Trimetazidine Improves Mitochondrial Dysfunction in SOD1G93A Cellular Models of Amyotrophic Lateral Sclerosis through Autophagy Activation.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.

TDP-43 Epigenetic Facets and Their Neurodegenerative Implications.

Since its initial involvement in numerous neurodegenerative pathologies in 2006, either as a principal actor or as a cofactor, new pathologies implicating transactive response (TAR) DNA-binding protein 43 (TDP-43) are regularly emerging also beyond the neuronal system. This reflects the fact that TDP-43 functions are particularly complex and broad in a great variety of human cells. In neurodegenerative diseases, this protein is often pathologically delocalized to the cytoplasm, where it irreversibly aggregates and is subjected to various post-translational modifications such as phosphorylation, polyubiquitination, and cleavage. Until a few years ago, the research emphasis has been focused particularly on the impacts of this aggregation and/or on its widely described role in complex RNA splicing, whether related to loss- or gain-of-function mechanisms. Interestingly, recent studies have strengthened the knowledge of TDP-43 activity at the chromatin level and its implication in the regulation of DNA transcription and stability. These discoveries have highlighted new features regarding its own transcriptional regulation and suggested additional mechanistic and disease models for the effects of TPD-43. In this review, we aim to give a comprehensive view of the potential epigenetic (de)regulations driven by (and driving) this multitask DNA/RNA-binding protein.

The Ying and Yang of Hydrogen Sulfide as a Paracrine/Autocrine Agent in Neurodegeneration: Focus on Amyotrophic Lateral Sclerosis.

Ever since its presence was reported in the brain, the nature and role of hydrogen sulfide (H2S) in the Central Nervous System (CNS) have changed. Consequently, H2S has been elected as the third gas transmitter, along with carbon monoxide and nitric oxide, and a number of studies have focused on its neuromodulatory and protectant functions in physiological conditions. The research on H2S has highlighted its many facets in the periphery and in the CNS, and its role as a double-faced compound, switching from protective to toxic depending on its concentration. In this review, we will focus on the bell-shaped nature of H2S as an angiogenic factor and as a molecule released by glial cells (mainly astrocytes) and non-neuronal cells acting on the surrounding environment (paracrine) or on the releasing cells themselves (autocrine). Finally, we will discuss its role in Amyotrophic Lateral Sclerosis, a paradigm of a neurodegenerative disease.

NOS1AP is a novel molecular target and critical factor in TDP-43 pathology.

Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.

NeuriTES. Monitoring neurite changes through transfer entropy and semantic segmentation in bright-field time-lapse microscopy.

One of the most challenging frontiers in biological systems understanding is fluorescent label-free imaging. We present here the NeuriTES platform that revisits the standard paradigms of video analysis to detect unlabeled objects and adapt to the dynamic evolution of the phenomenon under observation. Object segmentation is reformulated using robust algorithms to assure regular cell detection and transfer entropy measures are used to study the inter-relationship among the parameters related to the evolving system. We applied the NeuriTES platform to the automatic analysis of neurites degeneration in presence of amyotrophic lateral sclerosis (ALS) and to the study of the effects of a chemotherapy drug on living prostate cancer cells (PC3) cultures. Control cells have been considered in both the two cases study. Accuracy values of 93% and of 92% are achieved, respectively. NeuriTES not only represents a tool for investigation in fluorescent label-free images but demonstrates to be adaptable to individual needs.

Corrigendum: Very Early Involvement of Innate Immunity in Peripheral Nerve Degeneration in SOD1-G93A Mice.

[This corrects the article DOI: 10.3389/fimmu.2020.575792.].

Investigating Different Forms of Hydrogen Sulfide in Cerebrospinal Fluid of Various Neurological Disorders.

Over the past 30 years a considerable amount of data has accumulated on the multifaceted role of hydrogen sulfide (H2S) in the central nervous system. Depending on its concentrations, H2S has opposite actions, ranging from neuromodulator to neurotoxic. Nowadays, accurate determination of H2S is still an important challenge to understand its biochemistry and functions. In this perspective, this study aims to explore H2S levels in cerebrospinal fluid (CSF), key biofluid for neurological studies, and to assess alleged correlations with neuroinflammatory and neurodegenerative mechanisms. A validated analytical determination combining selective electrochemical detection with ion chromatography was developed to measure free and bound sulfur forms of H2S. A first cohort of CSF samples (n = 134) was analyzed from patients with inflammatory and demyelinating disorders (acute disseminated encephalomyelitis; multiple sclerosis), chronic neurodegenerative diseases (Alzheimer disease; Parkinson disease), and motor neuron disease (Amyotrophic lateral sclerosis). Given its analytical features, the chromatographic method resulted sensitive, reproducible and robust. We also explored low molecular weight-proteome linked to sulphydration by proteomics analysis on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). This study is a first clinical report on CSF H2S concentrations from neurological diseases and opens up new perspectives on the potential clinical relevance of H2S and its potential therapeutic application.

Cerebrospinal fluid from frontotemporal dementia patients is toxic to neurons.

Frontotemporal Lobar Degeneration (FTD) is a neurodegenerative disease characterized by a progressive deterioration of cognitive functions. Currently, no effective treatment exists. We have studied cytotoxicity and neuronal functionality in cortical and spinal cord cultures upon exposure to cerebrospinal fluid (CSF) from 39 FTD patients. FTD-CSF alters the miniature excitatory postsynaptic currents in the cortical cultures and it is toxic to spinal cord cultures, particularly to GABAergic+ and calbindin-D28k + neurons.

Very Early Involvement of Innate Immunity in Peripheral Nerve Degeneration in SOD1-G93A Mice.

Recent preclinical and clinical evidence suggest that immune system has a role in the progression and prognosis of Amyotrophic Lateral Sclerosis (ALS), but the identification of a clear mechanism and immune players remains to be elucidated. Here, we have investigated, in 30 and 60 days (presymptomatic) and 120 days (symptomatic) old SOD1-G93A mice, systemic, peripheral, and central innate and adaptive immune and inflammatory response, correlating it with the progression of the neurodegeneration in neuromuscular junction, sciatic nerves, and spinal cord. Surprisingly, we found a very initial (45-60 days) presence of IgG in sciatic nerves together with a gradual enhancement of A20/TNFAIP3 (protein controlling NF-κB signalling) and a concomitantly significant increase and activation of circulating mast cells (MCs) as well as MCs and macrophages in sciatic nerve and an enhancement of IL-6 and IL-10. This immunological frame coincided with a myelin aggregation. The 30-60 days old SOD1-G93A mice didn't show real elements of neuroinflammation and neurodegeneration in spinal cord. In 120 days old mice macrophages and monocytes are widely diffused in sciatic nerves, peripheral neurodegeneration reaches the tip, high circulating levels of TNFα and IL-2 were found and spinal cord exhibits clear signs of neural damage and infiltrating immune cells. Our results underpin a clear immunological disorder at the origin of ALS axonopathy, in which MCs are involved in the initiation and sustaining of inflammatory events. These data cannot be considered a mere epiphenomenon of motor neuron degeneration and reveal new potential selective immune targets in ALS therapy.

Crosstalk Between Oxidative Stress and Mitochondrial Damage: Focus on Amyotrophic Lateral Sclerosis.

Proteins oxidation by reactive species is implicated in the aetiology or progression of a panoply of disorders and diseases such as neurodegenerative disorders. It is becoming increasingly evident that redox imbalance in the brain mediates neurodegeneration. Free radicals, as reactive species of oxygen (ROS) but also reactive nitrogen species (RNS) and reactive sulfur species (RSS), are generated in vivo from several sources. Within the cell the mitochondria represent the main source of ROS and mitochondrial dysfunction is both the major contributor to oxidative stress (OS) as well its major consequence.To date there are no doubts that a condition of OS added to other factors as mitochondrial damage in mtDNA or mitochondrial respiratory chain, may contribute to trigger or amplify mechanisms leading to neurodegenerative disorders.In this chapter, we aim at illustrate the molecular interplay occurring between mitochondria and OS focusing on Amyotrophic Lateral Sclerosis, describing a phenotypic reprogramming mechanism of mitochondria in complex neurological disorder.

Impact of Pharmacological Inhibition of Hydrogen Sulphide Production in the SOD1G93A-ALS Mouse Model.

A number of factors can trigger amyotrophic lateral sclerosis (ALS), although its precise pathogenesis is still uncertain. In a previous study done by us, poisonous liquoral levels of hydrogen sulphide (H2S) in sporadic ALS patients were reported. In the same study very high concentrations of H2S in the cerebral tissues of the familial ALS (fALS) model of the SOD1G93A mouse, were measured. The objective of this study was to test whether decreasing the levels of H2S in the fALS mouse could be beneficial. Amino-oxyacetic acid (AOA)-a systemic dual inhibitor of cystathionine-ß-synthase and cystathionine-γ lyase (two key enzymes in the production of H2S)-was administered to fALS mice. AOA treatment decreased the content of H2S in the cerebral tissues, and the lifespan of female mice increased by approximately ten days, while disease progression in male mice was not affected. The histological evaluation of the spinal cord of the females revealed a significant increase in GFAP positivity and a significant decrease in IBA1 positivity. In conclusion, the results of the study indicate that, in the animal model, the inhibition of H2S production is more effective in females. The findings reinforce the need to adequately consider sex as a relevant factor in ALS.

Tissue degeneration in ALS affected spinal cord evaluated by Raman spectroscopy.

The Raman spectral features from spinal cord tissue sections of transgenic, ALS model mice and non-transgenic mice were compared using 457 nm excitation line, profiting from the favourable signal intensity obtained in the molecular fingerprint region at this wavelength. Transverse sections from four SOD1G93A mice at 75 days and from two at 90 days after birth were analysed and compared with sections of similarly aged control mice. The spectra acquired within the grey matter of tissue sections from the diseased mice is markedly different from the grey matter signature of healthy mice. In particular, we observe an intensity increase in the spectral windows 450-650 cm-1 and 1050-1200 cm-1, accompanied by an intensity decrease in the lipid contributions at ~1660 cm-1, ~1440 cm-1 and ~1300 cm-1. Axons demyelination, loss of lipid structural order and the proliferation and aggregation of branched proteoglycans are related to the observed spectral modifications. Furthermore, the grey and white matter components of the spinal cord sections could also be spectrally distinguished, based on the relative intensity of characteristic lipid and protein bands. Raman spectra acquired from the white matter regions of the SOD1G93A mice closely resembles those from control mice.

Activation of Phosphotyrosine-Mediated Signaling Pathways in the Cortex and Spinal Cord of SOD1G93A, a Mouse Model of Familial Amyotrophic Lateral Sclerosis.

Degeneration of cortical and spinal motor neurons is the typical feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease for which a pathogenetic role for the Cu/Zn superoxide dismutase (SOD1) has been demonstrated. Mice overexpressing a mutated form of the SOD1 gene (SOD1G93A) develop a syndrome that closely resembles the human disease. The SOD1 mutations confer to this enzyme a "gain-of-function," leading to increased production of reactive oxygen species. Several oxidants induce tyrosine phosphorylation through direct stimulation of kinases and/or phosphatases. In this study, we analyzed the activities of src and fyn tyrosine kinases and of protein tyrosine phosphatases in synaptosomal fractions prepared from the motor cortex and spinal cord of transgenic mice expressing SOD1G93A. We found that (i) protein phosphotyrosine level is increased, (ii) src and fyn activities are upregulated, and (iii) the activity of tyrosine phosphatases, including the striatal-enriched tyrosine phosphatase (STEP), is significantly decreased. Moreover, the NMDA receptor (NMDAR) subunit GluN2B tyrosine phosphorylation was upregulated in SOD1G93A. Tyrosine phosphorylation of GluN2B subunits regulates the NMDAR function and the recruitment of downstream signaling molecules. Indeed, we found that proline-rich tyrosine kinase 2 (Pyk2) and ERK1/2 kinase are upregulated in SOD1G93A mice. These results point out an involvement of tyrosine kinases and phosphatases in the pathogenesis of ALS.

Proteomics and Toxicity Analysis of Spinal-Cord Primary Cultures upon Hydrogen Sulfide Treatment.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter recognized as an essential body product with a dual, biphasic action. It can function as an antioxidant and a cytoprotective, but also as a poison with a high probability of causing brain damage when present at noxious levels. In a previous study, we measured toxic liquoral levels of H2S in sporadic amyotrophic lateral sclerosis (ALS) patients and in the familial ALS (fALS) mouse model, SOD1G93A. In addition, we experimentally demonstrated that H2S is extremely and selectively toxic to motor neurons, and that it is released by glial cells and increases Ca2+ concentration in motor neurons due to a lack of ATP. The presented study further examines the effect of toxic concentrations of H2S on embryonic mouse spinal-cord cultures. We performed a proteomic analysis that revealed a significant H2S-mediated activation of pathways related to oxidative stress and cell death, particularly the Nrf-2-mediated oxidative stress response and peroxiredoxins. Furthermore, we report that Na2S (a stable precursor of H2S) toxicity is, at least in part, reverted by the Bax inhibitor V5 and by necrostatin, a potent necroptosis inhibitor.

Cognitive impairment in amyotrophic lateral sclerosis, clues from the SOD1 mouse.

Amyotrophic lateral sclerosis (ALS) is now recognized as a multisystem disorder, in which the primary pathology is the degeneration of motor neurons, with cognitive and/or behavioral dysfunctions that constitutes the non-motor manifestations of ALS. The combination of clinical, neuroimaging, and neuropathological data, and detailed genetic studies suggest that ALS and frontotemporal dementia (FTD) might form part of a disease continuum, with pure ALS and pure FTD at the two extremes. Mutations in the superoxide dismutase 1 (SOD1) gene were the first genetic mutations linked to the insurgence of ALS. Since that discovery numerous animal models carrying SOD1 mutations have been created. Despite their limitations these animal models, particularly the mice, have broaden our knowledge on the system alterations occurring in the ALS spectrum of disorders. The present review aims at providing an overview of the data obtained with the SOD1 animal models first and foremost on the cortical and subcortical regions, the cortico-striatal and hippocampal synaptic plasticity, dendritic branching and glutamate receptors function.

Evidence of hydrogen sulfide involvement in amyotrophic lateral sclerosis.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease whose pathophysiological deficits, causing impairment in motor function, are largely unknown. Here we propose that hydrogen sulfide (H2 S), as a glial-released inflammatory factor, contributes to ALS-mediated motor neuron death. METHODS: H2 S concentrations were analyzed in the cerebrospinal fluid of 37 sporadic ALS patients and 14 age- and gender-matched controls, in tissues of a familial ALS (fALS) mouse model, and in spinal cord culture media by means of a specific and innovative high-performance liquid chromatography method. The effects of H2 S on motor neurons cultures was analyzed immunohistochemically and by patch clamp recordings and microfluorometry. RESULTS: We found a significantly high level of H2 S in the spinal fluid of the ALS patients. Consistently, we found increased levels of H2 S in the tissues and in the media from mice spinal cord cultures bearing the fALS mutation SOD1G93A. In addition, NaHS, an H2 S donor, added to spinal culture, obtained from control C57BL/6J mice, is toxic for motor neurons, and induces an intracellular Ca(2+) increase, attenuated by the intracytoplasmatic application of adenosine triphosphate. We further show that H2 S is mainly released by astrocytes and microglia. INTERPRETATION: This study unravels H2 S as an astroglial mediator of motor neuron damage possibly involved in the cellular death characterizing ALS.

Endothelin-1 is over-expressed in amyotrophic lateral sclerosis and induces motor neuron cell death.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor neurons (MNs) and astrogliosis. Recent evidence suggests that factors secreted by activated astrocytes might contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up-regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly expressed by reactive astrocytes in the spinal cord of the SOD1-G93A mouse model and sporadic ALS patients. To test if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100-200nM ET-1 for 48h resulting in 40-50% MN cell death. Importantly, ET-1 did not induce MN degeneration when administered on cultures treated with AraC (5µM) or grown in a serum-free medium that did not favor astrocyte proliferation and reactivity. We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 toxic effect was mimicked by ET-3 (100nM) and sarafotoxin S6c (10nM), two selective agonists of endothelin-B receptors, and was not additive with that of ET-3 suggesting the involvement of ETB receptors. Surprisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ-788 (200nM-2µM) and was slightly reversed by the ETA receptor antagonist BQ-123 (2µM), suggesting an atypical pharmacological profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20µM), indicating that it is not caused by an increased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute exposure to AMPA (50µM), which is indicative of two distinct pathways leading to neuronal death. Altogether these results indicate that ET-1 exerts a toxic effect on cultured MNs through mechanisms mediated by reactive astrocytes and suggest that ET-1 may contribute to MN degeneration in ALS. Thus, a treatment aimed at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow down MN degeneration in this devastating disease.

Role of the N-methyl-d-aspartate receptors complex in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease pathologically characterized by the massive loss of motor neurons in the spinal cord, brain stem and cerebral cortex. There is a consensus in the field that ALS is a multifactorial pathology and a number of possible mechanisms have been suggested. Among the proposed hypothesis, glutamate toxicity has been one of the most investigated. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated cell death and impairment of the glutamate-transport system have been suggested to play a central role in the glutamate-mediated motor neuron degeneration. In this context, the role played by the N-methyl-d-aspartate (NMDA) receptor has received considerable less attention notwithstanding its high Ca(2+) permeability, expression in motor neurons and its importance in excitotoxicity. This review overviews the critical role of NMDA-mediated toxicity in ALS, with a particular emphasis on the endogenous modulators of the NMDAR.

CREB selectively controls learning-induced structural remodeling of neurons.

The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons remains elusive. Using transgenic mice conditionally expressing a dominant-negative CREB (CREBS133A: mCREB) mutant, we found that inhibiting CREB function does not alter spine density, spine morphology, and levels of polymerized actin in naive CA1 neurons. CREB inhibition, however, impaired contextual fear conditioning and produced a learning-induced collapse of spines associated with a blockade of learning-dependent increase in actin polymerization. Blocking mCREB expression with doxycycline rescued memory and restored a normal pattern of learning-induced spines, demonstrating that CREB controls structural adaptations of neurons selectively involved in memory formation.

Protein repertoire impact of Ubiquitin-Proteasome System impairment: insight into the protective role of beta-estradiol.

The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal.