Amyotrophic Lateral Sclerosis Mice Research Articles

Host genetics and gut microbiota influence lipid metabolism and inflammation: potential implications for ALS pathophysiology in SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the progressive loss of motor neurons, with genetic and environmental factors contributing to its complex pathogenesis. Dysregulated immune responses and altered energetic metabolism are key features, with emerging evidence implicating the gut microbiota (GM) in disease progression. We investigated the interplay among genetic background, GM composition, metabolism, and immune response in two distinct ALS mouse models: 129Sv_G93A and C57Ola_G93A, representing rapid and slow disease progression, respectively.Using 16 S rRNA sequencing and fecal metabolite analysis, we characterized the GM composition and metabolite profiles in non-transgenic (Ntg) and SOD1G93A mutant mice of both strains. Our results revealed strain-specific differences in GM composition and functions, particularly in the abundance of taxa belonging to Erysipelotrichaceae and the levels of short and medium-chain fatty acids in fecal samples. The SOD1 mutation induces significant shifts in GM colonization in both strains, with C57Ola_G93A mice showing changes resembling those in 129 Sv mice, potentially affecting disease pathogenesis. ALS symptom progression does not significantly alter microbiota composition, suggesting stability.Additionally, we assessed systemic immunity and inflammatory responses revealing strain-specific differences in immune cell populations and cytokine levels.Our findings underscore the substantial influence of genetic background on GM composition, metabolism, and immune response in ALS mouse models. These strain-specific variations may contribute to differences in disease susceptibility and progression rates. Further elucidating the mechanisms underlying these interactions could offer novel insights into ALS pathogenesis and potential therapeutic targets.

Nanoparticles encapsulating phosphatidylinositol derivatives promote neuroprotection and functional improvement in preclinical models of ALS via a long-lasting activation of TRPML1 lysosomal channel

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease currently incurable, in which motor neuron degeneration leads to voluntary skeletal muscle atrophy. Molecularly, ALS is characterized by protein aggregation, synaptic and organellar dysfunction, and Ca2+ dyshomeostasis. Of interest, autophagy dysfunction is emerging as one of the main putative targets of ALS therapy. A tune regulation of this cleansing process is affordable by a proper stimulation of TRPML1, one of the main lysosomal channels. However, TRPML1 activation by PI(3,5)P2 has low open probability to remain in an active conformation. To overcome this drawback we developed a lipid-based formulation of PI(3,5)P2 whose putative therapeutic potential has been tested in in vitro and in vivo ALS models.Pharmacodynamic properties of PI(3,5)P2 lipid-based formulations (F1 and F2) on TRPML1 activity have been characterized by means of patch-clamp electrophysiology and Fura-2AM video-imaging in motor neuronal cells. Once selected for the ability to stabilize TRPML1 activity, the most effective preparation F1 was studied in vivo to measure neuromuscular function and survival of SOD1G93A ALS mice, thereby establishing its therapeutic profile.F1, but not PI(3,5)P2 alone, stabilized the open state of the lysosomal channel TRPML1 and increased the persistence of intracellular calcium concentration ([Ca2+]i). Then, F1 was effective in delaying motor neuron loss, improving innervated endplants and muscle performance in SOD1G93A mice, extending overall lifespan by an average of 10 days. Of note F1 prevented gliosis and autophagy dysfunction in ALS mice by restoring PI(3,5)P2 level.Our novel self-assembling lipidic formulation for PI(3,5)P2 delivery exerts a neuroprotective effect in preclinical models of ALS mainly regulating dysfunctional autophagy through TRPML1 activity stabilization.

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2 + transients and reactive oxygen species (i.e., H2O2). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes.

Exercise, disease state and sex influence the beneficial effects of Fn14-depletion on survival and muscle pathology in the SOD1G93A amyotrophic lateral sclerosis (ALS) mouse model

BackgroundAmyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1G93A ALS mice could lead to differential and potentially improved benefits.MethodsWe thus investigated the contribution of Fn14 to disease phenotypes in the SOD1G93A ALS mice. To do so, Fn14 knockout mice (Fn14−/−) were crossed onto the SOD1G93A background to generate SOD1G93A;Fn14−/− mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14−/−, SOD1G93A and SOD1G93A;Fn14−/−).ResultsHere, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1G93A mice. We then show that Fn14-depleted SOD1G93A mice display increased lifespan, myofiber size, neuromuscular junction endplate area as well as altered expression of known molecular effectors of the TWEAK/Fn14 pathway, without an impact on motor function. Importantly, we also observe a complex interaction between exercise (rotarod and grid test), genotype, disease state and sex that influences the overall effects of Fn14 deletion on survival, expression of known molecular effectors of the TWEAK/Fn14 pathway, expression of myosin heavy chain isoforms and myofiber size.ConclusionsOur study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease, sex and exercise. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism, sex and exercise will be of importance in future studies.

Gasdermin D is activated but does not drive neurodegeneration in SOD1 G93A model of ALS: Implications for targeting pyroptosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron loss, microgliosis, and neuroinflammation. While pyroptosis, an inflammatory form of programmed cell death, has been implicated in ALS, the specific role of Gasdermin D (GSDMD) - the primary executioner of pyroptosis - remains unexplored. In this study, we examined the function of GSDMD in the well-established SOD1 G93A mouse model of ALS. Our results showed robust GSDMD activation in the spinal cords of SOD1 G93A animals across two strain backgrounds, with elevated expression in Iba1+ microglia. To explore its role in disease progression, we bred B6.SOD1 G93A mice onto a GSDMD - deficient background. In comparing SOD1 G93A ; Gsdmd +/+ and SOD1 G93A ; Gsdmd -/- mice, we found that Gsdmd loss did not affect disease onset, weight loss, or grip strength decline in either male or female animals. Notably, GSDMD deficiency resulted in a modest but statistically significant increase in mortality in SOD1 G93A mice. Moreover, GSDMD absence had minimal impact on astrogliosis, microgliosis and motor neuron loss. These findings show that while GSDMD is activated in the ALS mouse model, its loss does not mitigate key ALS behavioral phenotypes, gliosis or motor neuron loss. This study provides insights into the potential therapeutic relevance of targeting pyroptosis and inflammatory pathways in ALS.

Human dental pulp MSCs attenuated motor neuron dysfunction and prolonged lifespan in ALS murine model

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that causes skeletal muscle weakness and atrophy, resulting in respiratory failure and a short lifespan. Considering the lack of effective treatment, this study investigated the effects of human dental pulp stem cells (hDPSCs) on the clinical symptoms and potential mechanisms in a mouse model of ALS superoxide dismutase 1 (SOD1-G93A). Neurological assessments, including neurological scoring, rotarod testing, and 7-T magnetic resonance imaging, were conducted to evaluate neurological impairments. Survival rates and body weight of the mice were also recorded. Immunofluorescence staining and flow cytometry analyses were performed to investigate the number of neurons and infiltrated inflammatory cells in the spinal cord as well as the central nervous system. The results indicate that infusion of hDPSCs increased the body weight, mitigated motor neuron dysfunction, and extended the lifespan of SOD1-G93A mice by approximately 15 days. Moreover, hDPSCs infusion reduced the degree of spinal cord atrophy. Results suggested that the number of neurons in the central nervous system of SOD1-G93A mice was significantly decreased, but hDPSCs infusion resulted in an increase in these numbers. However, hDPSCs infusion had no obvious effect on microglia phenotypes in SOD1-G93A mice. This study emphasizes the potential of hDPSCs to mitigate neuronal loss in an ALS mouse model, suggesting a promising therapeutic avenue for ALS.

Evidence for the potential role of m6A modification in regulating autophagy in models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. Research indicates that N6-methyladenosine (m6A) modification plays a crucial role in cellular autophagy during ALS development. This study investigates the role of autophagy in ALS, with a focus on the effect of messenger ribonucleic acid m6A methylation modification on disease progression. We compared m6A levels and regulatory molecule expressions in transgenic superoxide dismutase (SOD1)-G93A and non-transgenic mice, categorized into end-stage and control groups, using quantitative polymerase chain reaction and Western blotting. The NSC-34 cell line, which was modified to model ALS, enabled the investigation of apoptosis, autophagy, and autophagy disruption through terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assays, Western blotting, and fluorescent staining. Our findings indicate significantly elevated m6A methylation levels in ALS mice (0.262 ± 0.005) compared with the controls (0.231 ± 0.003) and in the ALS model cells (0.242±0.005) relative to those belonging to the wild-type control group (0.183 ± 0.007). Furthermore, the proteins involved in m6A RNA modification differed between groups, which suggest impaired autophagy flux in the ALS models. These results suggest that m6A methylation may accelerate ALS progression through the disruption of autophagic processes. Our study underscores the role of m6A methylation in the pathology of ALS and proposes the targeting of m6A methylation as a potential therapeutic strategy for disease treatment. Although this study primarily used transgenic SOD1-G93A mice and NSC-34 cell models to investigate ALS pathology, potential differences in disease mechanisms between animal models and humans must be considered. Although a correlation was detected between m6A methylation levels and autophagy disruption in ALS, the study primarily established an association rather than provided detailed mechanistic insights.

TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b−/− mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes

N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.

TwinF interface inhibitor FP802 prevents retinal ganglion cell loss in a mouse model of amyotrophic lateral sclerosis

Motor neuron loss is well recognized in amyotrophic lateral sclerosis (ALS), but research on retinal ganglion cells (RGCs) is limited. Ocular symptoms are generally not considered classic ALS symptoms, although RGCs and spinal motor neurons share certain cell pathologies, including hallmark signs of glutamate neurotoxicity, which may be triggered by activation of extrasynaptic NMDA receptors (NMDARs). To explore potential novel strategies to prevent ALS-associated death of RGCs, we utilized inhibition of the TwinF interface, a new pharmacological principle that detoxifies extrasynaptic NMDARs by disrupting the NMDAR/TRPM4 death signaling complex. Using the ALS mouse model SOD1G93A, we found that the small molecule TwinF interface inhibitor FP802 prevents the loss of RGCs, improves pattern electroretinogram (pERG) performance, increases the retinal expression of Bdnf, and restores the retinal expression of the immediate early genes, Inhibin beta A and Npas4. Thus, FP802 not only prevents, as recently described, death of spinal motor neurons in SOD1G93A mice, but it also mitigates ALS-associated retinal damage. TwinF interface inhibitors have great potential for alleviating neuro-ophthalmologic symptoms in ALS patients and offer a promising new avenue for therapeutic intervention.

Potential Therapeutic Interventions Targeting NAD+ Metabolism for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons. While there have been many potential factors implicated for ALS development, such as oxidative stress and mitochondrial dysfunction, no exact mechanism has been determined at this time. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in mammalian cells and is crucial for a broad range of cellular functions from DNA repair to energy homeostasis. NAD+ can be synthesized from three different intracellular pathways, but it is the NAD+ salvage pathway that generates the largest proportion of NAD+. Impaired NAD+ homeostasis has been connected to aging and neurodegenerative disease-related dysfunctions. In ALS mice, NAD+ homeostasis is potentially disrupted prior to the appearance of physical symptoms and is significantly reduced in the nervous system at the end stage. Treatments targeting NAD+ metabolism, either by administering NAD+ precursor metabolites or small molecules that alter NAD+-dependent enzyme activity, have shown strong beneficial effects in ALS disease models. Here, we review the therapeutic interventions targeting NAD+ metabolism for ALS and their effects on the most prominent pathological aspects of ALS in animal and cell models.

Amyotrophic Lateral Sclerosis and swim training affect copper metabolism in skeletal muscle in a mouse model of disease.

Swim training and regulation of copper metabolism result in clinical benefits in amyotrophic lateral sclerosis (ALS) mice. Therefore, the study aimed to determine whether swim training improves copper metabolism by modifying copper metabolism in the skeletal muscles of ALS mice. SOD1G93A mice (n = 6 per group) were used as the ALS model, and wild-type B6SJL (WT) mice as controls (n = 6). Mice with ALS were analyzed before the onset of ALS (ALS BEFORE), at baseline ALS (first disease symptoms, trained and untrained, ALS ONSET), and at the end of ALS (last stage disease, trained and untrained, ALS TERMINAL). Copper concentrations and the level of copper metabolism proteins in the skeletal muscles of the lower leg were determined. ALS disease caused a reduction in the copper concentration in ALS TERMINAL untrained mice compared with the ALS BEFORE (10.43 ± 1.81 and 38.67 ± 11.50 μg/mg, respectively, p = .0213). The copper chaperon for SOD1 protein, which supplies copper to SOD1, and ATPase7a protein (copper exporter), increased at the terminal stage of disease by 57% (p = .0021) and 34% (p = .0372), while the CTR1 protein (copper importer) decreased by 45% (p = .002). Swim training moderately affected the copper concentration and the concentrations of proteins responsible for copper metabolism in skeletal muscles. The results show disturbances in skeletal muscle copper metabolism associated with ALS progression, which is moderately affected by swim training. From a clinical point of view, exercise in water for ALS patients should be an essential element of rehabilitation for maintaining quality of life.

Acute stress differently modulates interneurons excitability and synaptic plasticity in the primary motor cortex of wild-type and SOD1G93A mouse model of ALS.

Primary motor cortex (M1) network stability depends on activity of inhibitory interneurons, for which susceptibility to stress was previously demonstrated in limbic regions. Hyperexcitability in M1 following changes in the excitatory/inhibitory balance is a key pathological hallmark of amyotrophic lateral sclerosis (ALS). Using electrophysiological approaches, we assessed the impact of acute restraint stress on inhibitory interneurons excitability and global synaptic plasticity in M1 of the SOD1G93A ALS mouse model at a late pre-symptomatic stage (10-12.5weeks). Based on their firing type (continuous, discontinuous, with accommodation or not) and electrophysiological characteristics (resting potential, rheobase, firing frequency), interneurons from M1slices were separated into four clusters, labelled from 1 to 4. Among them, only interneurons from the first cluster, presenting continuous firing with few accommodations, tended to show increased excitability in wild-type (WT) and decreased excitability in SOD1G93A animals following stress. In vivo analyses of evoked field potentials showed that stress suppressed the theta burst-induced plasticity of an excitatory component (N1) recorded in the superficial layers of M1 in WT, with no impact on an inhibitory complex (N2-P1) from the deeper layers. In SOD1G93A mice, stress did not affect N1 but suppressed the N2-P1 plasticity. These data suggest that stress can alter M1 network functioning in a different manner in WT and SOD1G93A mice, possibly through changes of inhibitory interneurons excitability and synaptic plasticity. This suggests that stress-induced activity changes in M1may therefore influence ALS outcomes. KEY POINTS: Disruption of the excitatory/inhibitory balance in the primary motor cortex (M1) has been linked to cortical hyperexcitability development, a key pathological hallmark of amyotrophic lateral sclerosis (ALS). Psychological stress was reported to influence excitatory/inhibitory balance in limbic regions, but very little is known about its influence on the M1 functioning under physiological or pathological conditions. Our study revealed that acute stress influences the excitatory/inhibitory balance within the M1, through changes in interneurons excitability along with network plasticity. Such changes were different in pathological (SOD1G93A ALS mouse model) vs. physiological (wild-type) conditions. The results of our study help us to better understand how stress modulates the M1 and highlight the need to further characterize stress-induced motor cortex changes because it may be of importance when evaluating ALS outcomes.

Schwann cell JUN expression worsens motor performance in an amyotrophic lateral sclerosis mouse model.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease characterized by motor neuron death and distal axonopathy. Despite its clinical severity and profound impact in the patients and their families, many questions about its pathogenesis remain still unclear, including the role of Schwann cells and axon-glial signaling in disease progression. Upon axonal injury, upregulation of JUN transcription factor promotes Schwann cell reprogramming into a repair phenotype that favors axon regrowth and neuronal survival. To study the potential role of repair Schwann cells on motoneuron survival in amyotrophic lateral sclerosis, we generated a mouse line that over-expresses JUN in the Schwann cells of the SOD1G93A mutant, a mouse model of this disease. Then, we explored disease progression by evaluating survival, motor performance and histology of peripheral nerves and spinal cord of these mice. We found that Schwann cell JUN overexpression does not prevent axon degeneration neither motor neuron death in the SOD1G93A mice. Instead, it induces a partial demyelination of medium and large size axons, worsening motor performance and resulting in more aggressive disease phenotype.

Increase of HCN current in SOD1-associated amyotrophic lateral sclerosis.

The clinical manifestations of sporadic amyotrophic lateral sclerosis (ALS) vary widely. However, the current classification of ALS is mainly based on clinical presentations, while the roles of electrophysiological and biomedical biomarkers remain limited. Herein, we investigated a group of patients with sporadic ALS and an ALS mouse model with superoxide dismutase 1 (SOD1)/G93A transgenes using nerve excitability tests (NET) to investigate axonal membrane properties and chemical precipitation, followed by enzyme-linked immunosorbent assay analysis to measure plasma misfolded protein levels. Six of 19 patients (31.6%) with sporadic ALS had elevated plasma misfolded SOD1 protein levels. In sporadic ALS patients, only those with elevated misfolded SOD1 protein levels showed an increased inward rectification in the current-threshold (I/V) curve and an increased threshold reduction in the hyperpolarizing threshold electrotonus (TE) in the NET study. Two familial ALS patients with SOD1 mutations also exhibited similar electrophysiological patterns of NET. For patients with sporadic ALS showing significantly increased inward rectification in the I/V curve, we noted an elevation in plasma misfolded SOD1 level, but not in total SOD1, misfolded C9orf72, or misfolded phosphorylated TDP43 levels. Computer simulations demonstrated that the aforementioned axonal excitability changes are likely associated with an increase in hyperpolarization-activated cyclic nucleotide-gated (HCN) current. In SOD1/G93A mice, NET also showed an increased inward rectification in the I/V curve, which could be reversed by a single injection of the HCN channel blocker, ZD7288. Daily treatment of SOD1/G93A mice with ZD7288 partially prevented the early motor function decline and spinal motor neuron death. In summary, sporadic ALS patients with elevated plasma misfolded SOD1 exhibited similar patterns of motor axonal excitability changes as familial ALS patients and ALS mice with mutant SOD1 genes, suggesting the existence of SOD1-associated sporadic ALS. The observed NET pattern of increased inward rectification in the I/V curve was attributable to an elevation in the HCN current in SOD1-associated ALS.

Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice

Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.

Effect of ultrasound-mediated blood-spinal cord barrier opening on survival and motor function in females in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. The limited efficacy of recent therapies in clinical development may be linked to lack of drug penetration to the affected motor neurons due to the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). In this work, the safety and efficacy of repeated short transient opening of the BSCB by low intensity pulsed ultrasound (US, sonication) was studied in females of an ALS mouse model (B6.Cg-Tg(SOD1∗G93A)1Gur/J). The BSCB was disrupted using a 1MHz ultrasound transducer coupled to the spinal cord, with and without injection of insulin-like growth factor 1 (IGF1), a neurotrophic factor that has previously shown efficacy in ALS models. Results in wild-type (WT) animals demonstrated that the BSCB can be safely disrupted and IGF1 concentrations significantly enhanced after a single session of transient BSCB disruption (176±32μg/g vs. 0.16±0.008μg/g, p<0.0001). Five repeated weekly US sessions performed in female ALS mice demonstrated a survival advantage in mice treated with IGF1 and US (US IGF1) compared to treatment with IGF1 alone (176 vs. 166 days, p=0.0038). Surprisingly, this survival advantage was also present in mice treated with US alone vs. untreated mice (178.5 vs. 166.5 days, p=0.0061). Muscle strength did not show difference among the groups. Analysis of glial cell immunoreactivity and microglial transcriptome showing reduced cell proliferation pathways, in addition to lymphocyte infiltration, suggested that the beneficial effect of US or US IGF1 could act through immune cell modulation. These results show the first step towards a possible beneficial impact of transient BSCB opening for ALS therapy and suggest implication of immune cells. Fondation pour la Recherche Médicale (FRM). Investissements d'avenirANR-10-IAIHU-06, Société Française de Neurochirurgie (SFNC), Fond d'étude et de Recherche du Corps Medical (FERCM), Aide à la Recherche des Maladies du Cerveau (ARMC), SLA Fondation Recherche (SLAFR), French Ministry for High Education and Research (MENR), Carthera, Laboratoire de Recherche en Technologies Chirurgicales Avancées (LRTCA).

Nicotinamide Adenine Dinucleotide Precursor Supplementation Modulates Neurite Complexity and Survival in Motor Neurons from Amyotrophic Lateral Sclerosis Models.

Aims: Increasing nicotinamide adenine dinucleotide (NAD+) availability has been proposed as a therapeutic approach to prevent neurodegeneration in amyotrophic lateral sclerosis (ALS). Accordingly, NAD+ precursor supplementation appears to exert neuroprotective effects in ALS patients and mouse models. The mechanisms mediating neuroprotection remain uncertain but could involve changes in multiple cell types. We investigated a potential direct effect of the NAD+ precursor nicotinamide mononucleotide (NMN) on the health of cultured induced pluripotent stem cell (iPSC)-derived human motor neurons and in motor neurons isolated from two ALS mouse models, that is, mice overexpressing wild-type transactive response DNA binding protein-43 (TDP-43) or the ALS-linked human superoxide dismutase 1 with the G93A mutation (hSOD1G93A). Results: NMN treatment increased the complexity of neuronal processes in motor neurons isolated from both mouse models and in iPSC-derived human motor neurons. In addition, NMN prevented neuronal death induced by trophic factor deprivation. In mouse and human motor neurons expressing ALS-linked mutant superoxide dismutase 1, NMN induced an increase in glutathione levels, but this effect was not observed in nontransgenic or TDP-43 overexpressing motor neurons. In contrast, NMN treatment normalized the TDP-43 cytoplasmic mislocalization induced by its overexpression. Innovation: NMN can directly act on motor neurons to increase the growth and complexity of neuronal processes and prevent the death induced by trophic factor deprivation. Conclusion: Our results support a direct beneficial effect of NAD+ precursor supplementation on the maintenance of the neuritic arbor in motor neurons. Importantly, this was observed in motor neurons isolated from two different ALS models, with and without involvement of TDP-43 pathology, supporting its therapeutic potential in sporadic and familial ALS. Antioxid. Redox Signal. 41, 573-589.

BI1 Activates Autophagy and Mediates TDP43 to Regulate ALS Pathogenesis.

Amyotrophic lateral sclerosis (ALS) is the most prevalent motor neuron disease in adults. Currently, there are no known drugs or clinical approaches that have demonstrated efficacy in treating ALS. Mitochondrial function and autophagy have been identified as crucial mechanisms in the development of ALS. While Bax inhibitor 1 (BI1) has been implicated in neurodegenerative diseases, its exact mechanism remains unknown. This study investigates the therapeutic impact of BI1 overexpression on ALS both in vivo and in vitro, revealing its ability to mitigate SOD1G93A-induced apoptosis, nuclear damage, mitochondrial dysfunction, and axonal degeneration of motor neurons. At the same time, BI1 prolongs onset time and lifespan of ALS mice, improves motor function, and alleviates neuronal damage, muscle damage, neuromuscular junction damage among other aspects. The findings indicate that BI1 can inhibit pathological TDP43 morphology and initially stimulate autophagy through interaction with TDP43. This study establishes a solid theoretical foundation for understanding the regulation of autophagy by BI1 and TDP43 while shedding light on the pathogenesis of ALS through their interaction - offering new concepts and targets for clinical implementation and drug development.

RNAi-mediated silencing of SOD1 profoundly extends survival and functional outcomes in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo. With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo. Administered intraventricularly, di-siRNASOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders.