Mutations in α-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type α-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia ...with Lewy bodies, implicating α-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with α-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human α-synuclein in dopamine neurons. The TG worms exhibit accumulation of α-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 α-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant α-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 β-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of α-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.
Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type α-synuclein as LB 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 10 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.
Mutations in α-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type α-synuclein as Lewy bodies
occurs as a hallmark lesion of sporadic Parkinson disease and dementia ...with Lewy bodies, implicating α-synuclein in the pathogenesis
of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration
associated with α-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human α-synuclein in dopamine neurons. The TG worms
exhibit accumulation of α-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 α-synuclein, without overt loss
of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant α-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 β-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of
α-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade
and therapeutic strategies for Parkinson disease.
Formalin-fixed, paraffin-embedded (FFPE) tissue specimens are stored around the world, and a lot of useful information about gene or protein expression is potentially retained in these samples. ...However, the FFPE samples have been exclusively used to the histochemical studies (e.g. immunostaining or in situ hybridization) because the remaining nucleic acids or proteins in FFPE samples were highly degraded and bridged. Here we performed RNA extraction from rat liver FFPE specimens and measured RNA levels by quantitative reverse transcription PCR (qRT-PCR). By using commercially available RNA purification system, we successfully extracted sufficient yield and purity of RNA from the liver sections (5μm-thick), and the extracted RNA was readily PCR-amplified. We then prepared both FFPE specimens and intact (RNA preservative solution-fixed) tissues from the liver that were administered by two chemical agents, Wy14643 (PPARα agonist) and thioacetamide. We quantitatively measured RNA levels in FFPE and intact tissues, and found that both samples showed similar expression profiles. We next examined RNA levels in FFPE specimens that have been stocked over three years. These specimens are the rat liver administered by another PPARα agonist LY-518674, and the expression profiles of the intact samples were examined three years ago. RNA expression profiles of the FFPE specimens were almost similar to those of intact samples, suggesting that RNAs in FFPE specimens are well-preserved for at least three years. We further tried to extract RNA from microdissected FFPE sections of rat liver administered by thioacetamide. The expression level of HO-1 was selectively increased in the centrilobular region, whereas that of IP-10 was increased both in the centrilobular and peripheral region of the liver. These results together raise the idea that gene expression studies using archived FFPE specimens are possible and that analyses of microdissected FFPE section is useful to reveal the link between pathological changes and gene expression profiles.
The deposition of alpha-synuclein (aS), a product of pathogenic gene for dominantly inherited familial Parkinson's disease (PD; park1), as fibrillary aggregates like Lewy bodies (LB), is a hallmark ...lesion of a set of neurodegenerative disorders termed synucleinopathies, including sporadic PD and dementia with Lewy bodies (DLB). We found that aS is the major component of LBs and further identified a specific phosphorylation of Ser129 of insoluble aS by mass spectrometric analysis. The roles of DJ-1 and PINK-1, products of pathogenic genes for autosomal recessive forms of early-onset parkinsonism, have subsequently been examined. Overexpression of DJ-1 conferred cultured cells resistance to oxidative stress, suggesting an antioxidant function of DJ-1. We also confirmed the anti-PTEN function of DJ-1 that may promote cell survival, showing decreased phosphorylation of Akt through upregulation of PTEN activity upon siRNA knockdown for DJ-1. PINK-1, that had been identified as a gene upregulated by PTEN overexpression, turned out to be a protein kinase localized in mitochondria. Collectively, information derived from studies on pathogenic genes for familial PD will open up the way toward the clarification of the pathogenesis of PD, underscoring the roles of protein aggregation, proteolysis, oxidative stress and protein phosphorylation in PD.
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