Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

Blood-based untargeted metabolomics in relapsing-remitting multiple sclerosis revealed the testable therapeutic target.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

Repeat intramuscular transplantation of human dental pulp stromal cells is more effective in sustaining Schwann cell survival and myelination for functional recovery after onset of diabetic neuropathy.

BACKGROUND AIMS: Mesenchymal stromal cell (MSC) therapy for diabetic neuropathy (DN) has been extensively researched in vitro and in pre-clinical studies; however, the clinical scenario thus far has been disappointing. Temporary recovery, a common feature of these studies, indicates that either the retention of transplanted cells deteriorates with time or recovery of supportive endogenous cells, such as bone marrow-derived MSCs (BM-MSCs), does not occur, requiring further replenishment. In DN, BM-MSCs are recognized mediators of Schwann cell regeneration, and we have earlier shown that they suffer impairment in the pre-neuropathy stage. In this study, we attempted to further elucidate the mechanisms of functional recovery by focusing on changes occurring at the cellular level in the sciatic nerve, in conjunction with the biodistribution and movement patterns of the transplanted cells, to define the interval between doses. METHOD & RESULTS: We found that two doses of 1 × 106 dental pulp stromal cells (DPSCs) transplanted intramuscularly at an interval of 4 weeks effectively improved nerve conduction velocity (NCV) and restored motor coordination through improving sciatic nerve architecture, Schwann cell survival and myelination. Despite very minimal recovery of endogenous BM-MSCs, a temporary restoration of NCV and motor function was achieved with the first dose of DPSC transplantation. However, this did not persist, and a repeat dose was needed to consolidate functional improvement and rehabilitate the sciatic nerve architecture. CONCLUSION: Thus, repeat intramuscular transplantation of DPSCs is more effective for maintenance of Schwann cell survival and myelination for functional recovery after onset of DN.

Reduction of phosphorylated α-synuclein through downregulation of casein kinase 2α alleviates dopaminergic-neuronal function.

Among the post-translational modifications of α-synuclein, phosphorylation has been reported to modulate the protein's nuclear localization, gene-expression and cytotoxicity. However, its effect on the functional performance of dopaminergic-neurons is not known. We aimed to evaluate the effect of siRNA-silencing of casein kinase (CK)2α in SH-SY5Y-cells overexpressing A53T α-synuclein, in alleviating phosphorylated α-synuclein serine129 (pSyn-129)-induced changes in intracellular Ca2+ ([Ca2+]i) response to physiological stimuli and vesicular-dopamine release. A53T transfection showed distinct increase in basal pSyn-129 expression with simultaneous nuclear localization, and CK2α siRNA decreased ROS-generation and pSyn-129 levels. A significant reduction was observed in KCl-induced ([Ca2+]i) response and vesicular-dopamine release in the A53T-transfected cells with a corresponding decrease in immunopositive-population of resting-vesicles (VMAT2). CK2α siRNA treatment showed recovery in [Ca2+]i rise with a corresponding upregulation of expression of voltage-gated Ca2+-channels (VGCC) CaV1.3 and CaV2.2 and RyR1 responsible for Ca2+ induced Ca2+ release from ER, VMAT2 expression and vesicular-dopamine release. Thus, using CK2α siRNA to reduce phosphorylation improved cellular-pathology in terms of ROS generation and pSyn-129 levels, as well as functional performance of DA-neuronal cells.

Impact of monomeric and aggregated wild-type and A30P/A53T double-mutant α-synuclein on antioxidant mechanism and glutamate metabolic profile of cultured astrocytes.

Serving as a source of glutathione and up-taking and metabolizing glutamate are the primary supportive role of astrocytes for the adjacent neurons. Despite the clear physical association between astrocytes and α-synuclein, the effect of extracellular α-synuclein on these astrocytic functions has not yet been elucidated. Hence, we aim to assess the effect of various forms of α-synuclein on antioxidant mechanism and glutamate metabolism. Wild-type and A53T/A30P double-mutant α-synuclein, both in monomeric and aggregated forms, were added extracellularly to media of midbrain rat astrocyte culture, with their survival, oxidative, and nitrative stress, glutathione and glutamate content, expression of enzymes associated with oxidative stress and glutamate metabolism, glutamate and glutathione transporters being assessed along with the association/engulfment of these peptides by astrocytes. A30P/A53T peptide associated more with astrocytes, and low-extracellular K+ concentration showed prominent reduction in the engulfment of the monomeric forms, suggesting that the association of the aggregated forms was greater with the membrane. The peptide-associated astrocytes showed lower survival and increased oxidative stress generation, owing to the decrease in nuclear localization of Nrf2 and increase in iNOS, and further aggravated by the decrease in glutathione content and related enzymes like glutathione synthetase, glutathione peroxidase, and glutathione reductase. Glutamate uptake increased in aggregate-treated cells due to the increase in GLAST1 expression, de novo synthesis of glutamate by pyruvate carboxylase, and/or glutamine synthase, bolstered by the differential glutamate dehydrogenase enzyme activity. We thus show for the first time that extracellular α-synuclein exposure leads to astrocytic dysfunction with respect to the antioxidant mechanism and glutamate metabolic profile. The impact was higher in the case of the aggregated and mutated peptide, with the highest dysfunction for the mutant aggregated α-synuclein treatment.

Recovery of Human Embryonic Stem Cells-Derived Neural Progenitors Exposed to Hypoxic-Ischemic-Reperfusion Injury by Indirect Exposure to Wharton's Jelly Mesenchymal Stem Cells Through Phosphatidyl-inositol-3-Kinase Pathway.

Increasing evidence suggests that mesenchymal stem cells(MSCs) have beneficial effects in hypoxic ischemic reperfusion injury, but the underlying mechanisms are unclear. Here, we first examined the effect of OGD reperfusion injury on the vulnerability of human NPs derived from human embryonic stem cells (hESCs) with regard to cell survival and oxidative stress. Cellular deregulation was assessed by measuring glutathione levels, basal calcium and intracellular calcium [Ca2+]i response under KCl stimulation, as well as the key parameters of proliferation, glial progenitor marker expression and migration. Next, the influence of WJ-MSCs in recovering these parameters was evaluated, and the role of Phosphatidyl-inositol-3-Kinase(PI3K) pathway in actuating the protective effect was assessed. OGD reperfusion injury induced significant increases in cell death, ROS generation, oxidative stress susceptibility and decreased glutathione levels in NPs, accompanied by rises in basal [Ca2+]i, KCl-induced [Ca2+]i, expression of K+ leak channel(TASK1), and declines in proliferation, migration potential and glial progenitor population. The introduction of WJ-MSCs(after 2 h of reperfusion) through a non-contact method brought about significant improvement in all these cellular parameters as observed after 24hrs, and the PI3K pathway played an important role in the neuroprotection process. Presence of WJ-MSCs increased the expression of survival signals like phosphorylated Akt/Akt and PI3K in the OGD-reperfused NPs. Our data clearly demonstrate for the first time that soluble factors from WJ-MSCs can not only ameliorate survival, proliferation, migration and glial progenitor expression of OGD-reperfused NPs, but also regulate their intracellular Ca2+ response to KCl stimulation and expression of TASK1 through the PI3K pathway.

An emerging potential of metabolomics in multiple sclerosis: a comprehensive overview.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the nervous system that primarily affects young adults. Although the exact etiology of the disease remains obscure, it is clear that alterations in the metabolome contribute to this process. As such, defining a reliable and disease-specific metabolome has tremendous potential as a diagnostic and therapeutic strategy for MS. Here, we provide an overview of studies aimed at identifying the role of metabolomics in MS. These offer new insights into disease pathophysiology and the contributions of metabolic pathways to this process, identify unique markers indicative of treatment responses, and demonstrate the therapeutic effects of drug-like metabolites in cellular and animal models of MS. By and large, the commonly perturbed pathways in MS and its preclinical model include lipid metabolism involving alpha-linoleic acid pathway, nucleotide metabolism, amino acid metabolism, tricarboxylic acid cycle, D-ornithine and D-arginine pathways with collective role in signaling and energy supply. The metabolomics studies suggest that metabolic profiling of MS patient samples may uncover biomarkers that will advance our understanding of disease pathogenesis and progression, reduce delays and mistakes in diagnosis, monitor the course of disease, and detect better drug targets, all of which will improve early therapeutic interventions and improve evaluation of response to these treatments.

Expression and regulatory roles of lncRNAs in G-CIMP-low vs G-CIMP-high Glioma: an in-silico analysis.

BACKGROUND: Clinically relevant glioma subtypes, such as the glioma-CpG island methylator phenotype (G-CIMP), have been defined by epigenetics. In this study, the role of long non-coding RNAs in association with the poor-prognosis G-CMIP-low phenotype and the good-prognosis G-CMIP-high phenotype was investigated. Functional associations of lncRNAs with mRNAs and miRNAs were examined to hypothesize influencing factors of the aggressive phenotype. METHODS: RNA-seq data on 250 samples from TCGA's Pan-Glioma study, quantified for lncRNA and mRNAs (GENCODE v28), were analyzed for differential expression between G-CIMP-low and G-CIMP-high phenotypes. Functional interpretation of the differential lncRNAs was performed by Ingenuity Pathway Analysis. Spearman rank order correlation estimates between lncRNA, miRNA, and mRNA nominated differential lncRNA with a likely miRNA sponge function. RESULTS: We identified 4371 differentially expressed features (mRNA = 3705; lncRNA = 666; FDR ≤ 5%). From these, the protein-coding gene TP53 was identified as an upstream regulator of differential lncRNAs PANDAR and PVT1 (p = 0.0237) and enrichment was detected in the "development of carcinoma" (p = 0.0176). Two lncRNAs (HCG11, PART1) were positively correlated with 342 mRNAs, and their correlation estimates diminish after adjusting for either of the target miRNAs: hsa-miR-490-3p, hsa-miR-129-5p. This suggests a likely sponge function for HCG11 and PART1. CONCLUSIONS: These findings identify differential lncRNAs with oncogenic features that are associated with G-CIMP phenotypes. Further investigation with controlled experiments is needed to confirm the molecular relationships.

Mesenchymal stromal cell therapy for coronavirus disease 2019: which? when? and how much?

Mesenchymal stromal cells (MSCs) are under active consideration as a treatment strategy for controlling the hyper-inflammation and slow disease progression associated with coronavirus disease 2019 (COVID-19). The possible mechanism of protection through their immunoregulatory and paracrine action has been reviewed extensively. However, the importance of process control in achieving consistent cell quality, maximum safety and efficacy-for which the three key questions are which, when and how much-remains unaddressed. Any commonality, if it exists, in ongoing clinical trials has yet to be analyzed and reviewed. In this review, the authors have therefore compiled study design data from ongoing clinical trials to address the key questions of "which" with regard to tissue source, donor profile, isolation technique, culture conditions, long-term culture and cryopreservation of MSCs; "when" with regard to defining the transplantation window by identifying and staging patients based on their pro-inflammatory profile; and "how much" with regard to the number of cells in a single administration, number of doses and route of transplantation. To homogenize MSC therapy for COVID-19 on a global scale and to make it readily available in large numbers, a shared understanding and uniform agreement with respect to these fundamental issues are essential.

Biodistribution of Intramuscularly-Transplanted Human Dental Pulp Stem Cells in Immunocompetent Healthy Rats through NIR Imaging.

Owing to their neural crest origin, dental pulp stem cells (DPSCs) are increasingly gaining prominence in treating nervous system disease conditions. However, as per the regulatory bodies [European-Medicines Agency (EMA), Indian-Council of Medical-Research (ICMR)], their biodistribution after transplantation needs to be evaluated for them to be considered for cell-based therapy for clinical trials. There are yet no studies describing the dynamic distribution of human origin DPSCs (hDPSCs) after transplantation in an immunocompetent, physiologically healthy animal model. Here, using near-infrared (NIR)-based whole animal and ex vivo tissue imaging, we assessed the biodistribution of intramuscularly transplanted hDPSCs in immunocompetent healthy Wistar rats. Further validation was done by quantifying gene expression of the human Alu gene in rat tissues. After 24 h of transplantation, an increase in signal intensity and area of signal was observed in the muscle of administration compared to 30 min and 6 h. At hour 24, neither increase in human Alu nor human Ki67 gene expression was seen in the rat muscle, thus confirming that the increase in signal area and intensity at hour 24 was not due to proliferation of the transplanted cells. Rather at hour 24, the NIR-signal intensity in bone marrow increased, suggesting that the NIR-tagged DPSCs have started entering into the blood vessels adjacent to the muscle, and the blood vessels being placed just beneath the subcutaneous layer might be responsible for an increase in signal intensity. Signal intensity increased distinctly in all organs at this timepoint, confirming that the cells entered the bloodstream by hour 24. Lung entrapment of DPSCs was not observed, since signal intensity was least in lungs as compared to the site of injection. Cells were retained for up to 28 days at the site of injection. These findings lay the basis to design the dosage for intramuscular delivery of hDPSCs for degenerative disease models and for future clinical trials.