RESUMO
Despite substantial progress, causal variants are identified only for a minority of familial Parkinson's disease (PD) cases, leaving high-risk pathogenic variants unidentified1,2. To identify such variants, we uniformly processed exome sequencing data of 2,184 index familial PD cases and 69,775 controls. Exome-wide analyses converged on RAB32 as a novel PD gene identifying c.213C > G/p.S71R as a high-risk variant presenting in ~0.7% of familial PD cases while observed in only 0.004% of controls (odds ratio of 65.5). This variant was confirmed in all cases via Sanger sequencing and segregated with PD in three families. RAB32 encodes a small GTPase known to interact with LRRK2 (refs. 3,4). Functional analyses showed that RAB32 S71R increases LRRK2 kinase activity, as indicated by increased autophosphorylation of LRRK2 S1292. Here our results implicate mutant RAB32 in a key pathological mechanism in PD-LRRK2 kinase activity5-7-and thus provide novel insights into the mechanistic connections between RAB family biology, LRRK2 and PD risk.
Assuntos
Predisposição Genética para Doença , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Proteínas rab de Ligação ao GTP , Humanos , Doença de Parkinson/genética , Proteínas rab de Ligação ao GTP/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Feminino , Masculino , Linhagem , Pessoa de Meia-Idade , Mutação , Exoma/genética , Sequenciamento do Exoma , Estudos de Casos e Controles , IdosoRESUMO
Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-ß1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/genética , Transporte Ativo do Núcleo Celular , Quinase 1 Relacionada a NIMA/genética , Proteínas , Neurônios Motores , Microtúbulos , HomeostaseRESUMO
Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5AΔExon27) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/genética , Transporte Axonal/genética , Mutação com Ganho de Função , Humanos , Cinesinas/genética , Mutação/genéticaRESUMO
Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease-linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.
Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Ácido Glutâmico/efeitos adversos , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Receptores de AMPA/metabolismo , Estresse Fisiológico , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica , Citoplasma , Demência Frontotemporal , Humanos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteína FUS de Ligação a RNA/genética , Receptores de AMPA/genéticaRESUMO
Aberrant translational repression is a feature of multiple neurodegenerative diseases. The association between disease-linked proteins and stress granules further implicates impaired stress responses in neurodegeneration. However, our knowledge of the proteins that evade translational repression is incomplete. It is also unclear whether disease-linked proteins influence the proteome under conditions of translational repression. To address these questions, a quantitative proteomics approach was used to identify proteins that evade stress-induced translational repression in arsenite-treated cells expressing either wild-type or amyotrophic lateral sclerosis (ALS)-linked mutant FUS. This study revealed hundreds of proteins that are actively synthesized during stress-induced translational repression, irrespective of FUS genotype. In addition to proteins involved in RNA- and protein-processing, proteins associated with neurodegenerative diseases such as ALS were also actively synthesized during stress. Protein synthesis under stress was largely unperturbed by mutant FUS, although several proteins were found to be differentially expressed between mutant and control cells. One protein in particular, COPBI, was downregulated in mutant FUS-expressing cells under stress. COPBI is the beta subunit of the coat protein I (COPI), which is involved in Golgi to endoplasmic reticulum (ER) retrograde transport. Further investigation revealed reduced levels of other COPI subunit proteins and defects in COPBI-relatedprocesses in cells expressing mutant FUS. Even in the absence of stress, COPBI localization was altered in primary and human stem cell-derived neurons expressing ALS-linked FUS variants. Our results suggest that Golgi to ER retrograde transport may be important under conditions of stress and is perturbed upon the expression of disease-linked proteins such as FUS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Neurônios Motores/metabolismo , Biossíntese de Proteínas , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Arsenitos/farmacologia , Linhagem Celular Tumoral , Complexo I de Proteína do Envoltório/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Humanos , Camundongos , Neurônios Motores/efeitos dos fármacos , Mutação , Biossíntese de Proteínas/efeitos dos fármacos , Proteômica , Proteína FUS de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress. RESULTS: We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved. CONCLUSIONS: Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Grânulos Citoplasmáticos/metabolismo , Estresse Oxidativo/fisiologia , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Western Blotting , Linhagem Celular , Grânulos Citoplasmáticos/patologia , Imunofluorescência , Humanos , Camundongos , Transdução GenéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Predisposição Genética para Doença/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Profilinas/genética , Profilinas/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Células Cultivadas , Exoma/genética , Feminino , Cones de Crescimento/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Judeus/genética , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Proteínas Mutantes/genética , Linhagem , Conformação Proteica , Ubiquitinação , População Branca/genéticaRESUMO
Axonemal dyneins are multisubunit molecular motors that provide the driving force for flagellar motility. Dynein light chain 1 (LC1) has been well studied in Chlamydomonas reinhardtii and is unique among all dynein components as the only protein known to bind directly to the catalytic motor domain of the dynein heavy chain. However, the role of LC1 in dynein assembly and/or function is unknown because no mutants have previously been available. We identified an LC1 homologue (TbLC1) in Trypanosoma brucei and have investigated its role in trypanosome flagellar motility using epitope tagging and RNAi studies. TbLC1 is localized along the length of the flagellum and partitions between the axoneme and soluble fractions following detergent and salt extraction. RNAi silencing of TbLC1 gene expression results in the complete loss of the dominant tip-to-base beat that is a hallmark of trypanosome flagellar motility and the concomitant emergence of a sustained reverse beat that propagates base-to-tip and drives cell movement in reverse. Ultrastructure analysis revealed that outer arm dyneins are disrupted in TbLC1 mutants. Therefore LC1 is required for stable dynein assembly and forward motility in T. brucei. Our work provides the first functional analysis of LC1 in any organism. Together with the recent findings in T. brucei DNAI1 mutants [Branche et al. (2006). Conserved and specific functions of axoneme components in trypanosome motility. J. Cell Sci. 119, 3443-3455], our data indicate functionally specialized roles for outer arm dyneins in T. brucei and C. reinhardtii. Understanding these differences will provide a more robust description of the fundamental mechanisms underlying flagellar motility and will aid efforts to exploit the trypanosome flagellum as a drug target.
Assuntos
Dineínas/fisiologia , Flagelos/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Animais , Dineínas/genética , Dineínas/metabolismo , Flagelos/genética , Flagelos/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the eukaryotic flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.