hnRNP A1 dysfunction in oligodendrocytes contributes to the pathogenesis of multiple sclerosis.

Oligodendrocyte (OL) damage and death are prominent features of multiple sclerosis (MS) pathology, yet mechanisms contributing to OL loss are incompletely understood. Dysfunctional RNA binding proteins (RBPs), hallmarked by nucleocytoplasmic mislocalization and altered expression, have been shown to result in cell loss in neurologic diseases, including in MS. Since we previously observed that the RBP heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was dysfunctional in neurons in MS, we hypothesized that it might also contribute to OL pathology in MS and relevant models. We discovered that hnRNP A1 dysfunction is characteristic of OLs in MS brains. These findings were recapitulated in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, where hnRNP A1 dysfunction was characteristic of OLs, including oligodendrocyte precursor cells and mature OLs in which hnRNP A1 dysfunction correlated with demyelination. We also found that hnRNP A1 dysfunction was induced by IFNγ, indicating that inflammation influences hnRNP A1 function. To fully understand the effects of hnRNP A1 dysfunction on OLs, we performed siRNA knockdown of hnRNP A1, followed by RNA sequencing. RNA sequencing detected over 4000 differentially expressed transcripts revealing alterations to RNA metabolism, cell morphology, and programmed cell death pathways. We confirmed that hnRNP A1 knockdown was detrimental to OLs and induced apoptosis and necroptosis. Together, these data demonstrate a critical role for hnRNP A1 in proper OL functioning and survival and suggest a potential mechanism of OL damage and death in MS that involves hnRNP A1 dysfunction.

PuraMatrix hydrogel enhances the expression of motor neuron progenitor marker and improves adhesion and proliferation of motor neuron-like cells.

OBJECTIVES: Cell therapy has provided clinical applications to the treatment of motor neuron diseases. The current obstacle in stem cell therapy is to direct differentiation of stem cells into neurons in the neurodegenerative disorders. Biomaterial scaffolds can improve cell differentiation and are widely used in translational medicine and tissue engineering. The aim of this study was to compare the efficiency of two-dimensional with a three-dimensional culture system in their ability to generate functional motor neuron-like cells from adipose-derived stem cells. MATERIALS AND METHODS: We compared motor neuron-like cells derived from rat adipose tissue in differentiation, adhesion, proliferation, and functional properties on two-dimensional with three-dimensional culture systems. Neural differentiation was analyzed by immunocytochemistry for immature (Islet1) and mature (HB9, ChAT, and synaptophysin) motor neuron markers. RESULTS: Our results indicated that the three-dimensional environment exhibited an increase in the number of Islet1. In contrast, two-dimensional culture system resulted in more homeobox gene (HB9), Choline Acetyltransferase (ChAT), and synaptophysin positive cells. The results of this investigation showed that proliferation and adhesion of motor neuron-like cells significantly increased in three-dimensional compared with two-dimensional environments. CONCLUSION: The findings of this study suggested that three-dimension may create a proliferative niche for motor neuron-like cells. Overall, this study strengthens the idea that three-dimensional culture may mimic neural stem cell environment for neural tissue regeneration.

Transplantation of human meningioma stem cells loaded on a self-assembling peptide nanoscaffold containing IKVAV improves traumatic brain injury in rats.

Traumatic brain injury (TBI) can result in permanent brain function impairment due to the poor regenerative ability of neural tissue. Tissue engineering has appeared as a promising approach to promote nerve regeneration and to ameliorate brain damage. The present study was designed to investigate the effect of transplantation of the human meningioma stem-like cells (hMgSCs) seeded in a promising three-dimensional scaffold (RADA4GGSIKVAV; R-GSIK) on the functional recovery of the brain and neuroinflammatory responses following TBI in rats. After induction of TBI, hMgSCs seeded in R-GSIK was transplanted within the injury site and its effect was compared to several control groups. Application of hMgSCs with R-GSIK improved functional recovery after TBI. A significant higher number of hMgSCs was observed in the brain when transplanted with R-GSIK scaffold compared to the control groups. Application of hMgSCs seeded in R-GSIK significantly decreased the lesion volume, reactive gliosis, and apoptosis at the injury site. Furthermore, treatment with hMgSCs seeded in R-GSIK significantly inhibited the expression of Toll-like receptor 4 and its downstream signaling molecules, including interleukin-1ß and tumor necrosis factor. These data revealed the potential for hMgSCs seeded in R-GSIK to improve the functional recovery of the brain after TBI; possibly via amelioration of inflammatory responses. STATEMENT OF SIGNIFICANCE: Tissue engineered scaffolds that mimic the natural extracellular matrix of the brain may modulate stem cell fate and contribute to tissue repair following traumatic brain injury (TBI). Among several scaffolds, self-assembly peptide nanofiber scaffolds markedly promotes cellular behaviors, including cell survival and differentiation. We developed a novel three-dimensional scaffold (RADA16GGSIKVAV; R-GSIK). Transplantation of the human meningioma stem-like cells seeded in R-GSIK in an animal model of TBI significantly improved functional recovery of the brain, possibly via enhancement of stem cell survival as well as reduction of the lesion volume, inflammatory process, and reactive gliosis at the injury site. R-GSIK is a suitable microenvironment for human stem cells and could be a potential biomaterial for the reconstruction of the injured brain after TBI.

Therapeutic potential of conditioned medium derived from oligodendrocytes cultured in a self-assembling peptide nanoscaffold in experimental autoimmune encephalomyelitis.

The use of neurotrophic factors is considered to be a novel therapeutic approach for restoring and/or maintaining neurological function in neurodegenerative disorders, such as multiple sclerosis (MS). Various studies have shown that conditioned medium produced by oligodendrocyte (OL-CM) contain a variety of neurotrophic factors. Here, we investigated the restorative effects of OL-CM, collected from oligodendrocytes cultured in a self-assembling peptide hydrogels scaffold (PuraMatrix), in experimental autoimmune encephalomyelitis (EAE) mouse model. Neural stem/progenitor cells, isolated from the embryonic mouse brain, were cultured and differentiated into oligodendrocyte. Cell viability and proliferation of oligodendrocytes were assessed by live/dead and MTT assays. Motor functions, myelination, cell infiltration, gliosis, and inflammatory process were assessed in EAE mice after intracranial injection of OL-CM at different concentrations. Application of OL-CM improved clinical score and neurological function in EAE mice and reduced the inflammatory cell infiltration and demyelination. Furthermore, administration of OL-CM reduced the expression of pro-inflammatory cytokines and suppressed the activation of NLRP3-inflammasome complex in EAE mice. These data suggest the potential therapeutic effect of OL-CM for MS treatment.

Human Neural Stem/Progenitor Cells Derived From Epileptic Human Brain in a Self-Assembling Peptide Nanoscaffold Improve Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is a disruption in the brain functions following a head trauma. Cell therapy may provide a promising treatment for TBI. Among different cell types, human neural stem cells cultured in self-assembling peptide scaffolds have been suggested as a potential novel method for cell replacement treatment after TBI. In the present study, we accessed the effects of human neural stem/progenitor cells (hNS/PCs) derived from epileptic human brain and human adipose-derived stromal/stem cells (hADSCs) seeded in PuraMatrix hydrogel (PM) on brain function after TBI in an animal model of brain injury. hNS/PCs were isolated from patients with medically intractable epilepsy undergone epilepsy surgery. hNS/PCs and hADSCs have the potential for proliferation and differentiation into both neuronal and glial lineages. Assessment of the growth characteristics of hNS/PCs and hADSCs revealed that the hNS/PCs doubling time was significantly longer and the growth rate was lower than hADSCs. Transplantation of hNS/PCs and hADSCs seeded in PM improved functional recovery, decreased lesion volume, inhibited neuroinflammation, and reduced the reactive gliosis at the injury site. The data suggest the transplantation of hNS/PCs or hADSCs cultured in PM as a promising treatment option for cell replacement therapy in TBI.

Apoptosis Following Cortical Spreading Depression in Juvenile Rats.

Repetitive cortical spreading depression (CSD) can lead to cell death in immature brain tissue. Caspases are involved in neuronal cell death in several CSD-related neurological disorders, such as stroke and epilepsy. Yet, whether repetitive CSD itself can induce caspase activation in adult or juvenile tissue remains unknown. Inducing repetitive CSD in somatosensory cortices of juvenile and adult rats in vivo, we thus aimed to investigate the effect of repetitive CSD on the expression caspase-3, caspase-8, caspase-9, and caspase-12 in different brain regions using immunohistochemistry and western blotting techniques. Higher numbers of dark neurons and TUNEL-positive cells were observed in the hippocampal CA1 and CA3 regions as well as in the entorhinal and somatosensory cortices after CSD in juvenile rats. This was accompanied by higher expressions of caspase-3, caspase-8, and caspase-9. Caspase-12 levels remained unchanged after CSD, suggesting that endoplasmic reticulum stress is not involved in CSD-triggered apoptosis. Changes in caspase expression were paralleled by a decrease of procaspase-3, procaspase-8, and procaspase-9 in juvenile rat brain tissue subjected to CSD. In contrast, repetitive CSD in adult rats did not result in the upregulation of caspase signaling. Our data points to a maturation-dependent vulnerability of brain tissue to repetitive CSD with a higher degree of apoptotic damage and caspase upregulation observed in juvenile tissue. Findings suggest a key role of caspase signaling in CSD-induced cell death in the immature brain. This implies that anti-apoptotic treatment may prevent CSD-related functional deficits in the immature brain.

Curcumin attenuates harmful effects of arsenic on neural stem/progenitor cells.

OBJECTIVE: Arsenic, an environmental pollutant, decreases neuronal migration as well as cellular maturation and inhibits the proliferation of neural progenitor cells. Curcumin has been described as an antioxidant and neuroprotective agent with strong therapeutic potential in some neurological disorders. Human adipose-derived stem cells (hADSCs), a source of multipotent stem cells, can self-renew and differentiate into neural cells. The aim of the present study was to investigate the preventive effect of curcumin against arsenic toxic effects on the viability, telomerase activity, and apoptosis of neural stem/progenitor cells (NSPCs) derived from hADSCs. MATERIALS AND METHODS: The characteristics of human adipose tissue were identified by immunocytochemistry for surface markers namely, CD105, CD73, and CD90. Using neurosphere assay, hADSCs were differentiated into neuronal cells. To characterize neural cells, expression of nestin, SOX2, MAP2, and GFAP were assessed by immunocytochemistry. Cytotoxicity and viability of NSPCs were evaluated by MTT assay. Reactive oxygen species (ROS) generated by arsenic exposure, were measured and caspase 3/7 activity and caspase-3 processing as well as the telomerase activity were determined. RESULTS: The isolated hADSCs positively expressed CD105, CD73, and CD90. Nestin, Sox2, GFAP, and MAP2 were expressed in the neurospheres derived from hADSCs. Curcumin/arsenic co-treatment significantly increased telomerase activity of NSPCs compared to arsenic group. Furthermore, curcumin significantly reduced arsenic-induced apoptosis (via inactivation of caspases) as well as arsenic-associated ROS generation. CONCLUSION: Our findings revealed that curcumin has the potential to prevent harmful effects of arsenic on neurogenesis.

miR-181 interacts with signaling adaptor molecule DENN/MADD and enhances TNF-induced cell death.

MicroRNAs are small noncoding RNAs, which regulate the expression of protein coding transcripts through mRNA degradation or translational inhibition. Numerous reports have highlighted the role of miRNAs in regulating cell death pathways including the expression of genes involved in the induction of apoptosis. Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine which can send pro-death signals through its receptor TNFR1. Diverse adaptor molecules including DENN/MADD adaptor protein have been shown to modulate TNF-α pro-death signaling via recruitment of MAP kinases to TNFR1 and activation of pro-survival NFκB signaling. Herein, we investigated the role of microRNA-181 (miR-181) in regulating DENN/MADD expression levels and its subsequent effects on TNF-α-induced cell death. Using bioinformatics analyses followed by luciferase reporter assays we showed that miR-181 interacts with the 3' UTR of DENN/MADD transcripts. miR-181 overexpression also led to decreased endogenous DENN/MADD mRNA levels in L929 murine fibroblasts. Flow cytometric analysis of miR-181 transfected cells showed this miRNA accentuates mitochondrial membrane potential loss caused by TNF-α. These findings were associated with enhanced apoptosis of L929 cells following TNF-α treatment. Overall, these data point to the potential role of miR-181 in regulating TNF-α pro-death signaling, which could be of importance from pathogenesis and therapeutic perspectives in inflammatory disorders associated with tissue degeneration and cell death.

Enhancement of Neural Stem Cell Survival, Proliferation, Migration, and Differentiation in a Novel Self-Assembly Peptide Nanofibber Scaffold.

Considerable efforts have been made to combine biologically active molecules into the self-assembling peptide in order to improve cells growth, survival, and differentiation. In this study, a novel three-dimensional scaffold (RADA4GGSIKVAV; R-GSIK) was designed by adding glycine and serine between RADA4 and IKVAV to promote the strength of the peptide. The cell adhesion, viability, proliferation, migration, and differentiation of rat embryonic neural stem cells (NSCs) in R-GSIK were investigated and compared to laminin-coated, two-dimensional, and Puramatrix cultures. The scanning electron microscopy studies of the R-GSIK showed an open porous structure and a suitable surface area available for cell interaction. R-GSIK promoted the cell adhesion, viability, proliferation, and migration compared to the other cultures. In addition, the R-GSIK enhanced NSCs differentiation into neuronal cells. The NSCs injected in R-GSIK had a lower glial differentiation rate than in the Puramatrix. The results suggest that R-GSIK holds great promise for cell therapies and neuronal tissue repair.