RESUMO
AIMS: Synaptic dysfunction in Parkinson's disease is caused by propagation of pathogenic α-synuclein between neurons. Previously, in multiple system atrophy (MSA), pathologically characterised by ectopic deposition of abnormal α-synuclein predominantly in oligodendrocytes, we demonstrated that the occurrence of memory impairment was associated with the number of α-synuclein-positive neuronal cytoplasmic inclusions (NCIs) in the hippocampus. In the present study, we aimed to investigate how abnormal α-synuclein in the hippocampus can lead to memory impairment. METHODS: We performed pathological and biochemical analyses using a mouse model of adult-onset MSA and human cases (MSA, N = 25; Parkinson's disease, N = 3; Alzheimer's disease, N = 2; normal controls, N = 11). In addition, the MSA model mice were examined behaviourally and physiologically. RESULTS: In the MSA model, inducible human α-synuclein was first expressed in oligodendrocytes and subsequently accumulated in the cytoplasm of excitatory hippocampal neurons (NCI-like structures) and their presynaptic nerve terminals with the development of memory impairment. α-Synuclein oligomers increased simultaneously in the hippocampus of the MSA model. Hippocampal dendritic spines also decreased in number, followed by suppression of long-term potentiation. Consistent with these findings obtained in the MSA model, post-mortem analysis of human MSA brain tissues showed that cases of MSA with memory impairment developed more NCIs in excitatory hippocampal neurons along with α-synuclein oligomers than those without. CONCLUSIONS: Our results provide new insights into the role of α-synuclein oligomers as a possible pathological cause of memory impairment in MSA.
Assuntos
Atrofia de Múltiplos Sistemas , Doença de Parkinson , Humanos , Atrofia de Múltiplos Sistemas/patologia , alfa-Sinucleína/metabolismo , Doença de Parkinson/patologia , Corpos de Inclusão/patologia , Neurônios/patologia , Encéfalo/patologiaRESUMO
Wilson's disease is an autosomal-recessive disorder of copper metabolism caused by mutations in ATP7B and associated with neurological, psychiatric, ophthalmological and hepatic manifestations. Decoppering treatments are used to prevent disease progression and reduce symptoms, but neurological outcomes remain mixed. In this article, we review the current understanding of pathogenesis, biomarkers and treatments for Wilson's disease from the neurological perspective, with a focus on recent advances. The genetic and molecular mechanisms associated with ATP7B dysfunction have been well characterised, but despite extensive efforts to identify genotype-phenotype correlations, the reason why only some patients develop neurological or psychiatric features remains unclear. We discuss pathological processes through which copper accumulation leads to neurodegeneration, such as mitochondrial dysfunction, the role of brain iron metabolism and the broader concept of selective neuronal vulnerability in Wilson's disease. Delayed diagnoses continue to be a major problem for patients with neurological presentations. We highlight limitations in our current approach to making a diagnosis and novel diagnostic biomarkers, including the potential for newborn screening programmes. We describe recent progress in developing imaging and wet (fluid) biomarkers for neurological involvement, including findings from quantitative MRI and other neuroimaging studies, and the development of a semiquantitative scoring system for assessing radiological severity. Finally, we cover the use of established and novel chelating agents, paradoxical neurological worsening, and progress developing targeted molecular and gene therapy for Wilson's disease, before discussing future directions for translational research.
Assuntos
Encéfalo/diagnóstico por imagem , ATPases Transportadoras de Cobre/genética , Cobre/metabolismo , Degeneração Hepatolenticular/diagnóstico , Biomarcadores/metabolismo , Encéfalo/metabolismo , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/terapia , Humanos , Ferro/metabolismo , Imageamento por Ressonância MagnéticaRESUMO
The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia, and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer's disease and Parkinson's disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition.
Assuntos
Estudo de Associação Genômica Ampla , Doença por Corpos de Lewy/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/genética , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Glucosilceramidase/genética , Humanos , Proteínas Nucleares/genética , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único , Proteínas Supressoras de Tumor/genética , alfa-Sinucleína/genéticaRESUMO
Impulsive compulsive behaviours in Parkinson's disease have been linked to increased dopaminergic release in the ventral striatum and excessive stimulation of dopamine D3 receptors. Thirty-one patients with impulsive compulsive behaviours and Parkinson's disease who donated their brains to the Queen Square Brain Bank for Neurological Disorders were assessed for α-synuclein neuropathological load and tyrosine hydroxylase levels in the nucleus accumbens, dorsal putamen and caudate using immunohistochemistry. Dopamine D2 and dopamine D3 receptors protein levels in the nucleus accumbens, frontal cortex and putamen were determined using western blotting. Results were compared to 29 Parkinson's disease cases without impulsive compulsive behaviours matched by age, sex, disease duration, age at Parkinson's disease onset and disease duration. The majority of patients with impulsive compulsive behaviours had dopamine dysregulation syndrome. Patients with Parkinson's disease and impulsive compulsive behaviours had lower α-synuclein load and dopamine D3 receptor levels in the nucleus accumbens. No differences were seen between groups in the other brain areas and in the analysis of tyrosine hydroxylase and dopamine D2 receptor levels. Lower α-synuclein load in the nucleus accumbens of individuals with Parkinson's disease and impulsive compulsive behaviours was confirmed on western blotting. Downregulation of the dopamine D3 receptor levels may have occurred either as a consequence of the degenerative process or of a pre-morbid trait. The lower levels of α-synuclein may have contributed to an excessive stimulation of the ventral striatum resulting in impulsive compulsive behaviours.
Assuntos
Comportamento Compulsivo/metabolismo , Comportamento Compulsivo/psicologia , Comportamento Impulsivo , Núcleo Accumbens/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/psicologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , alfa-Sinucleína/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Autopsia , Comportamento Compulsivo/patologia , Feminino , Humanos , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Núcleo Accumbens/patologia , Doença de Parkinson/patologia , Receptores de Dopamina D2/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Importance: Clinical subtyping of Parkinson disease at diagnosis is useful in estimating disease course and survival. Severity and rate of progression of neuropathologies are important determinants of clinical Parkinson subtypes. Objective: To provide longitudinal clinical disease-course data and neuropathologic correlation for newly proposed Parkinson disease subtypes. Design, Setting, and Participants: Retrospective cohort study of consecutive patients with autopsy-confirmed Parkinson disease who were regularly seen throughout their disease course by hospital specialists in the United Kingdom and donated their brain at death to the Queen Square Brain Bank between January 2009 and December 2017. Patients with additional neuropathologic diagnoses, monogenic forms of parkinsonism, or insufficiently detailed clinical information were excluded. Based on severity of motor symptoms, rapid eye movement sleep behavior disorder, and autonomic and cognitive function at diagnosis, patients were classified adapting a subtyping classification into mild-motor predominant, intermediate, or diffuse malignant subtypes. Main Outcomes and Measures: Time from diagnosis to disease milestones (recurrent falls, wheelchair dependence, dementia, and care home placement) and death were compared between subtypes, and their risk was estimated using Cox hazard regression models. Severity and distribution of Lewy pathology and Alzheimer disease-related pathology were assessed using staging systems. Results: From a total of 146 patients, 111 patients were included (67 men [60.4%]; mean [SD] age at diagnosis, 62.5 [11.5] years). The diffuse malignant subtype had earlier development of milestones and reduced survival. Cox proportional hazard regression showed an increased adjusted risk of any disease milestone (hazard ratio, 10.90; 95% CI, 5.51-21.58; P < .001) and death (hazard ratio, 3.65; 95% CI, 1.98-6.75; P < .001) in the diffuse malignant group. Age at diagnosis was the only additional variable with statistical significance (adjusted hazard ratio for death, 1.14; 95% CI, 1.11-1.17; P <.001). Staging of Lewy pathology and Alzheimer disease-related pathology did not differ between subtypes, although they showed different rates of progression, and the latter was associated with age at death. Conclusions and Relevance: Parkinson clinical subtypes at diagnosis may estimate disease course and survival, which may be useful in providing a more accurate prognosis in individual patients in clinical practice and helping to stratify subgroups in clinical trials. Different severity and progression of neuropathologies are important determinants of Parkinson subtypes, and age at diagnosis should be included in future subtype classifications.
Assuntos
Encéfalo/patologia , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Idade de Início , Idoso , Doenças do Sistema Nervoso Autônomo/etiologia , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Autopsia , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/fisiopatologia , Progressão da Doença , Feminino , Humanos , Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Doença de Parkinson/classificação , Doença de Parkinson/complicações , Placa Amiloide/patologia , Prognóstico , Modelos de Riscos Proporcionais , Transtorno do Comportamento do Sono REM/etiologia , Transtorno do Comportamento do Sono REM/fisiopatologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Taxa de SobrevidaRESUMO
LRRK2, the gene encoding the multidomain kinase Leucine-Rich Repeat Kinase 2 (LRRK2), has been linked to familial and sporadic forms of Parkinson's disease (PD), as well as cancer, leprosy and Crohn's disease, establishing it as a target for discovery therapeutics. LRRK2 has been associated with a range of cellular processes, however its physiological and pathological functions remain unclear. The most prevalent LRRK2 mutations in PD have been shown to affect macroautophagy in various cellular models while a role in autophagy signalling has been recapitulated in vivo. Dysregulation of autophagy has been implicated in PD pathology, and this raises the possibility that differential autophagic activity is relevant to disease progression in PD patients carrying LRRK2 mutations. To examine the relevance of LRRK2 to the regulation of macroautophagy in a disease setting we examined the levels of autophagic markers in the basal ganglia of G2019S LRRK2 PD post-mortem tissue, in comparison to pathology-matched idiopathic PD (iPD), using immunoblotting (IB). Significantly lower levels of p62 and LAMP1 were observed in G2019S LRRK2 PD compared to iPD cases. Similarly, an increase in ULK1 was observed in iPD but was not reflected in G2019S LRRK2 PD cases. Furthermore, examination of p62 by immunohistochemistry (IH) recapitulated a distinct signature for G2019S PD. IH of LAMP1, LC3 and ULK1 broadly correlated with the IB results. Our data from a small but pathologically well-characterized cases highlights a divergence of G2019S PD carriers in terms of autophagic response in alpha-synuclein pathology affected brain regions compared to iPD.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/fisiologia , Corpos de Lewy/metabolismo , Doença de Parkinson/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autofagia/fisiologia , Encéfalo/fisiopatologia , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Corpos de Lewy/patologia , Proteínas de Membrana Lisossomal/análise , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , alfa-Sinucleína/metabolismoRESUMO
BACKGROUND: Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has neuroprotective effects in preclinical models of Parkinson's disease. We investigated whether these effects would be apparent in a clinical trial. METHODS: In this single-centre, randomised, double-blind, placebo-controlled trial, patients with moderate Parkinson's disease were randomly assigned (1:1) to receive subcutaneous injections of exenatide 2 mg or placebo once weekly for 48 weeks in addition to their regular medication, followed by a 12-week washout period. Eligible patients were aged 25-75 years, had idiopathic Parkinson's disease as measured by Queen Square Brain Bank criteria, were on dopaminergic treatment with wearing-off effects, and were at Hoehn and Yahr stage 2·5 or less when on treatment. Randomisation was by web-based randomisation with a two strata block design according to disease severity. Patients and investigators were masked to treatment allocation. The primary outcome was the adjusted difference in the Movement Disorders Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor subscale (part 3) in the practically defined off-medication state at 60 weeks. All efficacy analyses were based on a modified intention-to-treat principle, which included all patients who completed any post-randomisation follow-up assessments. The study is registered at ClinicalTrials.gov (NCT01971242) and is completed. FINDINGS: Between June 18, 2014, and March 13, 2015, 62 patients were enrolled and randomly assigned, 32 to exenatide and 30 to placebo. Our primary analysis included 31 patients in the exenatide group and 29 patients in the placebo group. At 60 weeks, off-medication scores on part 3 of the MDS-UPDRS had improved by 1·0 points (95% CI -2·6 to 0·7) in the exenatide group and worsened by 2·1 points (-0·6 to 4·8) in the placebo group, an adjusted mean difference of -3·5 points (-6·7 to -0·3; p=0·0318). Injection site reactions and gastrointestinal symptoms were common adverse events in both groups. Six serious adverse events occurred in the exenatide group and two in the placebo group, although none in either group were judged to be related to the study interventions. INTERPRETATION: Exenatide had positive effects on practically defined off-medication motor scores in Parkinson's disease, which were sustained beyond the period of exposure. Whether exenatide affects the underlying disease pathophysiology or simply induces long-lasting symptomatic effects is uncertain. Exenatide represents a major new avenue for investigation in Parkinson's disease, and effects on everyday symptoms should be examined in longer-term trials. FUNDING: Michael J Fox Foundation for Parkinson's Research.
Assuntos
Doença de Parkinson/tratamento farmacológico , Peptídeos/administração & dosagem , Peçonhas/administração & dosagem , Adulto , Idoso , Método Duplo-Cego , Esquema de Medicação , Exenatida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Exame Neurológico/efeitos dos fármacos , Resultado do TratamentoRESUMO
INTRODUCTION: There is an urgent need to identify robust biomarkers for Parkinson's disease (PD). Previous studies have shown changes in composition and secretion of saliva in patients with PD, including an increase in salivary DJ-1 concentration. Autonomic dysfunction is a known feature of PD and could contribute to abnormal salivary gland function. METHODS: In this pilot cross-sectional study, characterisation of the saliva of 16 patients with PD and 22 age-matched controls was performed. Salivary DJ-1 concentration was measured with quantitative immunoblotting; total protein concentration with a BCA assay and spectrophotometry; amylase with an amylase activity assay; albumin with an ELISA and mucin concentration with periodic-acid Schiff staining of SDS-gels. RESULTS: Patient saliva showed an increase in both total protein concentration (8.4 vs 5.0 mg/ml, p = 0.0002) and DJ-1 concentration (0.84 vs 0.42 µg/ml, p = 0.001), but there was no difference in salivary DJ-1 after adjusting for total protein concentration. In patients, adjusted DJ-1 levels correlated with disease severity measured with the MDS-Unified Parkinson's Disease Rating Scale (p = 0.019). Patient saliva had elevated concentrations of amylase (127 vs 64 units/ml, p = 0.0005) and albumin (110 vs 47µg/ml, p = 0.0003) but not mucins. CONCLUSIONS: This study suggests that the saliva of patients with PD is different in composition to that of healthy age-matched controls, supporting the notion that saliva may be a good candidate for biomarker discovery in PD. The specific differences suggest that major salivary glands and gingival crevicular fluid may both be sources of additional DJ-1 and protein in patient saliva.
Assuntos
Biomarcadores/análise , Peptídeos e Proteínas de Sinalização Intracelular/análise , Proteínas Oncogênicas/análise , Doença de Parkinson/metabolismo , Saliva/química , Proteínas e Peptídeos Salivares/análise , Idoso , Western Blotting , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas/metabolismo , Projetos Piloto , Proteína Desglicase DJ-1 , Proteínas e Peptídeos Salivares/metabolismoRESUMO
The major pathway by which the brain obtains essential omega-3 fatty acids from the circulation is through a sodium-dependent lysophosphatidylcholine (LPC) transporter (MFSD2A), expressed in the endothelium of the blood-brain barrier. Here we show that a homozygous mutation affecting a highly conserved MFSD2A residue (p.Ser339Leu) is associated with a progressive microcephaly syndrome characterized by intellectual disability, spasticity and absent speech. We show that the p.Ser339Leu alteration does not affect protein or cell surface expression but rather significantly reduces, although not completely abolishes, transporter activity. Notably, affected individuals displayed significantly increased plasma concentrations of LPCs containing mono- and polyunsaturated fatty acyl chains, indicative of reduced brain uptake, confirming the specificity of MFSD2A for LPCs having mono- and polyunsaturated fatty acyl chains. Together, these findings indicate an essential role for LPCs in human brain development and function and provide the first description of disease associated with aberrant brain LPC transport in humans.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Microcefalia/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Animais , Sequência de Bases , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Células HEK293 , Humanos , Lactente , Lisofosfatidilcolinas/sangue , Masculino , Microcefalia/sangue , Mutação de Sentido Incorreto , Linhagem , Análise de Sequência de DNA , Simportadores , SíndromeRESUMO
Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.
Assuntos
Proteases Dependentes de ATP/genética , Endopeptidase Clp/genética , Exoma/genética , Genes Recessivos , Disgenesia Gonadal 46 XX/etiologia , Perda Auditiva Neurossensorial/etiologia , Mitocôndrias/enzimologia , Mutação/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Feminino , Homozigoto , Humanos , Hibridização In Situ , Masculino , Mitocôndrias/genética , Linhagem , Fenótipo , Adulto JovemRESUMO
DYT1 dystonia is caused by an autosomal dominant mutation that leads to a glutamic acid deletion in torsinA (TA), a member of the AAA+ ATPase superfamily. In this study, we identified a novel-binding partner of TA, the subunit 4 (CSN4) of CSN signalosome. TA binds CSN4 and the synaptic regulator snapin in neuroblastoma cells and in brain synaptosomes. CSN4 and TA are required for the stability of both snapin and the synaptotagmin-specific endocytic adaptor stonin 2, as downregulation of CSN4 or TA reduces the levels of both proteins. Snapin is phosphorylated by the CSN-associated kinase protein kinase D (PKD) and its expression is decreased upon PKD inhibition. In contrast, the stability of stonin 2 is regulated by neddylation, another CSN-associated activity. Overexpression of the pathological TA mutant (ΔE-TA) reduces stonin 2 expression, causing the accumulation of the calcium sensor synaptotagmin 1 on the cell surface. Retrieval of surface-stranded synaptotagmin 1 is restored by overexpression of stonin 2 in ΔE-TA-expressing cells, suggesting that the DYT1 mutation compromises the role of TA in protein stabilisation and synaptic vesicle recycling.
Assuntos
Proteínas de Transporte/metabolismo , Chaperonas Moleculares/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Encéfalo/metabolismo , Complexo do Signalossomo COP9 , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Distonia/genética , Distonia/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.
Assuntos
Endocitose/fisiologia , Chaperonas Moleculares/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Dopamina/genética , Dopamina/metabolismo , Distonia Muscular Deformante/genética , Distonia Muscular Deformante/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Células PC12 , Mutação Puntual , RNA Interferente Pequeno/genética , Ratos , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
SPG4, the gene encoding for spastin, a member of the ATPases associated with various cellular activities (AAA) family, is mutated in around 40% of cases of autosomal dominant hereditary spastic paraplegia (AD-HSP). This group of neurodegenerative diseases is characterized by a progressive spasticity and lower limb weakness with degeneration of terminal axons in cortico-spinal tracts and dorsal columns. Spastin has two main domains, a microtubule interacting and endosomal trafficking (MIT) domain at the N-terminus and the C-terminus AAA domain. Early studies suggested that spastin interacts with microtubules similarly to katanin, a member of the same subgroup of AAA. Recent evidence confirmed that spastin possesses microtubule-severing activity but can also bundle microtubules in vitro. Understanding the physiologic and pathologic involvement of these activities and their regulation is critical in the study of HSP.
Assuntos
Adenosina Trifosfatases/fisiologia , Microtúbulos/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Animais , Humanos , Doenças Neurodegenerativas/metabolismo , EspastinaRESUMO
Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP.
Assuntos
Adenosina Trifosfatases/fisiologia , Microtúbulos/fisiologia , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Adenilil Imidodifosfato/farmacologia , Linhagem Celular , Análise Mutacional de DNA/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Guanosina Trifosfato/farmacologia , Histidina/metabolismo , Humanos , Katanina , Microscopia Eletrônica de Varredura/métodos , Microtúbulos/ultraestrutura , Modelos Biológicos , Biologia Molecular/métodos , Mutagênese/fisiologia , Neuroblastoma , Paclitaxel/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Espastina , Fatores de Tempo , Transfecção/métodos , Tubulina (Proteína)/farmacologiaRESUMO
A single GAG deletion in the DYT1 gene causes primary early-onset, generalized torsion dystonia. The DYT1 protein product, torsinA, belongs to the AAA+ family of proteins. When overexpressed, wild-type torsinA localizes mainly to the endoplasmic reticulum, whereas the mutant forms inclusions of unclear biogenetic origin. In this study, overexpressed wild-type torsinA in human neuroblastoma (SH-SY5Y) cell lines was distributed throughout the cell body and colocalized with a marker for the endoplasmic reticulum, confirming it is an endoplasmic reticulum protein. However, mutant torsinA showed perinuclear staining and formed distinct globular inclusions, which did not colocalize with endoplasmic reticulum markers. Immunoelectron microscopy of the mutant torsinA inclusions revealed membrane whorls staining for torsinA, as well as labeling of lamellae, isolated bilayers, and perinuclear membranes. This finding shows that mutant torsinA redistributes to specific membranous structures, which may represent different stages of maturation of the intracellular inclusions. The mutant torsinA-containing bodies were immunoreactive for vesicular monoamine transporter 2 (VMAT2). VMAT2 expression is important for the exocytosis of bioactive monoamines in neurons. Abnormal processing, transport, or entrapment of VMAT2 within the mutant torsinA membranous inclusions, therefore, may affect cellular dopamine release, providing a potential pathogenic mechanism for the DYT1-dependent dystonia.
Assuntos
Distonia Muscular Deformante/genética , Distonia Muscular Deformante/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Mutação Puntual/genética , Fatores Etários , Western Blotting , Linhagem Celular Tumoral , DNA Complementar/genética , Imunofluorescência , Deleção de Genes , Humanos , Microscopia Eletrônica , Neuroblastoma/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Repetições de Trinucleotídeos/genética , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
There are two known autosomal dominant genes for the hereditary ulcero-mutilating neuropathies: SPTLC1 (hereditary sensory neuropathy type 1) and RAB7 (Charcot-Marie-Tooth disease type 2B). We report a family with autosomal dominant ulcero-mutilating neuropathy, developing in the teens and characterized by ulcers, amputations, sensory involvement in the feet but no motor features. Sequencing the RAB7 gene showed a novel heterozygous A to C mutation, changing asparagine to threonine at codon 161. The mutation is situated adjacent to a previously identified valine to methionine mutation at codon 162, implying a hotspot for mutations in the highly conserved C terminus of RAB7.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação , Proteínas rab de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Asparagina/genética , Biópsia/métodos , Proteínas de Transporte/metabolismo , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Análise Mutacional de DNA/métodos , Saúde da Família , Lateralidade Funcional/fisiologia , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Neuropatias Hereditárias Sensoriais e Autônomas/fisiopatologia , Humanos , Imuno-Histoquímica/métodos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Condução Nervosa/fisiologia , Coloração e Rotulagem/métodos , Nervo Sural/metabolismo , Nervo Sural/patologia , Nervo Sural/fisiopatologia , Nervo Sural/efeitos da radiação , Treonina/genética , proteínas de unión al GTP Rab7RESUMO
We have shown in a mouse model of motor neuron disease, the legs-at-odd-angles (Loa) mutant, and that mutations in the cytoplasmic dynein heavy chain gene (Dnchc1) cause motor neuron degeneration. Mice exhibiting the Loa phenotype suffer progressive loss of locomotor function and homozygous animals have neuronal inclusion bodies that are positive for SOD1, CDK5, neurofilament and ubiquitin proteins. As this phenotype models some aspects of human motor neuron degeneration disorders, we think there is a reasonable likelihood that dynein may be a causative gene or susceptibility factor in human motor neuron disease. Therefore we have screened exons of this gene in a set of human patients with familial forms of disparate motor neuron degeneration diseases, affecting both upper and lower motor neurons: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and hereditary spastic paraplegia. As part of this study, we have determined that DNCHC1 is a large gene of 78 exons spanning 86 kb genomic length. We have focused on the exons known to be mutated in Loa, and in a very similar mouse mutation, cramping 1 (Cra1); both mutations result in loss of anterior horn cells. The exons studied are highly conserved in a wide range of eukaryotes. We screened our patient samples by sequencing and although we detect single nucleotide polymorphisms, our results show these occur at the same frequency in our patient group as in control samples of unaffected individuals. Therefore we do not find any association between familial motor neuron disease and the genotypes presented here in the exons screened.
Assuntos
Dineínas/genética , Éxons , Doença dos Neurônios Motores/genética , Subunidades Proteicas/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Genômica , Genótipo , Humanos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , População Branca/genéticaRESUMO
Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous condition, characterised principally by progressive spasticity of the lower limbs. Forty percent of autosomal dominant (AD) pedigrees show linkage to the SPG4 locus on chromosome 2, which encodes spastin, an ATPase associated with diverse cellular activities (AAA) protein. We have performed a clinical and genetic study of three AD-HSP families linked to SPG4. Sequencing revealed three novel causative mutations. Two of the mutations were located in exon 5 (a 1-base pair (bp) insertion and a 5-bp deletion), resulting in frameshift and premature termination of translation, with the predicted protein lacking the entire AAA functional domain. The 5-bp deletion was associated with a later onset and mild cerebellar features. The third mutation was a 3-bp deletion in exon 9, resulting in the loss of a highly conserved phenylalanine residue within the AAA cassette and an apparently milder phenotype. This is the first example of a deletion of an amino acid in spastin.