Survival Of Neural Precursors Research Articles

Development and evaluation of hyaluronan nanocomposite conduits for neural tissue regeneration

Hyaluronan-based hydrogels are among the most promising neural tissue engineering materials because of their biocompatibility and the immunomodulation capabilities of their degradation byproducts. Despite these features, the problems related to their handling and mechanical properties have not yet been solved. In the present work it is proposed to address these drawbacks through the development of nanohybrid materials in which different nanometric phases (carbon nanotubes, mesoporous silica nanoparticles) are embedded in a crosslinked hyaluronan matrix. These nanohybrid matrices were next processed in the shape of cylindrical conduits aimed at promoting and improving neural stem cell differentiation and regeneration in neural tracts. These constructs could be of use specifically for peripheral nerve regeneration. Results of the study show that the inclusion of the different phases improved physico-chemical features of the gel such as its relative electrical permittivity, water intake and elastic modulus, giving hints on how the nanometric phase interacts with hyaluronan in the composite as well as for their potential in combined therapeutic approaches. Regarding the in vitro biological behavior of the hybrid tubular scaffolds, an improved early cell adhesion and survival of Schwann cells in their lumen was found, as compared to conduits made of pure hyaluronan gels. Furthermore, the differentiation and survival of neural precursors was not compromised, despite alleged safety concerns.

195 Development of a fetal porcine model for congenital viral infections in the developing CNS

To develop a porcine model for analysis of the effects of congenital viral infections on CNS and spinal (CSF) derived fetal neural precursor (NPC) and neural progenitor cells. Isolation of NPCs from the subventricular zone (SVZ) of the porcine brain or CSF derived from premature (day 104) and post-term (day 135) Cesarean-delivered piglets were plated in 12-well plates. Nonadherent plates were used to generate neurospheres with self-renewal capacity and for long term passages. After a 2-day incubation for neurosphere formation and removal of apoptotic debris, cells were seeded in various conditions including incubation in (1) 10% FBS, (2) Rock Inhibitor (RI)-specific inhibitor of apoptosis, (3) FBS and RI, and (4) Neurobasal media alone (control). After purification and identification of NPCs, cells were then infected with low MOI of clinically isolated first passage ZIKV HN16, then fixed and stained with Flavivirus-specific antibody (MAb 4G2) followed by FITC labeled secondary antibody staining visualization under fluorescence microscopy. Gene expression studies were also performed from RNA from ZIKV infected and mock infected samples to determine effects on genes related to NPC self-renewal and survival. The brain derived cells cultured for 4 days showed three distinct populations including NSCs, NPCs, and a mixed NSC/NPC population. After 21 days of culture, cells shifted from an NSC population toward later within the NPC lineage. Both brain and CSF cells showed relatively equal permissivity to ZIKV viral infection (Fig.1). Moreover, there was no difference in Vimentin gene expression in ZIKV infected cells, while SOX-2 and Nestin gene expression following ZIKV infection was significantly lower (p<0.05; Fig.2). The findings that Nestin expression is downregulated when comparing brain and CSF cells, particularly after ZIKA infection, is of interest since Nestin is postulated to play a role in axonal growth. This study demonstrated the ability to utilize porcine fetal NPCs in a safe and effective manner for the study of NPC development and effects of viral infections

Huwe1 as a therapeutic target for neural injury.

The ubiquitin-proteasome system (UPS) regulates many cellular processes, including protein stability, cell cycle control, DNA repair, transcription, signal transduction, and protein trafficking. In fact, UPS plays a key role in various stress conditions such as ischemia, glutamate toxicity, Alzheimer's disease, and Parkinson's disease. Huwe1, a homologous to E6-AP carboxy terminus (HECT) domain ubiquitin ligase, is now being regarded as a vital protein involved in neural stem cell differentiation, adult neurogenesis, and the DNA damage response pathway. In response to DNA damage, Huwe1 may have a dual function in arresting DNA replication and in ending checkpoint signaling. The proliferation and differentiation of neural stem cells regulated by Huwe1-mediated Notch signaling could also play an important role in neural protection following neural injury. Considering Huwe1 is required for neural precursor survival and the regulation of the DNA damage response pathway, there is growing evidence and considerable hope that Huwe1 might be a therapeutic target for neural injury.

Lysophosphatidic acid activates β-catenin/T cell factor signaling, which contributes to the suppression of apoptosis in H19-7 cells

Lysophosphatidic acid (LPA) is a lipid growth factor that regulates diverse cell functions, including cell proliferation, survival and apoptosis. LPA has been demonstrated to be involved in the regulation of cortical neurogenesis by increasing the survival of neural precursors. Previously, we reported that LPA stimulated the inactivation of glycogen synthase kinase 3 (GSK3) via the G protein-coupled LPA1 and LPA2 receptors, by which apoptosis is suppressed in H19-7 cells [an embryonic hippocampal progenitor cell (HPC) line]. Increasing numbers of studies have demonstrated that certain G protein-coupled receptors activate β-catenin/T cell factor (TCF) signaling independently of Wnt, which is involved in cell fate determination, cell proliferation and cell survival. To determine whether LPA activates β-catenin-mediated transcriptional activation pathways and whether β-catenin/TCF signaling is involved in neurogenesis by controlling the survival of neural precursors, β-catenin/TCF signaling cascades induced by LPA were investigated in the HPCs. Activation of β-catenin/TCF signaling was determined by the nuclear translocation of β-catenin and the transcriptional activation of a TCF reporter gene. The activation of β-catenin/TCF signaling was blocked by pertussis toxin (PTX) and a protein kinase C (PKC) inhibitor. The expression of a constitutively active mutated form of GSK3β activated β-catenin/TCF signaling to comparable levels to those induced by LPA, and protected against apoptosis in differentiating H19-7 cells. These results showed that LPA activates β-catenin/TCF signaling in a PTX- and PKC-dependent manner, which contributes to LPA-induced cell survival in the HPCs. Activation of β-catenin/TCF signaling by LPA may be involved in neurogenesis by controlling the survival of neural precursors.

Effective long-term immunosuppression in rats by subcutaneously implanted sustained-release tacrolimus pellet: Effect on spinally grafted human neural precursor survival

Achievement of effective, safe and long-term immunosuppression represents one of the challenges in experimental allogeneic and xenogeneic cell and organ transplantation. The goal of the present study was to develop a reliable, long-term immunosuppression protocol in Sprague–Dawley (SD) rats by: 1) comparing the pharmacokinetics of four different subcutaneously delivered/implanted tacrolimus (TAC) formulations, including: i) caster oil/saline solution, ii) unilamellar or multilamellar liposomes, iii) biodegradable microspheres, and iv) biodegradable 3-month lasting pellets; and 2) defining the survival and immune response in animals receiving spinal injections of human neural precursors at 6weeks to 3months after cell grafting. In animals implanted with TAC pellets (3.4mg/kg/day), a stable 3-month lasting plasma concentration of TAC averaging 19.1±4.9ng/ml was measured. Analysis of grafted cell survival in SOD+ or spinal trauma-injured SD rats immunosuppressed with 3-month lasting TAC pellets (3.4–5.1mg/kg/day) showed the consistent presence of implanted human neurons with minimal or no local T-cell infiltration. These data demonstrate that the use of TAC pellets can represent an effective, long-lasting immunosuppressive drug delivery system that is safe, simple to implement and is associated with a long-term human neural precursor survival after grafting into the spinal cord of SOD+ or spinal trauma-injured SD rats.

A zebrafish model of lethal congenital contracture syndrome 1 reveals Gle1 function in spinal neural precursor survival and motor axon arborization

In humans, GLE1 is mutated in lethal congenital contracture syndrome 1 (LCCS1) leading to prenatal death of all affected fetuses. Although the molecular roles of Gle1 in nuclear mRNA export and translation have been documented, no animal models for this disease have been reported. To elucidate the function of Gle1 in vertebrate development, we used the zebrafish (Danio rerio) model system. gle1 mRNA is maternally deposited and widely expressed. Altering Gle1 using an insertional mutant or antisense morpholinos results in multiple defects, including immobility, small eyes, diminished pharyngeal arches, curved body axis, edema, underdeveloped intestine and cell death in the central nervous system. These phenotypes parallel those observed in LCCS1 human fetuses. Gle1 depletion also results in reduction of motoneurons and aberrant arborization of motor axons. Unexpectedly, the motoneuron deficiency results from apoptosis of neural precursors, not of differentiated motoneurons. Mosaic analyses further indicate that Gle1 activity is required extrinsically in the environment for normal motor axon arborization. Importantly, the zebrafish phenotypes caused by Gle1 deficiency are only rescued by expressing wild-type human GLE1 and not by the disease-linked Fin(Major) mutant form of GLE1. Together, our studies provide the first functional characterization of Gle1 in vertebrate development and reveal its essential role in actively dividing cells. We propose that defective GLE1 function in human LCCS1 results in both neurogenic and non-neurogenic defects linked to the apoptosis of proliferative organ precursors.

Hormona de crecimiento: acciones y aplicaciones preventivas y terapéuticas

Growth hormone (GH) is a pleiotropic hormone, expressed at pituitary and peripheral level, which plays a number of different roles far beyond of those classically described. Among these effects it is remarkable the neurotropic role of GH: the hormone increases the proliferation and survival of neural precursors in response to neurological injuries. At the cardiovascular level, GH improves the lipidic profile and decreases the factors of cardiac risk; the hormone recovers the endothelial function, improves the cardiac function and potentiates revascularisation in ischemic territories. Differently to that occurring with autocrine GH, exogenous GH administration does not seem to be related to oncogenesis. According to its effects, there are multiple potential clinical applications of GH: acute treatment of brain injury, due to its antiapoptotic effect; central or peripheral neural regeneration; acute treatment of perinatal anoxia, prevention cerebral palsy; revascularisation of ischemic areas; decrease of the time of bone consolidation after a bone fracture; and torpid ulcer healing.

¿Existe envejecimiento precoz entre los consumidores de drogas de abuso?

The aging or senescence process that follows maturation is characterized by time-related functional decline due to genetic, biochemical, physiological and anatomical degeneration in tissues and organ systems with time. Oxidative damage to mitochondrial DNA (mtDNA) in the heart and brain is inversely related to maximum life span of mammals, suggesting that accumulation of mtDNA damage is involved in the various disorders associated with aging, cancer and neurodegeneration. The suppression of stem/progenitor cell proliferation also contributes to the aging process, by reducing tissue regeneration and repair and ultimately reducing longevity. Another important factor is the intracellular deposition of lipofuscin granules (age pigment), a non-degradable polymeric material accumulated within lysosomes, which ultimately exacerbate oxidative stress levels in senescent cells. Drugs of abuse can strongly contribute to these senescence accelerating factors in the brain. Methylenedioxymethamphetamine ('ecstasy') and methamphetamine were shown to promote deletions in brain mtDNA. Concerning stem/progenitor cells, it has been shown that several opiates and psychostimulants, including ecstasy, decrease the self-renewal capacity of the hippocampus by diminishing the rate of proliferation of neural progenitors and/or by impairing the long-term survival of neural precursors. Chronic alcohol consumption induces lipofuscin deposition in neurons and heart cells. These facts provide interesting hints on the potential of these drugs in accelerating brain senescence. While the extent and severity of the contribution of drugs of abuse for accelerated senescence remain uncertain, these putative aging effects add up to the dark side of drug addiction and undoubtedly require a strong research effort in the near future.

THE P53 FAMILY MEMBER, P63, REGULATES NEURAL PRECURSOR CELL SURVIVAL DURING CORTICAL DEVELOPMENT

Background: p63, a member of the p53 family of proteins, is involved in the regulation of naturally-occurring apoptosis in sympathetic neurons of the peripheral nervous system. Since data from our laboratory indicated that p63 is also expressed in stem cells and neurons within the developing brain, we hypothesized that p63 is involved in regulating the genesis and survival of developing neurons. Methods: As cortical neurogenesis is initiated at embryonic day 12, we knocked-down p63 levels in isolated murine cortical precursors byusing shRNA against p63 or by transfecting floxed-p63 precursors with Cre recombinase. We performed similar studies in vivo using in uteroelectroporation to express either p63 shRNA or Cre recombinase to acutely knockdown or genetically ablate p63. We then performed immunofluorescence for known markers of apoptosis, cell-division, and differentiation to assess the level of cell death, proliferation and neurogenesis. Results: Knock-down of p63 in vitro resulted in a 2-foldincrease in the death of precursors and neurons, associated with blunted neurogenesis but unaltered precursor proliferation. Coincident knock-down of p63 family members, p53, but not p73, rescued the elevated death suggesting that p63 and p53 antagonize each other to promote survival. Similar results were observed in vivo, where knockdown of p63 caused cell death and a decrease in the proportionof neurons in the cortical plate. Conclusions: These experiments indicate that p63 is required forthe survival of neural precursors and newly-born neurons, and for normal cortical development. Ongoing work will explore the environmental cues that regulate p63 during neurogenesis.

Regulation of neural precursor survival, proliferation and differentiation by BDNF

Regulation of neural precursor survival, proliferation and differentiation by BDNF

Binge administration of 3,4-methylenedioxymethamphetamine (“ecstasy”) impairs the survival of neural precursors in adult rat dentate gyrus

3,4-Methylenedioxymethamphetamine (MDMA) is a potent stimulant and hallucinogenic drug whose ability to regulate neurogenesis in the adult has not been previously investigated. We used 5′-bromo-2-deoxyuridine (BrdU) and Ki-67 as mitotic markers, and doublecortin (DCX) as a marker of immature neurons, to study proliferation, survival and maturation of adult-generated cells in the dentate gyrus (DG) of the hippocampus following binge administration of MDMA (8 injections of 5 mg/kg at 6 h intervals). The results showed that MDMA treatment did not affect cytogenesis in the DG, but significantly decreased the survival rate of cells incorporated after 2 weeks to the granular layer of the DG by ca. 50%, and of those remaining in the subgranular layer by ca. 30%. Two weeks after exposure to MDMA the length of dendritic arbors and the number of dendritic branches of immature DCX+ neurons were nearly identical to those of control rats, as was the level of colocalization of BrdU with DCX. These results demonstrate that binge MDMA administration does not affect the proliferation rates of progenitor cells in the DG, but has deleterious effects on adult neurogenesis by impairing the short-term survival of vulnerable neural precursors.

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors

Embryonic ectoderm is fated to become either neural or epidermal, depending on patterning processes that occur before and during gastrulation. It has been stated that epidermal commitment proceeds from a bone morphogenetic protein-4 (BMP-4)-dependent inhibition of dorsal ectoderm neuralization. We recently demonstrated that murine embryonic stem (ES) cells treated with BMP-4 undergo effective keratinocyte commitment and epidermogenesis. Focusing on the precise role of BMP-4 in the early choice between neural and epidermal commitment, we show here that BMP-4 treatment of ES cells leads to a dramatic apoptotic death of Sox-1+ neural precursors with concomitant epidermal engagement. In addition, neutralization of the Smad pathway prevents both the BMP-4 apoptotic process and the inhibition of neural differentiation. Our results suggest that, in mammals, BMP-4, as an active inducer of epidermal commitment, interferes with the survival of neural precursors through induction of their apoptotic cell death.

Dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) regulate apoptosis during neurogenesis by triggering the Akt signaling pathway in opposing ways

Dehydroepiandrosterone (DHEA) can function to protect neural precursors and their progeny targeted with toxic insults; however, the molecular mechanisms underlying the neuroprotective effects of DHEA are not understood. We cultured neural precursors from the embryonic forebrain of rats and examined the effects of DHEA and its sulfated derivative (DHEAS) on the activation of the serine-threonine protein kinase Akt, which is widely implicated in cell survival signaling. We found that DHEA activated Akt in neural precursor culture, in association with a decrease in apoptosis. In contrast, DHEAS decreased activated Akt levels and increased apoptosis. The effects of DHEA on neural cell survival and activation of Akt were not blocked by the steroid hormone antagonists flutamide and tamoxifen, but both were blocked by a PI3-K inhibitor, LY294002. These findings suggest that during neurogenesis in the developing cortex, DHEA and DHEAS regulate the survival of neural precursors and progeny through the Akt signaling pathway.

Gene targeting and development of the nervous system

Gene targeting provides a means of directly assaying the function of specific genes during mouse nervous system development. Generation of targeted mutant mice has provided the first evidence of developmental roles for genes whose function was suggested based on their expression, but for which appropriate assay systems were lacking. In other cases, where gene function was known, targeted mutations have revealed in which cell population, and at what developmental stage, particular genes are first indispensable. The existing targeted mutants suggest that an early mechanism of pattern formation in mammals involves regional control of proliferation or survival of neural precursors, and that later general functions, such as the control of differentiation of precursors, may be performed by different genes in distinct neural lineages. As many genes display complex temporal and spatial patterns of expression, analysis of the full range of functions of such genes will require the generation of a series of alleles, including stage- and tissue-specific mutations.