Response Of Neural Stem Cells Research Articles

Essential role of p21Waf1/Cip1 in the modulation of post-traumatic hippocampal Neural Stem Cells response

BackgroundTraumatic Brain Injury (TBI) represents one of the main causes of brain damage in young people and the elderly population with a very high rate of psycho-physical disability and death. TBI is characterized by extensive cell death, tissue damage and neuro-inflammation with a symptomatology that varies depending on the severity of the trauma from memory loss to a state of irreversible coma and death. Recently, preclinical studies on mouse models have demonstrated that the post-traumatic adult Neural Stem/Progenitor cells response could represent an excellent model to shed light on the neuro-reparative role of adult neurogenesis following damage. The cyclin-dependent kinase inhibitor p21Waf1/Cip1 plays a pivotal role in modulating the quiescence/activation balance of adult Neural Stem Cells (aNSCs) and in restraining the proliferation progression of progenitor cells. Based on these considerations, the aim of this work is to evaluate how the conditional ablation of p21Waf1/Cip1 in the aNSCS can alter the adult hippocampal neurogenesis in physiological and post-traumatic conditions.MethodsWe designed a novel conditional p21Waf1/Cip1 knock-out mouse model, in which the deletion of p21Waf1/Cip1 (referred as p21) is temporally controlled and occurs in Nestin-positive aNSCs, following administration of Tamoxifen. This mouse model (referred as p21 cKO mice) was subjected to Controlled Cortical Impact to analyze how the deletion of p21 could influence the post-traumatic neurogenic response within the hippocampal niche.ResultsThe data demonstrates that the conditional deletion of p21 in the aNSCs induces a strong increase in activation of aNSCs as well as proliferation and differentiation of neural progenitors in the adult dentate gyrus of the hippocampus, resulting in an enhancement of neurogenesis and the hippocampal-dependent working memory. However, following traumatic brain injury, the increased neurogenic response of aNSCs in p21 cKO mice leads to a fast depletion of the aNSCs pool, followed by declined neurogenesis and impaired hippocampal functionality.ConclusionsThese data demonstrate for the first time a fundamental role of p21 in modulating the post-traumatic hippocampal neurogenic response, by the regulation of the proliferative and differentiative steps of aNSCs/progenitor populations after brain damage.

CpG methylation changes in human mesenchymal and neural stem cells in response to in vitro niche modifications

Stem cell therapies hold promise in addressing the burden of neurodegenerative diseases with human embryonic neural stem cells (hNSC-H9s) and bone marrow-derived human mesenchymal stem cells (hMSCs) as viable candidates. The induction of hMSC neurospheres (hMSC-IN) generate a more lineage-restricted common neural progenitor-like cell population, potentially tunable by heparan sulfate proteoglycans (HSPGs). We examined CpG (5 mC) site methylation patterns using Illumina Infinium 850 K EPIC arrays in hNSC-H9, hMSCs and hMSC-IN cultures with HSPG agonist heparin at early and late phases of growth. We identified key regulatory CpG sites in syndecans (SDC2; SDC4) that potentially regulate gene expression in monolayers. Unique hMSC-IN hypomethylation in glypicans (GPC3; GPC4) underscore their significance in neural lineages with Sulfatase 1 and 2 (SULF1 &2) CpG methylation changes potentially driving the neurogenic shift. hMSC-INs methylation levels at SULF1 CpG sites and SULF2:cg25401628 were more closely aligned with hNSC-H9 cells than with hMSCs. We further suggest SOX2 regulation governed by lncSOX2-Overall Transcript (lncSOX2-OT) methylation changes with preferential activation of ENO2 over other neuronal markers within hMSC-INs. Our findings illuminate epigenetic dynamics governing neural lineage commitment of hMSC-INs offering insights for targeted mechanisms for regenerative medicine and therapeutic strategies.

Response of Neural Stem Cells to Developmental, Homeostatic and Aging cues

Response of Neural Stem Cells to Developmental, Homeostatic and Aging cues

Deletion of the adaptor protein p66ShcA prevents the apoptosis of neural stem cells in response to mitogen deprivation

AbstractBackgroundAlzheimer’s disease (AD) has been associated with altered hippocampal neurogenesis and the depletion of the neural stem cell (NSC) niche. NSCs rely on cues from their extracellular environment to mediate their survival and maturation. The Shc adaptor family mediates this mitogenic signaling in response to a wide range of growth factors and is necessary for embryonic neurogenesis. A member of this family, p66ShcA has been found to antagonize mitogenic signalling, however its role in neurogenesis has not been established. Moreover, the genetic ablation of p66ShcA, has a been reported to reverse cognitive decline in mouse models of AD, despite its minimal expression in mature neurons.MethodTo evaluate p66ShcA’s role in the fate of NSCs, we generated and characterized a p66ShcA‐null murine NSC line (p66KO‐NSC). The NSCs were characterized by assessing their differentiation competency and viability in response to alterations in epidermal growth factor (EGF) signalling.ResultThe p66KO‐NSCs exhibited enhanced neurogenesis in response to EGF withdrawal, relative to WT NSCs. The pharmacologic inhibition of either EGFR or ERK induced the apoptosis of the WT NSCs via the release of cytochrome C and caspase activation. Surprisingly, the inhibition of EGFR or ERK had a negligible effect on the survival of the p66KO‐NPCs and instead induced their neuronal differentiation.Conclusionp66ShcA mediates NSC apoptosis in response to the loss of mitogenic cues. The deletion of p66ShcA increases neurogenesis by facilitating the survival of NSCs during their neuronal differentiation.

Injury primes mutation-bearing astrocytes for dedifferentiation in later life

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.

Electronic to ionic transduction of the electric field applied to PEDOT:PSS substrates to the cell cultures on top

The potential of the electrical double layer (EDL) formed at the interface between cross-linked poly (3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) and phosphate-buffered saline (PBS) was measured with respect to a reference Ag/AgCl electrode, yielding a value of ca. 300 mV, which corresponds to a work function of 4.9 eV. More importantly, we report that the application of a voltage along the PEDOT:PSS substrate induces a modification of the EDL, which mirrors the potential applied to the PEDOT:PSS underneath. This is translated into an ionic electric field, tangential to the interface that images the electric field applied to the PEDOT:PSS. We propose that this modification of the EDL, via application of the electrical field away from the cell culture medium, is at origin of the neural stem cell response to that field, when cultured on top of PEDOT:PSS. Despite the comparatively low value of the Debye length (estimated around 1 nm) with respect to the much larger cell to PEDOT:PSS surface distance, we believe that the perturbation of the EDL is the likely source of the increase of neuronal differentiation of the neural stem cells. We discuss other possible implications of that EDL modulation.

The Serine Protease Homolog, Scarface, Is Sensitive to Nutrient Availability and Modulates the Development of the Drosophila Blood-Brain Barrier.

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as “brain sparing.” However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood–brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood–brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood–brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB.

Orexin-A Attenuates Inflammatory Responses in Lipopolysaccharide-Induced Neural Stem Cells by Regulating NF-KB and Phosphorylation of MAPK/P38/Erk Pathways.

BackgroundNeuronal damage is the main cause of neurological diseases. Neural stem cells (NSCs) have the functions of cell repair and replacement of neurons, secretion of neurotrophic factors, and immune regulation of the neural microenvironment.ObjectivePrevious study found that Orexin-A had a protective effect on neurons in the central nervous system, but it is lacking in making great efforts on the function of Orexin-A on NSCs. This study aimed to investigate the anti-inflammatory responses and signaling mechanisms of Orexin-A on lipopolysaccharide (LPS)-induced NSCs.MethodsQuantitative real-time polymerase chain reaction was used to detect the mRNA level. Signaling pathway-related protein expression was detected by Western blot. The proliferation and migration of NSCs were investigated by Cell Counting Kit-8 (CCK-8) detection kit and transwell assay. Besides, the staining of hematoxylin and eosin (HE) was performed to study the morphology of cell.ResultsOrexin-A decreased the pro-inflammatory cytokines of IL-1β, TNF-α, and IL-6 induced by LPS by regulating nuclear factor-k-gene binding (NF-kB) and phosphorylation of P38/Erk-mitogen-activated protein kinases (MAPKs) pathways, but not p-JNK signaling.ConclusionOur findings indicate that Orexin-A can alleviate the inflammatory response of NSC. It can provide beneficial help in neural stem cell therapy applications.

Mouse Models for Studying Hippocampal Adult Neural Stem Cell Biology.

The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells (NSCs), are important for learning and memory as well as mood control. During aging, the number and responses of NSCs to neurogenic stimuli diminish, leading to decreased neurogenesis and age-associated cognitive decline and psychiatric disorders. Thus, adult hippocampal neurogenesis has been the subject of intense investigation, generating both excitement and controversy. Identifying the core molecular machinery responsible for NSC preservation is of fundamental importance if we are to use neurogenesis to halt or reverse hippocampal age-related pathology. Here, we briefly overview themost frequently used mouse models to study hippocampal neurogenesis and then focus on a unique mouse model that allows NSC-specific studies based on their unique expression of lunatic fringe (Lfng). The Lfng-eGFP and Lfng(BAC)-CreERT2;RCL-tdT transgenic mice provide us with an excellent tool to resolve long-standing questions regarding the properties of NSCs, such as their specific molecular composition, potency, and plasticity, in isolation from any other cell in the hippocampal neurogenic niche.

SUN-LB56 Steroid and Sex Specific Responses of Neural Stem Cells to Prenatal Dexamethasone versus Betamethasone Administration

Synthetic glucocorticoids (sGCs) are widely administered to pregnant women for their anti-inflammatory, immunosuppressive and organ maturation properties. Worldwide, Dexamethasone (Dex) and Betamethasone (Beta) are the two most commonly administered prenatal sGCs to reduce morbidity and mortality associated with respiratory distress, intraventricular hemorrhage and necrotizing enterocolitis. Preterm administration of sGCs is associated with reduced birthweight and increased risk for hypertension, cardiovascular, metabolic, and neurological problems later in life. Adverse neurological outcome has been shown to depend on the type of sGCs used, the dose, timing of sGCs administration and sex. We have previously shown that the glucocorticoid receptor (GR) is expressed in the developing brain in stem and progenitor cells, neurons and glia from early developmental stages, and that prenatal Dex alters neural stem cell (NSC) biology and the developmental trajectory of the cerebral cortex, hypothalamus and adult behavior. To identify the molecular and cellular basis of the sex and steroid specific responses in the developing brain, we compared the consequence of Dex versus Beta exposure on embryonic cerebral cortical NSC biology. Murine NSC were isolated from the E14.5 cerebral cortex and exposed to 10-7 M Dex, 10-7 M Beta, or Vehicle for 4 or 24 hours and the immediate and long-term impact on transcription, proliferation and neuronal, glial and oligodendrocyte differentiation examined. Affymetrix complete genome transcriptional analyses reveal sex specific responses to Dex versus Beta within 4 hours. At >+/-1.5-fold change 548 genes were differentially regulated by Dex, 452 by Beta and 256 were altered by both Dex and Beta (P < 0.05). Distinct sex specific responses to Dex versus Beta were observed. At >+/-2-fold change 126 genes were significantly different in the Dex versus Beta female transcriptome, 146 in the male transcriptome with 18 genes unique to both male and female transcriptome. Ingenuity Pathway Analysis revealed that the most significantly altered pathway altered (Z score >2) with both sGCs is Inositol Phosphate metabolism. Cardiac hypertrophy, Tec kinase, and Th1 pathways were unique to Beta stimulation, whereas Melatonin, Neuropathic Pain and IL6 signaling pathways were specific to Dex stimulation. Both Dex and Beta significantly alter genes implicated in proliferation and differentiation as also described in other studies, therefore the biological response of NSC to sGCs stimulation was compared. Only Dex significantly decreased the rate of proliferation over a 72 hour. In-vitro differentiation studies reveal that both Dex and Beta reduced oligodendrocyte differentiation without altering neuronal differentiation when cells were exposed to sGCs as progenitors. However, when cells were exposed to sGCs during differentiation, Dex increased oligodendrocyte and neuronal maturation while Beta only increased oligodendrocyte differentiation. These results reveal gene targets, cellular pathways and processes that are differentially altered by prenatal Dex versus Beta exposure. Prenatal sGCs administration provides clear benefits for neonatal outcome, however, a detailed understanding of their targets in the brain is required to identify alternative sGCs drug regimens to reduce adverse neurological effects. Our finds may provide insights into the sex specific neurological outcomes observed in children exposed to sGCs in-utero.

Distinct modes of death in human neural stem and glioblastoma cells irradiated with carbon-ion radiation and gamma-rays

Purpose: Accumulated damage in neural stem cells (NSCs) during brain tumor radiotherapy causes cognitive dysfunction to the patients. Carbon-ion radiotherapy can reduce undesired irradiation of normal tissues more efficiently than conventional photon radiotherapy. This study elucidates the responses of NSCs to carbon-ion radiation.Methods: Human NSCs and glioblastoma A-172 cells were irradiated with carbon-ion radiation and γ-rays, which have different linear-energy-transfer (LET) values of 108 and 0.2 keV/μm, respectively. After irradiation, growth rates were measured, apoptotic cells were detected by flow cytometry, and DNA synthesizing cells were immunocytochemically visualized.Results: Growth rates of NSCs and A-172 cells were decreased after irradiation. The percentages of apoptotic cells were remarkably increased in NSCs but not in A-172 cells. In contrast, the fractions of DNA synthesizing A-172 cells were decreased in a dose-dependent manner. These results indicate that apoptosis induction and DNA synthesis inhibition contribute to the growth inhibition of NSCs and glioblastoma cells, respectively. In addition, high-LET carbon ions induced more profound effects than low-LET γ-rays.Conclusions: Apoptosis is an important clinical target to protect NSCs during brain tumor radiotherapy using carbon-ion radiation as well as conventional X-rays.

Chronic poly-drug administration damages adult mouse brain neural stem cells

Cocaine and ethanol are two commonly co-abused substances; however, the neuropathology following chronic dual consumption is poorly understood. Neural stem cells (NSCs) are a subpopulation of cells within the adult brain that are integral to brain maintenance and repair making them an appealing target to reverse neurodegeneration associated with abused substances. Yet, knowledge about NSC response to chronic poly-drug administration of ethanol and cocaine is minimal. Here, we developed a novel chronic poly-drug administration paradigm of ethanol and cocaine using a transgenic mouse model to trace endogenous NSC survival and differentiation in three brain regions from both male and female mice. We report significant and distinct patterns of NSC survival and differentiation among brain regions, as well as between sexes. Additionally, poly-drug administration had synergistic effects on NSC survival. Altered cognitive and hedonic behaviors were also observed, however the extent of these behavioral changes was not proportional to the NSC changes. With this mouse model we can effectively examine cognitive and behavioral changes and correlate them with pathological changes in the brain in response to chronic poly-drug administration, which is of great value in understanding the progression of neurodegeneration in polysubstance use disorders and evaluation potential therapeutics on neuroregeneration.

Abstract 3934: Bcl2-expressing quiescent type B neural stem cells in the SVZ are resistant to concurrent temozolomide/X-irradiation

Abstract The subventricular zone (SVZ) is the largest source of neural stem cells (NSCs) in the adult brain. Emerging research indicates that NSCs within the SVZ may be cells of origin for WHO grade IV astrocytoma (glioblastoma, GBM)1. GBM consists of multiple fractions of proliferative and/or quiescent stem-like cells that are thought to be lineally related. GBM located adjacent to the SVZ are very resistant to standard of care concurrent temozolomide (TMZ)/X-irradiation (XRT) therapy, a consequence, it is hypothesized, of their NSC origin2. An important, unanswered question is the origin of this resistance. While a significant effort has been undertaken to study proliferating cells, the origins of quiescent cell resistance are not well understood. Normal NSCs adjacent to the SVZ are mainly quiescent. We rationalized that a fundamental understanding of the response of quiescent NSCs to TMZ/XRT would be informative and aid in our understanding of GBM resistance. For 5 consecutive days cohorts of C57BL/6 mice were administered TMZ (0 or 50 mg.kg i.p.). One hr later 0 or 2 Gy was administered to the brain. Transcardial perfusion was performed on day 6 for half the mice. The remaining mice received adjuvant TMZ (100 mg/kg) or vehicle on days 19-22 and transcardial perfusion was performed on day 82. 10 µm coronal brain sections were obtained and immunostained for well characterized markers of type B NSCs (GFAP and Sox2) and type A neuroblasts (Dcx). Immunofluorescence was imaged using a Leica Aperio Versa 200 slide scanning microscope. Cell Profiler software was used to quantify type B and A cells in the SVZ in all cohorts. Proliferating type A cells were exquisitely sensitive to 5 days of concurrent TMZ/XRT treatment whereas quiescent NSCs located within 30 µm of a dorsal or dorsolateral ventricle were very resistant. NSCs in mice exposed to concurrent and adjuvant therapy were also resistant and importantly, able to repopulate type A cells to sham/control levels. 53BP1 foci formation, a surrogate for DNA DSBs, was quantified in Sox2- and Dcx-expressing cells using confocal microscopy following a single TMZ/XRT exposure. Foci formation, measured 6 min to 24 hrs after TMZ/XRT, was not statistically different between cell types (P&amp;gt;0.05). Because TMZ/XRT induced an apoptotic response in A but not in B cells, as marked by cleaved Caspase-3 staining, we investigated expression of Bax and Bcl2 on a per cell basis. Bax expression was not significantly different for type A or B cells (P&amp;gt;0.05). In contrast, type B NSCs expressed 5-fold more Bcl2 than type A neuroblasts (P&amp;lt; 0.001). In conclusion, we demonstrate that type A neuroblasts are sensitive to TMZ/XRT but can be repopulated by inherently resistant type B NSCs given sufficient time. The resistance of quiescent NSCs to TMZ/XRT is associated with high basal expression of anti-apoptotic proteins. 1Lee et al Nature 2018, 560:243-47; 2Smith et al J Neurooncol 2016, 128:207-16 Citation Format: Brent D. Cameron, Geri Traver, Joseph T. Roland, Daniel Dean, Levi Johnson, Kelli L. Boyd, Rebecca A. Ihrie, Jialiang Wang, Michael L. Freeman. Bcl2-expressing quiescent type B neural stem cells in the SVZ are resistant to concurrent temozolomide/X-irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3934.

Differentiation Induction as a Response to Irradiation in Neural Stem Cells In Vitro

Radiotherapy plays a significant role in brain cancer treatment; however, the use of this therapy is often accompanied by neurocognitive decline that is, at least partially, a consequence of radiation-induced damage to neural stem cell populations. Our findings describe features that define the response of neural stem cells (NSCs) to ionizing radiation. We investigated the effects of irradiation on neural stem cells isolated from the ventricular-subventricular zone of mouse brain and cultivated in vitro. Our findings describe the increased transcriptional activity of p53 targets and proliferative arrest after irradiation. Moreover, we show that most cells do not undergo apoptosis after irradiation but rather cease proliferation and start a differentiation program. Induction of differentiation and the demonstrated potential of irradiated cells to differentiate into neurons may represent a mechanism whereby damaged NSCs eliminate potentially hazardous cells and circumvent the debilitating consequences of cumulative DNA damage.

Loss of lipoprotein receptor LRP1 ablates neural stem cell migration to ischemic lesions

Brain damage due to stroke is a leading cause of death and long‐term disability in the US. Treatment options are limited, necessitating a better understanding of reparative and pathological processes after stroke. Ischemia stimulates the proliferation and migration of neural stem cells (NSCs) from the subventricular zone toward the ischemic lesion in mice. During this time, the migrating NSCs differentiate into GFAP positive cells and pro‐reparative glia. While the importance of this process is not fully elucidated, it is clear that limiting NSC response increases lesion size and worsens behavioral outcomes after ischemic stroke. We tested the effect of low‐density lipoprotein receptor related protein 1 (LRP1) on modulating NSC response and differentiation, and found that virtually every aspect of NSC physiology we tested was altered after deletion of LRP1; increased glial differentiation, proliferation, and self‐renewal were measured. After middle cerebral artery occlusion in mice, we observed a marked reduction in the migration of neuroblasts toward the site of the lesion, which correlated with increased gliosis and worsened behavioral outcomes. Loss of LRP1 also caused reduction in expression of the chemokine receptor CXCR4, which could account for the loss of migration. Ongoing studies involve elucidating the mechanism by which LRP1 regulates CXCR4 expression as well as understanding the effect on pro‐reparative processes after stroke.Support or Funding InformationThis research is supported by a pilot grant from the William and Ella Owen's Medical Research Foundation, and a Veteran's Health Administration Career Development Award K2BX003240.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Response of neural stem cells to ionizing radiation

Adult neurons are believed to be in a state of growth arrest. The generation of neurons is complete at the time of birth in most of the brain regions. However neurogenesis is present through life in the dentate gyrus of hippocampus and the lateral ventricles due to the presence of neural stem cells (NSC). This postnatal neurogenesis in hippocampus plays a critical role in cognitive development mainly in learning and memory functions. NSC are self-renewing, multipotent cells that generate the neurons and glia of the nervous system. Due to their high proliferation, NSC are highly sensitive to ionizing radiation. This review describes the current knowledge on impact of ionizing radiation on neural stem cells biology. Widening the knowledge of mechanisms involved in radiation-induced neurotoxicity at the level of NSC may help to overcome in the future the side effects occurring after anti-cancer therapies of the brain and help to protect and maintain neurogenesis.

Activation of GPR55 induces neuroprotection of hippocampal neurogenesis and immune responses of neural stem cells following chronic, systemic inflammation

New neurons are continuously produced by neural stem cells (NSCs) within the adult hippocampus. Numerous diseases, including major depressive disorder and HIV-1 associated neurocognitive disorder, are associated with decreased rates of adult neurogenesis. A hallmark of these conditions is a chronic release of neuroinflammatory mediators by activated resident glia. Recent studies have shown a neuroprotective role on NSCs of cannabinoid receptor activation. Yet, little is known about the effects of GPR55, a candidate cannabinoid receptor, activation on reductions of neurogenesis in response to inflammatory insult. In the present study, we examined NSCs exposed to IL-1β in vitro to assess inflammation-caused effects on NSC differentiation and the ability of GPR55 agonists to attenuate NSC injury. NSC differentiation and neurogenesis was determined via immunofluorescence and flow cytometric analysis of NSC markers (Nestin, Sox2, DCX, S100β, βIII Tubulin, GFAP). GPR55 agonist treatment protected against IL-1β induced reductions in neurogenesis rates. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55−/− mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4 µg/kg/day). Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55−/− animals manifesting in a prolonged inflammatory response (IL-1β, IL-6, TNFα) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult.

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site - demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Inhibition of Notch1 Signaling at the Subacute Stage of Stroke Promotes Endogenous Neurogenesis and Motor Recovery After Stroke.

Background and Purpose: It is still not clear whether Notch1 signaling inhibition can promote functional outcomes after stroke, given that it plays time-dependent roles in the sequential process of endogenous neurogenesis. The purpose of this study was to identify the appropriate time frame for Notch1 signaling inhibition according to the temporal evolution of Notch1 signaling activation and the responses of neural stem cells (NSCs), in order to target it for therapeutic intervention and stimulate neurorestorative strategies after stroke.Methods: Sprague-Dawley (SD) rats were subjected to 90-min of middle cerebral artery occlusion (MCAO). Rats were sacrificed before, and at day 1, day 2, day 3, day 4, and day 7 after ischemia for immunohistochemical analysis of the Notch intracellular domain (NICD), Nestin and doublecortin (Dcx). Next, MCAO rats were treated with the γ-secretase inhibitor N-[N-(3,5-di uorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT) or with saline at day 4 after ischemia, and subsequently evaluated with behavioral test analysis and magnetic resonance imaging (MRI). The rat brains were then harvested for immunohistochemical analysis of Dcx, NeuN and myelin basic protein (MBP) at 2, 3, 4, and 8 weeks.Results: Notch1 signaling was maximally activated at day 3 after ischemia in parallel with the temporal evolution of NSCs. Inhibiting Notch1 signaling at day 4 after reperfusion with DAPT further promoted recovery of MRI parameters of the corticospinal tract (CST) and the functional outcomes, concomitantly with an increase in neuroblasts, their migration to the ischemic boundary, and potential differentiation to mature neurons, as well as the amelioration of axonal bundle integrity.Conclusion: Inhibition of Notch1 signaling at the subacute stage of stroke could maximally promote endogenous neurogenesis and axonal reorganization.

The interplay of peptide affinity and scaffold stiffness on neuronal differentiation of neural stem cells

Cells are sensitive to physical cues in their environment, such as the stiffness of the substrate, peptide density, and peptide affinity. Understanding how neural stem cells (NSCs) sense and respond to these matrix cues has the potential to improve disease outcome, particularly if a regenerative response can be exploited. While the material properties are known to influence other stem cells, little is known about how NSC differentiation is altered by this interplay of mechanical, or bulk properties, with peptide concentration and affinity, or microscale properties. We are interested in the combined effect of bulk and microscale features in an in vitro hydrogel model and therefore we investigated NSC differentiation by focusing on integrin interactions via RGD peptide affinity and concentration. Our studies demonstrated that the peptide concentration affected adhesion as there were more cells on scaffolds with 1 mM RGD than 2.5 mM RGD. The hydrogel stiffness affected neurite length in differentiating NSCs, as 0.1–0.8 kPa substrates promoted greater neurite extension than 4.2–7.9 kPa substrates. The NSCs differentiated towards β-ΙΙΙ tubulin positive cells on scaffolds with RGD after 7 days and those scaffolds containing 1 mM linear or cyclic RGD had longer neurite extensions than scaffolds containing 0.1 or 2.5 mM RGD. While peptide affinity had a lesser effect on the NSC response in our hydrogel system, blocking actin, myosin II, or integrin interactions resulted in changes to the cell morphology and focal adhesion assembly. Overall, these results demonstrated NSCs are more responsive to a change in tissue stiffness than peptide affinity in the range of gels tested, which may influence design of materials for neural tissue engineering.