Neuroinflammation-mediated Degradation of NMDA receptors and Tau Dephosphorylation Mechanisms Related to Alzheimer’s Disease
Creators: name: Youngstrom, Diane
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2021
Subjects: Western blot
neuroinflammation
Alzheimer's disease

description: Current Alzheimer’s disease (AD) research highlights the importance of neuroinflammation and tau accumulation as drivers of AD onset and progression. To better understand the role of neuroinflammation in tau phosphorylation mechanisms in neurodegeneration, in vitro wildtype primary cortical rat neurons were treated with conditioned media (CM) generated from Aβ-oligomer challenged macrophages. This experimental paradigm enables an in vitro model to isolate how neurons respond to an inflammatory insult without the confounds and difficulties of co-culture or in vivo models. Following a time-course experiment to optimize the length of CM treatment, Western blot biochemical analysis of primary cortical neuron lysate with and without CM was performed to investigate changes in protein expression levels of NMDA receptor (NMDAR) subunits, AMPA receptor (AMPAR) subunits, tau epitopes, and other synaptic proteins. Subsequently, we used pharmacological inhibitors to dissect the molecular mechanisms of the observed biochemical changes. The dynamics of synchronous glutamate release under control, D-AP5, and CM conditions were investigated using widefield microscopy of transfected intensity-based glutamate-sensing fluorescent reporter (iGluSnFR). The neuroinflammatory response to (30 minute) CM treatment prior to the addition of any pharmacological inhibitors generally results in decreased expression and/or phosphorylation levels of many proteins perhaps through excitotoxic mechanisms. Blocking the phosphatases, particularly PP1 and PP2A (with okadaic acid), appears to block the tau dephosphorylation typically caused by CM as evidenced by significantly increased expression levels (to the point of hyperphosphorylation) of several phospho-Tau antibodies (AT8, AT100, AT180, pS396) when treated with and without CM. Meanwhile, inhibiting phosphatases doesn't protect the NMDA receptors (particularly the GluN2A and GluN2B subunits) from being degraded under the neuroinflammatory insult of CM, which suggests that the NMDA receptors are either upstream or independent from the phosphatases. Blocking the NMDA receptors with D-AP5 appears to rescue neurons from the neuroinflammatory and excitotoxic effects of CM as evidenced by recovered expression levels of NMDAR subunits (GluN2A, GluN2B) and phosphorylated AMPAR (GluA1) subunits, yet the expression levels of tau do not appear to recover with D-AP5 treatment under CM treatment. Live-cell imaging of neurons transfected with iGluSnFR corroborates the excitotoxic effect of CM as application of CM abolishes the synchronous glutamatergic activity. The results indicate that acute neuroinflammatory insult via CM treatment induces degradation of NMDARs and tau dephosphorylation. Whether the NMDAR and tau degradation mechanisms under neuroinflammatory insult are independent or through some unknown mechanistic link has yet to be determined. The neuroinflammatory factors secreted from glia challenged with Aβ-oligomers clearly impact both NMDAR and tau, linking AD behavioral pathology (e.g., memory impairments and cognitive deficits) with neuronal physiology. Further investigation of the mechanistic link between NMDAR and tau could be instrumental in identifying treatments for AD.