The cannabinoid system exerts functional regulation of neural stem cell (NSC) selfrenewal, proliferation, and differentiation during both homeostatic and pathologic conditions. Recent evidence suggests that cannabinoid signaling is neuroprotective against reduction in NSC proliferation and neurogenesis caused by a multitude of conditions including injury due to HIV-1 associated neurotoxic proteins, neuroinflammation, and stroke. Yet not all effects of cannabinoids or cannabinoid-like compounds on neurogenesis can be attributed to signaling through either of the classical cannabinoid receptors CB1 or CB2. The recently de-orphaned GPR55 is targeted by numerous cannabinoid compounds suggesting GPR55 may be causing these aberrant effects. Activation of GPR55 has shown immune-modulatory effects outside the central nervous system (CNS) and anti-inflammatory actions on microglia, the resident immune cells within the CNS. New evidence has confirmed that both human and murine NSCs express functional levels of GPR55 yet the effects that GPR55 activation has on adult neurogenesis or NSC responses to inflammation has not been elucidated. In the present study we sought to determine the role GPR55 signaling has on NSC proliferation and neurogenesis as well as possible neuroprotective mechanisms within the NSC pool in response to inflammatory insult. Activation of GPR55 increased human NSC proliferation in vitro as assessed by BrdU incorporation and flow cytometry. Neuronal differentiation was also upregulated by signaling through GPR55 under homeostatic conditions in both human and murine NSC samples. Expression of NSC differentiation markers (nestin, sox2, GFAP, S100b, DCX, bIII-tubulin) in vitro was determined by immunohistochemistry, qPCR, and flow cytometry. In vivo, C57BL/6 and GPR55-/- mice were administered the GPR55 agonist O-1602 (4 μg/kg/day) directly into the left hippocampus via stainless steel cannula connected to an osmotic mini-pump for a continuous 14 days. O-1602 treatment increased hippocampal NSC proliferation, survival, and immature neuron formation as compared to vehicle treated animals. These results were determined to be dependent on GPR55 activation as GPR55-/- animals did not show any response to agonist treatment. Interestingly, GPR55-/- mice displayed significantly reduced rates of hippocampal NSC proliferation and neuroblast formation as compared to C57BL/6 animals. Chronic production of inflammatory mediators, such as IL-1b seen in neuroinflammation, to NSCs is known to reduce proliferation rates and attenuate neurogenesis both in vitro and in vivo. Addition of GPR55 agonists to IL-1b (10 ng/mL) treated human and murine NSC samples in vitro protected against reductions in neuron formation as assessed by immunohistochemistry and flow cytometry. 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 (4μg/kg/day) in C57BL/6 mice but not in GPR55-/-mice. Neuroprotection by O-1602 treatment was not found to be microglia dependent as microglia activation was not altered by agonist administration. Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a prolonged inflammatory response (IL-1b, IL-6, TNFa) 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. / Biomedical Sciences

Neural stem/progenitor cells (NSPCs) play a crucial part in the evolutionary development of the human neocortex. During early postnatal development, NSPCs give rise to immature neurons called neuroblasts within the subventricular zone (SVZ) that utilize unique migratory streams to integrate widely in the cerebral cortex. However, the cellular mechanisms enabling these unique migratory routes through the compacted cellular landscape remain unknown. Special emphasis has been placed on understanding the susceptibility of these brain regions to severe conditions such as congenital heart disease (CHD), resulting in poor neurological outcomes. Owing to its reminiscent complexity to humans, the neonatal piglet (Sus scrofa domesticus), which possesses a highly evolved gyrencephalic neocortex and an expansive outer SVZ, provides a powerful translational model system for the study of how heart dysfunction impacts cortical development from both a modern and evolutionary perspective. The present study provides a detailed characterization of neuroblast migration along their associate substrates in the piglet cortex under normal physiological conditions and how reduced oxygenation (i.e., hypoxia) can impact their vulnerability and/or resistance to injury during a critical period of postnatal development. In this thesis, I investigated the spatiotemporal distribution and developmental origin of SVZ-derived neuroblasts. Following BrdU tracing, multiplex labeling, and confocal microscopy, I show that the porcine brain contains populations of newly generated (BrdU+/DCX+) neurons in the prefrontal cortex that are produced postnatally. Regional analyses using immunohistochemical staining for doublecortin (DCX), a marker expressed by immature neurons, revealed that DCX+ clusters co-express markers of neuronal cell migration (PSA-NCAM), GABAergic interneuron marker (GABA+), and specific transcription factors (SCGN+SP8+) associated with the caudal- and lateral ganglionic eminence progenitor domains in the ventral forebrain. Moreover, I found that DCX+ neuroblasts are encased by astrocytic processes and tightly associated with blood vessels in the SVZ. Additionally, this thesis describes the use of chronic hypoxia as a model to profile neuroblast migration along associated substrates in pathological conditions related to CHD. Together, this work serves as a framework for the functional utilization of the neonatal piglet to understand the impact of substrate-dependent neuronal migration on brain maturation and neurodevelopmental diseases. / Doctor of Philosophy / Congenital heart disease (CHD) remains a significant cause of abnormal fetal brain development, affecting 1-2% of live births per year. Although many surgical strategies have shown promise in increasing quality of life, the current challenges remain the long-term cognitive deficits and diverse neurodevelopmental disabilities due to CHD. Recent studies suggest that dysregulated neurogenesis, which is associated with impaired neocortical development in human fetuses of CHD, may be influenced by altered brain circulation of blood and oxygen deliverance during critical periods of prenatal cortical growth. The brain's subventricular zone (SVZ) niche is essential for producing new neurons following birth to restore, repair, and replace existing neurons in the developing brain. In addition, these newborn neurons undergo long-distance migration from the SVZ to reach their final cortical destinations and ultimately contribute to brain development/plasticity. This study seeks to characterize the migration patterns of newborn neurons and the substrates (e.g., blood vessels or astrocytes), enabling the movement along the unique migratory routes under normal and pathological (i.e., hypoxia) conditions. In short, we found that the vast majority of the SVZ-derived newborn neurons are inhibitory neurons (i.e., interneurons) that originate in the deep region of the brain called the telencephalon and migrate tangentially utilizing blood vessels as scaffolds to the cortex, which is likely to contribute to cortical plasticity. These postnatal piglet findings demonstrate that swine represent a powerful translational model system to study large-brained mammalian cortical development and neuronal migration as it correlates to humans in normal and diseased states.

The Reelin signaling pathway has been proven to play a critical role in human neural development, especially in the architectonic development of the central nervous system. Extracelltilar Reeiin binds to the Very Low Density Lipoprotein Receptor (VLDLR) or the Apolipoprotein -E Receptor Type 2 (ApoER2) on the neural cell membrane and then induces tyrosine phosphorylation· o( the adapter protein Disabled - 1 (Dab-1 ). The phosphorylated Dab-I then cross talk with Wnt pathway to regulate gene expression. Recent researches have shown the Reelin pathway, or more specifically, Dab 1 over expression inhibits Glycogen Synthase Kinase 3P (GSK-3P) of the Wnt pathway. Taken the effect that inhibition of GSK-3P frees and promotes the P-Catenin acumination in cell cytosol and nucleus, and demonstrated by recent researches, increased level of neuron differentiation of GSK-3 p inhibited cell, we suggest that Dab-1 's ability of inhibit GSK- 3P will also result in increase level of P-Catenin in the human neural stem cells (HNSC), thus inducing the HNSC cells to differentiate. Testing the HNSC cells separately with Reelin conditioned media treatment and Dab-1 over-expression show significant increasing of acumination of P-Catenin in cell nucleus. Furthermore, demonstrated by our studies, Dab-1 over-expression also increases the neuron differentiation in HNSCs.

LRP2/Megalin ist ein Rezeptor der LDL-Rezeptor Genfamilie mit essentieller Funktion in der Entwicklung des Zentralnervensystems. Der Rezeptor wird im Neuroepithel des Embryos exprimiert und steuert die Ausbildung der Vorderhirnstrukturen. In weiteren Untersuchungen an LRP2-defizienten Mäusen konnte ich zeigen, dass der Verlust der Expression des Rezeptors in adulten Tieren zu einer Beeinträchtigung der Proliferation neuronaler Vorläuferzellen in der subventrikulären Zone (SVZ) des lateralen Ventrikels führt. Infolgedessen war die Anzahl der Neuroblasten reduziert, die zum olfaktorischen Bulbus wandern und dort zu Interneuronen differenzieren. Anhand immunhistologischer Untersuchungen konnte ich nachweisen, dass der Verlust von LRP2 zu einer verminderten Anzahl GFAP-positiver Zellen, die auch die neuronalen Stammzellen (Typ B Zellen) umfassen, führt. Weiterhin wiesen LRP2 Mutanten eine Reduktion in den Signalen für Nestin, DLX2, PSA-NCAM und DCX. Meine derzeitige Hypothese besagt daher, dass LRP2 ebenso wie im embryonalen Neuroepithel auch in der adulten neuronalen Stammzellnische eine Rolle bei der Regulation der BMP4 Signalwege spielt. Dies wird möglicherweise durch Endozytose-vermittelte Aufnahme mit anschließendem Abbau des Morphogens durch LRP2 vermittelt, wie es bereits in vitro gezeigt werden konnte. BMP4 hat im Wesentlichen eine anti-proliferative Wirkung. Eine exakt gesteuerte Regulation der Konzentration dieses Morphogens ist daher eine Vorraussetzung für ein Neurogenese-permissives Milieu in der adulten neurogenen Stammzellnische. Die Ergebnisse meiner Arbeit entschlüsseln eine neue Rolle von LRP2 in der adulten Neurogenese. Die Expression des Rezeptors im Ependym des lateralen Ventrikels ist essentiell für die Regulation der BMP Signalwege in der neurogenen Stammzellnische. / LRP2 (also know as megalin) is a member of the low-density lipoprotein receptor gene family that plays an important role in regulation of neurogenesis in the embryonic neural tube. During early forebrain development, LRP2 deficiency leads to an increase in bone morphogenetic protein 4 (Bmp4) expression and signalling in the dorsal neuroepithelium, and a loss of sonic hedgehog (Shh) expression in the ventral forebrain. In this thesis I demonstrate that LRP2 is expressed in ependymal cells of the lateral ventricles in the adult brain. Intriguingly, expression is restricted to the ependyma that faces the stem cell niche. Expression is not seen in ependyma elsewhere in the lateral ventricles or in the dentate gyrus, the second neurogenic zone of the adult mouse brain. I further show that lack of LRP2 expression in adult mice results in impaired proliferation of neural precursor cells in the SVZ resulting in a decreased number of neuroblasts reaching the olfactory bulb. Using immunohistological detection of marker proteins, absence of LRP2 was shown mainly to affect the GFAP-positive neuronal precursor cell population in the SVZ (B cells). Furthermore, Lrp2 mutant mice also showed a decrease in the signals for nestin, DLX2, PSA-NCAM and DCX. Reduced neurogenesis in the SVZ in LRP2-deficient mice coincides with a significant increase in BMP2/4 expression and enhanced activation of downstream mediators Phospho-SMAD1/5/8 and ID3 in the stem cell niche. My findings revealed a novel regulatory pathway whereby LRP2 down-regulates BMP signaling to modulate the instructive microenvironment of the SVZ and to enable adult neurogenesis to proceed. Thus, LRP2 plays a crucial role in regulating BMP-signaling levels in the adult SVZ, highlighting the unique role of ependymal cells in this stem cell niche. The underlying mechanism of LRP2 action in control of neurogenesis may thus be conserved between the embryonic and adult brain.

Neurogenesis is the process of generation of neurons during embryonic development and adulthood. The focus of this doctoral work is the study of the cell biological aspects of neurogenesis and the mechanisms regulating the switch of neural stem cells from proliferation to differentiation. During embryonic development neurogenic divisions occur at the apical or basal side of the pseudostratified epithelium that forms the wall of the neural tube, the neuroepithelium. Apical asymmetric neurogenic divisions (AP) give rise to a neuron and a progenitor cell, while basal symmetric neurogenic divisions (BP) give rise to two neurons. The first part of this thesis is focused on the study of some cell biological aspects of BPs. We first validated the use of the Tis21-GFP knock in mouse line, previously generated in our laboratory. We found that the totality of neurogenic progenitors is marked by the expression of a nuclear GFP. We calculated the abundance of BPs overtime since the onset of neurogenesis showing that BPs overcome APs over development. We studied the loss of apical contact of the basal dividing cells. We found that both neurogenic and non-neurogenic basally dividing progenitors miss the apical contact; which is lost prior mitosis. We generated and characterized a second mouse line, the Tubb3-GFP line expressing a plasma membrane-localized GFP in neurons. These two lines were crossed to obtain a new line (TisTubb-GFP) allowing detection of neurogenic divisions and tracking daughter cells. Using this model: (i) we imaged symmetric neurogenic divisions of BPs, identifying daughter cells as neurons, during imaging; (ii) we compared the kinetics of betaIII-tubulin-GFP appearance after apical or basal mitosis, showing that daughters of BPs express betaIII-tubulin-GFP faster than daughters coming from apical divisions; (iii) we imaged neuronal migration and localization of the Golgi apparatus. Neurogenesis in the adult is confined to two specific regions in the telencephalon: the sub ependymal zone, lining the ventricle, and dentate gyrus of the hippocampus. The second part of this thesis focuses on the adult neurogenic progenitors lineage. Tis21-GFP expression was found and characterized in the two adult neurogenic regions from early postnatal to adulthood. Using a panel of markers for the adult neurogenic cell lineage and confocal imaging, we characterized Tis21-GFP expression, in the dentate gyrus. Tis21-GFP is first expressed in the neurogenic subpopulation of doublecortin positive cells. Tis21-GFP is inherited by the neurons and eventually degraded. Moreover, our data suggest that mitotic Tis21-GFP cells are an indicator of the levels of neurogenesis more accurate than doublecortin positive cells, in the early postnatal mouse. (Anlage Quick time movies 77,88 MB)

Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post-embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term.

Neurogenesis is the process of generation of neurons during embryonic development and adulthood. The focus of this doctoral work is the study of the cell biological aspects of neurogenesis and the mechanisms regulating the switch of neural stem cells from proliferation to differentiation. During embryonic development neurogenic divisions occur at the apical or basal side of the pseudostratified epithelium that forms the wall of the neural tube, the neuroepithelium. Apical asymmetric neurogenic divisions (AP) give rise to a neuron and a progenitor cell, while basal symmetric neurogenic divisions (BP) give rise to two neurons. The first part of this thesis is focused on the study of some cell biological aspects of BPs. We first validated the use of the Tis21-GFP knock in mouse line, previously generated in our laboratory. We found that the totality of neurogenic progenitors is marked by the expression of a nuclear GFP. We calculated the abundance of BPs overtime since the onset of neurogenesis showing that BPs overcome APs over development. We studied the loss of apical contact of the basal dividing cells. We found that both neurogenic and non-neurogenic basally dividing progenitors miss the apical contact; which is lost prior mitosis. We generated and characterized a second mouse line, the Tubb3-GFP line expressing a plasma membrane-localized GFP in neurons. These two lines were crossed to obtain a new line (TisTubb-GFP) allowing detection of neurogenic divisions and tracking daughter cells. Using this model: (i) we imaged symmetric neurogenic divisions of BPs, identifying daughter cells as neurons, during imaging; (ii) we compared the kinetics of betaIII-tubulin-GFP appearance after apical or basal mitosis, showing that daughters of BPs express betaIII-tubulin-GFP faster than daughters coming from apical divisions; (iii) we imaged neuronal migration and localization of the Golgi apparatus. Neurogenesis in the adult is confined to two specific regions in the telencephalon: the sub ependymal zone, lining the ventricle, and dentate gyrus of the hippocampus. The second part of this thesis focuses on the adult neurogenic progenitors lineage. Tis21-GFP expression was found and characterized in the two adult neurogenic regions from early postnatal to adulthood. Using a panel of markers for the adult neurogenic cell lineage and confocal imaging, we characterized Tis21-GFP expression, in the dentate gyrus. Tis21-GFP is first expressed in the neurogenic subpopulation of doublecortin positive cells. Tis21-GFP is inherited by the neurons and eventually degraded. Moreover, our data suggest that mitotic Tis21-GFP cells are an indicator of the levels of neurogenesis more accurate than doublecortin positive cells, in the early postnatal mouse. (Anlage Quick time movies 77,88 MB)

A complex regulatory framework underlies the generation of newborn neurons in the adult mammalian brain, including the lifelong maintenance of neural stem cell (NSC) quiescence and instructing NSC entry to and exit from quiescence. Future therapies targeting endogenous repair of the aging or afflicted brain, including neurodegenerative pathologies, rely on present efforts to define and characterize the mechanisms underlying the regulation of adult NSC fate. In this dissertation, we demonstrate a requirement for the Rb/E2F axis in the regulation of the molecular program instructing adult NSC quiescence and activation, with a potential role in the impaired hippocampal function observed in Alzheimer's disease pathology. While Rb plays a role in the production and survival of hippocampal newborn neurons, we identify a collective requirement for Rb family proteins — pRb, p107 and p130 — as well as their targets, E2F family transcriptional activators E2F1 and E2F3, in the regulation of NSC quiescence and activation. We further demonstrate that this is mediated through pivotal factors REST and ASCL1, identified as direct molecular targets of the Rb/E2F axis, and that REST inactivation can partially rescue NSC depletion following Rb family loss. We finally demonstrate impaired NSC activation and a return to quiescence in the 3xTG-AD model of Alzheimer's disease, with altered expression of Rb/E2F genes observed within cell population-specific defects. Ultimately, this work addresses the key issue of how transcriptional signatures of quiescence and activation among adult NSCs are co- ordinated with cell cycle control, and demonstrates that Rb family proteins serve as master regulators of the molecular program instructing adult NSC exit from and re-entry into quiescence.

A significant consequence of alcohol use disorders (AUDs) is hippocampal neurodegeneration. The hippocampus is responsible for learning and memory, and neurodegeneration in this brain region has been shown to result in cognitive deficits. Interestingly, some alcoholics demonstrate improvements in hippocampus-dependent functions, potentially due the phenomenon termed adult neurogenesis. Adult neurogenesis, the process by which neural stem cells (NSCs) proliferate, differentiate into neurons, migrate into the granule cell layer, and survive, occurs in two brain regions; however, this study examines only neurogenesis occurring in the subgranular zone of the hippocampal dentate gyrus. Four-day binge ethanol exposure in an animal model causes a decrease in neurogenesis during intoxication; however, there is a reactive increase in cell proliferation on day seven of abstinence. The purpose of this study was to determine the timing of increased cell proliferation. Furthermore, most alcoholics also smoke tobacco, and nicotine, the addictive component of tobacco, has also been shown to affect hippocampal neurogenesis. As many people initiate alcohol and tobacco use during adolescence, the second experiment herein examined the effect of nicotine coadministration on alcohol-induced reactive hippocampal cell proliferation.

Traumatic brain injury (TBI) is a prevalent CNS neurodegenerative condition that results in lasting neurological dysfunction, including potentially debilitating cognitive impairments. Despite the advancements in understanding the complex damage that can culminate in cellular dysfunction and loss, no therapeutic treatment has been effective in clinical trials, highlighting that new approaches are desperately needed. A therapy that limits cell death while simultaneously promoting reparative mechanisms, including post-traumatic neurogenesis, in the injured brain may have maximum effectiveness in improving recovery of function after TBI. Insulin-like growth factor-1 (IGF-1) is a potent growth factor that has previously been shown to promote recovery of function after TBI, but no studies have evaluated the efficacy of IGF-1 to promote cell survival and modulate neurogenesis following brain injury. Systemic infusion of IGF-1 resulted in undetectable levels of IGF-1 in the brain, but did promote increased cortical activation of Akt, a pro-survival downstream mediator of IGF-1 signaling, in mice subjected to controlled cortical impact (CCI), a well-established model of contusion TBI. However, systemic infusion of IGF-1 did not promote recovery of motor function in mice after CCI. A one week central infusion of IGF-1 elevated brain levels of IGF-1, increased Akt activation and improved motor and cognitive function after CCI. Central infusion of IGF-1 also significantly increased immature neuron density at 7 d post-injury for a range of doses and when administered with a clinically relevant delayed onset of 6 hr post-injury. To mitigate potential side effects of central infusion, an alternative conditional astrocyte-specific IGF-1 overexpressing mouse model was utilized to evaluate the efficacy of IGF-1 to promote post-traumatic neurogenesis. Overexpression of IGF-1 did not protect against acute immature neuron loss, but did increase immature neuron density above uninjured levels at 10 d post-injury. The increase in immature neuron density appeared to be driven by enhanced neuronal differentiation. In wildtype mice, immature neurons exhibited injury-induced reductions in dendritic arbor complexity following severe CCI, a previously unknown pathological phenomenon. Overexpression of IGF-1 in brain-injured mice promoted the restoration of dendritic arbor complexity to the dendritic morphology observed in uninjured mice. Together, these findings provide strong evidence that treatment with IGF-1 promotes the recovery of neurobehavioral function and enhances post-traumatic neurogenesis in a mouse model of contusion TBI.