Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease.

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aß1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aß1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aß1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

Is spinal muscular atrophy a disease of the motor neurons only: pathogenesis and therapeutic implications?

Spinal muscular atrophy (SMA) is a genetic neurological disease that causes infant mortality; no effective therapies are currently available. SMA is due to homozygous mutations and/or deletions in the survival motor neuron 1 gene and subsequent reduction of the SMN protein, leading to the death of motor neurons. However, there is increasing evidence that in addition to motor neurons, other cell types are contributing to SMA pathology. In this review, we will discuss the involvement of non-motor neuronal cells, located both inside and outside the central nervous system, in disease onset and progression. Even if SMN restoration in motor neurons is needed, it has been shown that optimal phenotypic amelioration in animal models of SMA requires a more widespread SMN correction. It has been demonstrated that non-motor neuronal cells are also involved in disease pathogenesis and could have important therapeutic implications. For these reasons it will be crucial to take this evidence into account for the clinical translation of the novel therapeutic approaches.

Minimally invasive transplantation of iPSC-derived ALDHhiSSCloVLA4+ neural stem cells effectively improves the phenotype of an amyotrophic lateral sclerosis model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently, there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS, and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study, we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity, low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS, treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span, in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms, including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases.

Pluripotent stem cell-based models of spinal muscular atrophy.

Motor neuron diseases, as the vast majority of neurodegenerative disorders in humans, are incurable conditions that are challenging to study in vitro, owing to the obstacles in obtaining the cell types majorly involved in the pathogenesis. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, have opened up the possibility of generating a substantial amount of disease-specific neuronal cells, including motor neurons and glial cells. The present review analyzes the practical implications of iPSCs, generated from fibroblasts of patients affected by spinal muscular atrophy (SMA), and discusses the challenges in the development and optimization of in vitro disease models. Research on patient-derived disease-specific cells may shed light on the pathological processes behind neuronal dysfunction and death in SMA, thus providing new insights for the development of novel effective therapies.

Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis.

Neurodegenerative disorders are characterized by the selective vulnerability and progressive loss of discrete neuronal populations. Non-neuronal cells appear to significantly contribute to neuronal loss in diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, and Alzheimer's disease. In ALS, there is deterioration of motor neurons in the cortex, brainstem, and spinal cord, which control voluntary muscle groups. This results in muscle wasting, paralysis, and death. Neuroinflammation, characterized by the appearance of reactive astrocytes and microglia as well as macrophage and T-lymphocyte infiltration, appears to be highly involved in the disease pathogenesis, highlighting the involvement of non-neuronal cells in neurodegeneration. There appears to be cross-talk between motor neurons, astrocytes, and immune cells, including microglia and T-lymphocytes, which are subsequently activated. Currently, effective therapies for ALS are lacking; however, the non-cell autonomous nature of ALS may indicate potential therapeutic targets. Here, we review the mechanisms of action of astrocytes, microglia, and T-lymphocytes in the nervous system in health and during the pathogenesis of ALS. We also evaluate the therapeutic potential of these cellular populations, after transplantation into ALS patients and animal models of the disease, in modulating the environment surrounding motor neurons from pro-inflammatory to neuroprotective. We also thoroughly discuss the recent advances made in the field and caveats that need to be overcome for clinical translation of cell therapies aimed at modulating non-cell autonomous events to preserve remaining motor neurons in patients.

Stem cell transplantation for amyotrophic lateral sclerosis: therapeutic potential and perspectives on clinical translation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by degeneration of upper and lower motor neurons. There are currently no clinically impactful treatments for this disorder. Death occurs 3-5 years after diagnosis, usually due to respiratory failure. ALS pathogenesis seems to involve several pathological mechanisms (i.e., oxidative stress, inflammation, and loss of the glial neurotrophic support, glutamate toxicity) with different contributions from environmental and genetic factors. This multifaceted combination highlights the concept that an effective therapeutic approach should counteract simultaneously different aspects: stem cell therapies are able to maintain or rescue motor neuron function and modulate toxicity in the central nervous system (CNS) at the same time, eventually representing the most comprehensive therapeutic approach for ALS. To achieve an effective cell-mediated therapy suitable for clinical applications, several issues must be addressed, including the identification of the most performing cell source, a feasible administration protocol, and the definition of therapeutic mechanisms. The method of cell delivery represents a major issue in developing cell-mediated approaches since the cells, to be effective, need to be spread across the CNS, targeting both lower and upper motor neurons. On the other hand, there is the need to define a strategy that could provide a whole distribution without being too invasive or burdened by side effects. Here, we review the recent advances regarding the therapeutic potential of stem cells for ALS with a focus on the minimally invasive strategies that could facilitate an extensive translation to their clinical application.

Molecular, genetic and stem cell-mediated therapeutic strategies for spinal muscular atrophy (SMA).

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell-mediated approaches for the treatment of SMA.

Polymorphism Pro64His within galectin-3 has functional consequences at proteome level in thyroid cells.

Introduction: The single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: LGALS3), specifying the variant proline (P64) to histidine (H64), is known to affect the protein's functions and has been associated with the risk of several types of cancer, including differentiated thyroid carcinoma (DTC). Materials and methods: To deepen our understanding of the biological effects of this SNP, we analyzed the proteome of two isogenic cell lines (NC-P64 vs. NA-H64) derived from the immortalized non-malignant thyrocyte cell line Nthy-Ori, generated through the CRISPR-Cas9 technique to differ by rs4644 genotype. We compared the proteome of these cells to detect differentially expressed proteins and studied their proteome in relation to their transcriptome. Results: Firstly, we found, consistently with previous studies, that gal-3-H64 could be detected as a monomer, homodimer, and heterodimer composed of one cleaved and one uncleaved monomer, whereas gal-3-P64 could be found only as a monomer or uncleaved homodimer. Moreover, results indicate that rs4644 influences the expression of several proteins, predominantly upregulated in NA-H64 cells. Overall, the differential protein expression could be attributed to the altered mRNA expression, suggesting that rs4644 shapes the function of gal-3 as a transcriptional co-regulator. However, this SNP also appeared to affect post-transcriptional regulatory mechanisms for proteins whose expression was oppositely regulated compared to mRNA expression. It is conceivable that the rs4644-dependent activities of gal-3 could be ascribed to the different modalities of self-dimerization. Conclusion: Our study provided further evidence that rs4644 could affect the gal-3 functions through several routes, which could be at the base of differential susceptibility to diseases, as reported in case-control association studies.

Direct reprogramming of human astrocytes into neural stem cells and neurons.

Generating neural stem cells and neurons from reprogrammed human astrocytes is a potential strategy for neurological repair. Here we show dedifferentiation of human cortical astrocytes into the neural stem/progenitor phenotype to obtain progenitor and mature cells with a neural fate. Ectopic expression of the reprogramming factors OCT4, SOX2, or NANOG into astrocytes in specific cytokine/culture conditions activated the neural stem gene program and induced generation of cells expressing neural stem/precursor markers. Pure CD44+ mature astrocytes also exhibited this lineage commitment change and did not require passing through a pluripotent state. These astrocyte-derived neural stem cells gave rise to neurons, astrocytes, and oligodendrocytes and showed in vivo engraftment properties. ASCL1 expression further promoted neuronal phenotype acquisition in vitro and in vivo. Methylation analysis showed that epigenetic modifications underlie this process. The restoration of multipotency from human astrocytes has potential in cellular reprogramming of endogenous central nervous system cells in neurological disorders.

Research advances in gene therapy approaches for the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons that causes progressive muscle weakness, paralysis, and premature death. No effective therapy is available. Research in the motor neuron field continues to grow, and recent breakthroughs have demonstrated the possibility of completely achieving rescue in animal models of spinal muscular atrophy, a genetic motor neuron disease. With adeno-associated virus (AAV) vectors, gene transfer can be achieved with systemic non-invasive injection and minimal toxicity. In the context of this success, we review gene therapy approaches for ALS, considering what has been done and the possible future directions for effective application of the latest generation of vectors for clinical translation. We focus on recent developments in the areas of RNA/antisense-mediated silencing of specific ALS causative genes like superoxide dismutase-1 and other molecular pathogenetic targets, as well as the administration of neuroprotective factors with viral vectors. We argue that gene therapy offers new opportunities to open the path for clinical progress in treating ALS.

New amidine-benzenesulfonamides as iNOS inhibitors for the therapy of the triple negative breast cancer.

Triple negative breast cancer (TNBC) is a specific breast cancer subtype, and poor prognosis is associated to this tumour when it is in the metastatic form. The overexpression of the inducible Nitric Oxide Synthase (iNOS) is considered a predictor of poor outcome in TNBC patients, and this enzyme is reported as a valuable molecular target to compromise TNBC progression. In this work, new amidines containing a benzenesulfonamide group were designed and synthesized as selective iNOS inhibitors. An in vitro biological evaluation was performed to assess compounds activity against both the inducible and constitutive NOSs. The most interesting compounds 1b and 2b were evaluated on MDA-MB-231 cells as antiproliferative agents, and 1b capability to counteract cell migration was also studied. Finally, an in-depth docking study was performed to shed light on the observed potency and selectivity of action of the most promising compounds.

Systemic transplantation of c-kit+ cells exerts a therapeutic effect in a model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disease characterized by the loss of motor neurons. Motor neuron degeneration is probably both a cell autonomous and a non-autonomous event. Therefore, manipulating the diseased microenvironment via non-neural cell replacement could be a therapeutic strategy. We investigated a cell therapy approach using intravascular injection to transplant a specific population of c-kit(+) stem/progenitor cells from bone marrow into the SOD1G93A mouse model of ALS. Transplanted cells engrafted within the host spinal cord. Cell transplantation significantly prolonged disease duration and lifespan in superoxide dismutase 1 mice, promoted the survival of motor neurons and improved neuromuscular function. Neuroprotection was mediated by multiple effects, in particular by the expression of primary astrocyte glutamate transporter GLT1 and by the non-mutant genome. These findings suggest that this type of somatic cell transplantation strategy merits further investigation as a possible effective therapy for ALS and other neurodegenerative diseases.

Embryonic stem cell-derived neural stem cells improve spinal muscular atrophy phenotype in mice.

Spinal muscular atrophy, characterized by selective loss of lower motor neurons, is an incurable genetic neurological disease leading to infant mortality. We previously showed that primary neural stem cells derived from spinal cord can ameliorate the spinal muscular atrophy phenotype in mice, but this primary source has limited translational value. Here, we illustrate that pluripotent stem cells from embryonic stem cells show the same potential therapeutic effects as those derived from spinal cord and offer great promise as an unlimited source of neural stem cells for transplantation. We found that embryonic stem cell-derived neural stem cells can differentiate into motor neurons in vitro and in vivo. In addition, following their intrathecal transplantation into spinal muscular atrophy mice, the neural stem cells, like those derived from spinal cord, survived and migrated to appropriate areas, ameliorated behavioural endpoints and lifespan, and exhibited neuroprotective capability. Neural stem cells obtained using a drug-selectable embryonic stem cell line yielded the greatest improvements. As with cells originating from primary tissue, the embryonic stem cell-derived neural stem cells integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in neural stem cell-mediated therapies and highlight potential safety improvements and benefits of drug selection for neuroepithelial cells.

Pitfalls and solutions in assaying anandamide transport in cells.

Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.

A new group of oxime carbamates as reversible inhibitors of fatty acid amide hydrolase.

A series of oxime carbamates have been identified as potent inhibitors of fatty acid amide hydrolase (FAAH), an important regulatory enzyme of the endocannabinoid signaling system. Kinetic analysis indicates that they behave as non-competitive, reversible inhibitors, and show remarkable selectivity for FAAH over the other components of the endocannabinoid system.

Changes in quality of life and dyspnoea after hospitalization in COVID-19 patients discharged at home.

BACKGROUND: To date, the effects of COVID-19 pneumonia on health-related quality of life (HRQoL) and dyspnoea are unknown. METHODS: In a real-life observational study, 20 patients with COVID-19-related pneumonia received usual care plus erdosteine (300 mg twice daily) for 15 days after hospital discharge following local standard operating procedures. At discharge (T0) and on Day 15 (T1), participants completed the St George's Respiratory Questionnaire (SGRQ), the modified Medical Research Council (mMRC) scale of dyspnoea during daily activity, the BORG scale for dyspnoea during exertion, and Visual Analogue Scale (VAS) for dyspnoea at rest. Paired t-tests compared scores at T0 and T1. RESULTS: The mean (SD) SGRQ total score decreased from 25.5 (15.5) at T0 to 16.9 (13.2) at T1 (p<0.01); 65% of patients achieved a clinically important change of ≥4 points. SGRQ domain scores (symptoms, activity, and impact) were also significantly reduced (all p<0.01). The mean (SD) VAS score decreased from 1.6 (1.7) to 1.4 (2.5); p<0.01. The mean mMRC score decreased significantly (p=0.031) and 30% of patients achieved a clinically important change of ≥1 point. The mean (SD) Borg score increased from 12.8 (4.2) to 14.3 (2.4); p<0.01. CONCLUSION: The present proof of concept study is the first to report HRQoL in patients with COVID-19. During 15 days after hospital discharge, patients reported significant improvements in HRQoL and dyspnoea at rest and during daily activities.

Fatty acid amide hydrolase: a gate-keeper of the endocannabinoid system.

The family of endocannabinoids contains several polyunsaturated fatty acid amides such as anandamide (AEA), but also esters such as 2-arachidonoylglycerol (2-AG). These compounds are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta9-tetrahydrocannabinol (Delta9-THC), the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA at its receptors is limited by cellular uptake, through a putative membrane transporter, followed by intracellular degradation by fatty acid amide hydrolase (FAAH). Growing evidence demonstrates that FAAH is the critical regulator of the endogenous levels of AEA, suggesting that it may serve as an attractive therapeutic target for the treatment of human disorders. In particular, FAAH inhibitors may be next generation therapeutics of potential value for the treatment of pathologies of the central nervous system, and of peripheral tissues. Investigations into the structure and function of FAAH, its biological and therapeutic implications, as well as a description of different families of FAAH inhibitors, are the topic of this chapter.

Type-1 cannabinoid receptors colocalize with caveolin-1 in neuronal cells.

Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed "endocannabinoids". Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-beta-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases.

Involvement of the endocannabinoid system in retinal damage after high intraocular pressure-induced ischemia in rats.

PURPOSE: To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult. METHODS: Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used. RESULTS: In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively). CONCLUSIONS: The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.

A case of Legionella pneumophila evaluated with CT and ultrasound.

A 36-year-old man was admitted to the emergency department of "SS Annunziata" hospital in Chieti complaining of a sharp chest pain arisen some hours before admission. On examination, the patient looked sweaty; his vital signs showed tachycardia and augmented breath rate; sinus tachycardia and normal ventricular repolarization were observed on ECG, and no abnormalities were observed in the echoscan of the hearth. According to the clinical and electrocardiographic findings, and to previous episode of DVT in anamnesis, a thorax CT scan was performed in order to rule out pulmonary embolism. It showed an "area of parenchymal consolidation involving almost all the left lower lobe with patent bronchial structures"; given the patient's CURB 65 score, he was then admitted to the pneumology ward where empiric treatment with levofloxacin (750 mg PO once daily) was initiated. Thoracic ultrasound was performed using a multifrequency convex transducer, and the posterior left area was examined through intercostal approach, placing the patient in a sitting position. A subpleural patchy hypoechoic lesion with irregular boundaries was detected; the maximum diameter was 11 cm, and the multiple hyperechoic spots inside it (elsewhere defined as "air bronchogram") showed no Doppler signal. Given the positive result of the Legionella urinary antigen test, antibiotic treatment was switched to Levofloxacin 1000 mg PO once daily and Claritromicin 500 mg PO twice daily. After 3 days, his clinical conditions improved dramatically. Ultrasound performed after 5 days from the diagnosis showed decreased dimensions of the lesion previously identified (maximum diameter 8.25 cm) and a marked reduction of the hyperechoic spots in it. The patient was discharged in good clinical conditions, and both thorax CT scan obtained after 1 and 4 months from the diagnosis showed radiological resolution of the parenchymal consolidation. The key to ultrasound visualization of pneumonia is its contact with the pleural surface (86-98% in cases of CAP) and the relative loss of aeration of the portion involved by the infection and a concomitant increase in the fluid content. A paradigmatic US image for parenchymal inflammatory infiltrate has not been established yet; anyway, some typical findings, when combined with the clinical features, can confirm the diagnostic hypothesis.