Data_Sheet_1_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.docx

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.

帕金森病（Parkinson’s disease, PD）发病机制的经典特征为路易小体（Lewy bodies）与路易神经突（Lewy neurites）内错误折叠的α-突触核蛋白（alpha-synuclein, αSyn）聚集，但其在小胶质细胞功能异常及由此引发的多巴胺能（dopaminergic, DAergic）神经毒性中的作用仍不明确。既往研究证实，蛋白激酶Cδ（protein kinase C delta, PKCδ）在死后PD脑组织及实验性帕金森模型中被激活，并参与反应性小胶质细胞增生；然而在α-突触核蛋白病（α-synucleinopathy）背景下，PKCδ激活、内质网应激（endoplasmic reticulum stress, ERS）与反应性小胶质细胞激活状态之间的关联仍鲜为人知。
本研究中，我们发现：在聚集型α-突触核蛋白（aggregated αSyn, αSynagg）刺激的原代小胶质细胞内，氧化应激、线粒体功能异常、NLR家族pyrin结构域包含蛋白3（NLR family pyrin domain containing 3, NLRP3）炎症小体激活及PKCδ激活，与包括激活转录因子4（activating transcription factor 4, ATF-4）、磷酸化丝氨酸/苏氨酸蛋白激酶/内质网必需核糖核酸酶1α（serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α, p-IRE1α）、磷酸化真核翻译起始因子2α（p-eukaryotic initiation factor 2α, p-eIF2α）在内的内质网应激标志物水平同步升高，同时IL-1β等神经毒性细胞因子的生成量亦显著增加。
值得注意的是，经αSynagg处理的小鼠原代小胶质细胞中，硫氧还蛋白相互作用蛋白（Thioredoxin-interacting protein, TXNIP）——硫氧还蛋白（thioredoxin, Trx）通路的内源性抑制剂，也是核心抗氧化蛋白系统的关键组分——的表达水平显著上调。此外，αSynagg可促进这些细胞内NLRP3与TXNIP的相互结合。
体外实验显示，通过小干扰RNA（small interfering RNA, siRNA）敲低PKCδ，可降低αSynagg刺激的小鼠小胶质细胞（mouse microglial cells, MMCs）的ERS水平，并减少TXNIP的表达与NLRP3的激活反应。同时，通过线粒体靶向夹竹桃素（mito-apocynin）清除线粒体活性氧（mitochondrial reactive oxygen species, mitoROS），或通过eIF2α抑制剂萨鲁布林（salubrinal, SAL）缓解ERS，均可抑制ERS/TXNIP/NLRP3信号轴的激活，提示线粒体功能异常与ERS可能协同介导αSynagg诱导的小胶质细胞激活过程。
与之相符的是，通过siRNA敲低TXNIP，同样可减弱αSynagg诱导的NLRP3炎症小体激活反应。最后，向野生型小鼠纹状体单侧注射α-突触核蛋白预形成纤丝（αSyn preformed fibrils, αSynPFF），可在酪氨酸羟化酶阳性（tyrosine hydroxylase-positive, TH+）神经元发生延迟丢失之前，显著上调黑质区p-PKCδ的表达、升高ERS标志物水平，并激活TXNIP/NLRP3炎症小体信号轴。
综上，本研究结果表明，靶向抑制ERS及其下游信号介质TXNIP与NLRP3，有望成为缓解PD及其他突触核蛋白病中小胶质细胞介导的神经炎症的新型治疗策略。