Α-synuclein In Dopaminergic Neurons Research Articles

SARS-CoV-2 Spike Protein 1 Causes Aggregation of α-Synuclein via Microglia-Induced Inflammation and Production of Mitochondrial ROS: Potential Therapeutic Applications of Metformin.

Abnormal aggregation of α-synuclein is the hallmark of neurodegenerative diseases, classified as α-synucleinopathies, primarily occurring sporadically. Their onset is associated with an interaction between genetic susceptibility and environmental factors such as neurotoxins, oxidative stress, inflammation, and viral infections. Recently, evidence has suggested an association between neurological complications in long COVID (sometimes referred to as 'post-acute sequelae of COVID-19') and α-synucleinopathies, but its underlying mechanisms are not completely understood. In this study, we first showed that SARS-CoV-2 Spike protein 1 (S1) induces α-synuclein aggregation associated with activation of microglial cells in the rodent model. In vitro, we demonstrated that S1 increases aggregation of α-synuclein in BE(2)M-17 dopaminergic neurons via BV-2 microglia-mediated inflammatory responses. We also identified that S1 directly affects aggregation of α-synuclein in dopaminergic neurons through increasing mitochondrial ROS, though only under conditions of sufficient α-Syn accumulation. In addition, we observed a synergistic effect between S1 and the neurotoxin MPP+ S1 treatment. Combined with a low dose of MPP+, it boosted α-synuclein aggregation and mitochondrial ROS production compared to S1 or the MPP+ treatment group. Furthermore, we evaluated the therapeutic effects of metformin. The treatment of metformin suppressed the S1-induced inflammatory response and α-synucleinopathy. Our findings demonstrate that S1 promotes α-synucleinopathy via both microglia-mediated inflammation and mitochondrial ROS, and they provide pathological insights, as well as a foundation for the clinical management of α-synucleinopathies and the onset of neurological symptoms after the COVID-19 outbreak.

Preclinical Model to Evaluate Outcomes of Amyloid Cross-Toxicity in the Rodent Brain.

The progress of neurodegenerative disorders correlates with the spread of their associated amyloidogenic proteins. Here, we investigated whether amyloid entry into nonconstitutive neurons could drive cross-toxic outcomes. Amyloid β (Aβ) was stereotaxically introduced into the rodent midbrain tegmentum, where it is not endogenously expressed. Postinfusion, rodent motor and sensorimotor capacities were assessed by standard behavioral tests at 3, 6, 9, and 12 months. The longitudinal study revealed no behavioral abnormalities. However, Aβ insult provoked intraneuronal inclusions positive for phosphorylated α-synuclein in dopaminergic neurons and were seen throughout the midbrain, a pathognomonic biomarker suggesting Parkinson's pathogenesis. These findings not only underscore the cross-toxic potential of amyloid proteins but also provide a mechanism by which they disrupt homeostasis in nonconstitutive neurons and cause neuronal corruption, injury, and demise. This study may help reconcile the large incidence of neurodegenerative comorbidity observed clinically.

Biomarker of Neuroinflammation in Parkinson's Disease.

Parkinson’s disease (PD) is caused by abnormal accumulation of α-synuclein in dopaminergic neurons of the substantia nigra, which subsequently causes motor symptoms. Neuroinflammation plays a vital role in the pathogenesis of neurodegeneration in PD. This neuroinflammatory neurodegeneration involves the activation of microglia, upregulation of proinflammatory factors, and gut microbiota. In this review, we summarized the recent findings on detection of PD by using inflammatory biomarkers, such as interleukin (IL)-1β, IL-2, IL-6, IL-10, tumor necrosis factor (TNF)-α; regulated upon activation, normal T cell expressed and presumably secreted (RANTES) and high-sensitivity c-reactive protein (hsCRP); and radiotracers such as [11C]PK11195 and [18F]-FEPPA, as well as by monitoring disease progression and the treatment response. Many PD-causing mutations in SNCA, LRRK2, PRKN, PINK1, and DJ-1 are also associated with neuroinflammation. Several anti-inflammatory medications, including nonsteroidal anti-inflammatory drugs (NSAID), inhibitors of TNF-α and NLR family pyrin domain containing 3 (NLRP3), agonists of nuclear factor erythroid 2-related factor 2 (NRF2), peroxisome proliferator-activated receptor gamma (PPAR-γ), and steroids, have demonstrated neuroprotective effects in in vivo or in vitro PD models. Clinical trials applying objective biomarkers are required to investigate the therapeutic potential of anti-inflammatory medications for PD.

Role of G Protein-Coupled Receptors in Microglial Activation: Implication in Parkinson's Disease.

Parkinson’s disease (PD) is one of the prevalent neurodegenerative diseases associated with preferential loss of dopaminergic (DA) neurons in the substantia nigra compacta (SNc) and accumulation of α-synuclein in DA neurons. Even though the precise pathogenesis of PD is not clear, a large number of studies have shown that microglia-mediated neuroinflammation plays a vital role in the process of PD development. G protein-coupled receptors (GPCRs) are widely expressed in microglia and several of them act as regulators of microglial activation upon corresponding ligands stimulations. Upon α-synuclein insults, microglia would become excessively activated through some innate immune receptors. Presently, as lack of ideal drugs for treating PD, certain GPCR which is highly expressed in microglia of PD brain and mediates neuroinflammation effectively could be a prospective source for PD therapeutic intervention. Here, six kinds of GPCRs and two types of innate immune receptors were introduced, containing adenosine receptors, purinergic receptors, metabotropic glutamate receptors, adrenergic receptors, cannabinoid receptors, and melatonin receptors and their roles in neuroinflammation; we highlighted the relationship between these six GPCRs and microglial activation in PD. Based on the existing findings, we tried to expound the implication of microglial GPCRs-regulated neuroinflammation to the pathophysiology of PD and their potential to become a new expectation for clinical therapeutics.

Ferruginol prevents degeneration of dopaminergic neurons by enhancing clearance of α-synuclein in neuronal cells

Lewy bodies are characteristic spherical inclusions in Parkinson's disease (PD) that are formed by α-synuclein fibrils. Ferruginol (Fer) is an amonomeric compound isolated from a traditional Chinese herb. Here, we show that Fer exerted potent neuroprotective effects in both in vitro and in vivo PD models. Neuronal cells transfected with A53T mutant (A53T) α-synuclein plasmids and treated with Fer exhibited attenuated the cytotoxicity induced by pathogenic A53T α-synuclein overexpression. Further, when we transfected neuronal cells with siRNA-SNCA (alpha-synuclein) plasmids and incubated them with Fer, the protective role of Fer decreased. We also found that Fer was a potent α-synuclein inhibitor in neuronal cells, which promotes the clearance of αsynuclein in dopaminergic neurons exposed to 1-Methyl-4-phenylpyridinium (MPP +). Fer could inhibit abnormal α-synuclein aggregation and dopaminergic neuron depletion in A53T-Tg mice, suggesting that a role for Fer in α-synuclein accumulation and nigrostriatal pathway injury. Our study revealed that Fer strongly alleviated neurodegeneration by promoting α-synuclein clearance, indicating a neuroprotective role against α-synuclein oligomer-induced neurodegeneration, which makes it a promising candidate for the treatment of PD and other neurodegenerative diseases.

Memantine exerts neuroprotective effects by modulating α-synuclein transmission in a parkinsonian model

Ample evidence has demonstrated that α-Synuclein can propagate from one area of the brain to others via cell-to-cell transmission, which might be the underlying mechanism for pathological propagation and the disease progression of Parkinson's disease (PD). Recent reports have demonstrated cell surface receptor-mediated cell-to-cell transmission of α-synuclein. Memantine decreased the levels of internalized cytosolic α-synuclein and led to attenuation in α-synuclein-induced cell death. Specifically, memantine attenuated α-synuclein-induced expression of clathrin and EEA1, and increased expression of NR2A subunits. Moreover, memantine inhibited propagation of extracellular α-synuclein and thus, decreased the expression of the phosphorylated form of α-synuclein in dopaminergic neurons of the substantia nigra, which was accompanied by increased survival of dopaminergic neurons with functional improvement of motor deficits. The present study demonstrated that memantine modulates extracellular α-synuclein propagation by inhibiting interactions between α-synuclein and NR2A subunits, which leads to neuroprotective effects on nigral dopaminergic neurons against α-synuclein-enriched conditions. The repositioning use of memantine in α-synuclein propagation needs to be further evaluated in patients with α-synucleinopathies as an effective therapeutic approach.

Caveolae formation is critical for fatty acid-binding protein 3-dependent uptake of α-synuclein in dopaminergic neurons

Caveolae formation is critical for fatty acid-binding protein 3-dependent uptake of α-synuclein in dopaminergic neurons

Long-term adenosine A1 receptor activation-induced sortilin expression promotes α-synuclein upregulation in dopaminergic neurons.

Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases. However, the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear. In this study, rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) for five weeks. The mobility of rats was evaluated by forced swimming test, while their cognitive capabilities were evaluated by Y-maze test. Expression of sortilin, α-synuclein, p-JUN, and c-JUN proteins in the substantia nigra were detected by western blot analysis. In addition, immunofluorescence staining of sortilin and α-synuclein was performed to detect expression in the substantia nigra. The results showed that, compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (5 mg/kg) + CPA co-treated rats, motor and memory abilities were reduced, surface expression of sortin and α-synuclein in dopaminergic neurons was reduced, and total sortilin and total α-synuclein were increased in CPA-treated rats. MN9D cells were incubated with 500 nM CPA alone or in combination with 10 μM SP600125 (JNK inhibitor) for 48 hours. Quantitative real-time polymerase chain reaction analysis of sortilin and α-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone, but the combination of CPA and SP600125 could inhibit this expression. Predictions made using Jasper, PROMO, and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents. A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction. After sortilin expression was inhibited by sh-SORT1, expression of p-JUN and c-JUN was detected by western blot analysis. Long-term adenosine A1 receptor activation levels upregulated α-synuclein expression at the post-transcriptional level by affecting sortilin expression. The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind to α-synuclein. Co-immunoprecipitation revealed an interaction between sortilin and α-synuclein in MN9D cells. Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression and α-synuclein accumulation, and dramatically improved host cognition and kineticism. This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan (approval No. AUP#20070090) in March 2007 and the Animals Ethics Committee of University of South China (approval No. LL0387-USC) in June 2017.

Effect of L-DOPA/Benserazide on Propagation of Pathological α-Synuclein

Parkinson’s disease (PD) and related disorders are characterized by filamentous or fibrous structures consisting of abnormal α-synuclein in the brains of patients, and the distributions and spread of these pathologies are closely correlated with disease progression. L-DOPA (a dopamine precursor) is the most effective therapy for PD, but it remains unclear whether the drug has any effect on the formation and propagation of pathogenic abnormal α-synuclein in vivo. Here, we tested whether or not L-DOPA influences the prion-like spread of α-synuclein pathologies in a wild-type (WT) mouse model of α-synuclein propagation. To quantitative the pathological α-synuclein in mice, we prepared brain sections stained with an anti-phosphoSer129 (PS129) antibody after pretreatments with autoclaving and formic acid, and carefully analyzed positive aggregates on multiple sections covering the areas of interest using a microscope. Notably, a significant reduction in the accumulation of phosphorylated α-synuclein was detected in substantia nigra of L-DOPA/benserazide (a dopamine decarboxylase inhibitor)-treated mice, compared with control mice. These results suggest that L-DOPA may slow the progression of PD in vivo by suppressing the aggregation of α-synuclein in dopaminergic neurons and the cell-to-cell propagation of abnormal α-synuclein. This is the first report describing the suppressing effect of L-DOPA/benserazide on the propagation of pathological α-synuclein. The experimental protocols and detection methods in this study are expected to be useful for evaluation of drug candidates or new therapies targeting the propagation of α-synuclein.

Depopulation of dense \u03b1-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson\u2019s disease model

Parkinson’s disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1–120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20–500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b’s function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD.

A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein.

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.

Silica nanoparticles induce alpha-synuclein induction and aggregation in PC12-cells

Silica nanoparticles (SiO2-NPs) are widely applied in diagnosis, imaging, and drug delivery of central nervous diseases. Previously, we found that SiO2-NPs enter the brain and, more specifically, the dopaminergic neurons in the striatum. Whether SiO2-NPs have neurotoxicity and contribute to development of Parkinson's disease (PD) remains unclear. In this study, we investigated the effect of SiO2-NPs on PC12 cells, a dopaminergic neuron-like cell line. We showed that SiO2-NPs up-regulated α-synuclein expression, and N-acetyl cysteine reduced α-synuclein. SiO2-NPs inhibited 20S proteasome activity and decreased ubiquitin, Parkin, and ubiquitin carboxy-terminal hydrolase L1 (UCHL1) protein levels in the ubiquitin-proteasome system (UPS). SiO2-NPs induced autophagy as shown by transmission electron microscopy, and elevated LC3-II and Beclin 1 levels in PC12 cells. SiO2-NPs inhibited phosphorylation of PI3K, Akt, mTOR, and P70S6. These data suggest that SiO2-NPs induce oxidative stress and α-synuclein aggregation by inhibiting the UPS. SiO2-NPs also induce autophagy through inhibiting PI3K-Akt-mTOR signaling, which is known to negatively regulate autophagy. Amyloid aggregates of α-synuclein in dopaminergic neurons of the midbrain are considered the hallmark of PD. Our findings indicate that SiO2-NPs exposure induces neurotoxicity and may be a significant risk factor for the development of PD.

Resveratrol alleviates MPTP-induced motor impairments and pathological changes by autophagic degradation of α-synuclein via SIRT1-deacetylated LC3.

The accumulation of misfolded α-synuclein in dopaminergic neurons is the leading cause of Parkinson's disease (PD). Resveratrol (RV), a polyphenolic compound derived from grapes and red wine, exerts a wide range of beneficial effects via activation of sirtuin 1 (SIRT1) and induction of vitagenes. Here, we assessed the role of RV in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mouse model of PD and explored its potential mechanisms. RV and EX527, a specific inhibitor of SIRT1, were administered before and after MPTP treatment. RV protected against MPTP-induced loss of dopaminergic neurons, and decreases in tyrosine hydroxylase and dopamine levels, as well as behavioral impairments. Meanwhile, RV administration activated SIRT1. Microtubule-associated protein 1 light chain 3 (LC3) was then deacetylated and redistributed from the nucleus to the cytoplasm, which provoked the autophagic degradation of α-synuclein in dopaminergic neurons. Furthermore, EX527 antagonized the neuroprotective effects of RV by reducing LC3 deacetylation and subsequent autophagic degradation of α-synuclein. We showed that RV ameliorated both motor deficits and pathological changes in MPTP-treated mice via activation of SIRT1 and subsequent LC3 deacetylation-mediated autophagic degradation of α-synuclein. Our observations suggest that RV may be a potential prophylactic and/or therapeutic agent for PD.

Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague–Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic–pituitary–adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to alterations in proteins involved in synaptic plasticity.

Emotional memory impairments induced by AAV-mediated overexpression of human α-synuclein in dopaminergic neurons of the ventral tegmental area

Parkinson’s disease (PD) is associated with extensive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta, but neuronal loss is also found in the ventral tegmental area (VTA). The VTA projects to areas involved in cognitive and emotional processes, including hippocampus, amygdala, nucleus accumbens and prefrontal cortex, and has thus been proposed to play a role in emotional memory impairments in PD. Since the formation of α-synuclein inclusions throughout the central nervous system is a pathological hallmark of PD, we studied the progressive effects of α-synuclein overexpression in the VTA on motor functions, emotional behaviour and emotional memory. Adeno-associated viral (AAV) vectors encoding either human α-synuclein or green fluorescent protein (GFP) were injected stereotactically into the VTA, and behaviour was monitored 3 and 8 weeks following AAV injection. At week 8, there was a 22% reduction of TH+ neurons in the VTA. We demonstrate that α-synuclein overexpression in dopaminergic neurons of the VTA induced mild motor deficits that appeared 3 weeks following AAV-α-synuclein injection and were aggravated at week 8. No depressive- or anxiety-like behaviours were found. To address emotional memory, we used the passive avoidance test, a one-trial associative learning paradigm based on contextual conditioning which requires minimal training. Interestingly, emotional memory impairments were found in α-synuclein overexpressing animals at week 8. These findings indicate that α-synuclein overexpression induces progressive memory impairments likely caused by a loss of function of mesolimbic dopaminergic projections.

Neuroprotective effects of mesenchymal stem cells through autophagy modulation in a parkinsonian model

Autophagy is a major degradation pathway for abnormal aggregated proteins and organelles that cause various neurodegenerative diseases. Current evidence suggests a central role for autophagy in pathogenesis of Parkinson's disease, and that dysfunction in the autophagic system may lead to α-synuclein accumulation. In the present study, we investigated whether mesenchymal stem cells (MSCs) would enhance autophagy and thus exert a neuroprotective effect through the modulation of α-synuclein in parkinsonian models. In MPP+-treated neuronal cells, coculture with MSCs increased cellular viability, attenuated expression of α-synuclein, and enhanced the number of LC3-II-positive autophagosomes compared with cells treated with MPP+ only. In an MPTP-treated animal model of Parkinson's disease, MSC administration significantly increased final maturation of late autophagic vacuoles, fusion with lysosomes. Moreover, MSC administration significantly reduced the level of α-synuclein in dopaminergic neurons, which was elevated in MPTP-treated mice. These results suggest that MSC treatment significantly enhances autophagolysosome formation and may modulate α-synuclein expression in parkinsonian models, which may lead to increased neuronal survival in the presence of neurotoxins.

Olfaction in Three Genetic and Two MPTP-Induced Parkinson’s Disease Mouse Models

Various genetic or toxin-induced mouse models are frequently used for investigation of early PD pathology. Although olfactory impairment is known to precede motor symptoms by years, it is not known whether it is caused by impairments in the brain, the olfactory epithelium, or both. In this study, we investigated the olfactory function in three genetic Parkinson’s disease (PD) mouse models and mice treated with MPTP intraperitoneally and intranasally. To investigate olfactory function, we performed electro-olfactogram recordings (EOGs) and an olfactory behavior test (cookie-finding test). We show that neither a parkin knockout mouse strain, nor intraperitoneal MPTP treated animals display any olfactory impairment in EOG recordings and the applied behavior test. We also found no difference in the responses of the olfactory epithelium to odorants in a mouse strain over-expressing doubly mutated α-synuclein, while this mouse strain was not suitable to test olfaction in a cookie-finding test as it displays a mobility impairment. A transgenic mouse expressing mutated α-synuclein in dopaminergic neurons performed equal to control animals in the cookie-finding test. Further we show that intranasal MPTP application can cause functional damage of the olfactory epithelium.

Dysregulated dopamine storage increases the vulnerability to α-synuclein in nigral neurons

Impairments in the capacity of dopaminergic neurons to handle cytoplasmic dopamine may be a critical factor underlying the selective vulnerability of midbrain dopamine neurons in Parkinson's disease. Furthermore, toxicity of α-synuclein in dopaminergic neurons has been suggested to be mediated by direct interaction between dopamine and α-synuclein through formation of abnormal α-synuclein species, although direct in vivo evidence to support this hypothesis is lacking. Here, we investigated the role of dopamine availability on α-synuclein mediated neurodegeneration in vivo. We found that overexpression of α-synuclein in nigral dopamine neurons in mice with deficient vesicular storage of dopamine led to a significant increase in dopaminergic neurodegeneration. Importantly, silencing the tyrosine hydroxylase enzyme – thereby reducing dopamine content in the nigral neurons – reversed the increased vulnerability back to the baseline level observed in wild-type littermates, but failed to eliminate it completely. Importantly, TH knockdown was not effective in altering the toxicity in the wild-type animals. Taken together, our data suggest that under normal circumstances, in healthy dopamine neurons, cytoplasmic dopamine is tightly controlled such that it does not contribute significantly to α-synuclein mediated toxicity. Dysregulation of the dopamine machinery in the substantia nigra, on the other hand, could act as a trigger for induction of increased toxicity in these neurons and could explain how these neurons become more vulnerable and die in the disease process.

Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in aDrosophilamodel of Parkinson’s disease

SUMMARYParkinson’s disease (PD) is the most common neurodegenerative movement disorder. Oxidative stress has been associated with the etiology of both sporadic and monogenic forms of PD. The transcription factor Nrf2, a conserved global regulator of cellular antioxidant responses, has been implicated in neuroprotection against PD pathology. However, direct evidence that upregulation of the Nrf2 pathway is sufficient to confer neuroprotection in genetic models of PD is lacking. Expression of the PD-linked gene encoding α-synuclein in dopaminergic neurons of Drosophila results in decreased locomotor activity and selective neuron loss in a progressive age-dependent manner, providing a genetically accessible model of PD. Here we show that upregulation of the Nrf2 pathway by overexpressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of α-synuclein-expressing flies. Similar benefits are observed upon RNA-interference-mediated downregulation of the prime Nrf2 inhibitor, Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the α-synuclein-induced dopaminergic neuron loss is suppressed by Maf-S overexpression or keap1 heterozygosity. Our data validate the sustained upregulation of the Nrf2 pathway as a neuroprotective strategy against PD. This model provides a genetically accessible in vivo system in which to evaluate the potential of additional Nrf2 pathway components and regulators as therapeutic targets.

Role of Matrix Metalloproteinase 3-mediated α-Synuclein Cleavage in Dopaminergic Cell Death

Evidence suggests that the C-terminal truncation of α-synuclein is equally important as aggregation of α-synuclein in Parkinson disease (PD). Our previous results showed that an endopeptidase, matrix metalloproteinase-3 (MMP3), was induced and activated in dopaminergic (DA) cells upon stress conditions. Here, we report that MMP3 cleaved α-synuclein in vitro and in vivo and that α-synuclein and MMP3 were co-localized in Lewy bodies (LB) in the postmortem brains of PD patients. Incubation of α-synuclein with the catalytic domain of MMP3 (cMMP3) resulted in generation of several peptides, and the peptide profiles of WT α-synuclein (WTsyn) and A53T mutant (A53Tsyn) were different. Combined analysis using mass spectrometry and N-terminal determination revealed that MMP3 generated C-terminally truncated peptides of amino acids 1-78, 1-91, and 1-93 and that A53Tsyn produced significantly higher quantities of these peptides. Similar sizes of peptides were detected in N27 DA cells under oxidative stress and RNA interference to knock down MMP3-attenuated peptide generation. Co-overexpression of cMMP3 with either WTsyn or A53Tsyn led to a reduction in Triton X-100-insoluble aggregates and an increase in protofibril-like small aggregates. In addition, overexpression of the 1-93-amino acid peptide in the substantia nigra led to DA neuronal loss without LB-like aggregate formation. The results strongly indicate that MMP3 digestion of α-synuclein in DA neurons plays a pivotal role in the progression of PD through modulation of α-synuclein in aggregation, LB formation, and neurotoxicity.