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. 2022 Jul 28;17(7):e0272085. doi: 10.1371/journal.pone.0272085

NXP031 prevents dopaminergic neuronal loss and oxidative damage in the AAV-WT-α-synuclein mouse model of Parkinson’s disease

Min Kyung Song 1,2, Levi Adams 1,2, Joo Hee Lee 3, Yoon-Seong Kim 1,2,4,*
Editor: Hemant K Paudel5
PMCID: PMC9333296  PMID: 35901090

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease characterized by inclusions of aggregated α-synuclein (α-Syn). Oxidative stress plays a critical role in nigrostriatal degeneration and is responsible for α-Syn aggregation in PD. Vitamin C or ascorbic acid acts as an effective antioxidant to prevent free radical damage. However, vitamin C is easily oxidized and often loses its physiological activity, limiting its therapeutic potential. The objective of this study was to evaluate whether NXP031, a new compound we developed consisting of Aptamin C and Vitamin C, is neuroprotective against α-synucleinopathy. To model α-Syn induced PD, we stereotactically injected AAV particles overexpressing human α-Syn into the substantia nigra (SN) of mice. One week after AAV injection, NXP031 was administered via oral gavage every day for eight weeks. We found that oral administration of NXP031 ameliorated motor deficits measured by the rotarod test and prevented the loss of nigral dopaminergic neurons caused by WT-α-Syn overexpression in the SN. Also, NXP031 blocked the propagation of aggregated α-Syn into the hippocampus by alleviating oxidative stress. These results indicate that NXP031 can be a potential therapeutic for PD.

Introduction

Parkinson’s disease (PD) is a common neurodegenerative disease. The neuropathological hallmarks of PD consist of the progressive loss of dopaminergic neurons in the substantia nigra (SN) and inclusions of aggregated α-synuclein (α-Syn), called Lewy bodies and Lewy neurites, in neurons [1, 2]. The Braak hypothesis postulates that α-Syn pathology can spread from the gut via the vagus nerve or anterior olfactory nucleus to the brain as the disease progresses [3]. α-Syn aggregates spread widely to the various interconnected brain regions by cell-to-cell propagation [4].

Oxidative stress occurs by an imbalance between reactive oxygen species production and antioxidants, resulting in excessive accumulation of ROS [5]. Increased ROS generates lipid peroxidation, protein modifications, and DNA damage, leading to mitochondrial dysfunction, autophagy deregulation, oxidative DNA injury, and neuroinflammation [6]. Although the exact pathogenesis of PD has not been elucidated, increasing evidence suggests that oxidative stress caused by excessive accumulation of ROS is a critical contributor to neurodegeneration of dopaminergic neurons. Also, ROS has been shown to increase pathological aggregation of α-Syn and neurodegeneration in disorders with Lewy bodies [7–9]. Therefore, antioxidant compounds that can reduce ROS levels and mitigate aggregation are attractive options for potential therapeutic development for PD.

Ascorbic acid (vitamin C) is a natural water-soluble vitamin and plays an essential role as a powerful antioxidant against free radicals [10]. Disappointingly, many studies have reported that vitamin C intake from diet and supplements had no effect on mitigating the risk of PD [11–13]. However, recently the Swedish National March Cohort long-term longitudinal study reported the positive effects of dietary vitamin C intake on PD risk. Participants in the highest tertile of dietary vitamin C showed reduced PD risk compared with those in the lowest tertile [14]. A recent study to confirm the effect of vitamin C in the MTPT-induced PD model demonstrated that vitamin C reduced dopaminergic neuronal loss by alleviating neuroinflammation makers such as microglial responses and astrocyte activation [15]. Additionally, vitamin C concentrations remain high in the central nervous system [16–18], and vitamin C has shown neuroprotective effects in vitro [19, 20]. Although there is still debate about the link between vitamin C and PD, it is clear that vitamin C is one of the excellent potential candidates for treating PD. Despite this, there are significant hurdles in developing Vitamin C-based therapeutic agents for neurodegenerative diseases. Vitamin C is rapidly oxidized and loses its antioxidant activity in the body, and it does not freely cross the blood-brain barrier.

We recently developed a new compound, NXP031, composed of Aptamin C and vitamin C, which may overcome the shortcomings of vitamin C. Aptamin C is a single-stranded DNA aptamer that maintains a stable tertiary structure and binds explicitly to vitamin C, delaying its oxidation. We recently demonstrated that NXP031 prevented nigrostriatal degeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model, suggesting a potential therapeutic intervention for PD [21]. Although this result was encouraging, MPTP-based models do not fully recapitulate α-synucleinopathy seen in PD. Previous work clearly show that AAV-mediated overexpression of either wild-type (WT) α-Syn or PD-related mutants (A30P or A53T α-Syn) in the SN leads to a progressive loss of nigrostriatal dopaminergic neurons and replicates many PD-like pathological features [22–26]. Here, we investigated the neuroprotective effects of NXP031 in this α-Syn overexpression model and confirmed the broad applicability of NXP031’s therapeutic potential.

Materials and methods

Animals

24 male C57BL/6J mice (7 weeks, 18–20 g) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animals were allowed to acclimate to the new environment for a week and were maintained in an environment with a temperature of 22 ± 2°C, the humidity of 50 ± 10% under a 12-h light-dark cycle. Water and food were freely available. Mice were randomly divided into 4 groups (n = 6–8 per group): AAV-Empty + saline group (AAV-GFP group), AAV-Empty + NXP031 group (AAV-GFP + NXP031 group), AAV-WT-α-SYN + saline group (AAV-WT-α-Syn group), and AAV-WT-α-SYN + NXP031 group (AAV-WT-α-Syn + NXP031 group). All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Central Florida (IACUC protocol # PROTO201900005).

NXP031 preparation

Development and characterization of Aptamin C has been previously described [27]. The purified DNA aptamer was obtained from Integrated DNA Technologies (IDT, IA, USA). DNA aptamer was dissolved in folding buffer, 1 mM MgCl2 in 0.01 M PBS, heated in boiling water at 90–95°C for 5 min, and cooled slowly at room temperature (RT) to fold into a tertiary structure. 1mg/ml Aptamin C stock was prepared and dilution with saline to adjust the concentration to 4mg/kg for oral gavage. L-ascorbic acid (ThermoFisher Scientific, MA, USA) was freshly mixed with a DNA aptamer in a ratio of 1:50 (w/w) before oral gavage.

Experimental designs

Recombinant AAV vectors were produced by modification of pAAV-IRES-hrGFP (Agilent) to express wild-type human α-Syn under control of the CMV promoter. Viral particles (AAV2) were produced at the University of Iowa Viral Vector Core according to their standard operating procedures (https://medicine.uiowa.edu/vectorcore/). For viral injection, the mice were deeply anesthetized with 3% isoflurane and placed in a stereotactic instrument (Stoelting, IL, USA). After making a midline incision of the scalp and a burr hole in the skull over the appropriate injection site for the SN, all mice were stereotactically injected unilaterally into the right SN with a microinjector at a rate of 0.25 μl/min with either 2 μl of GFP-only AAV or 2 μl of WT-α-Syn AAV at a concentration of 2 x 1012 viral genomes/mL. The needle was left in place for an additional 8 min before it was slowly withdrawn. Stereotaxic coordinates for the SN were as follows: anteroposterior (AP) -3.0 mm, mediolateral (ML) -1.4 mm, and dorsoventral (DV) -4.4 mm from the skull surface. 1 week following AAV injection, NXP031 (Vitamin C/Aptamin C 200 mg/4 mg/kg, dosage determined in previous studies [21]) was administered to the AAV-GFP + NXP031 and the AAV-WT-α-Syn + NXP031 groups via oral gavage every day for 8 weeks. The AAV-GFP and the AAV-WT-α-Syn groups were orally administered with saline. 8 weeks after NXP031 or saline treatment, rotarod test was carried out. All mice were deeply anesthetized with 3% isofluorane and were sacrificed by transcardial perfusion with saline followed by 4% PFA (Fig 1A).

Fig 1.

Fig 1

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(A) Experimental paradigm. (B) Effect of NXP031 on the rotarod test at 9 weeks after delivery of GFP-only or α-Syn AAV in the SN. Latency to fall time (sec) in the rotarod test. Data are presented as the mean ± S.E.M. (Two-way ANOVA followed by Tukey’s test, *p < 0.05, ***p < 0.001 compared to selected group, n = 6–8 mice per group).

Rotarod test

The rotarod test is widely used to assess the balance and motor coordination of rodents, as previously described [21]. A rotarod machine (Ugo Basile, Coerio, Italy) was used to record the latency time to fall off the rotating rod. All mice were trained on the rotarod for 2 consecutive days at a fixed speed of 10 rpm for 60 s. Mice were received 3 training trials per day with a 1 h inter-trial interval. The next day, mice were tested at an automatically accelerated speed from 0 to 30 rpm. When the mice fell off the rod, the latency time to fall was recorded, up to a maximum of 480 s.

Immunohistochemistry and immunofluorescence

The brain samples were cut at a thickness of 30 μm in the coronal plane on a freezing microtome (CM3050S; Leica, Nussloch, Germany). The brain tissues of the SN and hippocampus were selected, and the free-floating method was used for immunohistochemistry as previously described [28]. First, selected tissues were incubated with 0.3% hydrogen peroxide in 0.01 M PBS for 15 min at RT to quench endogenous peroxidase activity. Next, the tissues were blocked with 1% bovine serum albumin (BSA) in 0.01 M PBS for 1 h to reduce non-specific staining and then incubated with anti-tyrosine hydroxylase (TH, 1:500; sc-25269, Santa Cruz, CA, USA), anti-pS129 (1:200; ab51253, Abcam, MA, USA), anti-4-HNE (1:200; HNE11-S, Alpha Diagnostic International Inc, TX, USA), and anti-phosphorylation of histone H2A.X (γH2A.X) (1:500; 9718S, Cell signaling, MA, USA) diluted in 0.3% Triton X-100 and 0.5% BSA in 0.01 M PBS for overnight at 4°C. To visualize the primary antibody, anti-mouse or anti-rabbit biotinylated secondary antibody (1:500; Vector Laboratories, CA, USA) was applied for 2 h at RT, followed by avidin–biotin complex solution (Vector Elite ABC kit; Vector Laboratories, CA, USA) for 1 h at RT. Finally, the sections were developed with a 3.3′-diaminobenzidine tetrahydrochloride (DAB kit; Vector Laboratories, CA, USA). The stained samples were mounted onto slides, coverslipped, and imaged under an optical microscope (Leica DMi8; Leica, Bensheim, Germany). To quantify pS129-α-Syn, 4-HNE, and pH2A.X staining in the hippocampus, pS129-α-Syn, 4-HNE, and γH2A.X positive cells were counted in 2 sections per mouse. Cell counting was performed blind to the identity of groups by two researchers who did not participate in this experiment. The two researchers’ average values were taken as the representative values.

Fluorescence immunostaining was performed in the same procedure as above for immunohistochemistry. As secondary antibodies, the Alexa 488 conjugated anti-mouse (1:500; Invitrogen, CA, USA) and Alexa 647 conjugated anti-rabbit (1:500; Invitrogen, CA, USA) were used. Tissue sections were then stained with Hoechst (Tocris Bioscience, MO, USA) and cover-slipped for imaging using a Dragonfly 2000 confocal microscope.

Unbiased stereological counting

For unbiased stereological counting, we estimated the number of TH-positive neurons in the SN using Leica DM4B motorized microscope equipped with StereoInvestigator software (MicroBrightField Bioscience, Williston, VT, USA). The total number of TH-positive neurons was estimated according to the optical fractionator workflow probe. Every sixth section (30 μm thickness), from the anterior to the posterior midbrain, was analyzed. The ipsilateral SN was first outlined at low magnification (5x). The counting frame size was set 60 x 60 μm, the grid frame size was set 120 x 120 μm. Top and bottom guard zones were applied to each site, and an optical dissector height of 25 μm was used. Counts for TH-positive neurons were performed at high magnification (20x). An acceptable cell estimation had a coefficient of error (CE) using the Gunderson method (m = 1) less than 0.1.

Nissl staining

The brain tissues of SN were stained with FD cresyl violet solution (FD Neurotechnologies, Inc., MD, USA) and rinsed briefly in distilled water, then differentiated in 95% ethanol containing 0.1% glacial acetic acid for 1 min. The tissues were dehydrated in 100% ethanol for 2 min and placed in xylene, then coverslipped.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software Inc., CA, USA). Two-way analysis of variance (ANOVA) was used in comparing means of all the groups, followed by Tukey’s post-hoc tests for multiple comparisons. All data were presented as means ± S.E.M. The criterion for significance was set at p < 0.05.

Results

NXP031 attenuates motor deficits induced by AAV-mediated overexpression of human α-Syn in the SN

To model dopaminergic degeneration caused by α-synucleinopathy, AAV particles containing human α-Syn or GFP-only control were stereotactically injected unilaterally into the SN of mice. We performed TH immunostaining to check whether there was dopaminergic neuronal loss in the SN 1 week after the AAV-WT-α-Syn injection, and found no dopaminergic neuronal loss (S1 Fig). 1 week after AAV injection, NXP031 or saline was administered via oral gavage every day for 8 weeks. 9 weeks after delivery of AAV, we evaluated motor function using the rotarod test (Fig 1B). A two-way ANOVA revealed that there was a statistically significant interaction between the effects of AAV vector type (GFP-only or WT-α-Syn) and treatment (saline or NXP031) (F1, 24 = 11.00, p = 0.003). Mice in the AAV-WT-α-Syn group showed a shorter latency to fall than the AAV-GFP injected mice (p < 0.001), indicating that α-Syn overexpression led to motor deficits. However, the AAV-WT-α-Syn + NXP031 group exhibited a significant increase in latency to fall compared with the AAV-WT-α-Syn group (p = 0.031). Our finding demonstrates that NXP031 prevented motor dysfunction caused by α-Syn overexpression in the SN.

NXP031 blocks the loss of dopaminergic neurons against AAV-mediated overexpression of human α-Syn in the SN

At 9 weeks post-injection, we checked whether our AAV-GFP titer was neurotoxicity in the SN. The number of TH-positive neurons between the contralateral and ipsilateral sides was compared, showing no significant difference with slightly less number in the ipsilateral SN (S2 Fig) (t = 1.787, p = 0.104). To assess the protective effect of NXP031 on dopaminergic neurons/fibers in the striatum and SN, we performed TH immunohistochemistry (Fig 2A–2D). We observed significant differences in the optical density of TH-stained dopaminergic fiber in the striatum (a main effect of vector group F1, 24 = 50.00, p < 0.001; a main effect of treatment F1, 24 = 4.561, p = 0.043; without a significant interaction) and in the survival of nigral dopaminergic neurons in the SN (AAV vector type x treatment interaction F1, 24 = 21.81, p < 0.001). We observed drastic loss of dopaminergic neurons in the AAV-WT-α-Syn group, showing a 55–60% loss of TH-positive neurons in the SN compared to the AAV-GFP injected mice (p < 0.001). Interestingly, oral gavage of NXP031 for 8 weeks showed a significant protective effect against dopaminergic neuronal degeneration, preserving up to 65% of TH-positive neurons in the AAV-WT-α-Syn + NXP031 group compared to the AAV-WT-α-Syn group (p < 0.001) (Fig 2E). To confirm that the decreased TH immunoreactivity was caused by neuronal loss and not by transient decreases in TH expression changes, we performed Nissl staining (Fig 2F and 2G). We observed a similar reduction in the number of Nissl-positive cells in the SN at 9 weeks after α-Syn overexpression. Together with decreased TH-positive staining, this result indicates dopaminergic neuronal degeneration (AAV vector type x treatment interaction F1, 24 = 8.187, p = 0.009). The result implies that NXP031 efficiently blocked dopaminergic neuronal death caused by α-Syn overexpression in the SN.

Fig 2. Effect of NXP031 on TH-immunoreactivity in the striatum and SN at 9 weeks after delivery of GFP-only or α-Syn AAV.

Fig 2

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(A) Representative micrographs of TH immunostaining in the striatum. (B) The relative optical density of TH-stained dopaminergic fiber in the striatum (C, D) Representative micrographs of TH immunostaining in the SN. (E) The number of TH-positive neurons in the SN. (F) Representative micrographs of double staining with TH antibody and Nissl violet in the SN. (G) % of Nissl-positive neurons compared to the AAV-GFP group. Data are presented as the mean ± S.E.M. (Two-way ANOVA followed by Tukey’s test, *p < 0.05, **p < 0.01, ***p < 0.001 compared to selected group, n = 6–8 mice per group).

NXP031 prevents accumulation and propagation of phosphorylated α-Syn in the brain

Phosphorylation of α-Syn at the serine 129 residue (pS129-α-Syn) is known to be associated with the pathological changes of PD and enhances fibril formation and insoluble aggregation of α-Syn [29, 30]. We performed double immunostaining of TH and pS129-α-Syn, demonstrating that pS129-α-Syn staining is mostly co-stained with TH-positive neurons (Fig 3A). No pS129-α-Syn staining was observed in the AAV-GFP group, while the AAV-WT-α-Syn injected mice displayed pronounced staining of phosphorylated α-Syn in the SN (Fig 3B). However, contrary to our expectations, we observed that pS129-α-Syn levels were reduced in the areas with the highest dopaminergic neuronal loss. As previously reported, severe dopaminergic neuronal loss accounts for concomitant loss of cellular markers such as pS129-α-Syn [31]. However, this made it challenging to simply quantify pS129-α-Syn levels in the SN to assess the effects of NXP031 on α-Syn aggregation.

Fig 3. Effect of NXP031 on pS129-α-Syn expression in the SN and hippocampus after delivery of GFP-only or α-Syn AAV.

Fig 3

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(A) Representative micrographs of double immunostaining of TH and pS129-α-Syn in the SN. (B) Representative micrographs of pS129-α-Syn immunostaining in the SN. (C) Representative micrographs of pS129-α-Syn immunostaining in the hippocampus. (D) The number of pS129-α-Syn-positive cells in the hippocampal DG. (E) The number of pS129-α-Syn-positive cells in the hippocampal CA3. Data are presented as the mean ± S.E.M. (Two-way ANOVA followed by Tukey’s test, **p < 0.01, ***p < 0.001 compared to selected group, n = 6–8 mice per group).

To circumvent this issue, we examined the hippocampus of our mice. Propagation of α-Syn aggregates from the SN to the hippocampus over time is well known [32–34]. Our Nissl staining in the hippocampus showed that hippocampal neuronal loss was minimal even if α-Syn pathology was observed. We found significant differences in pS129-α-Syn levels in the hippocampal dentate gyrus (DG) and cornu ammonis 3 (CA3) regions (DG: AAV vector type x treatment interaction F1, 24 = 4.570, p = 0.043; CA3: AAV vector type x treatment interaction F1, 24 = 9.716, p = 0.005) (Fig 3C). The number of pS129-α-Syn-positive neurons in the AAV-WT-α-Syn group was largely increased compared to the AAV-GFP group (DG: p = 0.002; CA3: p < 0.001). However, oral administration of NXP031 significantly ameliorated the pS129-α-Syn levels in the hippocampal regions compared to the AAV-WT-α-Syn group (DG: p < 0.001; CA3: p < 0.001) (Fig 3D and 3E), and appeared similar to GFP-only (DG: p = 0.988, CA3: p = 0.285). The result suggests that the hippocampal regions are relatively resistant to α-Syn-mediated cell death, and NXP031 can effectively prevent the propagation of α-Syn phosphorylation and aggregation into the hippocampus.

NXP031 blocks oxidative stress and DNA double-strand breaks

4-hydroxynonenal (4-HNE) is a product of lipid peroxidation and is considered a biomarker of oxidative stress [35]. To estimate the oxidative stress induced by α-Syn overexpression, we measured the levels of 4-HNE expression in the SN and hippocampus (Fig 4A and 4B). 4-HNE immunoreactivity was similar to that of pS129-α-Syn. 4-HNE staining was not detected in the SN region with severe dopaminergic neuronal loss, so we examined the hippocampal regions. Two-way ANOVA of the number of 4-HNE positive cells in the hippocampal regions showed a significant difference among the whole experimental groups (DG: AAV vector type x treatment interaction F1, 24 = 6.557, p = 0.017; CA3: AAV vector type x treatment interaction F1, 24 = 5.325, p = 0.030). Compared with the AAV-GFP group, the AAV-WT-α-Syn group showed a dramatic increase in the number of 4-HNE positive cells in the hippocampal regions (DG: p < 0.001; CA3: p < 0.001). NXP031 treatments significantly reduced 4-HNE generation induced by the propagation of aggregated-α-Syn in the hippocampus (DG: p = 0.003; CA3: p = 0.011), with levels similar to control (DG: p = 0.642; CA3: p = 0.276) (Fig 4C and 4D).

Fig 4. Effect of NXP031 on oxidative stress in the SN and hippocampus of mice after delivery of GFP-only or α-Syn AAV.

Fig 4

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(A) Representative micrographs of 4-HNE immunostaining in the SN. (B) Representative micrographs of 4-HNE immunostaining in the hippocampus. (C) The number of 4-HNE positive cells in the hippocampal DG. (D) The number of 4-HNE positive cells in the hippocampal CA3. Data are presented as the mean ± S.E.M. (Two-way ANOVA followed by Tukey’s test, *p < 0.05, **p < 0.01, ***p < 0.001 compared to selected group, n = 6–8 mice per group).

Aside from lipid peroxidation, oxidative stress can also lead to DNA damage. γH2A.X occurs in response to DNA double-strand break formation as an indicator of DNA damage [36]. We investigated the levels of γH2A.X in the SN and hippocampus and found that its levels were well correlated with the 4-HNE staining pattern (DG: AAV vector type x treatment interaction F1, 24 = 4.729, p = 0.040; CA3: AAV vector type x treatment interaction F1, 24 = 7.154, p = 0.013) (Fig 5A and 5B). The AAV-WT-α-Syn group showed a marked increase in the number of γH2A.X-positive cells and stronger immunostaining in the hippocampus than the AAV-GFP group (DG: p < 0.001; CA3: p < 0.001). Administration of NXP031 inhibited DNA damage in the hippocampus, leading to a significant decrease in the number of γH2A.X-positive cells (DG: p = 0.016; CA3: p = 0.003), and was not significantly different than GFP-only (DG: p = 0.477; CA3: p = 0.171) (Fig 5C and 5D). These results suggest that NXP031 prevents oxidative stress and related DNA damage caused by α-Syn aggregate propagation.

Fig 5. Effect of NXP031 on DNA damage in the hippocampus of mice after delivery of GFP-only or α-Syn AAV.

Fig 5

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(A) Representative micrographs of γH2A.X immunostaining in the SN. (B) Representative micrographs of γH2A.X immunostaining in the hippocampus. (C) The number of γH2A.X positive cells in the hippocampal DG. (D) The number of γH2A.X positive cells in the hippocampal CA3. Data are presented as the mean ± S.E.M. (Two-way ANOVA followed by Tukey’s test, *p < 0.05, **p < 0.01, ***p < 0.001 compared to selected group, n = 6–8 mice per group).

Discussion

α-Syn is a central protein in PD pathology. PD is characterized by the loss of dopaminergic neurons in the SN and accumulation of aggregated of α-Syn in dopaminergic neurons [1, 37]. Thus, therapeutic interventions targeting abnormal α-Syn aggregation might be promising in preventing or slowing down the degeneration process of PD. In this study, we investigated the neuroprotective effects of NXP031, a compound combining Aptamin C and vitamin C, under neurodegenerative conditions induced by AAV-mediated overexpression of human α-Syn in the SN.

At 9 weeks after the human WT-α-Syn/AAV-injection into the SN, we assessed the motor function and dopaminergic neuronal survival at the injection site. Consistent with previous studies, AAV-mediated α-Syn overexpression in the SN of mice led to a significant reduction of TH-positive neurons, leading to motor dysfunction [38–40]. To make sure dopaminergic neuronal loss, we also used Nissl staining, a observed a loss of both TH and Nissl staining indicating dopaminergic neuronal degeneration. NXP031 treatments ameliorated motor deficits and dopaminergic neuronal death in the SN. These results support our previous study that NXP031 protects acute degeneration of nigral dopaminergic neurons in the MPTP-induced mitochondrial oxidative stress PD model [21] and expand its application to α-synucleinopathy-based PD models.

α-Syn aggregation is a biomarker for PD [2, 37]. Although dopaminergic neurons in the SN appear to be selectively vulnerable to α-Syn-induced degeneration, α-Syn pathology is not restricted to this region and propagates throughout the brain as PD progresses. In 2003, Braak and colleagues originally proposed the hypothesis that the aggregated α-Syn may start in the digestive tract and spread toward the central nervous system via the vagus nerve [3]. Eventually, the aggregated α-Syn arrives at the SN and extends to other brain areas. pS129-α-Syn is a reliable marker of α-Syn aggregates [41, 42]. We performed pS129-α-Syn immunostaining in the SN to assess the effects of NXP031 on the levels of α-Syn aggregation. AAV-mediated α-Syn overexpression resulted in pS129-α-Syn expression in the injection site. However, as dopaminergic neuronal loss in the SN increases, pS129 staining is concomitantly reduced [33]. This made it difficult to use pS129-α-Syn as an indicator of therapeutic efficacy of NXP031 for α-Syn aggregation in the SN. To circumvent this problem, we investigated other brain regions relatively resistant to α-Syn-mediated cell death. As previous studies have shown that α-Syn aggregates propagate from the midbrain to the hippocampus [32–34], we performed pS129-α-Syn staining in this region. Interestingly, the AAV-WT-α-Syn group showed significantly increased pS129-α-Syn levels compared to the AAV-GFP group even in this more distal region, which was blocked by NXP031 treatments, further highlighting the therapeutic potential of this compound.

Oxidative stress is one of the main factors contributing to dopaminergic neuronal degeneration in PD, inducing oxidative damage to proteins, lipids, and DNA [43, 44]. We then showed that NXP031 blocked oxidative stress induced by α-Syn overexpression in the SN. 4-HNE staining for toxic products of oxidative damage is a widely used biomarker for increased ROS levels, and we found that overexpression of α-Syn led to increased levels of 4-HNE staining. A previous study with immunohistochemical studies has indicated that 4-HNE-modified proteins were significantly increased in nigral melanized neurons in human post-mortem brain PD samples [45]. The A53T-α-synuclein rat model of PD has shown an increase in the 4-HNE staining signal in the SN of the AAV-A53T-a-Syn injected rat [39]. Previous studies are consistent with our results [39, 45], suggesting that oxidative stress may contribute to nigral neuronal cell death. Similar to pS129-α-Syn staining, severe dopaminergic neuronal loss caused by α-Syn overexpression made it difficult to detect 4-HNE in the SN. Alternatively, we found a significant increase in 4-HNE levels in the hippocampus caused by α-Syn aggregation’s propagation into the hippocampus. We observed that NXP031 treatments prevented the propagation of aggregated α-Syn into the hippocampus, resulting in a decreased level of 4-HNE expression in the hippocampus.

DNA double-strand breaks are the most harmful DNA damage, triggering activation of phosphorylation of the histone variant H2A.X, a significant component of DNA damage response [36]. In two different synucleinopathy models of PD, DNA damage markers such as γH2A.X, 53BP1, and pATM were upregulated, demonstrating that α-Syn-mediated oxidative stress results in DNA double-strand breaks [46]. We obtained γH2A.X staining results similar to 4-HNE staining in the SN and hippocampus. While PD has strong connections with reactive oxygen species, other neurodegenerative conditions such as Alzheimer’s disease and multiple amyotrophic lateral sclerosis have also been shown to have oxidative damage components. It will be worthwhile to investigate the potential of this compound on those conditions in future studies.

It is interesting to note that the oral route of administration of NXP031 exerts therapeutic effects in PD model. As increasing evidence suggests the critical contribution to the gut microbiome to PD pathogenesis, it will be interesting to investigate the effects of NXP031 on the intestinal flora and their metabolism. Combined with our previous study demonstrating that NXP031 can significantly prevent dopaminergic neuronal degeneration induced by the MPTP-induced PD model, the current study provides a strong foundation for deeper investigation into the potential benefits of this novel therapeutic strategy for PD. This promising initial result based on motor function and histological results is exciting, and in future studies we will expand our scope to include behavioral and cognitive tests. It will be interesting to see if similar protection can be seen against other PD symptoms such as cognitive decline.

In conclusion, our results demonstrate the neuroprotective promise of NXP031 in the AAV-WT-α-Syn mouse model of PD by preventing dopaminergic neuronal loss in the SN and inhibiting the propagation of α-Syn pathology further into the other brain regions through reducing oxidative stress. Thus, we suggest that NXP031 could represent a prospective therapeutic strategy for PD.

Supporting information

S1 Fig. Assessing dopaminergic neurons in the SN 1 weeks after delivery of AAV-WT-α-Syn.

Representative micrographs of TH immunostaining in the SN.

(TIF)

Click here for additional data file. (1.2MB, tif)
S2 Fig. Comparison of TH-positive neurons between the contralateral and ipsilateral sides in the SN at 9 weeks after delivery of GFP-only AAV.

(A) Representative micrographs of TH immunostaining in the SN. (B) % of TH-positive neurons in the SN compared to the contralateral side. Data are presented as the mean ± S.E.M. (Student t-test, n = 6 mice).

(TIF)

Click here for additional data file. (1.1MB, tif)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was fully supported by the Nexmos Co., Ltd., Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Hemant K Paudel

29 Mar 2022

PONE-D-22-01833NXP031 reduces dopaminergic neuronal loss and oxidative damage in the AAV-WT-α-synuclein mouse model of Parkinson’s diseaPLOS ONE

Dear Dr. Kim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Reviewers find that your manuscript is interesting and may have therapeutic potential. However, they raised several issues that need your attention. Data from the striatum, where aSyn injected in substantia nigra is eventually transported. aSyn aggregation and  some antioxidant response markers in the substantia nigra.In addition you need to demonstrate how far the AAV-alpha synuclein spread throughout the substantia nigra. You also needed to remove a group of animals 1 week after the AAV-a-syn infusion, and prior to the start of the NXP treatment, to determine if there was any loss of TH labeled neurons in the SNpc.  There are a  number of other comments made by reviewers.

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Reviewer #1: In the current manuscript, Kim et al. describe the effects of novel vitamin C derivative, NXP-031, on a Parkinson's disease mouse model based on AAV-alpha-synuclein injection on substantia nigra. Although the results are interesting, particularly the protective effect of NXP-031 on TH+ neuron count, there are several issues that needs to be addressed.

- My main concern is that the authors do not show any data from the striatum, where aSyn injected in substantia nigra is eventually transported? That is anyway the equally important area (or in early disease even more important) as substantia nigra in Parkinson’s disease pathology, and most of the endogenous aSyn is located in presynaptic area. I do understand hippocampus as one of the projection areas of substantia nigra where aSyn is also seen but why the authors did not study striatum and e.g. motor cortex that are more important for locomotor deficits. Moreover, as the authors use unilateral model of AAV-aSyn, a model measuring unilateral deficits e.g. cylinder test or staircase test would be probably more informative than rotarod assay, and gives usually more information on striatal damages than rotarod assay.

- Additionally, the authors do not provide any information about the aSyn aggregation itself, particularly when the pS129 aSyn did not show any reliable staining in the substantia nigra. If they consider that the mechanism-of-action for NXP031 is antioxidant mechanism, they should also show some antioxidant response markers in the substantia nigra, e.g. SOD1, catalase.

- The authors should also revise the manuscript more carefully, now there are several mistakes that make the interpretation of the results difficult: Figure 3, there are no details in the materials and methods about the pS129 aSyn analysis and that should be added. How many sections and brains were counted etc. The staining figures of pS129 aSyn also look puzzling. Why there is high amount of staining in the substantia nigra pars reticulata? I have some doubts about the antibody specificity, it appears to detect either normal aSyn as well or then some other unspecific protein. Moreover, Figure 5 should present the immunostaining of phosphorylated H2A in the hippocampus but it appears to present a Nissl staining? This needs to be corrected.

Other comments:

Introduction:

Page 3: “α-Syn aggregates spread from the gut via the vagus nerve or anterior olfactory nucleus to the brain as the disease progresses [3].” This is indeed seen in the experimental animals and certain support in clinical disease comes from Braak staging (2003) but I would still avoid such strong statements on this as aSyn may well start aggregating in brain neuronal cells without propagating species.

Page 3.” Additionally, vitamin C concentrations remain high in the central nervous system [15].” In the refence 15 (Nualart et al.) there were no neuronal cells used, and the publication is completely performed by using cell cultures and primary cells. How the authors can claim based on this reference that vitamin C concentrations remain high in the CNS?

Page 3. Several studies have shown that dietary supplements of vitamin C are not protective for PD, and authors should be careful not to draw conclusions about the beneficial effects of vitamin C on PD based on one study that has also raised some concerns (Tomoyuki Kawada; Reader Response: Dietary Antioxidants and the Risk of Parkinson Disease: The Swedish National March Cohort, 2021).

Materials and methods:

Page 4-5: Why there is no AAV-empty + NXP031 group? AAV-GFP can also be toxic for the TH+ neurons (Albert et al. J Neurosci Res 2019), was this seen in the GFP-group?

Page 5-6: How does the saline correlate with NXP031 solvent? Was NXP031 also dissolved to saline in the end?

Page 8: TH+ neuron count. More details are needed. Was stereology taken into account as the authors used 30 um sections?

Results:

Figure 4. The authors state that they didn’t detect 4-HNE staining in SNPc but in the Figure 4, there is clearly some staining visible. Why this was not analyzed or did the authors conclude that this is not specific?

Discussion:

TH loss does not always refer to loss of DAergic neurons, and this is also visible in Suppelementary Figure S1. This should be taken into account in the discussion.

Reviewer #2: March 22, 2022

Song et al report that 1 week following unilateral infusion of AAV-a-syn into the SN, daily oral administration of NXP031 for the next 8 weeks is neuroprotective in terms of blocking the alpha synuclein-induced motor dysfunction as determined using the rotorod, and blocking the alpha synuclein-induced loss of TH neurons in the SNpc. Although there was no evidence that NXP blocked the aggregation of alpha synuclein within the SNpc, since the aggregation was apparently located in non-neuronal cells, the authors quantified the aggregation within the hippocampus. This alpha synuclein-induced aggregation was blocked by prior administration of NXP within both the DG and CA3. Only within the hippocampus did NXP block the accumulation of 4-HNE, a marker of oxidative stress, and H2A.X (histone phosphorylation). Since this is a neuroprotective study, it is more appropriate to use the term, blocked, versus ameliorated. Although the aggregation extended from the SN to the hippocampus, the authors provide no behavioral data to suggest this aggregation was deleterious, which is a major limitation of this study. Also, NXP blocked the aggregation of alpha synuclein and did not reduce the aggregation. 'Reducing' implies that there was previous aggregation but this is incorrect, since this was a neuroprotective study design. In addition, since here are no prodromal biomarkers that can diagnose PD years prior to the emergence of the motor dysfunction, a neuroprotective study design is not translational to the clinic at this time. Therefore, the authors have over-interpreted their findings that NXP is a potential therapeutic for PD.

1.) Abstract: based on the comments above, the authors need to change the wording of the abstract (ie., reduced) and decrease the emphasis of the last sentence.

2.) Materials: Animals: the authors need to include an NXP only treated group that was originally infused with the AAV-empty vector group to determine if the drug has its own effects. Therefore, the 3 groups analyzed in this study is incomplete. Was the saline also administered orally? This should be included.

3.) Experimental design: The authors need to demonstrate how far the AAV-alpha synuclein spread throughout the SN. Using the GFP-only AAV, this needs to be carried out. The concern of this reviewer is that the authors needed to remove a group of animals 1 week after the AAV-a-syn infusion, and prior to the start of the NXP treatment, to determine if there was any loss of TH labeled neurons in the SNpc. This group needs to be included.

4.) Rotorod test: when did the animals undergo the 3 days of training sessions? Assume prior to the start of the AAV-infusion? Please clarify.

5.) Immunohistochemistry: although it was later explained that following the infusion of AAV-a-syn, the protein is distributed/transported to various regions, including the hippocampus, a clear experimental design flaw in this study was that there were no behavioral experiments to determine if the aggregation, increase in ROS and double stranded breaks in the hippocampus lead to a deficit in learning/memory. Considering the authors carried out rotorod testing, was there aggregation within the motor cortex? The hippocampal data, although of interest, has little to do with the neuroprotective effects in the SNpc.

For the SNpc/TH analysis, was the entire rostral-caudal extent of the SN sectioned and analyzed? This is important since it's not clear how far the AAV infusion spread along the SN and whether the infusion spread outside the SN. For the hippocampus, how many sections were analyzed?

6.) TH-positive cells counting: please provide more detail as to how the cell count was carried out. Was this done stereologically?

7.) Statistical analysis: please include the Degrees of Freedom in your ANOVA. In addition, with the needed inclusion of a 4th experimental group (VEH+NXP), the authors will need to carry out a 2-way ANOVA.

8.) Where is the Figure Legend for Figure #1? Please include.

9.) Line 191: were there rotorod motor difficulties 1 week following the infusion of AAV-a-syn but prior to the start of the NXP treatment? That would be important data to know.

10.) Line 200: similar to the comment in #9 above, how much loss of TH neurons were there 1 week after AAV-a-syn infusion but prior to the start of NXP treatment. Also please change the heading of that section to 'NXP blocks ....'.

11.) Line 206: in the S1 Figure, since these are double stained sections for TH and CV, the authors need to quantify the number of TH negative/CV positive neurons and not just show the photos. In the S1 figure, far right photographs, it appears there is loss of TH+ cells on the contralateral (left) side of the SNpc. The authors now need to also quantify the number of TH+ neurons on the contralateral side since it appears that there was possibly spread of the AAV-a-syn to the contralateral SN. In this S1 figure, the authors need to show a photo of the vehicle only control group.

12.) Line 224: the authors need to double label for p-129 and TH to determine if the p-129 is located in non-dopamine neurons.

13.) Line 230: as mentioned before, the lack of any learning/memory correlate to the increased aggregation in the DG/CA3 limits the interpretation of this finding and has little to do with the main theme of this study, which was to determine the effects of the NXP treatment in terms of PD.

14.) Line 239: Because no learning/memory behavioral studies were carried out, the authors cannot say that there was no toxicity associated with the aggregation. Toxicity does not necessarily imply cell loss. Aggregation within neurons in the DG/CA3 may have affected the function of the cells, which some would interpret as a toxic effect. This line needs to be changed or eliminated. The same goes for line 321 in the discussion section. This line needs to be changed or eliminated.

15.) Figure 3A: In the far right photo showing the effects of alpha synuclein, how do the authors know this is the SNpc? The authors need to show a much higher magnification. Since this reviewer is a basal ganglia morphologist, it's difficult to determine the exact location of the labeled cells. They actually appear to be dorsal to the SNpc. In addition, what cell type is labeled with p-129 in these photos?

16.) Line 250: need to change, suppresses, to 'blocks'. Also in Figure 4, there was no 4-HNE staining of the SN, it appears that the loss of TH cells following AAV-a-syn has nothing to do with oxidative stress or DNA double breaks. Please comment in the discussion section.

17.) In Figure 4 and Figure , how many hippocampal sections were taken for the cell counts? Did these sections cover the entire rostral-caudal extent of both the DG and CA3? This information should be included in the methods section. For Figure 5, where is the SN data?

18.) Discussion section: Page 16: As noted in comment #16 above, the authors need to provide an explanation as to their hypothesis as to how the dopamine cells are dying since it does not appear to be associated with either increased ROS or DNA double strand breaks. This is why a group of mice should have been removed from the study at the end of the first week following AAV-a-syn infusion, since it's possible the remaining SNpc neurons might have labeled for both markers. So how does NXP block the a-syn induced loss of DA/TH cells if not via blocking ROS or DNA double strand breaks? The authors should have carried out TH IHC in the striatum since it's highly possible that there might have been sprouting of new DA terminals in the striatum due to NXP treatment.

19.) Conclusion: the authors need to note the blocking of the aggregation was in the hippocampus and not the SNpc and that they need to tone down the idea that is drug could be used as a therapeutic since this was a neuroprotective study design.

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Reviewer #1: Yes: Timo Myöhänen

Reviewer #2: No

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PLoS One. 2022 Jul 28;17(7):e0272085. doi: 10.1371/journal.pone.0272085.r002

Author response to Decision Letter 0

26 May 2022

We appreciate the constructive comments made by reviewers and giving us the opportunity for revision. We have revisited the entire manuscript and thoroughly revised the manuscript accordingly, and we are confident that our manuscript satisfies reviewers’ concerns and is greatly improved.

Prior to point-by-point answers to each question raised by reviewers, we would like to clarify the AAV-WT-α-Syn PD model that we used in this study. We noticed common concerns regarding hippocampal vs. nigral pathology and related issues. The primary aim of using this model is to test if NXP031 effectively reduces or blocks α-Syn aggregation-induced dopaminergic (DA) neuronal loss in the SN (not hippocampal dysfunction or loss). To assess the therapeutic efficacy, we measured 1) the number of TH-positive neurons in the SN, 2) pS129-α-Syn as a marker for α-Syn aggregation, and 3) 4-HNE as oxidative lipid peroxidation, and 4) γH2A.X as a DNA double-strand break. DA neuronal loss in the saline ingested group was significantly obvious compared to the NXP031 treated group. This evident DA neuronal loss invited difficulty in measuring other pathology markers (2-4) as all markers express inside cells and disappear with degeneration. Therefore, we investigate the hippocampus as an alternative region for two reasons: 1) propagation of α-Syn aggregates from the SN to the hippocampus over time is well documented (ref below), 2) hippocampal neuronal loss is minimal even though α-Syn pathology is observed (shown in our nissl staining in the hippocampus in Fig. 5). In the hippocampus, we were able to measure changes in pathologic markers between groups, confirming NXP031 blocked lipid peroxidation, α-Syn aggregation, and DNA damage. As these pathologic markers were also similarly detected in some remaining neurons in the SN (Figure below), what we observed in the hippocampus could indicate the neuroprotective mechanisms underlying how NXP031 prevents α-Syn-mediated DA neuronal degeneration.

We have attached the answer to each reviewer's question as a word file.

Attachment

Submitted filename: Response to reviewers - Final.docx

Click here for additional data file. (18.3MB, docx)

Decision Letter 1

Hemant K Paudel

21 Jun 2022

PONE-D-22-01833R1NXP031 prevents dopaminergic neuronal loss and oxidative damage in the AAV-WT-α-synuclein mouse model of Parkinson’s diseasePLOS ONE

Dear Dr. Kim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewer has asked some clarification of figures and grammar corrections. 

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Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All points I have raised, have been responded by the authors. The manuscript can be accepted for publication.

Reviewer #2: June 19, 2022

Song et al have significantly revised their manuscript but this reviewer has a few more concerns that need to be addressed:

1.) Supplemental Figure 1: this reviewer found it of interest that following AAV-a-syn infusion into the SNpc, that only about 50% of the SNpc neurons were GFP labeled. I am assuming the 50% loss of TH cells after 9 weeks in the SNpc following alpha synuclein infusion were only those cells containing the alpha synuclein? Since the TH stereology was carried out throughout the entire rostral-caudal extent of the SNpc, the authors need to determine the percentage of SNpc neurons containing the alpha synuclein after just one week (since it was shown that there was essentially no loss of TH neurons at this early time period). Was the alpha synuclein induced loss of TH cells uniform throughout the entire SNpc or, since there is most likely complete loss of TH in the immediate area of the viral infusion, as essentially stated by the authors, was there significantly less loss further away from the injection site? These data are important in terms of interpreting the protective effects of the NXP031 drug administered. Since there was about a 50% loss of TH cells in this study, and if there was nearly 100% loss at the site of the infusion, this suggests that further away from the injection site, there must have been nearly no loss of TH cells. This needs to be clarified.

2.) Line 139: the authors state the animals were trained for 2 days before the start of the measurement. This reviewer assumes the mice were trained after the 9 weeks of AAV infusion? If that is the case, please state it more specifically. Initially the authors had stated the mice were trained for 3 days. Which is correct? Please clarify.

3.) Figure 2F/G: the legend states (line 244/245) the following: Representative micrographs of double staining with TH antibody and Nissl violet in the SN. (G) % of Nissl-positive neurons compared to the AAV GFP group. For this analysis, this reviewer assumes that the authors counted both TH positive and Nissl only positive cells, or were just the Nissl positive and TH NEGATIVE cells counted? In the original question asked by this reviewer, Point #11, only the Nissl positive and TH NEGATIVE cells should be counted in order to determine if there was a change in those numbers. The reason for this request is that this author has reported several times that following loss of nigrostriatal TH cells, there was an increase in the number of TH negative/Nissl positive cells in the SNpc, suggesting that some of the original TH positive cells had not died but simply were not expressing the TH protein at the moment. This reviewer has interpreted the data from Figure 2G as the authors counting all TH positive/Nissl stained cells. Please clarify.

4.) This author is still not convinced as to why the hippocampal data are included without any behavioral testing. The fact that there was no hippocampal cell loss, but there was still significant aggregation/increased p-alpha synuclein labeling, suggests to this reviewer that learning/memory deficits are most likely present. Of interest, there are recent data suggesting that during the training of a learning/memory task, there is an actual increase in DSBs in the hippocampus, suggesting that such breaks and increased repair are essential to the learning of certain tasks. Therefore, the testing of these alpha synuclein-infused mice in a L&M task is even more important. Why didn't the authors investigate p-alpha synuclein labeling in the striatum? This reviewer has found, using essentially the same AAV-alpha synuclein viral infusion into the SN, a clear increase in p-alpha synuclein in the striatum. Since the SNpc projects to the striatum, as it appears to also do to the hippocampus, it would have made more sense to investigate the striatum. Please comment.

5.) There are still some minor grammar issues that need to be corrected.

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2022 Jul 28;17(7):e0272085. doi: 10.1371/journal.pone.0272085.r004

Author response to Decision Letter 1

27 Jun 2022

We appreciate the constructive comments made by reviewers and for giving us the opportunity for revision. We have revisited the entire manuscript and thoroughly revised the manuscript accordingly, and we are confident that our manuscript satisfies reviewers’ concerns and is greatly improved. We performed grammar corrections mentioned by the reviewer and carefully conducted the reference check journal requirements.

We have attached a file to the response to the reviewer.

Attachment

Submitted filename: Response to reviewers 2nd.docx

Click here for additional data file. (1.7MB, docx)

Decision Letter 2

Hemant K Paudel

13 Jul 2022

NXP031 prevents dopaminergic neuronal loss and oxidative damage in the AAV-WT-α-synuclein mouse model of Parkinson’s disease

PONE-D-22-01833R2

Dear Dr. Kim,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hemant K. Paudel

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Hemant K Paudel

19 Jul 2022

PONE-D-22-01833R2

NXP031 prevents dopaminergic neuronal loss and oxidative damage in the AAV-WT-α-synuclein mouse model of Parkinson’s disease

Dear Dr. Kim:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Hemant K. Paudel

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Assessing dopaminergic neurons in the SN 1 weeks after delivery of AAV-WT-α-Syn.

    Representative micrographs of TH immunostaining in the SN.

    (TIF)

    Click here for additional data file. (1.2MB, tif)
    S2 Fig. Comparison of TH-positive neurons between the contralateral and ipsilateral sides in the SN at 9 weeks after delivery of GFP-only AAV.

    (A) Representative micrographs of TH immunostaining in the SN. (B) % of TH-positive neurons in the SN compared to the contralateral side. Data are presented as the mean ± S.E.M. (Student t-test, n = 6 mice).

    (TIF)

    Click here for additional data file. (1.1MB, tif)
    Attachment

    Submitted filename: Response to reviewers - Final.docx

    Click here for additional data file. (18.3MB, docx)
    Attachment

    Submitted filename: Response to reviewers 2nd.docx

    Click here for additional data file. (1.7MB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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