Neuroinflammation and α-Synuclein Dysfunction Potentiate Each Other, Driving Chronic Progression of Neurodegeneration in a Mouse Model of Parkinson’s Disease

Background Mechanisms whereby gene–environment interactions mediate chronic, progressive neurodegenerative processes in Parkinson’s disease (PD)—the second most common neurodegenerative disease—remain elusive. Objective We created a two-hit [neuroinflammation and mutant α-synuclein (α-syn) overexpression] animal model to investigate mechanisms through which mutant α-syn and inflammation work in concert to mediate chronic PD neurodegeneration. Methods We used an intraperitoneal injection of the inflammogen lipopolysaccharide (LPS; 3 × 106 EU/kg) to initiate systemic and brain inflammation in wild-type (WT) mice and transgenic (Tg) mice overexpressing human A53T mutant α-syn. We then evaluated nigral dopaminergic neurodegeneration, α-syn pathology, and neuroinflammation. Results After LPS injection, both WT and Tg mice initially displayed indistinguishable acute neuroinflammation; however, only Tg mice developed persistent neuroinflammation, chronic progressive degeneration of the nigrostriatal dopamine pathway, accumulation of aggregated, nitrated α-syn, and formation of Lewy body-like inclusions in nigral neurons. Further mechanistic studies indicated that 4-week infusion of two inhibitors of inducible nitric oxide synthase and NADPH oxidase, major free radical–generating enzymes in activated microglia, blocked nigral α-syn pathology and neurodegeneration in LPS-injected Tg mice. Conclusions Microglia-derived oxidative stress bridged neuroinflammation and α-syn pathogenic alteration in mediating chronic PD progression. Our two-hit animal model involving both a genetic lesion and an environmental trigger reproduced key features of PD and demonstrated synergistic effects of genetic predisposition and environmental exposures in the development of PD. The chronic progressive nature of dopaminergic neurodegeneration, which is absent in most existing PD models, makes this new model invaluable for the study of mechanisms of PD progression.

Parkinson's disease (PD), an age-related neurodegenerative disorder, progresses over decades. The mechanism for the gradual loss of nigral dopamine (DA) neurons and the chronic disease progression in PD is largely unknown. Emerging evidence has indicated that neuroinflammation contributes to PD ; Gao and Hong 2008;Hirsch and Hunot 2009). Morphologic changes of microglia (the resident brain immune cells) and accumulation of pro inflammatory factors are associated with degenerating DA neurons in the substantia nigra (SN) of PD patients and various PD animal models (Liberatore et al. 1999;McGeer et al. 1988;Sherer et al. 2003). DNA polymorphisms of several inflammatory cytokines and genetic variation in the HLA (human leukocyte antigen) region that contains numerous immune-related genes might increase the risk of PD (Hamza et al. 2010;Wahner et al. 2007). Moreover, inhibition of inflammation is associated with reduced neuronal impairment in various PD models (Gao et al. 2003;Wu et al. 2003). We and others previously reported that an intra peritoneal injection or chronic nigral infusion of relatively high doses of lipo polysaccharide (LPS; a common inflammogen) led to time-dependent degeneration of nigral DA neurons in rodents (Gao et al. 2002;Ling et al. 2006;Qin et al. 2007). However, mechanisms through which an acute neuro inflammatory response is transformed into a chronic inflammatory process, and chronic neuro inflammation influences progressive neuro degeneration, require further investigation.
Numerous mutations in multiple genes have been identified in familial PD. α-Synuclein (α-syn) is closely associated with both familial and sporadic PD. Missense mutations (A53T, A30P, and E46K) and multiplications of the α-syn gene (SNCA) cause autosomal-dominant PD (Farrer 2006;Krüger et al. 1998;Polymeropoulos et al. 1997). Disease severity in PD patients with SNCA multiplications positively correlates with gene copy number (Chartier-Harlin et al. 2004;Ibanez et al. 2004;Singleton et al. 2003). Common variability in SNCA has been associated with a greater risk of sporadic PD (Pals et al. 2004). Additionally, age-related increases of α-syn in monkeys and humans are linked to nigrostriatal DA depletion (Chu and Kordower 2007). Furthermore, α-syn aggregates are the primary component of Lewy bodies in both familial and sporadic PD (Spillantini et al. 1997). The occurrence of α-syn-positive inclusions in neurons grafted into the brains of PD patients and a transgenic (Tg) PD model (Desplats et al. 2009;Kordower et al. 2008;Li et al. 2008;Mendez et al. 2008) further emphasizes a pivotal role of α-syn in ongoing disease progression and host-to-graft disease propagation.
The current consensus suggests that PD develops from complex gene-environment interactions. For the present study, we created a two-hit, chronic, progressive model that we used to investigate mechanisms of PD progression. Specifically, we examined how mutant human α-syn and low-grade neuro inflammation inter acted and influenced PD neuro degeneration and disease progression in Tg mice over expressing A53T mutant human α-syn. We intraperitoneally injected a relatively low dose of LPS (3 × 10 6 EU/kg) to induce systemic inflammation that transferred into the brain to form a comparatively mild neuro inflammatory response.

Animals and the LPS injection.
Male homozygous α-syn Tg mice and B6C3F1/J mice [wild-type (WT) controls] were obtained from the Jackson Laboratory (Bar Harbor, ME) and treated humanely and with regard for alleviation of suffering following the NIH Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996). The Tg mice were generated on a C57BL/C3H background and expressed endogenous α-syn and human A53T mutant α-syn driven by a mouse prion protein promoter (Giasson et al. 2002). Seven-month-old WT and Tg mice Background: Mechanisms whereby gene-environment interactions mediate chronic, progressive neuro degenerative processes in Parkinson's disease (PD)-the second most common neuro degenerative disease-remain elusive. oBjective: We created a twohit [neuro inflammation and mutant αsynuclein (αsyn) over expression] animal model to investigate mechanisms through which mutant αsyn and inflamma tion work in concert to mediate chronic PD neuro degeneration. Methods: We used an intraperitoneal injection of the inflammogen lipopolysaccharide (LPS; 3 × 10 6 EU/kg) to initiate systemic and brain inflammation in wildtype (WT) mice and transgenic (Tg) mice over expressing human A53T mutant αsyn. We then evaluated nigral dopaminergic neuro degeneration, αsyn pathology, and neuro inflammation. results: After LPS injection, both WT and Tg mice initially displayed indistinguishable acute neuro inflammation; however, only Tg mice developed persistent neuro inflammation, chronic pro gressive degeneration of the nigrostriatal dopamine pathway, accumulation of aggregated, nitrated αsyn, and formation of Lewy bodylike inclusions in nigral neurons. Further mechanistic studies indicated that 4week infusion of two inhibitors of inducible nitric oxide synthase and NADPH oxidase, major free radical-generating enzymes in activated microglia, blocked nigral αsyn pathol ogy and neuro degeneration in LPSinjected Tg mice. conclusions: Microgliaderived oxidative stress bridged neuro inflammation and αsyn pathogenic alteration in mediating chronic PD progression. Our twohit animal model involving both a genetic lesion and an environmental trigger reproduced key features of PD and demonstrated synergistic effects of genetic pre disposition and environmental exposures in the development of PD. The chronic progressive nature of dopaminergic neuro degeneration, which is absent in most existing PD models, makes this new model invaluable for the study of mechanisms of PD progression.  an intra peritoneal injection of normal saline (NS) or LPS (Escherichia coli 0111:B4; 3 × 10 6 EU/kg). Four-week infusion of 1400W [an inhibitor of inducible nitric oxide synthase (iNOS); 50 μg/kg/hr] and diphenyleneiodonium (DPI; a NADPH oxidase inhibitor; 5 μg/kg/hr) or vehicle (phosphate-buffered saline containing 6% DMSO) via osmotic mini pumps (0.25 μL/hr; 90 μL total capacity; replaced once) was started 30 min before the intra peritoneal injection of NS/LPS. None of the Tg mice presented overt neuro logic impairment at the time of LPS/NS injection. Mice were euthanized at desired time points in an age-matched fashion.
Sequential biochemical fractiona tion and Western blotting. Sequential protein extraction initially removed highly soluble proteins using high-salt (HS) buffer. Proteins from insoluble pellets were then extracted using buffers with increasing solubilization strength: HST buffer (HS buffer containing 1% Triton X-100), RIPA (radio immuno precipitation assay) buffer, and 2% sodium dodecyl sulfate (SDS). For all other biochemical studies, we used the 2% SDS buffer to extract both soluble and aggregated proteins from stratum, SN, or whole brains Giasson et al. 2002).
Statistical analysis. All values are expressed as mean ± SE. Differences among means were analyzed using one-or two-way analysis of variance (ANOVA) with treatment or genotype as independent factors. When ANOVA showed significant differences, pairwise compari sons between means were tested by Newman-Keuls post hoc testing. In all analyses, the null hypothesis was rejected at the 0.05 level.

Results
Delayed progressive nigral neuro degeneration in α-Syn Tg mice, but not WT mice, after LPS challenge. Both α-syn and neuro inflammation are implicated in PD, but it is unclear how their inter action affects chronic progression of PD. Additionally, for unknown reasons, most available α-syn Tg mice fail to develop overt nigral DA neuro degeneration while exhibiting widespread α-syn pathology in the central nervous system (Giasson et al. 2002). We therefore created a chronic, two-hit mouse model to investigate whether inflammation enhances α-syn pathogenic alteration and accelerates neuro degeneration and disease progression. After LPS injection, Tg mice, but not WT mice, developed delayed, chronic and progressive degeneration of nigral DA neurons, as shown by time-dependent decreases in the number of TH-IR neurons ( Figure 1A-D). In particular, 5 months after LPS injection, Tg mice lost 58% of nigral DA neurons ( Figure 1A), whereas their striatal TH levels were reduced by 46% ( Figure 1B). In contrast, neither nigral DA neurons nor striatal TH levels were altered significantly in LPS-injected WT mice compared with NS-injected WT mice. We identified synergistic neuro toxicity of LPS treatment and α-syn over expression when the difference in cell loss between NS-injected WT mice and LPS-injected Tg mice was significantly greater than the sum of cell loss observed in NS-injected Tg mice (i.e., mice with the geno type but not the environmental exposure) and LPS-injected WT mice (i.e., mice with the environmental exposure only). TH immunostaining demonstrated the destruction of nigral DA neurons and striatal DA fibers in LPS-injected Tg mice, whereas the nigro striatal pathway in NS-injected Tg mice and all WT mice remained intact ( Figure 1C,D). The relative selectivity of nigral neuro degeneration was characterized by double-label immuno fluorescence ( Figure 1E). In the SNpc of LPS-injected Tg mice, the number of DA neurons (TH + /Neu-N + ) was decreased by 52 ± 5.6%, whereas non-DA neurons (TH -/Neu-N + ) were relatively spared, as indicated by a 9.2 ± 4.9% reduction. Here, the average number of DA neurons (TH + / Neu-N + ) and non-DA neurons (TH -/Neu-N + ) per SNpc region in the NS-injected Tg was 96 ± 7.4 and 51 ± 9.1, respectively. Moreover, Western blotting ( Figure 5) showed that in LPS-versus NS-injected Tg mice, there was a 53% reduction in nigral TH (indicating loss of DA neurons) and a 20% reduction in nigral Neu-N (indicating loss of DA neurons and probably some non-DA neurons in the SN) 5 months after LPS administration. Consistent with the pattern of neuronal loss observed in PD patients, the destruction of nigral DA neurons was not accompanied by significant damage to DA neurons in the adjacent ventral tegmental area. The numbers of TH-IR neurons in the ventral tegmental area of Tg mice 5 months after the injection were 159 ± 8.19 (NS; n = 4) and 154 ± 5.18 (LPS; n = 4). Collectively, this two-hit PD model reproduced the signature lesion of PD, the relatively selective degeneration of DA neurons in the SN.
Accumulation and pathologic modification of insoluble, aggregated α-syn in LPS-injected Tg mice. In addition to developing progressive neuro degeneration, LPS-injected Tg mice accumulated more α-syn in the whole brain 5 months after injection than did NS-injected Tg mice (Figure 2A). Next, sequential extraction using buffers with increasing solubilization strength detected highly insoluble α-syn in midbrain SDS fractions of Tg mice 5 months after LPS injection. By contrast, midbrain α-syn of NS-injected Tg mice and age-matched WT mice injected with NS/LPS was soluble in HS and HST buffers ( Figure 2B). Unlike midbrains, the cortex and spinal cord of 12-month-old Tg mice displayed age-related accumulation of insoluble α-syn in the absence of any treatment (data not shown), which was in accordance with earlier observations (Giasson et al. 2002). Immunofluorescence analysis revealed accumulation of α-syn-positive aggregates in peri nuclear locations in the SN of LPS-injected Tg mice ( Figure 2C). nSYN514 antibody (specific for nitrated human α-syn) positively stained these aggregates ( Figure 3A). Doublelabel immuno fluorescence demonstrated α-syn aggregates in TH-IR neurons in the SN of LPS-injected Tg mice ( Figure 3B). Moreover, nitrated α-syn accumulated in neurons to form cytoplasmic inclusions ( Figure 3C). In contrast, nigral α-syn immunoreactivity in NS-injected Tg mice was diffuse and barely visible ( Figures 2C and 3A-C). In midbrains of LPS-injected but not NS-injected Tg mice, immunoblotting also detected nitrated human α-syn, which appeared predominantly in HSand HST-insoluble fractions ( Figure 3D,E). Thus, in this two-hit model, LPS-induced inflammation accelerated α-syn accumulation, aggregation, and nitration.

Similar acute systemic and brain inflammatory reactions in WT and Tg mice after LPS injection.
To determine the basis for differential nigral α-syn pathology and neuro degeneration in Tg and WT mice after LPS exposure, we first examined acute inflammation in these mice. Tg and WT mice reacted to LPS in an indistinguishable way. Serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were greatly increased to similar levels after LPS injection in both genotypes, implying that geno type did not alter acute systemic inflammation in response to LPS [see Supplemental Material, Figure 1A (doi:10.1289/ehp.1003013)]. We chose TNF-α and IL-1β as representative indicators of systemic inflammation because both cyto kines are crucial for systemic effects of inflammation, including fever, increased heart rate, and sepsis, and because TNF-α was one of the major pro inflammatory cyto kines responsible for transferring peripheral inflammation to the brain when LPS was intra peritoneally injected into mice (Qin et al. 2007).
The absence of detectable differences between WT and Tg mice in the expression of Mac1 and Iba-1 in the brain 1 day after LPS injection suggests similar acute neuroinflammatory responses in both geno types. Microglia in the SN displayed similar acute morphologic transformation from resting to activated states in both types of mice [see Supplemental Material, Figure 1B Association of α-Syn pathology and progressive nigral neuro degeneration with prolonged neuro inflammation. We next detected chronic nigral neuro inflammation in Tg mice, but not WT mice (Figure 4). Five months after LPS injection, nigral microglia of Tg mice were activated, exhibiting enlarged cell bodies, elevated expression of Iba-1, and increased density in the SN. Microglia of LPS-injected WT mice revealed ramified resting morphology; levels of Iba-1 expression in microglia were similar in LPS-injected and NS-injected WT mice (Figure 4). Five months after LPS exposure, we observed marked up-regulation of multiple inflammatory markers, including Iba-1, Mac1, iNOS, COX-2, and gp91 phox , in both the SN and striatum of Tg mice, indicating prolonged neuro inflammation in these regions ( Figure 5). By repeatedly probing, stripping, and reprobing the same immuno blotting membranes, we observed that the persistent neuroinflammation was negatively correlated with TH levels and positively correlated with α-syn levels in both the SN and striatum of LPSinjected Tg mice ( Figure 5). In concurrence with elevated inflammatory mediators, α-syn levels in the SN and striatum showed 80% and 60% increases, respectively, and TH levels were decreased by approximately 50% (Figure 5). The more pronounced increase in α-syn expression in the SN and striatum (60-80%) than in the whole brain (28%; Figure 2A) of LPSchallenged Tg mice may be attributable, at least partially, to the high density of microglia (Kim et al. 2000) and more marked inflammatory responses in these brain regions.
Unlike LPS-injected Tg mice, NS-injected Tg mice and LPS-injected WT mice failed to develop chronic neuro inflammation (Figure 4), α-syn pathology (Figures 2 and 3), or detectable neuronal loss (Figures 1, 4, and 5) in the nigro striatal pathway. These data together demonstrate that chronic progression of PD was driven by synergistic effects of low-grade neuro inflammation and α-syn dysfunction.
In contrast, neither factor appeared to be sufficient to cause neuronal death in isolation.
Continuous inhibition of iNOS and NADPH oxidase blocked α-syn pathology and nigral neuro degeneration. NADPH oxidase and iNOS are major free radicalgenerating enzymes in activated microglia. The marked up-regulation of iNOS and gp91 phox (the catalytic subunit of NADPH oxidase) in the nigro striatal pathway of LPS-injected Tg mice ( Figure 5A,B) prompted us to hypothesize that microglia-derived free radicals (e.g., superoxide, nitric oxide, and their reaction product peroxy nitrite) caused the nitration of α-syn, which in turn promoted formation of α-syn aggregates and sensitized DA neurons to diverse insults. Inhibition of NADPH oxidase and iNOS by 4-week infusion of DPI and 1400W reduced chronic neuro inflammation  Figure 6A,B,E), blocked nigral α-syn pathology ( Figure 6A-C,E), and prevented dopaminergic neuro degeneration ( Figure 5A,B,D,E) in Tg mice 3 months after LPS exposure. Within the brain, both iNOS and NADPH oxidase are primarily located in microglia. Therefore, the attenuation of Mac1, gp91 phox , and iNOS up-regulation by 1400W and DPI implies that microglia-derived free radicals played a critical role in maintaining chronic neuroinflammation ( Figure 6A,B). The correlation of dampened neuro inflammation with abolished nitration of α-syn, reduced accumulation of insoluble α-syn, and attenuated neuronal impairment is consistent with a causative relationship between inflammation-mediated α-syn pathologic alterations and chronic dopaminergic neuro degeneration.

Discussion
The present study demonstrates that persistent neuro inflammation bridges α-syn pathologic alterations and progressive neuro degeneration in mediating chronic PD progression. The two-hit (neuro inflammation and mutant α-syn overexpression) progressive animal model created for this study mimics PD multifactorial etiology and reproduces the following key features of PD: a) chronic, progressive, and relatively selective degeneration of dopaminergic neurons and fibers in the nigro striatal pathway; b) Lewy body-like neuronal inclusions containing aggregated α-syn; c) persistent neuro inflammation, a common feature shared by all neurodegenerative diseases including PD; and d) a chronic progressive disease course that is absent in most available PD models. These key features of PD were replicated in LPS-injected α-syn Tg mice, but not in NS-injected Tg mice or in LPS-injected WT mice, indicating synergistic effects of environmental exposures and genetic predisposition in the pathogenesis of PD. α-Syn plays a prominent role in PD onset, on going disease progression, and hostto-graft disease propagation. In general, Tg over expression of human α-syn results in widespread α-syn pathologies and variable neuro degeneration in different animal species (Farrer 2006;Lee et al. 2002). Among different α-syn Tg mouse models, only a few exhibit overt degeneration of nigro striatal DA fibers and even fewer show loss of nigral DA neurons (Dawson et al. 2010;Lee et al. 2002;Masliah et al. 2000). The lack of DA neuron loss in most gene-based PD animal models and the dearth of α-syn pathology in most toxin-based PD animal models under score a crucial role of gene-environment inter actions in PD. The low rate of concordance for PD in mono zygotic and dizygotic twins (Marttila et al. 1988;Tanner 2003) strongly suggests the involvement of environmental factors in PD. Epidemiologic and case-control studies have implicated rural living, well water consumption, and pesticide exposure as potential environ mental risk factors for PD (Elbaz et al. 2009). Interestingly, it has been reported that over expression of mutant α-syn in mice protects against (Manning-Bog et al. 2003) or fails to affect paraquat neuro toxicity (Norris et al. 2007), augments neuro degeneration elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and LPS Nieto et al. 2005), or does not enhance rotenone-induced neuro toxicity (Nieto et al. 2005). These findings indicate that inter actions between α-syn and environ mental toxins on PD neurodegeneration require further investigation. Another newly developed two-hit mouse model combining a genetic lesion (Parkin -/-) and neuro inflammation has provided additional experimental evidence supporting a crucial role of gene-environment interactions in PD (Frank-Cannon et al. 2008).
Compared with a previous mouse model, generated by an intra peritoneal injection of a higher dose of LPS (5 mg/kg, equal to ~ 15 × 10 6 EU/kg) into WT mice (Qin et al. 2007), our two-hit model created by a moderately toxic dose of LPS (3 × 10 6 EU/kg) revealed accelerated DA neuro degeneration and provided additional insight into α-syn pathologic alterations under inflammatory states. Specifically, significant loss of DA neurons started to manifest at 7 months after LPS injection in the prior model (Qin et al. 2007) and at 2.5 months in our two-hit model; at these two time points, a 23% and 37% loss of DA neurons was observed, respectively. A Figure 5. Association of neurodegeneration with chronic α-syn accumulation and prolonged neuroinflammation. Five months after LPS injection, Tg mice displayed significant decreases in TH levels, marked up-regulation of multiple inflammatory markers (Mac1, Iba-1, gp91, iNOS, and COX-2), and accumulation of α-syn in the SN (A and B) and striatum (C and D). NS-injected 12-month-old Tg mice did not show overt changes in levels of TH, α-syn, or inflammatory markers, in contrast with 2-month-old Tg mice. Graphs (B and D) show the ratio of densitometry values (mean ± SE) of each examined protein, and β-actin was normalized to each respective NS-injected control. All immunoblot lanes are representative samples from among at least three different animals in each group. *p < 0.05 compared with corresponding NS-injected control mice.  single nigral injection of LPS resulted in acute, dramatic nigral neuro inflammation and DA neuron loss in mutant α-syn Tg mice , but that acute two-hit model did not replicate the chronic, progressive course of PD, and acute, robust, and localized nigral inflammation may not be highly relevant to potential etiologies or pathogenesis of PD. The present study therefore advances research to elucidate a mechanistic link among persistent neuro inflammation, chronic α-syn pathology, and progressive neuro degeneration in chronic PD progression. Persistent activation of iNOS and NADPH oxidase in LPS-treated Tg mice ( Figure 5) and blockage of nitrated α-syn formation by continuous inhibition of these enzymes ( Figure 6) indicated that increased nitric oxide, superoxide, and peroxy nitrite may nitrosylate and/or oxidize α-syn and lower its solubility to form aggregates (Figures 2 and 3). Aggregated α-syn may over whelm the ubiquitin-proteasome system and accumulate over time, leading to endoplasmic reticulum stress and mitochondrial impairment (Cooper et al. 2006). Loss of physiologic function of normal α-syn and oxidative insults from microglia-derived free radicals further exacerbate neuronal damage, eventually causing neuronal death. DA neurons in the SN are known to be uniquely vulnerable to oxidative insults (Jenner and Olanow 1998), and they encounter an excessively high level of oxidative stress during brain inflammation because of the high density of microglia in this region (Kim et al. 2000). The combination of these factors may be partially responsible for the relatively selective degenera tion of DA neurons in the SN in PD.
We have proposed that dying/dead neurons further activate microglia by releasing noxious compounds (e.g., α-syn) into the extra cellular milieu (Gao and Hong 2008). Indeed, monomeric and aggregated α-syn can be released from cultured cells and is recovered from the cerebro spinal fluid of patients with PD (Liu et al. 2009). Exogenous application of aggregated α-syn activates cultured microglia (Zhang et al. 2005). Therefore, ongoing neurodegeneration and chronic neuro inflammation cause a vicious self-propelling cycle that makes it possible for neuro inflammation to persist long after peripheral inflammation has abated, and for neuro degeneration to become a chronic progressive process. Without positive feedback from damaged neurons, the lowgrade acute neuro inflammation induced by LPS was not sustained in WT mice, and consequent effects on α-syn pathology and neurodegeneration were negligible.

Conclusions
Our two-hit progressive model underscores a central role of gene-environment inter actions in the development of PD. The attenuation of nigral α-syn pathology and dopaminergic neuro degeneration by the inhibition of inflammatory enzymes-NADPH oxidase and iNOS-strongly supports a causa tive relationship between inflammation-mediated α-syn pathologic alterations and chronic dopaminergic neuro degeneration. Microglia-derived free radicals were major factors that bridged neuro inflammation and α-syn dysfunction in mediating chronic PD neuro degeneration. Figure 6. Inhibition of iNOS and NADPH oxidase prevent chronic dopaminergic neuro degeneration and α-syn pathology in LPS-injected Tg mice. (A) Immunoblotting analysis of midbrain proteins extracted by 2% SDS buffer indicates that a 4-week infusion of 1400W and DPI blunted up-regulation of inflammatory markers (Mac1, iNOS, and gp91 phox ), attenuated loss of TH, and reduced accumulation of nitrated α-syn in Tg mice 3 months after LPS injection. (B) Densitometry analysis of proteins and β-actin examined in A; Results are mean ± SE normalized to the corresponding NS-injected control. (C) Accumulation of nitrated human α-syn with low solubility in RIPA and SDS fractions from midbrains of Tg mice 3 months after LPS injection, which was blocked by 1400W and DPI. (D) Blockage of chronic degeneration of nigral DA neurons in α-syn Tg mice 3 months after LPS injection by 1400W and DPI. (E) Representative brain images of α-syn Tg mice 3 months after LPS indicate that a 4-week infusion of 1400W and DPI dampened chronic microglial activation, attenuated accumulation of α-syn, and reduced nigral DA lesions. Insets are magnified photomicrographs of corresponding images. In insets, bar = 25 μm. For immuno blotting and immuno staining, n = 4 mice per group. *p < 0.05 compared with corresponding NS-injected control mice; # p < 0.05 compared with corresponding LPS-injected mice.