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Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the central nervous system (CNS). Mutations in the metalloenzyme SOD1 are associated with inherited forms of ALS and cause a toxic gain of function thought to be mediated by dimer destabilization and misfolding. SOD1 binds two Cu and two Zn ions in its homodimeric form. We have applied native ambient mass spectrometry imaging to visualize the spatial distributions of intact metal-bound SOD1G93A complexes in SOD1G93A transgenic mouse spinal cord and brain sections and evaluated them against disease pathology. The molecular specificity of our approach reveals that metal-deficient SOD1G93A species are abundant in CNS structures correlating with ALS pathology whereas fully metalated SOD1G93A species are homogenously distributed. Monomer abundance did not correlate with pathology. We also show that the dimer-destabilizing post-translational modification, glutathionylation, has limited influence on the spatial distribution of SOD1 dimers.
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Esclerose Lateral Amiotrófica , Encéfalo , Espectrometria de Massas , Camundongos Transgênicos , Medula Espinal , Superóxido Dismutase-1 , Animais , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/química , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Camundongos , Medula Espinal/metabolismo , Medula Espinal/patologia , Espectrometria de Massas/métodos , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Cobre/metabolismo , Zinco/metabolismo , Humanos , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/química , Mutação , Processamento de Proteína Pós-Traducional , Multimerização Proteica , Modelos Animais de Doenças , MasculinoRESUMO
Neuromuscular disorders are a group of conditions that can result in weakness of skeletal muscles. Examples include fatal diseases such as amyotrophic lateral sclerosis and conditions associated with high morbidity such as myopathies (muscle diseases). Many of these disorders are known to have abnormal protein folding and protein aggregates. Thus, easy to apply methods for the detection of such changes may prove useful diagnostic biomarkers. Raman spectroscopy has shown early promise in the detection of muscle pathology in neuromuscular disorders and is well suited to characterising the conformational profiles relating to protein secondary structure. In this work, we assess if Raman spectroscopy can detect differences in protein structure in muscle in the setting of neuromuscular disease. We utilise in vivo Raman spectroscopy measurements from preclinical models of amyotrophic lateral sclerosis and the myopathy Duchenne muscular dystrophy, together with ex vivo measurements of human muscle samples from individuals with and without myopathy. Using quantitative conformation profiling and matrix factorisation we demonstrate that quantitative 'conformational fingerprinting' can be used to identify changes in protein folding in muscle. Notably, myopathic conditions in both preclinical models and human samples manifested a significant reduction in α-helix structures, with concomitant increases in ß-sheet and, to a lesser extent, nonregular configurations. Spectral patterns derived through non-negative matrix factorisation were able to identify myopathy with a high accuracy (79% in mouse, 78% in human tissue). This work demonstrates the potential of conformational fingerprinting as an interpretable biomarker for neuromuscular disorders.
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Biomarcadores , Distrofia Muscular de Duchenne , Análise Espectral Raman , Análise Espectral Raman/métodos , Humanos , Animais , Biomarcadores/análise , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/diagnóstico , Músculo Esquelético/química , Músculo Esquelético/patologia , Camundongos , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/patologia , MasculinoRESUMO
Amyotrophic lateral sclerosis (ALS) is a poorly treated multifactorial neurodegenerative disease associated with multiple cell types and subcellular organelles. As with other multifactorial diseases, it is likely that drugs will need to target multiple disease processes and cell types to be effective. We review here the role of Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signalling in ALS, confirm the association of this signalling with fundamental ALS disease processes using the BenevolentAI Knowledge Graph, and demonstrate that inhibitors of this pathway could reduce the ALS pathophysiology in neurons, glia, muscle fibres, and blood cells. Specifically, we suggest that inhibition of the JAK enzymes by approved inhibitors known as Jakinibs could reduce STAT3 activation and modify the progress of this disease. Analysis of the Jakinibs highlights baricitinib as a suitable candidate due to its ability to penetrate the central nervous system and exert beneficial effects on the immune system. Therefore, we recommend that this drug be tested in appropriately designed clinical trials for ALS.
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Esclerose Lateral Amiotrófica , Inibidores de Janus Quinases , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Inibidores de Janus Quinases/uso terapêutico , Sistema Nervoso Central/metabolismo , Janus Quinases/metabolismo , Janus Quinases/uso terapêuticoRESUMO
INTRODUCTION/AIMS: Electromyography (EMG) remains a key component of the diagnostic work-up for suspected neuromuscular disease, but it does not provide insight into the molecular composition of muscle which can provide diagnostic information. Raman spectroscopy is an emerging neuromuscular biomarker capable of generating highly specific, molecular fingerprints of tissue. Here, we present "optical EMG," a combination of EMG and Raman spectroscopy, achieved using a single needle. METHODS: An optical EMG needle was created to collect electrophysiological and Raman spectroscopic data during a single insertion. We tested functionality with in vivo recordings in the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), using both transgenic (n = 10) and non-transgenic (NTg, n = 7) mice. Under anesthesia, compound muscle action potentials (CMAPs), spontaneous EMG activity and Raman spectra were recorded from both gastrocnemius muscles with the optical EMG needle. Standard concentric EMG needle recordings were also undertaken. Electrophysiological data were analyzed with standard univariate statistics, Raman data with both univariate and multivariate analyses. RESULTS: A significant difference in CMAP amplitude was observed between SOD1G93A and NTg mice with optical EMG and standard concentric needles (p = .015 and p = .011, respectively). Spontaneous EMG activity (positive sharp waves) was detected in transgenic SOD1G93A mice only. Raman spectra demonstrated peaks associated with key muscle components. Significant differences in molecular composition between SOD1G93A and NTg muscle were identified through the Raman spectra. DISCUSSION: Optical EMG can provide standard electrophysiological data and molecular Raman data during a single needle insertion and represents a potential biomarker for neuromuscular disease.
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Esclerose Lateral Amiotrófica , Análise Espectral Raman , Camundongos , Animais , Eletromiografia , Superóxido Dismutase-1/genética , Músculo Esquelético , Camundongos Transgênicos , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , Modelos Animais de Doenças , Superóxido DismutaseRESUMO
A p.Y374X truncation in TARDBP was recently shown to reduce expression of TDP43 in fibroblasts isolated from ALS cases. In this follow up study focused on assessing the downstream phenotypic consequences of loss of TDP43 in the context of the truncation, we have shown a striking effect on the fibroblast metabolic profile. Phenotypic metabolic screening uncovered a distinct metabolic profile in TDP43-Y374X fibroblasts compared to controls, which was driven by alterations in key metabolic checkpoint intermediates including pyruvate, alpha-ketoglutarate and succinate. These metabolic alterations were confirmed using transcriptomics and bioenergetic flux analysis. These data suggest that TDP43 truncation directly compromises glycolytic and mitochondrial function, identifying potential therapeutic targets for mitigating the effects of TDP43-Y374X truncation.
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Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.
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Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Animais , Camundongos , Dipeptídeos , Proteína C9orf72 , Transporte Ativo do Núcleo Celular , Células HEK293 , Peptídeos , Neurônios Motores , RNA , Fatores de Processamento de Serina-ArgininaRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating disease caused by degeneration of motor neurons. As with all major neurodegenerative disorders, development of disease-modifying therapies has proven challenging for multiple reasons. Nevertheless, ALS is one of the few neurodegenerative diseases for which disease-modifying therapies are approved. Significant discoveries and advances have been made in ALS preclinical models, genetics, pathology, biomarkers, imaging and clinical readouts over the last 10-15 years. At the same time, novel therapeutic paradigms are being applied in areas of high unmet medical need, including neurodegenerative disorders. These developments have evolved our knowledge base, allowing identification of targeted candidate therapies for ALS with diverse mechanisms of action. In this Review, we discuss how this advanced knowledge, aligned with new approaches, can enable effective translation of therapeutic agents from preclinical studies through to clinical benefit for patients with ALS. We anticipate that this approach in ALS will also positively impact the field of drug discovery for neurodegenerative disorders more broadly.
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Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Neurônios Motores , Descoberta de Drogas/métodos , BiomarcadoresRESUMO
We describe an autosomal dominant, multi-generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co-segregates with a heterozygous p.Y374X nonsense mutation within TDP-43. Mislocalization of TDP-43 and formation of insoluble TDP-43-positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP-43 pathology within lower motor neurons, but classical TDP-43-positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP-43 protein expression is reduced compared to wild-type controls. Despite absence of TDP-43-positive inclusions we confirmed deficient TDP-43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP-43 protein species but not typical C-terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP-43 combined with atypical TDP-43 protein species and absent C-terminal fragments extends the molecular phenotypes associated with TDP-43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP-43-mediated toxicity.
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Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , LinhagemRESUMO
Raman spectroscopy shows promise as a biomarker for complex nerve and muscle (neuromuscular) diseases. To maximise its potential, several challenges remain. These include the sensitivity to different instrument configurations, translation across preclinical/human tissues and the development of multivariate analytics that can derive interpretable spectral outputs for disease identification. Nonnegative matrix factorisation (NMF) can extract features from high-dimensional data sets and the nonnegative constraint results in physically realistic outputs. In this study, we have undertaken NMF on Raman spectra of muscle obtained from different clinical and preclinical settings. First, we obtained and combined Raman spectra from human patients with mitochondrial disease and healthy volunteers, using both a commercial microscope and in-house fibre optic probe. NMF was applied across all data, and spectral patterns common to both equipment configurations were identified. Linear discriminant models utilising these patterns were able to accurately classify disease states (accuracy 70.2-84.5%). Next, we applied NMF to spectra obtained from the mdx mouse model of a Duchenne muscular dystrophy and patients with dystrophic muscle conditions. Spectral fingerprints common to mouse/human were obtained and able to accurately identify disease (accuracy 79.5-98.8%). We conclude that NMF can be used to analyse Raman data across different equipment configurations and the preclinical/clinical divide. Thus, the application of NMF decomposition methods could enhance the potential of Raman spectroscopy for the study of fatal neuromuscular diseases.
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INTRODUCTION/AIMS: Raman spectroscopy is an emerging technique for the evaluation of muscle disease. In this study we evaluate the ability of in vivo intramuscular Raman spectroscopy to detect the effects of voluntary running in the mdx model of Duchenne muscular dystrophy (DMD). We also compare mdx data with muscle spectra from human DMD patients. METHODS: Thirty 90-day-old mdx mice were randomly allocated to an exercised group (48-hour access to a running wheel) and an unexercised group (n = 15 per group). In vivo Raman spectra were collected from both gastrocnemius muscles and histopathological assessment subsequently performed. Raman data were analyzed using principal component analysis-fed linear discriminant analysis (PCA-LDA). Exercised and unexercised mdx muscle spectra were compared with human DMD samples using cosine similarity. RESULTS: Exercised mice ran an average of 6.5 km over 48 hours, which induced a significant increase in muscle necrosis (P = .03). PCA-LDA scores were significantly different between the exercised and unexercised groups (P < .0001) and correlated significantly with distance run (P = .01). Raman spectra from exercised mice more closely resembled human spectra than those from unexercised mice. DISCUSSION: Raman spectroscopy provides a readout of the biochemical alterations in muscle in both the mdx mouse and human DMD muscle.
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Distrofia Muscular de Duchenne , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/patologia , Análise Espectral RamanRESUMO
Mice with transgenic expression of human SOD1G93A are a widely used model of ALS, with a caudal-rostral progression of motor impairment. Previous studies have quantified the progression of motoneuron (MN) degeneration based on size, even though alpha (α-) and gamma (γ-) MNs overlap in size. Therefore, using molecular markers and synaptic inputs, we quantified the survival of α-MNs and γ-MNs at the lumbar and cervical spinal segments of 3- and 4-month SOD1G93A mice, to investigate whether there is a caudal-rostral progression of MN death. By 3 months, in the cervical and lumbar spinal cord, there was α-MN degeneration with complete γ-MN sparing. At 3 months, the cervical spinal cord had more α-MNs per ventral horn than the lumbar spinal cord in SOD1G93A mice. A similar spatial trend of degeneration was observed in the corticospinal tract, which remained intact in the cervical spinal cord at 3- and 4- months of age. These findings agree with the corticofugal synaptopathy model that α-MNs and CST of the lumbar spinal cord are more susceptible to degeneration in SOD1G93A mice. Hence, there is a spatial and temporal caudal-rostral progression of α-MN and CST degeneration in SOD1G93A mice.
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BACKGROUND: Glutathione (GSH) is an important brain antioxidant and a number of studies have reported its measurement by edited and nonedited localized 1 H spectroscopy techniques within a range of applications in healthy volunteers and disease states. Good test-retest reproducibility is key when assessing the efficacy of treatments aimed at modulating GSH levels within the central nervous system or when noninvasively assessing changes in GSH content over time. PURPOSE: To evaluate the intraday (in vitro and in vivo) and 1-month apart (in vivo) test-retest reproducibility of GSH measurements from GSH-edited MEGA-PRESS acquisitions at 3 T in a phantom and in the brain of a cohort of middle-aged and older healthy volunteers. STUDY TYPE: Prospective. SUBJECTS/PHANTOMS: A phantom containing physiological concentrations of GSH and metabolites with overlapping spectral signatures and 10 healthy volunteers (4 F, 6 M, 55 ± 14 years old). FIELD STRENGTH/SEQUENCE: GSH-edited spectra were acquired at 3 T using the MEGA-PRESS sequence. ASSESSMENT: The phantom was scanned twice and the healthy subjects were scanned three times (on two separate days, 1 month apart). GSH was quantified from each acquisition, with the in vivo voxels placed at the primary motor cortex (PMC) and the occipital cortex (OCC). STATISTICAL TESTS: Mean coefficients of variation (CV) were used to assess short-term (in vitro and in vivo) and longer-term (in vivo) test-retest reproducibility. RESULTS: In vitro, the CV was 2.3%. In vivo, the mean intraday CV was 3.3% in the PMC and 2.4% in the OCC, while the CVs at 1 month apart were 4.6% in the PMC and 7.8% in the OCC. DATA CONCLUSION: GSH-edited MEGA-PRESS spectroscopy allows measurement of GSH with excellent precision. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.
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Córtex Motor , Adulto , Idoso , Encéfalo , Glutationa , Humanos , Pessoa de Meia-Idade , Lobo Occipital/diagnóstico por imagem , Estudos Prospectivos , Reprodutibilidade dos TestesRESUMO
Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066-1315 (emGFP-1066:TetC), 1093-1315 (emGFP-1093:TetC) and 1109-1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.
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Microscopia Confocal , Junção Neuromuscular/diagnóstico por imagem , Toxina Tetânica/toxicidade , Animais , Animais Recém-Nascidos , Axônios/efeitos dos fármacos , Axônios/metabolismo , Sítios de Ligação , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Tecido Nervoso/efeitos dos fármacos , Tecido Nervoso/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
GLP-1 receptor agonists used for the treatment of diabetes, have shown some neuroprotective effects in cellular and animal models of Alzheimer's disease (AD) and Parkinson's disease (PD). There are currently few studies investigating GLP-1 receptor agonists in the treatment of ALS, where these neuroprotective effects may be beneficial. Here we investigate the effects of liraglutide, a GLP-1 receptor agonist, in two well characterised transgenic mouse models of ALS (SOD1G93A and TDP-43Q331K) to determine if liraglutide could be a potential treatment in ALS patients. Doses of liraglutide previously shown to have efficacy in AD and PD mouse models were used. Behavioural testing was carried out to ascertain the effect of liraglutide on disease progression. Immunohistochemical analysis of tissue was used to determine any neuroprotective effects on the CNS. We found that liraglutide dosed animals showed no significant differences in disease progression when compared to vehicle dosed animals in either the SOD1G93A or TDP-43Q331K mouse models of ALS. We also observed no changes in motor neuron counts or glial activation in lumbar spinal cords of liraglutide treated mice compared to vehicle dosed mice. Overall, we found no evidence to support clinical evaluation of liraglutide as a potential candidate for the treatment of ALS.
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Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/genética , Progressão da Doença , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Liraglutida/farmacologia , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/sangue , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Glicemia/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismoRESUMO
The majority of preclinical studies in ALS have relied on transgenic models with overexpression of mutant human superoxide dismutase 1 (SOD1), widely regarded to have failed in terms of translation of therapeutic effects. However, there are still no widely accepted models of other genetic subtypes of ALS. The majority of patients show ubiquitinated cytoplasmic inclusions of TAR DNA binding protein of 43 kilodaltons (TDP-43) in spinal motor neurons at the end stage of disease and a small proportion have mutations in TARDBP, the gene encoding TDP-43. TDP-43 transgenic mouse models have been produced, but have not been widely adopted. Here, we characterised one of these models available from the Jackson Laboratory in detail. Compared to TDP-43WT mice, TDP-43Q331K mice had 43% less hindlimb muscle mass at 6 months and a 73% reduction in hindlimb compound muscle action potential at 8 months of age. Rotarod and gait analysis indicated motor system decline with elevated weight gain. At the molecular level, the lack of TDP-43 cellular pathology was confirmed with a surprising increase in nuclear TDP-43 in motor neurons. Power analysis indicated group sizes of 12-14 mice are needed to detect 10-20% changes in measured parameters with a power of 80%, providing valid readouts for preclinical testing. Overall, this model may represent a useful component of multi-model pre-clinical therapeutic studies for ALS.
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Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Transgênicos , Mutação PuntualRESUMO
Oxidative stress appears to be a key feature of many neurodegenerative diseases either as a cause or consequence of disease. A range of molecules are subject to oxidation, but in particular, proteins are an important target and measure of oxidative stress. Proteins are subject to a range of oxidative modifications at reactive cysteine residues, and depending on the level of oxidative stress, these modifications may be reversible or irreversible. A range of experimental approaches has been developed to characterize cysteine oxidation of proteins. In particular, mass spectrometry-based proteomic methods have emerged as a powerful means to identify and quantify cysteine oxidation sites on a proteome scale; however, their application to study neurodegenerative diseases is limited to date. Here we provide a guide to these approaches and highlight the under-exploited utility of these methods to measure oxidative stress in neurodegenerative diseases for biomarker discovery, target engagement and to understand disease mechanisms.
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Neuromuscular diseases result in muscle weakness, disability, and, in many instances, death. Preclinical models form the bedrock of research into these disorders, and the development of in vivo and potentially translational biomarkers for the accurate identification of disease is crucial. Spontaneous Raman spectroscopy can provide a rapid, label-free, and highly specific molecular fingerprint of tissue, making it an attractive potential biomarker. In this study, we have developed and tested an in vivo intramuscular fiber optic Raman technique in two mouse models of devastating human neuromuscular diseases, amyotrophic lateral sclerosis, and Duchenne muscular dystrophy (SOD1G93A and mdx, respectively). The method identified diseased and healthy muscle with high classification accuracies (area under the receiver operating characteristic curves (AUROC): 0.76-0.92). In addition, changes in diseased muscle over time were also identified (AUROCs 0.89-0.97). Key spectral changes related to proteins and the loss of α-helix protein structure. Importantly, in vivo recording did not cause functional motor impairment and only a limited, resolving tissue injury was seen on high-resolution magnetic resonance imaging. Lastly, we demonstrate that ex vivo muscle from human patients with these conditions produced similar spectra to those observed in mice. We conclude that spontaneous Raman spectroscopy of muscle shows promise as a translational research tool.
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Esclerose Lateral Amiotrófica , Distrofia Muscular de Duchenne , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético , Músculos , Análise Espectral RamanRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative condition for which new therapeutic options are urgently needed. We injected GFP+ adipose-derived stem cells (EGFP-ADSCs) directly into the cerebrospinal fluid (CSF) of transgenic SOD1G93A mice, a well-characterized model of familial ALS. Despite short-term survival of the injected cells and limited engraftment efficiency, EGFP-ADSCs improved motor function and delayed disease onset by promoting motor neuron (MN) survival and reducing glial activation. We then tested the in vitro neuroprotective potential of mouse ADSCs in astrocyte/MN co-cultures where ALS astrocytes show neurotoxicity. ADSCs were able to rescue MN death caused by ALS astrocytes derived from symptomatic SOD1G93A mice. Further, ADSCs were found to reduce the inflammatory signature of ALS astrocytes by inhibiting the release of pro-inflammatory mediators and inducing the secretion of neuroprotective factors. Finally, mouse ADSCs were able to protect MNs from the neurotoxicity mediated by human induced astrocytes (iAstrocytes) derived from patients with either sporadic or familial ALS, thus for the first time showing the potential therapeutic translation of ADSCs across the spectrum of human ALS. These data in two translational models of ALS show that, through paracrine mechanisms, ADSCs support MN survival and modulate the toxic microenvironment that contributes to neurodegeneration in ALS.
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Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping neurodegenerative disorders. ALS is more commonly seen in men than women and the same may be the case for FTD. Preclinical models demonstrating sex-specific vulnerability may help to understand female resistance to ALS-FTD and thereby identify routes to therapy. We previously characterised a TDP-43Q331K knock-in mouse, which demonstrated behavioural phenotypes reminiscent of ALS-FTD in males. Here we present our behavioural observations of female TDP-43Q331K mutants. Female TDP-43Q331K knock-in mice displayed increased weight relative to wild-type and increased food intake at 20 months of age, much later than previously observed in male mutants. Spontaneous digging behaviour was initially normal and only declined in mutants in the second year of life. Gait analysis using Catwalk ( https://www.noldus.com/catwalk-xt ) found significant deficits in the second year of life, while nocturnal running behaviour was attenuated from ~ 250 days of life. These results indicate that while female TDP-43Q331K knock-in mice do display progressive behavioural phenotypes, these are less severe than we previously noted in male mutants. Further studies of male and female TDP-43Q331K knock-in mice may help to unravel the mechanisms underlying sex-specific vulnerability in ALS-FTD.