Fibrillar α-synuclein induces neurotoxic astrocyte activation via rip kinase signaling and nf-κb

ABSTRACT Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopamine neurons. The pathogenesis of PD is poorly understood, though misfolded

and/or aggregated forms of the protein α-synuclein have been implicated in several neurodegenerative disease processes, including neuroinflammation and astrocyte activation. Astrocytes in

the midbrain play complex roles during PD, initiating both harmful and protective processes that vary over the course of the disease. However, despite their significant regulatory roles

during neurodegeneration, the cellular and molecular mechanisms that promote pathogenic astrocyte activity remain mysterious. Here, we show that α-synuclein preformed fibrils (PFFs) induce

pathogenic activation of human midbrain astrocytes, marked by inflammatory transcriptional responses, downregulation of phagocytic function, and conferral of neurotoxic activity. These

effects required the necroptotic kinases RIPK1 and RIPK3, but were independent of MLKL and necroptosis. Instead, both transcriptional and functional markers of astrocyte activation occurred

via RIPK-dependent activation of NF-κB signaling. Our study identifies a previously unknown function for α-synuclein in promoting neurotoxic astrocyte activation, as well as new cell

death-independent roles for RIP kinase signaling in the regulation of glial cell biology and neuroinflammation. Together, these findings highlight previously unappreciated molecular

mechanisms of pathologic astrocyte activation and neuronal cell death with implications for Parkinsonian neurodegeneration. SIMILAR CONTENT BEING VIEWED BY OTHERS MICROGLIA-SPECIFIC

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BY PARKINSON’S DISEASE NEUROTOXINS AND Α-SYNUCLEIN AGGREGATES Article 04 December 2020 INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disease after

Alzheimer’s disease [1]. Pathologically, PD is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), as well as axonal degeneration in the

nigrostriatal pathway and reduction of dopamine inputs into the striatum [2]. Though the pathogenesis of PD is poorly understood, growing evidence implicates aggregated forms of the protein

α-synuclein as an etiologic agent of Parkinsonian neurodegeneration [3, 4]. Misfolding of soluble α-synuclein monomers leads to the formation of insoluble aggregates that exert neurotoxic

and inflammatory activity, contributing to the neuronal death and degeneration observed in PD and other synucleinopathies [5, 6]. While the impact of aggregated α-synuclein on neurons has

been extensively described, roles for α-synuclein in astrocytes are comparatively poorly understood. Astrocytes serve key homeostatic functions, including promoting neurite outgrowth and

phagocytosing cellular debris [7]. However, the activation and proliferation of astrocytes has been associated with both protective and pathologic functions in many neurodegenerative

diseases, including PD [8, 9], Alzheimer’s disease [10], Huntington’s disease [11], amyotrophic lateral sclerosis [12, 13], and others [7, 14,15,16,17,18,19,20,21,22]. Previous work has

described at least two putative subtypes of reactive astrocytes, termed “A1,” which are pro-inflammatory and neurotoxic, and “A2,” which are generally anti-inflammatory and neurotrophic

[18]. These activation states have been distinguished by distinct transcriptional signatures and functional profiles, including the propensity to induce cell death in neurons (a marker of A1

astrocyte activity). While aggregated α-synuclein has been shown to contribute to inflammatory astrocyte activation [23,24,25], the transcriptional and functional consequences of this

effect have not been fully established. Moreover, the molecular mechanisms that promote inflammatory astrocyte activation downstream of pathogenic α-synuclein species are poorly defined.

During neurodegeneration, inflammatory signals induce multiple forms of programmed cell death in susceptible neural cells, including both apoptosis and necroptosis [26,27,28,29,30].

Necroptosis is a form of programmed cell death mediated by receptor-interacting protein kinases-1 (RIPK1) and −3 (RIPK3). These kinases coordinate activation of the executioner pseudokinase

mixed lineage kinase domain-like protein (MLKL), which permeabilizes cell membranes, resulting in necrotic cell death [31, 32]. However, aside from necroptosis, several groups have recently

described pleiotropic, cell death-independent functions for RIPK signaling [33,34,35,36,37,38,39]. We and others have shown that, in neurons, RIPK activation can initiate inflammatory

transcriptional responses without inducing MLKL oligomerization and host cell death following neurotropic viral infection [33, 34, 40]. However, whether necroptosis-independent functions for

RIPK signaling are relevant during sterile neurodegenerative diseases requires further investigation. Moreover, whether RIPK signaling promotes inflammatory signaling in nonneuronal cells

of the central nervous system (CNS), such as astrocytes, has yet to be fully addressed. In this study, we sought to define the impact of aggregated α-synuclein on astrocyte activation state.

We show that α-synuclein preformed fibrils (PFFs) induce robust inflammatory transcriptional signaling in human midbrain astrocytes, including transcripts associated with both the putative

A1 and A2 astrocyte activation states. Functional analyses demonstrated that α-synuclein PFFs conferred neurotoxic activity in midbrain astrocytes, while diminishing homeostatic phagocytic

activity. Each of these transcriptional and functional outcomes required the inflammatory transcription factor NF-κB. Importantly, NF-κB activation and subsequent inflammatory transcription

and neurotoxic activation could be rescued via pharmacological blockade of RIPK1 and RIPK3 signaling, while MLKL was dispensable for these effects, which occurred in the absence of

astrocytic cell death. These data identify a previously unknown necroptosis-independent function for RIPK signaling in promoting a neurotoxic activation state in astrocytes following

exposure to a pathogenic species of α-synuclein. MATERIALS AND METHODS Α-SYNUCLEIN PREFORMED FIBRILS Purified human α-synuclein monomers were purchased from Proteos, Inc. (Kalamazoo, MI,

#RP-003) and were used to generate PFFs according to established protocols [41]. Briefly, monomers were diluted to a concentration of 5 mg/mL with PBS and agitated on a thermomixer at 1000

RPM at 37 °C for 7 days. Fibrilization was confirmed by measuring fluorescence in the presence of thioflavin T (Supplementary Fig. 1), as previously described [42]. Briefly, 95 µL of

thioflavin T (25 mM) diluted in PBS was mixed with 2.5 µL of lab-generated PFF stocks or monomeric control stocks. Samples were mixed for 15 m at room temperature. Fluorescence was measured

using a SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA) at excitation 450 nm, emission 500 nm. PFF stocks were diluted to a concentration of 100 μg/mL in 1x HBSS buffer

followed by three 10 s pulses of sonication using a 1/8_″_ probe equipped QS5 Sonicator (Covaris, Woburn, MA) prior to use. For cell culture experiments, freshly sonicated PFFs were used at

0.1 μg/mL. INHIBITORS BAY 11–7085 (#1743), SR 11302 (#2476), and Pyridone 6 (#6577) were purchased from Tocris Bioscience (Bristol, UK). JSH-23 was purchased from Selleck Chemicals (#S7351,

Houston, TX). GSK963 (#SML2376), GSK872 (#530389), necrosulfonamide (#480073), and Z-VAD-FMK (#627610) were purchased from Millipore Sigma (Burlington, MA). All inhibitors were solubilized

in DMSO. BAY 11–7085, SR 11302, and Pyridone 6 were used at a final concentration of 100 μM for cell culture treatments. JSH-23 was used at 50 μM. GSK 963 and GSK 872 were used at 1 μM.

Necrosulfonamide was used at 10 μM. Z-VAD-FMK was used at 5 μM. HUMAN ASTROCYTE AND NEURONAL CULTURES Primary human midbrain astrocytes (#1850) were obtained from ScienCell Research

Laboratories (Carlsbad, CA) and cultured in astrocyte media (AM, #1801), supplemented with 2% heat-inactivated fetal bovine serum (#0010), astrocyte growth supplement (#1852), and

penicillin/streptomycin cocktail (# 0503). Cells were cultured in poly-l-lysine coated T75 flasks. Human neuronal cells SH-SY5Y (ATCC, Manassas, VA, #CRL-2266) were cultured in DMEM medium

(VWR, Radnor, PA, #0101–0500) supplemented with 10% FBS (Gemini Biosciences West Sacramento, CA, #100–106), nonessential amino acids (Hyclone, #SH30138.01), HEPES (Hyclone #30237.01),

penicillin, streptomycin, and antifungal (Gemini Biosciences #400–110, #100–104). SH-SY5Y cells were propagated in T75 flasks prior to the differentiation process. Low passage stocks (less

than 15 passages) were used for differentiation throughout the manuscript. Cells were regularly screened for mycoplasma contamination. SH-SY5Y DIFFERENTIATION SH-SY5Y neuroblastoma cells

were differentiated into mature neuron-like cells by treating with retinoic acid (4 μg/mL; Sigma-Aldrich, St. Louis, MO, #R2625) and BDNF (25 ng/mL, Sigma-Aldrich, #B3795) diluted in DMEM

supplemented with 2% heat-inactivated fetal bovine serum (FBS, Gemini Biosciences, West Sacramento, CA, #100–106), nonessential amino acids (1x; HyClone, #SH30238.01), HEPES buffer (10 mM;

HyClone, #SH30237.01), l-Glutamine, penicillin, streptomycin (Gemini Biosciences, #400–110), and antifungal amphotericin B (Gemini Biosciences, #100–104). Differentiated SH-SY5Y cultures

were used for experiments 7 days post-differentiation. CELL DEATH AND VIABILITY ASSAY Cell viability was assessed with the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega,

Madison, WI, #G7573), according to the manufacturer’s instructions. Luminescence signal was read with a SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA). CASPASE 3/7 ACTIVITY

ASSAY Caspase 3/7 activity was measured using a chromogenic DEVD cleavage assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, #K106-100). QUANTITATIVE

REAL-TIME PCR Total RNA from cultured cells was isolated with Qiagen RNeasy mini extraction kit (Qiagen, Valencia, CA, #74106) following the manufacturer’s protocol. RNA concentration was

measured with a Quick Drop device (Molecular Devices, San Jose, CA). cDNA was subsequently synthesized with qScript cDNA Synthesis Kit (Quantabio, Beverly, MA, #95047). qRT-PCR was performed

with SYBR Green Master Mix (Bio-Rad, Hercules, #CA1725125) using a QuantStudio5 instrument (Applied Biosystems, Foster City, CA). Cycle threshold (CT) values for analyzed genes were

normalized to CT values of the housekeeping gene _18_ _S_ (CTTarget − CT18S = ΔCT). Data were further normalized to baseline control values (ΔCTexperimental − ΔCTcontrol = ΔΔCT (DDCT).

Primers were designed using Primer3 (https://bioinfo.ut.ee/primer3/) against human genomic sequences. A list of all primer sequences in our study appears in Supplementary Table 1.

IMMUNOCYTOCHEMISTRY For imaging experiments, cells were grown on poly-d-lysine coated coverslips (Neuvitro, Vancouver, WA, #GG-12-PDL). Following experimental treatments, cells were fixed in

4% paraformaldehyde for 15 min, followed by three washes in 1x PBS, followed by incubation in blocking solution (10% goat serum, Gibco, Waltham, MA, #16210 and 0.1% Triton X-100) for 30 min

at room temperature. Cells were then incubated in primary antibody (rabbit-anti-p65/RELA; 2 ug/mL; ThermoFisher, Waltham, MA, #10745-1-AP) diluted in blocking solution for 1 h. After three

15 min washes in 1x PBS, coverslips were incubated in secondary antibody (goat-anti-rabbit IgG conjugated Alexa Fluor 594; 2 ug/mL; Invitrogen Waltham, MA, #A32740) and nuclear stain (DAPI;

10 ug/mL; Biotium, Fremont, CA, #40043) for 15 min at room temperature, followed by another series of washes with 1x PBS. Coverslips were then mounted using ProLong Diamond Antifade Mountant

(Invitrogen, #P36931) onto slides. Images were acquired with an Airyscan fluorescent confocal microscope (Carl Zeiss LSM 800). TUNEL ASSAY TUNEL was performed using a standard kit according

to the manufacturer’s protocol (TMR In Situ Cell Death Detection Kit, Sigma-Aldrich, #12156792910) in combination with nuclear staining using DAPI (10 ug/mL; Biotium, Fremont, CA, #40043).

Images were captured using a 20x objective. The numbers of TUNEL positive nuclei in each image were counted by a blinded operator. COLOCALIZATION ANALYSIS For each coverslip, three regions

with matched cell density were captured with the 63x objective. p65 colocalization with DAPI was quantified using the Colocalization Colormap plugin in ImageJ software (National Institute of

Health, Bethesda, MD). The plugin calculates normalized mean deviation product, an index of correlation between pixels. Fisher’s Z transformation was applied to the index of correlation

prior to comparison. NUCLEAR PROTEIN EXTRACTION Primary human midbrain astrocytes were cultured to confluency. Cells were washed twice with PBS followed by 5 min incubation in cold 5 mM

EDTA. Cells were then scraped into 15 mL conical tubes and centrifuged for 5 min at 1000 rpm. Nuclear extraction was performed using a standard kit according to the manufacturer’s protocol

(Nuclear Extraction Kit, Abcam, Cambridge, MA, #ab113474). NF-ΚB P65 TRANSCRIPTION FACTOR ASSAY Protein concentrations of nuclear extracts were determined using BCA assay (ThermoFisher,

#23227), according to the manufacturer’s instructions. Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit,

Abcam, Cambridge, MA, #ab133112). Absorbance at 450 nm was read with SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA). FLOW CYTOMETRIC ANALYSIS OF PHAGOCYTOSIS Differentiated

SH-SY5Y neuronal cells were labeled with CSFE, according to the manufacture protocol (Millipore Sigma, Burlington, MA, #SCT110) and lysed using repeated freeze-thaw cycles to generate

labeled debris. Unlabeled neuronal debris was used as staining control. Neuronal debris was stored at −80 °C until needed. To detect phagocytosis, CSFE-labeled neuronal debris was added to

astrocyte cultures at a ratio of 1:100 for 24 h. Un-phagocytosed neuronal debris was washed away with 1XPBS, and astrocytes were harvested with 5 mM EDTA followed by scraping of adherent

cells. Astrocytes were stained with Zombie NIR at 1:1000 in 1XPBS according to the manufacturer protocol (BioLegend, San Diego, CA, #423105), followed by fixation in 1% paraformaldehyde.

Data collection and analysis were performed using a Northern Lights flow cytometer (Cytek, Fremont, California) and FlowJo software (FlowJo LLC, Ashland, OR). STATISTICAL ANALYSIS Normally

distributed data were analyzed using appropriate parametric tests: two-way analysis of variance (ANOVA) with Sidak’s correction for multiple comparisons was performed using GraphPad Prism

Software v8 (GraphPad Software, San Diego, CA). _P_ < 0.05 was considered statistically significant. Analysis of publicly available microarray data was performed in GEO2R and the GO

Enrichment Analysis tool [43]. Corrected _p_ values (false discovery rate) were determined using the Benjamini & Hochberg procedure. All data points represent biological replicates

unless otherwise noted. RESULTS Α-SYNUCLEIN PFFS INDUCE NF-ΚB-DEPENDENT TRANSCRIPTIONAL ACTIVITY ASSOCIATED WITH ASTROCYTE ACTIVATION To assess the impact of α-synuclein aggregates on

astrocyte activation state, we treated primary cultures of human midbrain astrocytes with α-synuclein PFFs, which have been extensively shown to induce inflammatory activation and seed

aggregation of endogenous α-synuclein both in vitro and in vivo [41, 44, 45]. To profile the astrocyte activation state following this treatment, we performed a qPCR screen of transcripts

previously associated with the putative A1 (neurotoxic) and A2 (neurotrophic) astrocyte activation states [18], along with other general markers of inflammatory activity. To further

characterize the nature of the PFF-induced transcriptional response, we performed these experiments in the presence of inhibitors of three major inflammatory transcriptional pathways: BAY

11–7085 (BAY) is an irreversible inhibitor of IκB kinase (IKK), thereby blocking NF-κB activation; Pyridone 6 (PYR) is a pan-JAK inhibitor, thereby blocking signaling through STAT family

transcription factors; and SR 11302 (SR) is an inhibitor of the transcription factor AP1. Overnight treatment with PFFs induced increased expression of 8 of 11 A1-associated transcripts in

our screen (Fig. 1a and Supplementary Fig. 2a–h), including _HLA-A_, _SERPING1_, _SRGN_, _HLA-E_, _PSMB8_, _GBP2_, _FKBP5_, and _UGT1A1_, while expression of _GGTA1_, _FBLN5_, and _AMIGO2_

was not significantly impacted (Supplementary Fig. 2i–k). Notably, inhibition of NF-κB signaling with BAY rescued the upregulation of all eight A1-associated transcripts, while inhibition of

JAK/STAT or AP1 signaling had minimal effect. Surprisingly, however, PFF treatment also upregulated 5 of 11 A2-associated genes in our study, including _PTX3_, _PTGS2_, _SPHK1_, _TM4FS1_,

and _CLCF1_ (Fig. 1b and Supplementary Fig. 3a–e), while _S100A10_, _SLC10A6_, _B3GNT5_, _CD14_, _CD109_, and _EMP1_ each showed various degrees of upregulation that did not reach

statistical significance (Supplementary Fig. 3f–k). PFF treatment also increased the expression of a number of inflammatory chemokines, including _CCL2_, _CXCL1_, _CXCL10_ (Fig. 1c–e). The

upregulation of CXCL10 was also detected at the protein level via ELISA (Fig. 1f). In all cases, blockade of NF-κB signaling, but not JAK/STAT or AP1 signaling, prevented upregulation of

reactive astrocyte genes. Importantly, treatment of midbrain astrocytes with a matched concentration of α-synuclein monomers did not induce expression of either A1- or A2- associated

transcripts (Supplementary Fig. 4a, b). Inflammatory responses to PFFs were also independent of serum concentrations in astrocyte culture media, as we observed essentially identical results

in astrocytes grown in serum-free medium (Supplementary Fig. 5a–l). Together, these data suggest that α-synuclein PFFs strongly induce an NF-κB-dependent transcriptional response that

includes a broad array of genes associated with astrocyte activation and inflammatory activity. Α-SYNUCLEIN PFFS INDUCE BOTH EXPRESSION AND ACTIVATION OF NF-ΚB SIGNALING ELEMENTS We next

sought to more thoroughly assess the impact of α-synuclein PFFs on NF-κB activation in astrocytes. While BAY blocks NF-κB signaling by inhibiting upstream IKK activity, we next tested

whether direct blockade of NF-κB nuclear translocation would impact PFF-mediated gene expression using the inhibitor JSH-23, which also rescued the induction of several reactive astrocyte

genes (Fig. 2a–d). To directly confirm that PFFs induced NF-κB activation, we performed confocal microscopy to visualize nuclear translocation of the NF-κB component p65. PFF treatment

induced robust accumulation of p65 in the nucleus, as indicated by enhanced colocalization of p65 signal with nuclear DAPI staining (Fig. 2e, f). Importantly, treatment with BAY completely

blocked this effect, confirming that IKK inhibition effectively blocked NF-κB activation in our experiments. As a secondary confirmation of NF-κB activation, we extracted nuclear fractions

from astrocytes following treatment with PFFs or PBS control and performed ELISA to detect p65. These experiments also revealed a significant accumulation of nuclear p65 that was completely

blocked by cotreatment with BAY (Fig. 2g). We also questioned whether PFF treatment might influence NF-κB activity via upregulation of NF-κB signaling elements. To answer this, we first

turned to two publicly available transcriptomic databases. In one study, Lee and colleagues [24] exposed primary rat astrocytes to a conditioned medium from a human neuroblastoma cell line

(SH-SY5Y) that had been transduced to express either α-synuclein or a LacZ control. Secondary analysis of microarray data from this study revealed that exposure to α-synuclein-containing

conditioned medium significantly upregulated many genes associated with the “NF-κB signaling” gene ontology term (GO:0038061), including _Rela_ (p65), _Traf2_, _Ripk3_, _Nfkb1_, _Ikbke_,

_Nfkb2_, and _Chi3l1_ (Fig. 2h). We performed a similar analysis on a dataset originally published by Durrenberger and colleagues [46], who reported transcriptomic profiles of postmortem

samples of substantia nigra tissues from 12 PD patients and eight healthy controls. Secondary analysis of this dataset revealed significant upregulation of three genes associated with NF-κB

signaling, including _RELA_, _NFKB1_, and _TRAF2_ (Fig. 2i). In our own experimental system, we saw that treatment of human midbrain astrocytes with PFFs also significantly upregulated

expression of _IKBKE_, _NFKB1_, _NFKB2_, and _TRAF2_ (Fig. 2j–m). Notably, the induction of these genes was blocked by BAY, suggesting that PFFs may induce feed-forward amplification of

NF-κB signaling. Together, these data confirm that α-synuclein induces both expression and activation of NF-κB in astrocytes and that enhanced expression of NF-κB signaling elements in the

substantia nigra is a feature of PD in human patients. Α-SYNUCLEIN PFFS INDUCE NEUROTOXIC ACTIVITY IN MIDBRAIN ASTROCYTE CULTURES Neuroinflammatory astrocytes, generally, have been reported

to induce programmed cell death in neurons, thereby contributing to disease pathogenesis. To test if α-synuclein PFFs could confer neurotoxic activity to astrocytes, we treated midbrain

astrocyte cultures with PFFs or PBS control in the presence of transcription factor pathway inhibitors (Fig. 3a). We then collected astrocyte conditioned medium (ACM) from these cultures and

added it to cultures of differentiated SH-SY5Y neuroblastoma cells at a 1:1 ratio with a normal culture medium. We observed that 24 h treatment with ACM derived from astrocyte cultures

treated with PFFs significantly reduced the viability of SH-SY5Y cultures, as assessed via ATP luciferase assay (Fig. 3b). Blockade of NF-κB signaling in astrocytes with BAY rescued this

neurotoxic activity, while PYR and SR treatment did not. We saw similarly that NF-κB inhibition with JSH-23 also prevented neurotoxic activity in astrocytes following PFF treatment (Fig.

3c). As a secondary confirmation of cell death in SH-SY5Y cultures, we performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect DNA damage associated with

programmed cell death, which revealed a significant increase in TUNEL+ nuclei in SH-SY5Y cultures treated with ACM derived from PFF-treated astrocytes (Fig. 3d, e). However, the treatment of

astrocytes with BAY prevented this effect. Together, these data suggest that α-synuclein PFFs stimulate neurotoxic activation in midbrain astrocytes in an NF-κB-dependent manner.

Importantly, we confirmed that cell death in our study could not be explained by exposure to residual α-synuclein PFFs found in ACM samples, as direct treatment of SH-SY5Y with PFFs did not

impact their viability within the timeframe of our experiments (Supplementary Fig. 6). While apoptosis is the most commonly reported form of neuronal cell death during Parkinsonian

neurodegeneration, other cell death modalities, including necroptosis, have also been reported [27, 47]. We questioned which cell death modality was induced in differentiated SH-SY5Ycells in

our experiments. We first used a DEVD-cleavage assay to measure levels of executioner caspase (caspase 3 and caspase 7) activity, which revealed that ACM derived from PFF-treated astrocytes

induced robust Caspase 3/7 activity in SH-SY5Y cultures, and that this effect was blocked when astrocytes were cotreated with BAY (Fig. 3f). To more carefully determine if caspase-dependent

apoptosis was occurring, we modified our treatment paradigm such that, prior to the addition of ACM, SH-SY5Y cultures were pretreated with inhibitors of programmed cell death, including the

pan-caspase inhibitor zVAD-FMK (zVAD), the RIPK3 inhibitor GSK872, the RIPK1 inhibitor GSK963, or the MLKL inhibitor necrosulfonamide (NSA) (Fig. 3g). Blockade of caspase signaling with

zVAD completely rescued cell death in SH-SY5Y cultures following treatment with ACM derived from PFF-treated astrocytes, while inhibiting of necroptosis signaling components (RIPK1, RIPK3,

or MLKL) did not (Fig. 3h). These data suggest that the neurotoxic activity conferred by PFFs in astrocytes induces apoptosis rather than necroptosis in SH-SY5Y cells. Α-SYNUCLEIN PFFS

REDUCE HOMEOSTATIC PHAGOCYTIC ACTIVITY IN ASTROCYTES Neurotoxic astrocytes have been shown to downregulate key homeostatic functions, including phagocytosis. We thus questioned whether

α-synuclein PFFs would perturb the phagocytic function of midbrain astrocytes. We treated astrocyte cultures with PFFs for 24 h in the presence of transcription factor pathway inhibitors and

measured the expression of key genes known to be involved in astrocytic phagocytosis. PFF treatment downregulated expression of _GAS6_ (Fig. 4a), which encodes an important opsonin for

phagocytosis of apoptotic cells, as well as _MEGF10_ (Fig. 4b), which encodes a receptor for C1q, an important opsonin in the classical complement pathway. In contrast, expression of

_MERTK_, a GAS6 receptor, was increased by PFF treatment (Fig. 4c), which may represent a compensatory response to decreased GAS6 expression. Expression of _AXL_, which encodes an additional

GAS6 receptor, was not impacted (Fig. 4d). BAY treatment prevented all PFF-induced changes to phagocytic gene expression, while PYR and SR had marginal or no impact. To determine whether

changes to phagocytic gene expression actually impacted phagocytic activity, we measured uptake of fluorescently labeled zymosan, a yeast-associated glucan, in astrocyte cultures following

24 h treatment with PFFs and/or BAY (or respective controls). PFF-treated astrocytes phagocytosed significantly less zymosan compared to control cultures, and this effect was blocked in the

presence of BAY (Fig. 4e). These data suggest that, in addition to inducing inflammatory gene transcription and neurotoxic activity, NF-κB activation downstream of PFF treatment reduces the

homeostatic phagocytic capacity of astrocytes. Α-SYNUCLEIN PFFS INDUCE RIPK-DEPENDENT TRANSCRIPTIONAL ACTIVATION IN ASTROCYTES, INDEPENDENTLY OF NECROPTOSIS While our data clearly identified

NF-κB as a molecular driver of astrocyte activation in our system, the upstream inputs into this pathway remained unclear. We previously described a role for RIPK signaling in inflammatory

transcriptional activation in neurons that was independent of necroptotic cell death [34]. Moreover, RIPK signaling is a known activator of the NF-κB pathway [48,49,50,51]. We thus

questioned whether RIPK signaling was required for PFF-mediated astrocyte activation. Treatment of midbrain astrocyte cultures with PFFs for 24 h in the presence of inhibitors of either

RIPK3 or RIPK1 revealed that both kinases were required for induction of the PFF-mediated transcriptional response, including genes associated with astrocyte activation (Fig. 5a–c) and

inflammatory chemokines (Fig. 5d–f). Blockade of RIPK signaling also prevented the induction of CXCL10 protein expression, as confirmed by ELISA (Fig. 5g). We also observed that inhibition

of both RIPK3 and RIPK1 prevented transcriptional induction of NF-κB associated genes (Fig. 5h–k). Notably, these effects were independent of necroptosis, as inhibition of MLKL had no effect

on PFF-mediated transcriptional activation (Fig. 5a–f, h–k), nor did it impact protein expression of CXCL10 (Fig. 5g). Moreover, neither PFFs nor any of the inhibitors in our study induced

detectable levels of cell death in astrocytes (Supplementary Fig. 7), further confirming that necroptosis was not the source of transcriptional activation downstream of astrocytic RIPK

signaling. Blockade of caspase signaling was also not required for these effects, as treatment with zVAD prior to PFF exposure had no effect on gene expression (Supplementary Fig. 8a). These

data suggest that RIPK1 and RIPK3 engage inflammatory transcriptional activity in astrocytes following PFF treatment via a cell death-independent mechanism. RIPK SIGNALING IS REQUIRED FOR

NF-ΚB ACTIVATION DOWNSTREAM OF Α-SYNUCLEIN PFFS We next confirmed that RIPK signaling was required for NF-κB activation in PFF-treated astrocytes. Confocal microscopic analysis of p65

expression revealed that blockade of either RIPK1 or RIPK3 was sufficient to prevent nuclear accumulation of p65, while blockade of MLKL had no effect (Fig. 6a, b). We observed similar

findings following the detection of p65 in isolated nuclear fractions using ELISA (Fig. 6c). To further assess the relatedness of the RIPK3- and NF-κB-dependent transcriptional responses to

PFFs, we performed a more thorough time-course analysis of gene expression associated with astrocyte activation in the presence of RIPK3 and NF-κB inhibitors. Remarkably, blockade of both

molecules had essentially identical effects, as both inhibitors completely blocked the upregulation of astrocyte activation-associated genes following PFF treatment (Fig. 6d–j). Notably,

however, these experiments revealed that the putative A1 genes _SERPING1_, _HLA_-_E_, _SRGN_, and _PSMB8_ remained highly expressed up to 48 h following PFF exposure (Fig. 6d–g), while the

A2 genes _PTGS2_, _PTX3_, and _TM4SF1_ exhibited a distinct profile of robust early expression that largely resolved by 48 h (Fig. 6h–j). While RIPK3 and NF-κB did not appear to influence

this temporally distinct expression pattern, these data suggest that the nature of astrocyte activation following exposure to α-synuclein aggregates may vary significantly over time. RIPK

SIGNALING IS REQUIRED FOR FUNCTIONAL MARKERS OF ASTROCYTE ACTIVATION DOWNSTREAM OF Α-SYNUCLEIN PFFS To confirm that RIPK signaling was also required for functional indications of astrocyte

activation, we treated midbrain astrocytes with PFFs or PBS control for 24 h following 30 min pretreatment with RIPK1, RIPK3, or MLKL inhibitors. We then treated differentiated cultures of

SH-SY5Y cells with the ACM from these astrocyte cultures at a 1:1 ratio with a normal culture medium (Fig. 7a). As expected, ACM derived from PFF-treated astrocytes greatly reduced the

viability of SH-SY5Y cultures. However, this effect could be blocked by inhibition of either RIPK1 or RIPK3, but not MLKL or caspase signaling, in astrocytes (Fig. 7b and Supplementary Fig.

8b, c). We next assessed whether RIPK3 activation downstream of PFFs influenced astrocyte phagocytic activity. To do so, we labeled differentiated SH-SY5Y cultures with CSFE, then used these

cultures to generate neuronal debris by subjecting cells to rapid freeze-thaw lysis. We then treated astrocytes with PFFs or PBS control following pretreatment with RIPK3 or NF-κB

inhibitors. Following PFF treatment, we exposed astrocytes to CSFE-labeled neuronal debris for 24 h and measured uptake via flow cytometric analysis. PFF treatment significantly reduced

uptake of neuronal debris, as measured by geometric mean fluorescence intensity (GMFI) of CSFE within astrocytes (Fig. 7c, d). However, blockade of either RIPK3 or NF-κB completely abolished

this effect, confirming that both molecules are required for downregulation of phagocytic activity following exposure to PFFs. These data suggest that necroptosis-independent RIPK activity

engages both transcriptional and functional activation of astrocytes following exposure to fibrillar α-synuclein. We thus returned to our secondary analysis of gene expression in the

substantia nigra of Parkinson’s patients in order to see if there was evidence of increased expression of this pathway in human PD. We observed significant upregulation of _RIPK3_ in PD

patients compared to normal controls, while expression of both _RIPK1_ and _MLKL_ did not reach statistical significance (Fig. 7e). Together, these data identify a previously unknown

function for the RIPKs in the promotion of a neurotoxic activation state and suggest further work is needed to identify roles for necroptosis-independent RIPK signaling in Parkinsonian

neurodegeneration and other synucleinopathies. DISCUSSION Abnormal aggregation of α-synuclein is a pathological hallmark of PD [52,53,54]. However, the effects of pathogenic α-synuclein

species on astrocytes have not been thoroughly studied. Previous work has shown that astrocytes can protect dopaminergic neurons from α-synuclein deposition and degeneration [55]. However,

α-synuclein aggregates are also frequently observed in PD patients, where they disturb vital homeostatic functions and thereby exacerbate disease pathology in neurons [56]. We show that

α-synuclein PFFs induce a potently inflammatory transcriptional program in human midbrain astrocytes that is associated with neurotoxic activity and decreased homeostatic phagocytic

function. Notably, this effect requires RIPK signaling, but does not result in necroptosis, the canonical function of this pathway. RIPK3 activation in astrocytes has been observed in mouse

models of PD [28], as well as postmortem human patient samples [27]. Moreover, both genetic and pharmacological ablation of RIPK3 signaling have been shown to be protective in mouse models

of neurodegeneration [57, 58]. Our findings suggest that therapies targeting RIPK signaling may serve to limit the pathologic consequences of inflammatory astrocyte activation in PD and

other neurodegenerative diseases. Since the initial description of the A1 and A2 astrocyte subtypes, transcriptional profiling of astrocytes as an indication of their A1 or A2 activation

state has grown increasingly popular, though whether the respective transcriptomes of these states are stable across disease stimuli or reliably indicate neurotoxic vs. neurotrophic

functional activity is controversial [17, 59]. In our study, treatment with α-synuclein PFFs induced neurotoxic activity and diminished phagocytic capacity, in line with previous

descriptions of the A1 activation state [18, 60]. However, while α-synuclein PFFs induced many putative A1-associated genes, we also saw marked upregulation of A2-associated genes as well,

though each gene set did display somewhat different expression kinetics. Together, our data suggest that expression of both A1 and A2 genes is associated with clear functional changes in

neurotoxic and phagocytic activity following exposure to α-synuclein PFFs, supporting other recent work demonstrating that gene expression profiling alone is insufficient to predict the

functional outcomes of astrocyte activation [59]. RIPK1 and RIPK3 are activators of necroptotic cell death [61, 62], which has been shown to exert complex functions in diverse disease

states, including infection [39, 63, 64], cancer [65], and sterile injury [66]. In the CNS, RIPK signaling has principally been connected to deleterious neuroinflammation and necroptosis in

the context of neurodegenerative diseases, including PD [67, 68]. However, we and others recently described a necroptosis-independent function for RIPK signaling in neurons in which a

RIPK-dependent transcriptional program induced protective neuroinflammation in response to viral infection [33, 34]. Here, we show that necroptosis-independent RIPK signaling can also

coordinate inflammatory transcription in astrocytes, which is associated with the conferral of neurotoxic activity. These findings suggest that cell death-independent functions of this

pathway in the CNS may extend beyond neurons, possibly to all cells of neuroectodermal lineage. While we have identified a previously unknown function for RIPK signaling and NF-κB in

promoting a neurotoxic astrocyte activation state, several questions remain. For example, the mechanisms by which aggregated α-synuclein is sensed by astrocytes prior to the induction of

inflammatory activation is poorly understood. While some studies have suggested that α-synuclein aggregates engage TLR4-mediated innate sensing pathways [23], whether adult astrocytes

express TLR4 in vivo is a matter of debate [18, 69, 70]. Further work, particularly in rodent models, will help further refine our understanding of the upstream signaling events that promote

RIPK3 activation in astrocytes in the context of synucleinopathy. Finally, while several groups have now shown that activated astrocytes express some secreted factor that exerts neurotoxic

activity, the identity of this factor (or factors) and its mechanism of action have been difficult to discern [16]. Ongoing work characterizing the secretomes of activated astrocytes is

needed to answer these important questions. DATA AVAILABILITY Microarray results are derived from secondary analysis of two previously published datasets, which can be accessed via NCBI’s

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2014;34:11929–47. Article  PubMed  PubMed Central  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank Drs. Gabriella D’Arcangelo, Max Tischfield, and Jessica

Williams for feedback on this work. This work was supported by a research grant from the American Parkinson’s Disease Association, NIH R01 NS120895, and startup funds from Rutgers University

(all to B.P.D.). M.K. was supported in part by a Parkinson’s Foundation Summer Student Fellowship. A.P.P. and P.-L.K. were supported in part by Division of Life Sciences Summer

Undergraduate Research Fellowships from Rutgers University. AUTHOR INFORMATION Author notes * These authors contributed equally: Tsui-Wen Chou, Nydia P. Chang. AUTHORS AND AFFILIATIONS *

Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA Tsui-Wen Chou, Nydia P. Chang, Medha Krishnagiri, Aisha P. Patel, Marissa Lindman, Juan P. Angel, Po-Lun

Kung, Colm Atkins & Brian P. Daniels Authors * Tsui-Wen Chou View author publications You can also search for this author inPubMed Google Scholar * Nydia P. Chang View author

publications You can also search for this author inPubMed Google Scholar * Medha Krishnagiri View author publications You can also search for this author inPubMed Google Scholar * Aisha P.

Patel View author publications You can also search for this author inPubMed Google Scholar * Marissa Lindman View author publications You can also search for this author inPubMed Google

Scholar * Juan P. Angel View author publications You can also search for this author inPubMed Google Scholar * Po-Lun Kung View author publications You can also search for this author

inPubMed Google Scholar * Colm Atkins View author publications You can also search for this author inPubMed Google Scholar * Brian P. Daniels View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization: T.-W.C., N.P.C., C.A. and B.P.D.; Investigation and analysis: T.-W.C., N.P.C., M.K., A.P.P., M.L., J.P.A., P.-L.K., C.A.

and B.P.D.; Writing- original draft: T.-W.C., N.P.C., C.A. and B.P.D.; Writing- review and editing: T.-W.C., N.P.C., J.P.A., C.A. and B.P.D.; Supervision: C.A. and B.P.D.; Funding

acquisition: B.P.D. CORRESPONDING AUTHOR Correspondence to Brian P. Daniels. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ETHICS STATEMENT No

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α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB. _Cell Death Dis_ 12, 756 (2021). https://doi.org/10.1038/s41419-021-04049-0 Download citation *

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