Α-synuclein Knockdown Research Articles

MALAT1 Mediates α-Synuclein Expression through miR-23b-3p to Induce Autophagic Impairment and the Inflammatory Response in Microglia to Promote Apoptosis in Dopaminergic Neuronal Cells.

Parkinson's disease (PD) is a very common neurodegenerative disease that adversely affects the physical and mental health of many patients, but there is currently no effective treatment. To this end, this study focused on investigating the potential mechanisms leading to dopaminergic neuronal apoptosis in PD. Rotenone induces damage in dopaminergic neuronal MN9D cells. Apoptosis was detected by flow cytometry, and the expression of apoptosis-related proteins was detected by western blot. RT-qPCR was used to detect the expression of MALAT1 and miR-23b-3p. The expression of α-synuclein was detected by ELISA. A dual luciferase gene reporter assay was used to determine the targeted regulatory relationship between MALAT1 and miR-23b-3p and miR-23b-3p and α-synuclein. MN9D supernatant was cocultured with BV-2 cells, or BV-2 cells were treated with exogenous α-synuclein and then treated with an autophagy inhibitor (3-MA) and autophagy activator (RAPA). The expression of α-synuclein in BV-2 cells was detected by immunofluorescence. The expression of MIP-1α, a marker of microglial activation, was detected by ELISA. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The expression of proinflammatory cytokines was detected by ELISA. Western blotting was used to detect the expression of autophagy-related proteins. Apoptosis of MN9D cells was detected after coculture of BV-2 supernatant with MN9D. The expression of MALAT1 and α-synuclein was upregulated, while the expression of miR-23b-3p was downregulated in damaged MN9D cells, resulting in cell apoptosis. MALAT1 can negatively regulate the expression of miR-23b-3p, while miR-23b-3p negatively regulates the expression of α-synuclein. α-synuclein can enter BV-2 cells through cell phagocytosis. Coculture of BV-2 cells with α-synuclein or with MN9D supernatant overexpressing MALAT1 resulted in a decrease in the autophagy level of BV-2 cells and an inflammatory reaction. However, miR-23b-3p mimics and knockdown of α-synuclein reversed the effect of MALAT1 on autophagy and the inflammatory response of BV-2 cells. In addition, after coculture of BV-2 cells with α-synuclein, the level of autophagy further decreased when 3-MA was added, while the opposite result occurred when RAPA was added. After coculture of α-synuclein-treated BV-2 cell supernatant with MN9D cells, autophagy-impaired BV-2 promoted the apoptosis of MN9D cells, and 3-MA aggravated the autophagy disorder of BV-2 and further promoted the apoptosis of MN9D cells, while RAPA reversed the autophagy disorder of BV-2 and alleviated the apoptosis of MN9D cells. MALAT1 can promote α-synuclein expression by regulating miR-23b-3p, thereby inducing microglial autophagy disorder and an inflammatory response leading to apoptosis of dopaminergic neurons. This newly discovered molecular mechanism may provide a potential target for the treatment of PD.

α-Synuclein amplifies cytoplasmic peroxide flux and oxidative stress provoked by mitochondrial inhibitors in CNS dopaminergic neurons in vivo

Convergent evidence implicates impaired mitochondrial function and α-Synuclein accumulation as critical upstream events in the pathogenesis of Parkinson's disease, but comparatively little is known about how these factors interact to provoke neurodegeneration. We previously showed that α-Synuclein knockdown protected rat substantia nigra dopaminergic neurons from systemic exposure to the mitochondrial complex I inhibitor rotenone. Here we show that motor abnormalities prior to neuronal loss in this model are associated with extensive α-Synuclein-dependent cellular thiol oxidation. In order to elucidate the underlying events in vivo we constructed novel transgenic zebrafish that co-express, in dopaminergic neurons: (i) human α-Synuclein at levels insufficient to provoke neurodegeneration or neurobehavioral abnormalities; and (ii) genetically-encoded ratiometric fluorescent biosensors to detect cytoplasmic peroxide flux and glutathione oxidation. Live intravital imaging of the intact zebrafish CNS at cellular resolution showed unequivocally that α-Synuclein amplified dynamic cytoplasmic peroxide flux in dopaminergic neurons following exposure to the mitochondrial complex I inhibitors MPP+ or rotenone. This effect was robust and clearly evident following either acute or prolonged exposure to each inhibitor. In addition, disturbance of the resting glutathione redox potential following exogenous hydrogen peroxide challenge was augmented by α-Synuclein. Together these data show that α-Synuclein is a critical determinant of the redox consequences of mitochondrial dysfunction in dopaminergic neurons. The findings are important because the mechanisms underlying α-Synuclein-dependent reactive oxygen species fluxes and antioxidant suppression might provide a pharmacological target in Parkinson's disease to prevent progression from mitochondrial dysfunction and oxidative stress to cell death.

Specific Knockdown of α-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity

α-Synuclein (α-syn) overload is strongly associated with Parkinson disease (PD), and reduction of the α-syn level by targeting the peptide-based system through the autophagy-lysosomal pathway (ALP) is a promising strategy to delay PD progression. However, if the ALP is comprised, targeting the peptide-based proteasomal degradation system would be a good alternative. In this study, we designed a fusion peptide containing an α-syn-binding domain and a short strong proteasome-targeting motif. Ourresults reveal that this peptide could specifically bind to α-syn, and direct it to the proteasomes for degradation in a recombinant expression system. Furthermore, by adding a membrane-penetrating motif to this fusion peptide, we demonstrated that it could penetrate into cells and consequently suppress the cellular α-syn level through proteasome degradation in a dose- and time-dependent manner. Functionally, these effects rescued the mitochondrial dysfunction and cellular defects caused by α-syn overexpression in the cultured cells and primary neurons.

Glial HMOX1 expression promotes central and peripheral α-synuclein dysregulation and pathogenicity in parkinsonian mice.

α-Synuclein is a key player in the pathogenesis of Parkinson disease (PD). Expression of human heme oxygenase-1 (HO-1) in astrocytes of GFAP.HMOX1 transgenic (TG) mice between 8.5 and 19 months of age results in a parkinsonian phenotype characterized by neural oxidative stress, nigrostriatal hypodopaminergia associated with locomotor incoordination, and overproduction of α-synuclein. We identified two microRNAs (miR-), miR-153 and miR-223, that negatively regulate α-synuclein in the basal ganglia of male and female GFAP.HMOX1 mice. Serum concentrations of both miRNAs progressively declined in the wild-type (WT) and GFAP.HMOX1 mice between 11 and 19 months of age. Moreover, at each time point surveyed, circulating levels of miR-153 were significantly lower in the TG animals compared to WT controls, while α-synuclein protein concentrations were elevated in erythrocytes of the GFAP.HMOX1 mice at 19 months of age relative to WT values. Primary WT neurons co-cultured with GFAP.HMOX1 astrocytes exhibited enhanced protein oxidation, mitophagy and apoptosis, aberrant expression of genes regulating the dopaminergic phenotype, and an imbalance in gene expression profiles governing mitochondrial fission and fusion. Many, but not all, of these neuronal abnormalities were abrogated by small interfering RNA (siRNA) knockdown of α-synuclein, implicating α-synuclein as a potent, albeit partial, mediator of HO-1's neurodystrophic effects in these parkinsonian mice. Overexpression of HO-1 in stressed astroglia has previously been documented in the substantia nigra of idiopathic PD and may promote α-synuclein production and toxicity by downmodulating miR-153 and/or miR-223 both within the CNS and in peripheral tissues.

Long-term RNAi knockdown of α-synuclein in the adult rat substantia nigra without neurodegeneration

α-Synuclein plays a central role in the pathogenesis of Parkinson's disease (PD); interventions that decrease its expression appear neuroprotective in PD models. Successful translation of these observations into effective therapies will be dependent on the safety of suppressing α-synuclein expression in the adult brain. We investigated long-term α-synuclein knockdown in the adult rat CNS. 8-month old animals received either AAV-sh[Snca] (an RNA interference vector targeting the Snca mRNA transcript) or AAV-sh[Ctrl] (a control vector) unilaterally into the substantia nigra. No signs of systemic toxicity or motor dysfunction were observed in either experimental group over 12 months. Viral transgene expression persisted to 12 months post-inoculation, at which point Snca mRNA expression in substantia nigra dopaminergic neurons of animals that received AAV-sh[Snca] was decreased by ≈90%, and α-synuclein immunoreactivity by >70% relative to the control side. Stereological quantification of Nissl-labeled neurons showed no evidence of neurodegeneration in the substantia nigra 12 months after inoculation with either vector, and we observed abundant dopaminergic neurons with minimal α-synuclein immunoreactivity that appeared otherwise unremarkable in the AAV-sh[Snca] group. Despite the absence of neurodegeneration, some loss of TH expression was evident in nigral neurons after transduction with either vector, presumably a non-specific consequence of vector delivery, cellular transduction, or expression of shRNA or GFP. We conclude that long-term α-synuclein knockdown in the substantia nigra does not cause significant functional deficits in the ascending dopaminergic projection, or neurodegeneration. These findings are encouraging that it may be feasible to target α-synuclein expression therapeutically in PD.

P.2.023 - Impairment of learning and memory in mice overexpressing α- and γ-synuclein in dopaminergic neurons- implication in Parkinson’s disease

Introduction Idiopathic Parkinson’s disease (PD) is characterized by progressive brain pathology affecting several neurotransmitter systems, leading to a diverse profile of autonomic, motor, cognitive and psychiatric dysfunctions. Multiple convergent lines of evidence implicate the aggregation and accumulation of α-synuclein in the PD pathogenesis [1]. Moreover, post-translational changes of γ-synuclein increase the α-synuclein aggregation [2]. α- and γ-synuclein are members of the synuclein family of small cytoplasmic neuron-specific proteins, and have been suggested to modulate the function of monoamine transporters (DAT, SERT, NET) [3]. Previously, we reported that α-synuclein knockdown mice showed increased extracellular dopamine and serotonin levels –compared to control mice– in caudate-putamen (CPu) and medial prefrontal cortex (mPFC) after DAT or SERT blockade with nomifensine or citalopram, respectively (Alarcon-Aris et al., in review). Aims Given the role of monoamine neurotransmission in cognitive processes, in the present study we assessed the effects of over-expressing α- and γ-synuclein in mouse dopaminergic neurons on spatial and working memory. Methods We generated a mouse model overexpressing human wild-type α-synucleinor endogenous γ-synuclein in dopaminergic neurons from the substantia nigra compacta (SNc) and ventral tegmental area (VTA) using adeno-associated virus type-5 (AAV5-human-α-synuclein) or type-10 (AAV10-mouse-γ-synuclein) unilaterally injected in SNc/VTA. Behavioral tests were performed in mice at 4, 8 and 16 weeks post-infection using cognitive (novel object recognition, Morris water maze, passive avoidance tests) and motor paradigms (cylinder test, rotarod). Extracellular striatal dopamine levels were measured by in vivo microdialysis after implanting concentric dialysis probes, and analyzed by high precision liquid chromatography with electrochemical detection (+0.75 V). In situ hybridization was performed on fixed mice tissues, with 33P-dATP labeled probes in order to detect α- and γ-synuclein transcripts through autoradiography. Results AAV5-α-synuclein mice showed increased human α-synuclein mRNA levels in the ipsilateral SNc over time reaching a 278% of sham mice at 8 weeks post-infection. Similarly, AAV10-γ-synuclein mice reached to a maximal of 250% of sham mice. Overexpression of α- or γ-synuclein led to lower extracellular dopamine levels in CPu and mPFC compared to sham mice after local veratridine (sodium chanel activator agent, 50 µM) or nomifensine (DAT inhibitor, 1-10-50 µM) administration at 4 weeks post-infection. Moreover, overexpression of α- or γ-synuclein led to the impairment of working memory in mice as revealed by passive avoidance test (retention memory 35.5% and 45.9% shorter in AAV5-α-synuclein andAAV10-γ-synuclein mice, respectively) and novel object recognition test (reduction of 10% in time spent exploring novel object in both infected models) at 4-8 weeks post-infection. Likewise, these mice showed deficits in the spatial learning using the Morris water maze (F(1,28)=5.711, P=0.02 in AAV5-α-synuclein; F(1,28)=11.43, P=0.0021 in AAV10-γ-synuclein). These non-motor symptoms appeared earlier than motor deficits assessed by the cylinder test and rotarod. Conclusions Overall, these findings indicate that 1) α- and γ-synuclein are directly involved in the regulation of cognitive functions, likely through changes in monoamine functions, and 2) overexpression of α- or γ-synuclein in dopaminergic neurons mimics the temporal progression of clinical symptoms in PD, with early cognitive deficits appearing before motor symptoms.

Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson’s Disease

Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%-40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD.

Alpha-synuclein contributes to malignant progression of human meningioma via the Akt/mTOR pathway.

BackgroundThe aim of this study is to explore the expression of alpha-synuclein (α-synuclein) in benign, atypical, and anaplastic meningiomas and determine its role in the malignant progression of meningiomas.MethodsExpression of α-synuclein was measured in 44 meningioma samples by real-time PCR analysis. The effects of overexpression or knockdown of α-synuclein on meningioma cell growth, invasiveness, and tumorigenicity were determined.ResultsAtypical and anaplastic meningiomas displayed significantly greater levels of α-synuclein mRNA, relative to benign tumors. Depletion of α-synuclein decreased cell proliferation and colony formation and promoted apoptosis in IOMM-Lee meningioma cells, whereas overexpression of α-synuclein facilitated cell proliferation and colony formation in CH-157MN meningioma cells. Silencing of α-synuclein attenuated IOMM-Lee cell migration and invasion. In contrast, ectopic expression of α-synuclein increased the invasiveness of CH-157MN cells. In vivo studies further demonstrated that downregulation of α-synuclein significantly retarded meningioma growth in nude mice. At the molecular level, the phosphorylation levels of Akt, mTOR, p70S6K and 4EBP were significantly decreased in α-synuclein-depleted IOMM-Lee cells.ConclusionsIn conclusion, α-synuclein upregulation contributes to aggressive phenotypes of meningiomas via the Akt/mTOR pathway and thus represents a potential therapeutic target for malignant meningiomas.

G2019S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons.

Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.

Knockdown of α-synuclein in cerebral cortex improves neural behavior associated with apoptotic inhibition and neurotrophin expression in spinal cord transected rats.

Spinal cord injury (SCI) often causes severe functional impairment with poor recovery. The treatment, however, is far from satisfaction, and the mechanisms remain unclear. By using proteomics and western blot, we found spinal cord transection (SCT) resulted in a significant down-regulation of α-synuclein (SNCA) in the motor cortex of SCT rats at 3days post-operation. In order to detect the role of SNCA, we used SNCA-ORF/shRNA lentivirus to upregulate or knockdown SNCA expression. In vivo, SNCA-shRNA lentivirus injection into the cerebral cortex motor area not only inhibited SNCA expression, but also significantly enhanced neurons' survival, and attenuated neuronal apoptosis, as well as promoted motor and sensory function recovery in hind limbs. While, overexpression SNCA exhibited the opposite effects. In vitro, cortical neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival and neurite outgrowth, while it was accompanied by reverse efficiency in SNCA-ORF group. In molecular level, SNCA silence induced the upregulation of Bcl-2 and the downregulation of Bax, and the expression of NGF, BDNF and NT3 was substantially upregulated in cortical neurons. Together, endogenous SNCA play a crucial role in motor and sensory function regulation, in which, the underlying mechanism may be linked to the regulation of apoptosis associated with apoptotic gene (Bax, Bcl2) and neurotrophic factors expression (NGF, BDNF and NT3). These finds provide novel insights to understand the role of SNCA in cerebral cortex after SCT, and it may be as a novel treatment target for SCI repair in future clinic trials.

ShRNA targeting α-synuclein prevents neurodegeneration in a Parkinson's disease model.

Multiple convergent lines of evidence implicate both α-synuclein (encoded by SCNA) and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson's disease (PD). Occupational exposure to the mitochondrial complex I inhibitor rotenone increases PD risk; rotenone-exposed rats show systemic mitochondrial defects but develop specific neuropathology, including α-synuclein aggregation and degeneration of substantia nigra dopaminergic neurons. Here, we inhibited expression of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus-mediated delivery of a short hairpin RNA (shRNA) targeting the endogenous rat Snca transcript. Knockdown of α-synuclein by ~35% did not affect motor function or cause degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-exposed rats, progressive motor deficits were substantially attenuated contralateral to α-synuclein knockdown. Correspondingly, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites, and their striatal terminals was decreased ipsilateral to α-synuclein knockdown. These data show that α-synuclein knockdown is neuroprotective in the rotenone model of PD and indicate that endogenous α-synuclein contributes to the specific vulnerability of dopaminergic neurons to systemic mitochondrial inhibition. Our findings are consistent with a model in which genetic variants influencing α-synuclein expression modulate cellular susceptibility to environmental exposures in PD patients. shRNA targeting the SNCA transcript should be further evaluated as a possible neuroprotective therapy in PD.

Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

Protective effect of alpha-synuclein knockdown on methamphetamine-induced neurotoxicity in dopaminergic neurons.

The over-expression of α-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson's disease. In the present study, α-synuclein knockdown rats were created by injecting α-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraperitoneally with methamphetamine to establish the model of Parkinson's disease. Expression of α-synuclein mRNA and protein in the right striatum of the injected rats was significantly downregulated. Food intake and body weight were greater in α-synuclein knockdown rats, and water intake and stereotyped behavior score were lower than in model rats. Striatal dopamine and tyrosine hydroxylase levels were significantly elevated in α-synuclein knockdown rats. Moreover, superoxide dismutase activity was greater in α-synuclein knockdown rat striatum, but the levels of reactive oxygen species, malondialdehyde, nitric oxide synthase and nitrogen monoxide were lower compared with model rats. We also found that α-synuclein knockdown inhibited methamphetamine-induced neuronal apoptosis. These results suggest that α-synuclein has the capacity to reverse methamphetamine-induced apoptosis of dopaminergic neurons in the rat striatum by inhibiting oxidative stress and improving dopaminergic system function.

α-Synuclein Overexpression Represses 14-3-3θ Transcription

Previous gene microarray studies have shown that expression of 14-3-3θ is significantly decreased in an α-synuclein transgenic mouse model. In this study, we tested whether α-synuclein can regulate 14-3-3θ transcription. We demonstrate that the 14-3-3θ mRNA level is decreased in SH-SY5Y cells overexpressing α-synuclein. Luciferase activity under the control of the 14-3-3θ promoter is reduced both in stable SH-SY5Y cells constitutively overexpressing α-synuclein and in doxycycline-inducible SH-SY5Y cells upon α-synuclein induction, suggesting that the regulation of 14-3-3θ by α-synuclein occurs at the transcriptional level. Knockdown of α-synuclein by RNA interference does not increase the 14-3-3θ mRNA level. These findings suggest that α-synuclein represses 14-3-3θ transcription under pathologic conditions, but that regulation of 14-3-3θ expression is not a function of endogenous α-synuclein at baseline.

Role of Alpha-Synuclein Protein Levels in Mitochondrial Morphology and Cell Survival in Cell Lines

α-Synuclein is highly associated with some neurodegeneration and malignancies. Overexpressing wild-type or mutant α-synuclein promotes neuronal death by mitochondrial dysfunction, the underlying mechanisms of which remain poorly defined. It was recently reported that α-synuclein expression could directly lead to mitochondrial fragmentation in vitro and in vivo, which may be due to α-synuclein localization on mitochondria. Here, we applied a double staining method to demonstrate mitochondrial morphogenetic changes in cells overexpressed with α-synuclein. We show that mitochondrial localization of α-synuclein was increased following its overexpression in three distinct cell lines, including HeLa, SH-SY5Y, and PC12 cells, but no alteration in mitochondrial morphology was detected. However, α-synuclein knockdown prevents MPP+-induced mitochondrial fragmentation in SH-SY5Y and PC12 cells. These data suggest that α-synuclein protein levels hardly affect mitochondrial morphology in normal cell lines, but may have some influence on that under certain environmental conditions.

Knockdown of α-Synuclein Enhances Susceptibility to Staurosporine-Induced Apoptosis in Human Melanoma SK-MEL28 Cells

Knockdown of α-Synuclein Enhances Susceptibility to Staurosporine-Induced Apoptosis in Human Melanoma SK-MEL28 Cells

Manganese induces the overexpression of α-synuclein in PC12 cells via ERK activation

Manganese has been known to induce neurological disorders. In the present study, we determined the effect of manganese on the expression of α-synuclein in PC12 cells and its role in manganese-induced cytotoxicity. We also investigated the relationship between α-synuclein expression and the change of ERK1/2 MAPK activity. In our research, manganese exposure induced the overexpression of α-synuclein, while siRNA knockdown of α-synuclein reversed manganese-induced cytotoxicity. Furthermore, manganese induced the activation of ERK1/2 MAPK. The MEK1 inhibitor PD98059, which inhibits the activation of ERK MAPK, attenuated the overexpression of α-synuclein and the cytotoxicity induced by manganese. In conclusion, our studies show that manganese may induce the overexpression of α-synuclein via ERK1/2 activation, which may play a role in manganese-induced cytotoxicity.

α-Synuclein knockdown attenuates MPP+ induced mitochondrial dysfunction of SH-SY5Y cells

α-Synuclein is one of the main constituents of Lewy bodies and plays an important role in the pathology of Parkinson's disease. Mutation or overexpression of α-synuclein causes Parkinson's disease, and downregulation of α-synuclein resists MPP+-induced cell death, but the mechanism remains elusive. In this study, we attempted to explore the effect of α-synuclein knockdown on mitochondrial function in MPP+-treated SH-SY5Y cells. We reconstructed the short hairpin RNA expression vector, pGenesil-2, specially targeting α-synuclein mRNA, and it was stably transfected into SH-SY5Y cells. Cell viability, nuclear morphology, and mitochondrial membrane potential were then detected, and the expression of α-synuclein, cytochrome c, Bcl-2 and Bax were analyzed by Western blotting. The results showed that after exposure to 500 μM MPP+ for 24 h, about 41.0±1.5% control cells showed low mitochondrial membrane potential. However, the percentage was 13.6±1.2% in MPP+ treated α-synuclein knockdown cells. MPP+ induced cytochrome c release significantly, which was about 3.1-fold compared with that of control. However, in α-synuclein knockdown cells, the release of cytochrome c was blocked, which was about 1.4-fold compared with that of control. The Bcl-2/Bax ratio of SH-SY5Y cells reduced to 35.5±3.8% after MPP+ treatment, and this ratio was 85.2±3.0% in MPP+ treated α-synuclein knockdown cells. These data suggest that knockdown of α- synuclein might be an effective means in rescuing MPP+-induced mitochondrial dysfunction of SH-SY5Y cells.