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a-Synuclein antibody

RRID:AB_398107

Antibody ID

AB_398107

Target Antigen

a-Synuclein mouse, rat, human, mouse, rat

Proper Citation

(BD Biosciences Cat# 610786, RRID:AB_398107)

Clonality

monoclonal antibody

Comments

Immunofluorescence, Western blot

Host Organism

mouse

Vendor

BD Biosciences Go To Vendor

Sensitivity and specificity of phospho-Ser129 α-synuclein monoclonal antibodies.

  • Delic V
  • J. Comp. Neurol.
  • 2018 Aug 15

Literature context:


Abstract:

α-Synuclein (α-syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α-syn is phosphorylated at the serine-129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho-specific antibodies often cross-react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF-R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129-α-syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129-α-syn inclusions in the brain that were induced by preformed α-syn fibrils in wild-type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF-R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α-syn knockout mice as well as mice and rats injected with monomeric α-syn, with some nonspecific staining titrating with pS129-α-syn inclusions. By immunoblot, all the clones cross-reacted with proteins other than α-syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129-α-syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129-α-syn.

Funding information:
  • NIGMS NIH HHS - GM08347(United States)
  • NINDS NIH HHS - P20 NS092530()
  • NINDS NIH HHS - R01 NS064934()
  • NINDS NIH HHS - R21 NS097643()
  • NINDS NIH HHS - R33 NS097643()

Stimulation of synaptoneurosome glutamate release by monomeric and fibrillated α-synuclein.

  • Sarafian TA
  • J. Neurosci. Res.
  • 2018 Apr 17

Literature context:


Abstract:

The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.

Funding information:
  • NIDDK NIH HHS - P30 DK063491()

α-Synuclein-Dependent Calcium Entry Underlies Differential Sensitivity of Cultured SN and VTA Dopaminergic Neurons to a Parkinsonian Neurotoxin.

  • Lieberman OJ
  • eNeuro
  • 2017 Nov 28

Literature context:


Abstract:

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra (SN). Although mitochondrial dysfunction and dysregulated α-synuclein (aSyn) expression are postulated to play a role in PD pathogenesis, it is still debated why neurons of the SN are targeted while neighboring dopaminergic neurons of the ventral tegmental area (VTA) are spared. Using electrochemical and imaging approaches, we investigated metabolic changes in cultured primary mouse midbrain dopaminergic neurons exposed to a parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). We demonstrate that the higher level of neurotoxicity in SN than VTA neurons was due to SN neuron-specific toxin-induced increase in cytosolic dopamine (DA) and Ca2+, followed by an elevation of mitochondrial Ca2+, activation of nitric oxide synthase (NOS), and mitochondrial oxidation. The increase in cytosolic Ca2+ was not caused by MPP+-induced oxidative stress, but rather depended on the activity of both L-type calcium channels and aSyn expression, suggesting that these two established pathogenic factors in PD act in concert.

Funding information:
  • Intramural NIH HHS - ZIA CL002119-04(United States)

Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease.

  • Taguchi YV
  • J. Neurosci.
  • 2017 Oct 4

Literature context:


Abstract:

Glucocerebrosidase 1 (GBA) mutations responsible for Gaucher disease (GD) are the most common genetic risk factor for Parkinson's disease (PD). Although the genetic link between GD and PD is well established, the underlying molecular mechanism(s) are not well understood. We propose that glucosylsphingosine, a sphingolipid accumulating in GD, mediates PD pathology in GBA-associated PD. We show that, whereas GD-related sphingolipids (glucosylceramide, glucosylsphingosine, sphingosine, sphingosine-1-phosphate) promote α-synuclein aggregation in vitro, glucosylsphingosine triggers the formation of oligomeric α-synuclein species capable of templating in human cells and neurons. Using newly generated GD/PD mouse lines of either sex [Gba mutant (N370S, L444P, KO) crossed to α-synuclein transgenics], we show that Gba mutations predispose to PD through a loss-of-function mechanism. We further demonstrate that glucosylsphingosine specifically accumulates in young GD/PD mouse brain. With age, brains exhibit glucosylceramide accumulations colocalized with α-synuclein pathology. These findings indicate that glucosylsphingosine promotes pathological aggregation of α-synuclein, increasing PD risk in GD patients and carriers.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a prevalent neurodegenerative disorder in the aging population. Glucocerebrosidase 1 mutations, which cause Gaucher disease, are the most common genetic risk factor for PD, underscoring the importance of delineating the mechanisms underlying mutant GBA-associated PD. We show that lipids accumulating in Gaucher disease, especially glucosylsphingosine, play a key role in PD pathology in the brain. These data indicate that ASAH1 (acid ceramidase 1) and GBA2 (glucocerebrosidase 2) enzymes that mediate glucosylsphingosine production and metabolism are attractive therapeutic targets for treating mutant GBA-associated PD.

Funding information:
  • NIAMS NIH HHS - R01 AR065932()
  • NIGMS NIH HHS - T32 GM007223()
  • NINDS NIH HHS - R01 NS064963()
  • NINDS NIH HHS - R01 NS083846()
  • NINDS NIH HHS - T32 NS007224()

Randomized CRISPR-Cas Transcriptional Perturbation Screening Reveals Protective Genes against Alpha-Synuclein Toxicity.

  • Chen YC
  • Mol. Cell
  • 2017 Oct 5

Literature context:


Abstract:

The genome-wide perturbation of transcriptional networks with CRISPR-Cas technology has primarily involved systematic and targeted gene modulation. Here, we developed PRISM (Perturbing Regulatory Interactions by Synthetic Modulators), a screening platform that uses randomized CRISPR-Cas transcription factors (crisprTFs) to globally perturb transcriptional networks. By applying PRISM to a yeast model of Parkinson's disease (PD), we identified guide RNAs (gRNAs) that modulate transcriptional networks and protect cells from alpha-synuclein (αSyn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of αSyn toxicity reported previously, highlighting PRISM's ability to identify modulators of important phenotypes. Gene expression profiling revealed genes differentially modulated by this strong protective gRNA that rescued yeast from αSyn toxicity when overexpressed. Human homologs of top-ranked hits protected against αSyn-induced cell death in a human neuronal PD model. Thus, high-throughput and unbiased perturbation of transcriptional networks via randomized crisprTFs can reveal complex biological phenotypes and effective disease modulators.

Funding information:
  • NIDDK NIH HHS - P30 DK043351()

Parkinson Sac Domain Mutation in Synaptojanin 1 Impairs Clathrin Uncoating at Synapses and Triggers Dystrophic Changes in Dopaminergic Axons.

  • Cao M
  • Neuron
  • 2017 Feb 22

Literature context:


Abstract:

Synaptojanin 1 (SJ1) is a major presynaptic phosphatase that couples synaptic vesicle endocytosis to the dephosphorylation of PI(4,5)P2, a reaction needed for the shedding of endocytic factors from their membranes. While the role of SJ1's 5-phosphatase module in this process is well recognized, the contribution of its Sac phosphatase domain, whose preferred substrate is PI4P, remains unclear. Recently a homozygous mutation in its Sac domain was identified in early-onset parkinsonism patients. We show that mice carrying this mutation developed neurological manifestations similar to those of human patients. Synapses of these mice displayed endocytic defects and a striking accumulation of clathrin-coated intermediates, strongly implicating Sac domain's activity in endocytic protein dynamics. Mutant brains had elevated auxilin (PARK19) and parkin (PARK2) levels. Moreover, dystrophic axonal terminal changes were selectively observed in dopaminergic axons in the dorsal striatum. These results strengthen evidence for a link between synaptic endocytic dysfunction and Parkinson's disease.

Funding information:
  • NCATS NIH HHS - UL1 TR001863()
  • NIDA NIH HHS - P30 DA018343()
  • NIGMS NIH HHS - P41 GM103412()
  • NINDS NIH HHS - R01 NS036251()
  • NINDS NIH HHS - R01 NS036942()
  • NINDS NIH HHS - R37 NS036251()
  • NINDS NIH HHS - R37 NS036942()

Dopaminergic lesioning impairs adult hippocampal neurogenesis by distinct modification of α-synuclein.

  • Schlachetzki JC
  • J. Neurosci. Res.
  • 2016 Jan 28

Literature context:


Abstract:

Nonmotor symptoms of cognitive and affective nature are present in premotor and motor stages of Parkinson's disease (PD). Neurogenesis, the generation of new neurons, persists throughout the mammalian life span in the hippocampal dentate gyrus. Adult hippocampal neurogenesis may be severely affected in the course of PD, accounting for some of the neuropsychiatric symptoms such as depression and cognitive impairment. Two important PD-related pathogenic factors have separately been attributed to contribute to both PD and adult hippocampal neurogenesis: dopamine depletion and accumulation of α-synuclein (α-syn). In the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model, altered neurogenesis has been linked merely to a reduced dopamine level. Here, we seek to determine whether a distinct endogenous α-syn expression pattern is associated, possibly contributing to the hippocampal neurogenic deficit. We observed a persistent reduction of striatal dopamine and a loss of tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta in contrast to a complete recovery of tyrosine hydroxylase-immunoreactive dopaminergic fibers within the striatum. However, dopamine levels in the hippocampus were significantly decreased. Survival of newly generated neurons was significantly reduced and paralleled by an accumulation of truncated, membrane-associated, insoluble α-syn within the hippocampus. Specifically, the presence of truncated α-syn species was accompanied by increased activity of calpain-1, a calcium-dependent protease. Our results further substantiate the broad effects of dopamine loss in PD-susceptible brain nuclei, gradually involved in the PD course. Our findings also indicate a detrimental synergistic interplay between dopamine depletion and posttranslational modification of α-syn, contributing to impaired hippocampal plasticity in PD.

α-Synuclein-independent histopathological and motor deficits in mice lacking the endolysosomal Parkinsonism protein Atp13a2.

  • Kett LR
  • J. Neurosci.
  • 2015 Apr 8

Literature context:


Abstract:

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.

Funding information:
  • NINDS NIH HHS - R01 NS090390(United States)

MicroRNA-7 protects against 1-methyl-4-phenylpyridinium-induced cell death by targeting RelA.

  • Choi DC
  • J. Neurosci.
  • 2014 Sep 17

Literature context:


Abstract:

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Mitochondrial complex I impairment in PD is modeled in vitro by the susceptibility of dopaminergic neurons to the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). In the present study, we demonstrate that microRNA-7 (miR-7), which is expressed in tyrosine hydroxylase-positive nigral neurons in mice and humans, protects cells from MPP+-induced toxicity in dopaminergic SH-SY5Y cells, differentiated human neural progenitor ReNcell VM cells, and primary mouse neurons. RelA, a component of nuclear factor-κB (NF-κB), was identified to be downregulated by miR-7 using quantitative proteomic analysis. Through a series of validation experiments, it was confirmed that RelA mRNA is a target of miR-7 and is required for cell death following MPP+ exposure. Further, RelA mediates MPP+-induced suppression of NF-κB activity, which is essential for MPP+-induced cell death. Accordingly, the protective effect of miR-7 is exerted through relieving NF-κB suppression by reducing RelA expression. These findings provide a novel mechanism by which NF-κB suppression, rather than activation, underlies the cell death mechanism following MPP+ toxicity, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for this disease.

Funding information:
  • NCRR NIH HHS - RR-17072(United States)

Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death.

  • Martin LJ
  • J. Comp. Neurol.
  • 2007 Jan 1

Literature context:


Abstract:

The mechanisms of human mutant superoxide dismutase-1 (mSOD1) toxicity to motor neurons (MNs) are unresolved. We show that MNs in G93A-mSOD1 transgenic mice undergo slow degeneration lacking similarity to apoptosis structurally and biochemically. It is characterized by somal and mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in nuclear and mitochondrial DNA. p53 and p73 are activated in degenerating MNs, but without nuclear import. The MN death is independent of activation of caspases-1, -3, and -8 or apoptosis-inducing factor within MNs, with a blockade of apoptosis possibly mediated by Aven up-regulation. MN swelling is associated with compromised Na,K-ATPase activity and aggregation. mSOD1 mouse MNs accumulate mitochondria from the axon terminals and generate higher levels of superoxide, nitric oxide, and peroxynitrite than MNs in control mice. Nitrated and aggregated cytochrome c oxidase subunit-I and alpha-synuclein as well as nitrated SOD2 accumulate in mSOD1 mouse spinal cord. Mitochondria in mSOD1 mouse MNs accumulate NADPH diaphorase and inducible nitric oxide synthase (iNOS)-like immunoreactivity, and iNOS gene deletion extends significantly the life span of G93A-mSOD1 mice. Prior to MN loss, spinal interneurons degenerate. These results identify novel mechanisms for mitochondriopathy and MN degeneration in amyotrophic lateral sclerosis (ALS) mice involving blockade of apoptosis, accumulation of MN mitochondria with enhanced toxic potential from distal terminals, NOS localization in MN mitochondria and peroxynitrite damage, and early degeneration of alpha-synuclein(+) interneurons. The data support roles for oxidative stress, protein nitration and aggregation, and excitotoxicity as participants in the process of MN degeneration caused by mSOD1.

Funding information:
  • NHGRI NIH HHS - R01 HG004069-03(United States)