REGULATED RELEASE OF HYPER-PHOSPHORYLATED, AGGREGATED AND SEEDING COMPETENT TAU FROM RODENT BRAINS AND HUMAN SYNAPTOSOMES

Abstract

Tau is a microtubule binding protein expressed in neuronal axons and nerve terminals. Mutations in tau are known to cause different neurological diseases and tau pathology, in the form of aggregates and tangles, is a hallmark of Alzheimer's disease. Tau pathology in AD starts in the entorhinal cortex but “spreads” along synaptically connected areas as the disease progresses. Understanding how tau is trafficked to the nerve terminal, secreted, and taken up by postsynaptic neurons could lead to the identification of novel mechanisms aimed at halting disease progression in AD. Synaptic structures (synaptosomes) were purified from the brains of transgenic mice harbouring pathological human tau (P301S and tg4510) and from human control and AD subjects. Western blots were used to detect synaptic proteins and tau. STED microscopy was used to evalaute tau sub-cellular localisation. The ability of synaptic tau to “seed” pathological tau aggregation was evaluated in transfected HEK293 and rat cortical neurons. Calcium dependent release was induced by KCl or ionomycin, while botulinum toxins and pharmacological agents were used to cleave SNARE proteins and block exocytosis. Pathologically hyper-phosphorylated (AT8+) aggregated (Sarcosyl-insoluble) and seeding-competent tau was found in synaptosomes from both transgenic mice and human AD brains. Pathological tau was enriched in synaptosomes of the most affected brain areas in the mice, while in human brain synaptosomes the levels of pathological tau were correlated with the patient's Braak stage. STED analysis demonstrated that pathological tau was enriched in the presynaptic compartment. Pathological tau was released in a calcium dependent manner by both KCl and ionomycin. Botulinum toxin A and D were found to cleave synaptosomal SNAP25 and synaptobrevin, respectively, and at the same time inhibit calcium-dependent tau release. All together these data build on previous evidence of pathological tau presence in presynaptic terminals and demonstrate its calcium-dependent release. Pathological tau release requires intact SNARE proteins, suggesting a vesicular, exocytotic, process. This could represent a critical step in the spreading of tau in AD brain. Therapeutic interventions aimed at blocking tau release could be beneficial in reducing tau propagation and disease progression in AD.