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The Sonic Hedgehog (SHH) signaling pathway is related to the progression of various tumors and nervous system diseases. Still, its specific role in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), remains studied. This research investigates the role of SHH and PI3K/AKT signaling pathway proteins on ALS development in a SOD1-G93A transgenic mouse model. After injection of SHH and PI3K/AKT signaling pathway inhibitors or agonists in hSOD1-G93A (9 weeks of age) transgenic mice, we studied skeletal muscle pathology using immunohistochemical staining and Western blot methods. In addition, recorded data on rotation time, weight, and survival were analyzed for these mice. Our study showed that the expression of SHH, Gli-1 and p-AKT in ALS mice decreased with the progression of the disease. The expression of p-AKT changed together with Gli-1 while injecting PI3K/AKT signaling pathway inhibitor or agonist; SHH and Gli-1 protein expression remained unchanged; p-AKT protein expression significantly decreased while injecting PI3K/AKT signaling pathway inhibitor. These results indicate that SHH has a regulatory effect on PI3K/AKT signaling pathway. In behavioral experiments, we found that the survival time of hSOD1-G93A mice was prolonged by injection of SHH agonist while shortened by injection of SHH inhibitor. In conclusion, we confirmed that the SHH pathway played a neuroprotective role in ALS by mediating PI3K/AKT signaling pathway.

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 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 motor symptoms. Despite this, fluorescent -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 , 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.

Background Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. Protein O-linked β- N -acetylglucosamine (O-GlcNAc) modification has been found to affect the processing of several important proteins implicated in ALS. However, the overall level and cellular localization of O-GlcNAc during ALS progression are incompletely understood, and large-scale profiling of O-GlcNAcylation sites in this context remains unexplored. Methods By using immunostaining analysis and chemoenzymatic labeling-based quantitative chemoproteomics, we assayed O-GlcNAcylation dynamics of lumbar spinal cords from SOD-G93A mice and their non-transgenic (NTG) littermates, the most widely used animal model for studying ALS pathogenesis. Results We discovered that the global O-GlcNAcylation was significantly reduced at the disease end stage. Correlatively, a great increase of OGA was observed. Immunohistochemistry and immunofluorescence analysis showed a higher proportion of O-GlcNAc-positive neurons in the NTG group, while O-GlcNAc colocalization with astrocytes/microglia was elevated in SOD1-G93A mice. Moreover, we reported the identification of 568 high-confidence O-GlcNAc sites from end-stage SOD1-G93A and NTG mice. Of the 568 sites, 226—many of which occurred on neuronal function and structure-related proteins—were found to be dynamically regulated. Conclusion These data provide a valuable resource for dissecting the functional role of O-GlcNAcylation in ALS and shed light on promising therapeutic avenues for ALS. The chemoenzymatic labeling-based chemoproteomic approach is applicable for probing O-GlcNAc dynamics in various pathological processes.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by the degeneration of motor neurons that leads to muscle wasting and atrophy. Epidemiological and experimental evidence suggests a causal relationship between ALS and physical activity (PA). However, the impact of PA on motor neuron loss and sarcopenia is still debated, probably because of the heterogeneity and intensities of the proposed exercises. With this study, we aimed to clarify the effect of intense endurance exercise on the onset and progression of ALS in the SOD1-G93A mouse model. We randomly selected four groups of twelve 35-day-old female mice. SOD1-G93A and WT mice underwent intense endurance training on a motorized treadmill for 8 weeks, 5 days a week. During the training, we measured muscle strength, weight, and motor skills and compared them with the corresponding sedentary groups to define the disease onset. At the end of the eighth week, we analyzed the skeletal muscle-motor neuron axis by histological and molecular techniques. Intense endurance exercise anticipates the onset of the disease by 1 week (age of the onset: trained SOD1-G93A = 63.17 ± 2.25 days old; sedentary SOD1-G93A = 70.75 ± 2.45 days old). In SOD1-G93A mice, intense endurance exercise hastens the muscular switch to a more oxidative phenotype and worsens the denervation process by dismantling neuromuscular junctions in the tibialis anterior, enhancing the Wallerian degeneration in the sciatic nerve, and promoting motor neuron loss in the spinal cord. The training exacerbates neuroinflammation, causing immune cell infiltration in the sciatic nerve and a faster activation of astrocytes and microglia in the spinal cord. Intense endurance exercise, acting on skeletal muscles, worsens the pathological hallmarks of ALS, such as denervation and neuroinflammation, brings the onset forward, and accelerates the progression of the disease. Our findings show the potentiality of skeletal muscle as a target for both prognostic and therapeutic strategies; the preservation of skeletal muscle health by specific intervention could counteract the dying-back process and protect motor neurons from death. The physiological characteristics and accessibility of skeletal muscle further enhance its appeal as a therapeutic target.

Oxidant toxicity has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), an insidiously progressive neurodegenerative disorder involving upper and lower motor neurons. Here, we investigated the cellular and molecular mechanisms underlying the neuroprotective effects of an anti-oxidant genistein in SOD1-G93A transgenic mouse model of ALS. Rotarod test, hanging wire test and hindlimb clasping test were used to determined disease onset and assess motor performance. Immunostaining together with neuronal size measurement were used to count viable motor neurons. In addition, immunostaining procedure and ELISA kit were used to assess the inflammatory response in the spinal cord. Our results showed that Genistein administration suppressed the production of pro-inflammatory cytokines and alleviated gliosis in the spinal cord of SOD1-G93A mice. In addition, genistein administration induced autophagic processes and enhanced the viability of spinal motor neurons. As a result, genistein alleviated ALS-related symptoms and slightly prolonged the lifespan of SOD1-G93A mice. Taken together, our results indicate that genistein is neuroprotective in SOD1-G93A mice, suggesting genistein could be a promising treatment for human ALS. Graphical Abstract Genistein protects impariments in SOD1-G93A transgenic mouse model.

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu 2+ /Zn 2+ superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression.

Microglial activation plays a crucial role in the disease progression in amyotrophic lateral sclerosis (ALS). Interleukin receptor-associated kinases-M (IRAK-M) is an important negative regulatory factor in the Toll-like receptor 4 (TLR4) pathway during microglia activation, and its mechanism in this process is still unclear. In the present study, we aimed to investigate the dynamic changes of IRAK-M and its protective effects for motor neurons in SOD1-G93A mouse model of ALS. qPCR (Real-time Quantitative PCR Detecting System) were used to examine the mRNA levels of IRAK-M in the spinal cord in both SOD1-G93A mice and their age-matched wild type (WT) littermates at 60, 100 and 140 days of age. We established an adeno-associated virus 9 (AAV9)-based platform by which IRAK-M was targeted mostly to microglial cells to investigate whether this approach could provide a protection in the SOD1-G93A mouse. Compared with age-matched WT mice, IRAK-M mRNA level was elevated at 100 and 140 days in the anterior horn region of spinal cords in the SOD1-G93A mouse. AAV9-IRAK-M treated SOD1-G93A mice showed reduction of IL-1β mRNA levels and significant improvements in the numbers of spinal motor neurons in spinal cord. Mice also showed previously reduction of muscle atrophy. Our data revealed the dynamic changes of IRAK-M during ALS pathological progression and demonstrated that an AAV9-IRAK-M delivery was an effective and translatable therapeutic approach for ALS. These findings may help identify potential molecular targets for ALS therapy.

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disease where activated glia release pro-inflammatory cytokines that trigger a vicious cycle of neurodegeneration in the absence of resolution of inflammation. Given the well-established role of histamine as a neuron-to-glia alarm signal implicated in brain disorders, the aim of this study was to investigate the expression and regulation of the histaminergic pathway in microglial activation in ALS mouse model and in humans. By examining the contribution of the histaminergic system to ALS, we found that particularly H1 and H4 receptors, histamine promoted an anti-inflammatory profile in microglia from SOD1-G93A mice by modulating their activation state. A decrease in NF-κB and NADPH oxidase 2 with an increase in arginase 1 and P2Y12 receptor was induced by histamine only in the ALS inflammatory environment, but not in the healthy microglia, together with an increase in IL-6, IL-10, CD163, and CD206 phenotypic markers in SOD1-G93A cells. Moreover, histaminergic H1, H2, H3, and H4 receptors, and histamine metabolizing enzymes histidine decarboxylase, histamine -methyltransferase, and diamine oxidase were found deregulated in spinal cord, cortex, and hypothalamus of SOD1-G93A mice during disease progression. Finally, by performing a meta-analysis study, we found a modulated expression of histamine-related genes in cortex and spinal cord from sporadic ALS patients. Our findings disclose that histamine acts as anti-inflammatory agent in ALS microglia and suggest a dysregulation of the histaminergic signaling in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal condition marked by the degeneration of motor neurons. ALS has been linked to numerous genes with diverse biological roles, reflecting a highly intricate and multifaceted disease process. This diversity poses significant challenges in developing universally effective and bioavailable treatments. Advancing therapeutic strategies require uncovering molecular pathways that are major drivers of ALS. We conducted proteomic analyses of human iPSC‐derived motor neurons carrying C9ORF72 mutations, alongside spinal ventral horns from mice with pathogenic C9orf72‐mutations. This cross‐species approach revealed disruptions in synaptic vesicle release, endoplasmic reticulum (ER) and mitochondrial stress responses as conserved ALS pathogenic mechanisms. Disease progression was associated with accumulation of cytotoxic protein aggregates and oxidative stress. We analyzed the potential of GM1, an established neuroprotective molecule, to reverse these pathogenic features. To enhance the pharmacokinetics of GM1, we developed Talineuren (TLN), a nanoliposome‐based formulation of the active pharmaceutical ingredient GM1 ganglioside that improves its bioavailability. GM1 stabilized mitochondrial Ca2⁺ handling, improved energy metabolism, and alleviated ER stress, preventing protein aggregation and restoring cellular proteostasis and counteracted behavioral deficits in C9orf72 and SOD1‐G93A mouse models. Together, these findings underscore the central, convergent role for cellular disruptions in ALS and position TLN as a promising therapeutic candidate. Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease driven by genetic and molecular disruptions affecting energy balance, protein homeostasis, and stress responses in nerve cells. Studies using human and rodent models identified convergent defects in mitochondria and synaptic function. GM1‐based nanotherapy, “Talineuren” restored cellular health and motor function, showing strong therapeutic promise.