Neural Stem Cell Compartment Research Articles

PTBP1-mediated repression of neuron-specific CDC42 splicing constitutes a genomic alteration-independent, developmentally conserved vulnerability in IDH-wildtype glioblastoma.

Gene co-expression networks may encode hitherto inadequately recognized vulnerabilities for adult gliomas. By identifying evolutionally conserved gene co-expression modules around EGFR (EM) or PDGFRA (PM), we recently proposed an EM/PM classification scheme, which assigns IDH-wildtype glioblastomas (GBM) into the EM subtype committed in neural stem cell compartment, IDH-mutant astrocytomas and oligodendrogliomas into the PM subtype committed in early oligodendrocyte lineage. Here, we report the identification of EM/PM subtype-specific gene co-expression networks and the characterization of hub gene polypyrimidine tract-binding protein 1 (PTBP1) as a genomic alteration-independent vulnerability in IDH-wildtype GBM. Supervised by the EM/PM classification scheme, we applied weighted gene co-expression network analysis to identify subtype-specific global gene co-expression modules. These gene co-expression modules were characterized for their clinical relevance, cellular origin and conserved expression pattern during brain development. Using lentiviral vector-mediated constitutive or inducible knockdown, we characterized the effects of PTBP1 on the survival of IDH-wildtype GBM cells, which was complemented with the analysis of PTBP1-depedent splicing pattern and overexpression of splicing target neuron-specific CDC42 (CDC42-N) isoform. Transcriptomes of adult gliomas can be robustly assigned into 4 large gene co-expression modules that are prognostically relevant and are derived from either malignant cells of the EM/PM subtypes or tumor microenvironment. The EM subtype is associated with a malignant cell-intrinsic gene module involved in pre-mRNA splicing, DNA replication and damage response, and chromosome segregation, and a microenvironment-derived gene module predominantly involved in extracellular matrix organization and infiltrating immune cells. The PM subtype is associated with two malignant cell-intrinsic gene modules predominantly involved in transcriptional regulation and mRNA translation, respectively. Expression levels of these gene modules are independent prognostic factors and malignant cell-intrinsic gene modules are conserved during brain development. Focusing on the EM subtype, we identified PTBP1 as the most significant hub for the malignant cell-intrinsic gene module. PTBP1 is not altered in most glioma genomes. PTBP1 represses the conserved splicing of CDC42-N. PTBP1 knockdown or CDC42-N overexpression disrupts actin cytoskeleton dynamics, causing accumulation of reactive oxygen species and cell apoptosis. PTBP1-mediated repression of CDC42-N splicing represents a potential genomic alteration-independent, developmentally conserved vulnerability in IDH-wildtype GBM.

Low-density lipoprotein receptor-related protein-1 (LRP1) in the glial lineage modulates neuronal excitability.

The low-density lipoprotein related protein receptor 1 (LRP1), also known as CD91 or α-Macroglobulin-receptor, is a transmembrane receptor that interacts with more than 40 known ligands. It plays an important biological role as receptor of morphogens, extracellular matrix molecules, cytokines, proteases, protease inhibitors and pathogens. In the CNS, it has primarily been studied as a receptor and clearance agent of pathogenic factors such as Aβ-peptide and, lately, Tau protein that is relevant for tissue homeostasis and protection against neurodegenerative processes. Recently, it was found that LRP1 expresses the Lewis-X (Lex) carbohydrate motif and is expressed in the neural stem cell compartment. The removal of Lrp1 from the cortical radial glia compartment generates a strong phenotype with severe motor deficits, seizures and a reduced life span. The present review discusses approaches that have been taken to address the neurodevelopmental significance of LRP1 by creating novel, lineage-specific constitutive or conditional knockout mouse lines. Deficits in the stem cell compartment may be at the root of severe CNS pathologies.

Temporal and spatial stability of the EM/PM molecular subtypes in adult diffuse glioma.

Detailed characterizations of genomic alterations have not identified subtype-specific vulnerabilities in adult gliomas. Mapping gliomas into developmental programs may uncover new vulnerabilities that are not strictly related to genomic alterations. After identifying conserved gene modules co-expressed with EGFR or PDGFRA (EM or PM), we recently proposed an EM/PM classification scheme for adult gliomas in a histological subtype- and grade-independent manner. By using cohorts of bulk samples, paired primary and recurrent samples, multi-region samples from the same glioma, single-cell RNA-seq samples, and clinical samples, we here demonstrate the temporal and spatial stability of the EM and PM subtypes. The EM and PM subtypes, which progress in a subtype-specific mode, are robustly maintained in paired longitudinal samples. Elevated activities of cell proliferation, genomic instability and microenvironment, rather than subtype switching, mark recurrent gliomas. Within individual gliomas, the EM/PM subtype was preserved across regions and single cells. Malignant cells in the EM and PM gliomas were correlated to neural stem cell and oligodendrocyte progenitor cell compartment, respectively. Thus, while genetic makeup may change during progression and/or within different tumor areas, adult gliomas evolve within a neurodevelopmental framework of the EM and PM molecular subtypes. The dysregulated developmental pathways embedded in these molecular subtypes may contain subtype-specific vulnerabilities.

Region-Specific Effects of Fractionated Low-Dose Versus Single-Dose Radiation on Hippocampal Neurogenesis and Neuroinflammation.

Despite technical advances in hippocampus-sparing radiotherapy, radiation-induced injury to neural stem cell compartments may affect neurocognitive functions. In pre-clinical mouse models with fractionated low-dose radiation (FLDR) and single-dose radiation (SDR), the accurate response to radiation-induced injury was analyzed in different hippocampal subregions. Adult and juvenile C57BL/6NCrl mice were exposed to FLDR (20 × 0.1 Gy, daily exposure from Monday to Friday for 4 weeks) or SDR (1 × 2 Gy). In addition, 72 h after the last exposure, neuroglia (astrocytes and microglia) and neuroprogenitor cells were characterized and quantified in the hippocampal cornu ammonis (CA) and dentate gyrus (DG) by immunofluorescence studies. After analyzing different hippocampal subregions, it was observed that radiation responses varied between non-neurogenic CA, with no detectable inflammatory alterations, and neurogenic DG, characterized by impaired neurogenesis and subsequent neuroinflammation. Age-dependent differences in radiosensitivity appeared to depend on the varying proliferative potential of neural stem cell niches. Using the same overall dose for FLDR and SDR (2 Gy), both the cumulative dose over time and also the single dose fraction have decisive impacts on hippocampal damage. Region-specific effects of radiation-induced hippocampal injury relies primarily on cell deaths of proliferating neuroprogenitors. Dose per fraction defines the extent of neuronal injury, and subsequently activated microglia and reactive astrocytes modulate dynamic processes of neuroinflammation. Thus, limiting both cumulative doses and dose fractions to hippocampal DG is an important issue of clinical radiotherapy to preserve neurocognitive functions.

Fractionated Low-Dose Radiation Induces Long-Lasting Inflammatory Responses in the Hippocampal Stem Cell Niche

Despite major technical advances in hippocampus-sparing radiation therapy, radiation-induced injury to the neural stem cell compartment may affect neurocognitive functions. In the brain, glial cells modulate neuronal functions and are major mediators of neuroinflammation. In a preclinical mouse model with fractionated low-dose radiation (LDR), the complex response to radiation-induced injury was analyzed in the hippocampal stem cell compartment over a period of 6 months. Adult and juvenile C57BL/6NCrl mice were exposed to low doses of ionizing radiation (IR; 20 fractions of 0.1 Gy, for up to 4 weeks) daily. At 72 hours and 1, 3, and 6 months after fractionated LDR, magnetic resonance imaging (9.4 T) was conducted to detect structural and functional abnormalities in the hippocampal region. Using immunofluorescence and histologic studies, neuroglia cells (astrocytes, microglia, oligodendrocytes) were quantified and neuroinflammatory responses were characterized in the hippocampal dentate gyrus. Using in vivo bromodeoxyuridine labeling, the cell fate of newly generated progenitor cells was tracked in the subgranular zone of the dentate gyrus during fractionated LDR. Low doses of IR induced long-lasting inflammatory responses with local increases of activated microglia and reactive astrocytes, which were most pronounced in the juvenile hippocampus within the first months after LDR. Glial activation with the consequent release of proinflammatory mediators increased local blood flow and vascular permeability in the hippocampal region. Cell fate mapping of progenitors located in the subgranular zone revealed a transient shift from neurogenesis to gliogenesis. Glial cell activation and transient neuroinflammation may reflect radiation-induced neuronal damage in the hippocampal stem cell niche. The increased proliferative capacity of the developing brain may explain the enhanced hippocampal radiosensitivity, with stronger inflammatory reactions in the juvenile hippocampus. Thus, limiting the radiation dose to the hippocampal region is an important issue of clinical radiation therapy at all ages to preserve neurocognitive functions.

Abstract 3068: Everolimus mediated Radioprotection on surrounding healthy tissue in glioblastoma microbrain models

Abstract Background: After surgical resection, ionizing radiation alone or in combination with chemotherapy is the main treatment modality for brain tumors including glioblastoma (GBM). Although the effect of radiation-induced (RI) damage improves the patient survival ratio, injury to the neural stem and progenitor cell (NSPC) compartments and damage to NSPC populations is hypothesized to be central to the pathogenesis of RI cognitive decline in the patients. Studies on sensitivity of neural cell populations to radiation have shown that mature neurons and astrocytes have substantial radio resistance; in contrast, NSPC are highly sensitive to radiation, as exposure induces DNA double-strand breaks that when unrepaired can lead to cell death. Therefore, protection of NPCs is an important countermeasure against radiotherapy in normal tissues. Rapamycin, a small molecule inhibitor of mammalian target of rapamycin (mTOR), has been shown to exert a profoundly protective effect on epithelial and hematopoietic stem cells upon exposure to radiation therapy. Recently, several pharmacokinetically improved rapamycin analogs have secured FDA approval. In this study we investigate the potential of Everolimus as radio-protector for NSPCs in a GBM environment. Materials and Methods: NSPCs generated from healthy donors and GBM cells were treated with different doses of Everolimus 24 h before and after a single dose 4 Gy irradiation. Survival was monitored using Cell Titer Glo assay. Western Blots, qPCR and immunofluorescence were used to assess mTOR pathway activation and NPC differentiation in 2D and 3D culture. Co-culture of NSPCs and GFP-tagged GBM cells were treated with Everolimus and irradiated with subsequent followup by confocal imaging to check the relative survival of these cells. Results: Low doses of Everolimus restored survival and proliferation of irradiated NSPCs, while GBM cells remained radiosensitive. qPCR analysis of Nestin, SOX2 and FOXG1 revealed that NSPCs maintained their stemness after treatment. MAP2 and βIII tubulin analysis demonstrated that irradiated NSPCs were able to differentiate to neurons upon treatment. Everolimus treatment also reduced γH2A and phospho-PP2A levels and promoted phosphorylation of Akt suggesting that mTOR inhibition with Everolimus could activate DNA repair in irradiated NPCs. Conclusions: Our findings suggest that pretreatment with low-dose Everolimus may protect NSPCs from radiation induced injuries after radiation therapy, while maintaining radiation sensitivity to GBM cells. This study suggests low-dose Everolimus as a potential radio-protector for clinical treatment of glioblastoma and other brain malignancies. Citation Format: Tithi Ghosh Halder, Ryan Rodriguez del Villar, Jerome Lacombe, Kevin Drenner, Serina Ng, Trason Thode, Alexis Weston, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. Everolimus mediated Radioprotection on surrounding healthy tissue in glioblastoma microbrain models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3068.

Epigenetic Delay in the Neurodevelopmental Trajectory of DNA Methylation States in Autism Spectrum Disorders.

Autism spectrum disorders (ASD) are hypothesized to originate in utero from perturbations in neural stem cell niche regions of the developing brain. Dynamic epigenetic processes including DNA methylation are integral to coordinating typical brain development. However, the extent and consequences of alterations to DNA methylation states in neural stem cell compartments in ASD are unknown. Here, we report significant DNA methylation defects in the subventricular zone of the lateral ventricles from postmortem brain of 17 autism diagnosed compared to 17 age- and gender-matched typically developing individuals. Both array- and sequencing-based genome-wide methylome analyses independently revealed that these alterations were preferentially targeted to intragenic and bivalently modified chromatin domains of genes predominately involved in neurodevelopment, which associated with aberrant precursor messenger RNA splicing events of ASD-relevant genes. Integrative analysis of our ASD and typically developing postmortem brain methylome datasets with that from fetal brain at different neurodevelopmental stages revealed that the methylation states of differentially methylated loci associated with ASD remarkably resemble the methylation states at earlier time points in fetal brain development. This observation was confirmed using additional methylome datasets from three other brain regions. Altogether, these findings implicate an epigenetic delay in the trajectory of normal DNA methylation states during the course of brain development that may consequently lead to deleterious transcriptomic events in ASD and support the hypothesis of an early developmental origin of ASD.

Effects of Radiation Therapy on Neural Stem Cells.

Brain and nervous system cancers in children represent the second most common neoplasia after leukemia. Radiotherapy plays a significant role in cancer treatment; however, the use of such therapy is not without devastating side effects. The impact of radiation-induced damage to the brain is multifactorial, but the damage to neural stem cell populations seems to play a key role. The brain contains pools of regenerative neural stem cells that reside in specialized neurogenic niches and can generate new neurons. In this review, we describe the advances in radiotherapy techniques that protect neural stem cell compartments, and subsequently limit and prevent the occurrence and development of side effects. We also summarize the current knowledge about neural stem cells and the molecular mechanisms underlying changes in neural stem cell niches after brain radiotherapy. Strategies used to minimize radiation-related damages, as well as new challenges in the treatment of brain tumors are also discussed.

NRG Oncology CC003: A randomized phase II/III trial of prophylactic cranial irradiation with or without hippocampal avoidance for small cell lung cancer.

TPS8578 Background: Multiple clinical trials have shown that prophylactic cranial irradiation (PCI) prevents brain metastases and may prolong survival in small cell lung cancer (SCLC). However,prophylactic cranial irradiation can lead to decline in cognitive function. Preclinical evidence suggests that the pathogenesis of this toxicity includes inflammatory injury to proliferating neuronal progenitor cells in the peri-hippocampal stem cell niches. We hypothesized that conformal avoidance of the hippocampal neural stem cell compartment during brain irradiation using intensity-modulated radiotherapy (IMRT) would decrease the likelihood and/or severity of this toxicity. This hypothesis was recently validated by positive results from NRG Oncology CC001, a phase III trial of hippocampal avoidance during whole-brain radiotherapy for patients with brain metastases. NRG Oncology CC003 is an ongoing randomized phase II/III trial of hippocampal avoidance during prophylactic cranial irradiation (HA-PCI) for small cell lung cancer, conducted in parallel with NRG Oncology CC001. Methods: The primary endpoints of the phase IIR and III components are 12-month intracranial relapse rate and 6-month deterioration in Hopkins Verbal Learning Test-Revised (HVLT-R) Delayed Recall, respectively. This is a seamless phase IIR/III trial, with the phase IIR designed to demonstrate non-inferiority. If the non-inferiority margin of the phase IIR component is not exceeded, then the trial would transition to the phase III component. Following accrual of 182 of planned 172 patients on the phase IIR component, the trial closed to accrual on 10/13/17 to assess the phase IIR primary endpoint. The DSMB evaluated the IIR outcomes, and on 1/9/19, the trial was reactivated to accrue an additional 122 patients to the phase III component. Eligibility criteria include: 1) small cell lung cancer with at least partial response to chemotherapy; 2) contrast-enhanced thin-slice volumetric MRI scan; and, 3) Zubrod performance status 0-2. Supported by grant UG1CA189867 (NCORP) from the National Cancer Institute. Clinical trial information: NCT02635009.

Distinct response of adult neural stem cells to low versus high dose ionising radiation

Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible.Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50–500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.

MicroRNAs Engage in Complex Circuits Regulating Adult Neurogenesis.

The finding that the adult mammalian brain is still capable of producing neurons has ignited a new field of research aiming to identify the molecular mechanisms regulating adult neurogenesis. An improved understanding of these mechanisms could lead to the development of novel approaches to delay cognitive decline and facilitate neuroregeneration in the adult human brain. Accumulating evidence suggest microRNAs (miRNAs), which represent a class of post-transcriptional gene expression regulators, as crucial part of the gene regulatory networks governing adult neurogenesis. This review attempts to illustrate how miRNAs modulate key processes in the adult neurogenic niche by interacting with each other and with transcriptional regulators. We discuss the function of miRNAs in adult neurogenesis following the life-journey of an adult-born neuron from the adult neural stem cell (NSCs) compartment to its final target site. We first survey how miRNAs control the initial step of adult neurogenesis, that is the transition of quiescent to activated proliferative adult NSCs, and then go on to discuss the role of miRNAs to regulate neuronal differentiation, survival, and functional integration of the newborn neurons. In this context, we highlight miRNAs that converge on functionally related targets or act within cross talking gene regulatory networks. The cooperative manner of miRNA action and the broad target repertoire of each individual miRNA could make the miRNA system a promising tool to gain control on adult NSCs in the context of therapeutic approaches.

FOXO in Neural Cells and Diseases of the Nervous System.

The evolutionarily conserved FOXO family of transcription factors has emerged as a significant arbiter of neural cell fate and function in mammals. From the neural stem cell (NSC) state through mature neurons under both physiological and pathological conditions, they have been found to modulate neural cell survival, stress responses, lineage commitment, and neuronal signaling. Lineage-specific FOXO knockout mice have provided an invaluable tool for the dissection of FOXO biology in the nervous system. Within the NSC compartments of the brain, FOXOs are required for the maintenance of NSC quiescence and for the clearance of reactive oxygen species. Within mature neurons, FOXO transcriptional activity is essential for the prevention of age-dependent axonal degeneration. Acutely, FOXO3 has been found to cause axonal degeneration upon withdrawal of neurotrophic factors. In more active neural signaling, FOXO6 promotes increased dendritic spine density of hippocampal neurons and is required for the consolidation of memories. In addition to the central nervous system (CNS), FOXOs also influence the functionality of the peripheral nervous system (PNS). FOXO1 knockout within the PNS results in a reduction of sympathetic tone and decreased levels of brain-derived norepinephrine and lower energy expenditure. FOXO3 knockout mice have impaired hearing which may be due to defects in synapse localization within the ear. Given the scope of FOXO activities in both the CNS and PNS, it will be of interest to study FOXOs within the context of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis. From within the nervous system, FOXOs may also regulate important parameters such as whole-body metabolism, motor function, and catecholamine production, making FOXOs key players in physiologic homeostasis.

Analyses of distribution and dosimetry of brain metastases in small cell lung cancer with relation to the neural stem cell regions: feasibility of sparing the hippocampus in prophylactic cranial irradiation

BackgroundThis work aims to assess the feasibility of selectively sparing the hippocampus during prophylactic cranial irradiation (PCI) for small cell lung cancer (SCLC).MethodsSCLC patients with brain metastases (BMs) diagnosed with MRI were enrolled. Lesions localized to the neural stem cell (NSC) compartments [subventricular zone (SVZ) or hippocampus] were analyzed. Patients were categorized by the total number of intracranial metastases, the therapy processes and the symptoms. Hippocampi and enhanced lesions within 15 mm from the hippocampus were contoured. IMRT treatment plans were generated for hippocampal avoidance (HA)-PCI (25Gy in 10 fractions).ResultsFrom Jan 2011 to Oct 2014, 1511 metastases were identified in 238 patients. The overall ratio of metastatic lesions located in NSC regions was 2.0% in the 1511 total metastases and 9.7% in the 238 overall patients. Among the NSC region metastases, 15 (1.0%) lesions involved the HA region of 14 (5.9%) patients and another 15 (1.0%) involved the SVZ of 15 (6.3%) patients. The involvement of HA region or SVZ was significantly different between patients with oligometastatic and non-oligometastatic BMs (P < 0.05). Based on the dosimetric analysis, 26 (10.9%) patients with 41 (2.7%) metastases within 15 mm from the hippocampus had inadequate dosage in case that HA-PCI was applied.ConclusionsOur retrospective review of 1511 metastases in 238 patients (among whom 89.5% were male) suggests that the metastatic involvement of the NSC regions (especially hippocampus) is unusual and limited primarily to patients with non-oligometastatic disease in SCLC. Also, dosimetric analysis shows that about 10% of patients may have adequate dosage due to HA-PCI treatment. But we believe that this is still an acceptable clinical treatment strategy for SCLC.

High-dose Neural Stem Cell Radiation May Not Improve Survival in Glioblastoma

AimsTo evaluate the effect of radiotherapy dose-volume parameters of neural stem cell (NSC) compartment on progression-free survival (PFS) and overall survival after post-resection chemoradiation in newly diagnosed glioblastoma. Materials and methodsSixty-one patients with unifocal glioblastoma were included. Ipsilateral (NSC_Ipsi), contralateral (NSC_Contra) and combined NSC (NSC_Combined) were contoured on radiotherapy planning computerised tomography datasets. NSC dose-volume parameters were correlated with PFS and overall survival. Serial magnetic resonance imaging scans were assessed to understand the frequency of pre- and post-treatment involvement of the NSC by contrast enhancing lesions (CELs). ResultsBaseline involvement of NSC with CELs was seen in 67.2% and 95.9% had CELs and FLAIR abnormalities at progression. With a median follow-up of 14.1 months (interquartile range 9.4–20.6 months), median PFS and overall survival were 14.5 (95% confidence interval 11.6–17.5) and 16.2 (95% confidence interval 13.3–19.2) months, respectively. Poor Eastern Cooperative Oncology Group performance score, advanced recursive partitioning analysis class, unmethylated O6-methylguanine methyltransferase (MGMT) status, higher than median of mean NSC_Ipsi dose were associated with significantly inferior PFS and overall survival on univariate analysis. On multivariate analysis, unmethylated MGMT status, higher than median of mean doses to NSC_Ipsi and poor compliance to adjuvant temozolomide were independent predictors of inferior survival. ConclusionsIn this cohort, 67.2% of newly diagnosed glioblastoma patients had NSC involved with CELs at presentation and 95.9% at progression. This might be an imaging surrogate of the current notion of gliomagenesis and progression from NSC rests. A high radiation dose to NSC_Ipsi was significantly associated with inferior survival. This could be a function of larger tumours and planning target volumes in those with pre-treatment NSC involvement. Methylated MGMT and good compliance to adjuvant temozolomide were independent predictors of better survival. Until further evidence brings hope for glioblastoma, elective, partial NSC irradiation remains experimental.

Abstract WMP42: Neuro and Gliogenesis in a Nonhuman Primate Model of Ischemic Stroke with Impaired Dexterous Movements

Ischemic stroke is the leading cause of upper extremity motor impairments. Well-characterized experimental stroke models for upper extremity motor impairment remain underdeveloped. Cortical representation of dexterous movements in nonhuman primates (NHP) is functionally and topographically similar to that in humans. We recently reported the characterization of an NHP model of focal ischemia reperfusion with a defined syndrome, impaired arm function and finger dexterity. In this study, we investigated the cellular changes in the neural stem cell compartment and glial cell populations in this NHP model. NHPs were subjected to transient cerebral ischemia by temporarily occluding the M3 segment of the left side middle cerebral artery (MCA). Motor and cognitive functions following the stroke were evaluated using the object retrieval task with barrier-detour. Postmortem analysis included magnetic resonance imaging (MRI) and immunohistopathology to map the infarct and characterize the neurogenic and gliogenic changes. The MCA occlusion produced significant loss of fine motor function characterized by impaired dexterity. Immunocytochemical analysis revealed significant increase of Sox2+ neural stem cells in the subventricular zone, and of GFAP+ astrocytes (P&lt;0.0001) and Iba-1+ microglia (P&lt;0.0001) in the infarct region. In addition, there was a 42% increase in doublecortin positive cells (P&lt;0.0001) compared to non-ischemic hemisphere. This study describes the cellular composition of the endogenous changes in the neural stem cell compartment and in the stroke region. These data may help reveal the cellular identity mediating neural plasticity and the cellular mechanisms mediating behavioral deficits and post-stroke recovery.

RTHP-15. NEURAL STEM CELL NICHE INVOLVEMENT AND PATTERNS OF FAILURE IN GLIOBLASTOMA MULTIFORME

To evaluate the impact of magnetic resonance imaging (MRI) dynamics of glioblastoma, in context of pre and post treatment involvement of the neural stem cell compartment (NSC) on progression free (PFS) and overall survival (OS). Baseline and followup MRI images were serially evaluated in 61 patients treated with standard post resection chemoradiation and temozolomide between 2011 to 2015. MRI morphology groups were based on any combination(s) of NSC involvement, cortical involvement or both by contrast enhancing lesions (CELs). MRI images at recurrence were registered with the radiation treatment planning datasets in order to assess the patterns of failure in context of the previously contoured NSC volumes. With a median followup of 14.1 months (interquartile range 9.4 – 20.6 months), the median [95% confidence interval (CI)] PFS and OS were 14.5 (95% CI 11.6 – 17.5 months) and 16.2 (95% CI 13.3 – 19.2 months) months, respectively. Baseline involvement of NSC with CELs with varying degrees of cortical involvement was seen in 41/61 patients. At progression, 95.9% patients had progressive involvement of previously contoured NSC with CELs and FLAIR abnormalities. In patients with CELs involving the NSC at presentation, log-rank univariate analyses revealed a non significant decline in PFS (13.01 vs 15.18 months, p = 0.51) and OS (14.62 vs 17.08 months, p = 0.62). However, there was a significantly higher chance of distant brain progression (40.5%, p = 0.05) and multifocal progression (36%, p = 0.05) in patients with pretreatment NSC involvement with CELs compared to those without. In our cohort, pre treatment NSC involvement was associated with a significantly higher incidence of distant brain failure and multifocal recurrences. Though it did not reach significance, baseline NSC involvement might be associated with inferior PFS and OS. Prospective validation in a larger cohort is necessary.

In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation.

The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5–14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4Y288C), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4Y288C embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4Y288C mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis.

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells

ABSTRACT The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV ( Lig4 Y288C ), ataxia telangiectasia mutated ( Atm − / − ) and double mutant Atm − / − / Lig4 Y288C mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig 4 Y288C mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4 Y288C mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4 Y288C mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.

Early Inflammatory Responses Following Cell Grafting in the CNS Trigger Activation of the Subventricular Zone: A Proposed Model of Sequential Cellular Events.

While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells.

ABSTRACTThe embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4Y288C), ataxia telangiectasia mutated (Atm−/−) and double mutant Atm−/−/Lig4Y288C mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4Y288C mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4Y288C mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4Y288C mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.