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In JoVE (1)
Other Publications (10)
- Experimental Eye Research
- Proceedings of the National Academy of Sciences of the United States of America
- Proceedings of the National Academy of Sciences of the United States of America
- Molecular Brain
- Nature Genetics
- Nature Chemical Biology
- Disease Models & Mechanisms
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Human Molecular Genetics
- Science (New York, N.Y.)
Articles by Shusei Hamamichi in JoVE
JoVE General
Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes
Department of Biological Sciences, University of Alabama
This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.
Other articles by Shusei Hamamichi on PubMed
Involvement of Hepatic Glucocorticoid Receptor-mediated Functions in Steroid-induced Cataract Formation
Determination of whether the steroid-induced cataract formation is caused through glucocorticoid (GC) receptor-mediated process was conducted by using GC antagonist (RU486) and anti-GC receptor antibody, and by sucrose density gradient ultracentrifugation analysis. (1) When 15 day-old chick embryos were treated with dexamethasone (DEX, 0.025 micromol per egg), their lenses started to form an opaque ring around the peri-nuclear region (stage II-III) after 12 hr and developed into nuclear-like cataract (stage IV-V) after 44 hr. The cataract formation examined at the 44 hr could be effectively prevented by administration of RU486 (0.2 micromol per egg) ranging from 2 hr before to 12 hr after the DEX administration. (2) GC receptor was present in liver, but could not be determined in lens by western blot analysis using monoclonal anti-GC receptor antibody. (3) Sucrose gradient ultracentrifugation analysis indicated that the receptor (9S) in the liver could be transformed to the 4S form after 0.4M NaCl treatment. Combined with our previous data, this suggests that changes in hepatic functions mediated by the GC receptor after the GC administration may be involved in the process of the cataract formation.
The Parkinson's Disease Protein Alpha-synuclein Disrupts Cellular Rab Homeostasis
alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.
Hypothesis-based RNAi Screening Identifies Neuroprotective Genes in a Parkinson's Disease Model
Genomic multiplication of the locus-encoding human alpha-synuclein (alpha-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of alpha-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent alpha-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.
Lysosomal Enzyme Cathepsin D Protects Against Alpha-synuclein Aggregation and Toxicity
α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.
Alpha-synuclein is Part of a Diverse and Highly Conserved Interaction Network That Includes PARK9 and Manganese Toxicity
Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn.
Rapid Selection of Cyclic Peptides That Reduce Alpha-synuclein Toxicity in Yeast and Animal Models
Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of alpha-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human alpha-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease.
Compounds from an Unbiased Chemical Screen Reverse Both ER-to-Golgi Trafficking Defects and Mitochondrial Dysfunction in Parkinson's Disease Models
alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.
C. Elegans As a Model Organism to Investigate Molecular Pathways Involved with Parkinson's Disease
Parkinson's disease (PD) is an age-related movement disorder resulting, in part, from selective loss of dopaminergic neurons. Both invertebrate and mammalian models have been developed to study the cellular mechanisms altered during disease progression; nevertheless there are limitations within each model. Mammalian models remain invaluable in studying PD, but are expensive and time consuming. Here, we review genetic and environmental factors associated with PD, and describe how the nematode roundworm, Caenorhabditis elegans, has been used as a model organism for studying various aspects of this neurodegenerative disease. Both genetic and chemical screens have been conducted in C. elegans to identify molecular pathways, proteins, and small molecules that can impact PD pathology. Lastly, we highlight future areas of investigation, in the context of emerging fields in biology, where the nematode can be exploited to provide mechanistic insights and potential strategies to accelerate the path toward possible therapeutic intervention for PD.
Inhibitors of LRRK2 Kinase Attenuate Neurodegeneration and Parkinson-like Phenotypes in Caenorhabditis Elegans and Drosophila Parkinson's Disease Models
Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.
Functional Links Between Aβ Toxicity, Endocytic Trafficking, and Alzheimer's Disease Risk Factors in Yeast
Aβ (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.
