Zinc metabolism and its role in immunity status in subjects with trisomy 21: chromosomal dosage effect.

Introduction: Trisomy 21 (T21), which causes Down syndrome (DS), is the most common chromosomal aneuploidy in humankind and includes different clinical comorbidities, among which the alteration of the immune system has a heavy impact on patient's lives. A molecule with an important role in immune response is zinc and it is known that its concentration is significantly lower in children with T21. Different hypotheses were made about this metabolic alteration and one of the reasons might be the overexpression of superoxide dismutase 1 (SOD1) gene, as zinc is part of the SOD1 active enzymatic center. Methods: The aim of our work is to explore if there is a linear correlation between zinc level and immune cell levels measured in a total of 217 blood samples from subjects with T21. Furthermore, transcriptome map analyses were performed using Transcriptome Mapper (TRAM) software to investigate whether a difference in gene expression is detectable between subjects with T21 and euploid control group in tissues and cells involved in the immune response such as lymphoblastoid cells, thymus and white blood cells. Results: Our results have confirmed the literature data stating that the blood zinc level in subjects with T21 is lower compared to the general population; in addition, we report that the T21/control zinc concentration ratio is 2:3, consistent with a chromosomal dosage effect due to the presence of three copies of chromosome 21. The transcriptome map analyses showed an alteration of some gene's expression which might explain low levels of zinc in the blood. Discussion: Our data suggest that zinc level is not associated with the levels of immunity cells or proteins analyzed themselves and rather the main role of this ion might be played in altering immune cell function.

Perineuronal nets are phagocytosed by MMP-9 expressing microglia and astrocytes in the SOD1G93A ALS mouse model.

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.

Vitamin D3 exacerbates steatosis while calcipotriol inhibits inflammation in non-alcoholic fatty liver disease in Sod1 knockout mice: a comparative study of two forms of vitamin D.

The role of vitamin D (VD) in non-alcoholic fatty liver disease (NAFLD) remains controversial, possibly due to the differential effects of various forms of VD. In our study, Sod1 gene knockout (SKO) mice were utilized as lean NAFLD models, which were administered 15 000 IU VD3 per kg diet, or intraperitoneally injected with the active VD analog calcipotriol for 12 weeks. We found that VD3 exacerbated hepatic steatosis in SKO mice, with an increase in the levels of Cd36, Fatp2, Dgat2, and CEBPA. However, calcipotriol exerted no significant effect on hepatic steatosis. Calcipotriol inhibited the expression of Il-1a, Il-1b, Il-6, Adgre1, and TNF, with a reduction of NFκB phosphorylation in SKO mice. No effect was observed by either VD3 or calcipotriol on hepatocyte injury and hepatic fibrosis. Co-immunofluorescence stains of CD68, a liver macrophage marker, and VDR showed that calcipotriol reduced CD68 positive cells, and increased the colocalization of VDR with CD68. However, VD3 elevated hepatocyte VDR expression, with no substantial effect on the colocalization of VDR with CD68. Finally, we found that VD3 increased the levels of serum 25(OH)D3 and 24,25(OH)2D3, whereas calcipotriol decreased both. Both VD3 and calcipotriol did not disturb serum calcium and phosphate levels. In summary, our study found that VD3 accentuated hepatic steatosis, while calcipotriol diminished inflammation levels in SKO mice, and the difference might stem from their distinct cellular selectivity in activating VDR. This study provides a reference for the application of VD in the treatment of lean NAFLD.

Association of SOD1 gene variants (50 bp Ins/Del, rs4817415, rs2070424, rs1041740, rs17880135) with plasma protein levels in vitiligo patients and their analysis in silico.

OBJECTIVE: Vitiligo is a common systemic, idiopathic autoimmune disease. The aim of this study was to analyze the frequency of variants of the superoxide dismutase 1 (SOD1) gene (50 bp Ins/Del, rs4817415, rs2070424, rs1041740, rs17880135) and circulating plasma protein levels through in-silico analysis. PATIENTS AND METHODS: Blood samples were collected from adult patients of both sexes with a clinical diagnosis of vitiligo. ELISA tests for SOD and analysis of gene variants by qPCR were compared to a disease-free reference group. RESULTS: The population analyzed was young people between 29 and 37 years old, with a higher percentage of women. The population was found in the Hardy-Weinberg equilibrium (HWE). The 50 bp Ins/Del, rs4817415, and rs2070424 variants showed no significant difference between groups (p > 0.05). Although, in the dominant model, the CT and CTTT genotypes of the rs1041740 and rs17880135 variants showed an association with susceptibility to vitiligo compared to the control. Plasma SOD levels showed significant differences between the groups, and when stratified according to the genotypes of each variant, there was a significant difference, except with the rs17880135 variant. The haplotypes InsCGTC and InsAGCC are shown to be risk factors for susceptibility to vitiligo. The in-silico analysis demonstrated that the rs4817415, rs2070424, rs1041740, and rs17880135 variants of the SOD1 gene participate in the modification of selected regulatory elements for differentiating the protein, transcription factors, and long non-coding RNA. CONCLUSIONS: Information regarding the pathogenesis of vitiligo helps recognize risk factors and identify the relationship of diagnostic markers of cell damage inherent to the disease. This will help improve aspects of prevention and the choice of treatment alternatives appropriate to each case.

[SOD1 gene therapy delays ALS disease progression]. / Precisionsmedicinsk genterapi har bromsat utveckling av ALS.

We present a patient with familial amyotrophic lateral sclerosis caused by an aggressive A4S mutation in the SOD1 gene. In 2020, the patient was enrolled in the VALOR SOD1 gene therapy phase-3 trial. At screening, the ALSFRS-R score was 41 (48 is normal) and the level of CSF-neurofilament L (an indicator of ongoing neuronal damage) was 11 000 ng/L (ref <650 ng/L). In the four years following enrollment, the patient received monthly intrathecal treatment with tofersen, an antisense oligonucleotide compound that inhibits SOD1 protein expression and hence lowers the synthesis of toxic SOD1 protein species. Side effects have been minimal and mostly attributed to the spinal taps. The patient remains ambulatory with an active social lifestyle. The ALSFRS-R score has in the past 18 months stabilized around 35-37, CSF-NfL is 1 290 ng/L and plasma-NfL is 12 (reference <13). This is the first documented arresting intervention in a patient with ALS in Sweden.

Discovery of a novel homozygous SOD1 truncating variant bolsters infantile SOD1 deficiency syndrome.

OBJECTIVE: Superoxide dismutase 1 (SOD1) is an important antioxidant enzyme whose main function is to neutralise superoxide free radicals in the cytoplasm. Heterozygous variants in SOD1 are responsible for a substantial percentage of familial amyotrophic lateral sclerosis (ALS) cases. Recently, several reports have shown that biallelic loss of SOD1 function results in a novel phenotype called infantile SOD1 deficiency syndrome, which is consistent with a recessive pattern of inheritance and can be distinguished from typical (adult-onset) ALS. METHODS: We documented detailed family histories and clinical data, followed by whole-exome sequencing and family co-segregation analysis through Sanger sequencing. To facilitate comparisons, relevant data from fifteen previously reported patients with SOD1-related neurodevelopmental disorders were included. RESULTS: This study presents a new Turkish family with two affected children exhibiting severe delayed motor development, infancy-onset loss of motor skills, axial hypotonia, tetraspasticity, and impaired cognitive functions. Genetic analysis revealed a novel homozygous frameshift variant in SOD1 (c.248dupG [p.Asp84Argfs*8]), with computational biochemical studies shedding light on the mechanistic aspects of SOD1 dysfunction. CONCLUSIONS: Our findings contribute an affirmative report of a fourth biallelic variant resulting in a severe clinical phenotype, reminiscent of those induced by previously identified homozygous loss-of-function SOD1 variants. This research not only advances our understanding of the pathogenesis of this debilitating neurological syndrome but also aligns with ongoing intensive efforts to comprehend and address SOD1-linked ALS.

Binge alcohol induces NRF2-related antioxidant response in the skeletal muscle of female mice.

BACKGROUND: Chronic alcohol enhances oxidative stress, but the temporal response of antioxidant genes in skeletal muscle following a binge drinking episode remains unknown. METHODS: Experiment 1: C57BL/6Hsd female mice received an IP injection of saline (CON; n = 39) or ethanol (ETOH; n = 39) (5 g/kg). Gastrocnemius muscles were collected from baseline (untreated; n = 3), CON (n = 3), and ETOH (n = 3) mice every 4 h for 48 h. Experiment 2: Gastrocnemius muscles were collected from control-fed (CON-FED; n = 17), control-fasted (CON-FAST; n = 18), or alcohol-fed (ETOH-FED; n = 18) mice every 4hrs for 20hrs after saline or ethanol (5 g/kg). RESULTS: EtOH enhanced Superoxide dismutase 1 (Sod1) and NADPH Oxidase 4 (Nox4) from 24 to 48hr after the binge, while Sod2 and Nox2 were suppressed. Nuclear factor erythroid-derived 2-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) increased 12hrs after intoxication. Cytochrome P450 oxidoreductase (Por), Heme oxygenase 1 (Ho1), Peroxiredoxin 6 (Prdx6), Glutamate-cysteine ligase catalytic subunit (Gclc), Glutamate-cysteine ligase modifier subunit (Gclm), and Glutathione-disulfide reductase (Gsr) were increased by ETOH starting 12-16hrs post-binge. Fasting had similar effects on Nrf2 compared to alcohol, but downstream targets of NRF2, including Por, Ho1, Gclc, and Gclm, were differentially altered with fasting and EtOH. CONCLUSION: These data suggest that acute alcohol intoxication induced markers of oxidative stress and antioxidant signaling through the NRF2 pathway and that there were effects of alcohol independent of a possible decrease in food intake caused by binge intoxication.

SOD1-high fibroblasts derived exosomal miR-3960 promotes cisplatin resistance in triple-negative breast cancer by suppressing BRSK2-mediated phosphorylation of PIMREG.

Platinum-based neoadjuvant therapy represented by cisplatin is widely employed in treating Triple-Negative Breast Cancer (TNBC), a particularly aggressive subtype of breast cancer. Nevertheless, the emergence of cisplatin resistance presents a formidable challenge to clinical chemotherapy efficacy. Herein, we revealed the critical role of tumor microenvironment (TME) derived exosomal miR-3960 and phosphorylation at the S16 site of PIMREG in activating NF-κB signaling pathway and promoting cisplatin resistance of TNBC. Detailed regulatory mechanisms revealed that SOD1-upregulated fibroblasts secrete miR-3960 and are then transported into TNBC cells via exosomes. Within TNBC cells, miR-3960 targets and inhibits the expression of BRSK2, an AMPK protein kinase family member. Furthermore, we emphasized that BRSK2 contributes to ubiquitination degradation of PIMREG and modulates subsequent activation of the NF-κB signaling pathway by mediating PIMREG phosphorylation at the S16 site, ultimately affects the cisplatin resistance of TNBC. In conclusion, our research demonstrated the crucial role of SOD1high fibroblast, exosomal miR-3960 and S16 site phosphorylated PIMREG in regulating the NF-κB signaling pathway and cisplatin resistance of TNBC. These findings provided significant potential as biomarkers for accurately diagnosing cisplatin-resistant TNBC patients and guiding chemotherapy strategy selection.

VEGF expression disparities in brainstem motor neurons of the SOD1G93A ALS model: Correlations with neuronal vulnerability.

Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease characterized by severe muscle weakness mainly due to degeneration and death of motor neurons. A peculiarity of the neurodegenerative processes is the variable susceptibility among distinct neuronal populations, exemplified by the contrasting resilience of motor neurons innervating the ocular motor system and the more vulnerable facial and hypoglossal motor neurons. The crucial role of vascular endothelial growth factor (VEGF) as a neuroprotective factor in the nervous system is well-established since a deficit of VEGF has been related to motoneuronal degeneration. In this study, we investigated the survival of ocular, facial, and hypoglossal motor neurons utilizing the murine SOD1G93A ALS model at various stages of the disease. Our primary objective was to determine whether the survival of the different brainstem motor neurons was linked to disparate VEGF expression levels in resilient and susceptible motor neurons throughout neurodegeneration. Our findings revealed a selective loss of motor neurons exclusively within the vulnerable nuclei. Furthermore, a significantly higher level of VEGF was detected in the more resistant motor neurons, the extraocular ones. We also examined whether TDP-43 dynamics in the brainstem motor neuron of SOD mice was altered. Our data suggests that the increased VEGF levels observed in extraocular motor neurons may potentially underlie their resistance during the neurodegenerative processes in ALS in a TDP-43-independent manner. Our work might help to better understand the underlying mechanisms of selective vulnerability of motor neurons in ALS.

Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease influenced by genetic, epigenetic, and environmental factors, resulting in dysfunction in cellular and molecular pathways. The limited efficacy of current treatments highlights the need for combination therapies targeting multiple aspects of the disease. Niclosamide, an anthelminthic drug listed as an essential medicine, has been repurposed in clinical trials for different diseases due to its anti-inflammatory and anti-fibrotic properties. Niclosamide can inhibit various molecular pathways (e.g., STAT3, mTOR) that are dysregulated in ALS, suggesting its potential to disrupt these altered mechanisms associated with the pathology. We administered niclosamide intraperitoneally to two transgenic murine models, SOD1-G93A and FUS mice, mimicking key pathological processes of ALS. The treatment was initiated at the onset of symptoms, and we assessed disease progression by neurological scores, rotarod and wire tests, and monitored survival. Furthermore, we investigated cellular and molecular mechanisms affected by niclosamide in the spinal cord and muscle of ALS mice. In both models, the administration of niclosamide resulted in a slowdown of disease progression, an increase in survival rates, and an improvement in tissue pathology. This was characterised by reduced gliosis, motor neuron loss, muscle atrophy, and inflammatory pathways. Based on these results, our findings demonstrate that niclosamide can impact multiple pathways involved in ALS. This multi-targeted approach leads to a slowdown in the progression of the disease, positioning niclosamide as a promising candidate for repurposing in the treatment of ALS.

Novel Pathogenic Variants Leading to Sporadic Amyotrophic Lateral Sclerosis in Greek Patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease that affects motor neurons, leading to paralysis and death usually 3-5 years after the onset of symptoms. The investigation of both sporadic and familial ALS highlighted four main genes that contribute to the pathogenesis of the disease: SOD1, FUS, TARDBP and C9orf72. This study aims to provide a comprehensive investigation of genetic variants found in SOD1, FUS and TARDBP genes in Greek sporadic ALS (sALS) cases. Our sequencing analysis of the coding regions of the abovementioned genes that include the majority of the variants that lead to ALS in 32 sALS patients and 3 healthy relatives revealed 6 variants in SOD1, 19 variants in FUS and 37 variants in TARDBP, of which the SOD1 p.D90A and the FUS c.*356G>A (rs886051940) variants have been previously associated with ALS, while two novel nonsense pathogenic variants were also identified, namely FUS p.R241* and TDP-43 p.Y214*. Our study contributes to the worldwide effort toward clarifying the genetic basis of sALS to better understand the disease's molecular pathology.

Structural insights into the modulation Of SOD1 aggregation By a fungal metabolite Phialomustin-B: Therapeutic potential in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal human motor neuron disease leading to muscle atrophy and paralysis. Mutations in superoxide dismutase 1 (SOD1) are associated with familial ALS (fALS). The SOD1 mutants in ALS have a toxic-gain of function by destabilizing the functional SOD1 homodimer, consequently inducing fibril-like aggregation with a cytotoxic non-native trimer intermediate. Therefore, reducing SOD1 oligomerization via chemical modulators is an optimal therapy in ALS. Here, we report the discovery of Phialomustin-B, an unsaturated secondary metabolite from the endophytic fungus Phialophora mustea, as a modulator of SOD1 aggregation. The crystal structure of the SOD1-Phialomustin complex refined to 1.90 Å resolution demonstrated for the first time that the ligand binds to the dimer interface and the lateral region near the electrostatic loop. The aggregation analyses of SOD1WT and the disease mutant SOD1A4V revealed that Phialomustin-B reduces cytotoxic trimerization. We propose that Phialomustin-B is a potent lead molecule with therapeutic potential in fALS.

Cortical hyperexcitability in mouse models and patients with amyotrophic lateral sclerosis is linked to noradrenaline deficiency.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by the death of upper (UMN) and lower motor neurons (LMN) in the motor cortex, brainstem, and spinal cord. Despite decades of research, ALS remains incurable, challenging to diagnose, and of extremely rapid progression. A unifying feature of sporadic and familial forms of ALS is cortical hyperexcitability, which precedes symptom onset, negatively correlates with survival, and is sufficient to trigger neurodegeneration in rodents. Using electrocorticography in the Sod1G86R and FusΔNLS/+ ALS mouse models and standard electroencephalography recordings in patients with sporadic ALS, we demonstrate a deficit in theta-gamma phase-amplitude coupling (PAC) in ALS. In mice, PAC deficits started before symptom onset, and in patients, PAC deficits correlated with the rate of disease progression. Using mass spectrometry analyses of CNS neuropeptides, we identified a presymptomatic reduction of noradrenaline (NA) in the motor cortex of ALS mouse models, further validated by in vivo two-photon imaging in behaving SOD1G93A and FusΔNLS/+ mice, that revealed pronounced reduction of locomotion-associated NA release. NA deficits were also detected in postmortem tissues from patients with ALS, along with transcriptomic alterations of noradrenergic signaling pathways. Pharmacological ablation of noradrenergic neurons with DSP-4 reduced theta-gamma PAC in wild-type mice and administration of a synthetic precursor of NA augmented theta-gamma PAC in ALS mice. Our findings suggest theta-gamma PAC as means to assess and monitor cortical dysfunction in ALS and warrant further investigation of the NA system as a potential therapeutic target.

Novel insights into RAGE signaling pathways during the progression of amyotrophic lateral sclerosis in RAGE-deficient SOD1 G93A mice.

Amyotrophic lateral sclerosis (ALS) is neurodegenerative disease characterized by a progressive loss of motor neurons resulting in paralysis and muscle atrophy. One of the most prospective hypothesis on the ALS pathogenesis suggests that excessive inflammation and advanced glycation end-products (AGEs) accumulation play a crucial role in the development of ALS in patients and SOD1 G93A mice. Hence, we may speculate that RAGE, receptor for advanced glycation end-products and its proinflammatory ligands such as: HMGB1, S100B and CML contribute to ALS pathogenesis. The aim of our studies was to decipher the role of RAGE as well as provide insight into RAGE signaling pathways during the progression of ALS in SOD1 G93A and RAGE-deficient SOD1 G93A mice. In our study, we observed alternations in molecular pattern of proinflammatory RAGE ligands during progression of disease in RAGE KO SOD1 G93A mice compared to SOD1 G93A mice. Moreover, we observed that the amount of beta actin (ACTB) as well as Glial fibrillary acidic protein (GFAP) was elevated in SOD1 G93A mice when compared to mice with deletion of RAGE. These data contributes to our understanding of implications of RAGE and its ligands in pathogenesis of ALS and highlight potential targeted therapeutic interventions at the early stage of this devastating disease. Moreover, inhibition of the molecular cross-talk between RAGE and its proinflammatory ligands may abolish neuroinflammation, gliosis and motor neuron damage in SOD1 G93A mice. Hence, we hypothesize that attenuated interaction of RAGE with its proinflammatory ligands may improve well-being and health status during ALS in SOD1 G93A mice. Therefore, we emphasize that the inhibition of RAGE signaling pathway may be a therapeutic target for neurodegenerative diseases.

Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS.

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Communication defects with astroglia contribute to early impairments in the motor cortex plasticity of SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, involving the selective degeneration of cortical upper synapses in the primary motor cortex (M1). Excitotoxicity in ALS occurs due to an imbalance between excitation and inhibition, closely linked to the loss/gain of astrocytic function. Using the ALS SOD1G93A mice, we investigated the astrocytic contribution for the electrophysiological alterations observed in the M1 of SOD1G93A mice, throughout disease progression. Results showed that astrocytes are involved in synaptic dysfunction observed in presymptomatic SOD1G93A mice, since astrocytic glutamate transport currents are diminished and pharmacological inhibition of astrocytes only impaired long-term potentiation and basal transmission in wild-type mice. Proteomic analysis revealed major differences in neuronal transmission, metabolism, and immune system in upper synapses, confirming early communication deficits between neurons and astroglia. These results provide valuable insights into the early impact of upper synapses in ALS and the lack of supportive functions of cortical astrocytes, highlighting the possibility of manipulating astrocytes to improve synaptic function.

Excitatory action of low frequency depolarizing GABA/glycine synaptic inputs is prevalent in prenatal spinal SOD1G93A motoneurons.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. Recent evidence suggests the dysfunction of inhibitory signalling in ALS motor neurons. We have shown that embryonic day (E)17.5 spinal motoneurons (MNs) of the SOD1G93A mouse model of ALS exhibit an altered chloride homeostasis. At this prenatal stage, inhibition of spinal motoneurons (MNs) is mediated by depolarizing GABAergic/glycinergic postsynaptic potentials (dGPSPs). Here, using an ex vivo preparation and patch clamp recording from MNs with a chloride equilibrium set below spike threshold, we report that low input resistance (Rin ) E17.5 MNs from the SOD1G93A ALS mouse model do not correctly integrate dGPSPs evoked by electrical stimulations of GABA/glycine inputs at different frequencies. Indeed, firing activity of most wild-type (WT) MNs with low Rin was inhibited by incoming dGPSPs, whereas low Rin SOD1G93A MNs were excited or exhibited a dual response (excited by low frequency dGPSPs and inhibited by high frequency dGPSPs). Simulation highlighted the importance of the GABA/glycine input density and showed that pure excitation could be obtained in SOD-like MNs by moving GABA/glycine input away from the cell body to dendrites. This was in agreement with confocal imaging showing a lack of peri-somatic inhibitory terminals in SOD1G93A MNs compared to WT littermates. Putative fast ALS-vulnerable MNs with low Rin are therefore lacking functional inhibition at the near-term prenatal stage. KEY POINTS: We analysed the integration of GABAergic/glycinergic synaptic events by embryonic spinal motoneurons (MNs) in a mouse model of the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. We found that GABAergic/glycinergic synaptic events do not properly inhibit ALS MNs with low input resistance, most probably corresponding to future vulnerable MNs. We used a neuron model to highlight the importance of the GABA/glycine terminal location and density in the integration of the GABAergic/glycinergic synaptic events. Confocal imaging showed a lack of GABA/glycine terminals on the cell body of ALS MNs. The present study suggests that putative ALS vulnerable MNs with low Rin lack functional inhibition at the near-term stage.

Oxidized SOD1 accelerates cellular senescence in neural stem cells.

BACKGROUND: Neural stem cells (NSCs), especially human NSCs, undergo cellular senescence characterized by an irreversible proliferation arrest and loss of stemness after prolonged culture. While compelling correlative data have been generated to support the oxidative stress theory as one of the primary determinants of cellular senescence of NSCs, a direct cause-and-effect relationship between the accumulation of oxidation-mediated damage and cellular senescence of NSCs has yet to be firmly established. Human SOD1 (hSOD1) is susceptible to oxidation. Once oxidized, it undergoes aberrant misfolding and gains toxic properties associated with age-related neurodegenerative disorders. The present study aims to examine the role of oxidized hSOD1 in the senescence of NSCs. METHODS: NSCs prepared from transgenic mice expressing the wild-type hSOD1 gene were maintained in culture through repeated passages. Extracellular vesicles (EVs) were isolated from culture media at each passage. To selectively knock down oxidized SOD1 in NSCs and EVs, we used a peptide-directed chaperone-mediated protein degradation system named CT4 that we developed recently. RESULTS: In NSCs expressing the hSOD1 from passage 5, we detected a significant increase of oxidized hSOD1 and an increased expression of biomarkers of cellular senescence, including upregulation of P53 and SA-ß-Gal and cytoplasmic translocation of HMGB1. The removal of oxidized SOD1 remarkably increased the proliferation and stemness of the NSCs. Meanwhile, EVs derived from senescent NSCs carrying the wild-type hSOD1 contained high levels of oxidized hSOD1, which could accelerate the senescence of young NSCs and induce the death of cultured neurons. The removal of oxidized hSOD1 from the EVs abolished their senescence-inducing activity. Blocking oxidized SOD1 on EVs with the SOD1 binding domain of the CT4 peptide mitigated its toxicity to neurons. CONCLUSION: Oxidized hSOD1 is a causal factor in the cellular senescence of NSCs. The removal of oxidized hSOD1 is a strategy to rejuvenate NSCs and to improve the quality of EVs derived from senescent cells.

Changes in glial cell activation and extracellular vesicles production precede the onset of disease symptoms in transgenic hSOD1G93A pigs.

SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.

Sex-Dependent Changes to the Intestinal and Hepatic Abundance of Drug Transporters and Metabolizing Enzymes in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.