Essential functions of discrete Pannexin-1 N-terminal domains and their relevance for Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is a common form of dementia associated with accumulation of soluble amyloid beta oligomers (AβOs) resulting in a cascade of events that eventually lead to neurodegeneration and loss of memory. During the early stages of AD, AβOs are known to impair the function of excitatory glutamatergic synapses in the hippocampus. Here, NMDA subtype of glutamate receptors (NMDARs), that are important for learning and memory, are known to play a role in excitotoxicity induced by AβOs. Targeting NMDARs directly for AD therapy has been difficult hence discovering NMDAR signalling effectors as an alternate drug target is important. In this regard, pannexin1 (Panx1) ion channels, that are activated downstream of NMDAR stimulation, are known to contribute towards neurodegeneration in ischemia and epilepsy. Thus, my thesis investigates the mechanisms underlying NMDAR-stimulated Panx1 activation and the extent to which NMDAR-Panx1 coupling contributes to impaired synaptic function in AD. I demonstrate that endoplasmic reticulum resident stromal interaction molecules (STIM) link Ca2+ influx from NMDARs to Panx1 activation under physiological conditions. Using reverse genetics, electrophysiology, quantitative fluorescence imaging, western blotting and co-immunoprecipitation assays, we show that STIMs physically interact with Panx1 N-terminal (1-18 region) to regulate channel opening downstream of NMDAR stimulation in hippocampal neurons. To establish a means of disrupting STIM-Panx1 interaction in intact cells, we demonstrate that a novel cell penetrating, Tat-conjugated Panx1 mimetic peptide (Tat-Panx19-18; designed by us) can prevent NMDAR-stimulated Panx1 activation. Further, I demonstrate that NMDAR-STIM-Panx1 coupling contributes towards AβO-mediated excitatory synaptic deficits and show that Tat-Panx19-18 treatment can reverse the synaptotoxic effects of AβO. Lastly, I show that Panx1 N-terminal is a regulator of multi-modal Panx1 functions governing small pore, large pore and surface localization of Panx1 channels. To conclude, this thesis highlights the broad implications of NMDAR-STIM-Panx1 coupling in physiology and establishes its contribution during early stages of AD.