The Key Role of Astrocytes in Amyotrophic Lateral Sclerosis and Their Commitment to Glutamate Excitotoxicity.

In the last two decades, there has been increasing evidence supporting non-neuronal cells as active contributors to neurodegenerative disorders. Among glial cells, astrocytes play a pivotal role in driving amyotrophic lateral sclerosis (ALS) progression, leading the scientific community to focus on the "astrocytic signature" in ALS. Here, we summarized the main pathological mechanisms characterizing astrocyte contribution to MN damage and ALS progression, such as neuroinflammation, mitochondrial dysfunction, oxidative stress, energy metabolism impairment, miRNAs and extracellular vesicles contribution, autophagy dysfunction, protein misfolding, and altered neurotrophic factor release. Since glutamate excitotoxicity is one of the most relevant ALS features, we focused on the specific contribution of ALS astrocytes in this aspect, highlighting the known or potential molecular mechanisms by which astrocytes participate in increasing the extracellular glutamate level in ALS and, conversely, undergo the toxic effect of the excessive glutamate. In this scenario, astrocytes can behave as "producers" and "targets" of the high extracellular glutamate levels, going through changes that can affect themselves and, in turn, the neuronal and non-neuronal surrounding cells, thus actively impacting the ALS course. Moreover, this review aims to point out knowledge gaps that deserve further investigation.

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.

Micro-RNAs Shuttled by Extracellular Vesicles Secreted from Mesenchymal Stem Cells Dampen Astrocyte Pathological Activation and Support Neuroprotection in In-Vitro Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes' neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes' neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes' activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes' reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.

mTOR Inhibition via Rapamycin Treatment Partially Reverts the Deficit in Energy Metabolism Caused by FH Loss in RPE Cells.

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including aging, genetics, and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation, and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), which leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. Although the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress, and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of which are associated with UPS. As inhibition of mTORC1 was previously shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

Reassessing neurodegenerative disease: immune protection pathways and antagonistic pleiotropy.

The antagonistic pleiotropy (AP) theory posits that adaptive evolutionary changes, which facilitate reproduction and individual fitness early in life, can enhance detrimental aging-related processes. Several genes associated with human brain diseases play a protective role in infection, suggesting the relevance of AP in the context of brain aging and neurodegeneration. Relatedly, genetic variants that confer immune protection against pathogens may lead to uncontrolled brain inflammation later in life. Here, we propose a conceptual framework suggesting that the pleiotropic roles of genes in infections and host-pathogen interactions should be considered when studying neurological illnesses. We reinterpret recent findings regarding the impact of neurological disease-associated genetic traits on infections and chronic inflammatory diseases. Identifying the AP pathways shared among these seemingly unrelated conditions might provide further insights into the detrimental role of the immune system in brain disease as well as the mechanisms involved in chronic infections.

Blocking glutamate mGlu5 receptors with the negative allosteric modulator CTEP improves disease course in SOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: The pathogenesis of amyotrophic lateral sclerosis (ALS) is not fully clarified, although excessive glutamate (Glu) transmission and the downstream cytotoxic cascades are major mechanisms for motor neuron death. Two metabotropic glutamate receptors (mGlu1 and mGlu5 ) are overexpressed in ALS and regulate cellular disease processes. Expression and function of mGlu5 receptors are altered at early symptomatic stages in the SOD1G93A mouse model of ALS and knockdown of mGlu5 receptors in SOD1G93A mice improved disease progression. EXPERIMENTAL APPROACH: We treated male and female SOD1G93A mice with 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), an orally available mGlu5 receptor negative allosteric modulator (NAM), using doses of 2 mg·kg-1 per 48 h or 4 mg·kg-1 per 24 h from Day 90, an early symptomatic disease stage. Disease progression was studied by behavioural and histological approaches. KEY RESULTS: CTEP dose-dependently ameliorated clinical features in SOD1G93A mice. The lower dose increased survival and improved motor skills in female mice, with barely positive effects in male mice. Higher doses significantly ameliorated disease symptoms and survival in both males and females, females being more responsive. CTEP also reduced motor neuron death, astrocyte and microglia activation, and abnormal glutamate release in the spinal cord, with equal effects in male and female mice. No differences were also observed in CTEP access to the brain. CONCLUSION AND IMPLICATIONS: Our results suggest that mGlu5 receptors are promising targets for the treatment of ALS and highlight mGlu5 receptor NAMs as effective pharmacological tools with translational potential.

Redefining Microglial Identity in Health and Disease at Single-Cell Resolution.

Microglia have long been considered a homogenous cell population that uniformly responds to extrinsic factors. Here, we describe how the recent development of single-cell technologies has revealed the heterogeneity of both human and mouse microglia and identified distinct microglial states linked to specific developmental, aging, and disease stages. We discuss progress and future developments in data analysis, essential tools for the comprehension of big data derived from single-cell omics, and the necessity of integrating such data with functional studies to correlate genetic cues with the relevant biological functions of microglia. Defining the functional correlates of distinct microglia states is fundamental to dissecting the 'microglial etiology' of aging and complex neurological diseases and identifying novel therapeutic and diagnostic targets.

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 glucose catabolism in the presynaptic and perisynaptic compartments of SOD1G93A mouse spinal cord and motor cortex indicates that mitochondria are the site of bioenergetic imbalance in ALS.

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops because of motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, whereas perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analyzed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment.

In-vivo genetic ablation of metabotropic glutamate receptor type 5 slows down disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease due to motor neuron (MN) loss. The mechanisms causing selective MN death are largely unknown, thus prejudicing successful pharmacological treatments. Major causes of MN damage are effects downstream of the abnormal glutamate (Glu) neurotransmission. Group I metabotropic Glu receptors (mGluR1, mGluR5) actively contribute to the excitotoxicity in ALS and represent druggable molecular targets. We previously demonstrated that halving mGluR1 or mGluR5 expression in the widely studied SOD1G93A mouse model of ALS had a positive impact on disease onset, clinical progression and survival, as well as on cellular and biochemical parameters altered in ALS. Whereas these effects were similar in female and male mGluR1 heterozygous SOD1G93Amice, only male mGluR5 heterozygous SOD1G93A mice showed improved motor skills during disease progression. To further validate the role of Group I mGluRs in ALS, we generated in this study mGluR1 or mGluR5 null mice expressing the SOD1G93A mutation (SOD1G93AGrm1crv4/crv4 or SOD1G93AGrm5-/-, respectively). SOD1G93AGrm1crv4/crv4 mice showed early and progressive motor impairments and died even before SOD1G93A mice, while SOD1G93AGrm5-/- mice exhibited delayed disease onset, longer survival, and ameliorated motor skills than SOD1G93A mice. No difference between female and male SOD1G93AGrm5-/- mice were observed. These effects were associated with enhanced MN preservation and decreased astrocytic and microglial activation. Our results strongly support the assumption that constitutively lowering of mGluR5 expression has a positive impact in mice with ALS by counteracting the abnormal Glu transmission and this could be a potentially effective pharmacological target in ALS.

Exenatide once-weekly improves metabolic parameters, endothelial dysfunction and carotid intima-media thickness in patients with type-2 diabetes: An 8-month prospective study.

AIM: To evaluate the effect of exenatide long acting release (LAR) on carotid intima-media thickness (IMT) and endothelial function in patients with type 2 diabetes mellitus. METHODS: Sixty subjects with type 2 diabetes mellitus were treated with exenatide LAR as add-on to stable doses of metformin for 8 months in an open label study. Anthropometric variables, lipid profile and glycemic parameters were assessed by routine analysis. Carotid IMT by Doppler ultrasound and endothelial function by flow-mediated dilation of the brachial artery were also assessed. RESULTS: Exenatide significantly improved fasting glycaemia (from 8.8 ±â€¯2.8 to 7.3 ±â€¯2.2 mmol/L, p < 0.0001), HbA1c (from 8.0 ±â€¯0.4 to 6.9 ±â€¯1.1%, p < 0.0001), body mass index (from 33 ±â€¯9 to 31 ±â€¯6 kg/m2, p = 0.0348) and waist circumference (from 109 ±â€¯13 to 106 ±â€¯13 cm, p = 0.0105). There was a significant improvement of the lipid profile, except in triglyceride level where no changes were observed. Carotid IMT and flow-mediated dilation were also improved (from 0.98 ±â€¯0.14 to 0.87 ±â€¯0.15 mm and from 5.8 ±â€¯1.3 to 6.8 ±â€¯1.7%, respectively; p < 0.0001 for both). CONCLUSIONS: Treatment with exenatide LAR led to improved cardio-metabolic parameters, including carotid IMT and flow-mediated dilation, independently of glucometabolic control. These results may help to explain, at least in part, the cardiovascular safety of exenatide LAR, as recently reported in cardiovascular outcome trials.

Efficacy and Renal Safety of Dapagliflozin in Patients with Type 2 Diabetes Mellitus Also Receiving Metformin: A Real-Life Experience.

INTRODUCTION: This study aimed at evaluating the efficacy and safety of dapagliflozin in patients with type 2 diabetes (T2D) who also received metformin in clinical practice in Italy. METHODS: This was a retrospective observational study and it included data from patients who received dapagliflozin 10 mg once daily in conjunction with metformin for 12 months (DAPA + MET). In those with inadequate glycemic control, insulin or glimepiride was added after 30 days (DAPA + MET + other glucose-lowering drugs). Efficacy assessments included glycosylated hemoglobin (HbA1c) levels at 6 and 12 months, as well as body mass index (BMI) and lipid parameters at 12 months. Safety was also assessed. RESULTS: Data on 66 patients were included. In both groups, HbA1c was significantly reduced at 6 and 12 months compared with baseline and significant reductions in HbA1c were observed at 12 months compared with 6 months. Over the 12-month treatment period, dapagliflozin significantly reduced BMI in both groups. No significant changes in lipid parameters were observed in either group and no detrimental effects on renal function were detected. CONCLUSIONS: Dapagliflozin is effective and safe in patients with T2D also receiving metformin. Glycemic control was already achieved with dapagliflozin + metformin, and add-on therapy was not associated with further improvements.

Characterization of the Mitochondrial Aerobic Metabolism in the Pre- and Perisynaptic Districts of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disease characterized by muscle wasting, weakness, and spasticity due to a progressive degeneration of cortical, brainstem, and spinal motor neurons. The etiopathological causes are still largely obscure, although astrocytes definitely play a role in neuronal damage. Several mechanisms have been proposed to concur to neurodegeneration in ALS, including mitochondrial dysfunction. We have previously shown profound modifications of glutamate release and presynaptic plasticity in the spinal cord of the SOD1G93A mouse model of ALS. In this work, we characterized, for the first time, the aerobic metabolism in two specific compartments actively involved in neurotransmission (i.e. the presynaptic district, using purified synaptosomes, and the perisynaptic astrocyte processes, using purified gliosomes) in SOD1G93A mice at different stages of the disease. ATP/AMP ratio was lower in synaptosomes isolated from the spinal cord, but not from other brain areas, of SOD1G93A vs. control mice. The energy impairment was linked to altered oxidative phosphorylation (OxPhos) and increment of lipid peroxidation. These metabolic dysfunctions were present during disease progression, starting at the very pre-symptomatic stages, and did not depend on a different number of mitochondria or a different expression of OxPhos proteins. Conversely, gliosomes showed a reduction of the ATP/AMP ratio only at the late stages of the disease and an increment of oxidative stress also in the absence of a significant decrement in OxPhos activity. Data suggest that the presynaptic neuronal moiety plays a pivotal role for synaptic energy metabolism dysfunctions in ALS. Changes in the perisynaptic compartment seem subordinated to neuronal damage.

In-vivo effects of knocking-down metabotropic glutamate receptor 5 in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1G93A mice with reduced expression of mGluR5 (SOD1G93AGrm5-/+) by crossing the SOD1G93A mutant mouse with the mGluR5 heterozigous Grm5-/+ mouse. SOD1G93AGrm5-/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1G93AGrm5-/+ mice. Unexpectedly, only male SOD1G93AGrm5-/+ mice showed improved motor skills during disease progression vs. SOD1G93A mice, while SOD1G93AGrm5-/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease.

The effectiveness and durability of an early insulin pump therapy in children and adolescents with type 1 diabetes mellitus.

BACKGROUND: This study evaluated the predictors of effectiveness and durability of insulin pump therapy in children and adolescents who have initiated continuous subcutaneous insulin infusion (CSII) within 2 years after the diagnosis of type 1 diabetes mellitus (T1DM). SUBJECTS AND METHODS: The charts of individuals with T1DM using insulin pumps who were treated at our center were reviewed, including subjects with age at onset of <22 years, interval between onset and insulin pump commencement (interval onset-commencement) of <2 years, use of pumps of >1 year, and use of glucose sensors for <4 weeks/year. The primary end point was the mean glycosylated hemoglobin (HbA1c) value (MHbA1c) throughout the follow-up. RESULTS: From 684 patients treated with insulin pumps, 119 met the inclusion criteria, and 113 were selected for statistical analysis (60 females; age at diabetes onset, 8.9±5.6 years [mean±SD]; follow-up, 4.0±1.8 years; range, 1-8 years; baseline HbA1c, 9.3±1.8%). Only the interval onset-commencement was a linear predictor of the MHbA1c (P=0.01; R(2)=0.089). A significant reduction of the mean yearly HbA1c from baseline throughout all the follow-up was observed (P<0.001). Categorizing the sample into four quartiles on the basis of an increasing interval onset-commencement resulted in levels of MHbA1c significantly lower in the first and second quartiles in comparison with the fourth quartile (7.6±0.8% and 7.8±1.0%, respectively, versus 8.5±0.8%; P<0.001 and P=0.004, respectively). CONCLUSIONS: The present study suggests that early pump commencement in children and adolescents with T1DM provides lower and more durable HbA1c values than a late commencement. It is possible that an early pump commencement could prolong the honeymoon phase, but we cannot confirm or exclude this hypothesis because the lack of data about C-peptide levels during the follow-up.