RESUMO
The delineation of the molecular pathology underlying amyotrophic lateral sclerosis (ALS) is being hampered by the lack of suitable biomarkers. We have previously reported that bromocriptine upregulates the endogenous antioxidative factor, neuronal apoptosis inhibitory protein (NAIP), sustains motor function and slows disease progression in ALS patients, implying the NAIP's implication in ALS. Here, we aimed to verify a correlation of NAIP level with disease progression in ALS patients. The amount of NAIP in mononuclear cells (MNC) from peripheral blood from ALS patients (n = 18) and the age matched healthy controls (n = 12) was validated by NAIP-Dot blotting. Notably, the MNC-NAIP level in ALS patients (0.62 ± 0.29 ng) was nearly half of that in the healthy controls (1.34 ± 0.61 ng, P = 0.0019). Furthermore, the MNC-NAIP level in ALS patients and their ALS Functional Rating Scale-Revised (ALSFRS-R) score were evaluated through 1 year. Regression analysis of the MNC-NAIP vs ALSFRS-R indicated that a higher amount of MNC-NAIP was associated with a smaller change in ALSFRS-R at 12 months (R2 = 0.799; P = 0.016), suggesting that a progressive increment of the MNC-NAIP led to slower ALS progression. Our present report implies that NAIP will have broad implications for ALS symptoms as a risk factor and a promising prognostic biomarker.
Assuntos
Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/metabolismo , Proteína Inibidora de Apoptose Neuronal/genética , Proteína Inibidora de Apoptose Neuronal/metabolismo , Idoso , Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Testes de Função Respiratória , Avaliação de SintomasRESUMO
Therapeutic agents to the central nervous system (CNS) need to be efficiently delivered to the target site of action at appropriate therapeutic levels. However, a limited number of effective drugs for the treatment of neurological diseases has been developed thus far. Further, the pharmacological mechanisms by which such therapeutic agents can protect neurons from cell death have not been fully understood. We have previously reported the novel small-molecule compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316), as a unique neuroprotectant against oxidative injury and a highly promising remedy for the treatment of amyotrophic lateral sclerosis (ALS). One of the remarkable characteristics of WN1316 is that its efficacious doses in ALS mouse models are much less than those against oxidative injury in cultured human neuronal cells. It is also noted that the WN1316 cytoprotective activity observed in cultured cells is totally dependent upon the addition of fetal bovine serum in culture medium. These findings led us to postulate some serum factors being tightly linked to the WN1316 efficacy. In this study, we sieved through fetal bovine serum proteins and identified two N-linked glycoproteins, alpha-2-HS-glycoprotein (AHSG) and hemopexin (HPX), requisites to exert the WN1316 cytoprotective activity against oxidative injury in neuronal cells in vitro. Notably, the removal of glycan chains from these molecules did not affect the WN1316 cytoprotective activity. Thus, two glycoproteins, AHSG and HPX, represent a pivotal glycoprotein of the cytoprotective activity for WN1316, showing a concrete evidence for the novel glycan-independent function of serum glycoproteins in neuroprotective drug efficacy.
Assuntos
Acetamidas/administração & dosagem , Sistema Nervoso Central/efeitos dos fármacos , Meios de Cultura/química , Hemopexina/metabolismo , Imidazóis/administração & dosagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , alfa-2-Glicoproteína-HS/metabolismo , Animais , Bovinos , Morte Celular/efeitos dos fármacos , Sistema Nervoso Central/patologia , Hemopexina/química , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Estresse Oxidativo/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , alfa-2-Glicoproteína-HS/químicaRESUMO
OBJECTIVE: Bromocriptine mesylate (BRC), a dopamine D2 receptor agonist has been shown to confer neuroprotection, sustained motor function and slowed disease progression in mouse models of amyotrophic lateral sclerosis (ALS) Here we report a first in human trial in ALS. DESIGN: A multicenter, Riluzole add-on, randomized, double-blind, placebo controlled 102-week extension BRC clinical trial. METHODS: The trial was conducted between January 2009 and March 2012 on 36 Japanese ALS patients. A 12-week treatment with Riluzole observational period was followed by combined treatment (Riluzole + BRC; n = 29 or Riluzole + placebo; n = 7). The dosing commenced at 1.25 mg/day increasing in steps at two weeks intervals to a maximum of 15 mg/day. The efficacy of BRC was evaluated by comparing BRC and placebo groups upon completion of stepwise dosing at 14 weeks 2 points (1st endpoint) and upon completion or discontinuation of the study (2nd endpoint) of the dosing. RESULTS: Statistics analyses revealed a marginal BRC treatment efficacy with Pâ¦20%to placebo by 1st and 2nd endpoint analysis. In the 1st endpoint analysis, BRC group was significantly effective on the scores of ALSAQ40-communicaton (P = 1.2%), eating and drinking (P = 2.2%), ALSFRS-R total (P = 17.6%), grip strength (P = 19.8%) compared to the placebo group. In the 2nd endpoint analysis, differences between the scores of Limb Norris Scale (P = 18.3%), ALSAQ40-communication (P = 11.9%), eating and drinking (P = 13.6%), and neck forward-bent test (P = 15.4%) of BRC group were detected between the two groups. There was no significant difference between the treatment groups for adverse events or serious drug reactions incidence. CONCLUSIONS: BRC sustains motoneuronal function at least in part through BRC treatment. Further analysis involving a Phase 2b or 3 clinical trial is required but BRC currently shows promise for ALS treatment. TRIAL REGISTRATION: UMIN Clinical Trials UMIN000008527.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Bromocriptina/uso terapêutico , Idoso , Bromocriptina/efeitos adversos , Método Duplo-Cego , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Riluzol/uso terapêutico , Resultado do TratamentoRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron degenerative disease. Given that oxidative stress and resulting chronic neuronal inflammation are thought to be central pathogenic, anti-oxidative agents and modulators of neuronal inflammation could be potential therapies for ALS. We report here that the novel small molecular compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316) selectively suppresses oxidative stress-induced cell death and neuronal inflammation in the late-stage ALS mice. WN1316 has high blood-brain-barrier permeability and water solubility, and boosts both neuronal apoptosis inhibitory protein (NAIP) and NF-E2-related factor 2 (Nrf2) which governed glutathione (GSH)-related anti-oxidation pathway protecting motor neurons against oxidative injuries. Post-onset oral administration of low dose (1-100 µg/kg/day) WN1316 in ALS(SOD1(H46R)) and ALS(SOD1(G93A)) mice resulted in sustained improved motor function and post onset survival rate. Immunohistochemical analysis revealed less DNA oxidative damage and motor neuronal inflammation as well as repression of both microgliosis and astrocytosis, concomitant down regulation of interleukin-1ß and inducible nitric oxide synthase, and preservation of the motoneurons in anterior horn of lumbar spinal cord and skeletal muscle (quadriceps femoris). Thus, WN1316 would be a novel therapeutic agent for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Imidazóis/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Medula Espinal , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Modelos Animais de Doenças , Imidazóis/química , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
Antioxidant defense is crucial in restoring cellular redox homeostasis. Recent findings have suggested that oxidative stress plays pivotal roles in the pathogenesis of many neurodegenerative diseases. Thus, an anti-oxidative stress remedy might be a promising means for the treatment of such disorders. In this study, we employed a novel ligand-based virtual screening system and identified a novel small molecule, N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide (CPN-9), which selectively suppressed oxidative stress-induced cell death in a cell-type-independent manner. CPN-9 upregulates NF-E2-related factor 2 (Nrf2), a key transcriptional regulator of the expression of phase II detoxification enzymes and antioxidant proteins, and Nrf2-regulated factors such as heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase modifier subunit (GCLM). The CPN-9-mediated upregulation of HO-1, NQO1, and GCLM was abolished by Nrf2 knockdown. Moreover, the antioxidant N-acetylcysteine reduced the protective effect of CPN-9 against oxidative stress-induced cell death with concomitant diminishing of Nrf2 nuclear translocation. These results indicate that CPN-9 exerts its activity via the reactive oxygen species-dependent activation of the Nrf2 signaling pathway in cultured cells. It is noteworthy that the postonset systemic administration of CPN-9 to a transgenic ALS mouse model carrying the H46R mutation in the human Cu/Zn superoxide dismutase (SOD1) gene sustained motor functions and delayed disease progression after onset. Collectively, CPN-9 is a novel Nrf2 activator and a neuroprotective candidate for the treatment of neurodegenerative diseases, including ALS.
Assuntos
Acetamidas/farmacologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Elementos de Resposta Antioxidante , Apoptose/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tiazóis/farmacologia , Acetilcisteína/farmacologia , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutamato-Cisteína Ligase/metabolismo , Células HeLa , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peroxidação de Lipídeos , Masculino , Desintoxicação Metabólica Fase II/genética , Camundongos , Camundongos Transgênicos , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by a selective loss of upper and lower motor neurons. Since oxidative stress plays a crucial role in the progression of motor neuron loss observed in ALS, anti-oxidative agents could be an important therapeutic means for the ALS treatment. We have previously developed a drug screening system allowing the identification of small chemical compounds that upregulate endogenous neuronal apoptosis inhibitory protein (NAIP), an oxidative stress-induced cell death suppressor. Using this system, we identified the dopamine D2 receptor agonist bromocriptine (BRC) as one of NAIP-upregulating compounds. In this study, to prove the efficacy of BRC in ALS, we conducted a set of preclinical studies using a transgenic ALS mouse model carrying the H46R mutation in the human Cu/Zn superoxide dismutase (SOD1) gene ALS(SOD1(H46R)) by the post-onset administration of BRC. ALS(SOD1(H46R)) mice receiving BRC showed sustained motor functions and modest prolonged survival after onset. Further, BRC treatment delayed anterior horn cell loss, and reduced the number of reactive astrocytes and the level of inflammatory factors such as inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α in the spinal cord of late symptomatic mice. In vitro study showed the reduced level of extracellular TNF-α after lipopolysaccharide (LPS) exposure in BRC-treated mouse astrocytes. BRC-treated ALS(SOD1(H46R)) mice also showed a reduced level of oxidative damage in the spinal cord. Notably, BRC treatment resulted in an upregulation of anti-oxidative stress genes, activating transcription factor 3 (ATF3) and heme oxygenase-1 (HO-1), and the generation of a glutathione (GSH) in SH-SY5Y cultured neuronal cells in a dopamine receptor-independent manner. These results imply that BRC protects motor neurons from the oxidative injury via suppression of astrogliosis in the spinal cord of ALS(SOD1(H46R)) mice. Thus, BRC might be a promising therapeutic agent for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Células do Corno Anterior/efeitos dos fármacos , Bromocriptina/farmacologia , Agonistas de Dopamina/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/imunologia , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Células do Corno Anterior/patologia , Modelos Animais de Doenças , Progressão da Doença , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Proteína Inibidora de Apoptose Neuronal/metabolismo , Receptores de Dopamina D2/agonistas , Medula Espinal/efeitos dos fármacos , Medula Espinal/imunologia , Medula Espinal/fisiopatologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacosRESUMO
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
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1(H46R)); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1(H46R) transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Antagonistas de Dopamina/uso terapêutico , Microglia/efeitos dos fármacos , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Medula Espinal/citologia , Esclerose Lateral Amiotrófica/patologia , Animais , Movimento Celular/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Antagonistas de Dopamina/farmacologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Piridinas/farmacologia , Pirróis/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologiaRESUMO
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
In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.
Assuntos
Oócitos/citologia , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Animais , Diferenciação Celular , Citocinas/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Camundongos , Oócitos/ultraestrutura , Folículo Ovariano/citologia , Folículo Ovariano/ultraestrutura , Ovário/citologia , Ovário/ultraestruturaRESUMO
DNA helicases are known to play important roles in the maintenance of genome integrity including the replication of trinucleotide repeats in the cells. Here, we report the HFM1 gene, which encodes the putative human DNA helicase. The HFM1 gene comprises 39 exons mapping to human chromosome 1p22.2. The HFM1 cDNA encompasses 4931 nucleotides with a single open reading frame (ORF) of 1435 amino acid residues encoding a predicted 172 kDa protein (hHFM1). The deduced protein sequence shares similar domain and motif structures to those of Mer3, a DNA helicase of Saccharomyces cerevisiae; seven consecutive motifs conserved among the DEXH-box type of DNA/RNA helicases at the N-terminal and a single putative zinc finger motif at the C-terminal regions of the protein. Further, the HFM1 transcript is preferentially expressed in testis and ovary. Collectively, hHFM1 is the evolutionally conserved putative human DNA helicase, which may function as a modulator for genome integrity in germ-line tissues.
Assuntos
DNA Helicases/genética , Células Germinativas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Helicases/metabolismo , DNA Complementar/metabolismo , Éxons , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Dedos de ZincoRESUMO
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
Neuronal apoptosis inhibitory protein (NAIP/BIRC1), the inhibitor of apoptosis protein (IAP) family member, suppresses neuronal cell death induced by a variety of insults, including cell death from ischemia and stroke. The goal of the present study was to develop an efficient method for identification of compounds with the ability to upregulate endogenous NAIP and to determine the effects on these compounds on the cellular response to ischemia. A novel NAIP-enzyme-linked immunosorbent assay (ELISA)-based in vitro drug-screening system is established. Use of this system identified an antagonist of dopamine D4 receptor, termed L-745,870, with a potent NAIP upregulatory effect. L-745,870-mediated NAIP upregulation in neuronal and nonneuronal cultured cells resulted in decreased vulnerability to oxidative stress-induced apoptosis. Reducing NAIP expression via RNA interference techniques resulted in prevention of L-745,870-mediated protection from oxidative stress. Further, systemic administration of L-745,870 attenuated ischemia-induced damage of the hippocampal CA1 neurons and upregulated NAIP expression in the rescued hippocampal CA1 neurons in a gerbil model. These data suggest that the NAIP upregulating compound, L-745,870, has therapeutic potential in acute ischemic disorders and that our NAIP-ELISA-based drug screening may facilitate the discovery of novel neuroprotective compounds.
Assuntos
Antagonistas dos Receptores de Dopamina D2 , Isquemia/metabolismo , Isquemia/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Piridinas/farmacologia , Pirróis/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Gerbillinae , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Proteína Inibidora de Apoptose Neuronal , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Piridinas/administração & dosagem , Pirróis/administração & dosagem , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4 , Vitamina K 3/antagonistas & inibidores , Vitamina K 3/farmacologiaRESUMO
ALS2 is a causative gene product for a form of the familial motor neuron diseases. Computational genomic analysis identified ALS2CL, which is a novel protein highly homologous to the C-terminal region of ALS2. Both proteins contain the VPS9 domain, which is a hallmark for all known members of the guanine nucleotide exchange factors for Rab5 (Rab5GEF), and are known to act as novel factors modulating the Rab5-mediated endosome dynamics in the cells. It has also been reported that oligomerization of ALS2 is one of the fundamental features of its biochemical and physiological function involving endosome dynamics. This chapter describes methods, including purification of the recombinant ALS2 and ALS2CL, and Rab5GEF assay, which have been utilized to clarify the molecular function for ALS2 and ALS2CL.