CtBP Neuroprotective Role in Toxin-Based Parkinson’s Disease Models: From Expression Pattern to Dopaminergic Survival

C-terminal binding proteins (CtBP) are transcriptional co-repressors regulating gene expression. CtBP promote neuronal survival through repression of pro-apoptotic genes, and may represent relevant targets for neurodegenerative disorders, such as Parkinson’s disease (PD). Nevertheless, evidence of the role of CtBP1 and CtBP2 in neurodegeneration are scarce. Herein, we showed that CtBP1 and CtBP2 are expressed in neurons, dopaminergic neurons, astrocytes, and microglia in the substantia nigra (SN) and striatum of adult mice. Old mice showed a lower expression of CtBP1 in the SN and higher expression of CtPB2 in the SN and striatum compared with adult mice. In vivo models for PD (paraquat, MPTP, 6-OHDA) showed increased expression of CtBP1 in the SN and striatum while CtBP2 expression was increased in the striatum of paraquat-treated rats only. Moreover, an increased expression of both CtBP was found in a dopaminergic cell line (N27) exposed to 6-OHDA. In the 6-OHDA PD model, we found a dual effect using an unspecific ligand of CtBP, the 4-methylthio 2-oxobutyric acid (MTOB): higher concentrations (e.g. 2500 µM, 1000 µM) inhibited dopaminergic survival, while at 250 μM it counteracted cell death. In vitro, this latter protective role was absent after the siRNA silencing of CtBP1 or CtBP2. Altogether, this is the first report exploring the cellular and regional expression pattern of CtBP in the nigrostriatal pathway and the neuroprotective role in PD toxin-based models. CtBP could counteract dopaminergic cell death in the 6-OHDA PD model and, therefore, CtBP function and therapeutic potential in PD should be further explored. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-023-03331-w.


Introduction
C-terminal binding proteins (CtBP) are highly conserved proteins whose primary function is to repress transcription [1]. CtBP regulate gene expression by targeting various chromatin-modifying factors to the promoter-bound repressors. The CtBP corepressor complex mediates histone modifications by deacetylation and methylation [1]. In vertebrates, CtBP are encoded by two different genes, CtBP1 and CtBP2, and each produces different isoforms. CtBP share the conserved amino acid motif Pro-X-Asp-Leu-Ser (PXDLS) and the RRTGXPPXL (RRT motif) domains, which are essential to recruiting the core co-repressor machinery [2][3][4], and the dehydrogenase domain, which has an affinity for both nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide hydride (NADH) [5]. CtBP have environmental and metabolic sensing capability, exhibiting oxygen sensing and glycolysis-regulated transcriptional activities. CtBP perform both transcriptional and non-transcriptional functions. In fact, the long splice forms of CtBP proteins (CtBP1/2-L) have co-repression activity and are mainly localized in the nuclear compartment [6,7]. Nevertheless, the smaller isoform of CtBP1 (CtBP1-S) is mainly cytosolic [7]. For example, CtBP1 has been described in synaptic ribbons of sensory neurons and pre-synaptic neuronal terminals [8][9][10] and reported to be important for membranal traffic and Golgi partitioning during mitosis [11]. Likewise, a CtBP2 isoform, called RIBEYE, has also been described as important for forming ribbon synapses of sensory neurons and bipolar cell development [12,13].
Gene knockout of CtBP1 or CtBP2 in mice resulted in severe embryonic defects or lethality, demonstrating their crucial role during development. CtBP1-null mice were 30% smaller although viable, while CtBP2-null mice exhibit embryonic lethal phenotype, and different degrees of combined allele CtBP1 and CtBP2 mutations resulted in differential developmental defects, indicating that CtBP have both unique and overlapping functions [3]. CtBP2 has been linked to cortical development, with its overexpression in cortical cells causing impairments in the migration of neurons [14]. On the other hand, CtBP1 interacts with Hes1 to suppress neurogenesis in the chick dorsal spinal cord [15]. Interestingly, a missense mutation on CtBP1 was found in patients presenting neurodevelopmental deficits (e.g., ataxia, intellectual disability), further supporting the role of CtBP in central nervous system development [16]. Recently, we have demonstrated that CtBP are expressed in subventricular zone neural stem/progenitor cells, and the exposure of neonatal neural stem cell cultures to an unspecific ligand of CtBP, the 4-methylthio 2-oxobutyric acid (MTOB), resulted in enhanced neurogenesis and neuronal complexity as well as increased oligodendrogenesis indicating their potential for regenerative therapies [17].
In the adult mice brain, CtBP1 expression is found in the substantia nigra (SN), forebrain, cerebellum, diencephalon, dorsal thalamus, globus pallidus, ventral pallidum, cerebral cortex, and hippocampus, while CtBP2 is highly expressed in the olfactory bulb, cerebellum, cerebral cortex, and hippocampus [18]. Regarding cell survival, it has been shown in epithelial cells and fibroblasts that CtBP promote downregulation of pro-apoptotic genes, including p53 effector related to pmp-22 (PERP), p21, Bax, and Noxa [19][20][21], while activation of caspase-3, due to ultraviolet irradiation, was more pronounced in Ctbp1-and Ctbp2-null cells [19,22]. CtBP also seem to promote the survival of cerebellar granule neurons and dopaminergic neuron-like cells in vitro [23]. Moreover, the overexpression of CtBP1 in hippocampal and cortical neurons triggers neuroprotection in rat models of Alzheimer's disease [24]. In Huntington's disease, the mutant huntingtin (a hallmark of the pathology) showed less affinity to CtBP than its wild-type counterpart in human fibroblasts, which may cause weaker repression of pro-apoptotic genes [25]. Altogether these data points to a relevant role of CtBP in neuronal survival and development, indicating that these proteins could be valuable targets for developing novel therapeutics against neurological disorders such as PD. To date, no studies have assessed the role of CtBP in PD neurodegeneration or aging in vivo. In this sense, we hypothesized that CtBP could improve dopaminergic survival in PD models. First, the cellular expression (neurons and glial cells) of CtBP was evaluated in the SN and striatum (ST) of adult mice, the most susceptible regions in PD. Then, CtBP protein levels were assessed in the SN and ST in vivo both in physiological conditions (in young adult and old mice) and in experimental models mimicking PD. Then, the effect of CtBP on dopaminergic cell survival was investigated by using a non-specific substrate (MTOB) and siRNAs for each isoform. We first showed the cellular and regional pattern of CtBP expression in the nigrostriatal pathway in healthy young adult and aged mice and in PD preclinical models. Then, we reveal a novel neuroprotective role for CtBP in a 6-OHDA model for PD. Understanding the expression patterns of CtBP in PD neurodegeneration and targeting its function will boost its translation into the clinic.

N27 Cell Cultures and Cell Treatments
The immortalized rat mesencephalic dopaminergic cell line (N27 cells; a kind gift from Dr. Yoon-Seong Kim, Burnett School of Biomedical Sciences, University of Central Florida) was grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) containing 2 g/L sodium bicarbonate, 10% fetal bovine serum (FBS; Millipore) and 1 mL/L of penicillin/streptomycin (GIBCO), in a humidified atmosphere of 5% CO 2 at 37 °C.

MTT Reduction Assay
To assess cell viability, MTT reduction was performed as described previously [27] with some modifications. Briefly, after 24 h of cell treatments, 0.5 mg/mL of MTT (Acros Organics) was added to cells for 4 h at 37 °C. Then, the resultant precipitate was dissolved in 10% SDS and quantified at the wavelength of 570 nm, using a XMark™ Microplate Spectrophotometer (Bio-Rad).

In Vivo Studies
All mice and rats were bred and handled following institutional, national, and European Community guidelines (2010/63/EU). Young adult (2-4 months old) and aged (26 months old) male C57BL/6 mice, as well as male Wistar rats (8-10 weeks old), were housed in appropriate cages under a controlled environment (12 h light/dark cycles and 22 °C) and with ad libitum food and water access.

Paraquat (PQ) Rat Model
The chronic administration of PQ was carried out using osmotic minipumps (Alzet Durect, Cupertino, CA) at a dose of 2.5 mg/kg/day with a fluid delivery rate of 0.25 µL/h for a period of four weeks (Alzet model 2004, large pumps) -PQ group. The animals of the control groups were implanted with a minipump filled with sterile saline, the vehicle used to dissolve PQ. The pumps were implanted subcutaneously on the back, slightly posterior to the scapulae (shoulder blades), after rats were anesthetized with intraperitoneal (i.p.) injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Animals were euthanized 5 weeks after the minipumps were implanted [28].

Tissue Preparation
For western blotting experiments, mice were euthanized by cervical dislocation, while rats were euthanized by i.p. injection of pentobarbital (30 mg/kg). Brains were removed, and the SN and ST were dissected and frozen in liquid nitrogen. The tissue was stored at -80 °C until further processing.
For immunohistochemistry experiments, mice were anesthetized and transcardially perfused with 0.9% NaCl, followed by perfusion with 4% paraformaldehyde (PFA). The brains were collected and further fixed in 4% PFA overnight, followed by soaking in a 30% sucrose solution until sunk. Next, brains were cryopreserved and then coronally sectioned with a thickness of 40 µm using a cryostat (CM 3050S, Leica Microsystems). The slices were kept in an anti-freeze solution (30% ethylene glycol and 30% glycerol in phosphate buffer) until used for immunohistochemistry.

Immunohistochemistry for Tyrosine Hydroxylase (TH)
To assess dopaminergic neuronal survival, we performed TH-positive (TH + ) cell counting as described previously [27,29]. First, the sections were incubated with a blocking solution (0.1% Triton X-100 and 10% FBS in PBS), then the endogenous peroxidases were inhibited with H 2 O 2 and later incubated overnight at 4 °C with the primary antibody mouse anti-TH (1:500, Transduction Laboratories). After several rinses, the slices were incubated with biotinylated goat anti-mouse secondary antibody (1:200, Vector Laboratories) for 1 h at room temperature. Subsequently, the Vectastain ABC kit was added, and the resulting product was visualized by adding DAB to the slices until color developed (5-10 min). Afterward, the sections were counterstained with Nissl (0.25% Cresyl Violet dissolved in acetate buffer) for 4 min, washed in tap water, air-dried, cleaned with xylene, and mounted with Entellan™ (Merck).
Quantification of the TH + neurons in the mice SN was performed in five consecutive coronal sections separated by 240 µm. The SN was carefully delineated, and the number of TH + cells in each condition was counted. Images were acquired under the magnification of 10 × at the Zeiss Axiovert 200 imaging microscope (Axiobserver Z1, Zeiss), and the number of TH + cells was counted using the ImageJ program.

Western Blotting
N27 cells and brain tissue lysates were obtained using RIPA buffer (50 mM Tris, pH = 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and a cocktail of protease inhibitors) and mechanical dissociation. Lysates were then centrifuged, and the supernatant was collected and quantified using a Pierce bicinchoninic acid protein assay Kit (Thermo Scientific) according to the manufacturer's instructions. Then, 40 µg of lysate protein was loaded in a 10% SDS polyacrylamide gel (at 110 V until the samples reached the end of the gel). After electrophoresis and transfer into a polyvinylidene difluoride membrane (1.0 A, 25 V for 25 min at room temperature, using a Trans-blot Turbo System (Bio-Rad)), specific protein bands were detected by using appropriate primary antibodies (mouse anti-CtBP1 and mouse anti-CtBP2, 1:2500; mouse anti-GAPDH, 1:5000, Millipore) and secondary antibodies (goat anti-mouse antibody conjugated with horseradish peroxidase, 1:5000, Santa Cruz Biotechnology) followed by incubation with Luminata Crescendo Western HRP Substrate (Millipore) for 5 min. Protein bands were detected using the ChemiDocTM MP Imaging System (Bio-Rad) and quantified by densitometry analyses using the Image Lab 5.1 software (Bio-Rad Laboratories).

Statistical Analysis
All data are expressed as mean ± SEM of at least three independent experiments performed in triplicate (in vitro) or at least three different animals (in vivo) used for assessing TH-immunoreactivity and CtBP expression levels. Statistical analysis was performed using one-way ANOVA followed by the Dunnett's or Sidak's multiple comparisons test or by unpaired two-tailed Student's t-test. Values of P < 0.05 were considered significant. Statistical analysis was made using the GraphPad Prism 8.0 Software (GraphPad Sotware Inc.).

CtBP1 and CtBP2 Cellular Localization and Expression Levels in the Healthy Adult Mouse Brain
To better understand if CtBP modulation might regulate PD initiation and/or progression, we first performed a comprehensive analysis of the cellular and subcellular localization and expression levels of CtBP1 (Fig. 1) and CtBP2 (Fig. 2) in the SN and ST (most affected areas in PD) of young adult mice. CtBP1 was found to be expressed in all the cell types analyzed in both SN and ST, including dopaminergic neurons (TH + cells; SN), mature neurons (NeuN + cells; ST), microglia (CD11b + cells; SN and ST), and astrocytes (GFAP + cells; SN and ST), as shown in Fig. 1. Not surprisingly, CtBP1 was shown to be majorly expressed in the cell nucleus in both brain regions, although cytosolic expression was also seen. Like CtBP1, CtBP2 was expressed in all the cell types analyzed in SN and ST (dopaminergic neurons, mature neurons, astrocytes, and microglia; Fig. 2). Nevertheless, CtBP2 only displayed nuclear localization.

Discussion
Over the past decades, most information regarding the roles of CtBP1 and CtBP2 focused on embryonic development, oncogenesis, and apoptosis. Overall, studies have shown that CtBP exhibit pro-survival activity in both non-neuronal and neuronal cells [15,23,[34][35][36]. Therefore, CtBP emerge as potential targets for treating neurodegenerative diseases. However, few studies to date in the SN. Data are expressed as a percentage of control ± SEM. The control condition was set to 100%. n = 5-9, *P < 0.05, **P < 0.01 and ****P < 0.0001 when compared to control; ns, non-significant and $ P < 0.05 when compared with 6-OHDA using the one-way ANOVA, followed by the Dunnett's multiple comparison test. c) Representative images of the dopaminergic neurons in the SN of saline mice, 6-OHDA-exposed mice, and 6-OHDA-and MTOB-exposed mice. TH is shown in brown. Scale bar 200 µm have explored the role of CtBP in the development and/or progression of neurodegenerative disorders. In particular, no study has focused on the role of CtBP in PD models. Herein, we first explored the subcellular, cellular, and regional expression levels of CtBP isoforms in vitro (dopaminergic cell line) and in vivo (SN and ST of adult or aged mice) in physiological conditions and toxin-based rodent models of PD. Then, we evaluated the putative neuroprotective effect of CtBP in the 6-OHDA-induced PD model.
Regarding cellular and subcellular expression, we have found that both CtBP were expressed in the neurons, particularly dopaminergic neurons, astrocytes, and microglia both in the ST and SN in vivo. CtBP2 was present almost exclusively in the nucleus, while CtBP1 was present in neuronal and glial processes and also in the nucleus. These findings are in line with previous studies suggesting that both isoforms may overlap but also show distinct subcellular locations [9,18,37]. Several transcription factors, such as BKLF, can bind CtBP1 and sequester it to the nucleus, acting as a transcriptional corepressor [7]. On the contrary, interaction with neuronal nitric acid synthase (nNOS) and increased neuronal activity [9,37] directs CtBP1 towards the cytoplasm and presynaptic terminals, thereby hindering the function of CtBP1 as a corepressor. In the cytoplasm, CtBP play essential roles, such as in Golgi fission and membrane fission [38] and in the regulation of presynaptic neurotransmitter release and synaptic plasticity in neurons [9,10]. Our study contributes to a clearer understanding of the cellular and subcellular expression of both isoforms, particularly in the SN and ST, the most vulnerable regions in PD.
Expression levels of CtBP were also analyzed in the SN and ST of healthy (young adult and aged mice) and toxininjured animals. In the brains of 26-month-old mice (i.e. aged mice), a significant decrease in CtBP1 expression in the SN and a significant increase of CtBP2 expression in the SN and ST were observed. These data suggest that CtBP1 and CtBP2 proteins might have non-overlapping roles in the aged brain. Downregulation of CtBP1 and CtBP2 in a proteasomal-or caspase-dependent way was observed in apoptotic neurons [23]. Interestingly, studies in Caenorhabditis elegans showed that CtBP1 is expressed in their central nervous system through development and adulthood, and its downregulation prolongs the lifespan of these nematodes [39,40]. Therefore, lower levels of CtBP1 in SN may represent a protection mechanism for the dopaminergic neurons that populate the SN and project their fibers into the dorsal ST, which are more susceptible to degeneration or result from neurodegeneration occurring during aging. So far, there is no information about the role of CtBP2 in brain aging, so the interpretation of these data warrants additional experiments that are out of this manuscript's scope. Still, these data are important to open new avenues for studying the role of CtBP in aging.
In all the rodent models of PD assessed (i.e. PQ, MPTP, and 6-OHDA), a significant increase in CtBP1 levels in the SN was observed. Contrary to those observed in aged brains, this increase in PD models suggests a compensatory response to dopaminergic neuronal injury. CtBP1 levels were only augmented in the ST of 6-OHDA-challenged mice, justified probably by the local administration compared with the systemic administration paradigm of the PQ and MPTP models. This observation might also indicate that CtBP display a lower need for compensatory mechanisms in the ST than the SN, which may reflect the mechanisms and extent of neurodegeneration. CtBP2 levels remained unchanged in both SN and ST of MPTP and 6-OHDA mice models, except in the ST of PQ-based rat model, which was found increased. PQ-challenged animals are the only rat model, and they also have a different administration paradigm (chronic) compared to the other two toxins (acute). This model represents an earlier stage of human PD pathology. At week 5, it presents only mild neurodegeneration signals, including alpha-synuclein pathology and mild reduction in striatal dopamine levels [28]. Additionally, PQ is highly toxic and can cause peripheral organ damage, such as liver, kidney, and lung, which induces high lethality in this model [41,42]. The overall central and peripheral toxicity induced by PQ may explain the increased expression of both CtBP1 and CtBP2. Another fact one cannot exclude is differences related to the species [43]. Regarding the in vitro PD model, we have found increased CtBP1 and CtBP2 expression in the rat dopaminergic cell line N27 exposed to 6-OHDA. Interestingly, Stankiewic and collaborators showed that 6-OHDA induced a decrease in CtBP expression in N27 cells in vitro. Stankiewic et al. analyzed both alive and dead N27 cells after exposure to the 6-OHDA showing a significant decrease of CtBP1 and 2 in compromised N27 cells [23]. In the present study, we only consider cells that survived the 6-OHDA treatment, which may explain the contradictory results. Moreover, our in vitro data were also corroborated by in vivo analysis in the SN and ST lesioned regions. Altogether, the increased CtBP expression found in our PD models may suggest a compensatory response to dopaminergic neuronal injury, which seems more specific for CtBP1. Others have reported compensatory mechanisms that occur in the pre-symptomatic stages of the disease, including increased dopaminergic activity, enhanced TH activity and dopamine release in the ST, elevated levels of antioxidant enzymes, among others [44,45]. The pathophysiology and impact of these compensation mechanisms in the progression of the disease remain unclear and poorly investigated. Importantly, CtBP act as metabolic and redox sensors and react upon neuronal activity [9], suggesting its involvement in expression and/or activity in those earlier events. However, a correlation between the metabolic and redox state of the cells and CtBP expression/activity has not been definitively established in the context of neurodegenerative diseases and warrants further investigation. Understanding these mechanisms could lead to the development of disease-modifying therapeutic approaches and should be investigated in the future.
Next, we evaluated the effects of CtBP on dopaminergic survival by using MTOB, a substrate of both CtBP, which so far presents the highest affinity to CtBP due to the presence of a tryptophan residue in CtBP active site, unique to this dehydrogenase [33,46]. It acts as an inhibitor for CtBP at high concentrations (millimolar range), inhibiting the recruitment of CtBP to several target promoters, including pro-apoptotic promoters, and, therefore, decreasing the survival of several tumor cell lines and tumor growth in vivo [35,36]. So far, MTOB was used in brain cells mainly at high concentrations (1-5 mM), leading to apoptosis [17,23]. In a previous report, we showed that low levels of MTOB (up 100 µM) were not toxic to neural stem cell cultures and promoted neuronal differentiation and maturation, and oligodendrogenesis [17], suggesting that CtBP may co-activate genes beneficial for brain regeneration. Herein, we observed a toxic effect of MTOB per se at high concentrations, namely at 1000 µM and 2500 µM. Conversely, MTOB was able to counteract 6-OHDA-induced cell death at low concentrations (50 µM to 250 µM). MTOB at low concentrations can act as a CtBP substrate, as described previously [33], leading to a rapid increase in cell viability, probably by repressing some pro-apoptotic genes [19,21,36,47]. For example, Kim and collaborators have shown that CtBP knockout increases Bax transcription and reduces mitochondrial activities in fibroblast cell lines, suggesting that CtBP are important to maintain mitochondrial activities [21], which is also relevant in the context of PD. In another study, the glycolytic inhibitor, 2-deoxy-D-glucose, suppressed seizure activity in a model of temporal lobe epilepsy via an NRSF/CtBP-dependent repression of the brain-derived neurotrophic factor gene promoter [48]. Recently, it was also shown that overexpression of CtBP1 in Alzheimer's disease rat models induced neuroprotection of hippocampal and cortical neurons and enhanced neuronal activity [24]. Our study supports a neuroprotective role for CtBP in PD, which is corroborated by these previous studies demonstrating that CtBP are key factors for neuronal survival and brain function. Results obtained with low MTOB concentrations were confirmed by CtBP silencing with siRNA, which caused a loss of the neuroprotective effect achieved with these MTOB concentrations. CtBP isoforms have specific but also overlapping functions [3]. Our silencing data obtained in vitro suggest that in these experimental conditions, both isoforms act redundantly, both contributing to the survival of dopaminergic neurons. Nevertheless, one cannot exclude some MTOB effects in a CtBP-independent fashion since it can also interact with other proteins and is part of the methionine salvage pathway. Besides the putative direct effects observed in dopaminergic neurons, we cannot exclude an indirect effect on glial cells in vivo since CtBP1 and CtBP2 are expressed in microglia cells and astrocytes in the ST and SN of healthy mice. These cells participate in immune responses, and both CtBP have been reported to promote the expression of pro-inflammatory genes in primary microglia and astrocyte cultures upon lipopolysaccharide activation [49]. Moreover, Saijo et al. demonstrated that 5-androsten-3β,17β-diol (ADIOL) mediates the recruitment of CtBP to the promoter's region of inflammatory responsive genes together with c-Jun/c-Fos AP1heterodimers, repressing the transcription of these genes [50]. In our paradigm, relatively low MTOB concentrations may also recruit CtBP to repress pro-inflammatory genes and prevent an inflammatory response by microglia and astrocytes. Thus, enhancing CtBP function in both neurons and glial cells may offer a novel and largely unexplored therapeutic target for treating neurodegenerative diseases. This putative anti-inflammatory role of CtBP in PD should be explored in future studies to further evolve into its clinical application in neurodegenerative diseases. In fact, it has been used in the clinic to treat uremic patients [51,52]. So, better knowledge of the expression and function of CtBP in neurodegenerative conditions is essential to better understand the pathophysiology, identify novel therapeutic targets, and advance into clinical translation.

Conclusions
CtBP are expressed in neurons, dopaminergic neurons, astrocytes, and microglia cells in the SN and ST of wild-type adult mice. CtBP showed distinct expression patterns in the SN and ST of young adult, aged, and injured animals. CtBP1 expression in the SN is decreased in aged animals and increased in all toxin-injured animals compared with healthy adult animals. CtBP2 is increased in aged animals in both SN and ST and in the ST of PQ-intoxicated animals. In vitro, both CtBP isoforms showed increased expression in N27 cells treated with 6-OHDA. N27 cells treated with MTOB showed a dual effect: high concentrations inhibited CtBP and induced cell death while low concentrations acted as an agonist, counteracting 6-OHDA-induced cell death. The protective effect mediated by low concentrations of MTOB was confirmed by CtBP siRNA silencing, which led to a loss of the ability to stimulate these proteins and, consequently, hindered MTOB's ability to protect against 6-OHDA-induced cell death. Thus, synthesizing and/or optimizing drugs able to regulate CtBP activity, such as MTOB, could be potentially explored for developing future therapies for PD patients. However, further detailed investigation of the mechanisms mediated by CtBP in PD models, including genetic PD models and human-derived models, and its clinical usefulness is warranted. In sum, our study demonstrates that CtBP are relevant targets for toxinbased PD models, which might translate into future therapeutic targets in PD and other neurodegenerative diseases.