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Silencing alpha-synuclein gene expression enhances tyrosine hydroxylase activity in MN9D cells.

ABSTRACT: alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Previous studies have shown that alpha-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed alpha-synuclein stably silenced MN9D/alpha-SYN(-) cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/alpha-SYN(-) and MN9D/CON cells, suggesting that silencing alpha-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (L-DOPA) and DA levels were observed in MN9D/alpha-SYN(-) cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of alpha-synuclein, suggesting that alpha-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional alpha-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.

SUBMITTER: Liu D

PROVIDER: S-EPMC2413118 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Silencing alpha-synuclein gene expression enhances tyrosine hydroxylase activity in MN9D cells.

Liu Dongmei D Jin Ling L Wang Hao H Zhao Huanying H Zhao Chunli C Duan Chunli C Lu Lingling L Wu Bo B Yu Shun S Chan Piu P Li Yaohua Y Yang Hui H

Neurochemical research 20080321 7

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Previous studies have shown that alpha-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed alpha-synuclein stably silenced MN9D/alpha-SYN(-) cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were ...[more]

PMID: 18357527

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Project description:Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.

Project description:BackgroundThe characteristic progression of Lewy pathology in Parkinson's disease likely involves intercellular exchange and the accumulation of misfolded α-synuclein amplified by a prion-like self-templating mechanism. Silencing of the α-synuclein gene could provide long-lasting disease-modifying benefits by reducing the requisite substrate for the spreading aggregation.ObjectivesAs a result of the poor penetration of viral vectors across the blood-brain barrier, gene therapy for central nervous system disorders requires direct injections into the affected brain regions, and invasiveness is further increased by the need for bilateral delivery to multiple brain regions. Here we test a noninvasive approach by combining low-intensity magnetic resonance-guided focused ultrasound and intravenous microbubbles that can transiently increase the access of brain impermeant therapeutic macromolecules to targeted brain regions.MethodsTransgenic mice expressing human α-synuclein were subjected to magnetic resonance-guided focused ultrasound targeted to 4 brain regions (hippocampus, substantia nigra, olfactory bulb, and dorsal motor nucleus) in tandem with intravenous microbubbles and an adeno-associated virus serotype 9 vector bearing a short hairpin RNA sequence targeting the α-synuclein gene.ResultsOne month following treatment, α-synuclein immunoreactivity was decreased in targeted brain regions, whereas other neuronal markers such as synaptophysin or tyrosine hydroxylase were unchanged, and cell death and glial activation remained at basal levels.ConclusionsThese results demonstrate that magnetic resonance-guided focused ultrasound can effectively, noninvasively, and simultaneously deliver viral vectors targeting α-synuclein to multiple brain areas. Importantly, this approach may be useful to alter the progression of Lewy pathology along selected neuronal pathways, particularly as prodromal PD markers improve early diagnoses. © 2018 International Parkinson and Movement Disorder Society.

Project description:PURPOSE: Alpha-synuclein is a Parkinson's disease-linked protein of ubiquitous expression in the central nervous system. It has a proposed role in the modulation of neurotransmission and synaptic function. This study was aimed at analyzing expression of the alpha-synuclein gene in the normal retina, and characterizing its pattern of distribution in the different retinal cell types and layers in a variety of vertebrates, ranging from fish to humans. METHODS: Reverse transcriptase-polymerase chain reaction and immunoblotting were used to assess alpha-synuclein expression at both mRNA and protein levels. Its retinal distribution profile was characterized by immunohistochemical methods. With this purpose, retinal sections were analyzed under fluorescent confocal microscopy using specific antibodies against alpha-synuclein, alone and in double or triple combinations with a set of antibodies to molecular markers for the distinct retinal neuronal types. Also, synaptophysin was used as a marker for synaptic vesicles in the retina. RESULTS: Alpha-synuclein mRNA and protein were expressed by both retinal pigment epithelium (RPE) and neural retinal cells. The pattern of alpha-synuclein distribution in the retina was quite consistent across all vertebrate species examined. A strong immunoreactivity was found in the outer segments (OS) of photoreceptors and in their axon terminals (cone pedicles and rod spherules) in the outer plexiform layer (OPL) of the retina. Alpha-synuclein was also present in rod and cone bipolar cells, as well as in GABAergic and glycinergic amacrines, distributing along a complex plexus throughout the inner plexiform layer (IPL). Additionally, colocalization was found between alpha-synuclein and synaptophysin at presynaptic terminals of the retina. Alpha-synuclein-positive phagosome-like structures were observed in the cytoplasm of RPE cells. CONCLUSIONS: An involvement of alpha-synuclein can be postulated in neurotransmission at axon terminals of photoreceptors in the OPL, and at presynaptic endings of bipolar and amacrine cells in the IPL. As well, this protein could have a role in the function as well as the maintenance of photoreceptor OS. Alpha-synuclein contained in RPE cells should derive not only from protein expression by this cell type, but also from their phagocytosis of OS disc membranes.

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Silencing alpha-synuclein gene expression enhances tyrosine hydroxylase activity in MN9D cells.

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Silencing alpha-synuclein gene expression enhances tyrosine hydroxylase activity in MN9D cells.

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