EST79232 and EST79376, Two Novel Sigma-1 Receptor Ligands, Exert Neuroprotection on Models of Motoneuron Degeneration.

Motor neuron diseases (MNDs) include sporadic and hereditary neurological disorders characterized by progressive degeneration of motor neurons (MNs). Sigma-1 receptor (Sig-1R) is a protein enriched in MNs, and mutations on its gene lead to various types of MND. Previous studies have suggested that Sig-1R is a target to prevent MN degeneration. In this study, two novel synthesized Sig-1R ligands, coded EST79232 and EST79376, from the same chemical series, with the same scaffold and similar physicochemical properties but opposite functionality on Sig-1R, were evaluated as neuroprotective compounds to prevent MN degeneration. We used an in vitro model of spinal cord organotypic cultures under chronic excitotoxicity and two in vivo models, the spinal nerve injury and the superoxide dismutase 1 (SOD1)G93A mice, to characterize the effects of these Sig-1R ligands on MN survival and modulation of glial reactivity. The antagonist EST79376 preserved MNs in vitro and after spinal nerve injury but was not able to improve MN death in SOD1G93A mice. In contrast, the agonist EST79232 significantly increased MN survival in the three models of MN degeneration evaluated and had a mild beneficial effect on motor function in SOD1G93A mice. In vivo, Sig-1R ligand EST79232 had a more potent effect on preventing MN degeneration than EST79376. These data further support the interest in Sig-1R as a therapeutic target for neurodegeneration.

Prolonged NCX activation prevents SOD1 accumulation, reduces neuroinflammation, ameliorates motor behavior and prolongs survival in a ALS mouse model.

Imbalance in cellular ionic homeostasis is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Sodium-calcium exchanger (NCX) is a membrane antiporter that, operating in a bidirectional way, couples the exchange of Ca2+ and Na + ions in neurons and glial cells, thus controlling the intracellular homeostasis of these ions. Among the three NCX genes, NCX1 and NCX2 are widely expressed within the CNS, while NCX3 is present only in skeletal muscles and at lower levels of expression in selected brain regions. ALS mice showed a reduction in the expression and activity of NCX1 and NCX2 consistent with disease progression, therefore we aimed to investigate their role in ALS pathophysiology. Notably, we demonstrated that the pharmacological activation of NCX1 and NCX2 by the prolonged treatment of SOD1G93A mice with the newly synthesized compound neurounina: (1) prevented the reduction in NCX activity observed in spinal cord; (2) preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice; (3) prevented the spinal cord accumulation of misfolded SOD1; (4) reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord; (5) improved the lifespan and mitigated motor symptoms of ALS mice. The present study highlights the significant role of NCX1 and NCX2 in the pathophysiology of this neurodegenerative disorder and paves the way for the design of a new pharmacological approach for ALS.

A Novel Anti-Inflammatory d-Peptide Inhibits Disease Phenotype Progression in an ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by selective neuronal death in the brain stem and spinal cord. The cause is unknown, but an increasing amount of evidence has firmly certified that neuroinflammation plays a key role in ALS pathogenesis. Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and has been implicated as driver of disease progression. Here, we describe a treatment study demonstrating the therapeutic potential of a tandem version of the well-known all-d-peptide RD2 (RD2RD2) in a transgenic mouse model of ALS (SOD1*G93A). Mice were treated intraperitoneally for four weeks with RD2RD2 vs. placebo. SOD1*G93A mice were tested longitudinally during treatment in various behavioural and motor coordination tests. Brain and spinal cord samples were investigated immunohistochemically for gliosis and neurodegeneration. RD2RD2 treatment in SOD1*G93A mice resulted not only in a reduction of activated astrocytes and microglia in both the brain stem and lumbar spinal cord, but also in a rescue of neurons in the motor cortex. RD2RD2 treatment was able to slow progression of the disease phenotype, especially the motor deficits, to an extent that during the four weeks treatment duration, no significant progression was observed in any of the motor experiments. Based on the presented results, we conclude that RD2RD2 is a potential therapeutic candidate against ALS.

Overexpression of NDRG2 in skeletal muscle does not ameliorate the effects of stress in vivo.

NEW FINDINGS: What is the central question of this study? Do elevated levels of the stress-response protein NDRG2 protect against fasting and chronic disease in mouse skeletal muscle? What is the main finding and its importance? NDRG2 levels increased in the tibialis anterior muscle in response to fasting and the effects of motor neurone disease. No alleviation of the stress-related and proteasomal pathways, mitochondrial dysfunction or muscle mass loss was observed even with the addition of exogenous NDRG2 indicating that the increase in NDRG2 is a normal adaptive response. ABSTRACT: Skeletal muscle mass loss and dysfunction can arise from stress, which leads to enhanced protein degradation and metabolic impairment. The expression of N-myc downstream-regulated gene 2 (NDRG2) is induced in response to different stressors and is protective against the effects of stress in some tissues and cell types. Here, we investigated the endogenous NDRG2 response to the stress of fasting and chronic disease in mice and whether exogenous NDRG2 overexpression through adeno-associated viral (AAV) treatment ameliorated the response of skeletal muscle to these conditions. Endogenous levels of NDRG2 increased in the tibialis anterior muscle in response to 24 h fasting and with the development of the motor neurone disease, amyotrophic lateral sclerosis, in SOD1G93A transgenic mice. Despite AAV-induced overexpression and increased expression with fasting, NDRG2 was unable to protect against the activation of proteasomal and stress pathways in response to fasting. Furthermore, NDRG2 was unable to reduce muscle mass loss, mitochondrial dysfunction and elevated oxidative and endoplasmic reticulum stress levels in SOD1G93A mice. Conversely, elevated NDRG2 levels did not exacerbate these stress responses. Overall, increasing NDRG2 levels might not be a useful therapeutic strategy to alleviate stress-related disease pathologies in skeletal muscle.

The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice.

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNß and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1G93A mice.

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A2A receptor agonist (CGS21680), antagonist (KW6002) or the A1 receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A2A receptors modified the progressive loss of motor skills or survival of mSOD1G93A mice. Conversely, blockade of adenosine A1 receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

Urate mitigates oxidative stress and motor neuron toxicity of astrocytes derived from ALS-linked SOD1G93A mutant mice.

Dominant mutations in an antioxidant enzyme superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease characterized by loss of motor neurons. Oxidative stress has also been linked to many of the neurodegenerative diseases and is likely a central mechanism of motor neuron death in ALS. Astrocytes derived from mutant SOD1G93A mouse models or patients play a significant role in the degeneration of spinal motor neurons in ALS through a non-cell-autonomous process. Here we characterize the neuroprotective effects and mechanisms of urate (a.k.a. uric acid), a major endogenous antioxidant and a biomarker of favorable ALS progression rates, in a cellular model of ALS. Our results demonstrate a significant protective effect of urate against motor neuron injury evoked by mutant astrocytes derived from SOD1G93A mice or hydrogen peroxide induced oxidative stress. Overall, these results implicate astrocyte dependent protective effect of urate in a cellular model of ALS. These findings together with our biomarker data may advance novel targets for treating motor neuron disease.

Enhanced Function and Overexpression of Metabotropic Glutamate Receptors 1 and 5 in the Spinal Cord of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis during Disease Progression.

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.

Altered mechanisms underlying the abnormal glutamate release in amyotrophic lateral sclerosis at a pre-symptomatic stage of the disease.

Abnormal Glu release occurs in the spinal cord of SOD1(G93A) mice, a transgenic animal model for human ALS. Here we studied the mechanisms underlying Glu release in spinal cord nerve terminals of SOD1(G93A) mice at a pre-symptomatic disease stage (30days) and found that the basal release of Glu was more elevated in SOD1(G93A) with respect to SOD1 mice, and that the surplus of release relies on synaptic vesicle exocytosis. Exposure to high KCl or ionomycin provoked Ca(2+)-dependent Glu release that was likewise augmented in SOD1(G93A) mice. Equally, the Ca(2+)-independent hypertonic sucrose-induced Glu release was abnormally elevated in SOD1(G93A) mice. Also in this case, the surplus of Glu release was exocytotic in nature. We could determine elevated cytosolic Ca(2+) levels, increased phosphorylation of Synapsin-I, which was causally related to the abnormal Glu release measured in spinal cord synaptosomes of pre-symptomatic SOD1(G93A) mice, and increased phosphorylation of glycogen synthase kinase-3 at the inhibitory sites, an event that favours SNARE protein assembly. Western blot experiments revealed an increased number of SNARE protein complexes at the nerve terminal membrane, with no changes of the three SNARE proteins and increased expression of synaptotagmin-1 and ß-Actin, but not of an array of other release-related presynaptic proteins. These results indicate that the abnormal exocytotic Glu release in spinal cord of pre-symptomatic SOD1(G93A) mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanisms.

Immune response in peripheral axons delays disease progression in SOD1G93A mice.

BACKGROUND: Increasing evidence suggests that the immune system has a beneficial role in the progression of amyotrophic lateral sclerosis (ALS) although the mechanism remains unclear. Recently, we demonstrated that motor neurons (MNs) of C57SOD1G93A mice with slow disease progression activate molecules classically involved in the cross-talk with the immune system. This happens a lot less in 129SvSOD1G93A mice which, while expressing the same amount of transgene, had faster disease progression and earlier axonal damage. The present study investigated whether and how the immune response is involved in the preservation of motor axons in the mouse model of familial ALS with a more benign disease course. METHODS: First, the extent of axonal damage, Schwann cell proliferation, and neuromuscular junction (NMJ) denervation were compared between the two ALS mouse models at the disease onset. Then, we compared the expression levels of different immune molecules, the morphology of myelin sheaths, and the presence of blood-derived immune cell infiltrates in the sciatic nerve of the two SOD1G93A mouse strains using immunohistochemical, immunoblot, quantitative reverse transcription PCR, and rotating-polarization Coherent Anti-Stokes Raman Scattering techniques. RESULTS: Muscle denervation, axonal dysregulation, and myelin disruption together with reduced Schwann cell proliferation are prominent in 129SvSOD1G93A compared to C57SOD1G93A mice at the disease onset, and this correlates with a faster disease progression in the first strain. On the contrary, a striking increase of immune molecules such as CCL2, MHCI, and C3 was seen in sciatic nerves of slow progressor C57SOD1G93A mice and this was accompanied by heavy infiltration of CD8+ T lymphocytes and macrophages. These phenomena were not detectable in the peripheral nervous system of fast-progressing mice. CONCLUSIONS: These data show for the first time that damaged MNs in SOD1-related ALS actively recruit immune cells in the peripheral nervous system to delay muscle denervation and prolong the lifespan. On the contrary, the lack of this response has a negative impact on the disease course.

Chronic administration of AMD3100 increases survival and alleviates pathology in SOD1(G93A) mice model of ALS.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease, involving both upper and lower motor neurons. The disease is induced by multifactorial pathologies, and as such, it requires a multifaceted therapeutic approach. CXCR4, a chemokine receptor widely expressed in neurons and glial cells and its ligand, CXCL12, also known as stromal-cell-derived factor (SDF1), modulate both neuronal function and apoptosis by glutamate release signaling as well as hematopoietic stem and progenitor cells (HSPCs) migration into the blood and their homing towards injured sites. Inhibition approaches towards the CXCR4/CXCL12 signaling may result in preventing neuronal apoptosis and alter the HSPCs migration and homing. Such inhibition can be achieved by means of treatment with AMD3100, an antagonist of the chemokine receptor CXCR4. METHODS: We chronically treated male and female transgenic mice model of ALS, SOD1(G93A) mice, with AMD3100. Mice body weight and motor function, evaluated by Rotarod test, were recorded once a week. The most effective treatment regimen was repeated for biochemical and histological analyses in female mice. RESULTS: We found that chronic administration of AMD3100 to SOD1(G93A) mice led to significant extension in mice lifespan and improved motor function and weight loss. In addition, the treatment significantly improved microglial pathology and decreased proinflammatory cytokines in spinal cords of treated female mice. Furthermore, AMD3100 treatment decreased blood-spinal cord barrier (BSCB) permeability by increasing tight junction proteins levels and increased the motor neurons count in the lamina X area of the spinal cord, where adult stem cells are formed. CONCLUSIONS: These data, relevant to the corresponding disease mechanism in human ALS, suggest that blocking CXCR4 by the small molecule, AMD3100, may provide a novel candidate for ALS therapy with an increased safety.

Exocytosis regulates trafficking of GABA and glycine heterotransporters in spinal cord glutamatergic synapses: a mechanism for the excessive heterotransporter-induced release of glutamate in experimental amyotrophic lateral sclerosis.

The impact of synaptic vesicle endo-exocytosis on the trafficking of nerve terminal heterotransporters was studied by monitoring membrane expression and function of the GABA transporter-1 (GAT-1) and of type-1/2 glycine (Gly) transporters (GlyT-1/2) at spinal cord glutamatergic synaptic boutons. Experiments were performed by inducing exocytosis in wild-type (WT) mice, in amphiphysin-I knockout (Amph-I KO) mice, which show impaired endocytosis, or in mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1(G93A)), a model of human amyotrophic lateral sclerosis showing constitutively excessive Glu exocytosis. Exposure of spinal cord synaptosomes from WT mice to a 35mM KCl pulse increased the expression of GAT-1 at glutamatergic synaptosomal membranes and enhanced the GAT-1 heterotransporter-induced [(3)H]d-aspartate ([(3)H]d-Asp) release. Similar results were obtained in the case of GlyT-1/2 heterotransporters. Preventing depolarization-induced exocytosis normalized the excessive GAT-1 and GlyT-1/2 heterotransporter-induced [(3)H]d-Asp release in WT mice. Impaired endocytosis in Amph-I KO mice increased GAT-1 membrane expression and [(3)H]GABA uptake in spinal cord synaptosomes. Also the GAT-1 heterotransporter-evoked release of [(3)H]d-Asp was augmented in Amph-I KO mice. The constitutively excessive Glu exocytosis in SOD1(G93A) mice resulted in augmented GAT-1 expression at glutamatergic synaptosomal membranes and GAT-1 or GlyT-1/2 heterotransporter-mediated [(3)H]d-Asp release. Thus, endo-exocytosis regulates the trafficking of GAT-1 and GlyT-1/2 heterotransporters sited at spinal cord glutamatergic nerve terminals. As a consequence, it can be hypothesized that the excessive GAT-1 and GlyT-1/2 heterotransporter-mediated Glu release, in the spinal cord of SOD1(G93A) mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted by the excessive Glu exocytosis.

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission.

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

Preferential potentiation of AMPA-mediated currents in brainstem hypoglossal motoneurons by subchronic exposure of mice expressing the human superoxide dismutase 1 G93A gene mutation to neurotoxicant methylmercury in vivo.

The exact causes of Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurological disorder due to loss of upper and/or lower motoneurons, remain elusive. Gene-environment interactions are believed to be an important factor in the development of ALS. We previously showed that in vivo exposure of mice overexpressing the human superoxide dismutase 1 (hSOD1) gene mutation (hSOD1G93A; G93A), a mouse model for ALS, to environmental neurotoxicant methylmercury (MeHg) accelerated the onset of ALS-like phenotype. Here we examined the time-course of effects of MeHg on AMPA receptor (AMPAR)-mediated currents in hypoglossal motoneurons in brainstem slices prepared from G93A, hSOD1wild-type (hWT) and non-carrier WT mice following in vivo exposure to MeHg. Mice were exposed daily to 3 ppm (approximately 0.7 mg/kg/day) MeHg via drinking water beginning at postnatal day 28 (P28) and continued until P47, 64 or 84, then acute brainstem slices were prepared, and spontaneous excitatory postsynaptic currents (sEPSCs) or AMPA-evoked currents were examined using whole cell patch-clamp recording technique. Brainstem slices of untreated littermates were prepared at the same time points to serve as control. MeHg exposure had no significant effect on either sEPSCs or AMPA-evoked currents in slices from hWT or WT mice during any of those exposure time periods under our experimental conditions. MeHg also did not cause any significant effect on sEPSCs or AMPA-currents in G93A hypoglossal motoneurons at P47 and P64. However, at P84, MeHg significantly increased amplitudes of both sEPSCs and AMPA-evoked currents in hypoglossal motineurons from G93A mice (p < 0.05), but not the sEPSC frequency, suggesting a postsynaptic action on AMPARs. MeHg exposure did not cause any significant effect on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs). Therefore, MeHg exposure in vivo caused differential effects on AMPARs in hypoglossal motoneurons from mice with different genetic backgrounds. MeHg appears to preferentially stimulate the AMPAR-mediated currents in G93A hypoglossal motoneurons in an exposure time-dependent manner, which may contribute to the AMPAR-mediated motoneuron excitotoxicity, thereby facilitating development of ALS-like phenotype.

Endurance exercise has a negative impact on the onset of SOD1-G93A ALS in female mice and affects the entire skeletal muscle-motor neuron axis.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

IRAK-M Plays A Role in the Pathology of Amyotrophic Lateral Sclerosis Through Suppressing the Activation of Microglia.

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-1ß 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.

Genetic ablation of phospholipase C delta 1 increases survival in SOD1(G93A) mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, resulting in selective motor neuron degeneration and paralysis. Patients die approximately 3-5 years after diagnosis. Disease pathophysiology is multifactorial, including excitotoxicity, but is not yet fully understood. Genetic analysis has proven fruitful in the past to further understand genes modulating the disease and increase knowledge of disease mechanisms. Here, we revisit a previously performed microsatellite analysis in ALS and focus on another hit, PLCD1, encoding phospholipase C delta 1 (PLCδ1), to investigate its role in ALS. PLCδ1 may contribute to excitotoxicity as it increases inositol 1,4,5-trisphosphate (IP3) formation, which releases calcium from the endoplasmic reticulum through IP3 receptors. We find that expression of PLCδ1 is increased in ALS mouse spinal cord and in neurons from ALS mice. Furthermore, genetic ablation of this protein in ALS mice significantly increases survival, but does not affect astrogliosis, microgliosis, aggregation or the amount of motor neurons at end stage compared to ALS mice with PLCδ1. Interestingly, genetic ablation of PLCδ1 prevents nuclear shrinkage of motor neurons in ALS mice at end stage. These results indicate that PLCD1 contributes to ALS and that PLCδ1 may be a new target for future studies.

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, S100ß 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-1ß, 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.