RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Recently, genome-wide association studies identified KIF5A as a new ALS-causing gene. KIF5A encodes a protein of the kinesin-1 family, allowing the anterograde transport of cargos along the microtubule rails in neurons. In ALS patients, mutations in the KIF5A gene induce exon 27 skipping, resulting in a mutated protein with a new C-terminal region (KIF5A Δ27). To understand how KIF5A Δ27 underpins the disease, we developed an ALS-associated KIF5A Drosophila model. When selectively expressed in motor neurons, KIF5A Δ27 alters larval locomotion as well as morphology and synaptic transmission at neuromuscular junctions in both males and females. We show that the distribution of mitochondria and synaptic vesicles is profoundly disturbed by KIF5A Δ27 expression. That is consistent with the numerous KIF5A Δ27-containing inclusions observed in motor neuron soma and axons. Moreover, KIF5A Δ27 expression leads to motor neuron death and reduces life expectancy. Our in vivo model reveals that a toxic gain of function underlies the pathogenicity of ALS-linked KIF5A mutant.SIGNIFICANCE STATEMENT Understanding how a mutation identified in patients with amyotrophic lateral sclerosis (ALS) causes the disease and the loss of motor neurons is crucial to fight against this disease. To this end, we have created a Drosophila model based on the motor neuron expression of the KIF5A mutant gene, recently identified in ALS patients. KIF5A encodes a kinesin that allows the anterograde transport of cargos. This model recapitulates the main features of ALS, including alterations of locomotion, synaptic neurotransmission, and morphology at neuromuscular junctions, as well as motor neuron death. KIF5A mutant is found in cytoplasmic inclusions, and its pathogenicity is because of a toxic gain of function.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Masculino , Animais , Feminino , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Estudo de Associação Genômica Ampla , Doenças Neurodegenerativas/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Mutação/genética , Drosophila/metabolismo , Corpos de Inclusão/metabolismoRESUMO
We investigate the etiology of amyotrophic lateral sclerosis (ALS) in a 35-year-old woman presenting with progressive weakness in her left upper limb. Prior to sequencing, a comprehensive neurological work-up was performed, including neurological examination, electrophysiology, biomarker assessment, and brain and spinal cord MRI. Six months before evaluation, the patient experienced weakness and atrophy in her left hand, accompanied by brisk reflexes and Hoffman sign in the same arm. Electroneuromyography revealed lower motor neuron involvement in three body regions. Neurofilament light chains were elevated in her cerebrospinal fluid. Brain imaging showed asymmetrical T2 hyperintensity of the corticospinal tracts and T2 linear hypointensity of the precentral gyri. Trio genome sequencing identified a likely pathogenic de novo variant in the KIF1A gene (NM_001244008.2): c.574A>G, p.(Ile192Val). Pathogenic variants in KIF1A have been associated with a wide range of neurological manifestations called KIF1A-associated neurological diseases (KAND). This report describes a likely pathogenic de novo variant in KIF1A associated with ALS, expanding the phenotypic spectrum of KAND and our understanding of the pathophysiology of ALS.
Assuntos
Esclerose Lateral Amiotrófica , Cinesinas , Mutação de Sentido Incorreto , Humanos , Cinesinas/genética , Esclerose Lateral Amiotrófica/genética , Feminino , Adulto , Extremidade Superior/fisiopatologia , Extremidade Superior/patologia , Imageamento por Ressonância MagnéticaRESUMO
Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motoneuron diseases affecting adults and infants, respectively. ALS and SMA are both characterized by the selective degeneration of motoneurons. Although different in their genetic etiology, growing evidence indicates that they share molecular and cellular pathogenic signatures that constitute potential common therapeutic targets. We previously described a motoneuron-specific death pathway elicited by the Fas death receptor, whereby vulnerable ALS motoneurons show an exacerbated sensitivity to Fas activation. However, the mechanisms that drive the loss of SMA motoneurons remains poorly understood. Here, we describe an in vitro model of SMA-associated degeneration using primary motoneurons derived from Smn2B/- SMA mice and show that Fas activation selectively triggers death of the proximal motoneurons. Fas-induced death of SMA motoneurons has the molecular signature of the motoneuron-selective Fas death pathway that requires activation of p38 kinase, caspase-8, -9 and -3 as well as upregulation of collapsin response mediator protein 4 (CRMP4). In addition, Rho-associated Kinase (ROCK) is required for Fas recruitment. Remarkably, we found that exogenous activation of Fas also promotes axonal elongation in both wildtype and SMA motoneurons. Axon outgrowth of motoneurons promoted by Fas requires the activity of ERK, ROCK and caspases. This work defines a dual role of Fas signaling in motoneurons that can elicit distinct responses from cell death to axonal growth.
Assuntos
Esclerose Lateral Amiotrófica , Atrofia Muscular Espinal , Humanos , Camundongos , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Axônios/patologiaRESUMO
In amyotrophic lateral sclerosis (ALS) caused by SOD1 gene mutations, both cell-autonomous and noncell-autonomous mechanisms lead to the selective degeneration of motoneurons (MN). Here, we evaluate the therapeutic potential of gene therapy targeting mutated SOD1 in mature astrocytes using mice expressing the mutated SOD1G93A protein. An AAV-gfaABC1 D vector encoding an artificial microRNA is used to deliver RNA interference against mutated SOD1 selectively in astrocytes. The treatment leads to the progressive rescue of neuromuscular junction occupancy, to the recovery of the compound muscle action potential in the gastrocnemius muscle, and significantly improves neuromuscular function. In the spinal cord, gene therapy targeting astrocytes protects a small pool of the most vulnerable fast-fatigable MN until disease end stage. In the gastrocnemius muscle of the treated SOD1G93A mice, the fast-twitch type IIB muscle fibers are preserved from atrophy. Axon collateral sprouting is observed together with muscle fiber type grouping indicative of denervation/reinnervation events. The transcriptome profiling of spinal cord MN shows changes in the expression levels of factors regulating the dynamics of microtubules. Gene therapy delivering RNA interference against mutated SOD1 in astrocytes protects fast-fatigable motor units and thereby improves neuromuscular function in ALS mice.
Assuntos
Esclerose Lateral Amiotrófica , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/terapia , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Interferência de RNA , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis (ALS). CD4+ T cells that regulate a protective immunity during the neurodegenerative process have received the most attention. CD8+ T cells are also observed in the spinal cord of patients and ALS mice although their contribution to the disease still remains elusive. Here, we found that activated CD8+ T lymphocytes infiltrate the central nervous system (CNS) of a mouse model of ALS at the symptomatic stage. Selective ablation of CD8+ T cells in mice expressing the ALS-associated superoxide dismutase-1 (SOD1)G93A mutant decreased spinal motoneuron loss. Using motoneuron-CD8+ T cell coculture systems, we found that mutant SOD1-expressing CD8+ T lymphocytes selectively kill motoneurons. This cytotoxicity activity requires the recognition of the peptide-MHC-I complex (where MHC-I represents major histocompatibility complex class I). Measurement of interaction strength by atomic force microscopy-based single-cell force spectroscopy demonstrated a specific MHC-I-dependent interaction between motoneuron and SOD1G93A CD8+ T cells. Activated mutant SOD1 CD8+ T cells produce interferon-γ, which elicits the expression of the MHC-I complex in motoneurons and exerts their cytotoxic function through Fas and granzyme pathways. In addition, analysis of the clonal diversity of CD8+ T cells in the periphery and CNS of ALS mice identified an antigen-restricted repertoire of their T cell receptor in the CNS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Expressão Gênica , Neurônios Motores/metabolismo , Mutação , Superóxido Dismutase-1/genética , Linfócitos T Citotóxicos/metabolismo , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Comunicação Celular/imunologia , Morte Celular , Sobrevivência Celular/genética , Modelos Animais de Doenças , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Neurônios Motores/imunologia , Fenótipo , Índice de Gravidade de Doença , Medula Espinal/citologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease characterized by the progressive degeneration of upper and lower motoneurons. Most ALS cases are sporadic but approximately 10% of ALS cases are due to inherited mutations in identified genes. ALS-causing mutations were identified in over 30 genes with superoxide dismutase-1 (SOD1), chromosome 9 open reading frame 72 (C9orf72), fused in sarcoma (FUS), and TAR DNA-binding protein (TARDBP, encoding TDP-43) being the most frequent. In the last few decades, Drosophila melanogaster emerged as a versatile model for studying neurodegenerative diseases, including ALS. In this review, we describe the different Drosophila ALS models that have been successfully used to decipher the cellular and molecular pathways associated with SOD1, C9orf72, FUS, and TDP-43. The study of the known fruit fly orthologs of these ALS-related genes yielded significant insights into cellular mechanisms and physiological functions. Moreover, genetic screening in tissue-specific gain-of-function mutants that mimic ALS-associated phenotypes identified disease-modifying genes. Here, we propose a comprehensive review on the Drosophila research focused on four ALS-linked genes that has revealed novel pathogenic mechanisms and identified potential therapeutic targets for future therapy.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologiaRESUMO
Liver X Receptors (LXR) alpha and beta are two members of nuclear receptor superfamily documented as endogenous cholesterol sensors. Following conversion of cholesterol in oxysterol, both LXR isoforms detect intracellular concentrations and act as transcription factors to promote expression of target genes. Among their numerous physiological roles, they act as central cholesterol-lowering factors. In the central nervous system (CNS), cholesterol has been shown to be an essential determinant of brain function, particularly as a major constituent of myelin and membranes. In the brain, LXRs act as cholesterol central regulators, and, beyond this metabolic function, LXRs have additional roles such as providing neuroprotective effects and lowering neuroinflammation. In many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis (MS), dysregulations of cholesterol and oxysterol have been reported. In this paper, we propose to focus on recent advances in the knowledge of the LXRs roles on brain cholesterol and oxysterol homeostasis, neuroinflammation, neuroprotection, and their putative involvement in neurodegenerative disorders. We will discuss their potential use as candidates for both molecular diagnosis and as promising pharmacological targets in the treatment of ALS, AD, or MS patients.
Assuntos
Encéfalo/metabolismo , Colesterol/metabolismo , Metabolismo dos Lipídeos , Animais , Suscetibilidade a Doenças , Homeostase , Humanos , Ligantes , Receptores X do Fígado/química , Receptores X do Fígado/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Oxisteróis/metabolismo , Relação Estrutura-AtividadeRESUMO
The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592-605.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ácido Glutâmico/farmacologia , Ácido Láctico/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Imageamento por Ressonância Magnética , Redes e Vias Metabólicas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Análise de Componente Principal , Medula Espinal/citologia , Superóxido Dismutase/metabolismo , Trítio/metabolismoRESUMO
Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.
Assuntos
Agenesia do Corpo Caloso/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Simportadores/deficiência , Transmissão Sináptica/fisiologia , Agenesia do Corpo Caloso/tratamento farmacológico , Agenesia do Corpo Caloso/patologia , Animais , Carbamazepina/farmacologia , Células Cultivadas , Cloretos/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Neurotransmissores/farmacologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/patologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/patologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Simportadores/genética , Transmissão Sináptica/efeitos dos fármacosRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that primarily affects motoneurons in the brain and spinal cord. Astrocyte and microglia activation as well as skeletal muscle atrophy are also typical hallmarks of the disease. However, the functional relationship between astrocytes, microglia and skeletal muscle in the pathogenic process remains unclear. Here, we report that the tumor necrosis factor-like weak inducer of apoptosis (Tweak) and its receptor Fn14 are aberrantly expressed in spinal astrocytes and skeletal muscle of SOD1(G93A) mice. We show that Tweak induces motoneuron death, stimulates astrocytic interleukin-6 release and astrocytic proliferation in vitro. The genetic ablation of Tweak in SOD1(G93A) mice significantly reduces astrocytosis, microgliosis and ameliorates skeletal muscle atrophy. The peripheral neutralization of Tweak through antagonistic anti-Tweak antibody ameliorates muscle pathology and notably, decreases microglial activation in SOD1(G93A) mice. Unexpectedly, none of these approaches improved motor function, lifespan and motoneuron survival. Our work emphasizes the multi-systemic aspect of ALS, and suggests that a combinatorial therapy targeting multiple cell types will be instrumental to halt the neurodegenerative process.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Gliose/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Fatores de Necrose Tumoral/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Morte Celular , Proliferação de Células , Citocina TWEAK , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Interleucina-6/biossíntese , Expectativa de Vida , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Placa Motora/genética , Placa Motora/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Receptor de TWEAK , Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
Cholesterol plays a central role in numerous nervous system functions. Cholesterol is the major constituent of myelin sheaths, is essential for synapse and dendrite formation, axon guidance as well as neurotransmission. Among regulators of cholesterol homeostasis, liver X receptors (LXRs), two members of the nuclear receptor superfamily, play a determinant role. LXRs act as cholesterol sensors and respond to high intracellular cholesterol concentration by decreasing plasmatic and intracellular cholesterol content. Beyond their cholesterol-lowering role, LXRs have been proposed as regulators of immunity and anti-inflammatory factors. Dysregulation of cholesterol metabolism combined to neuroinflammatory context have been described in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). ALS is characterized by the progressive loss of motoneurons in the brain and spinal cord, leading to severe paralytic condition and death of patients in a median time of 3 years. Motoneuron degeneration is accompanied by chronic neuroinflammatory response, involving microglial and astrocytic activation, infiltration of blood-derived immune cells and release of pro-inflammatory factors. We propose to discuss here the role of LXRs as a molecular link between the central nervous system cholesterol metabolism, neuroinflammation, motoneuron survival and their potential as promising therapeutic candidates for ALS therapy.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Colesterol/metabolismo , Receptores X do Fígado/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neuroproteção , Esclerose Lateral Amiotrófica/patologia , Animais , Humanos , Receptores X do Fígado/química , Modelos BiológicosRESUMO
A receptor-ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type-specific post-receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death-triggering factor in motoneurons through LT-ßR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT-induced axonal elongation and branching require ERK and caspase-9 pathways. This distinct response involves a compartment-specific activation of LIGHT signals, with somatic activation-inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.
Assuntos
Axônios/fisiologia , Neurônios Motores/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Linfócitos B/imunologia , Butadienos/farmacologia , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/metabolismo , Camundongos , Camundongos Knockout , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/biossínteseRESUMO
Although the precise signaling mechanisms underlying the vulnerability of some sub-populations of motoneurons in ALS remain unclear, critical factors such as metallo-proteinase 9 expression, neuronal activity and endoplasmic reticulum stress have been shown to be involved. In the context of SOD1(G93A) ALS mouse model, we previously showed that a two-fold decrease in calreticulin (CRT) is occurring in the vulnerable fast motoneurons. Here, we asked to which extent the decrease in CRT levels was causative to muscle denervation and/or motoneuron degeneration. Toward this goal, a hemizygous deletion of the crt gene in SOD1(G93A) mice was generated since the complete ablation of crt is embryonic lethal. We observed that SOD1(G93A);crt(+/-) mice display increased and earlier muscle weakness and muscle denervation compared to SOD1(G93A) mice. While CRT reduction in motoneurons leads to a strong upregulation of two factors important in motoneuron dysfunction, ER stress and mTOR activation, it does not aggravate motoneuron death. Our results underline a prevalent role for CRT levels in the early phase of muscle denervation and support CRT regulation as a potential therapeutic approach.
Assuntos
Esclerose Lateral Amiotrófica/complicações , Calreticulina/metabolismo , Regulação da Expressão Gênica/genética , Neurônios Motores/patologia , Doenças Musculares/etiologia , Degeneração Neural/etiologia , Fator 6 Ativador da Transcrição/metabolismo , Fatores Etários , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Calreticulina/genética , Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Debilidade Muscular/etiologia , Doenças Musculares/patologia , Receptores Colinérgicos/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Serina-Treonina Quinases TOR , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
BACKGROUND: auto-antibodies against the potassium channel inward rectifying potassium channel 4.1 (Kir4.1) have previously been identified in 46% of patients with multiple sclerosis (MS). OBJECTIVES: to confirm these findings. METHODS: we evaluated the presence of anti-Kir4.1 antibodies by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence in 268 MS patients, 46 patients with other neurological diseases (OND) and 45 healthy controls. RESULTS: anti-Kir4.1 antibodies were found in 7.5% of MS patients, 4.3% of OND patients and 4.4% of healthy controls. Immunofluorescence analysis did not identify any specific staining. CONCLUSIONS: we confirmed the presence of anti-Kir4.1 antibodies in MS patients, but at a much lower prevalence than previously reported.
Assuntos
Autoanticorpos/sangue , Esclerose Múltipla/imunologia , Canais de Potássio Corretores do Fluxo de Internalização/imunologia , Adulto , Autoanticorpos/imunologia , Autoantígenos/imunologia , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Masculino , Esclerose Múltipla/sangueRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Cinesinas , Neurônios Motores , Animais , Cinesinas/genética , Cinesinas/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Mutação , Humanos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Drosophila melanogaster/genética , Transmissão Sináptica/genética , Modelos Animais de Doenças , Axônios/metabolismo , Axônios/patologia , Larva/genética , Larva/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is predominantly associated with motor cortex, corticospinal tract (CST), brainstem, and spinal cord degeneration, and cerebellar involvement is much less well characterized. However, some of the cardinal clinical features of ALS, such as dysarthria, dysphagia, gait impairment, falls, and impaired dexterity, are believed to be exacerbated by coexisting cerebellar pathology. Cerebellar pathology may also contribute to cognitive, behavioral, and pseudobulbar manifestations. Our objective was to systematically assess both intracerebellar pathology and cerebrocerebellar connectivity alterations in a genetically stratified cohort of ALS. METHODS: A prospective, multimodal neuroimaging study was conducted to evaluate the longitudinal evolution of intracerebellar pathology and cerebrocerebellar connectivity, using structural and functional measures. RESULTS: A total of 113 healthy controls and 212 genetically stratified individuals with ALS were included: (1) C9orf72 hexanucleotide carriers ("C9POS"), (2) sporadic patients who tested negative for ALS-associated genetic variants, and (3) intermediate-length CAG trinucleotide carriers in ATXN2 ("ATXN2"). Flocculonodular lobule (padj = 0.014, 95% CI -5.06e-5 to -3.98e-6) and crura (padj = 0.031, 95% CI -1.63e-3 to -5.55e-5) volume reductions were detected at baseline in sporadic patients. Cerebellofrontal and cerebelloparietal structural connectivity impairment was observed in both C9POS and sporadic patients at baseline, and both projections deteriorated further over time in sporadic patients (padj = 0.003, t(249) = 3.04 and padj = 0.05, t(249) = 1.93). Functional cerebelloparietal uncoupling was evident in sporadic patients at baseline (padj = 0.004, 95% CI -0.19 to -0.03). ATXN2 patients exhibited decreased cerebello-occipital functional connectivity at baseline (padj = 0.004, 95% CI -0.63 to -0.06), progressive cerebellotemporal functional disconnection (padj = 0.025, t(199) = -2.26), and progressive flocculonodular lobule degeneration (padj = 0.017, t(249) = -2.24). C9POS patients showed progressive ventral dentate atrophy (padj = 0.007, t(249) = -2.75). The CSTs (padj < 0.001, 95% CI 4.89e-5 to 1.14e-4) and transcallosal interhemispheric fibers (padj < 0.001, 95% CI 5.21e-5 to 1.31e-4) were affected at baseline in C9POS and exhibited rapid degeneration over the 4 time points. The rate of decline in CST and corpus callosum integrity was faster than the rate of cerebrocerebellar disconnection (padj = 0.001, t(190) = 6.93). DISCUSSION: ALS is associated with accruing intracerebellar disease burden as well as progressive corticocerebellar uncoupling. Contrary to previous suggestions, we have not detected evidence of compensatory structural or functional changes in response to supratentorial degeneration. The contribution of cerebellar disease burden to dysarthria, dysphagia, gait impairment, pseudobulbar affect, and cognitive deficits should be carefully considered in clinical assessments, monitoring, and multidisciplinary interventions.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Cerebelo , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Masculino , Feminino , Pessoa de Meia-Idade , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Idoso , Proteína C9orf72/genética , Estudos Prospectivos , Ataxina-2/genética , Imageamento por Ressonância Magnética , Progressão da Doença , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Adulto , Estudos LongitudinaisRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The immunosuppressive functions of regulatory T lymphocytes (Tregs) are impaired in ALS, and correlate to disease progression. The phase 2a IMODALS trial reported an increase in Treg number in ALS patients following the administration of low-dose (ld) interleukin-2 (IL-2). We propose a pharmacometabolomics approach to decipher metabolic modifications occurring in patients treated with ld-IL-2 and its relationship with Treg response. Blood metabolomic profiles were determined on days D1, D64, and D85 from patients receiving 2 MIU of IL-2 (n = 12) and patients receiving a placebo (n = 12). We discriminated the three time points for the treatment group (average error rate of 42%). Among the important metabolites, kynurenine increased between D1 and D64, followed by a reduction at D85. The percentage increase of Treg number from D1 to D64, as predicted by the metabolome at D1, was highly correlated with the observed value. This study provided a proof of concept for metabolic characterization of the effect of ld-IL-2 in ALS. These data could present advances toward a personalized medicine approach and present pharmacometabolomics as a key tool to complement genomic and transcriptional data for drug characterization, leading to systems pharmacology.
Assuntos
Esclerose Lateral Amiotrófica , Interleucina-2 , Metabolômica , Linfócitos T Reguladores , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Humanos , Interleucina-2/administração & dosagem , Interleucina-2/metabolismo , Metabolômica/métodos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Masculino , Pessoa de Meia-Idade , Feminino , Cinurenina/metabolismo , Idoso , Metaboloma/efeitos dos fármacosRESUMO
Cellular responses to protein misfolding are thought to play key roles in triggering neurodegeneration. In the mutant superoxide dismutase (mSOD1) model of amyotrophic lateral sclerosis (ALS), subsets of motoneurons are selectively vulnerable to degeneration. Fast fatigable motoneurons selectively activate an endoplasmic reticulum (ER) stress response that drives their early degeneration while a subset of mSOD1 motoneurons show exacerbated sensitivity to activation of the motoneuron-specific Fas/NO pathway. However, the links between the two mechanisms and the molecular basis of their cellular specificity remained unclear. We show that Fas activation leads, specifically in mSOD1 motoneurons, to reductions in levels of calreticulin (CRT), a calcium-binding ER chaperone. Decreased expression of CRT is both necessary and sufficient to trigger SOD1(G93A) motoneuron death through the Fas/NO pathway. In SOD1(G93A) mice in vivo, reductions in CRT precede muscle denervation and are restricted to vulnerable motor pools. In vitro, both reduced CRT and Fas activation trigger an ER stress response that is restricted to, and required for death of, vulnerable SOD1(G93A) motoneurons. Our data reveal CRT as a critical link between a motoneuron-specific death pathway and the ER stress response and point to a role of CRT levels in modulating motoneuron vulnerability to ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Calreticulina/antagonistas & inibidores , Calreticulina/metabolismo , Estresse do Retículo Endoplasmático/genética , Neurônios Motores/metabolismo , Receptor fas/genética , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Morte Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/enzimologia , Neurônios Motores/patologia , Mutação/genética , Transdução de Sinais/genética , Superóxido Dismutase/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable paralytic disorder caused by the progressive death of upper and lower motoneurons. Although numerous strategies have been developed to slow disease progression and improve life quality, to date only a few therapeutic treatments are available with still unsatisfactory therapeutic benefits. The secretome of dental pulp stem cells (DPSCs) contains numerous neurotrophic factors that could promote motoneuron survival. Accordingly, DPSCs confer neuroprotective benefits to the SOD1G93A mouse model of ALS. However, the mode of action of DPSC secretome on motoneurons remains largely unknown. Here, we used conditioned medium of human DPSCs (DPSCs-CM) and assessed its effect on survival, axonal length, and electrical activity of cultured wildtype and SOD1G93A motoneurons. To further understand the role of individual factors secreted by DPSCs and to circumvent the secretome variability bias, we focused on GDF15 and HB-EGF whose neuroprotective properties remain elusive in the ALS pathogenic context. DPSCs-CM rescues motoneurons from trophic factor deprivation-induced death, promotes axon outgrowth of wildtype but not SOD1G93A mutant motoneurons, and has no impact on the spontaneous electrical activity of wildtype or mutant motoneurons. Both GDF15 and HB-EGF protect SOD1G93A motoneurons against nitric oxide-induced death, but not against death induced by trophic factor deprivation. GDF15 and HB-EGF receptors were found to be expressed in the spinal cord, with a two-fold increase in expression for the GDF15 low-affinity receptor in SOD1G93A mice. Therefore, the secretome of DPSCs appears as a new potential therapeutic candidate for ALS.
RESUMO
Aim: The aim of this study was to detect misfolded Cu/Zn SOD1 as a potential biomarker for amyotrophic lateral sclerosis (ALS). Materials & methods: Two ultrasensitive immunodetection assays were developed for the quantification of total and misfolded SOD1. Results: The detection of total and misfolded SOD1 was possible in human serum and cerebrospinal fluid. Total SOD1 was increased in cerebrospinal fluid from ALS patients. Misfolded SOD1 had low and variable expression in both control and ALS patient samples. Conclusion: These assays hold promise for improving our understanding of ALS and its detection, and could lead to more effective treatment options in the future. Further studies in larger cohorts are now required.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with protein misfolding, including Cu/Zn SOD1. In this study, we set up a method for detecting normal and pathological misfolded SOD1 in human serum and cerebrospinal fluid. SOD1 was increased in ALS and misfolded SOD1 had low and variable expression in both control and ALS. These assays holds promise for improving our understanding of ALS and its diagnosis.