RESUMO
Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.
Assuntos
Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto/genética , Fagossomos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Loss-of-function mutations in human ALS2 account for several juvenile recessive motor neuron diseases (MNDs). To understand the molecular basis underlying motor dysfunction in ALS2-linked MNDs, several lines of Als2(-/-) mice with a mixed genetic background were thus far generated, and their phenotypes were thoroughly characterized. However, several phenotypic discrepancies among different Als2-deficient lines became evident. To investigate whether genetic backgrounds are associated with such discrepancies, we here generated congenic lines of Als2(-/-) mice on two different genetic backgrounds; C57BL/6 (B6) and FVB/N (FVB), and investigated their gross phenotypes. Both B6 and FVB congenic lines were viable and fertile with no evidences for obvious abnormalities. There were no differences in growth curves between wild-type and Als2(-/-) mice on each genetic background. Remarkably, Als2(-/-) mice on a FVB, but not a B6, background exhibited a shorter life span than wild-type litters. Further, B6 female, but not male, Als2(-/-) mice showed a significantly lower spontaneous rearing activity than wild-type litters. These genetic background- and/or gender-specific findings suggest the presence of modifiers for life span and motor activities in Als2(-/-) mice. These congenic mice should provide a useful means to understand the molecular and genetic basis for variable expression of pathological phenotypes in MNDs.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fenótipo , Caracteres Sexuais , Esclerose Lateral Amiotrófica/mortalidade , Análise de Variância , Animais , Peso Corporal/genética , Modelos Animais de Doenças , Feminino , Longevidade/genética , Masculino , Camundongos , Camundongos Congênicos , Camundongos Knockout , Atividade Motora/genética , Desempenho Psicomotor/fisiologia , Análise de SobrevidaRESUMO
BACKGROUND: ALS2/alsin is a guanine nucleotide exchange factor for the small GTPase Rab5 and involved in macropinocytosis-associated endosome fusion and trafficking, and neurite outgrowth. ALS2 deficiency accounts for a number of juvenile recessive motor neuron diseases (MNDs). Recently, it has been shown that ALS2 plays a role in neuroprotection against MND-associated pathological insults, such as toxicity induced by mutant Cu/Zn superoxide dismutase (SOD1). However, molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: To address this issue, we investigated the molecular and pathological basis for the phenotypic modification of mutant SOD1-expressing mice by ALS2 loss. Genetic ablation of Als2 in SOD1(H46R), but not SOD1(G93A), transgenic mice aggravated the mutant SOD1-associated disease symptoms such as body weight loss and motor dysfunction, leading to the earlier death. Light and electron microscopic examinations revealed the presence of degenerating and/or swollen spinal axons accumulating granular aggregates and autophagosome-like vesicles in early- and even pre-symptomatic SOD1(H46R) mice. Further, enhanced accumulation of insoluble high molecular weight SOD1, poly-ubiquitinated proteins, and macroautophagy-associated proteins such as polyubiquitin-binding protein p62/SQSTM1 and a lipidated form of light chain 3 (LC3-II), emerged in ALS2-deficient SOD1(H46R) mice. Intriguingly, ALS2 was colocalized with LC3 and p62, and partly with SOD1 on autophagosome/endosome hybrid compartments, and loss of ALS2 significantly lowered the lysosome-dependent clearance of LC3 and p62 in cultured cells. CONCLUSIONS/SIGNIFICANCE: Based on these observations, although molecular basis for the distinctive susceptibilities to ALS2 loss in different mutant SOD1-expressing ALS models is still elusive, disturbance of the endolysosomal system by ALS2 loss may exacerbate the SOD1(H46R)-mediated neurotoxicity by accelerating the accumulation of immature vesicles and misfolded proteins in the spinal cord. We propose that ALS2 is implicated in endolysosomal trafficking through the fusion between endosomes and autophagosomes, thereby regulating endolysosomal protein degradation in vivo.
Assuntos
Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Lisossomos/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase/metabolismo , Animais , Autofagia , Axônios/metabolismo , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neuritos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase-1RESUMO
Loss of function mutations in the ALS2 gene account for a number of juvenile/infantile recessive motor neuron diseases, indicating that its gene product, ALS2/alsin, plays a crucial role in maintenance and survival for a subset of neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5 and is implicated in endosome dynamics in cells. However, the role of ALS2 in neurons remains unclear. To elucidate the neuronal ALS2 functions, we investigate cellular phenotypes of ALS2-deficient primary cultured neurons derived from Als2-knockout (KO) mice. Here, we show that ALS2 deficiency results not only in the delay of axon outgrowth in hippocampal neurons, but also in a decreased level of the fluid phase horseradish peroxidase (HRP) uptake, which represents the activity for macropinocytic endocytosis, in cortical neurons. Thus, ALS2 may act as a modulator in neuronal differentiation and/or development through regulation of membrane dynamics.
Assuntos
Axônios/fisiologia , Diferenciação Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Pinocitose , Animais , Axônios/metabolismo , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Hipocampo/química , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Pinocitose/genética , Pseudópodes/química , Pseudópodes/metabolismoRESUMO
ALS2 is a causative gene for a juvenile autosomal recessive form of motor neuron diseases (MNDs), including amyotrophic lateral sclerosis 2 (ALS2), juvenile primary lateral sclerosis, and infantile-onset ascending hereditary spastic paralysis. These disorders are characterized by ascending degeneration of the upper motor neurons with or without lower motor neuron involvement. Thus far, a total of 12 independent ALS2 mutations, which include a small deletion, non-sense mutation, or missense mutation spreading widely across the entire coding sequence, are reported. They are predicted to result in either premature termination of translation or substitution of an evolutionarily conserved amino acid. Thus, a loss of functions in the ALS2-coded protein accounts for motor dysfunction and/or degeneration in the ALS2-linked MNDs. The ALS2 gene encodes a novel 184kDa protein of 1657 amino acids, ALS2 or alsin, comprising three predicted guanine nucleotide exchange factor (GEF) domains: the N-terminal RCC1-like domain, the central Dbl homology and pleckstrin homology (DH/PH) domains, and the C-terminal vacuolar protein sorting 9 (VPS9) domain. In addition, eight consecutive membrane occupation and recognition nexus (MORN) motifs are noted in the region between DH/PH and VPS9 domains. ALS2 activates Rab5 small GTPase and involves in endosome/membrane trafficking and fusions in the cells, and also promotes neurite outgrowth in neuronal cultures. Further, a neuroprotective role for ALS2 against cytotoxicity; i.e., the mutant Cu/Zn-superoxide dismutase 1 (SOD1)-mediated toxicity, oxidative stress, and excitotoxicity, has recently been implied. This review outlines current understandings of the molecular and cellular functions of ALS2 and its related proteins on safeguarding the integrity of motor neurons, and sheds light on the molecular pathogenesis of MNDs as well as other conditions of neurodegenerative diseases.
Assuntos
Sistema Nervoso Central/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/fisiopatologia , Citoproteção/fisiologia , Predisposição Genética para Doença/genética , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/fisiopatologia , Neurônios Motores/patologia , Mutação/genética , Estrutura Terciária de Proteína/fisiologia , Vesículas Transportadoras/genéticaRESUMO
Mutations in the ALS2 gene cause a number of recessive motor neuron diseases, indicating that the ALS2 protein (ALS2/alsin) is vital for motor neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for Rab5 (Rab5GEF) and is involved in endosome dynamics. However, the spatiotemporal regulation of the ALS2-mediated Rab5 activation is unclear. Here we identified an upstream activator for ALS2 and showed a functional significance of the ALS2 activation in endosome dynamics. ALS2 preferentially interacts with activated Rac1. In the cells activated Rac1 recruits cytoplasmic ALS2 to membrane ruffles and subsequently to nascent macropinosomes via Rac1-activated macropinocytosis. At later endocytic stages macropinosomal ALS2 augments fusion of the ALS2-localized macropinosomes with the transferrin-positive endosomes, depending on the ALS2-associated Rab5GEF activity. These results indicate that Rac1 promotes the ALS2 membranous localization, thereby rendering ALS2 active via Rac1-activated endocytosis. Thus, ALS2 is a novel Rac1 effector and is involved in Rac1-activated macropinocytosis. All together, loss of ALS2 may perturb macropinocytosis and/or the following membrane trafficking, which gives rise to neuronal dysfunction in the ALS2-linked motor neuron diseases.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios Motores/metabolismo , Neuropeptídeos/metabolismo , Pinocitose/fisiologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Endossomos/genética , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Neuropeptídeos/genética , Transporte Proteico/fisiologia , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTPRESUMO
ALS2, the causative gene product for a number of recessive motor neuron diseases, is a guanine-nucleotide exchange factor for Rab5, and acts as a modulator for endosome dynamics. Recently, we have identified a novel ALS2 homolog, ALS2CL, which is highly homologous to the C-terminal half of ALS2. In this study, we investigate the molecular features of ALS2CL and its functional relationship with ALS2. A majority of ALS2CL is present as a homo-dimeric form, which can interact with the ALS2-oligomer, resulting in the formation of the large ALS2/ALS2CL heteromeric complex. In cultured cells, overexpressed ALS2CL is colocalized with ALS2 onto membranous compartments. Further, ALS2CL dominantly suppresses the endosome enlargement induced by a constitutively active form of ALS2, and results in an extensive perinuclear tubulo-membranous phenotype, which are dependent upon the ALS2CL-ALS2 interaction. Collectively, ALS2CL is a novel ALS2-interacting protein and is implicated in ALS2-mediated endosome dynamics.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Chlorocebus aethiops , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Células HeLa , Humanos , Fenótipo , Frações Subcelulares/metabolismoRESUMO
ALS2/alsin is a member of guanine nucleotide exchange factors for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through 21 months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal growth factor (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null fibroblasts, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.
Assuntos
Proteínas de Transporte/genética , Endossomos/fisiologia , Doenças do Sistema Nervoso/genética , Fatores Etários , Análise de Variância , Animais , Transporte Biológico/fisiologia , Southern Blotting , Western Blotting , Primers do DNA , Eletrofisiologia , Fator de Crescimento Epidérmico/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios Motores/patologia , Células de Purkinje/patologiaRESUMO
ALS2, the causative gene product for juvenile recessive amyotrophic lateral sclerosis (ALS2), is a guanine-nucleotide exchange factor for the small GTPase Rab5. Here, we report a novel ALS2 homologous gene, ALS2 C-terminal like (ALS2CL), which encodes a 108-kD ALS2CL protein. ALS2CL exhibited a specific but a relatively weak Rab5-GEF activity with accompanying rather strong Rab5-binding properties. In HeLa cells, co-expression of ALS2CL and Rab5A resulted in a unique tubulation phenotype of endosome compartments with significant colocalization of ALS2CL and Rab5A. These results suggest that ALS2CL is a novel factor modulating the Rab5-mediated endosome dynamics in the cells.
Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Transporte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Frações Subcelulares/metabolismo , Distribuição Tecidual , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Mutations in the ALS2 gene have been known to account for a juvenile recessive form of amyotrophic lateral sclerosis (ALS2), a rare juvenile recessive form of primary lateral sclerosis, and a form of hereditary spastic paraplegia (HSP), indicating that the ALS2 protein is essential for the maintenance of motor neurons. Recently, we have demonstrated that the ALS2 protein specifically binds to the small GTPase Rab5 and acts as a GEF (guanine nucleotide exchange factor) for Rab5. We have also shown that its Rab5GEF-requisite domain resides within the C-terminal 640-amino acid region spanning membrane occupation and recognition nexus motifs and the vacuolar protein sorting 9 domain. Transiently expressed ALS2 localized onto early endosomal compartments and stimulated endosome fusions in neuronal and non-neuronal cells in an Rab5GEF activity-dependent manner. These results indicate that the C-terminal region of ALS2 plays a crucial role in endosomal dynamics by its Rab5GEF activity. Here we delineate a molecular feature of the ALS2-associated function through the C-terminal region-mediated homo-oligomerization. A yeast two-hybrid screen for interacting proteins with the ALS2 C-terminal portion identified ALS2 itself. ALS2 forms a homophilic oligomer through its distinct C-terminal regions. This homo-oligomerization is crucial for the Rab5GEF activity in vitro and the ALS2-mediated endosome enlargement in the cells. Taken together, these results indicate that oligomerization of the ALS2 protein is one of the fundamental features for its physiological function involving endosome dynamics in vivo.
Assuntos
Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas rab5 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Western Blotting , Células COS , Membrana Celular/metabolismo , Cromatografia em Gel , Dimerização , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia Confocal , Microscopia de Fluorescência , Neurônios/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismoRESUMO
ALS2 mutations account for a number of recessive motor neuron diseases including forms of amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. Although computational predictions suggest that ALS2 encodes a protein containing multiple guanine nucleotide exchange factor (GEF) domains [RCC1-like domain (RLD), the Dbl homology and pleckstrin homology (DH/PH), and the vacuolar protein sorting 9 (VPS9)], the functions of the ALS2 protein have not been revealed as yet. Here we show that the ALS2 protein specifically binds to small GTPase Rab5 and functions as a GEF for Rab5. Ectopically expressed ALS2 protein localizes with Rab5 and early endosome antigen-1 (EEA1) onto early endosomal compartments and stimulates the enlargement of endosomes in cultured cortical neurons. The carboxy-terminus of ALS2 protein carrying a VPS9 domain mediates not only the activation of Rab5 via a guanine-nucleotide exchanging reaction but also the endosomal localization of the ALS2 protein, while the amino-terminal half containing RLD acts suppressive in its membranous localization. Further, the DH/PH domain in the middle portion of ALS2 protein enhances the VPS9 domain-mediated endosome fusions. Taken together, the ALS2 protein as a novel Rab5-GEF, ALS2rab5GEF seems to be implicated in the endosomal dynamics in vivo. Notably, a feature common to eight reported ALS2 mutations among motor neuron diseases is the loss of VPS9 domain, resulting in the failure of Rab5 activation. Thus, a perturbation of endosomal dynamics caused by loss of ALS2 rab5GEF activity might underlie neuronal dysfunction and degeneration in a number of motor neuron diseases.
Assuntos
Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Proteínas de Transporte VesicularRESUMO
The cellular repressor of E1A-stimulated genes, CREG, is a secreted glycoprotein that enhances differentiation of pluripotent stem cells. Here we report two novel members of the CREG family, human CREG2 and mouse Creg2 cDNAs. The predicted human and mouse protein sequences exhibit 35% identity with CREG protein. Northern blot analyses demonstrate specific CREG2 and Creg2 transcription in brain-in the case of CREG2, mainly in the limbic system of the brain. Both mouse and human CREG2 fused to the carboxy terminus of EGFP in NIH3T3 cells localize to the perinuclear region, which demonstrates implicit endoplasmic reticulum and Golgi localization. Human and mouse CREG2 are N-glycosylated in HeLa cells and deletion of amino-terminal sequences completely abolishes N-glycosylation, indicating that the N termini of both proteins may function as signal sequences. Thus, human and mouse CREG2 are putative secreted glycoproteins and may be novel neuronal extracellular molecules.