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1.
Nat Commun ; 15(1): 3606, 2024 May 02.
Article in English | MEDLINE | ID: mdl-38697975

ABSTRACT

Amyotrophic Lateral Sclerosis (ALS), like many other neurodegenerative diseases, is highly heritable, but with only a small fraction of cases explained by monogenic disease alleles. To better understand sporadic ALS, we report epigenomic profiles, as measured by ATAC-seq, of motor neuron cultures derived from a diverse group of 380 ALS patients and 80 healthy controls. We find that chromatin accessibility is heavily influenced by sex, the iPSC cell type of origin, ancestry, and the inherent variance arising from sequencing. Once these covariates are corrected for, we are able to identify ALS-specific signals in the data. Additionally, we find that the ATAC-seq data is able to predict ALS disease progression rates with similar accuracy to methods based on biomarkers and clinical status. These results suggest that iPSC-derived motor neurons recapitulate important disease-relevant epigenomic changes.


Subject(s)
Amyotrophic Lateral Sclerosis , Induced Pluripotent Stem Cells , Motor Neurons , Humans , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/metabolism , Induced Pluripotent Stem Cells/metabolism , Motor Neurons/metabolism , Motor Neurons/pathology , Male , Female , Middle Aged , Case-Control Studies , Chromatin/metabolism , Chromatin/genetics , Aged , Epigenomics/methods , Chromatin Immunoprecipitation Sequencing/methods , Disease Progression , Epigenesis, Genetic
2.
Neuropathol Appl Neurobiol ; 50(3): e12982, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38742276

ABSTRACT

AIMS: Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated the spatio-temporal timeline of PNN breakdown and the contributing cellular factors in the SOD1G93A strain, a fast-onset ALS mouse model. METHODS: This was conducted at the presymptomatic (P30), onset (P70), mid-stage (P130), and end-stage disease (P150) using immunofluorescent microscopy, as this characterisation has not been conducted in the SOD1G93A strain. RESULTS: We observed a significant breakdown of PNNs around α-motor neurons in the ventral horn of onset and mid-stage disease SOD1G93A mice compared with wild-type controls. This was observed with increased numbers of microglia expressing matrix metallopeptidase-9 (MMP-9), an endopeptidase that degrades PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Further increases in microglia and astrocyte number, MMP-9 expression, and engulfment of PNN components by glia were observed in mid-stage SOD1G93A mice. This was observed with increased expression of fractalkine, a signal for microglia engulfment, within α-motor neurons of SOD1G93A mice. Following PNN breakdown, α-motor neurons of onset and mid-stage SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death. CONCLUSIONS: Our observations suggest that increased numbers of MMP-9 expressing glia and their subsequent engulfment of PNNs around α-motor neurons render these neurons sensitive to oxidative damage and eventual death in the SOD1G93A ALS model mouse.


Subject(s)
Amyotrophic Lateral Sclerosis , Astrocytes , Disease Models, Animal , Matrix Metalloproteinase 9 , Mice, Transgenic , Microglia , Animals , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/genetics , Microglia/metabolism , Microglia/pathology , Mice , Matrix Metalloproteinase 9/metabolism , Astrocytes/metabolism , Astrocytes/pathology , Motor Neurons/pathology , Motor Neurons/metabolism , Phagocytosis/physiology , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/pathology
3.
Nat Commun ; 15(1): 4120, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38750052

ABSTRACT

5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.


Subject(s)
Brain , Killer Cells, Natural , Motor Neurons , Muscular Atrophy, Spinal , Oligonucleotides , Humans , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/pathology , Muscular Atrophy, Spinal/genetics , Motor Neurons/drug effects , Motor Neurons/pathology , Motor Neurons/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/drug effects , Brain/pathology , Brain/drug effects , Female , Male , Survival of Motor Neuron 2 Protein/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 1 Protein/metabolism , Single-Cell Analysis , Cytotoxicity, Immunologic/drug effects , Infant , Child, Preschool , Child , Transcriptome
4.
PLoS One ; 19(5): e0303053, 2024.
Article in English | MEDLINE | ID: mdl-38776297

ABSTRACT

OBJECTIVE: To describe the protocol of a prospective study to test the validity of intermuscular coherence (IMC) as a diagnostic tool and biomarker of upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). METHODS: This is a multicenter, prospective study. IMC of muscle pairs in the upper and lower limbs is gathered in ∼650 subjects across three groups using surface electrodes and conventional electromyography (EMG) machines. The following subjects will be tested: 1) neurotypical controls; 2) patients with symptomatology suggestive for early ALS but not meeting probable or definite ALS by Awaji Criteria; 3) patients with a known ALS mimic. The recruitment period is between 3/31/2021 and 12/31/2025. Written consent will be sought from the subject or the subject's legally authorized representative during enrollment. RESULTS: The endpoints of this study include: 1) whether adding IMC to the Awaji ALS criteria improve its sensitivity in early ALS and can allow for diagnosis earlier; 2) constructing a database of IMC across different ages, genders, and ethnicities. SIGNIFICANCE: This study may validate a new inexpensive, painless, and widely available tool for the diagnosis of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Biomarkers , Electromyography , Amyotrophic Lateral Sclerosis/diagnosis , Amyotrophic Lateral Sclerosis/physiopathology , Humans , Prospective Studies , Electromyography/methods , Biomarkers/analysis , Male , Female , Middle Aged , Muscle, Skeletal/physiopathology , Muscle, Skeletal/pathology , Motor Neurons/pathology , Aged , Adult
5.
Cells ; 13(10)2024 May 07.
Article in English | MEDLINE | ID: mdl-38786016

ABSTRACT

The primary neural circuit affected in Amyotrophic Lateral Sclerosis (ALS) patients is the corticospinal motor circuit, originating in upper motor neurons (UMNs) in the cerebral motor cortex which descend to synapse with the lower motor neurons (LMNs) in the spinal cord to ultimately innervate the skeletal muscle. Perturbation of these neural circuits and consequent loss of both UMNs and LMNs, leading to muscle wastage and impaired movement, is the key pathophysiology observed. Despite decades of research, we are still lacking in ALS disease-modifying treatments. In this review, we document the current research from patient studies, rodent models, and human stem cell models in understanding the mechanisms of corticomotor circuit dysfunction and its implication in ALS. We summarize the current knowledge about cortical UMN dysfunction and degeneration, altered excitability in LMNs, neuromuscular junction degeneration, and the non-cell autonomous role of glial cells in motor circuit dysfunction in relation to ALS. We further highlight the advances in human stem cell technology to model the complex neural circuitry and how these can aid in future studies to better understand the mechanisms of neural circuit dysfunction underpinning ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Motor Neurons , Amyotrophic Lateral Sclerosis/physiopathology , Amyotrophic Lateral Sclerosis/pathology , Humans , Motor Neurons/pathology , Motor Neurons/physiology , Animals , Nerve Net/physiopathology , Nerve Net/pathology , Neuromuscular Junction/physiopathology , Neuromuscular Junction/pathology , Disease Models, Animal , Motor Cortex/physiopathology , Motor Cortex/pathology
6.
Georgian Med News ; (348): 87-90, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38807398

ABSTRACT

Selective degeneration of motoneurons is the pathological hallmark of amyotrophic lateral sclerosis (ALS). Does serotonin (5-HT) play a role in progression or development of disease is under the research. The topic of the present paper is pressing as there is no data available regarding the spread of amyotrophic lateral sclerosis. It is also noteworthy that previous studies have indicated that the pathogenesis of ALS is closely linked to 5-hydroxytryptamine (5-HT). The clinical research was conducted in Georgia. During the last five years, 60 patients from different parts of Georgia have been studied, searched, and examined by us. Including from Samegrolo, Kartli, Adjara, Abkhazia, Guria, Kakheti regions. The Georgian Neurologists Corps participated and helped us in finding patients. Brain MRI and electromyography were also performed. 60 patients with different forms of ALS participated in the study, including 34 (56.66%) men and 26 (43.33%) women. Their age ranges from 30 to 81 years. The study was conducted after obtaining the written consent of the patients, taking into account the ethical requirements for the study. We also compared the results of the serotonin level of patients with amyotrophic lateral sclerosis with a control group of 20 people (aged 18 to 50 years) who had no neurological disease in past medical history. Patients of the first group, with LMN damage, are observed with decreased amount of serotonin (61.3) %, compared to other pairs, followed by patients of the upper neuron and bulbar syndrome groups, the level of serotonin in the control group is quite high. Thus, the level of serotonin in the group of patients with bulbar events is higher than in the other groups. Low serotonin requires further investigation. According to our research, the longer the anamnesis of amyotrophic lateral sclerosis patients is, the lower the level of serotonin is observed. It should also be taken into account that a low level of serotonin may be due to the presence of depression, which requires additional research. We speculate that 5-HT could therefore be a potential therapeutic target for amyotrophic lateral sclerosis.


Subject(s)
Amyotrophic Lateral Sclerosis , Serotonin , Humans , Amyotrophic Lateral Sclerosis/pathology , Serotonin/metabolism , Middle Aged , Female , Male , Adult , Aged , Aged, 80 and over , Adolescent , Motor Neurons/pathology , Electromyography , Magnetic Resonance Imaging
7.
Sci Adv ; 10(22): eadk3229, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38820149

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of somatic motor neurons. A major focus has been directed to motor neuron intrinsic properties as a cause for degeneration, while less attention has been given to the contribution of spinal interneurons. In the present work, we applied multiplexing detection of transcripts and machine learning-based image analysis to investigate the fate of multiple spinal interneuron populations during ALS progression in the SOD1G93A mouse model. The analysis showed that spinal inhibitory interneurons are affected early in the disease, before motor neuron death, and are characterized by a slow progressive degeneration, while excitatory interneurons are affected later with a steep progression. Moreover, we report differential vulnerability within inhibitory and excitatory subpopulations. Our study reveals a strong interneuron involvement in ALS development with interneuron specific degeneration. These observations point to differential involvement of diverse spinal neuronal circuits that eventually may be determining motor neuron degeneration.


Subject(s)
Amyotrophic Lateral Sclerosis , Disease Models, Animal , Interneurons , Mice, Transgenic , Motor Neurons , Spinal Cord , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Motor Neurons/metabolism , Motor Neurons/pathology , Mice , Interneurons/metabolism , Interneurons/pathology , Spinal Cord/pathology , Spinal Cord/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Humans , Disease Progression , Nerve Degeneration/pathology
8.
Sci Transl Med ; 16(748): eadk1358, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38776392

ABSTRACT

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.


Subject(s)
Blood-Brain Barrier , Endothelial Cells , Gain of Function Mutation , Motor Neurons , TRPV Cation Channels , Animals , TRPV Cation Channels/metabolism , TRPV Cation Channels/genetics , Motor Neurons/pathology , Motor Neurons/metabolism , Endothelial Cells/metabolism , Endothelial Cells/pathology , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Mice , Nerve Degeneration/pathology , Nerve Degeneration/genetics , Phenotype , Spinal Cord/pathology , Spinal Cord/metabolism
9.
Cell Rep Med ; 5(5): 101546, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38703766

ABSTRACT

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.


Subject(s)
Amyotrophic Lateral Sclerosis , Macrophage Migration-Inhibitory Factors , Motor Neurons , Superoxide Dismutase-1 , Macrophage Migration-Inhibitory Factors/metabolism , Macrophage Migration-Inhibitory Factors/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/therapy , Amyotrophic Lateral Sclerosis/pathology , Animals , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Mice , Induced Pluripotent Stem Cells/metabolism , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/genetics , Mice, Transgenic , Dependovirus/genetics , Disease Models, Animal , Male , Mutation/genetics , Female , Protein Folding
10.
Neurobiol Dis ; 197: 106534, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38759931

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, yet effective treatment is lacking. Moreover, the underlying pathomechanisms of ALS remain unclear, with impaired mitophagy function being increasingly recognized as a contributing factor. FUN14 domain-containing protein 1 (FUNDC1) is an autophagy receptor localized to the outer mitochondrial membrane and a mitochondrial membrane protein that mediates mitophagy and therefore considered as important factor in neurodegenerative diseases. However, its specific role in ALS is not yet clear. Therefore, this study aimed to investigate the regulatory role of FUNDC1 in ALS and determine its regulatory mechanisms. ALS transgenic mice were obtained and maintained under standard conditions. Cell lines were generated by stable transfection with hSOD1G93A or control vectors. Mice received intrathecal injections of AAV9 vectors expressing FUNDC1 or EGFP. Motor function was assessed through behavioral tests, and histological and immunostaining analyses were performed. Colocalization analysis was conducted in transfected cells, and protein expression was evaluated via western blotting. We first observed that FUNDC1 was significantly downregulated in the spinal cord tissues of SOD1G93A mice. FUNDC1 overexpression considerably improved locomotor activity and prolonged survival time in SOD1G93A mice. Mechanistically, reduced expression of FUNDC1 resulted in decreased mitophagy, as indicated by decreased recruitment through LC3 in SOD1G93A mice and cellular models. Consequently, this led to increased mitochondrial accumulation and cell apoptosis, exacerbating the ALS phenotype. Furthermore, we identified transcription factor FOXD3 as an essential upstream factor of FUNDC1, resulting in reduced transcription of FUNDC1 in ALS lesions. This study suggests a novel strategy of targeting FUNDC1-mediated mitophagy for developing therapeutic interventions to mitigate disease progression and improve outcomes for ALS patients.


Subject(s)
Amyotrophic Lateral Sclerosis , Disease Models, Animal , Mice, Transgenic , Mitochondrial Proteins , Mitophagy , Motor Neurons , Animals , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/genetics , Mitophagy/physiology , Motor Neurons/metabolism , Motor Neurons/pathology , Mice , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Humans , Spinal Cord/metabolism , Spinal Cord/pathology
11.
J Vis Exp ; (206)2024 Apr 19.
Article in English | MEDLINE | ID: mdl-38709037

ABSTRACT

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.


Subject(s)
Diaphragm , Motor Neurons , Phrenic Nerve , Animals , Motor Neurons/pathology , Rats , Diaphragm/innervation , Diaphragm/physiopathology , Biomarkers/analysis , Biomarkers/metabolism , Action Potentials/physiology , Nerve Degeneration/pathology , Rats, Sprague-Dawley
12.
Exp Neurol ; 376: 114772, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38599366

ABSTRACT

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S- and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt- and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S- and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt- and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.


Subject(s)
Mice, Transgenic , Motor Neurons , Tremor , Animals , Motor Neurons/pathology , Motor Neurons/physiology , Mice , Tremor/genetics , Tremor/physiopathology , Muscle Fibers, Slow-Twitch/pathology , Muscle Fibers, Fast-Twitch/pathology , Muscular Diseases/physiopathology , Muscular Diseases/pathology , Muscular Diseases/etiology , Muscle Fatigue/physiology , Posture/physiology , Animals, Newborn , Disease Models, Animal
13.
Ecotoxicol Environ Saf ; 276: 116327, 2024 May.
Article in English | MEDLINE | ID: mdl-38626605

ABSTRACT

Roxithromycin (ROX), a commonly used macrolide antibiotic, is extensively employed in human medicine and livestock industries. Due to its structural stability and resistance to biological degradation, ROX persists as a resilient environmental contaminant, detectable in aquatic ecosystems and food products. However, our understanding of the potential health risks to humans from continuous ROX exposure remains limited. In this study, we used the zebrafish as a vertebrate model to explore the potential developmental toxicity of early ROX exposure, particularly focusing on its effects on locomotor functionality and CaP motoneuron development. Early exposure to ROX induces marked developmental toxicity in zebrafish embryos, significantly reducing hatching rates (n=100), body lengths (n=100), and increased malformation rates (n=100). The zebrafish embryos treated with a corresponding volume of DMSO (0.1%, v/v) served as vehicle controls (veh). Moreover, ROX exposure adversely affected the locomotive capacity of zebrafish embryos, and observations in transgenic zebrafish Tg(hb9:eGFP) revealed axonal loss in motor neurons, evident through reduced or irregular axonal lengths (n=80). Concurrently, abnormal apoptosis in ROX-exposed zebrafish embryos intensified alongside the upregulation of apoptosis-related genes (bax, bcl2, caspase-3a). Single-cell sequencing further disclosed substantial effects of ROX on genes involved in the differentiation of motor neuron progenitor cells (ngn1, olig2), axon development (cd82a, mbpa, plp1b, sema5a), and neuroimmunity (aplnrb, aplnra) in zebrafish larvae (n=30). Furthermore, the CaP motor neuron defects and behavioral deficits induced by ROX can be rescued by administering ngn1 agonist (n=80). In summary, ROX exposure leads to early-life abnormalities in zebrafish motor neurons and locomotor behavior by hindering the differentiation of motor neuron progenitor cells and inducing abnormal apoptosis.


Subject(s)
Cell Differentiation , Motor Neurons , Zebrafish , Animals , Motor Neurons/drug effects , Motor Neurons/pathology , Cell Differentiation/drug effects , Apoptosis/drug effects , Water Pollutants, Chemical/toxicity , Anti-Bacterial Agents/toxicity , Embryo, Nonmammalian/drug effects , Locomotion/drug effects , Stem Cells/drug effects , Animals, Genetically Modified , Behavior, Animal/drug effects
14.
J Neurol Sci ; 460: 123021, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38653115

ABSTRACT

BACKGROUND: Late-onset Pompe disease (LOPD) patients may still need ventilation support at some point of their disease course, despite regular recombinant human alglucosidase alfa treatment. This suggest that other pathophysiological mechanisms than muscle fibre lesion can contribute to the respiratory failure process. We investigate through neurophysiology whether spinal phrenic motor neuron dysfunction could contribute to diaphragm weakness in LOPD patients. MATERIAL AND METHODS: A group of symptomatic LOPD patients were prospectively studied in our centre from January 2022 to April 2023. We collected both demographic and clinical data, as well as neurophysiological parameters. Phrenic nerve conduction studies and needle EMG sampling of the diaphragm were perfomed. RESULTS: Eight treated LOPD patients (3 males, 37.5%) were investigated. Three patients (37.5%) with no respiratory involvement had normal phrenic nerve motor responses [median phrenic compound muscle action potential (CMAP) amplitude of 0.49 mV; 1st-3rd interquartile range (IQR), 0.48-0.65]. Those with respiratory failure (under nocturnal non-invasive ventilation) had abnormal phrenic nerve motor responses (median phrenic CMAP amplitude of 0 mV; 1st-3rd IQR, 0-0.15), and were then investigated with EMG. Diaphragm needle EMG revealed both myopathic and neurogenic changes in 3 (60%) and myopathic potentials in 1 patient. In the last one, no motor unit potentials could be recruited. CONCLUSIONS: Our study provide new insights regarding respiratory mechanisms in LOPD, suggesting a contribution of spinal phrenic motor neuron dysfunction for diaphragm weakness. If confirmed in further studies, our results recommend the need of new drugs crossing the blood-brain barrier.


Subject(s)
Diaphragm , Electromyography , Glycogen Storage Disease Type II , Motor Neurons , Muscle Weakness , Phrenic Nerve , Humans , Glycogen Storage Disease Type II/complications , Glycogen Storage Disease Type II/physiopathology , Male , Diaphragm/physiopathology , Female , Middle Aged , Muscle Weakness/etiology , Muscle Weakness/physiopathology , Phrenic Nerve/physiopathology , Motor Neurons/physiology , Motor Neurons/pathology , Adult , Neural Conduction/physiology , Muscle Fibers, Skeletal/pathology , Muscle Fibers, Skeletal/physiology , Aged , Respiratory Insufficiency/etiology , Respiratory Insufficiency/physiopathology , Prospective Studies , Action Potentials/physiology
15.
Biochim Biophys Acta Mol Basis Dis ; 1870(5): 167192, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38657911

ABSTRACT

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2-5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration. To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R). We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected. The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.


Subject(s)
Amyotrophic Lateral Sclerosis , Animals, Genetically Modified , Chromosome Aberrations , Drosophila melanogaster , Mutation , Superoxide Dismutase-1 , Animals , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Humans , Drosophila melanogaster/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Motor Neurons/metabolism , Motor Neurons/pathology , Disease Models, Animal , Neuroinflammatory Diseases/genetics , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/pathology , Neurons/metabolism , Neurons/pathology
16.
PLoS One ; 19(4): e0298006, 2024.
Article in English | MEDLINE | ID: mdl-38669239

ABSTRACT

BACKGROUND: As a leading cause of mortality and long-term disability, acute ischemic stroke can produce far-reaching pathophysiological consequences. Accumulating evidence has demonstrated abnormalities in the lower motor system following stroke, while the existence of Transsynaptic degeneration of contralateral spinal cord ventral horn (VH) neurons is still debated. METHODS: Using a rat model of acute ischemic stroke, we analyzed spinal cord VH neuron counts contralaterally and ipsilaterally after stroke with immunofluorescence staining. Furthermore, we estimated the overall lower motor unit abnormalities after stroke by simultaneously measuring the modified neurological severity score (mNSS), compound muscle action potential (CMAP) amplitude, repetitive nerve stimulation (RNS), spinal cord VH neuron counts, and the corresponding muscle fiber morphology. The activation status of microglia and extracellular signal-regulated kinase 1/2 (ERK 1/2) in the spinal cord VH was also assessed. RESULTS: At 7 days after stroke, the contralateral CMAP amplitudes declined to a nadir indicating lower motor function damage, and significant muscle disuse atrophy was observed on the same side; meanwhile, the VH neurons remained intact. At 14 days after focal stroke, lower motor function recovered with alleviated muscle disuse atrophy, while transsynaptic degeneration occurred on the contralateral side with elevated activation of ERK 1/2, along with the occurrence of neurogenic muscle atrophy. No apparent decrement of CMAP amplitude was observed with RNS during the whole experimental process. CONCLUSIONS: This study offered an overview of changes in the lower motor system in experimental ischemic rats. We demonstrated that transsynaptic degeneration of contralateral VH neurons occurred when lower motor function significantly recovered, which indicated the minor role of transsynaptic degeneration in lower motor dysfunction during the acute and subacute phases of focal ischemic stroke.


Subject(s)
Anterior Horn Cells , Animals , Rats , Male , Anterior Horn Cells/pathology , Rats, Sprague-Dawley , Synapses/pathology , Synapses/physiology , Disease Models, Animal , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Motor Neurons/pathology , Motor Neurons/physiology , Brain Ischemia/pathology , Brain Ischemia/physiopathology , Microglia/pathology , Action Potentials/physiology
17.
Biochemistry (Mosc) ; 89(Suppl 1): S34-S56, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38621743

ABSTRACT

Mutations that disrupt the function of the DNA/RNA-binding protein FUS could cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. One of the key features in ALS pathogenesis is the formation of insoluble protein aggregates containing aberrant isoforms of the FUS protein in the cytoplasm of upper and lower motor neurons. Reproduction of human pathology in animal models is the main tool for studying FUS-associated pathology and searching for potential therapeutic agents for ALS treatment. In this review, we provide a systematic analysis of the role of FUS protein in ALS pathogenesis and an overview of the results of modelling FUS-proteinopathy in animals.


Subject(s)
Amyotrophic Lateral Sclerosis , Animals , Humans , Amyotrophic Lateral Sclerosis/genetics , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Motor Neurons/metabolism , Motor Neurons/pathology , Cytoplasm/metabolism , Mutation , Disease Models, Animal
18.
Acta Neuropathol Commun ; 12(1): 69, 2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38664831

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex specimens. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development.


Subject(s)
Amyotrophic Lateral Sclerosis , C9orf72 Protein , Frontotemporal Dementia , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/metabolism , Humans , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , Frontotemporal Dementia/metabolism , Male , Motor Neurons/pathology , Motor Neurons/metabolism , Spinal Cord/pathology , Spinal Cord/metabolism , Nuclear Envelope/metabolism , Nuclear Envelope/pathology , Female , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Middle Aged , Aged , Motor Cortex/pathology , Motor Cortex/metabolism
19.
AJNR Am J Neuroradiol ; 45(4): 494-503, 2024 Apr 08.
Article in English | MEDLINE | ID: mdl-38548305

ABSTRACT

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving rapid motor neuron degeneration leading to brain, primarily precentral, atrophy. Neurofilament light chains are a robust prognostic biomarker highly specific to ALS, yet associations between neurofilament light chains and MR imaging outcomes are not well-understood. We investigated the role of neurofilament light chains as mediators among neuroradiologic assessments, precentral neurodegeneration, and disability in ALS. MATERIALS AND METHODS: We retrospectively analyzed a prospective cohort of 29 patients with ALS (mean age, 56 [SD, 12] years; 18 men) and 36 controls (mean age, 49 [SD, 11] years; 18 men). Patients underwent 3T (n = 19) or 7T (n = 10) MR imaging, serum (n = 23) and CSF (n = 15) neurofilament light chains, and clinical (n = 29) and electrophysiologic (n = 27) assessments. The control group had equivalent 3T (n = 25) or 7T (n = 11) MR imaging. Two trained neuroradiologists performed blinded qualitative assessments of MR imaging anomalies (n = 29 patients, n = 36 controls). Associations between precentral cortical thickness and neurofilament light chains and clinical and electrophysiologic data were analyzed. RESULTS: We observed extensive cortical thinning in patients compared with controls. MR imaging analyses showed significant associations between precentral cortical thickness and bulbar or arm impairment following distributions corresponding to the motor homunculus. Finally, uncorrected results showed positive interactions among precentral cortical thickness, serum neurofilament light chains, and electrophysiologic outcomes. Qualitative MR imaging anomalies including global atrophy (P = .003) and FLAIR corticospinal tract hypersignal anomalies (P = .033), correlated positively with serum neurofilament light chains. CONCLUSIONS: Serum neurofilament light chains may be an important mediator between clinical symptoms and neuronal loss according to cortical thickness. Furthermore, MR imaging anomalies might have underestimated prognostic value because they seem to indicate higher serum neurofilament light chain levels.


Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Male , Humans , Middle Aged , Amyotrophic Lateral Sclerosis/diagnostic imaging , Retrospective Studies , Prospective Studies , Intermediate Filaments , Motor Neurons/pathology , Magnetic Resonance Imaging/methods , Atrophy/pathology
20.
J Exp Med ; 221(5)2024 May 06.
Article in English | MEDLINE | ID: mdl-38517332

ABSTRACT

Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD.


Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Animals , Humans , Mice , Amyotrophic Lateral Sclerosis/pathology , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Motor Neurons/pathology , Mutation , Neuroinflammatory Diseases , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism
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