Abstract
A classical hallmark of Parkinson’s disease (PD) pathogenesis is the accumulation of misfolded alpha-synuclein (αSyn) 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 (PKCδ) is activated in post mortem PD brains and experimental Parkinsonism and that it participates in reactive microgliosis; however, the relationship between PKCδ activation, endoplasmic reticulum stress (ERS) and the reactive microglial activation state in the context of α-synucleinopathy is largely unknown. Herein, we show that oxidative stress, mitochondrial dysfunction, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, and PKCδ activation increased concomitantly with ERS markers, including the activating transcription factor 4 (ATF-4), serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α (p-IRE1α), p-eukaryotic initiation factor 2 (eIF2α) as well as increased generation of neurotoxic cytokines, including IL-1β in aggregated αSynagg-stimulated primary microglia. Importantly, in mouse primary microglia-treated with αSynagg we observed increased expression of Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the thioredoxin (Trx) pathway, a major antioxidant protein system. Additionally, αSynagg promoted interaction between NLRP3 and TXNIP in these cells. In vitro knockdown of PKCδ using siRNA reduced ERS and led to reduced expression of TXNIP and the NLRP3 activation response in αSynagg-stimulated mouse microglial cells (MMCs). Additionally, attenuation of mitochondrial reactive oxygen species (mitoROS) via mito-apocynin and amelioration of ERS via the eIF2α 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 αSynagg-induced microglial activation response. Likewise, knockdown of TXNIP by siRNA attenuated the αSynagg-induced NLRP3 inflammasome activation response. Finally, unilateral injection of αSyn preformed fibrils (αSynPFF) into the striatum of wild-type mice induced a significant increase in the expression of nigral p-PKCδ, 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.
Highlights
Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder (Mhyre et al, 2012)
Given that αSyn aggregation reportedly induces mitochondrial dysfunction, thereby contributing to disease pathology (Smith et al, 2005) we investigated the temporal pattern of mitochondrial reactive oxygen species (mitoROS) generation, mitochondrial membrane potential (MMP) collapse, and PKCδ activation in mouse primary microglia stimulated with 1 μM αSynagg based on previous reports (Boza-Serrano et al, 2014; Lee et al, 2018; Panicker et al, 2019) for increasing durations (6, 12, 18, 24 h; Hoffmann et al, 2016; Sarkar et al, 2020)
We found that there was a significant upregulation of endoplasmic reticulum stress (ERS) markers including eIF2α, ATF-4, Thioredoxin-interacting protein (TXNIP), and NLRP3 protein expression, as well as pro-inflammatory cytokine mRNA expression (IL-1β and TNF-α) that was accompanied by an upregulation of Trx expression in mouse microglial cell (MMC) cells treated with αSynagg, which was markedly reduced by treatment of mitoapocynin (Figures 5A–C)
Summary
Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder (Mhyre et al, 2012). PD originates from the progressive loss of nigral dopaminergic (DAergic) neurons, which is accompanied by the accumulation of misfolded aggregated αSyn (αSynagg) within Lewy bodies and Lewy neurites which is a classical pathological hallmark of this disease (Dickson, 2012). Numerous mechanisms have been implicated in PD pathogenesis, including mitochondrial dysfunction, endoplasmic reticulum stress (ERS), impaired proteostasis, and chronic neuroinflammation (Block et al, 2007) yet the exact mechanisms underlying increased vulnerability of nigral DAergic neurons to PD pathogenesis remains undefined. Emerging evidence shows that the chronic microglia-driven inflammatory response plays a significant role in the progressive loss of nigral DAergic neurons and disease progression in animal models of PD (Gao et al, 2002; Qin et al, 2013; Duffy et al, 2018; Olanow et al, 2019).
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