SOD1G93A Mice Research Articles

Genetic Downregulation of the Metabotropic Glutamate Receptor Type 5 Dampens the Reactive and Neurotoxic Phenotype of Adult ALS Astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.

Divergent functional outcomes of NLRP3 blockade downstream of multi-inflammasome activation: therapeutic implications for ALS.

NOD-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome modulation has emerged as a potential therapeutic approach targeting inflammation amplified by pyroptotic innate immune cell death. In diseases characterized by non-cell autonomous neurodegeneration including amyotrophic lateral sclerosis (ALS), the activation of several inflammasomes has been reported. Since functional redundancy can exist among inflammasome pathways, here we investigate the effects of NLRP3 inhibition on NLRP3, NLR family CARD Domain Containing 4 (NLRC4) and non-canonical pathways to understand whether NLRP3 blockade alone can mitigate pro-inflammatory cytokine release and pyroptotic cell death in contexts where single or multiple inflammasome pathways independent of NLRP3 are activated. In this study we do not limit our insights into inflammasome biology by solely relying on the THP-1 monocytic line under the LPS/nigericin-mediated NLRP3 pathway activation paradigm. We assess therapeutic potential and limitations of NLRP3 inhibition in multi-inflammasome activation contexts utilizing various human cellular systems including cell lines expressing gain of function (GoF) mutations for several inflammasomes, primary human monocytes, macrophages, healthy and Amyotrophic Lateral Sclerosis (ALS) patient induced pluripotent stem cells (iPSC)-derived microglia (iMGL) stimulated for canonical and non-canonical inflammasome pathways. We demonstrate that NLRP3 inhibition can modulate the NLRC4 and non-canonical inflammasome pathways; however, these effects differ between immortalized, human primary innate immune cells, and iMGL. We extend our investigation in more complex systems characterized by activation of multiple inflammasomes such as the SOD1G93A mouse model. Through deep immune phenotyping by single-cell mass cytometry we demonstrate that acute NLRP3 inhibition does not ameliorate spinal cord inflammation in this model. Taken together, our data suggests that NLRP3 inhibition alone may not be sufficient to address dynamic and complex neuroinflammatory pathobiological mechanisms including dysregulation of multiple inflammasome pathways in neurodegenerative disease such as ALS.

The cholesterol depleting agent, (2-Hydroxypropyl)-ß-cyclodextrin, does not affect disease progression in SOD1G93A mice

Objective Previously, we demonstrated that Amyloid Precursor Protein (APP) contributes to pathology in the SOD1G93A mouse model of ALS and that genetic ablation of APP in SOD1G93A mice significantly improved multiple disease parameters, including muscle innervation and motor neuron survival. We also observed elevated levels of potentially neurotoxic Aß peptides that have been implicated in Alzheimer’s Disease (AD) pathogenesis, within motor neurons and astrocytes in SOD1G93A mice. More recently, it has been shown that blocking Aß production improves outcome measures in SOD1G93A mice. The cyclodextrin, (2-Hydroxypropyl)-ß-cyclodextrin (HP-β-CD), has previously been shown to deplete intraneuronal unesterified cholesterol, resulting in effective reduction of Aß production and amelioration of disease progression in mouse models of AD and Niemann Pick Type C (NPC) disease. Here, we tested whether HP-β-CD could also improve phenotypic progression in SOD1G93A mice. Methods Pre-symptomatic male SOD1G93A mice were randomly assigned to the following treatment groups: HP-β-CD (4000mg/kg, n = 9) or vehicle (saline; n = 10), delivered by weekly subcutaneous injection, commencing at 67 days of age. Longitudinal grip-strength and body mass analysis was performed until late-stage disease (120 days of age), followed by in vivo bilateral isometric muscle tension analysis of tibialis anterior (TA) and extensor digitorum longus (EDL) muscles. Results: HP-β-CD administration had no effect on body mass or grip-strength compared to vehicle treated SOD1G93A mice. Similarly, HP-β-CD treatment had no effect on muscle force, contractile properties or motor unit number estimates (MUNE) at late-stage disease in SOD1G93A mice. Conclusion This study shows that HP-β-CD does not confer any therapeutic benefit in SOD1G93A mice. However, the absence of detrimental effects is informative, given the common use of cyclodextrins as complexing agents for other pharmaceutical products, their standalone therapeutic potential and the emerging association between dyslipidaemia and ALS progression.

Impaired ATF3 signaling involves SNAP25 in SOD1 mutant ALS patients

Epigenetic remodeling is emerging as a critical process for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Genetics alone fails to explain the etiology of ALS, the investigation of the epigenome might therefore provide novel insights into the molecular mechanisms of the disease. In this study, we interrogated the epigenetic landscape in peripheral blood mononuclear cells (PBMCs) of familial ALS (fALS) patients with either chromosome 9 open reading frame 72 (C9orf72) or superoxide dismutase 1 (SOD1) mutation and aimed to identify key epigenetic footprints of the disease. To this end, we used an integrative approach that combines chromatin immunoprecipitation targeting H3K27me3 (ChIP-Seq) with the matching gene expression data to gain new insights into the likely impact of blood-specific chromatin remodeling on ALS-related molecular mechanisms. We demonstrated that one of the hub molecules that modulates changes in PBMC transcriptome in SOD1-mutant ALS patients is ATF3, which has been previously reported in an SOD1G93A mouse model. We also identified potential suppression of SNAP25, with impaired ATF3 signaling in SOD1-mutant ALS blood. Together, our study shed light on the mechanistic underpinnings of SOD1 mutations in ALS.

Combining electromyography and Raman spectroscopy: optical EMG.

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. 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. 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. Optical EMG can provide standard electrophysiological data and molecular Raman data during a single needle insertion and represents a potential biomarker for neuromuscular disease.

CXCR2 Is Deregulated in ALS Spinal Cord and Its Activation Triggers Apoptosis in Motor Neuron-Like Cells Overexpressing hSOD1-G93A.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by progressive depletion of motor neurons (MNs). Recent evidence suggests a role in ALS pathology for the C-X-C motif chemokine receptor 2 (CXCR2), whose expression was found increased at both mRNA and protein level in cortical neurons of sporadic ALS patients. Previous findings also showed that the receptor inhibition is able to prevent iPSC-derived MNs degeneration in vitro and improve neuromuscular function in SOD1-G93A mice. Here, by performing transcriptional analysis and immunofluorescence studies, we detailed the increased expression and localization of CXCR2 and its main ligand CXCL8 in the human lumbar spinal cord of sporadic ALS patients. We further investigated the functional role of CXCR2/ligands axis in NSC-34 motor neuron-like cells expressing human wild-type (WT) or mutant (G93A) SOD1. A significant expression of CXCR2 was found in doxycycline-induced G93A-SOD1-expressing cells, but not in WT cells. In vitro assays showed CXCR2 activation by GROα and MIP2α, two murine endogenous ligands and functional homologs of CXCL8, reduces cellular viability and triggers apoptosis in a dose dependent manner, while treatment with reparixin, a non-competitive allosteric CXCR2 inhibitor, effectively counteracts GROα and MIP2α toxicity, significantly inhibiting the chemokine-induced cell death. Altogether, data further support a role of CXCR2 axis in ALS etiopathogenesis and confirm its pharmacological modulation as a candidate therapeutic strategy.

Glycogen accumulation modulates life span in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord. Glial cells, including astrocytes and microglia, have been shown to contribute to neurodegeneration in ALS, and metabolic dysfunction plays an important role in the progression of the disease. Glycogen is a soluble polymer of glucose found at low levels in the central nervous system that plays an important role in memory formation, synaptic plasticity, and the prevention of seizures. However, its accumulation in astrocytes and/or neurons is associated with pathological conditions and aging. Importantly, glycogen accumulation has been reported in the spinal cord of human ALS patients and mouse models. In the present work, using the SOD1G93A mouse model of ALS, we show that glycogen accumulates in the spinal cord and brainstem during symptomatic and end stages of the disease and that the accumulated glycogen is associated with reactive astrocytes. To study the contribution of glycogen to ALS progression, we generated SOD1G93A mice with reduced glycogen synthesis (SOD1G93A GShet mice). SOD1G93A GShet mice had a significantly longer life span than SOD1G93A mice and showed lower levels of the astrocytic pro-inflammatory cytokine Cxcl10, suggesting that the accumulation of glycogen is associated with an inflammatory response. Supporting this, inducing an increase in glycogen synthesis reduced life span in SOD1G93A mice. Altogether, these results suggest that glycogen in reactive astrocytes contributes to neurotoxicity and disease progression in ALS.

Comparing the effects of microgravity and amyotrophic lateral sclerosis on mouse dorsal root ganglia

Introduction: Microgravity (MG) exposure causes motor deficits and decreased neuronal activity, effects that resemble the ones observed in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Several recent studies have shown that exposure to MG and ALS also impacts the sensory systems. Yet, the role of sensory impairment in this degenerative process of exposure to MG and ALS remains unknown. In this study, we aimed at elucidating how the sensory system is affected by exposure to MG and ALS.Methods: To this end, we compared gene expression in the mouse lumbar dorsal root ganglia (DRG) of MG-exposed animals with that of control animals that remained under artificial gravity conditions. We then investigated the effects of the human superoxide dismutase 1 (SOD1) G93A mutation in a mouse model of ALS (SOD1G93A mice) on gene expression in the DRG.Results: The overlap of genes with negatively correlated expression was greater than those with positively correlated expression between the DRG of MG-exposed and SOD1G93A mice. Additionally, genes related to Imoonglia (characteristics of both immune and glial cells) and macrophage increased in response to MG exposure, while satellite glial cell genes were expressed in response to SOD1 mutation. Next, we examined genes related to sensory neuron subtypes in the DRG. We found altered gene expression in genes related to proprioceptive and mechanoreceptive neurons in the DRG of MG-exposed and SOD1G93A mice. Remarkably, the expression of Atf3 and genes related to nociceptive neurons in the DRG of SOD1G93A mice at postnatal day (P) 120 was considerably altered, whereas MG-exposed and SOD1G93A mice at P30 presented little changes.Discussion: These results indicate that exposure to MG and ALS affect gene expression in genes related to neurons and non-neuronal cells in the DRG, with significant differences between the effects of MG and the SOD1 mutation. Elucidation of the impact of exposure to MG and ALS pathogenesis in the DRG, including identification of the molecular pathways that regulate DRG dysfunction, will help better understand the differences in vulnerability and the triggering processes of impaired motor function associated with MG and ALS.

Transplanted Human Bone Marrow Endothelial Progenitor Cells Prolong Functional Benefits and Extend Survival of ALS Mice Likely via Blood-Spinal Cord Barrier Repair.

Amyotrophic lateral sclerosis (ALS) is a multifactorial disease with one of these factors being an impaired blood-spinal cord barrier (BSCB). In order to block harmful components in systemic circulation from accessing the CNS, barrier damage needs alleviation. Recently, we found that symptomatic ALS animals treated with intravenously delivered human bone marrow-derived CD34+ (hBM34+) cells or endothelial progenitor cells (hBMEPCs) showed delayed disease progression for 4 weeks post-transplant via BSCB repair. However, despite noted benefits from transplanted human bone marrow-derived stem cells, long-term effects of transplanted cells in ALS mice remain undetermined. This study aimed to determine prolonged effects of single equal doses of hBM34+ cells and hBMEPCs systemically transplanted into symptomatic G93A SOD1 mice on behavioral disease outcomes and mouse lifespan. Results showed that transplanted hBMEPCs better ameliorated disease behavioral outcomes than hBM34 + cells until near end-stage disease and significantly increased lifespan vs. media-treated mice. These results provide important evidence that transplanted hBMEPCs prolonged functional benefits and extended survival of ALS mice, potentially by repairing the damaged BSCB. However, due to modestly increased lifespan of hBMEPC-treated mice, repeated cell transplants into symptomatic ALS mice may more effectively delay motor function deficit and extend lifespan by continuous reparative processes via replacement of damaged endothelial cells during disease progression.

Ibrutinib Delays ALS Installation and Increases Survival of SOD1G93A Mice by Modulating PI3K/mTOR/Akt Signaling.

Amyotrophic lateral sclerosis (ALS) is a fatal multisystem degenerative disorder with minimal available therapeutic. However, some recent studies showed promising results of immunological-based treatment. Here, we aimed to evaluate the efficacy of ibrutinib against ALS-associated abnormalities by targeting inflammation and muscular atrophy. Ibrutinib was administrated orally to SOD1 G93A mice from 6 to 19 weeks for prophylactic administration and 13 to 19 weeks for therapeutic administration. Our results demonstrated that ibrutinib treatment significantly delayed ALS-like symptom onset in the SOD1 G93A mice, as shown by improved survival time and reduced behavioral impairments. Ibrutinib treatment significantly reduced muscular atrophy by increasing muscle/body weight and decreasing muscular necrosis. The ibrutinib treatment also considerably reduced pro-inflammatory cytokine production, IBA-1, and GFAP expression, possibly mediated by mTOR/Akt/Pi3k signaling in the medulla, motor cortex and spinal cord of the ALS mice. In conclusion, our study demonstrated that ibrutinib could delay ALS onset, increase survival time, and reduce ALS progression by targeting inflammation and muscular atrophy via mTOR/Akt/PI3K modulation.

Development of abnormalities at the neuromuscular junction in the SOD1-G93A mouse model of ALS: dysfunction then disruption of postsynaptic structure precede overt motor symptoms.

The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS. We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages. Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint. Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.

Sympathetic neuropathology is revealed in muscles affected by amyotrophic lateral sclerosis.

Rationale: The anatomical substrate of skeletal muscle autonomic innervation has remained underappreciated since it was described many decades ago. As such, the structural and functional features of muscle sympathetic innervation are largely undetermined in both physiology and pathology, mainly due to methodological limitations in the histopathological analysis of small neuronal fibers in tissue samples. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease which mainly targets motor neurons, and despite autonomic symptoms occurring in a significant fraction of patients, peripheral sympathetic neurons (SNs) are generally considered unaffected and, as such, poorly studied. Purpose: In this research, we compared sympathetic innervation of normal and ALS muscles, through structural analysis of the sympathetic network in human and murine tissue samples. Methods and Results: We first refined tissue processing to circumvent methodological limitations interfering with the detection of muscle sympathetic innervation. The optimized "Neuro Detection Protocol" (NDP) was validated in human muscle biopsies, demonstrating that SNs innervate, at high density, both blood vessels and skeletal myofibers, independent of the fiber metabolic type. Subsequently, NDP was exploited to analyze sympathetic innervation in muscles of SOD1G93A mice, a preclinical ALS model. Our data show that ALS murine muscles display SN denervation, which has already initiated at the early disease stage and worsened during aging. SN degeneration was also observed in muscles of MLC/SOD1G93A mice, with muscle specific expression of the SOD1G93A mutant gene. Notably, similar alterations in SNs were observed in muscle biopsies from an ALS patient, carrying the SOD1G93A mutation. Conclusion: We set up a protocol for the analysis of murine and, more importantly, human muscle sympathetic innervation. Our results indicate that SNs are additional cell types compromised in ALS and suggest that dysfunctional SOD1G93A muscles affect their sympathetic innervation.

A Glutamate Scavenging Protocol Combined with Deanna Protocol in SOD1-G93A Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive disease of neuronal degeneration in the motor cortex, brainstem, and spinal cord, resulting in impaired motor function and premature demise as a result of insufficient respiratory drive. ALS is associated with dysfunctions in neurons, neuroglia, muscle cells, energy metabolism, and glutamate balance. Currently, there is not a widely accepted, effective treatment for this condition. Prior work from our lab has demonstrated the efficacy of supplemental nutrition with the Deanna Protocol (DP). In the present study, we tested the effects of three different treatments in a mouse model of ALS. These treatments were the DP alone, a glutamate scavenging protocol (GSP) alone, and a combination of the two treatments. Outcome measures included body weight, food intake, behavioral assessments, neurological score, and lifespan. Compared to the control group, DP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan despite a greater loss of weight. GSP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan. DP+GSP had a significantly slower decline in neurological score with a trend toward increased lifespan, despite a greater loss of weight. While each of the treatment groups fared better than the control group, the combination of the DP+GSP was not better than either of the individual treatments. We conclude that the beneficial effects of the DP and the GSP in this ALS mouse model are distinct, and appear to offer no additional benefit when combined.

Integrative analysis of metabolomics and proteomics unravels purine metabolism dysregulation in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive paralysis of limbs and bulb in patients, the cause of which remains unclear. Accumulating studies suggest that motor neuron degeneration is associated with systemic metabolic impairment in ALS. However, the metabolic reprogramming and underlying mechanism in the longitudinal progression of the disease remain poorly understood. In this study, we aimed to investigate the molecular changes at both metabolic and proteomic levels during disease progression to identify the most critical metabolic pathways and underlying mechanisms involved in ALS pathophysiological changes. Utilizing liquid chromatography-mass spectrometry-based metabolomics, we analyzed the metabolites' levels of plasma, lumbar spinal cord, and motor cortex from SOD1G93A mice and wildtype (WT) littermates at different stages. To elucidate the regulatory network underlying metabolic changes, we further analyzed the proteomics profile in the spinal cords of SOD1G93A and WT mice. A group of metabolites implicated in purine metabolism, methionine cycle, and glycolysis were found differentially expressed in ALS mice, and abnormal expressions of enzymes involved in these metabolic pathways were also confirmed. Notably, we first demonstrated that dysregulation of purine metabolism might contribute to the pathogenesis and disease progression of ALS. Furthermore, we discovered that fatty acid metabolism, TCA cycle, arginine and proline metabolism, and folate-mediated one‑carbon metabolism were also significantly altered in this disease. The identified differential metabolites and proteins in our study could complement existing data on metabolic reprogramming in ALS, which might provide new insight into the pathological mechanisms and novel therapeutic targets of ALS.

Analysis of Feeding Behavior Characteristics in the Cu/Zn Superoxide Dismutase 1 (SOD1) SOD1G93A Mice Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a progressive disease affecting upper and lower motor neurons. Feeding disorders are observed in patients with ALS. The mastication movements and their systemic effects in patients with ALS with feeding disorders remain unclear. Currently, there is no effective treatment for ALS. However, it has been suggested that treating feeding disorders and improving nutritional status may prolong the lives of patients with ALS. Therefore, this study elucidates feeding disorders observed in patients with ALS and future therapeutic agents. We conducted a temporal observation of feeding behavior and mastication movements using an open-closed mouth evaluation artificial intelligence (AI) model in an ALS mouse model. Furthermore, to determine the cause of masticatory rhythm modulation, we conducted electrophysiological analyses of mesencephalic trigeminal neurons (MesV). Here, we observed the modulation of masticatory rhythm with a prolonged open phase in the ALS mouse model from the age of 12 weeks. A decreased body weight was observed simultaneously, indicating a correlation between the prolongation of the open phase and the decrease observed. We found that the percentage of firing MesV was markedly decreased. This study partially clarifies the role of feeding disorders in ALS.

Intramuscular IL-10 Administration Enhances the Activity of Myogenic Precursor Cells and Improves Motor Function in ALS Mouse Model.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult motor neuron disease, with a poor prognosis, a highly unmet therapeutic need, and a burden on health care costs. Hitherto, strategies aimed at protecting motor neurons have missed or modestly delayed ALS due to a failure in countering the irreversible muscular atrophy. We recently provided direct evidence underlying the pivotal role of macrophages in preserving skeletal muscle mass. Based on these results, we explored whether the modulation of macrophage muscle response and the enhancement of satellite cell differentiation could effectively promote the generation of new myofibers and counteract muscle dysfunction in ALS mice. For this purpose, disease progression and the survival of SOD1G93A mice were evaluated following IL-10 injections in the hindlimb skeletal muscles. Thereafter, we used ex vivo methodologies and in vitro approaches on primary cells to assess the effect of the treatment on the main pathological signatures. We found that IL-10 improved the motor performance of ALS mice by enhancing satellite cells and the muscle pro-regenerative activity of macrophages. This resulted in delayed muscle atrophy and motor neuron loss. Our findings provide the basis for a suitable adjunct multisystem therapeutic approach that pinpoints a primary role of muscle pathology in ALS.

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3-5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

Protective effects of intrathecal injection of AAV9-RabGGTB-GFP+ in SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that widely affects motor neurons of the CNS. About 20% of patients with ALS have familial ALS (fALS). One of the classic models of ALS are SOD1G93A mice. Misfolded SOD1 protein can be overexpressed in motor neurons, which results in progressive paralysis of the limbs of mice. There is still no effective treatment for ALS. In recent years, the treatment of ALS by regulating autophagy has become a research hotspot. Autophagy obstacles have been confirmed to be one of the early pathological events of ALS. Rab7 is a member of the Ras superfamily and plays a key role in the late stage of autophagy. In our previous studies, we found that prenoylation of Rab7 was inhibited in the ALS model. Prenylation is a post-translational modification in which farnesyl or geranylgeranyl groups are covalently linked to target proteins. Based on these findings, we proposed the novel idea that the regulation of RabGGTB (the β-subunit of RabGGTase) mediated prenylation modification of Rab7, and that this can be used as a prevention and treatment of ALS associated with abnormal protein accumulation. In the present study, RabGGTB was overexpressed in mouse spinal cord motoneurons by using adeno-associated virus as vector. Then immunofluorescence quantitative analysis was used for pathological study. The body weight, footprint analysis, the accelerating rotarod test, and neurological deficits score were used to evaluate animal behavior. Our results show that the protein level of RabGGTB was significantly increased in the lumbar and thoracic regions of spinal cord motoneurons of injected mice. Furthermore, the onset time and survival time of SOD1G93A mice injected with AAV9-RabGGTB-GFP+ were delayed compared with those of mice without overexpression. At the same time, we also observed a decrease in SOD1 misfolded and glial overactivation in the lumbar spinal cord of these SOD1G93A mice. The findings reported here show that RabGGTB plays a significant role in the pathogenesis of SOD1G93A mice and with great therapeutic potential for reducing abnormal aggregation of SOD1 in ALS.

Immune-mediated myogenesis and acetylcholine receptor clustering promote a slow disease progression in ALS mouse models

BackgroundAmyotrophic lateral sclerosis (ALS) is a heterogeneous disease in terms of onset and progression rate. This may account for therapeutic clinical trial failure. Transgenic SOD1G93A mice on C57 or 129Sv background have a slow and fast disease progression rate, mimicking the variability observed in patients.Based on evidence inferring the active influence of skeletal muscle on ALS pathogenesis, we explored whether dysregulation in hindlimb skeletal muscle reflects the phenotypic difference between the two mouse models.MethodsEx vivo immunohistochemical, biochemical, and biomolecular methodologies, together with in vivo electrophysiology and in vitro approaches on primary cells, were used to afford a comparative and longitudinal analysis of gastrocnemius medialis between fast- and slow-progressing ALS mice.ResultsWe reported that slow-progressing mice counteracted muscle denervation atrophy by increasing acetylcholine receptor clustering, enhancing evoked currents, and preserving compound muscle action potential. This matched with prompt and sustained myogenesis, likely triggered by an early inflammatory response switching the infiltrated macrophages towards a M2 pro-regenerative phenotype. Conversely, upon denervation, fast-progressing mice failed to promptly activate a compensatory muscle response, exhibiting a rapidly progressive deterioration of muscle force.ConclusionsOur findings further pinpoint the pivotal role of skeletal muscle in ALS, providing new insights into underestimated disease mechanisms occurring at the periphery and providing useful (diagnostic, prognostic, and mechanistic) information to facilitate the translation of cost-effective therapeutic strategies from the laboratory to the clinic.

Ultrasound–enhanced brain delivery of edaravone provides additive amelioration on disease progression in an ALS mouse model

BackgroundAlthough amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease and unfortunately incurable yet, incremental attention has been drawn to targeting the health of corticospinal motor neurons. Focused ultrasound combined with systemically circulating microbubbles (FUS/MB) is an emerging modality capable of site-specific molecular delivery temporarily and noninvasively within a range of appropriate parameters. ObjectiveTo investigate the effect of FUS/MB–enhanced delivery of therapeutics to the motor cortex on the disease progression by using a transgenic mouse model of ALS. MethodsMultiple FUS/MB–enhanced deliveries of Edaravone (Eda) to the motor cortex were performed on the SOD1G93A mouse model of ALS. The motor function of the animals was evaluated by gait analysis, grip strength and wire hanging tests. Corticospinal and spinal motor neuronal health, misfolded SOD1 protein and neuroinflammation after treatments were evaluated by histological examination. ResultsUltrasound–enhanced delivery of Eda in the targeted motor cortex was achieved by a two-fold increase without gross tissue damage. Compared with the ALS mice administered Eda treatments only, the animals given additionally FUS/MB–enhanced brain delivery of Eda (FUS/MB + Eda) exhibited further improvements in neuromuscular functions characterized by gait patterns, muscular strength, and motor coordination along with rescued muscle atrophy. FUS/MB + Eda treatments conferred remarkable neuroprotection to both upper and lower motor neurons revealed by normalized neuronal morphology with increasing cell body size and profoundly alleviated neuroinflammation and misfolded SOD1 protein in the brains and lumbar spinal cords. ConclusionWe report a pilot study that non-invasive ultrasound–enhanced brain delivery of Eda provides additive amelioration on disease progression of ALS and suggest that broadening the target from spinal to cortical network functions using the FUS/MB–enhanced delivery can be a rational therapeutic strategy of this debilitating disorder.