Journal: Frontiers in Aging Neuroscience

Article Title: 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

doi: 10.3389/fnagi.2021.661505

Figure Lengend Snippet: Time-dependent mitochondrial dysfunction, PKCδ activation, and endoplasmic reticulum stress (ERS) markers in αSyn agg -stimulated mouse primary microglia. Mouse primary microglial cells were exposed to 1 μM of αSyn agg for increasing durations (6, 12, 18, and 24 h) and mitochondrial superoxide generation was measured using MitoSOX dye-based spectrofluorometric analysis. (A) Generation of human preformed fibrils (PFF) αSyn and validation of uptake of αSyn agg by primary microglia. Representative scanning transmission electron microscopy (STEM) images of αSyn PFF before scale bar = 200 (nm) and after scale bar = 200 (nm) sonication. Even after sonication the αSyn PFF still retain their beta-sheet conformation. (B) Internalization of αSyn agg in primary microglia. Representative immunohistochemical images of mouse primary microglia depicting uptake of hu-αSyn as determined using antibodies against human αSyn (red) and microglial marker-IBA-1 (green) in primary microglia treated with αSyn agg for 1 h. Internalized human αSyn, visualized as red intracellular puncta), Scale bars, 15 μm. (C) MitoROS generation increased in a time-dependent manner in αSyn agg -treated mouse primary microglia cells. Data shown are the mean ± SEM from at least three independent experiments. (D) Assessment of mitochondrial membrane potential [MMP (ΔΨm)] using JC-1 spectrophotometric plate reader analysis. MMP decreased time-dependently post mouse PMG stimulation with αSyn agg . Data shown are the mean ± SEM from at least three independent experiments. (E) Representative immunoblots and densitometric evaluation of p-PKCδ (Y311) in the whole-cell lysates indicating a time-dependent increase in PKCδ phosphorylation in αSyn agg -treated mouse PMG. Data shown are the mean ± SEM from at least three independent experiments. (F) Representative immunoblots and densitometric analyses of mouse PMG cells treated with or without αSyn agg for 24 h revealing significantly increased ATF-4, p-IRE1α, and and CHOP expression levels. The immunoblot is representative of at least three independent experiments. (G) Representative images from dual immunohistochemical staining for p-eIF2α (Red) IBA1 (green) in primary microglia treated with αSyn agg for 24 h. Nuclei were counterstained with Hoechst stain (Blue). Results represent three independent experiments. Scale bar = 20 μm. Data were analyzed using one-way ANOVA followed by Bonferroni’s post hoc analysis and two-tailed t -test. Asterisks (*** p < 0.001, ** p < 0.01 and * p ≤ 0.05) indicate significant differences between control and treatment groups.

Article Snippet: The PKCδ siRNA was purchased from Santa Cruz Biotech (#SC-36246; Dallas, TX, USA), and TXNIP siRNA was purchased from life technologies (Ambion #4390771; Carlsbad, CA, USA).

Techniques: Activation Assay, Biomarker Discovery, Transmission Assay, Electron Microscopy, Sonication, Immunohistochemical staining, Marker, Membrane, Western Blot, Phospho-proteomics, Expressing, Staining, Two Tailed Test, Control

Journal: Frontiers in Aging Neuroscience

Article Title: 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

doi: 10.3389/fnagi.2021.661505

Figure Lengend Snippet: siRNA-mediated PKCδ knockdown alle via tes αSyn agg -induced ERS, TXNIP, and NLRP3 inflammasome activation in mouse microglial cells (MMCs). MMCs were transfected with scrambled siRNA (Scr siRNA) or PKCδ siRNA for 48 h and were then treated with αSyn agg for another 24 h before being processed for Western blot and qRT-PCR analyses. (A) Representative immunoblot and densitometric quantification revealing that PKCδ-silencing diminished the αSyn agg -mediated activation of p-eIF2α, BIP, TXNIP, and NLRP3 and reversed the αSyn agg -induced suppression of TrX. Data shown are the mean ± SEM from at least three independent experiments. (B) qRT-qPCR analysis depicting that PKCδ gene depletion significantly attenuated the αSyn agg -induced mRNA expression of the pro-inflammatory markers IL-1β and TNF-α. Data shown are the mean ± SEM from at least three independent experiments. Data were analyzed using one-way ANOVA followed by Bonferroni’s post hoc analysis. Asterisks (*** p < 0.001, ** p < 0.01, and * p < 0.05) indicate significant differences between control and treatment groups.

Article Snippet: The PKCδ siRNA was purchased from Santa Cruz Biotech (#SC-36246; Dallas, TX, USA), and TXNIP siRNA was purchased from life technologies (Ambion #4390771; Carlsbad, CA, USA).

Techniques: Knockdown, Activation Assay, Transfection, Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Frontiers in Aging Neuroscience

Article Title: 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

doi: 10.3389/fnagi.2021.661505

Figure Lengend Snippet: siRNA-mediated TXNIP knockdown mitigates NLRP3 inflammasome activation markers in primary microglial cells stimulated with αSyn agg . (A) Expression of caspase-1 and NLRP3 inflammasome activation marker in mouse PMG transfected with TXNIP siRNA for 48 followed by stimulation with αSyn agg for another 24 h. Western blot analysis and quantification reveals inhibitory effects of TXNIP siRNA on αSyn agg -induced upregulation of NLRP3 and cleaved caspase-1 in mouse PMG as compared with scramble siRNA transfected cells. β-actin used as an internal control. (B) qRT-PCR analysis of IL-1β and TNF-α in mouse PMG transfected with or without TXNIP siRNA with or without stimulation with αSyn agg for 24 h. TXNIP siRNA attenuated αSyn agg -induced upregulation of the aforementioned proinflammatory cytokine gene expression as compared to scramble transfected mouse PMG. Data shown are the mean ± SEM from at least three independent experiments. Significance is based on two-way ANOVA followed by a Bonferroni post hoc test. *** p < 0.001 and * p < 0.05 vs. controls.

Article Snippet: The PKCδ siRNA was purchased from Santa Cruz Biotech (#SC-36246; Dallas, TX, USA), and TXNIP siRNA was purchased from life technologies (Ambion #4390771; Carlsbad, CA, USA).

Techniques: Knockdown, Activation Assay, Expressing, Marker, Transfection, Western Blot, Control, Quantitative RT-PCR, Gene Expression

Journal: Frontiers in Aging Neuroscience

Article Title: 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

doi: 10.3389/fnagi.2021.661505

Figure Lengend Snippet: Chronic early activation of PKCδ, endoplasmic reticulum stress (ERS), and the TXNIP/NLRP3 signaling axis precedes the delayed tyrosine hydroxylase (TH) neuronal loss in the αSyn PFF mouse model of PD. C57BL/6 mice were intrastriatally infused with either 2 μl of αSyn PFF or saline (PBS) via stereotaxic injection. (A,B) Increased colocalization of ERS or inflammation-related marker in the nigral microglia of αSyn PFF infused mice. Representative immunohistochemical images showing colocalization of either TXNIP or p-eIF2α within IBA-1 positive microglia in the nigra of αSyn PFF infused mice as compared to the PBS-injected mice. Nigral brain sections were double immunolabeled for p-eIF2α/IBA-1 or TXNIP/IBA-1, Nuclei were counterstained for hoechest. (C) Western blots showing increased expression of PKCδ and p-eIF2α in the nigra of αSyn PFF inoculated mice. (D) Representative immunoblots showing that increased expression of CHOP, BIP, ATF-4, NLRP3, TXNIP, and downregulation of TrX in the SNpc of αSyn PFF inoculated mice. Quantification of the relative expression of the aforementioned proteins at 60 dpi. (E) qRT-PCR analysis showing increased expression of IL-6, TNF-α, and IL-1β in the striata of αSyn PFF -infused mice as compared to PBS-infused mice. (F) Representative photomicrographs of diaminobenzidine (DAB) immunostaining of TH in coronal midbrain sections of SN from perfused mouse brains at 180 dpi showing a dramatic reduction of TH-positive neurons in αSyn PFF -treated mice vs. PBS-treated mice. Data are the mean ± SEM; n = 4 independent mice. Data shown are the mean ± SEM from at least three independent experiments. Asterisks (*** p < 0.001, ** p < 0.01 and * p ≤ 0.05) indicate significant differences between control and treatment groups. NS: not significant.

Article Snippet: The PKCδ siRNA was purchased from Santa Cruz Biotech (#SC-36246; Dallas, TX, USA), and TXNIP siRNA was purchased from life technologies (Ambion #4390771; Carlsbad, CA, USA).

Techniques: Activation Assay, Saline, Injection, Marker, Immunohistochemical staining, Immunolabeling, Western Blot, Expressing, Quantitative RT-PCR, Immunostaining, Control

Journal: Frontiers in Aging Neuroscience

Article Title: 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

doi: 10.3389/fnagi.2021.661505

Figure Lengend Snippet: Schematic representation of the role of mitoROS-PKCδ-mediated endoplasmic reticulum stress (ERS)-dependent induction of the TXNIP/NLRP3 signaling axis in response to the αSyn agg -induced reactive microglial activation state. The treatment of microglial cells with αSyn agg triggers mitoROS and PKCδ activation resulting in ERS-dependent activation of proinflamamtory signaling events including the TXNIP/NLRP3 activation response. Both mitochondria-driven oxidative stress and PKCδ activation contribute to the αSyn agg -induced reactive microglial activation state via ERS through a feed-forward mechanism. An association between TXNIP and NLRP3 may partly explain the NLRP3 inflammasome-associated innate immune response. Moreover, αSyn agg -induced microglial ERS causes indirect DAergic neurotoxicity. Importantly, Mitoapo and PKCδ-silencing suppress ERS and the associated TXNIP/NLRP3 signaling axis in response to αSyn agg , thereby attenuating the reactive microglial activation state. Finally, salubrinal (SAL) attenuated the indirect DAergic neurotoxicity elicited by αSyn agg , further highlighting the pivotal role of ERS in the expression of a neurodegenerative microglial phenotype and associated DAergic neurotoxicity.

Article Snippet: The PKCδ siRNA was purchased from Santa Cruz Biotech (#SC-36246; Dallas, TX, USA), and TXNIP siRNA was purchased from life technologies (Ambion #4390771; Carlsbad, CA, USA).

Techniques: Activation Assay, Expressing