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.

Correction: Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

[This corrects the article DOI: 10.1371/journal.pone.0170825.].

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.

Functional and Molecular Changes in the Prefrontal Cortex of the Chronic Mild Stress Rat Model of Depression and Modulation by Acute Ketamine.

Stress is a primary risk factor in the onset of neuropsychiatric disorders, including major depressive disorder (MDD). We have previously used the chronic mild stress (CMS) model of depression in male rats to show that CMS induces morphological, functional, and molecular changes in the hippocampus of vulnerable animals, the majority of which were recovered using acute subanesthetic ketamine in just 24 h. Here, we focused our attention on the medial prefrontal cortex (mPFC), a brain area regulating emotional and cognitive functions, and asked whether vulnerability/resilience to CMS and ketamine antidepressant effects were associated with molecular and functional changes in the mPFC of rats. We found that most alterations induced by CMS in the mPFC were selectively observed in stress-vulnerable animals and were rescued by acute subanesthetic ketamine, while others were found only in resilient animals or were induced by ketamine treatment. Importantly, only a few of these modifications were also previously demonstrated in the hippocampus, while most are specific to mPFC. Overall, our results suggest that acute antidepressant ketamine rescues brain-area-specific glutamatergic changes induced by chronic stress.

Dopamine-Dependent Ketamine Modulation of Glutamatergic Synaptic Plasticity in the Prelimbic Cortex of Adult Rats Exposed to Acute Stress.

Traumatic stress is the main environmental risk factor for the development of psychiatric disorders. We have previously shown that acute footshock (FS) stress in male rats induces rapid and long-lasting functional and structural changes in the prefrontal cortex (PFC), which are partly reversed by acute subanesthetic ketamine. Here, we asked if acute FS may also induce any changes in glutamatergic synaptic plasticity in the PFC 24 h after stress exposure and whether ketamine administration 6 h after stress may have any effect. We found that the induction of long-term potentiation (LTP) in PFC slices of both control and FS animals is dependent on dopamine and that dopamine-dependent LTP is reduced by ketamine. We also found selective changes in ionotropic glutamate receptor subunit expression, phosphorylation, and localization at synaptic membranes induced by both acute stress and ketamine. Although more studies are needed to understand the effects of acute stress and ketamine on PFC glutamatergic plasticity, this first report suggests a restoring effect of acute ketamine, supporting the potential benefit of ketamine in limiting the impact of acute traumatic stress.

The Physio-Pathological Role of Group I Metabotropic Glutamate Receptors Expressed by Microglia in Health and Disease with a Focus on Amyotrophic Lateral Sclerosis.

Microglia cells are the resident immune cells of the central nervous system. They act as the first-line immune guardians of nervous tissue and central drivers of neuroinflammation. Any homeostatic alteration that can compromise neuron and tissue integrity could activate microglia. Once activated, microglia exhibit highly diverse phenotypes and functions related to either beneficial or harmful consequences. Microglia activation is associated with the release of protective or deleterious cytokines, chemokines, and growth factors that can in turn determine defensive or pathological outcomes. This scenario is complicated by the pathology-related specific phenotypes that microglia can assume, thus leading to the so-called disease-associated microglia phenotypes. Microglia express several receptors that regulate the balance between pro- and anti-inflammatory features, sometimes exerting opposite actions on microglial functions according to specific conditions. In this context, group I metabotropic glutamate receptors (mGluRs) are molecular structures that may contribute to the modulation of the reactive phenotype of microglia cells, and this is worthy of exploration. Here, we summarize the role of group I mGluRs in shaping microglia cells' phenotype in specific physio-pathological conditions, including some neurodegenerative disorders. A significant section of the review is specifically focused on amyotrophic lateral sclerosis (ALS) since it represents an entirely unexplored topic of research in the field.

Changes at glutamate tripartite synapses in the prefrontal cortex of a new animal model of resilience/vulnerability to acute stress.

Stress represents a main risk factor for psychiatric disorders. Whereas it is known that even a single trauma may induce psychiatric disorders in humans, the mechanisms of vulnerability to acute stressors have been little investigated. In this study, we generated a new animal model of resilience/vulnerability to acute footshock (FS) stress in rats and analyzed early functional, molecular, and morphological determinants of stress vulnerability at tripartite glutamate synapses in the prefrontal cortex (PFC). We found that adult male rats subjected to FS can be deemed resilient (FS-R) or vulnerable (FS-V), based on their anhedonic phenotype 24 h after stress exposure, and that these two populations are phenotypically distinguishable up to two weeks afterwards. Basal presynaptic glutamate release was increased in the PFC of FS-V rats, while depolarization-evoked glutamate release and synapsin I phosphorylation at Ser9 were increased in both FS-R and FS-V. In FS-R and FS-V rats the synaptic expression of GluN2A and apical dendritic length of prelimbic PFC layers II-III pyramidal neurons were decreased, while BDNF expression was selectively reduced in FS-V. Depolarization-evoked (carrier-mediated) glutamate release from astroglia perisynaptic processes (gliosomes) was selectively increased in the PFC of FS-V rats, while GLT1 and xCt levels were higher and GS expression reduced in purified PFC gliosomes from FS-R. Overall, we show for the first time that the application of the sucrose intake test to rats exposed to acute FS led to the generation of a novel animal model of resilience/vulnerability to acute stress, which we used to identify early determinants of maladaptive response related to behavioral vulnerability to stress.

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.

Pharmacological Profile of MP-101, a Novel Non-racemic Mixture of R- and S-dimiracetam with Increased Potency in Rat Models of Cognition, Depression and Neuropathic Pain.

The racemic mixture dimiracetam negatively modulates NMDA-induced glutamate release in rat spinal cord synaptosomal preparations and is orally effective in models of neuropathic pain. In this study, we compared the effects of dimiracetam, its R- or S-enantiomers, and the R:S 3:1 non-racemic mixture (MP-101). In vitro, dimiracetam was more potent than its R- or S-enantiomers in reducing the NMDA-induced [3H]D-aspartate release in rat spinal cord synaptosomes. Similarly, acute oral administration of dimiracetam was more effective than a single enantiomer in the sodium monoiodoacetate (MIA) paradigm of painful osteoarthritis. Then, we compared the in vitro effects of a broad range of non-racemic enantiomeric mixtures on the NMDA-induced [3H]D-aspartate release. Dimiracetam was a more potent blocker than each isolated enantiomer but the R:S 3:1 non-racemic mixture (MP-101) was even more potent than dimiracetam, with an IC50 in the picomolar range. In the chronic oxaliplatin-induced neuropathic pain model, MP-101 showed a significantly improved anti-neuropathic profile, and its effect continued one week after treatment suspension. MP-101 also performed better than dimiracetam in animal models of cognition and depression. Based on the benign safety and tolerability profile previously observed with racemic dimiracetam, MP-101 appears to be a novel, promising clinical candidate for the prevention and treatment of several neuropathic and neurological disorders.

MicroRNA Alteration, Application as Biomarkers, and Therapeutic Approaches in Neurodegenerative Diseases.

MicroRNAs (miRNAs) are essential post-transcriptional gene regulators involved in various neuronal and non-neuronal cell functions and play a key role in pathological conditions. Numerous studies have demonstrated that miRNAs are dysregulated in major neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington's disease. Hence, in the present work, we constructed a comprehensive overview of individual microRNA alterations in various models of the above neurodegenerative diseases. We also provided evidence of miRNAs as promising biomarkers for prognostic and diagnostic approaches. In addition, we summarized data from the literature about miRNA-based therapeutic applications via inhibiting or promoting miRNA expression. We finally identified the overlapping miRNA signature across the diseases, including miR-128, miR-140-5p, miR-206, miR-326, and miR-155, associated with multiple etiological cellular mechanisms. However, it remains to be established whether and to what extent miRNA-based therapies could be safely exploited in the future as effective symptomatic or disease-modifying approaches in the different human neurodegenerative disorders.

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease.

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knock-in mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular re-localization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

Acute Ketamine Facilitates Fear Memory Extinction in a Rat Model of PTSD Along With Restoring Glutamatergic Alterations and Dendritic Atrophy in the Prefrontal Cortex.

Stress represents a major risk factor for psychiatric disorders, including post-traumatic stress disorder (PTSD). Recently, we dissected the destabilizing effects of acute stress on the excitatory glutamate system in the prefrontal cortex (PFC). Here, we assessed the effects of single subanesthetic administration of ketamine (10 mg/kg) on glutamate transmission and dendritic arborization in the PFC of footshock (FS)-stressed rats, along with changes in depressive, anxious, and fear extinction behaviors. We found that ketamine, while inducing a mild increase of glutamate release in the PFC of naïve rats, blocked the acute stress-induced enhancement of glutamate release when administered 24 or 72 h before or 6 h after FS. Accordingly, the treatment with ketamine 6 h after FS also reduced the stress-dependent increase of spontaneous excitatory postsynaptic current (sEPSC) amplitude in prelimbic (PL)-PFC. At the same time, ketamine injection 6 h after FS was found to rescue apical dendritic retraction of pyramidal neurons induced by acute stress in PL-PFC and facilitated contextual fear extinction. These results show rapid effects of ketamine in animals subjected to acute FS, in line with previous studies suggesting a therapeutic action of the drug in PTSD models. Our data are consistent with a mechanism of ketamine involving re-establishment of synaptic homeostasis, through restoration of glutamate release, and structural remodeling of dendrites.

Insights into Human-Induced Pluripotent Stem Cell-Derived Astrocytes in Neurodegenerative Disorders.

Most neurodegenerative disorders have complex and still unresolved pathology characterized by progressive neuronal damage and death. Astrocytes, the most-abundant non-neuronal cell population in the central nervous system, play a vital role in these processes. They are involved in various functions in the brain, such as the regulation of synapse formation, neuroinflammation, and lactate and glutamate levels. The development of human-induced pluripotent stem cells (iPSCs) reformed the research in neurodegenerative disorders allowing for the generation of disease-relevant neuronal and non-neuronal cell types that can help in disease modeling, drug screening, and, possibly, cell transplantation strategies. In the last 14 years, the differentiation of human iPSCs into astrocytes allowed for the opportunity to explore the contribution of astrocytes to neurodegenerative diseases. This review discusses the development protocols and applications of human iPSC-derived astrocytes in the most common neurodegenerative conditions.

Nearly 30 Years of Animal Models to Study Amyotrophic Lateral Sclerosis: A Historical Overview and Future Perspectives.

Amyotrophic lateral sclerosis (ALS) is a fatal, multigenic, multifactorial, and non-cell autonomous neurodegenerative disease characterized by upper and lower motor neuron loss. Several genetic mutations lead to ALS development and many emerging gene mutations have been discovered in recent years. Over the decades since 1990, several animal models have been generated to study ALS pathology including both vertebrates and invertebrates such as yeast, worms, flies, zebrafish, mice, rats, guinea pigs, dogs, and non-human primates. Although these models show different peculiarities, they are all useful and complementary to dissect the pathological mechanisms at the basis of motor neuron degeneration and ALS progression, thus contributing to the development of new promising therapeutics. In this review, we describe the up to date and available ALS genetic animal models, classified by the different genetic mutations and divided per species, pointing out their features in modeling, the onset and progression of the pathology, as well as their specific pathological hallmarks. Moreover, we highlight similarities, differences, advantages, and limitations, aimed at helping the researcher to select the most appropriate experimental animal model, when designing a preclinical ALS study.

The Role of Endoplasmic Reticulum in the Differential Endurance against Redox Stress in Cortical and Spinal Astrocytes from the Newborn SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Recent studies reported that the uptake of [18F]-fluorodeoxyglucose (FDG) is increased in the spinal cord (SC) and decreased in the motor cortex (MC) of patients with ALS, suggesting that the disease might differently affect the two nervous districts with different time sequence or with different mechanisms. Here we show that MC and SC astrocytes harvested from newborn B6SJL-Tg (SOD1G93A) 1Gur mice could play different roles in the pathogenesis of the disease. Spectrophotometric and cytofluorimetric analyses showed an increase in redox stress, a decrease in antioxidant capacity and a relative mitochondria respiratory uncoupling in MC SOD1G93A astrocytes. By contrast, SC mutated cells showed a higher endurance against oxidative damage, through the increase in antioxidant defense, and a preserved respiratory function. FDG uptake reproduced the metabolic response observed in ALS patients: SOD1G93A mutation caused a selective enhancement in tracer retention only in mutated SC astrocytes, matching the activity of the reticular pentose phosphate pathway and, thus, of hexose-6P dehydrogenase. Finally, both MC and SC mutated astrocytes were characterized by an impressive ultrastructural enlargement of the endoplasmic reticulum (ER) and impairment in ER-mitochondria networking, more evident in mutated MC than in SC cells. Thus, SOD1G93A mutation differently impaired MC and SC astrocyte biology in a very early stage of life.

Visual Cortex Engagement in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a family of inherited disorders caused by the progressive degeneration of retinal photoreceptors. There is no cure for RP, but recent research advances have provided promising results from many clinical trials. All these therapeutic strategies are focused on preserving existing photoreceptors or substituting light-responsive elements. Vision recovery, however, strongly relies on the anatomical and functional integrity of the visual system beyond photoreceptors. Although the retinal structure and optic pathway are substantially preserved at least in early stages of RP, studies describing the visual cortex status are missing. Using a well-established mouse model of RP, we analyzed the response of visual cortical circuits to the progressive degeneration of photoreceptors. We demonstrated that the visual cortex goes through a transient and previously undescribed alteration in the local excitation/inhibition balance, with a net shift towards increased intracortical inhibition leading to improved filtering and decoding of corrupted visual inputs. These results suggest a compensatory action of the visual cortex that increases the range of residual visual sensitivity in RP.

Axonal GABAA stabilizes excitability in unmyelinated sensory axons secondary to NKCC1 activity.

KEY POINTS: GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABAA receptor. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to Na-K-Cl cotransporter type 1 (NKCC1) loading of axonal chloride. Activation of axonal GABAA receptor stabilized C-fibre excitability during prolonged low frequency (2.5 Hz) firing. NKCC1 maintains intra-axonal chloride to provide feed-forward stabilization of C-fibre excitability and thus support sustained firing. ABSTRACT: GABAA receptor (GABAA R)-mediated depolarization of dorsal root ganglia (DRG) axonal projections in the spinal dorsal horn is implicated in pre-synaptic inhibition. Inhibition, in this case, is predicated on an elevated intra-axonal chloride concentration and a depolarizing GABA response. In the present study, we report that the peripheral axons of DRG neurons are also depolarized by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABAA R agonists increased axonal excitability, whereas GABA excitability responses were blocked by GABAA R antagonists and were absent in mice lacking the GABAA R ß3 subunit selectively in DRG neurons (AdvillinCre or snsCre ). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5-2.5 Hz). However, during Na-K-Cl cotransporter type 1 (NKCC1) blockade, the electrical stimulation rate did not affect GABA response size, suggesting that NKCC1 regulation of axonal chloride is coupled to action potential firing. To examine this, activity-dependent conduction velocity slowing (activity-dependent slowing; ADS) was used to quantify C-fibre excitability loss during a 2.5 Hz challenge. ADS was reduced by GABAA R agonists and exacerbated by either GABAA R antagonists, ß3 deletion or NKCC1 blockade. This illustrates that activation of GABAA R stabilizes C-fibre excitability during sustained firing. We posit that NKCC1 acts in a feed-forward manner to maintain an elevated intra-axonal chloride in C-fibres during ongoing firing. The resulting chloride gradient can be utilized by GABAA R to stabilize axonal excitability. The data imply that therapeutic strategies targeting axonal chloride regulation at peripheral loci of pain and itch may curtail aberrant firing in C-fibres.

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.

Role of miRNAs shuttled by mesenchymal stem cell-derived small extracellular vesicles in modulating neuroinflammation.

Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for inflammatory/neurodegenerative diseases, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is release of extracellular vesicles that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We identified nine miRNAs significantly dysregulated in IFN-γ-primed MSCs, but present at different levels in their derived small extracellular vesicles (s-EV). We show that miR-467f and miR-466q modulate the pro-inflammatory phenotype of activated N9 microglia cells and of primary microglia acutely isolated from late symptomatic SOD1G93A mice, a murine ALS model, by downregulating Tnf and Il1b expression. Further analysis of the mode of action of miR-467f and miR-466q indicated that they dampen the pro-inflammatory phenotype of microglia by modulating p38 MAPK signaling pathway via inhibition of expression of their target genes, Map3k8 and Mk2. Finally, we demonstrated that in vivo administration of s-EV leads to decreased expression of neuroinflammation markers in the spinal cord of EAE-affected mice, albeit without affecting disease course. Overall, our data suggest that MSC-derived exosomes could affect neuroinflammation possibly through specific immunomodulatory miRNAs acting on microglia.

A multistationary loop model of ALS unveils critical molecular interactions involving mitochondria and glucose metabolism.

Amyotrophic lateral sclerosis (ALS) is a poor-prognosis disease with puzzling pathogenesis and inconclusive treatments. We develop a mathematical model of ALS based on a system of interactive feedback loops, focusing on the mutant SOD1G93A mouse. Misfolded mutant SOD1 aggregates in motor neuron (MN) mitochondria and triggers a first loop characterized by oxidative phosphorylation impairment, AMP kinase over-activation, 6-phosphofructo-2-kinase (PFK3) rise, glucose metabolism shift from pentose phosphate pathway (PPP) to glycolysis, cell redox unbalance, and further worsening of mitochondrial dysfunction. Oxidative stress then triggers a second loop, involving the excitotoxic glutamatergic cascade, with cytosolic Ca2+ overload, increase of PFK3 expression, and further metabolic shift from PPP to glycolysis. Finally, cytosolic Ca2+ rise is also detrimental to mitochondria and oxidative phosphorylation, thus closing a third loop. These three loops are overlapped and positive (including an even number of inhibitory steps), hence they form a candidate multistationary (bistable) system. To describe the system dynamics, we model the interactions among the functional agents with differential equations. The system turns out to admit two stable equilibria: the healthy state, with high oxidative phosphorylation and preferential PPP, and the pathological state, with AMP kinase activation, PFK3 over expression, oxidative stress, excitotoxicity and MN degeneration. We demonstrate that the loop system is monotone: all functional agents consistently act toward the healthy or pathological condition, depending on low or high mutant SOD1 input. We also highlight that molecular interactions involving PFK3 are crucial, as their deletion disrupts the system's bistability leading to a single healthy equilibrium point. Hence, our mathematical model unveils that promising ALS management strategies should be targeted to mechanisms that keep low PFK3 expression and activity within MNs.