Regulation of Postnatal Dentate Gyrus Neurogenesis and its Alteration in Experimental Epilepsy.

Medial temporal lobe epilepsy (mTLE) is a common, intractable epilepsy of unknown cause. In rodent mTLE, prolonged seizures, termed status epilepticus, stimulate neurogenesis, but many newborn dentate granule cells (DGCs) migrate and integrate aberrantly. We hypothesized that adult-born, rather than pre-existing, neurons contribute to epileptic pathology in the hippocampal dentate gyrus. Using the rat pilocarpine model, which recapitulates many human mTLE features, we tested our hypothesis by injecting green fluorescent protein-carrying retrovirus to label neural stem cells, or by killing dividing stem cells to suppress neurogenesis pre or post-epileptic injury. We found that an epilepsy-inciting injury differentially influences developing neurons depending upon their ages. Fully mature neurons at the time of injury are resistant, whereas developing neurons of differing maturities contribute to distinct, specific epileptic pathologies. To address the molecular underpinnings of this phenomenon, we investigated the Reelin pathway. Reelin is secreted into the extracellular matrix, binds its receptors and is internalized, leading to phosphorylation of disabled-1 (Dab1), an adaptor protein. Evidence implicates the Reelin/Dab1 pathway in DGC development, and mice with Reelin, Dab1, or Reelin receptor mutations have abnormalities similar to those in experimental mTLE. To better understand the role of Dab1 in neurogenesis, we suppressed Dab1 expression using a mouse line with conditional Dab1 knock-in alleles, or by injecting retroviral vector carrying a Dab1 shRNA into the adult rat dentate gyrus. In Dab1-conditional knock-in mice, Dab1 deletion led to aberrant DGC migration, impaired dendritic maturation and axonal disorganization. In rats, retroviral Dab1 shRNA delivery decreased dendritic arborization of DGCs and induced numerous labeled glia in the hilus and DGC layer that were not seen with control shRNA. Aberrant neurogenesis is a proposed cause of seizures and cognitive deficits common in mTLE, and this work supports an important role for Dab1 signaling in both neuronal migration, dendrite formation, and directing DGC progenitors to a neuronal fate. Taken together, these data suggest that neurons of distinct maturities contribute to specific epileptic abnormalities and that the Reelin/Dab1 pathway may underlie some of these aberrations. Correcting signaling of this pathway after epileptogenic insult may offer a novel strategy to prevent mTLE.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91485/1/mkron_1.pd

Examining reelin expression and neural plasticity in animal models of depression

Stress is an important risk factor for the development of depression, but little is known about the neurobiological mechanisms by which stress might promote depressive symptomatology. The hippocampus and amygdala are susceptible to the detrimental effects of prolonged elevated stress hormone levels and neuroplastic changes within these brain regions have been linked to the onset of depression. Some of the neurobiological changes associated with prolonged elevated glucocorticoids include decreased neurogenesis, synaptic plasticity, dendritic morphology, and spine density within the hippocampus and increased dendritic morphology and spine density in the amygdala. Interestingly, recent evidence has described a regulatory role for the extracellular matrix protein reelin in synaptic plasticity, hippocampal neurogenesis, dendritic arborization, and spine density. Moreover, reelin has been shown to be decreased in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Combined, these results suggest that reelin may be an interesting protein to examine in regard to the pathogenesis of depression and to further elucidate potential therapeutic targets for the treatment of this disorder. The goal of the current research was to provide a comprehensive examination into the role of repeated stress on reelin and neural plasticity in the pathogenesis of depression through multiple preclinical studies. Given the association between reelin and hippocampal plasticity, in chapter 2 the effects of repeated exposure to corticosterone (CORT) or physical restraint on reelin expression in specific hippocampal regions were examined. Results revealed that there was a significant decrease in the number of reelin-positive cells in the CA1 stratum lacunosum and the subgranular zone of the dentate gyrus in rats that received CORT, but not in rats that received restraint. Interestingly, these results parallel our laboratory’s previous observation that CORT increases depression-like behavior but physical restraint does not. As reelin was decreased in the subgranular zone, it suggests that this protein is in a prime location to influence neurogenesis. Accordingly, chapter 3 focused on assessing the effects of different durations of CORT on behavior, hippocampal reelin expression, and neurogenesis, by subjecting rats to 7, 14 or 21 days of repeated CORT injections (40 mg/kg, s.c.) or vehicle injections. Results revealed that both the 14-day and 21-day CORT-treated rats showed increased depressive-like behavior in the forced swim test, significantly fewer reelin-positive cells and decreased neurogenesis compared to the control rats. In chapter 4, mice with a genetic deficit in reelin expression were used to examine their vulnerability to the depressogenic effects of CORT. We hypothesized that heterozygous reeler mice (HRM), with approximately 50% normal levels of reelin, would be more sensitive to the depressogenic effects of CORT than wild-type mice (WTM). Mice received injections of either vehicle, 5 mg/kg, 10 mg/kg, or 20 mg/kg of CORT, and then were assessed for changes in depression-like behavior, reelin expression, and neurogenesis. The effects of CORT on behavior, the number of reelin-positive cells, and hippocampal neurogenesis were more pronounced in the HRM than in the WTM, providing support for the idea that mice with impaired reelin signaling are more vulnerable to the deleterious effects of glucocorticoids. As reelin is expressed in GABAergic interneurons and our previous studies consistently revealed decreases in reelin number following CORT exposure, in chapter 5 the effects of repeated CORT and restraint stress on GABAergic and glutamatergic markers in the hippocampus and amygdala were examined. Western blotting analyses revealed that CORT significantly decreased the GABAergic markers, GAD65 and the α2 receptor subunit, and increased the vesicular glutamate transporter VGLUT2 within the hippocampus. We also found that corticosterone decreased the GABAergic markers, GAD67 and the α2 receptor subunit, in the amygdala. Restraint stress had no significant effect in either of these areas. These findings suggest that the depressogenic effects of CORT may be related to alterations in GABAergic and glutamatergic neurotransmission within these structures. Together these results support a relationship between glucocorticoid-induced depressive-like behavior and decreases in reelin, neurogenesis and GABAergic signaling and provide support for investigating reelin as a novel therapeutic target for the treatment of depression

Aberrant structural and functional plasticity in the adult hippocampus of amygdala kindled rats

Amygdala kindling is commonly used to study the neural mechanisms of temporal lobe epilepsy and its behavioral consequences. The repetitive seizure activity that occurs during kindling is thought to induce an extensive array of structural and functional modifications within the brain, particularly in the hippocampus and dentate gyrus regions. Some of these changes include the growth or sprouting of new axonal connections as well as the birth and integration of new neurons into hippocampal circuits. Previous work has shown that these changes in structural and functional plasticity are not necessarily beneficial events. For instance, the growth and reorganization of synaptic terminals in the hippocampus and other brain regions might serve as a substrate that enhances hyperexcitability and seizure generation. In addition, although seizures induce the birth of new neurons, many of these newly generated cells migrate and function improperly within the hippocampal networks. Considering the prominent role of the hippocampus in a variety of behaviours, including learning, memory, and mood regulation, it would appear that alterations involving the structural and functional properties of both mature and newly born neurons in this region could impact these hippocampal-dependent functions. However, to date, the role of kindling-induced changes in hippocampal structural plasticity and neurogenesis on behaviour is incomplete, and the molecular mechanisms that govern these pathological events are poorly understood. The aim of this dissertation is to gain a better understanding of the changes in synaptic plasticity and neurogenesis within the hippocampus that occur after amygdala kindling. In chapter 2, we will examine if kindling alters the expression of synapsin I, a molecular marker of synaptic growth and activity, in both the hippocampus and other brain regions. In addition, we will also set out to determine if changes in synapsin I are related to the development of behavioural impairments associated with kindling. In chapter 3, the effect of kindling on hippocampal neurogenesis will be examined. In addition, we will also evaluate the effect of kindling on the expression of Reelin and Disrupted-in-Schizophrenia 1 (DISC1), two proteins instrumental for mediating proper neuronal migrational and maturation during development. In chapter 4, the effect of altered DISC1 expression in the dentate gyrus after kindling will be examined more extensively. We will examine whether altered DISC1 expression in the dentate contributes to some of the pathological features associated with seizure-induced hippocampal neurogenesis, such as ectopic cell migration and dentate granule cell layer dispersion. Finally, in chapter 5, the impact of aberrant seizure-induced neurogenesis on behaviour will be examined by determining if seizure-generated neurons functionally integrate and participate in hippocampal circuits related to memory processing. The results of this dissertation enhances our understanding of the functional consequences that altered hippocampal synaptic plasticity and neurogenesis may have on the development of epilepsy and emergence of cognitive impairments associated with chronic seizures

The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt–Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS

Cell-autonomous inactivation of the reelin pathway impairs adult neurogenesis in the hippocampus

Adult hippocampal neurogenesis is thought to be essential for learning and memory, and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of Disabled-1, an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain-of-function and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adultgenerated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus, and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders. © 2012 the authors.This project was supported by Grant BFU2008-3980 from the Ministerio de Ciencia e Innovación (MICINN), Spain; by grants from the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Caixa Catalunya-Obra Social Foundations to E.S.; by grants from the Spanish Ministry of Science and Innovation (SAF2009-07367 and CONSOLIDER CSD2007-00023) to V.B.; by the Fred Annegers Fellowship from the Epilepsy Foundation (M.M.K.); and by NIH Grant NS058585 to J.M.P. I.R. was recipient of a Formación de Personal Universitario predoctoral fellowship from MINECO (Spain).Peer Reviewe

Non-receptor tyrosine kinase Src is required for ischemia-stimulated neuronal cell proliferation via Raf/ERK/CREB activation in the dentate gyrus

<p>Abstract</p> <p>Background</p> <p>Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism.</p> <p>Results</p> <p>Src kinase activated continuously in the DG 24 h and 72 h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72 h of reperfusion, suggesting a role of Src kinase as an enhancer on neuronal cell proliferation in the DG via modifying the Raf/ERK/CREB cascade. This hypothesis is supported by further findings that U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf.</p> <p>Conclusion</p> <p>Src kinase increase numbers of newborn neuronal cells in the DG via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia.</p

Factors Regulating Neurogenesis in the Adult Dentate Gyrus

The dentate gyrus (DG), an important part of the hippocampus, plays a critical role in consolidation of information from short-term to long-term memory, and also in spatial navigation. Neural stem/progenitor cells (NSPCs) exist throughout life in the subgranular zone (SGZ) of the DG, where they develop into granular cells and establish synaptic connections with nearby cells. Granular cells of the DG sprout axons targeting neurons in the cornu ammonis 3 (CA3) area of the hippocampus, forming a neural trisynaptic circuit, an important part of the neural network in the hippocampus. Thus, the DG and the neurogenic cells it contains are of importance in controlling formation of memories, learned behaviors, and also in the maintenance and restoration of functions of the hippocampus. According to reports, both in vivo and in vitro neurogenesis in the DG are regulated by a variety of endogenous and exogenous factors at different stages. Therefore, a better understanding of the factors in NSPC niches and the intracellular molecules regulating/directing adult DG neurogenesis is needed to fully realize the potential of NSPCs in the treatment of hippocampal-related disorders. This chapter systematically summarizes the factors reported in regulating adult DG neurogenesis in mammals. Specifically, neurotransmitters, hormones, trophic factors, and others will be discussed

Adult neurogenesis and neurodegenerative diseases: A systems biology perspective

New neurons are generated throughout adulthood in two regions of the brain, the olfactory bulb and dentate gyrus of the hippocampus, and are incorporated into the hippocampal network circuitry; disruption of this process has been postulated to contribute to neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Known modulators of adult neurogenesis include signal transduction pathways, the vascular and immune systems, metabolic factors, and epigenetic regulation. Multiple intrinsic and extrinsic factors such as neurotrophic factors, transcription factors, and cell cycle regulators control neural stem cell proliferation, maintenance in the adult neurogenic niche, and differentiation into mature neurons; these factors act in networks of signaling molecules that influence each other during construction and maintenance of neural circuits, and in turn contribute to learning and memory. The immune system and vascular system are necessary for neuronal formation and neural stem cell fate determination. Inflammatory cytokines regulate adult neurogenesis in response to immune system activation, whereas the vasculature regulates the neural stem cell niche. Vasculature, immune/support cell populations (microglia/astrocytes), adhesion molecules, growth factors, and the extracellular matrix also provide a homing environment for neural stem cells. Epigenetic changes during hippocampal neurogenesis also impact memory and learning. Some genetic variations in neurogenesis related genes may play important roles in the alteration of neural stem cells differentiation into new born neurons during adult neurogenesis, with important therapeutic implications. In this review, we discuss mechanisms of and interactions between these modulators of adult neurogenesis, as well as implications for neurodegenerative disease and current therapeutic research

FGF-2-Responsive Neural Stem Cell Proliferation Requires CCg, a Novel Autocrine/Paracrine Cofactor

AbstractWe have purified and characterized a factor, from the conditioned medium of neural stem cell cultures, which is required for fibroblast growth factor 2's (FGF-2) mitogenic activity on neural stem cells. This autocrine/paracrine cofactor is a glycosylated form of cystatin C (CCg), whose N-glycosylation is required for its activity. We further demonstrated that, both in vitro and in vivo, neural stem cells undergoing cell division are immunopositive for cystatin C. Finally, we showed in vivo functional activity of CCg by demonstrating that the combined delivery of FGF-2 and CCg to the adult dentate gyrus stimulated neurogenesis. We propose that the process of neurogenesis is controlled by the cooperation between trophic factors and autocrine/paracrine cofactors, of which CCg is a prototype

Antidepressant-like Effects of Peripheral Reelin Administration in a Preclinical Model of Depression

Depression is a serious psychiatric disorder characterized by a range of debilitating symptoms. Long-term exposure to stress is a significant risk factor for the onset and maintenance of depression. Rats exposed to repeated treatment with the stress hormone corticosterone (CORT), a well-established rodent model of depression, begin to display depression-like symptoms. The development of depression-like symptoms with prolonged exposure to CORT is accompanied by reductions in the number and maturation rate of immature dentate granule cells within the hippocampus. Furthermore, these changes are paralleled by gradual decreases in reelin-expressing cells in the dentate subgranular zone of the hippocampus. Reelin is a large extracellular matrix protein that has been implicated in a number of neuropsychiatric disorders, including szchiphrenia, bipolar disorder, autism, as well as major depression. It holds important roles in learning and memory, cell migration and integration, synaptic contact formation, and adult neurogenesis. Mice deficient in reelin are more susceptible to CORT-induced impairments in hippocampal neurogenesis and the development of a depressive phenotype. Previous work has shown that intra-hippocampal infusions of reelin into the hippocampus reverse CORT-induced increases in depression-like behavior in rats, while restoring accompanying impairments in hippocampal neurogenesis. Reelin is also expressed in peripheral organs and tissues, though its roles here are not well understood. However, reelin-deficient mice show peripheral alterations in the clustering pattern of the serotonin transporter (SERT) in membranes of blood lymphocytes. The serotonin transporter is one of the main targets of antidepressant action, and importantly, this altered pattern of SERT clustering in reelin-deficient mice is mirrored both in patients with depression and in rats exposed to prolonged CORT treatment. Based on the previous findings, we were motivated to examine whether peripheral injections of reelin could restore CORT-induced increases in depression-like behavior. To investigate possible mechanisms, we examined (i) the SERT clustering pattern in peripheral lymphocyte membranes, and (ii) the maturation rate of immature hippocampal neurons. 40mg/kg of CORT was administered subcutaneously once per day for 21 consecutive days. In conjunction, we utilized a novel reelin injection paradigm, where reelin was delivered via the lateral tail vein at either 3μg/ml or 5μg/ml every 5 or 10 days during the period of CORT injections. Depression-like behavior was measured using the forced swim test the day following the last CORT injection. The open field test was included as a measure of locomotive and anxiety-like behavior. Importantly, peripheral reelin at all dosages administered restored CORT-induced increases in depression-like behavior in the forced swim test, normalizing both immobility and swimming behaviors. Neither CORT nor reelin impacted open field behavior. As expected, CORT-treated rats displayed alterations in SERT membrane protein clustering, and importantly this was restored by all doses of reelin administered. Peripheral reelin did not significantly reverse the CORT-induced deficits in immature neuron maturation rate. These novel findings demonstrate that reelin has antidepressant-like actions when given peripherally and provide evidence for the regulation of serotonin transporter clustering in lymphocyte membranes as a mechanism for the antidepressant action of reelin