RESUMO
An 8-year-old neutered male Bengal cat was referred because of a 1-year history of progressive and relapsing generalized muscle weakness and muscle atrophy. Before referral, the cat was treated with immunosuppressive doses of oral prednisolone, intermittently for 6 mo, and had responded well when the immunosuppressive dose was maintained. Generalized paresis, diffuse muscle atrophy, and diminished spinal reflexes were present in all limbs, consistent with a generalized lower motor neuron disease. Histopathologic evaluation of muscle biopsies confirmed a pattern of muscle fiber atrophy consistent with chronic and severe denervation. No specific abnormalities were identified in the nerve biopsy or within intramuscular nerve branches. A presumptive antemortem diagnosis of an adult-onset motor neuron degeneration resembling amyotrophic lateral sclerosis (ALS) or spinal muscle atrophy was suspected. However, given the response to immunosuppressive doses of corticosteroids, an autoimmune process or other degenerative process could not be definitively excluded. Key clinical message: In this case, an adult cat had a chronic, progressive history of lower motor neuron weakness and absent spinal reflexes; biopsies revealed a neurogenic pattern of muscle fiber atrophy and histologically normal peripheral nerve and intramuscular nerve branches. Although reports of motor neuron disease are rare in the veterinary literature, this case report highlights the importance of muscle and nerve biopsies that lead to a presumptive diagnosis of motor neuron degeneration.
Dégénérescence présumée des neurones moteurs chez un chat adulteUn chat Bengal mâle castré de 8 ans a été référé en raison d'un an d'antécédents de faiblesse musculaire généralisée progressive et récidivante et d'atrophie musculaire. Avant le transfert, le chat a été traité avec des doses immunosuppressives de prednisolone orale, par intermittence pendant 6 mois, et a bien répondu lorsque la dose immunosuppressive a été maintenue. Une parésie généralisée, une atrophie musculaire diffuse et des réflexes spinaux diminués étaient présents dans tous les membres, compatibles avec une maladie généralisée des neurones moteurs inférieurs. L'évaluation histopathologique des biopsies musculaires a confirmé un schéma d'atrophie des fibres musculaires compatible avec une dénervation chronique et sévère. Aucune anomalie spécifique n'a été identifiée dans la biopsie nerveuse ou dans les branches nerveuses intramusculaires. Un diagnostic antemortem présomptif d'une dégénérescence des neurones moteurs d'apparition adulte ressemblant à la sclérose latérale amyotrophique (SLA) ou à une atrophie musculaire spinale a été suspecté. Cependant, compte tenu de la réponse aux doses immunosuppressives de corticostéroïdes, un processus auto-immun ou un autre processus dégénératif ne pouvait être définitivement exclu.Message clinique clé :Dans ce cas, un chat adulte avait des antécédents chroniques et progressifs de faiblesse des neurones moteurs inférieurs et d'absence de réflexes spinaux; les biopsies ont révélé un schéma neurogène d'atrophie des fibres musculaires et des branches nerveuses périphériques et intramusculaires histologiquement normales. Bien que les rapports de maladie des neurones moteurs soient rares dans la littérature vétérinaire, ce rapport de cas souligne l'importance des biopsies musculaires et nerveuses qui conduisent à un diagnostic présomptif de dégénérescence des neurones moteurs.(Traduit par Dr Serge Messier).
Assuntos
Doenças do Gato , Doença dos Neurônios Motores , Gatos , Masculino , Animais , Doenças do Gato/patologia , Doenças do Gato/diagnóstico , Doenças do Gato/tratamento farmacológico , Doença dos Neurônios Motores/veterinária , Doença dos Neurônios Motores/patologia , Doença dos Neurônios Motores/diagnóstico , Prednisolona/uso terapêutico , Imunossupressores/uso terapêutico , Neurônios Motores/patologia , Atrofia Muscular/veterinária , Atrofia Muscular/patologia , Músculo Esquelético/patologiaRESUMO
Decoding the activity of individual neural cells during natural behaviours allows neuroscientists to study how the nervous system generates and controls movements. Contrary to other neural cells, the activity of spinal motor neurons can be determined non-invasively (or minimally invasively) from the decomposition of electromyographic (EMG) signals into motor unit firing activities. For some interfacing and neuro-feedback investigations, EMG decomposition needs to be performed in real time. Here, we introduce an open-source software that performs real-time decoding of motor neurons using a blind-source separation approach for multichannel EMG signal processing. Separation vectors (motor unit filters) are optimised for each motor unit from baseline contractions and then re-applied in real time during test contractions. In this way, the firing activity of multiple motor neurons can be provided through different forms of visual feedback. We provide a complete framework with guidelines and examples of recordings to guide researchers who aim to study movement control at the motor neuron level. We first validated the software with synthetic EMG signals generated during a range of isometric contraction patterns. We then tested the software on data collected using either surface or intramuscular electrode arrays from five lower limb muscles (gastrocnemius lateralis and medialis, vastus lateralis and medialis, and tibialis anterior). We assessed how the muscle or variation of contraction intensity between the baseline contraction and the test contraction impacted the accuracy of the real-time decomposition. This open-source software provides a set of tools for neuroscientists to design experimental paradigms where participants can receive real-time feedback on the output of the spinal cord circuits.
Assuntos
Eletromiografia , Neurônios Motores , Software , Eletromiografia/métodos , Humanos , Neurônios Motores/fisiologia , Músculo Esquelético/fisiologia , Processamento de Sinais Assistido por Computador , Adulto , Masculino , Feminino , Adulto JovemRESUMO
Motor circuits represent the main output of the central nervous system and produce dynamic behaviors ranging from relatively simple rhythmic activities like swimming in fish and breathing in mammals to highly sophisticated dexterous movements in humans. Despite decades of research, the development and function of motor circuits remain poorly understood. Breakthroughs in the field recently provided new tools and tractable model systems that set the stage to discover the molecular mechanisms and circuit logic underlying motor control. Here, we describe recent advances from both vertebrate (mouse, frog) and invertebrate (nematode, fruit fly) systems on cellular and molecular mechanisms that enable motor circuits to develop and function and highlight conserved and divergent mechanisms necessary for motor circuit development.
Assuntos
Neurônios Motores , Animais , Humanos , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Rede Nervosa/crescimento & desenvolvimentoRESUMO
Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Íntrons , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Orientação de Axônios/genética , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Regulação da Expressão GênicaRESUMO
Cerebral white matter lesions prevent cortico-spinal descending inputs from effectively activating spinal motoneurons, leading to loss of motor control. However, in most cases, the damage to cortico-spinal axons is incomplete offering a potential target for therapies aimed at improving volitional muscle activation. Here we hypothesize that, by engaging direct excitatory connections to cortico-spinal motoneurons, stimulation of the motor thalamus could facilitate activation of surviving cortico-spinal fibers thereby immediately potentiating motor output. To test this hypothesis, we identify optimal thalamic targets and stimulation parameters that enhance upper-limb motor-evoked potentials and grip forces in anesthetized monkeys. This potentiation persists after white matter lesions. We replicate these results in humans during intra-operative testing. We then design a stimulation protocol that immediately improves strength and force control in a patient with a chronic white matter lesion. Our results show that electrical stimulation targeting surviving neural pathways can improve motor control after white matter lesions.
Assuntos
Estimulação Elétrica , Potencial Evocado Motor , Córtex Motor , Neurônios Motores , Tálamo , Animais , Tálamo/fisiologia , Córtex Motor/fisiologia , Humanos , Potencial Evocado Motor/fisiologia , Masculino , Neurônios Motores/fisiologia , Estimulação Elétrica/métodos , Macaca mulatta , Feminino , Força da Mão/fisiologia , Substância Branca/fisiologia , Substância Branca/fisiopatologia , Medula Espinal/fisiologiaRESUMO
The recent assembly of the adult Drosophila melanogaster central brain connectome, containing more than 125,000 neurons and 50 million synaptic connections, provides a template for examining sensory processing throughout the brain1,2. Here we create a leaky integrate-and-fire computational model of the entire Drosophila brain, on the basis of neural connectivity and neurotransmitter identity3, to study circuit properties of feeding and grooming behaviours. We show that activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation4. In addition, using the model to activate neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing5-a testable hypothesis that we validate by optogenetic activation and behavioural studies. Activating different classes of gustatory neurons in the model makes accurate predictions of how several taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. Additionally, we applied this model to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit, and accurately describes the circuit response upon activation of different mechanosensory subtypes6-10. Our results demonstrate that modelling brain circuits using only synapse-level connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can describe complete sensorimotor transformations.
Assuntos
Encéfalo , Drosophila melanogaster , Modelos Neurológicos , Paladar , Animais , Drosophila melanogaster/fisiologia , Encéfalo/fisiologia , Encéfalo/citologia , Paladar/fisiologia , Comportamento Alimentar/fisiologia , Asseio Animal/fisiologia , Sinapses/fisiologia , Simulação por Computador , Optogenética , Neurônios Motores/fisiologia , Feminino , Masculino , ConectomaRESUMO
In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.
Assuntos
Esclerose Lateral Amiotrófica , Modelos Animais de Doenças , Endocitose , Neurônios Motores , Vesículas Sinápticas , Animais , Camundongos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Diafragma/inervação , Diafragma/metabolismo , Diafragma/fisiopatologia , Endocitose/fisiologia , Corantes Fluorescentes/metabolismo , Imidazóis/farmacologia , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Terminações Nervosas/metabolismo , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Transmissão Sináptica/fisiologia , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismoRESUMO
INTRODUCTION: Reliable, noninvasive early diagnostics of neuromuscular function in Bell's palsy, which causes facial paralysis and reduced quality of life, remain to be established. Here, we aimed to evaluate the utility of the motor unit number index (MUNIX) for the quantitative electrophysiological assessment of early-stage Bell's palsy, its correlation with clinical assessments, changes following treatment, and association with clinical prognosis. METHODS: MUNIX measures were recorded from the bilateral zygomaticus, orbicularis oculi, and orbicularis oris muscles of 10 healthy individuals and 64 patients with Bell's palsy. The patients were assessed by two specialist neurologists using the House-Brackmann and Sunnybrook Facial Grading Systems. Repeat assessments were performed on 20 patients with Bell's palsy who received treatment. Additionally, the 64 patients were reassessed using clinical scales after a 1-month interval. RESULTS: The MUNIX values of the main affected muscles on the affected side were lower than those on the healthy side in patients with Bell's palsy (p < .05). The MUNIX measurements significantly correlated with the clinical facial nerve palsy scale scores (p < .05). Significant improvements were observed in the MUNIX values on repeat testing following treatment (p < .05). The baseline motor unit size index (the compound muscle action potential amplitude divided by MUNIX) was positively associated with improved clinical presentation after 1 month (p < .05). CONCLUSION: MUNIX can be used as an electrophysiological biomarker for the quantitative assessment of facial nerve palsy and treatment response, and as a prognostic biomarker, in patients with early Bell's palsy, and is recommended as a complement to conventional neurophysiological examinations.
Assuntos
Paralisia de Bell , Eletromiografia , Humanos , Paralisia de Bell/fisiopatologia , Paralisia de Bell/diagnóstico , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Eletromiografia/métodos , Músculos Faciais/fisiopatologia , Adulto Jovem , Idoso , Biomarcadores , Neurônios Motores/fisiologia , Diagnóstico Precoce , Potenciais de Ação/fisiologiaRESUMO
Motor neurons (MNs) are the final output of circuits driving fundamental behaviors, such as respiration and locomotion. Hox proteins are essential in generating the MN diversity required for accomplishing these functions, but the transcriptional mechanisms that enable Hox paralogs to assign distinct MN subtype identities despite their promiscuous DNA binding motif are not well understood. Here we show that Hoxa5 modifies chromatin accessibility in all mouse spinal cervical MN subtypes and engages TALE co-factors to directly bind and regulate subtype-specific genes. We identify a paralog-specific interaction of Hoxa5 with the phrenic MN-specific transcription factor Scip and show that heterologous expression of Hoxa5 and Scip is sufficient to suppress limb-innervating MN identity. We also demonstrate that phrenic MN identity is stable after Hoxa5 downregulation and identify Klf proteins as potential regulators of phrenic MN maintenance. Our data identify multiple modes of Hoxa5 action that converge to induce and maintain MN identity.
Assuntos
Proteínas de Homeodomínio , Neurônios Motores , Fatores de Transcrição , Animais , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas Relacionadas à Autofagia , Cisteína Endopeptidases , Proteínas de Ligação a DNA , Proteínas Associadas aos Microtúbulos , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Masculino , Medula Espinal/metabolismo , Medula Espinal/patologia , Autofagia/fisiologia , Camundongos Knockout , Splicing de RNA/genética , Feminino , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Oligonucleotídeos Antissenso/farmacologiaRESUMO
West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.
West Nile virus, but not Usutu virus, can productively infect human motor neurons as a possible route of neuroinvasion.
Assuntos
Flavivirus , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Vírus do Nilo Ocidental , Humanos , Vírus do Nilo Ocidental/fisiologia , Vírus do Nilo Ocidental/patogenicidade , Neurônios Motores/virologia , Células Cultivadas , Flavivirus/fisiologia , Células-Tronco Pluripotentes Induzidas/virologia , Infecções por Flavivirus/virologia , Febre do Nilo Ocidental/virologia , Animais , Medula Espinal/virologiaRESUMO
The potassium-chloride cotransporter KCC2 is the main extruder of Cl- in neurons. It plays a fundamental role in the activity of the inhibitory neurotransmitters (GABA and glycine) since low levels of KCC2 promote intracellular Cl- accumulation, leading to the depolarizing activity of GABA and glycine. The downregulation of this cotransporter occurs in neurological disorders characterized by hyperexcitability, such as epilepsy, neuropathic pain, and spasticity. KCC2 is also downregulated after axotomy. If muscle reinnervation is allowed, the KCC2 levels recover in motoneurons. Therefore, we argued that target-derived neurotrophic factors might be involved in the regulation of KCC2 expression. For this purpose, we performed the axotomy of extraocular motoneurons via the monocular enucleation of adult rats, and a pellet containing either VEGF or BDNF was chronically implanted in the orbit. Double confocal immunofluorescence of choline acetyl-transferase (ChAT) and KCC2 was carried out in the brainstem sections. Axotomy led to a KCC2 decrease in the neuropil and somata of extraocular motoneurons, peaking at 15 days post-lesion, with the exception of the abducens motoneuron somata. VEGF administration prevented the axotomy-induced KCC2 downregulation. By contrast, BDNF either maintained or reduced the KCC2 levels following axotomy, suggesting that BDNF is involved in the axotomy-induced KCC2 downregulation in extraocular motoneurons. The finding that VEGF prevents KCC2 decrease opens up new possibilities for the treatment of neurological disorders coursing with neuronal hyperactivity due to KCC2 downregulation.
Assuntos
Axotomia , Fator Neurotrófico Derivado do Encéfalo , Cotransportadores de K e Cl- , Neurônios Motores , Simportadores , Fator A de Crescimento do Endotélio Vascular , Animais , Masculino , Ratos , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Regulação para Baixo , Neurônios Motores/metabolismo , Ratos Wistar , Simportadores/metabolismo , Simportadores/genética , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in Smn-deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase Kdm6b transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including Kdm6b transcripts. Gemin5 depletion reduces Kdm6b mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in Smn-deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates Kdm6b expression with implications for motor neuron diseases and therapy.
Assuntos
Flunarizina , Histona Desmetilases com o Domínio Jumonji , Neurônios Motores , Atrofia Muscular Espinal , Proteínas do Complexo SMN , Animais , Camundongos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Flunarizina/farmacologia , Neurônios Motores/metabolismo , Neurônios Motores/efeitos dos fármacos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas do Complexo SMN/metabolismo , Proteínas do Complexo SMN/genética , Neuroproteção/efeitos dos fármacos , Humanos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Many RNA-binding proteins (RBPs) are linked to the dysregulation of RNA metabolism in motor neuron diseases (MNDs). However, the molecular mechanisms underlying MN vulnerability have yet to be elucidated. Here, we found that such an RBP, Quaking5 (Qki5), contributes to formation of the MN-specific transcriptome profile, termed "MN-ness," through the posttranscriptional network and maintenance of the mature MNs. Immunohistochemical analysis and single-cell RNA sequencing (scRNA-seq) revealed that Qki5 is predominantly expressed in MNs, but not in other neuronal populations of the spinal cord. Furthermore, comprehensive RNA sequencing (RNA-seq) analyses revealed that Qki5-dependent RNA regulation plays a pivotal role in generating the MN-specific transcriptome through pre-messenger ribonucleic acid (mRNA) splicing for the synapse-related molecules and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathways. Indeed, MN-specific ablation of the Qki5 caused neurodegeneration in postnatal mice and loss of Qki5 function resulted in the aberrant activation of stress-responsive JNK/SAPK pathway both in vitro and in vivo. These data suggested that Qki5 plays a crucial biological role in RNA regulation and safeguarding of MNs and might be associated with pathogenesis of MNDs.
Assuntos
Neurônios Motores , Proteínas de Ligação a RNA , Medula Espinal , Transcriptoma , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Neurônios Motores/metabolismo , Camundongos , Medula Espinal/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Splicing de RNA , Camundongos KnockoutRESUMO
Peripheral nerve and large-scale muscle injuries result in significant disability, necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials, graphene is favored for its high conductivity, but its low bioactivity requires enhancement through biomimetic components. In this study, we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds, aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia, observing significant improvements in axon outgrowth. Next, we tested the scaffolds with C2C12 mouse and human primary myoblasts, finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally, using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts, we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration, offering robust support for the growth and development of diverse neuromuscular tissues.
Assuntos
Técnicas de Cocultura , Matriz Extracelular , Grafite , Células-Tronco Pluripotentes Induzidas , Alicerces Teciduais , Grafite/química , Animais , Alicerces Teciduais/química , Camundongos , Humanos , Matriz Extracelular/química , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/fisiologia , Neurônios Motores/citologia , Axônios/fisiologia , Mioblastos/citologia , Engenharia Tecidual/métodos , Crescimento Neuronal/efeitos dos fármacos , Crescimento Neuronal/fisiologia , Músculo Esquelético/fisiologia , Músculo Esquelético/citologia , Diferenciação Celular , Junção Neuromuscular/fisiologiaRESUMO
The GGGGCC hexanucleotide repeat expansion (HRE) of the C9orf72 gene is the most frequent cause of amyotrophic lateral sclerosis (ALS), a devastative neurodegenerative disease characterized by motor neuron degeneration. C9orf72 HRE is associated with lowered levels of C9orf72 expression and its translation results in the production of dipeptide-repeats (DPRs). To recapitulate C9orf72-related ALS disease in vivo, we developed a zebrafish model where we expressed glycine-proline (GP) DPR in a c9orf72 knockdown context. We report that C9orf72 gain- and loss-of-function properties act synergistically to induce motor neuron degeneration and paralysis with poly(GP) accumulating preferentially within motor neurons along with Sqstm1/p62 aggregation indicating macroautophagy/autophagy deficits. Poly(GP) levels were shown to accumulate upon c9orf72 downregulation and were comparable to levels assessed in autopsy samples of patients carrying C9orf72 HRE. Chemical boosting of autophagy using rapamycin or apilimod, is able to rescue motor deficits. Proteomics analysis of zebrafish-purified motor neurons unravels mitochondria dysfunction confirmed through a comparative analysis of previously published C9orf72 iPSC-derived motor neurons. Consistently, 3D-reconstructions of motor neuron demonstrate that poly(GP) aggregates colocalize to mitochondria, thus inducing their elongation and swelling and the failure of their processing by mitophagy, with mitophagy activation through urolithin A preventing locomotor deficits. Finally, we report apoptotic-related increased amounts of cleaved Casp3 (caspase 3, apoptosis-related cysteine peptidase) and rescue of motor neuron degeneration by constitutive inhibition of Casp9 or treatment with decylubiquinone. Here we provide evidence of key pathogenic steps in C9ALS-FTD that can be targeted through pharmacological avenues, thus raising new therapeutic perspectives for ALS patients.
Assuntos
Esclerose Lateral Amiotrófica , Apoptose , Autofagia , Proteína C9orf72 , Dipeptídeos , Mitofagia , Neurônios Motores , Peixe-Zebra , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Mitofagia/genética , Apoptose/genética , Humanos , Autofagia/genética , Autofagia/fisiologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/genética , Dipeptídeos/farmacologia , Dipeptídeos/metabolismo , Mutação com Perda de Função/genética , Mitocôndrias/metabolismo , Modelos Animais de DoençasRESUMO
A butterfly-like robot swims using an electronic device to stimulate human-derived motor neurons and cardiac muscle cells.
Assuntos
Neurônios Motores , Robótica , Natação , Robótica/instrumentação , Humanos , Natação/fisiologia , Neurônios Motores/fisiologia , Animais , Desenho de Equipamento , Miócitos Cardíacos/fisiologia , Biomimética/instrumentaçãoRESUMO
BACKGROUND: Hereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST. OBJECTIVE: To determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism. METHODS: A four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted. RESULTS: A novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration. CONCLUSION: The adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.
Assuntos
Neurônios Motores , Mutação , Linhagem , Sítios de Splice de RNA , Espastina , Peixe-Zebra , Espastina/genética , Espastina/metabolismo , Animais , Peixe-Zebra/genética , Humanos , Masculino , Feminino , Mutação/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Sítios de Splice de RNA/genética , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologia , Sequência de Bases , Pessoa de Meia-Idade , Adulto , Íntrons/genética , HeterozigotoRESUMO
Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease worldwide and is characterized by progressive muscle atrophy. There are currently two approved treatments, but they only relieve symptoms briefly and do not cure the disease. The main hindrance to research is the complex cause of ALS, with its pathogenesis not yet fully elucidated. Retinoids (vitamin A derivatives) appear to be essential in neuronal cells and have been implicated in ALS pathogenesis. This study explores 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)ethylnyl]benzoic acid (Ellorarxine, or DC645 or NVG0645), a leading synthetic retinoic acid, discussing its pharmacological mechanisms, neuroprotective properties, and relevance to ALS. The potential therapeutic effect of Ellorarxine was analyzed in vitro using the WT and SOD1G93A NSC-34 cell model of ALS at an administered concentration of 0.3-30 nM. Histological, functional, and biochemical analyses were performed. Elorarxine significantly increased MAP2 expression and neurite length, increased AMPA receptor GluA2 expression and raised intracellular Ca2+ baseline, increased level of excitability, and reduced Ca2+ spike during depolarization in neurites. Ellorarxine also displayed both antioxidant and anti-inflammatory effects. Overall, these results suggest Ellorarxine shows relevance and promise as a novel therapeutic strategy for treatment of ALS.
Assuntos
Fármacos Neuroprotetores , Animais , Camundongos , Fármacos Neuroprotetores/farmacologia , Retinoides/farmacologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Linhagem Celular , Humanos , Receptores de AMPA/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Benzoatos/farmacologia , Doença dos Neurônios Motores/tratamento farmacológico , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Cálcio/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismoRESUMO
A single neuronal population in the spinal cord is crucial to restore walking after paralysis.