Akihiko Koyama - Academia.edu (original) (raw)
Papers by Akihiko Koyama
Biochem Biophys Res Commun, 2005
Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), ... more Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson’s disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin–protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of α-synuclein (α-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and α-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increa... more Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid  peptides ending at residue 42 (A42). To identify a PS subdomain responsible for overproduction of A42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in A42 secretion, and A42 overproduction was de-pendent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of A42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
The Journal of Neuroscience, Dec 15, 1999
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increa... more Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid  peptides ending at residue 42 (A42). To identify a PS subdomain responsible for overproduction of A42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in A42 secretion, and A42 overproduction was de-pendent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of A42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dem... more Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies, and the deposition of wild-type α-synuclein as Lewy bodies occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating α-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of α-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human α-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed α-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified ten genes that, upon knockdown, caused severe growth/motor abnormalities selectively in α-synuclein Tg worms. Among these were four genes (i.e., apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing α-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. α-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of α-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated α-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and α-synuclein-induced neurotoxicity in synucleinopathy. by guest on August 11, 2015 http://hmg.oxfordjournals.org/ Downloaded from by guest on August 11, 2015 http://hmg.oxfordjournals.org/ Downloaded from
PloS one, 2011
Many neurodegenerative diseases are increasing in prevalence and cannot be prevented or cured. If... more Many neurodegenerative diseases are increasing in prevalence and cannot be prevented or cured. If they shared common pathogenic mechanisms, treatments targeting such mechanisms might be of benefit in multiple conditions. The tau protein has been implicated in the pathogenesis of diverse neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). Tau reduction prevents cognitive deficits, behavioral abnormalities and other pathological changes in multiple AD mouse models. Here we examined whether tau reduction also prevents motor deficits and pathological alterations in two mouse models of PD, generated by unilateral striatal injection of 6-hydroxydopamine (6-OHDA) or transgene-mediated neuronal expression of human wildtype α-synuclein. Both models were evaluated on Tau(+/+), Tau(+/-) and Tau(-/-) backgrounds in a variety of motor tests. Tau reduction did not prevent motor deficits caused by 6-OHDA and slightly worsened one of them. Tau reduc...
Neuroscience letters, Jan 23, 2003
alpha-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson'... more alpha-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson's disease (PD) as well as of neuronal/glial inclusions in a subset of neurodegenerative disorders collectively termed synucleinopathies. Here we studied by immunohistochemistry the accumulation of alpha-synuclein in transgenic (TG) Drosophila overexpressing wild-type (WT) or familial PD-linked mutant (i.e. A30P and A53T) alpha-synuclein in neurons, with special reference to the phosphorylation at Ser129, that is characteristic of human synucleinopathy lesions. Progressive accumulation of human alpha-synuclein was widely observed in the cell bodies and neurites of major neuronal nuclei in TG Drosophila brains, and phosphorylation of alpha-synuclein at Ser129 was detected in a limited subset of neurons approximately 1 week after alpha-synuclein immunoreactivity was first detected. Phosphorylated alpha-synuclein was most abundant in A53T mutant, followed by A30P and WT Drosophila. These res...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1999
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by in... more Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
PLoS ONE, 2012
Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest path... more Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer's disease (AD). It can occur before significant Aβ deposition and appears to "spread" into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.
Neuroscience Letters, 2003
a-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson's diseas... more a-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson's disease (PD) as well as of neuronal/glial inclusions in a subset of neurodegenerative disorders collectively termed synucleinopathies. Here we studied by immunohistochemistry the accumulation of a-synuclein in transgenic (TG) Drosophila overexpressing wildtype (WT) or familial PD-linked mutant (i.e. A30P and A53T) a-synuclein in neurons, with special reference to the phosphorylation at Ser129, that is characteristic of human synucleinopathy lesions. Progressive accumulation of human a-synuclein was widely observed in the cell bodies and neurites of major neuronal nuclei in TG Drosophila brains, and phosphorylation of a-synuclein at Ser129 was detected in a limited subset of neurons ,1 week after asynuclein immunoreactivity was first detected. Phosphorylated a-synuclein was most abundant in A53T mutant, followed by A30P and WT Drosophila. These results suggest that accumulation and phosphorylation of a-synuclein is recapitulated in neurons of a-synuclein transgenic Drosophila, that underscores the relevance of this model to human synucleinopatheis. q
Journal of Biological Chemistry, 2000
␣-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal ... more ␣-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and ␣-synuclein was found to be a major constituent of Lewy bodies. We have analyzed ␣-synuclein expression in transfected cell lines. In pulse-chase experiments ␣-synuclein appeared to be stable over long periods (t1 ⁄2 54 h) and no endoproteolytic processing was observed. ␣-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of ␣-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that ␣-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of ␣-synuclein is regulated by phosphorylation/dephosphorylation.
Journal of Biological Chemistry, 2006
Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wil... more Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.
Human Molecular Genetics, 2008
Mutations or multiplications in a-synuclein gene cause familial forms of Parkinson disease or dem... more Mutations or multiplications in a-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type a-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating a-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of a-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human a-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed a-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in a-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing a-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. a-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of a-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated a-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and a-synuclein-induced neurotoxicity in synucleinopathy.
Biomedical Chromatography, 2008
It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in... more It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease (PD), leads to neurodegeneration in PD models. In this study, the changes in protein expression between the transgenic over-expressing human alpha-synuclein wild type (alpha-synWT) and the control Caenorhabditis elegans were elucidated by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) proteome analysis, which is a highly selective, sensitive, repeatable and quantitative method for protein identification. Because the alpha-synuclein wild-type worms showed moderate levels of dopamine loss without overt behavioral abnormalities, it was suggested that the changes in proteins in the alpha-synWT are related in the sequence of the formation of Lewy bodies. Among more than 400 protein peaks detected, actin and several ribosomal proteins were identified for the first time as negative markers at early PD stages. Actin was suggested to be one of the important targets in the elucidation of the etiology of neuronal diseases such as PD or other synucleinopathies.
Biochemical and Biophysical Research Communications, 2005
Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), ... more Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of ParkinsonÕs disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of a-synuclein (a-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and a-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.
![Research paper thumbnail of Corrigendum to “Common anti-apoptotic roles of parkin and α-synuclein in human dopaminergic cells” [Biochem. Biophys. Res. Commun. 332 (2005) 233–240]](https://attachments.academia-assets.com/52980669/thumbnails/1.jpg)
](Biochemical and Biophysical Research Communications, 2005
Alzheimer's & Dementia, 2013
Alzheimer's & Dementia, 2006
alpha-Synuclein is a component of Lewy bodies in affected neurons of patients with Parkinson's di... more alpha-Synuclein is a component of Lewy bodies in affected neurons of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and missense mutations or multiplications in alpha-synuclein gene have been identified in pedigrees of familial PD (FPD)/DLB, implicating alpha-synuclein in the pathogenesis of PD and DLB. To gain insights into the mechanism whereby deposition of alpha-synuclein causes neurodegeneration, we established transgenic C. elegans (TG worms) that overexpress wild-type (wt) or FPD-mutant (A53T, A30P) human alpha-synuclein in dopamine neurons. TG worms expressing A53T or A30P FPD mutant alpha-synuclein showed significant defects in dopamine neuron function, the latter being reflected by an adoptive response for feeding (i.e., decrease in bending frequency upon entry into bacterial lawn), whereas those expressing wt alpha-synuclein were normal. We next generated TG worms that overexpress human alpha-synuclein in touch neurons under the control of a mec-7 promoter. These TG worms, especially those expressing A30P FPD mutant alpha-synuclein, showed impaired touch response, whereas those expressing wt alpha-synuclein showed a normal response. To examine the pathogenic significance of alpha-synuclein phosphorylation at Ser129, a characteristic feature of alpha-synuclein deposited in synucleinopathy brains, we next generated TG worms that pan-neuronally overexpress human G protein-coupled receptor kinase 5 (GRK5), previously reported as one of the alpha-synuclein kinases, and crossed them with the alpha-synculein TG worms. Phosphorylation of alpha-synuclein was upregulated in dopamine or touch neurons of double TG worms compared to those in TG worms that singly overexpress alpha-synuclein, as determined by immunohistochemisty. Behavioral examinations showed aggravations in functions of dopamine neurons or touch neurons overexpressing wt alpha-synuclein upon co-expression of GRK5, which were not observed upon co-expression of kinase-inactive mutant GRK5 (K215R). These results support the notion that missense mutations as well as Ser129 phosphorylation of alpha-synuclein contribute to neuronal dysfunction in PD and DLB.
Annals of Clinical and Translational Neurology, 2015
Objective: Dementia with Lewy bodies (DLB) is associated with the accumulation of wild-type human... more Objective: Dementia with Lewy bodies (DLB) is associated with the accumulation of wild-type human a-synuclein (SYN) in neurons and with prominent slowing of brain oscillations on electroencephalography (EEG). However, it remains uncertain whether the EEG abnormalities are actually caused by SYN. Methods: To determine whether SYN can cause neural network abnormalities, we performed EEG recordings and analyzed the expression of neuronal activitydependent gene products in SYN transgenic mice. We also carried out comparative analyses in humans with DLB. Results: We demonstrate that neuronal expression of SYN in transgenic mice causes a left shift in spectral power that closely resembles the EEG slowing observed in DLB patients. Surprisingly, SYN mice also had seizures and showed molecular hippocampal alterations indicative of aberrant network excitability, including calbindin depletion in the dentate gyrus. In postmortem brain tissues from DLB patients, we found reduced levels of calbindin mRNA in the dentate gyrus. Furthermore, nearly one quarter of DLB patients showed myoclonus, a clinical sign of aberrant network excitability that was associated with an earlier age of onset of cognitive impairments. In SYN mice, partial suppression of epileptiform activity did not alter their shift in spectral power. Furthermore, epileptiform activity in human amyloid precursor protein transgenic mice was not associated with a left shift in spectral power. Interpretation: We conclude that neuronal accumulation of SYN slows brain oscillations and, in parallel, causes aberrant network excitability that can escalate into seizure activity. The potential role of aberrant network excitability in DLB merits further investigation.
Biochem Biophys Res Commun, 2005
Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), ... more Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson’s disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin–protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of α-synuclein (α-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and α-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increa... more Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid  peptides ending at residue 42 (A42). To identify a PS subdomain responsible for overproduction of A42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in A42 secretion, and A42 overproduction was de-pendent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of A42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
The Journal of Neuroscience, Dec 15, 1999
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increa... more Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid  peptides ending at residue 42 (A42). To identify a PS subdomain responsible for overproduction of A42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in A42 secretion, and A42 overproduction was de-pendent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of A42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dem... more Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies, and the deposition of wild-type α-synuclein as Lewy bodies occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating α-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of α-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human α-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed α-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified ten genes that, upon knockdown, caused severe growth/motor abnormalities selectively in α-synuclein Tg worms. Among these were four genes (i.e., apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing α-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. α-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of α-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated α-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and α-synuclein-induced neurotoxicity in synucleinopathy. by guest on August 11, 2015 http://hmg.oxfordjournals.org/ Downloaded from by guest on August 11, 2015 http://hmg.oxfordjournals.org/ Downloaded from
PloS one, 2011
Many neurodegenerative diseases are increasing in prevalence and cannot be prevented or cured. If... more Many neurodegenerative diseases are increasing in prevalence and cannot be prevented or cured. If they shared common pathogenic mechanisms, treatments targeting such mechanisms might be of benefit in multiple conditions. The tau protein has been implicated in the pathogenesis of diverse neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). Tau reduction prevents cognitive deficits, behavioral abnormalities and other pathological changes in multiple AD mouse models. Here we examined whether tau reduction also prevents motor deficits and pathological alterations in two mouse models of PD, generated by unilateral striatal injection of 6-hydroxydopamine (6-OHDA) or transgene-mediated neuronal expression of human wildtype α-synuclein. Both models were evaluated on Tau(+/+), Tau(+/-) and Tau(-/-) backgrounds in a variety of motor tests. Tau reduction did not prevent motor deficits caused by 6-OHDA and slightly worsened one of them. Tau reduc...
Neuroscience letters, Jan 23, 2003
alpha-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson'... more alpha-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson's disease (PD) as well as of neuronal/glial inclusions in a subset of neurodegenerative disorders collectively termed synucleinopathies. Here we studied by immunohistochemistry the accumulation of alpha-synuclein in transgenic (TG) Drosophila overexpressing wild-type (WT) or familial PD-linked mutant (i.e. A30P and A53T) alpha-synuclein in neurons, with special reference to the phosphorylation at Ser129, that is characteristic of human synucleinopathy lesions. Progressive accumulation of human alpha-synuclein was widely observed in the cell bodies and neurites of major neuronal nuclei in TG Drosophila brains, and phosphorylation of alpha-synuclein at Ser129 was detected in a limited subset of neurons approximately 1 week after alpha-synuclein immunoreactivity was first detected. Phosphorylated alpha-synuclein was most abundant in A53T mutant, followed by A30P and WT Drosophila. These res...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1999
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by in... more Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
PLoS ONE, 2012
Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest path... more Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer's disease (AD). It can occur before significant Aβ deposition and appears to "spread" into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.
Neuroscience Letters, 2003
a-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson's diseas... more a-Synuclein is a major component of Lewy bodies in the brains of patients with Parkinson's disease (PD) as well as of neuronal/glial inclusions in a subset of neurodegenerative disorders collectively termed synucleinopathies. Here we studied by immunohistochemistry the accumulation of a-synuclein in transgenic (TG) Drosophila overexpressing wildtype (WT) or familial PD-linked mutant (i.e. A30P and A53T) a-synuclein in neurons, with special reference to the phosphorylation at Ser129, that is characteristic of human synucleinopathy lesions. Progressive accumulation of human a-synuclein was widely observed in the cell bodies and neurites of major neuronal nuclei in TG Drosophila brains, and phosphorylation of a-synuclein at Ser129 was detected in a limited subset of neurons ,1 week after asynuclein immunoreactivity was first detected. Phosphorylated a-synuclein was most abundant in A53T mutant, followed by A30P and WT Drosophila. These results suggest that accumulation and phosphorylation of a-synuclein is recapitulated in neurons of a-synuclein transgenic Drosophila, that underscores the relevance of this model to human synucleinopatheis. q
Journal of Biological Chemistry, 2000
␣-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal ... more ␣-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and ␣-synuclein was found to be a major constituent of Lewy bodies. We have analyzed ␣-synuclein expression in transfected cell lines. In pulse-chase experiments ␣-synuclein appeared to be stable over long periods (t1 ⁄2 54 h) and no endoproteolytic processing was observed. ␣-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of ␣-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that ␣-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of ␣-synuclein is regulated by phosphorylation/dephosphorylation.
Journal of Biological Chemistry, 2006
Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wil... more Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.
Human Molecular Genetics, 2008
Mutations or multiplications in a-synuclein gene cause familial forms of Parkinson disease or dem... more Mutations or multiplications in a-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type a-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating a-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of a-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human a-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed a-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in a-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing a-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. a-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of a-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated a-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and a-synuclein-induced neurotoxicity in synucleinopathy.
Biomedical Chromatography, 2008
It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in... more It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease (PD), leads to neurodegeneration in PD models. In this study, the changes in protein expression between the transgenic over-expressing human alpha-synuclein wild type (alpha-synWT) and the control Caenorhabditis elegans were elucidated by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) proteome analysis, which is a highly selective, sensitive, repeatable and quantitative method for protein identification. Because the alpha-synuclein wild-type worms showed moderate levels of dopamine loss without overt behavioral abnormalities, it was suggested that the changes in proteins in the alpha-synWT are related in the sequence of the formation of Lewy bodies. Among more than 400 protein peaks detected, actin and several ribosomal proteins were identified for the first time as negative markers at early PD stages. Actin was suggested to be one of the important targets in the elucidation of the etiology of neuronal diseases such as PD or other synucleinopathies.
Biochemical and Biophysical Research Communications, 2005
Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), ... more Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of ParkinsonÕs disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of a-synuclein (a-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and a-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.
Biochemical and Biophysical Research Communications, 2005
Alzheimer's & Dementia, 2013
Alzheimer's & Dementia, 2006
alpha-Synuclein is a component of Lewy bodies in affected neurons of patients with Parkinson's di... more alpha-Synuclein is a component of Lewy bodies in affected neurons of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and missense mutations or multiplications in alpha-synuclein gene have been identified in pedigrees of familial PD (FPD)/DLB, implicating alpha-synuclein in the pathogenesis of PD and DLB. To gain insights into the mechanism whereby deposition of alpha-synuclein causes neurodegeneration, we established transgenic C. elegans (TG worms) that overexpress wild-type (wt) or FPD-mutant (A53T, A30P) human alpha-synuclein in dopamine neurons. TG worms expressing A53T or A30P FPD mutant alpha-synuclein showed significant defects in dopamine neuron function, the latter being reflected by an adoptive response for feeding (i.e., decrease in bending frequency upon entry into bacterial lawn), whereas those expressing wt alpha-synuclein were normal. We next generated TG worms that overexpress human alpha-synuclein in touch neurons under the control of a mec-7 promoter. These TG worms, especially those expressing A30P FPD mutant alpha-synuclein, showed impaired touch response, whereas those expressing wt alpha-synuclein showed a normal response. To examine the pathogenic significance of alpha-synuclein phosphorylation at Ser129, a characteristic feature of alpha-synuclein deposited in synucleinopathy brains, we next generated TG worms that pan-neuronally overexpress human G protein-coupled receptor kinase 5 (GRK5), previously reported as one of the alpha-synuclein kinases, and crossed them with the alpha-synculein TG worms. Phosphorylation of alpha-synuclein was upregulated in dopamine or touch neurons of double TG worms compared to those in TG worms that singly overexpress alpha-synuclein, as determined by immunohistochemisty. Behavioral examinations showed aggravations in functions of dopamine neurons or touch neurons overexpressing wt alpha-synuclein upon co-expression of GRK5, which were not observed upon co-expression of kinase-inactive mutant GRK5 (K215R). These results support the notion that missense mutations as well as Ser129 phosphorylation of alpha-synuclein contribute to neuronal dysfunction in PD and DLB.
Annals of Clinical and Translational Neurology, 2015
Objective: Dementia with Lewy bodies (DLB) is associated with the accumulation of wild-type human... more Objective: Dementia with Lewy bodies (DLB) is associated with the accumulation of wild-type human a-synuclein (SYN) in neurons and with prominent slowing of brain oscillations on electroencephalography (EEG). However, it remains uncertain whether the EEG abnormalities are actually caused by SYN. Methods: To determine whether SYN can cause neural network abnormalities, we performed EEG recordings and analyzed the expression of neuronal activitydependent gene products in SYN transgenic mice. We also carried out comparative analyses in humans with DLB. Results: We demonstrate that neuronal expression of SYN in transgenic mice causes a left shift in spectral power that closely resembles the EEG slowing observed in DLB patients. Surprisingly, SYN mice also had seizures and showed molecular hippocampal alterations indicative of aberrant network excitability, including calbindin depletion in the dentate gyrus. In postmortem brain tissues from DLB patients, we found reduced levels of calbindin mRNA in the dentate gyrus. Furthermore, nearly one quarter of DLB patients showed myoclonus, a clinical sign of aberrant network excitability that was associated with an earlier age of onset of cognitive impairments. In SYN mice, partial suppression of epileptiform activity did not alter their shift in spectral power. Furthermore, epileptiform activity in human amyloid precursor protein transgenic mice was not associated with a left shift in spectral power. Interpretation: We conclude that neuronal accumulation of SYN slows brain oscillations and, in parallel, causes aberrant network excitability that can escalate into seizure activity. The potential role of aberrant network excitability in DLB merits further investigation.