- Email: [email protected]
Experimental models of genetic prion diseases
Symposium S1-02: Related Dementias metabolic risk states, which may precede or result from underlying dementiarelated neuropathologies. This is exemplified during the prodromal phase of dementia, as vascular and metabolic parameters decline in relation to dementia development, and potentially in a different way from ‘normal’ aging. While published reports discuss mid-life and late-life relationships between vascular risk factors and dementia, continuous data from longitudinal studies also point to a natural history of vascular and metabolic parameters in relationship to dementia onset. New genetic findings enrich the evidence base for a vascular and metabolic etiology, as well as provide a broader evidence base for potential involvement of peripheral metabolism in health of the brain. Methods: To evaluate the epidemiologic evidence for vascular and metabolic risk in dementia etiology and progression, a review of the epidemiologic literature is provided. Longitudinal population data are presented from different ages and stages of adult life to illustrate the evolutionary role of these factors in dementia. Potential biological mechanisms are discussed. Results: Studies suggest that the combined influence of vascular and metabolic factors contribute to risk and progression of dementia. Traditional vascular risk factors mediate their effects not only through effects on the vasculature, but via their roles in altering metabolism. In addition, neuropathologies accompanying dementia influence key regulatory axes modulating vascular and metabolic risk. Conclusions: Dementia is a disorder involving both brain and periphery and including both vascular and metabolic risk components. SUNDAY, JULY 11, 2010 SYMPOSIUM S1-02 RELATED DEMENTIAS S1-02-01
EXPERIMENTAL MODELS OF LRRK2 DYSFUNCTION IN PARKINSON’S DISEASE
Darren J. Moore, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland. Contact e-mail: [email protected] Background: Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson’s disease (PD) and also contribute to sporadic PD. The LRRK2 gene encodes a large multi-domain protein that functions as both a GTPase and a serine-/threonine-directed protein kinase. Disease-associated mutant forms of human LRRK2 induce neuronal toxicity through a mechanism dependent on both GTPase and kinase activity. However, the molecular mechanisms underlying LRRK2-linked disease are poorly understood. Methods: Here we describe two distinct models that we have developed to probe the pathophysiology of LRRK2. Firstly, we have developed a simple model of LRRK2-induced cytotoxicity in the baker’s yeast, Saccharomyces cerevisiae. Results: In this model, a key role for the GTPase domain is revealed in mediating the detrimental effects of LRRK2. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is associated with defects in vesicular trafficking pathways. LRRK2-induced toxicity acts through a mechanism distinct from toxicity induced by human alpha-synuclein in yeast. Genome-wide screening has identified a number of genetic interactions that enhance or suppress LRRK2induced toxicity. This yeast model will provide novel insight into the molecular mechanisms underlying LRRK2-induced cellular toxicity. To model LRRK2-linked disease in vivo, we have also developed transgenic mice expressing full-length human LRRK2 variants from a hybrid CMV-enhanced human PDGF-beta promoter. These mice exhibit widespread transgene expression throughout the brain, including the nigrostriatal dopaminergic pathway. Mutant LRRK2 transgenic mice display subtle motoric and neurochemical abnormalities. However, G2019S mutant LRRK2 mice exhibit a loss of substantia nigra dopaminergic neurons and neuritic processes at advanced ages concomitant with ultrastructural cytopathology. Conclusions: These LRRK2 transgenic mice provide important tools for understanding the molecular mechanisms through which disease-associated LRRK2 mutations induce neurodegeneration in PD. The general biology and pathophysiology of LRRK2 will be discussed.
S1-02-02
S65
EXPERIMENTAL MODELS OF GENETIC PRION DISEASES
Roberto Chiesa, Mario Negri Institute for Pharmacological Research, Milan, Italy. Contact e-mail: [email protected] Background: Approximately 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to insertional or point mutations in the gene encoding PrP. Different PrP mutations are associated with different disease phenotypes: Creutzfeldt-Jakob disease (CJD), recognized by dementia and motor abnormalities, Gerstmann-Stra¨usslerScheinker syndrome (GSS), in which ataxia is the prominent sign, and fatal familial insomnia (FFI), primarily characterized by sleep disruption and autonomic failure. It is thought that different mutations encode misfolded PrP variants selectively toxic to specific neuronal populations. Yet, the mechanism by which PrP misfolding leads to neuronal dysfunction is unknown. Methods: We have generated transgenic mice expressing mouse PrP homologues of a 72-amino acid insertion associated with GSS, and the D178N mutation linked to either CJD or FFI. Results: These mice accumulate in their brains misfolded forms of mutant PrP, and develop clinical features of the corresponding human disorders, including motor, cognitive and neurophysiological abnormalities. Conclusions: Our results indicate that mutant PrP transgenic mice recapitulate the phenotypic diversity of human prion diseases, providing experimental systems for a comprehensive dissection of pathophysiology and mechanisms of neuronal dysfunction.
S1-02-03
GENERATION AND CHARACTERIZATION OF A MOUSE MODEL OF PROGRANULIN DEFICIENCY
Terri Petkau, Scott J. Neal, Austen Milnerwood, Paul C. Orban, Howard H. Feldman, Lynn Raymond, Ian R. A. Mackenzie, Blair R. Leavitt, The University of British Columbia, Vancouver, BC, Canada. Contact e-mail: [email protected] Background: Frontotemporal dementia (FTD) is the second most common form of presenile dementia after Alzheimer’s disease. In 2006, mutations were identified in the progranulin gene (GRN) that cause a common type of familial FTD associated with a specific pattern of protein aggregation in the brain (formerly FTLD-U, now FTLD-TDP). All of the disease-causing GRN mutations result in significantly decreased levels of progranulin protein. We are developing a mouse model of human progranulin insufficiency by producing mice that lack the gene that makes progranulin. The development of such a model will be crucial for future studies examining how the loss of progranulin causes the dysfunction and death of neurons in FTD. Objective: We aim to characterize the phenotype of mice with both conditional and constitutive deletion of the mouse progranulin gene (grn). Methods: We created conditionally-targeted grn mice. These mice were generated with a second-generation gene-trap targeting vector that features Flp recombinase sites flanking a lacZ/neomycin bgeo fusion protein cassette - comprising a consensus splice acceptor, an internal ribosome entry site preceding the bgeo coding sequence, and a polyadenylation signal following the stop codon such that it will be spliced downstream of grn exons 1-4. The b-galactosidase reporter gene is expressed under the native grn promoter and was used to assess grn expression in vivo. Integration of our gene-trap vector generated two independent mouse lines with a null allele of the grn gene. Results: We have characterized the cellular and subcellular localization of progranulin in the developing and adult mouse CNS using both immunocytochemistry and b-galactosidase reporter expression in our grn-targeted mice. We will also present our behavioral, electrophysiologic and neuropathologic characterization of aged grn-deficient mice. Conclusions: Constitutively and conditionally-targeted grn mice will allow us to dissect the role of progranulin in distinct cell types and at defined times during development, and ultimately help enable us to understand how loss of progranulin function leads to CNS disease.
EXPERIMENTAL MODELS OF LRRK2 DYSFUNCTION IN PARKINSON’S DISEASE
Darren J. Moore, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland. Contact e-mail: [email protected] Background: Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson’s disease (PD) and also contribute to sporadic PD. The LRRK2 gene encodes a large multi-domain protein that functions as both a GTPase and a serine-/threonine-directed protein kinase. Disease-associated mutant forms of human LRRK2 induce neuronal toxicity through a mechanism dependent on both GTPase and kinase activity. However, the molecular mechanisms underlying LRRK2-linked disease are poorly understood. Methods: Here we describe two distinct models that we have developed to probe the pathophysiology of LRRK2. Firstly, we have developed a simple model of LRRK2-induced cytotoxicity in the baker’s yeast, Saccharomyces cerevisiae. Results: In this model, a key role for the GTPase domain is revealed in mediating the detrimental effects of LRRK2. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is associated with defects in vesicular trafficking pathways. LRRK2-induced toxicity acts through a mechanism distinct from toxicity induced by human alpha-synuclein in yeast. Genome-wide screening has identified a number of genetic interactions that enhance or suppress LRRK2induced toxicity. This yeast model will provide novel insight into the molecular mechanisms underlying LRRK2-induced cellular toxicity. To model LRRK2-linked disease in vivo, we have also developed transgenic mice expressing full-length human LRRK2 variants from a hybrid CMV-enhanced human PDGF-beta promoter. These mice exhibit widespread transgene expression throughout the brain, including the nigrostriatal dopaminergic pathway. Mutant LRRK2 transgenic mice display subtle motoric and neurochemical abnormalities. However, G2019S mutant LRRK2 mice exhibit a loss of substantia nigra dopaminergic neurons and neuritic processes at advanced ages concomitant with ultrastructural cytopathology. Conclusions: These LRRK2 transgenic mice provide important tools for understanding the molecular mechanisms through which disease-associated LRRK2 mutations induce neurodegeneration in PD. The general biology and pathophysiology of LRRK2 will be discussed.
S1-02-02
S65
EXPERIMENTAL MODELS OF GENETIC PRION DISEASES
Roberto Chiesa, Mario Negri Institute for Pharmacological Research, Milan, Italy. Contact e-mail: [email protected] Background: Approximately 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to insertional or point mutations in the gene encoding PrP. Different PrP mutations are associated with different disease phenotypes: Creutzfeldt-Jakob disease (CJD), recognized by dementia and motor abnormalities, Gerstmann-Stra¨usslerScheinker syndrome (GSS), in which ataxia is the prominent sign, and fatal familial insomnia (FFI), primarily characterized by sleep disruption and autonomic failure. It is thought that different mutations encode misfolded PrP variants selectively toxic to specific neuronal populations. Yet, the mechanism by which PrP misfolding leads to neuronal dysfunction is unknown. Methods: We have generated transgenic mice expressing mouse PrP homologues of a 72-amino acid insertion associated with GSS, and the D178N mutation linked to either CJD or FFI. Results: These mice accumulate in their brains misfolded forms of mutant PrP, and develop clinical features of the corresponding human disorders, including motor, cognitive and neurophysiological abnormalities. Conclusions: Our results indicate that mutant PrP transgenic mice recapitulate the phenotypic diversity of human prion diseases, providing experimental systems for a comprehensive dissection of pathophysiology and mechanisms of neuronal dysfunction.
S1-02-03
GENERATION AND CHARACTERIZATION OF A MOUSE MODEL OF PROGRANULIN DEFICIENCY
Terri Petkau, Scott J. Neal, Austen Milnerwood, Paul C. Orban, Howard H. Feldman, Lynn Raymond, Ian R. A. Mackenzie, Blair R. Leavitt, The University of British Columbia, Vancouver, BC, Canada. Contact e-mail: [email protected] Background: Frontotemporal dementia (FTD) is the second most common form of presenile dementia after Alzheimer’s disease. In 2006, mutations were identified in the progranulin gene (GRN) that cause a common type of familial FTD associated with a specific pattern of protein aggregation in the brain (formerly FTLD-U, now FTLD-TDP). All of the disease-causing GRN mutations result in significantly decreased levels of progranulin protein. We are developing a mouse model of human progranulin insufficiency by producing mice that lack the gene that makes progranulin. The development of such a model will be crucial for future studies examining how the loss of progranulin causes the dysfunction and death of neurons in FTD. Objective: We aim to characterize the phenotype of mice with both conditional and constitutive deletion of the mouse progranulin gene (grn). Methods: We created conditionally-targeted grn mice. These mice were generated with a second-generation gene-trap targeting vector that features Flp recombinase sites flanking a lacZ/neomycin bgeo fusion protein cassette - comprising a consensus splice acceptor, an internal ribosome entry site preceding the bgeo coding sequence, and a polyadenylation signal following the stop codon such that it will be spliced downstream of grn exons 1-4. The b-galactosidase reporter gene is expressed under the native grn promoter and was used to assess grn expression in vivo. Integration of our gene-trap vector generated two independent mouse lines with a null allele of the grn gene. Results: We have characterized the cellular and subcellular localization of progranulin in the developing and adult mouse CNS using both immunocytochemistry and b-galactosidase reporter expression in our grn-targeted mice. We will also present our behavioral, electrophysiologic and neuropathologic characterization of aged grn-deficient mice. Conclusions: Constitutively and conditionally-targeted grn mice will allow us to dissect the role of progranulin in distinct cell types and at defined times during development, and ultimately help enable us to understand how loss of progranulin function leads to CNS disease.