Pathomechanism Characterization and Potential Therapeutics Identification for Parkinson’s Disease Targeting Neuroinflammation

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons and the presence of α-synuclein-containing Lewy bodies. The unstructured α-synuclein forms insoluble fibrils and aggregates that result in increased reactive oxygen species (ROS) and cellular toxicity in PD. Neuroinflammation engaged by microglia actively contributes to the pathogenesis of PD. In this study, we showed that VB-037 (a quinoline compound), glycyrrhetic acid (a pentacyclic triterpenoid), Glycyrrhiza inflata (G. inflata, a Chinese herbal medicine), and Shaoyao Gancao Tang (SG-Tang, a formulated Chinese medicine) suppressed the nitric oxide (NO) production and interleukin (IL)-1β maturation in α-synuclein-stimulated BV-2 cells. Mouse inflammation antibody array further revealed increased IL-1α, IL-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in α-synuclein-inflamed BV-2 cells and compound pretreatment effectively reduced the expression and release of these pro-inflammatory mediators. The test compounds and herbal medicines further reduced α-synuclein aggregation and associated oxidative stress, and protected cells against α-synuclein-induced neurotoxicity by downregulating NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), caspase 1, IL-1β, IL-6, and associated nuclear factor (NF)-κB inhibitor alpha (IκBα)/NF-κB P65 subunit (P65), c-Jun N-terminal kinase (JNK)/proto-oncogene c-Jun (JUN), mitogen-activated protein kinase 14 (P38)/signal transducer and activator of transcription 1 (STAT1) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathways in dopaminergic neurons derived from α-synuclein-expressing SH-SY5Y cells. Our findings indicate the potential of VB-037, glycyrrhetic acid, G. inflata, and SG-Tang through mitigating α-synuclein-stimulated neuroinflammation in PD, as new drug candidates for PD treatment.


Introduction
Parkinson's disease (PD), one of the most common neurodegenerative disorders, has the clinical features of resting tremor, rigidity, bradykinesia, and postural instability. The pathology is characterized mainly by progressive loss of the nigro-striatal dopaminergic neurons and the presence of cytoplasmic inclusion bodies (Lewy bodies) containing α-synuclein in the ventral midbrain [1]. Alpha-synuclein (SNCA), parkin RBR E3 ubiquitin protein ligase (PRKN), parkinsonism associated deglycase (DJ1), PTEN induced kinase 1 (PINK1), leucine rich repeat kinase 2 (LRRK2), ATPase cation transporting 13A2 (ATP13A2), VPS35 retromer complex component (VPS35), eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), phospholipase A2 group VI (PLA2G6), F-box protein 7 (FBXO7), synaptojanin 1 (SYNJ1) and DnaJ heat shock protein family (Hsp40) member C6 (DNAJC6) have been identified to be the causative genes for familiar and early-onset PD (EOPD) [2]. Owing to the discovery of the causative genes, several pathogenic pathways were identified, which includes accumulation of aberrant or misfolded proteins, mitochondrial dysfunction, increased oxidative stress, impaired ubiquitin-proteasome function, failure of autophagy-lysosome and mitophagy, deficits in endosomal trafficking and inflammation (see review in [3]). Several genome wide-association studies (GWAS) also have identified novel genetic associations with PD and these genes are linked to the previously known pathogenic pathways as well as other less recognized pathways, such as endocytosis, transcriptional dysregulation, inflammation, and cytokine-mediated signaling [4,5]. PD brain at post-mortem has shown CD8 + and CD4 + T-cell infiltration and accumulations of microglia cells and astrocytes in substantia nigra [6]. Substantial evidence has also shown that microglial activation, nuclear factor kappa B (NF-κB) induced neuroinflammation and release of inflammatory factors may play an important role in the neurodegeneration of PD [7][8][9]. Aggregated α-synuclein could activate microglia, which leads to disease progression of PD [10]. Direct injection of α-synuclein into the substantia nigra resulted in the upregulation of mRNA expression of proinflammatory cytokines, the expression of endothelial markers of inflammation and microglial activation [11]. Neuroinflammation is also likely to play a key role in propagation of misfolded α-synuclein in a "prion-like" fashion in PD [12]. Gut microbiota promotes α-synuclein-dependent microglia activation, leading to neuroinflammatory damage in brains of mice [13]. Furthermore, several studies have suggested protective effects of anti-inflammatory drugs for PD animal models and epidemiological studies [14][15][16].
The nucleotide-binding oligomerization domain-like receptor (NLR) family of proteins (NLRPs) is involved in the regulation of innate immunity responses. Inflammasomes are composed of the NLRP sensor, the signaling adapter apoptosis associated speck-like protein containing a caspase recruitment domain (PYD and CARD domain containing, ASC), and the caspase 1 protease [17]. Caspase 1 is activated within the inflammasome multiprotein complex through interaction with ASC. Activation of caspase 1 leads to the processing interleukin (IL)-1β precursor (pro-IL-1β) and maturation of IL-1β [17]. Increased reactive oxygen species (ROS) promote α-synuclein to form fibrils or aggregates that can be uptaken by microglia to activate the microglial NLR family pyrin domain containing 3 (NLRP3) inflammasome, which further contributes to the generation of mitochondrial ROS [18]. While reactive microglia are the major source of NLRP3 inflammasome, NLR family pyrin domain containing 1 (NLRP1) is mainly generated by neurons. It has been shown that hyperglycemia inducing neuroinflammation through activation of NLRP1 causes diabetes-associated neuron injury [19].
We proposed that α-synuclein can be up-taken by microglia to activate microglia, which will then release IL-1β, IL-6, and TNF-α and can activate NLRP1 and NLRP3 in PD cellular models, both of which contribute to neuronal cytotoxicity. Previously, we have discovered Chinese herbal medicines (CHMs) Shaoyao Gancao Tang (SG-Tang) and Glycyrrhiza inflata (G. inflata), as well as pure compounds glycyrrhetic acid (C 30 H 46 O 4 ) and VB-037 (C 24 H 20 N 4 O 3 ) with anti-inflammatory and anti-oxidative effects. SG-Tang, a formulated CHM made of Paeonia lactiflora and Glycyrrhiza uralensis at 1:1 ratio, protects neurons from tau oligomers/aggregates-induced inflammatory damage [25] and from expanded polyglutamine-induced cytotoxicity [26]. We have also shown that extract of G. inflata inhibits aggregation by upregulating PPARGC1A and NFE2L2-ARE pathways in cell models of spinocerebellar ataxia 3 [27], and reduces Aβ misfolding and provides neuroprotection through anti-oxidative and anti-inflammatory action in cell models of Alzheimer's disease [28]. Glycyrrhetic acid is a hydrolytic product of glycyrrhizic acid. As an active constituent of G. inflata [27] and SG-Tang [25], glycyrrhizic acid has been used to treat inflammatory diseases [29]. VB-037 is a quinoline compound protecting neurons against Aβ aggregates-induced cytotoxicity through reduction of P38-and JNK-mediated inflammation [30]. In addition, anandamide transport inhibitor AM404 (C 26 H 37 NO 2 ) displays anti-inflammatory and neuroprotection effects on N-methyl-D-aspartic acid (NMDA)induced excitotoxicity [31]. In the present study, we examined the anti-inflammatory effects of AM404, VB-037, glycyrrhizic acid, SG-Tang, and G. inflata on BV-2 microglia and inducible A53T SNCA-GFP-expressing SH-SY5Y cells. We also explored if these CHMs or compounds exert their effects through mitigating the NLRP1/3, caspase 1, IL-1β, IL-6, and associated IκBα/P65, JNK/JUN, P38/STAT1, and JAK2/STAT3 pathways.

Discussion
Several lines of evidence have shown neuroinflammation contributes to neurodegeneration in PD [45]. However, which and how inflammatory pathways are involved in the pathogenesis remain to be explored. In this study, we provided evidence that BV-2 cells are activated by α-synuclein fibrils to secrete NO and promote maturation of IL-1β. IL-1β maturation is inhibited by VB-037, glycyrrhetic acid, G. inflata, and SG-Tang. Results of mouse cytokine antibody arrays demonstrate that expression of IL-1α, IL-1β, TNF-α, IFN-γ, IL-6, GM-CSF and G-CSF are increased in α-synuclein-stimulated BV-2 cells, which can be attenuated by VB-037 or glycyrrhetic acid. The release of IL-1β, TNF-α, GM-CSF, IL-6 and G-CSF cytokines from BV-2 cells to culture medium is also reduced by VB-037 or glycyrrhetic acid. It is noted that low IC 50 cytotoxicity of VB-037, glycyrrhetic acid, G. inflata, and SG-Tang in cells indicate their potential as agents for treatment of neurodegenerative diseases including PD.
We then showed that α-synuclein fibrils could provoke aggregation and increase oxidative stress, leading to impaired neurite outgrowth and apoptosis by activating NLRP1/NLRP3 inflammasome, IL-1β-mediated IκBα/P65, JNK/JUN, P38/STAT1, and IL-6-mediated JAK2/STAT3 pathways in DAergic neurons derived from α-synucleinexpressing SH-SY5Y cells. Furthermore, the aggregation, oxidative stress, neurite outgrowth deficits, apoptosis and neuroinflammation can be ameliorated by treatment with the tested compounds or CHM. VB-037 has been shown to inhibit LPS/IFN-γ-induced activation of BV-2 cells and attenuate IL-1β-, caspase 1-, P38-and JNK-mediated inflammatory damage caused by Aβ aggregates [30]. This study further confirms its anti-inflammation effect on α-synuclein-activated BV-2 and α-synuclein-expressing SH-SY5Y cells. Antiinflammatory activity of SG-Tang and G. inflata has also been shown in cell models induced by tau or Aβ misfolding [25,28]. Here, we demonstrated further evidence that G. inflata and SG-Tang provide neuroprotection through anti-inflammatory activity in PD cellular models. Glycyrrhetinic acid (glycyrrhetic acid) has been shown to act like a dopamine receptor D3 agonist to restore dopaminergic function [46]. Although glycyrrhizic acid reduced IL-1β, IL-6, and TNF-α, and the number of activated astrocytes and microglia in rotenone-injected animals [47], whether its metabolite glycyrrhetic acid also has antiinflammation effects on PD models is not known. Here we showed that glycyrrhetic acid exhibits anti-inflammatory action to mitigate neurotoxicity induced by α-synuclein fibrils in both BV-2 and A53T SNCA-GFP SH-SY5Y cells.
While NLRP3 is mainly produced by microglia and its role in contributing to neurodegeneration has been well shown in PD models [18,39], NLRP1 is majorly activated in neurons by different toxic stimuli and its involvement in neuroinflammation is less investigated [48,49]. NLRP1 receptor is increased in human AD brains and neurons, and NLRP1 up-regulation to activate caspase 1 to cleave pro-IL-1β has been shown in serumdeprived neurons [50]. Aβ oligomers have been reported to induce ATP leakage in cells and cause an overexpression of P2 × 7 purinergic receptors, leading to NLRP1 activation in both of microglia and hippocampal neurons [51,52]. Aβ aggregates induce neuronal NLRP1 inflammasome that drives IL-1β maturation and subsequent neuroinflammation in an AD mouse model [40]. In accordance with the result of NLRP1 upregulated by Aβ oligomers in neurons, we found that α-synuclein fibrils increase NLRP1, NLRP3, ASC, and caspase 1 that subsequently promote IL-1β maturation in α-synuclein-expressing SH-SY5Y neurons. Caspase 1 can be activated by known inflammasome-stimulators to directly cleave α-synuclein to generate truncated species that are prone to form aggregates in a neuronal PD cell model, and silencing of caspase 1 expression rescues neurotoxicity caused by α-synuclein, suggesting that under certain toxic stimuli, inflammasome, and caspase 1 cause PD pathology in neurons [53]. Substantial evidence has shown that ROS activate NLRP3 inflammasome through release of the ROS-sensitive NLRP3 ligand thioredoxininteracting protein (TXNIP) from its inhibitor thioredoxin and antioxidants can attenuate NLRP3-mediated inflammatory cytotoxicity [54][55][56]. We therefore suggest that α-synuclein fibrils may act like an inflammasome-stimulator or produce ROS to activate NLRP1, ASC, caspase 1, and IL-1β, leading to increased α-synuclein aggregates and neurotoxicity, all of which can be mitigated by the tested compounds and CHM as shown in the present study. However, future studies are warranted to provide direct evidence of the role of α-synuclein fibrils as an inflammasome-stimulator.
Previously, studies have shown sustained IL-1β expression at pro-inflammatory level confers profound toxic effects on the substantia nigra in two different animal models [57,58]. As shown above in our study results, mature IL-1β can be released by activated BV-2 cells stimulated by α-synuclein fibrils, or produced by neurons probably through NLRP1caspase 1 pathway. Although increased IL-1β has been shown in the brains of human PD and mouse PD models [59][60][61], whether the downstream signaling pathways of IL-1β in neurons overexpressing α-synuclein are involved is not clear. Responding to IL-1β binding signal, JNK, or P38 is activated and translocates to the nucleus to phosphorylate transcription factors such as JUN, FOS, STAT1, and MYC, which subsequently up-regulate the expression of pro-apoptotic genes [23,62]. Cytokines and p-P38 are increased by 6-OHDA and Toona sinensis seeds exert anti-inflammatory effects through suppressing p-P38 in a PD rat model [63]. Nuclear activation of p-P65 has been shown to mediate inflammatory toxicity in MPTP-, LPS-and rotenone-induced PD animal models and agents provide neuroprotection via inhibiting p-P65 activation [64]. Downstream pathways of IL-1β including p-IκBα/p-P65, p-JNK/p-JUN and p-P38/p-STAT1 were augmented in A53T SNCA-GFP SH-SY5Y cells added with α-synuclein fibrils, which are attenuated by VB-037, glycyrrhetic acid, G. inflata and SG-Tang, indicating the anti-inflammatory effects of the tested compounds and CHM on A53T SNCA-GFP SH-SY5Y cells via targeting IL-1β and its downstream signaling pathways.
IL-6 has been identified as an important cytokine to coordinate the acute phase response and the activation of immunocompetent glia within the brain. IL-6 is expressed not only by glial cells but also by neurons and can be activated by IL-1β [65][66][67]. The proinflammatory effects of IL-6 are mediated through autoactivation of JAK2/JAK3, which phosphorylate STAT3/STAT1 [24]. STAT3 and STAT1 are latent transcription factors and once phosphorylated, they translocate into the nucleus and induce the transcription of IL-6responsive genes, whereas SOCS3 counteracts JAK2/STAT3 signaling to diminish the IL-6mediated inflammation [24]. IL-6-mediated JAK2/STAT3 signaling cascade has been shown to contribute to neurodegeneration in AD and Huntington's disease models [68]. Increased IL-6 has been shown in the brains, CSF and plasma of human PD, and brains of mouse PD models [59,69,70], whether the downstream signaling pathways of IL-6 are involved, remains to be investigated. Overexpression of α-synuclein activates STAT3 in the substantia nigra in a mouse PD model, and pro-inflammatory cytokines and cytotoxicity are reduced by miR-let-7a that suppresses STAT3 [71]. Similarly, our study has shown that IL-6 and JAK2/STAT3 are induced and SOCS3 is reduced by α-synuclein fibrils, both of which can be rescued by VB-037, glycyrrhetic acid, G. inflata, and SG-Tang, indicating these compounds and CHM protect A53T SNCA-GFP SH-SY5Y cells against inflammatory damage via targeting IL-6 and its downstream signaling pathway. However, another study shows that bisdemethoxycurcumin protects neurons from rotenone-induced PD pathology via enhancing JAK2/STAT3 signaling [72]. These results suggest the role of IL-6/JAK2/STAT3 signaling pathway in PD pathogenesis remains to be clarified by future studies.

Cytokine qRT-PCR Assay
To measure GM-CSF, IL-6 and G-CSF RNA in BV-2 cells, total RNA was extracted using TRIzol reagent, treated with DNase to remove chromosomal DNA, and used for cDNA synthesis with SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Relative cytokine RNA expression was analyzed in 100 ng cDNA through real-time PCR (StepOnePlus Real-time PCR system; Applied Biosystems, Foster City, CA, USA) with TaqMan fluorogenic probes Mm01290062_m1 for Csf2 (GM-CSF), Mm00446190_m1 for IL-6, Mm00438334_m1 for Csf3 (G-CSF), and Mm00607939_s1 for β-actin control (Thermo Fisher Scientific). Fold change was calculated using the formula 2 ∆Ct , ∆C T = C T (control) -C T (target), in which C T indicates cycle threshold.

Cytokine ELISA
The levels of GM-CSF, IL-6, and G-CSF in BV-2 cell lysates and IL-1β, TNF-α, GM-CSF, IL-6, and G-CSF in cell culture media were determined using ELISA. Specifically, mouse IL-1β Instant ELISA, mouse IL-6, TNF-α, and GM-CSF (Csf2) Platinum ELISA, and mouse G-CSF (Csf3) ELISA kits (Thermo Fisher Scientific) were used. All experimental procedures were performed following the corresponding manufacturers' instructions. The optical density at 450 nm was detected using a microplate reader (Multiskan Go, Thermo Fisher Scientific).

Caspase 1 and 3 Activities and LDH Release Assays
TPA-differentiated A53T SNCA-GFP SH-SY5Y cells (4 × 10 5 on 6-well dishes for caspase 1 activity and LDH release assays; 5 × 10 4 on 12-well dishes for caspase 3 activity assay) were pretreated with test compounds or herbs and A53T SNCA-GFP expression was induced in the presence of α-synuclein fibrils as described. For LDH release assay, cell culture media were collected on day 14 and the release of LDH was examined by using LDH cytotoxicity assay kit (Cayman, Ann Arbor, MI, USA). The absorbance was read at 490 nm with Multiskan GO microplate reader. For caspase 1 and 3 activity assays, cells were lysed by repeated cycles of freezing and thawing. Caspase 1/3 activities were measured with the caspase 1 (BioVision, Milpitas, CA, USA) and caspase 3 (Sigma-Aldrich) fluorimetric assay kits, with 420/50 nm excitation and 485/20 nm emission (caspase 1 assay) or 360/40 nm excitation and 460/40 nm emission (caspase 3 assay) (FLx800 fluorescence microplate reader, Bio-Tek).

Statistical Analysis
For each data set, the experiments are performed three times and data were expressed as the means ± standard deviation (SD). Differences between groups were evaluated by Student's t test (comparing two groups) or one-way analysis of variance (ANOVA) with a post hoc Tukey test where appropriate (comparing several groups). All p values were two-tailed, with values lower than 0.05 to be considered statistically significant.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.