed in AD, which are thought to indicate an early stage of axonal degeneration. I did not observe overt axonal degeneration, or cell loss. However, the number of intact excitatory synapses was reduced in the AD-tau treated cultures. Furthermore, I used multielectrode arrays to measure the effects of the tau inclusions on neuronal function (e.g. action potential firing rate and network burst frequency). At baseline, the activity of the AD-tau treated cultures was normal. However, upon treatment with glutamate, the AD-tau treated cultures showed a significantly greater increase in network burst frequency compared to the control cultures. Furthermore, the AD-tau treated cultures showed a significantly greater decrease in network burst frequency upon treatment with an NMDA receptor (NMDAR) antagonist, suggesting that the hypersynchrony observed after glutamate addition was mediated by NMDARs. Notably, hyperexcitability, hyperactivity, and hypersynchrony are all phenotypes observed early in AD. These data demonstrate that I developed a model that recapitulates features of early neuronal dysfunction from intracellular AD-associated pathological tau. The lack of overt cell loss or axonal degeneration makes this model valuable for studying early mechanisms in AD pathogenesis, and for testing approaches to ameliorate early dysfunction and potentially prevent overt toxicity and neuronal loss in humans.
Read
