PKC Delta Activation Promotes Endoplasmic Reticulum Stress (ERS) and NLR Family Pyrin Domain-Containing 3 (NLRP3) Inflammasome Activation Subsequent to Asynuclein-Induced Microglial Activation: Involvement of Thioredoxin-Interacting Protein (TXNIP)/Thioredoxin (Trx) Redoxisome Pathway

A classical hallmark of Parkinson's disease (PD) pathogenesis is the accumulation of misfolded alpha-synuclein (alpha Syn) within Lewy bodies and Lewy neurites, although its role in microglial dysfunction and resultant dopaminergic (DAergic) neurotoxicity is still elusive. Previously, we identified that protein kinase C delta (PKC delta) is activated in post mortem PD brains and experimental Parkinsonism and that it participates in reactive microgliosis; however, the relationship between PKC delta activation, endoplasmic reticulum stress (ERS) and the reactive microglial activation state in the context of alpha-synucleinopathy is largely unknown. Herein, we show that oxidative stress, mitochondrial dysfunction, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, and PKC delta activation increased concomitantly with ERS markers, including the activating transcription factor 4 (ATF-4), serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 alpha (p-IRE1 alpha), p-eukaryotic initiation factor 2 (eIF2 alpha) as well as increased generation of neurotoxic cytokines, including IL-1 beta in aggregated alpha Syn(agg)-stimulated primary microglia. Importantly, in mouse primary microglia-treated with alpha Syn(agg) we observed increased expression of Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the thioredoxin (Trx) pathway, a major antioxidant protein system. Additionally, alpha Syn(agg) promoted interaction between NLRP3 and TXNIP in these cells. In vitro knockdown of PKC delta using siRNA reduced ERS and led to reduced expression of TXNIP and the NLRP3 activation response in alpha Syn(agg)-stimulated mouse microglial cells (MMCs). Additionally, attenuation of mitochondrial reactive oxygen species (mitoROS) via mito-apocynin and amelioration of ERS via the eIF2 alpha inhibitor salubrinal (SAL) reduced the induction of the ERS/TXNIP/NLRP3 signaling axis, suggesting that mitochondrial dysfunction and ERS may act in concert to promote the alpha Syn(agg)-induced microglial activation response. Likewise, knockdown of TXNIP by siRNA attenuated the alpha Syn(agg)-induced NLRP3 inflammasome activation response. Finally, unilateral injection of alpha Syn preformed fibrils (alpha Syn(PFF)) into the striatum of wild-type mice induced a significant increase in the expression of nigral p-PKC delta, ERS markers, and upregulation of the TXNIP/NLRP3 inflammasome signaling axis prior to delayed loss of TH+ neurons. Together, our results suggest that inhibition of ERS and its downstream signaling mediators TXNIP and NLRP3 might represent novel therapeutic avenues for ameliorating microglia-mediated neuroinflammation in PD and other synucleinopathies.