Matthew V. Covey

Activation of the Mammalian Target of Rapamycin (mTOR) Is Essential for Oligodendrocyte Differentiation

Although both extrinsic and intrinsic factors have been identified that orchestrate the differentiation and maturation of oligodendrocytes, less is known about the intracellular signaling pathways that control the overall commitment to differentiate. Here, we provide evidence that activation of the mammalian target of rapamycin (mTOR) is essential for oligodendrocyte differentiation. Specifically, mTOR regulates oligodendrocyte differentiation at the late progenitor to immature oligodendrocyte transition as assessed by the expression of stage specific antigens and myelin proteins including MBP and PLP. Furthermore, phosphorylation of mTOR on Ser 2448 correlates with myelination in the subcortical white matter of the developing brain. We demonstrate that mTOR exerts its effects on oligodendrocyte differentiation through two distinct signaling complexes, mTORC1 and mTORC2, defined by the presence of the adaptor proteins raptor and rictor, respectively. Disrupting mTOR complex formation via siRNA mediated knockdown of raptor or rictor significantly reduced myelin protein expression in vitro. However, mTORC2 alone controlled myelin gene expression at the mRNA level, whereas mTORC1 influenced MBP expression via an alternative mechanism. In addition, investigation of mTORC1 and mTORC2 targets revealed differential phosphorylation during oligodendrocyte differentiation. In OPC-DRG cocultures, inhibiting mTOR potently abrogated oligodendrocyte differentiation and reduced numbers of myelin segments. These data support the hypothesis that mTOR regulates commitment to oligodendrocyte differentiation before myelination.

Sustained neocortical neurogenesis after neonatal hypoxic/ischemic injury

Neocortical neurons are sensitive to hypoxic‐ischemic (H‐I) injuries at term and their demise contributes to neurological disorders. Here we tested the hypothesis that the subventricular zone of the immature brain regenerates neocortical neurons, and that this response is sustained.

Leukemia inhibitory factor participates in the expansion of neural stem/progenitors after perinatal hypoxia/ischemia

Subsequent to perinatal hypoxia/ischemia there is an increase in the number of neural stem/progenitor cells (NSP) within the subventricular zone (SVZ). Gene expression analyses have implicated Notch signaling in the expansion of these tripotential cells but there are limited data as to which signals are stimulating Notch activation. There is evidence that the leukemia inhibitory factor receptor (LIFR)/gp130 receptor heterodimer induces Notch1 to maintain NSP populations during normal development. LIF and ciliary neurotrophic factor (CNTF) bind to these receptor components and they coordinate injury responses in the CNS. Therefore, the aim of these studies was to investigate whether CNTF and/or leukemia inhibitory factor (LIF) participate in NSP expansion in the rat SVZ after hypoxia/ischemia (H/I) as well as to characterize the downstream events that regulate NSP numbers. We report that LIF mRNA is induced 48 h post-insult by 13-fold but that it returns almost to baseline by 72 h. Commensurate with increased LIF expression there is a corresponding increase in phosphorylated Stat-3 within the SVZ. Modeling the changes that occur in vivo, we show that LIF induces Stat-3 phosphorylation in neurospheres to enhance Delta-like-1 and Notch1 expression as well as to increase Notch1 activation. LIF also expands neurosphere number and size in vitro. Whereas CNTF can mimic the effects of LIF in vitro, CNTF expression in the SVZ was unchanged during recovery from H/I. Cumulatively, these data implicate LIF and not CNTF in the expansion of NSPs in the rat SVZ after perinatal brain injury. As both LIF expression and the endogenous regenerative response after brain injury are time-delimited, these findings provide insights into strategies to expand the endogenous pool of NSPs to repopulate the damaged brain.

Defining the Critical Period for Neocortical Neurogenesis after Pediatric Brain Injury

Pediatric traumatic brain injury (TBI) is a significant and underappreciated societal problem. Whereas many TBI studies have evaluated the mechanisms of cell death after TBI, fewer studies have evaluated the extent to which regeneration is occurring. Here we used a cryoinjury model to damage the somatosensory cortex of rats at postnatal day 6 (P6), P10 and P21. We evaluated the production of new neocortical neurons using a combination of 5-bromo-2-deoxyuridine (BrdU) labeling combined with staining for doublecortin (DCX). BrdU+/DCX+ bipolar cells were observed adjacent to the neocortical lesion, with their processes oriented perpendicular to the pial surface. As the animals aged, both the overall proliferative response as well as the production of neocortical neuroblasts diminished, with P6 animals responding most robustly, P10 animals less strongly, and P21 animals showing a very modest proliferative response and virtually no evidence of neocortical neurogenesis. When BrdU was administered at increasingly delayed intervals after the injury at P6, there was a clear difference in the number of new neuroblasts produced as a function of age, with the greatest number of new neocortical neurons produced between 4 and 7 days after the injury. These studies demonstrate that the immature brain has the capacity to produce neocortical neurons after traumatic injury, but this capacity diminishes as the brain continues to develop. Furthermore, in contrast to moderate hypoxic/ischemic brain damage in the P6 rat, where neurogenesis persists for at least 2 months, the response to cryoinjury is quite different as the neurogenic response diminishes over time.

Effect of the mitochondrial antioxidant, Mito Vitamin E, on hypoxic-ischemic striatal injury in neonatal rats: a dose-response and stereological study.

A mitochondria-targeted antioxidant, Mito Vitamin E (MitoVit E), has previously been shown to prevent mitochondrial oxidative damage. The aim of this study was to investigate the effect of MitoVit E on neuronal survival in the rat striatum after acute perinatal hypoxia-ischemia. Continuous striatal infusion with 4.35 microM, 43.5 microM, or 148 microM of MitoVit E before, during, and after hypoxia-ischemia was not neuroprotective for striatal medium-spiny neurons. Pre- or posttreatment with 435 microM MitoVit E was neurotoxic. These results suggest that MitoVit E is not significantly neuroprotective for striatal medium-spiny neurons after acute perinatal hypoxic-ischemic brain injury. The results also suggest that mitochondrial oxidative damage does not contribute significantly to the death of striatal medium-spiny neurons after perinatal hypoxia-ischemia.

ADHD-like hyperactivity, with no attention deficit, in adult rats after repeated hypoxia during the equivalent of extreme prematurity

The most common behavioural disorder seen in children and adolescents born extremely prematurely is attention deficit hyperactivity disorder (ADHD). The hyperactive/impulsive sub-type of ADHD or the inattentive sub-type or the hyperactive/impulsive/inattentive sub-type can be evident. These sub-types of ADHD can persist into adulthood. The aim of this study was to investigate the relevance of a new immature rat model of repeated hypoxic exposure to these behavioural characteristics of extreme prematurity. More specifically, this study aimed to measure ADHD-like hyperactivity in response to delayed reward, and inattention, in repeated hypoxic versus repeated normoxic rats. Sprague-Dawley rats were exposed to either repeated hypoxia or repeated normoxia during postnatal days (PN) 1-3. The rat brain during PN1-3 is generally considered to be developmentally equivalent to the human brain during extreme prematurity. The rats were then behaviourally tested at 16 months-of-age on a multiple component fixed interval-extinction test. This test detects ADHD-like hyperactivity in response to delayed reward, as well as inattention. It was found that the repeated hypoxic rats exhibited ADHD-like hyperactivity in response to delayed reward, but no attention deficit, when compared to repeated normoxic rats. These findings provide a new animal model to investigate the biological mechanisms and treatment of ADHD-like hyperactivity due to repeated hypoxia during the equivalent of extreme prematurity.

Effect of hypothermic post-treatment on hypoxic-ischemic striatal injury, and normal striatal development, in neonatal rats: a stereological study.

Fundamental questions remain about the optimal temperature, duration, and mode of delivery that provide the best striatal neuroprotection from hypothermia after perinatal hypoxia-ischemia. This study used stereological methods to investigate whether a mild (i.e. 2°C) or a moderate (5°C) decrease in whole body temperature, for 6 h immediately postinsult, was neuroprotective for striatal medium-spiny neurons after perinatal hypoxia-ischemia in the rat. This study also investigated whether moderate hypothermia had any effect on normal striatal development. Hypoxia-ischemia or sham hypoxia-ischemia was induced on postnatal day (PN) 7. Pups were kept either normothermic, mildly hypothermic, or moderately hypothermic for 6 h immediately postinsult. The absolute number of striatal medium-spiny neurons was calculated using modern stereological methods. There was no significant difference in the absolute number of medium-spiny neurons in the right striatum after either mild hypothermia or moderate hypothermia. There was also no significant difference in the absolute number of medium-spiny neurons between the control normothermic and the control moderately hypothermic pups. The latter results suggest that moderate hypothermia for 6 h immediately postinsult may be a safe treatment for striatal medium-spiny neurons. Yet, neither mild nor moderate hypothermia alone for 6 h immediately posthypoxia-ischemia is neuroprotective for striatal medium-spiny neurons.

Spectrum of Short- and Long-Term Brain Pathology and Long-Term Behavioral Deficits in Male Repeated Hypoxic Rats Closely Resembling Human Extreme Prematurity

Brain injury in the premature infant is associated with a high risk of neurodevelopmental disability. Previous small-animal models of brain injury attributable to extreme prematurity typically fail to generate a spectrum of pathology and behavior that closely resembles that observed in humans, although they provide initial answers to numerous cellular, molecular, and therapeutic questions. We tested the hypothesis that exposure of rats to repeated hypoxia from postnatal day 1 (P1) to P3 models the characteristic white matter neuropathological injury, gray matter volume loss, and memory deficits seen in children born extremely prematurely. Male Sprague Dawley rats were exposed to repeated hypoxia or repeated normoxia from P1 to P3. The absolute number of pre-oligodendrocytes and mature oligodendrocytes, the surface area and g-ratio of myelin, the absolute volume of cerebral white and gray matter, and the absolute number of cerebral neurons were quantified stereologically. Spatial memory was investigated on a radial arm maze. Rats exposed to repeated hypoxia had a significant loss of (1) pre-oligodendrocytes at P4, (2) cerebral white matter volume and myelin at P14, (3) cerebral cortical and striatal gray matter volume without neuronal loss at P14, and (4) cerebral myelin and memory deficits in adulthood. Decreased myelin was correlated with increased attention deficit hyperactivity disorder-like hyperactivity. This new small-animal model of extreme prematurity generates a spectrum of short- and long-term pathology and behavior that closely resembles that observed in humans. This new rat model provides a clinically relevant tool to investigate numerous cellular, molecular, and therapeutic questions on brain injury attributable to extreme prematurity.

Astrocyte-produced leukemia inhibitory factor expands the neural stem/progenitor pool following perinatal hypoxia–ischemia

Brain injuries, such as cerebral hypoxia–ischemia (H‐I), induce a regenerative response from the neural stem/progenitors (NSPs) of the subventricular zone (SVZ); however, the mechanisms that regulate this expansion have not yet been fully elucidated. The Notch‐ Delta‐Serrate‐Lag2 (DSL) signaling pathway is considered essential for the maintenance of neural stem cells, but it is not known if it is necessary for the expansion of the NSPs subsequent to perinatal H‐I injury. Therefore, the aim of this study was to investigate whether this pathway contributes to NSP expansion in the SVZ after H‐I and, if so, to establish whether this pathway is directly induced by H‐I or regulated by paracrine factors. Here we report that Notch1 receptor induction and one of its ligands, Delta‐like 1, precedes NSP expansion after perinatal H‐I in P6 rat pups and that this increase occurs specifically in the most medial cell layers of the SVZ where the stem cells reside. Pharmacologically inhibiting Notch signaling in vivo diminished NSP expansion. With an in vitro model of H‐I, Notch1 was not induced directly by hypoxia, but was stimulated by soluble factors, specifically leukemia inhibitory factor, produced by astrocytes within the SVZ. These data confirm the importance both of the Notch‐DSL signaling pathway in the expansion of NSPs after H‐I and in the role of the support cells in their niche. They further support the body of evidence that indicates that leukemia inhibitory factor is a key injury‐induced cytokine that is stimulating the regenerative response of the NSPs.