RESUMO
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). SMN-restoring therapies have recently emerged; however, preclinical and clinical studies revealed a limited therapeutic time window and systemic aspects of the disease. This raises a fundamental question of whether SMA has presymptomatic, developmental components to disease pathogenesis. We have addressed this by combining micro-computed tomography (µCT) and comparative proteomics to examine systemic pre-symptomatic changes in a prenatal mouse model of SMA. Quantitative µCT analyses revealed that SMA embryos were significantly smaller than littermate controls, indicative of general developmental delay. More specifically, cardiac ventricles were smaller in SMA hearts, whilst liver and brain remained unaffected. In order to explore the molecular consequences of SMN depletion during development, we generated comprehensive, high-resolution, proteomic profiles of neuronal and non-neuronal organs in SMA mouse embryos. Significant molecular perturbations were observed in all organs examined, highlighting tissue-specific prenatal molecular phenotypes in SMA. Together, our data demonstrate considerable systemic changes at an early, presymptomatic stage in SMA mice, revealing a significant developmental component to SMA pathogenesis.
Assuntos
Atrofia Muscular Espinal/genética , Miocárdio/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Coração/fisiopatologia , Humanos , Fígado/metabolismo , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/patologia , Miocárdio/patologia , Fenótipo , Diagnóstico Pré-Natal , Proteômica , Microtomografia por Raio-XRESUMO
The neuromuscular junction (NMJ)-a synapse formed between lower motor neuron and skeletal muscle fibre-represents a major focus of both basic neuroscience research and clinical neuroscience research. Although the NMJ is known to play an important role in many neurodegenerative conditions affecting humans, the vast majority of anatomical and physiological data concerning the NMJ come from lower mammalian (e.g. rodent) animal models. However, recent findings have demonstrated major differences between the cellular anatomy and molecular anatomy of human and rodent NMJs. Therefore, we undertook a comparative morphometric analysis of the NMJ across several larger mammalian species in order to generate baseline inter-species anatomical reference data for the NMJ and to identify animal models that better represent the morphology of the human NMJ in vivo. Using a standardized morphometric platform ('NMJ-morph'), we analysed 5,385 individual NMJs from lower/pelvic limb muscles (EDL, soleus and peronei) of 6 mammalian species (mouse, cat, dog, sheep, pig and human). There was marked heterogeneity of NMJ morphology both within and between species, with no overall relationship found between NMJ morphology and muscle fibre diameter or body size. Mice had the largest NMJs on the smallest muscle fibres; cats had the smallest NMJs on the largest muscle fibres. Of all the species examined, the sheep NMJ had the most closely matched morphology to that found in humans. Taken together, we present a series of comprehensive baseline morphometric data for the mammalian NMJ and suggest that ovine models are likely to best represent the human NMJ in health and disease.
Assuntos
Mamíferos/anatomia & histologia , Junção Neuromuscular/anatomia & histologia , Animais , Gatos , Cães , Humanos , CamundongosRESUMO
Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo.
Assuntos
Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Fosfoglicerato Quinase/genética , Medula Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Trifosfato de Adenosina/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Metabolismo Energético , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Mitocôndrias/metabolismo , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Fosfoglicerato Quinase/antagonistas & inibidores , Prazosina/administração & dosagem , Prazosina/análogos & derivados , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/patologia , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Neurodegenerative and neuromuscular disorders can manifest throughout the lifespan of an individual, from infant to elderly individuals. Axonal and synaptic degeneration are early and critical elements of nearly all human neurodegenerative diseases and neural injury, however the molecular mechanisms which regulate this process are yet to be fully elucidated. Furthermore, how the molecular mechanisms governing degeneration are impacted by the age of the individual is poorly understood. Interestingly, in mice which are under 3â¯weeks of age, the degeneration of axons and synapses following hypoxic or traumatic injury is significantly slower. This process, known as Wallerian degeneration (WD), is a molecularly and morphologically distinct subtype of neurodegeneration by which axons and synapses undergo distinct fragmentation and death following a range of stimuli. In this study, we first use an ex-vivo model of axon injury to confirm the significant delay in WD in neonatal mice. We apply tandem mass-tagging quantitative proteomics to profile both nerve and muscle between P12 and P24 inclusive. Application of unbiased in silico workflows to relevant protein identifications highlights a steady elevation in oxidative phosphorylation cascades corresponding to the accelerated degeneration rate. We demonstrate that inhibition of Complex I prevents the axotomy-induced rise in reactive oxygen species and protects axons following injury. Furthermore, we reveal that pharmacological activation of oxidative phosphorylation significantly accelerates degeneration at the neuromuscular junction in neonatal mice. In summary, we reveal dramatic changes in the neuromuscular proteome during post-natal maturation of the neuromuscular system, and demonstrate that endogenous dynamics in mitochondrial bioenergetics during this time window have a functional impact upon regulating the stability of the neuromuscular system.
Assuntos
Mitocôndrias/metabolismo , Junção Neuromuscular/metabolismo , Fosforilação Oxidativa , Degeneração Walleriana/metabolismo , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/patologia , Degeneração Walleriana/patologiaRESUMO
α-Synuclein plays a central role in Parkinson's disease, where it contributes to the vulnerability of synapses to degeneration. However, the downstream mechanisms through which α-synuclein controls synaptic stability and degeneration are not fully understood. Here, comparative proteomics on synapses isolated from α-synuclein-/- mouse brain identified mitochondrial proteins as primary targets of α-synuclein, revealing 37 mitochondrial proteins not previously linked to α-synuclein or neurodegeneration pathways. Of these, sideroflexin 3 (SFXN3) was found to be a mitochondrial protein localized to the inner mitochondrial membrane. Loss of SFXN3 did not disturb mitochondrial electron transport chain function in mouse synapses, suggesting that its function in mitochondria is likely to be independent of canonical bioenergetic pathways. In contrast, experimental manipulation of SFXN3 levels disrupted synaptic morphology at the Drosophila neuromuscular junction. These results provide novel insights into α-synuclein-dependent pathways, highlighting an important influence on mitochondrial proteins at the synapse, including SFXN3. We also identify SFXN3 as a new mitochondrial protein capable of regulating synaptic morphology in vivo.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Sinapses/metabolismo , alfa-Sinucleína/metabolismo , Animais , Drosophila melanogaster/metabolismo , Metabolismo Energético , Ontologia Genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Junção Neuromuscular/metabolismoRESUMO
Deafferentation of motor neurons as a result of defective sensory-motor connectivity is a critical early event in the pathogenesis of spinal muscular atrophy, but the underlying molecular pathways remain unknown. We show that restoration of ubiquitin-like modifier-activating enzyme 1 (UBA1) was sufficient to correct sensory-motor connectivity in the spinal cord of mice with spinal muscular atrophy. Aminoacyl-tRNA synthetases, including GARS, were identified as downstream targets of UBA1. Regulation of GARS by UBA1 occurred via a non-canonical pathway independent of ubiquitylation. Dysregulation of UBA1/GARS pathways in spinal muscular atrophy mice disrupted sensory neuron fate, phenocopying GARS-dependent defects associated with Charcot-Marie-Tooth disease. Sensory neuron fate was corrected following restoration of UBA1 expression and UBA1/GARS pathways in spinal muscular atrophy mice. We conclude that defective sensory motor connectivity in spinal muscular atrophy results from perturbations in a UBA1/GARS pathway that modulates sensory neuron fate, thereby highlighting significant molecular and phenotypic overlap between spinal muscular atrophy and Charcot-Marie-Tooth disease.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Atrofia Muscular Espinal/patologia , Vias Neurais/patologia , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/metabolismo , Vias Neurais/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
The incidence of neurodegenerative diseases in the developed world has risen over the last century, concomitant with an increase in average human lifespan. A major challenge is therefore to identify genes that control neuronal health and viability with a view to enhancing neuronal health during ageing and reducing the burden of neurodegeneration. Analysis of gene expression data has recently been used to infer gene functions for a range of tissues from co-expression networks. We have now applied this approach to transcriptomic datasets from the mammalian nervous system available in the public domain. We have defined the genes critical for influencing neuronal health and disease in different neurological cell types and brain regions. The functional contribution of genes in each co-expression cluster was validated using human disease and knockout mouse phenotypes, pathways and gene ontology term annotation. Additionally a number of poorly annotated genes were implicated by this approach in nervous system function. Exploiting gene expression data available in the public domain allowed us to validate key nervous system genes and, importantly, to identify additional genes with minimal functional annotation but with the same expression pattern. These genes are thus novel candidates for a role in neurological health and disease and could now be further investigated to confirm their function and regulation during ageing and neurodegeneration.
Assuntos
Perfilação da Expressão Gênica , Estudos de Associação Genética , Doenças do Sistema Nervoso/genética , Sistema Nervoso/metabolismo , Transcriptoma , Animais , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Sistema Nervoso/patologia , Doenças do Sistema Nervoso/patologiaRESUMO
Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of â¼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Doenças dos Cavalos/genética , Doenças Neurodegenerativas/genética , Deficiências na Proteostase/genética , Ubiquitina/genética , Proteínas tau/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Feminino , Gânglios Sensitivos/química , Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/metabolismo , Doenças dos Cavalos/patologia , Cavalos , Masculino , Anotação de Sequência Molecular , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica , Deficiências na Proteostase/diagnóstico , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Ubiquitina/metabolismo , Proteínas tau/metabolismoRESUMO
INTRODUCTION: Proteomic techniques offer insights into the molecular perturbations occurring in muscular-dystrophies (MD). Revisiting published datasets can highlight conserved downstream molecular alterations, which may be worth re-assessing to determine whether their experimental manipulation is capable of modulating disease severity. AREAS COVERED: Here, we review the MD literature, highlighting conserved molecular insights warranting mechanistic investigation for therapeutic potential. We also describe a workflow currently proving effective for efficient identification of biomarkers & therapeutic targets in other neurodegenerative conditions, upon which future MD proteomic investigations could be modelled. Expert commentary: Studying disease models can be useful for identifying biomarkers and model specific degenerative cascades, but rarely offer translatable mechanistic insights into disease pathology. Conversely, direct analysis of human samples undergoing degeneration presents challenges derived from complex chronic degenerative molecular processes. This requires a carefully planed & reproducible experimental paradigm accounting for patient selection through to grouping by disease severity and ending with proteomic data filtering and processing.
Assuntos
Biomarcadores , Distrofias Musculares/genética , Proteoma/genética , Proteômica , Humanos , Distrofias Musculares/patologiaRESUMO
Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi-quantitative results. Visualising infrared (IR) 'tags', with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR-based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR-based scanning technology yields comparable area-based quantification to those obtained from a modern high-resolution digital slide scanner. Secondly, IR-based dual target visualisation and expression-based quantification is rapid and simple. Thirdly, IR-based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR-based QFIHC provides an alternative method of rapid whole-tissue section low-resolution imaging for the production of reliable and accurate quantitative data.
Assuntos
Encéfalo/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Raios Infravermelhos , Microscopia/métodos , Animais , Camundongos , Camundongos Endogâmicos BALB C , Modelos AnimaisRESUMO
Postnatal synapse elimination plays a critical role in sculpting and refining neural connectivity throughout the central and peripheral nervous systems, including the removal of supernumerary axonal inputs from neuromuscular junctions (NMJs). Here, we reveal a novel and important role for myelinating glia in regulating synapse elimination at the mouse NMJ, where loss of a single glial cell protein, the glial isoform of neurofascin (Nfasc155), was sufficient to disrupt postnatal remodeling of synaptic circuitry. Neuromuscular synapses were formed normally in mice lacking Nfasc155, including the establishment of robust neuromuscular synaptic transmission. However, loss of Nfasc155 was sufficient to cause a robust delay in postnatal synapse elimination at the NMJ across all muscle groups examined. Nfasc155 regulated neuronal remodeling independently of its canonical role in forming paranodal axo-glial junctions, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Rather, high-resolution proteomic screens revealed that loss of Nfasc155 from glial cells was sufficient to disrupt neuronal cytoskeletal organization and trafficking pathways, resulting in reduced levels of neurofilament light (NF-L) protein in distal axons and motor nerve terminals. Mice lacking NF-L recapitulated the delayed synapse elimination phenotype observed in mice lacking Nfasc155, suggesting that glial cells regulate synapse elimination, at least in part, through modulation of the axonal cytoskeleton. Together, our study reveals a glial cell-dependent pathway regulating the sculpting of neuronal connectivity and synaptic circuitry in the peripheral nervous system.
Assuntos
Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/fisiologia , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/fisiologia , Junção Neuromuscular/fisiologia , Sinapses/fisiologia , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/fisiologia , Citoesqueleto/metabolismo , Camundongos , Camundongos Knockout , Placa Motora/crescimento & desenvolvimento , Neurônios Motores/metabolismo , Fatores de Crescimento Neural/genética , Condução Nervosa/genética , Condução Nervosa/fisiologia , Proteínas de Neurofilamentos/metabolismo , Neuroglia/metabolismo , Junção Neuromuscular/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Proteômica , Células de Schwann/metabolismo , Sinapses/genética , Transmissão Sináptica/fisiologiaRESUMO
Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.
Assuntos
Lesões Encefálicas , Drosophila , Degeneração Neural , Sinapses , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Axônios/fisiologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Calbindina 2 , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Proteômica , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Sinapses/metabolismo , Sinapses/patologia , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Low levels of survival of motor neuron (SMN) protein cause the neuromuscular disease spinal muscular atrophy (SMA), characterized by degeneration of lower motor neurons and atrophy of skeletal muscle. Recent work demonstrated that low levels of SMN also trigger pathological changes in Schwann cells, leading to abnormal axon myelination and disrupted deposition of extracellular matrix proteins in peripheral nerve. However, the molecular pathways linking SMN depletion to intrinsic defects in Schwann cells remained unclear. Label-free proteomics analysis of Schwann cells isolated from SMA mouse peripheral nerve revealed widespread changes to the Schwann cell proteome, including disruption to growth/proliferation, cell death/survival, and molecular transport pathways. Functional clustering analyses revealed significant disruption to a number of proteins contributing to ubiquitination pathways, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). Pharmacological suppression of Uba1 in Schwann cells was sufficient to reproduce the defective myelination phenotype seen in SMA. These findings demonstrate an important role for SMN protein and ubiquitin-dependent pathways in maintaining Schwann cell homeostasis and provide significant additional experimental evidence supporting a key role for ubiquitin pathways and, Uba1 in particular, in driving SMA pathogenesis across a broad range of cells and tissues.
Assuntos
Homeostase/fisiologia , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Proteômica/métodos , Células de Schwann/patologia , Ubiquitina/metabolismo , Animais , Análise por Conglomerados , Homeostase/genética , Camundongos , Nervos Periféricos/citologia , Nervos Periféricos/metabolismo , Células de Schwann/metabolismo , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidoresRESUMO
AIM: Levels of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) are robustly increased in spinal muscular atrophy (SMA) patient fibroblasts and mouse models. We therefore wanted to establish whether changes in UCHL1 contribute directly to disease pathogenesis, and to assess whether pharmacological inhibition of UCHL1 represents a viable therapeutic option for SMA. METHODS: SMA mice and control littermates received a pharmacological UCHL1 inhibitor (LDN-57444) or DMSO vehicle. Survival and weight were monitored daily, a righting test of motor performance was performed, and motor neurone loss, muscle fibre atrophy and neuromuscular junction pathology were all quantified. Ubiquitin-like modifier activating enzyme 1 (Uba1) was then pharmacologically inhibited in neurones in vitro to examine the relationship between Uba1 levels and UCHL1 in SMA. RESULTS: Pharmacological inhibition of UCHL1 failed to improve survival, motor symptoms or neuromuscular pathology in SMA mice and actually precipitated the onset of weight loss. LDN-57444 treatment significantly decreased spinal cord mono-ubiquitin levels, further exacerbating ubiquitination defects in SMA mice. Pharmacological inhibition of Uba1, levels of which are robustly reduced in SMA, was sufficient to induce accumulation of UCHL1 in primary neuronal cultures. CONCLUSION: Pharmacological inhibition of UCHL1 exacerbates rather than ameliorates disease symptoms in a mouse model of SMA. Thus, pharmacological inhibition of UCHL1 is not a viable therapeutic target for SMA. Moreover, increased levels of UCHL1 in SMA likely represent a downstream consequence of decreased Uba1 levels, indicative of an attempted supportive compensatory response to defects in ubiquitin homeostasis caused by low levels of SMN protein.
Assuntos
Indóis/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/enzimologia , Oximas/uso terapêutico , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Homeostase/efeitos dos fármacos , Humanos , Indóis/administração & dosagem , Indóis/efeitos adversos , Camundongos , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/patologia , Oximas/administração & dosagem , Oximas/efeitos adversosRESUMO
BACKGROUND: Idiopathic Parkinson's disease is marked by degeneration of dopamine neurons projecting from the substantia nigra to the striatum. Although proteins expressed by the target striatum can positively affect the viability and growth of dopaminergic neurons, very little is known about the molecular response of the striatum as nigro-striatal denervation progresses. Here, iTRAQ labelling and MALDI TOF/TOF mass spectrometry have been used to quantitatively compare the striatal proteome of rats before, during, and after 6-OHDA induced dopamine denervation. RESULTS: iTRAQ analysis revealed the differential expression of 50 proteins at 3 days, 26 proteins at 7 days, and 34 proteins at 14 days post-lesioning, compared to the unlesioned striatum. While the denervated striatum showed a reduced expression of proteins associated with the loss of dopaminergic input (e.g., TH and DARPP-32), there was an increased expression of proteins associated with regeneration and growth of neurites (e.g., GFAP). In particular, the expression of guanine deaminase (GDA, cypin) - a protein known to be involved in dendritic branching - was significantly increased in the striatum at 3, 7 and 14 days post-lesioning (a finding verified by immunohistochemistry). CONCLUSIONS: Together, these findings provide evidence to suggest that the response of the normal mammalian striatum to nigro-striatal denervation includes the increased expression of proteins that may have the capacity to facilitate repair and growth of neuronal circuitry.
RESUMO
BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.
Assuntos
Diferenciação Celular , Ácido Hialurônico , Células-Tronco Mesenquimais , Células-Tronco Pluripotentes , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Ácido Hialurônico/farmacologia , Ácido Hialurônico/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Meios de Cultura Livres de Soro/farmacologia , Linhagem da Célula , Células Cultivadas , Técnicas de Cultura de Células/métodos , Técnicas de CoculturaRESUMO
Glucocorticoids modulate glucose homeostasis, acting on metabolically active tissues such as liver, skeletal muscle, and adipose tissue. Intracellular regulation of glucocorticoid action in adipose tissue impacts metabolic responses to obesity. ATP-binding cassette family C member 1 (ABCC1) is a transmembrane glucocorticoid transporter known to limit the accumulation of exogenously administered corticosterone in adipose tissue. However, the role of ABCC1 in the regulation of endogenous glucocorticoid action and its impact on fuel metabolism has not been studied. Here, we investigate the impact of Abcc1 deficiency on glucocorticoid action and high-fat-diet (HFD)-induced obesity. In lean male mice, deficiency of Abcc1 increased endogenous corticosterone levels in skeletal muscle and adipose tissue but did not impact insulin sensitivity. In contrast, Abcc1-deficient male mice on HFD displayed impaired glucose and insulin tolerance, and fasting hyperinsulinaemia, without alterations in tissue corticosterone levels. Proteomics and bulk RNA sequencing revealed that Abcc1 deficiency amplified the transcriptional response to an obesogenic diet in adipose tissue but not in skeletal muscle. Moreover, Abcc1 deficiency impairs key signalling pathways related to glucose metabolism in both skeletal muscle and adipose tissue, in particular those related to OXPHOS machinery and Glut4. Together, our results highlight a role for ABCC1 in regulating glucose homeostasis, demonstrating diet-dependent effects that are not associated with altered tissue glucocorticoid concentrations.
Assuntos
Tecido Adiposo , Corticosterona , Dieta Hiperlipídica , Resistência à Insulina , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Músculo Esquelético , Obesidade , Animais , Masculino , Dieta Hiperlipídica/efeitos adversos , Camundongos , Obesidade/metabolismo , Obesidade/genética , Obesidade/etiologia , Tecido Adiposo/metabolismo , Resistência à Insulina/fisiologia , Corticosterona/sangue , Corticosterona/metabolismo , Músculo Esquelético/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Glucose/metabolismoRESUMO
BACKGROUND: Cardiac repair and remodeling following myocardial infarction (MI) is a multifactorial process involving pro-reparative inflammation, angiogenesis and fibrosis. Noninvasive imaging using a radiotracer targeting these processes could be used to elucidate cardiac wound healing mechanisms. The alpha7 nicotinic acetylcholine receptor (É7nAChR) stimulates pro-reparative macrophage activity and angiogenesis, making it a potential imaging biomarker in this context. We investigated this by assessing in vitro cellular expression of É7nAChR, and by using a tritiated version of the PET radiotracer [18F]NS14490 in tissue autoradiography studies. RESULTS: É7nAChR expression in human monocyte-derived macrophages and vascular cells showed the highest relative expression was within macrophages, but only endothelial cells exhibited a proliferation and hypoxia-driven increase in expression. Using a mouse model of inflammatory angiogenesis following sponge implantation, specific binding of [3H]NS14490 increased from 3.6 ± 0.2 µCi/g at day 3 post-implantation to 4.9 ± 0.2 µCi/g at day 7 (n = 4, P < 0.01), followed by a reduction at days 14 and 21. This peak matched the onset of vessel formation, macrophage infiltration and sponge fibrovascular encapsulation. In a rat MI model, specific binding of [3H]NS14490 was low in sham and remote MI myocardium. Specific binding within the infarct increased from day 14 post-MI (33.8 ± 14.1 µCi/g, P ≤ 0.01 versus sham), peaking at day 28 (48.9 ± 5.1 µCi/g, P ≤ 0.0001 versus sham). Histological and proteomic profiling of É7nAChR positive tissue revealed strong associations between É7nAChR and extracellular matrix deposition, and rat cardiac fibroblasts expressed É7nAChR protein under normoxic and hypoxic conditions. CONCLUSION: É7nAChR is highly expressed in human macrophages and showed proliferation and hypoxia-driven expression in human endothelial cells. While NS14490 imaging displays a pattern that coincides with vessel formation, macrophage infiltration and fibrovascular encapsulation in the sponge model, this is not the case in the MI model where the É7nAChR imaging signal was strongly associated with extracellular matrix deposition which could be explained by É7nAChR expression in fibroblasts. Overall, these findings support the involvement of É7nAChR across several processes central to cardiac repair, with fibrosis most closely associated with É7nAChR following MI.
RESUMO
Apolipoprotein E (apoE) is a 34 kDa glycoprotein with three distinct isoforms in the human population (apoE2, apoE3 and apoE4) known to play a major role in differentially influencing risk to, as well as outcome from, disease and injury in the central nervous system. In general, the apoE4 allele is associated with poorer outcomes after disease or injury, whereas apoE3 is associated with better responses. The extent to which different apoE isoforms influence degenerative and regenerative events in the peripheral nervous system (PNS) is still to be established, and the mechanisms through which apoE exerts its isoform-specific effects remain unclear. Here, we have investigated isoform-specific effects of human apoE on the mouse PNS. Experiments in mice ubiquitously expressing human apoE3 or human apoE4 on a null mouse apoE background revealed that apoE4 expression significantly disrupted peripheral nerve regeneration and subsequent neuromuscular junction re-innervation following nerve injury compared with apoE3, with no observable effects on normal development, maturation or Wallerian degeneration. Proteomic isobaric tag for relative and absolute quantitation (iTRAQ) screens comparing healthy and regenerating peripheral nerves from mice expressing apoE3 or apoE4 revealed significant differences in networks of proteins regulating cellular outgrowth and regeneration (myosin/actin proteins), as well as differences in expression levels of proteins involved in regulating the blood-nerve barrier (including orosomucoid 1). Taken together, these findings have identified isoform-specific roles for apoE in determining the protein composition of peripheral nerve as well as regulating nerve regeneration pathways in vivo.
Assuntos
Apolipoproteínas E/metabolismo , Regeneração Nervosa/fisiologia , Sistema Nervoso Periférico/fisiologia , Isoformas de Proteínas/metabolismo , Animais , Apolipoproteínas E/genética , Axônios/metabolismo , Axônios/ultraestrutura , Western Blotting , Eletrofisiologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Orosomucoide/metabolismo , Sistema Nervoso Periférico/lesões , Isoformas de Proteínas/genética , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Low levels of full-length survival motor neuron (SMN) protein cause the motor neuron disease, spinal muscular atrophy (SMA). Although motor neurons undoubtedly contribute directly to SMA pathogenesis, the role of muscle is less clear. We demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic severe SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomic data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. Robust upregulation of voltage-dependent anion-selective channel protein 2 (Vdac2) and downregulation of parvalbumin in severe SMA mice was confirmed in a milder SMA mouse model and in human patient muscle biopsies. Molecular pathology of skeletal muscle was ameliorated in mice treated with the FDA-approved histone deacetylase inhibitor, suberoylanilide hydroxamic acid. We conclude that intrinsic pathology of skeletal muscle is an important and reversible event in SMA and also suggest that muscle proteins have the potential to act as novel biomarkers in SMA.