RESUMO
Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Mutação Puntual , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/enzimologia , Mutação de Sentido Incorreto , Superóxido Dismutase/metabolismo , Superóxido Dismutase/toxicidade , Superóxido Dismutase-1RESUMO
Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation (head-bob, hb) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function.
RESUMO
Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture.
Assuntos
Sono REM , Sono , Animais , Encéfalo/fisiologia , Fenômenos Eletrofisiológicos , Camundongos , Sono/genética , Sono REM/genética , Vigília/genéticaRESUMO
Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.
Assuntos
Envelhecimento/genética , Testes Genéticos , Mutagênese/genética , Animais , Cóclea/metabolismo , Modelos Animais de Doenças , Epitélio/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Audição/genética , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , Linhagem , FenótipoRESUMO
The circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep.
Assuntos
Envelhecimento/genética , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Sono/genética , Sono/fisiologia , Acuidade Visual/genética , Acuidade Visual/fisiologia , Animais , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
Deficits in motor function are debilitating features in disorders affecting neurological, neuromuscular and musculoskeletal systems. Although these disorders can vary greatly with respect to age of onset, symptomatic presentation, rate of progression and severity, the study of these disease models in mice is confined to the use of a small number of tests, most commonly the rotarod test. To expand the repertoire of meaningful motor function tests in mice, we tested, optimised and validated an automated home-cage-based running-wheel system, incorporating a conventional wheel with evenly spaced rungs and a complex wheel with particular rungs absent. The system enables automated assessment of motor function without handler interference, which is desirable in longitudinal studies involving continuous monitoring of motor performance. In baseline studies at two test centres, consistently significant differences in performance on both wheels were detectable among four commonly used inbred strains. As further validation, we studied performance in mutant models of progressive neurodegenerative diseases--Huntington's disease [TgN(HD82Gln)81Dbo; referred to as HD mice] and amyotrophic lateral sclerosis [Tg(SOD1G93A)(dl)1/GurJ; referred to as SOD1 mice]--and in a mutant strain with subtle gait abnormalities, C-Snap25(Bdr)/H (Blind-drunk, Bdr). In both models of progressive disease, as with the third mutant, we could reliably and consistently detect specific motor function deficits at ages far earlier than any previously recorded symptoms in vivo: 7-8 weeks for the HD mice and 12 weeks for the SOD1 mice. We also conducted longitudinal analysis of rotarod and grip strength performance, for which deficits were still not detectable at 12 weeks and 23 weeks, respectively. Several new parameters of motor behaviour were uncovered using principal component analysis, indicating that the wheel-running assay could record features of motor function that are independent of rotarod performance. This represents a powerful new method to detect motor deficits at pre-symptomatic stages in mouse disease models and should be considered as a valid tool to investigate the efficacy of therapeutic agents.
Assuntos
Modelos Animais de Doenças , Doença de Huntington/fisiopatologia , Laboratórios , Atividade Motora , Condicionamento Físico Animal , Corrida , Animais , Automação , Comportamento Animal , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Análise de Componente Principal , Padrões de Referência , Reprodutibilidade dos Testes , Teste de Desempenho do Rota-Rod , Superóxido Dismutase/metabolismoRESUMO
The recent identification of multiple dominant mutations in the gene encoding ß-catenin in both humans and mice has enabled exploration of the molecular and cellular basis of ß-catenin function in cognitive impairment. In humans, ß-catenin mutations that cause a spectrum of neurodevelopmental disorders have been identified. We identified de novo ß-catenin mutations in patients with intellectual disability, carefully characterized their phenotypes, and were able to define a recognizable intellectual disability syndrome. In parallel, characterization of a chemically mutagenized mouse line that displays features similar to those of human patients with ß-catenin mutations enabled us to investigate the consequences of ß-catenin dysfunction through development and into adulthood. The mouse mutant, designated batface (Bfc), carries a Thr653Lys substitution in the C-terminal armadillo repeat of ß-catenin and displayed a reduced affinity for membrane-associated cadherins. In association with this decreased cadherin interaction, we found that the mutation results in decreased intrahemispheric connections, with deficits in dendritic branching, long-term potentiation, and cognitive function. Our study provides in vivo evidence that dominant mutations in ß-catenin underlie losses in its adhesion-related functions, which leads to severe consequences, including intellectual disability, childhood hypotonia, progressive spasticity of lower limbs, and abnormal craniofacial features in adults.
Assuntos
Anormalidades Craniofaciais/genética , Deficiência Intelectual/genética , Mutação , beta Catenina/genética , Adolescente , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Encéfalo/patologia , Caderinas/química , Pré-Escolar , Anormalidades Craniofaciais/patologia , DNA/genética , Modelos Animais de Doenças , Feminino , Genes Dominantes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Fenótipo , Homologia de Sequência de Aminoácidos , Síndrome , Adulto Jovem , beta Catenina/química , beta Catenina/metabolismoRESUMO
Prion infections of the central nervous system (CNS) are characterized by a reactive gliosis and the subsequent degeneration of neuronal tissue. The activation of glial cells, which precedes neuronal death, is likely to be initially caused by the deposition of misfolded, in part proteinase K-resistant, isoforms (termed PrP(TSE)) of the normal cellular prion protein (PrP(c)) in the brain. Proinflammatory cytokines and chemokines released by PrP(TSE)-activated glial cells and stressed neurons may contribute directly or indirectly to the disease development by enhancement and generalization of the gliosis and via cytotoxicity for neurons. Recent studies have illustrated that interfering with inflammatory responses may represent a therapeutic approach to slow down the course of disease development. Hence, a better understanding of driving factors in neuroinflammation may well contribute to the development of improved strategies for treatment of prion diseases.
Assuntos
Encéfalo/patologia , Doenças Priônicas/patologia , Encéfalo/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Doenças Priônicas/metabolismo , Príons/metabolismoRESUMO
Axon destruction represents one aspect of prion disease-associated neurodegeneration. We characterized here the scrapie infection of Wld(S)-mice in comparison to wild-type C57Bl/6 controls to determine whether mechanisms involved in Wallerian degeneration contribute to disease development in this murine model system. The Wld(S) mutation had neither an effect on survival times, nor on typical hallmarks of a prion infection like deposition of misfolded PrP(Sc) and glia activation. At the ultrastructural level, axonal damage like loss of axoplasms and disintegration of myelin sheaths was evident. Moreover, lysosomes accumulated in neuronal cell bodies. These alterations occured however similarly in Wld(S)- and wild-type mice. In conclusion, it appears unlikely that axonal damage of the kind, which is slowed down in Wld(S)-mice, contributes significantly to disease progression. These findings distinguish the neurodegeneration occuring in this prion model from chronic neurodegenerative diseases, in which the Wld(S)-mutation provides axon protection and greatly improves the clinical outcome.
Assuntos
Encéfalo/patologia , Degeneração Neural/patologia , Neurônios/ultraestrutura , Scrapie/patologia , Degeneração Walleriana/patologia , Animais , Western Blotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/patologiaRESUMO
Prion diseases are fatal and at present there are neither cures nor therapies available to delay disease onset or progression in humans. Inspired in part by therapeutic approaches in the fields of Alzheimer's disease and amyotrophic lateral sclerosis, we tested five different drugs, which are known to efficiently pass through the blood-brain barrier, in a murine prion model. Groups of intracerebrally prion-challenged mice were treated with the drugs curcumin, dapsone, ibuprofen, memantine and minocycline. Treatment with antibiotics dapsone and minocycline had no therapeutic benefit. Ibuprofen-treated mice showed severe adverse effects, which prevented assessment of therapeutic efficacy. Mice treated with low- but not high-dose curcumin and mice treated with memantine survived infections significantly longer than untreated controls (P<0.01). These results encourage further research efforts to improve the therapeutic effect of these drugs.
Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Ibuprofeno/efeitos adversos , Doenças Priônicas/tratamento farmacológico , Animais , Doenças do Sistema Nervoso Central/mortalidade , Curcumina/farmacologia , Curcumina/uso terapêutico , Estudos de Avaliação como Assunto , Ibuprofeno/farmacologia , Ibuprofeno/uso terapêutico , Memantina/farmacologia , Memantina/uso terapêutico , Camundongos , Minociclina/farmacologia , Minociclina/uso terapêutico , Doenças Priônicas/mortalidadeRESUMO
Galectin-3 is a multi-functional protein and participates in mediating inflammatory reactions. The pronounced overexpression of galectin-3 in prion-infected brain tissue prompted us to study the role of this protein in a murine prion model. Immunofluorescence double-labelling identified microglia as the major cell type expressing galectin-3. Ablation of galectin-3 did not affect PrP(Sc)-deposition and development of gliosis. However, galectin-3(-/-)-mice showed prolonged survival times upon intracerebral and peripheral scrapie infections. Moreover, protein levels of the lysosomal activation marker LAMP-2 were markedly reduced in prion-infected galectin-3(-/-)-mice suggesting a role of galectin-3 in regulation of lysosomal functions. Lower mRNA levels of Beclin-1 and Atg5 in prion-infected wild-type and galectin-3(-/-)-mice indicated an impairment of autophagy although autophagosome formation was unchanged. The results point towards a detrimental role of galectin-3 in prion infections of the CNS and suggest that endo-/lysosomal dysfunction in combination with reduced autophagy may contribute to disease development.