Characterization of the ventricular-subventricular stem cell niche during human brain development.

Human brain development proceeds via a sequentially transforming stem cell population in the ventricular-subventricular zone (V-SVZ). An essential, but understudied, contributor to V-SVZ stem cell niche health is the multi-ciliated ependymal epithelium, which replaces stem cells at the ventricular surface during development. However, reorganization of the V-SVZ stem cell niche and its relationship to ependymogenesis has not been characterized in the human brain. Based on comprehensive comparative spatiotemporal analyses of cytoarchitectural changes along the mouse and human ventricle surface, we uncovered a distinctive stem cell retention pattern in humans as ependymal cells populate the surface of the ventricle in an occipital-to-frontal wave. During perinatal development, ventricle-contacting stem cells are reduced. By 7 months few stem cells are detected, paralleling the decline in neurogenesis. In adolescence and adulthood, stem cells and neurogenesis are not observed along the lateral wall. Volume, surface area and curvature of the lateral ventricles all significantly change during fetal development but stabilize after 1 year, corresponding with the wave of ependymogenesis and stem cell reduction. These findings reveal normal human V-SVZ development, highlighting the consequences of disease pathologies such as congenital hydrocephalus.

EphA4 Regulates Neuroblast and Astrocyte Organization in a Neurogenic Niche.

Significant migration cues are required to guide and contain newly generated rodent subventricular zone (SVZ) neuroblasts as they transit along the lateral ventricles and then through the anterior forebrain to their ultimate site of differentiation in the olfactory bulbs (OBs). These cues enforce strict neuroblast spatial boundaries within the dense astroglial meshwork of the SVZ and rostral migratory stream (RMS), yet are permissive to large-scale neuroblast migration. Therefore, the molecular mechanisms that define these cues and control dynamic interactions between migratory neuroblasts and surrounding astrocytes are of particular interest. We found that deletion of EphA4 and specifically ablation of EphA4 kinase activity resulted in misaligned neuroblasts and disorganized astrocytes in the RMS/SVZ, linking EphA4 forward signaling to SVZ and RMS spatial organization, orientation, and regulation. In addition, within a 3 week period, there was a significant reduction in the number of neuroblasts that reached the OB and integrated into the periglomerular layer, revealing a crucial role for EphA4 in facilitating efficient neuroblast migration to the OB. Single-cell analysis revealed that EPHA4 and its EFN binding partners are expressed by subpopulations of neuroblasts and astrocytes within the SVZ/RMS/OB system resulting in a cell-specific mosaic, suggesting complex EphA4 signaling involving both homotypic and heterotypic cell-cell interactions. Together, our studies reveal a novel molecular mechanism involving EphA4 signaling that functions in stem cell niche organization and ultimately neuroblast migration in the anterior forebrain.SIGNIFICANCE STATEMENT The subventricular zone neurogenic stem cell niche generates highly migratory neuroblasts that transit the anterior forebrain along a defined pathway to the olfactory bulb. Postnatal and adult brain organization dictates strict adherence to a narrow migration corridor. Subventricular zone neuroblasts are aligned in tightly bundled chains within a meshwork of astrocytes; however, the cell-cell cues that organize this unique, cell-dense migration pathway are largely unknown. Our studies show that forward signaling through the EphA4 tyrosine kinase receptor, mediated by ephrins expressed by subpopulations of neuroblasts and astrocytes, is required for compact, directional organization of neuroblasts and astrocytes within the pathway and efficient transit of neuroblasts through the anterior forebrain to the olfactory bulb.

Ventricular and Periventricular Anomalies in the Aging and Cognitively Impaired Brain.

Ventriculomegaly (expansion of the brain's fluid-filled ventricles), a condition commonly found in the aging brain, results in areas of gliosis where the ependymal cells are replaced with dense astrocytic patches. Loss of ependymal cells would compromise trans-ependymal bulk flow mechanisms required for clearance of proteins and metabolites from the brain parenchyma. However, little is known about the interplay between age-related ventricle expansion, the decline in ependymal integrity, altered periventricular fluid homeostasis, abnormal protein accumulation and cognitive impairment. In collaboration with the Baltimore Longitudinal Study of Aging (BLSA) and Alzheimer's Disease Neuroimaging Initiative (ADNI), we analyzed longitudinal structural magnetic resonance imaging (MRI) and subject-matched fluid-attenuated inversion recovery (FLAIR) MRI and periventricular biospecimens to map spatiotemporally the progression of ventricle expansion and associated periventricular edema and loss of transependymal exchange functions in healthy aging individuals and those with varying degrees of cognitive impairment. We found that the trajectory of ventricle expansion and periventricular edema progression correlated with degree of cognitive impairment in both speed and severity, and confirmed that areas of expansion showed ventricle surface gliosis accompanied by edema and periventricular accumulation of protein aggregates, suggesting impaired clearance mechanisms in these regions. These findings reveal pathophysiological outcomes associated with normal brain aging and cognitive impairment, and indicate that a multifactorial analysis is best suited to predict and monitor cognitive decline.

Development and aging of a brain neural stem cell niche.

In the anterior forebrain, along the lateral wall of the lateral ventricles, a neurogenic stem cell niche is found in a region referred to as the ventricular-subventricular zone (V-SVZ). In rodents, robust V-SVZ neurogenesis provides new neurons to the olfactory bulb throughout adulthood; however, with increasing age stem cell numbers are reduced and neurogenic capacity is significantly diminished, but new olfactory bulb neurons continue to be produced even in old age. Humans, in contrast, show little to no new neurogenesis after two years of age and whether V-SVZ neural stem cells persist in the adult human brain remains unclear. Here, we review functional and organizational differences in the V-SVZ stem cell niche of mice and humans, and examine how aging affects the V-SVZ niche and its associated functions.

Repeated mild traumatic brain injury causes focal response in lateral septum and hippocampus.

AIM: To advance our understanding of regional and temporal cellular responses to repeated mild traumatic brain injury (rmTBI), we used a mouse model of rmTBI that incorporated acceleration, deceleration and rotational forces. MATERIALS & METHODS: A modified weight-drop method was used to compare two inter-injury intervals, rmTBI-short (five hits delivered over 3 days) and rmTBI-long (five hits delivered over 15 days). Regional investigations of forebrain and midbrain histological alterations were performed at three post-injury time points (immediate, 2 weeks and 6 weeks). RESULTS: The rmTBI-short protocol generated an immediate, localized microglial and astroglial response in the dorsolateral septum and hippocampus, with the astroglial response persisting in the dorsolateral septum. The rmTBI-long protocol showed only a transitory astroglial response in the dorsolateral septum. CONCLUSION: Our results indicate that the lateral septum and hippocampus are particularly vulnerable regions in rmTBI, possibly contributing to memory and emotional impairments associated with repeated concussions.

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Ventriculomegaly associated with ependymal gliosis and declines in barrier integrity in the aging human and mouse brain.

Age-associated ventriculomegaly is typically attributed to neurodegeneration; however, additional factors might initiate or contribute to progressive ventricular expansion. By directly linking postmortem human MRI sequences with histological features of periventricular tissue, we show that substantial lateral ventricle surface gliosis is associated with ventriculomegaly. To examine whether loss of ependymal cell coverage resulting in ventricle surface glial scarring can lead directly to ventricle enlargement independent of any other injury or degenerative loss, we modeled in mice the glial scarring found along the lateral ventricle surface in aged humans. Neuraminidase, which cleaves glycosidic linkages of apical adherens junction proteins, was administered intracerebroventricularly to denude areas of ependymal cells. Substantial ependymal cell loss resulted in reactive gliosis rather than stem cell-mediated regenerative repair of the ventricle lining, and the gliotic regions showed morphologic and phenotypic characteristics similar to those found in aged humans. Increased levels of aquaporin-4, indicative of edema, observed in regions of periventricular gliosis in human tissue were also replicated in our mouse model. 3D modeling together with volume measurements revealed that mice with ventricle surface scarring developed expanded ventricles, independent of neurodegeneration. Through a comprehensive, comparative analysis of the lateral ventricles and associated periventricular tissue in aged humans and mouse, followed by modeling of surface gliosis in mice, we have demonstrated a direct link between lateral ventricle surface gliosis and ventricle enlargement. These studies highlight the importance of maintaining an intact ependymal cell lining throughout aging.

Spatiotemporal changes to the subventricular zone stem cell pool through aging.

Through adulthood, the rodent subventricular zone (SVZ) stem cell niche generates new olfactory bulb interneurons. We had previously reported that the number of new neurons produced in the SVZ declines through aging; however, age-related changes attributable specifically to the SVZ neural stem cell (NSC) population have not been fully characterized. Here, we conducted a spatiotemporal evaluation of adult SVZ NSCs. We assessed ventricle-contacting NSCs, which together with ependymal cells form regenerative units (pinwheels) along the lateral wall of the lateral ventricle. Based on their apical GFAP-expressing process, individual NSCs were identified across the ventricle surface using serial reconstruction of the SVZ. We observed an 86% decline in total NSCs/mm² of intact ependyma in 2-year old versus 3-month-old mice, with fewer NSC processes within each aged pinwheel. This resulted in an associated 78% decline in total pinwheel units/mm². Regional analysis along the lateral ventricle surface revealed that the age-dependent decline of NSCs and pinwheels is spatially uniform and ultimately maintains the conserved ratio of olfactory bulb interneuron subtypes generated in young mice. However, the overall neurogenic output of the aged SVZ is reduced. Surprisingly, we found no significant change in the number of actively proliferating NSCs per mm² of ventricle surface. Instead, our data reveal that, although the total NSC number, pinwheel units and NSCs per pinwheel decline with age, the percentage of actively, mitotic NSCs increases, indicating that age-related declines in SVZ-mediated olfactory bulb neurogenesis occur downstream of NSC proliferation.

Changes in nucleus accumbens and neostriatal c-Fos and DARPP-32 immunoreactivity during different stages of food-reinforced instrumental training.

Nucleus accumbens is involved in several aspects of instrumental behavior, motivation and learning. Recent studies showed that dopamine (DA) release in the accumbens shell was significantly increased on the first day of training on a fixed ratio (FR) 5 schedule (i.e. the transition from FR1 to FR5) compared with those rats that continued FR1 training, even though the rats on their first day of FR5 training received less food reinforcement than rats continuing on the FR1 schedule. Additionally, the second day of FR5 responding was marked by a significant increase in DA release in accumbens core. The present studies employed immunohistochemical methods to characterize the changes in cellular markers of accumbens and neostriatal neural activity that occur during various stages of food-reinforced FR5 training. c-Fos and DARPP-32 immunoreactivity in accumbens shell was significantly increased on the first day of FR5 training, while core c-Fos and DARPP-32 expression showed large increases on the second day of FR5 training. Additional studies showed that c-Fos and DARPP-32 expression in neostriatum increased after more extensive training. Double-labeling studies with immunofluorescence methods indicated that increases in accumbens c-Fos and DARPP-32 expression were primarily seen in substance-P-positive neurons. These increases in accumbens c-Fos and DARPP-32 immunoreactivity seen during the initial phases of FR training may reflect several factors, including novelty, learning, stress or the presentation of a work-related challenge to the organism. Moreover, it appears that the separate subregions of the striatal complex are differentially activated at distinct phases of instrumental training.

AGING OF THE SUBVENTRICULAR ZONE NEURAL STEM CELL NICHE.

The persistence of an active subventricular zone neural stem cell niche in the adult mammalian forebrain supports its continued role in the production of new neurons and in generating cells to function in repair through adulthood. Unfortunately, with increasing age the niche begins to deteriorate, compromising these functions. The reasons for this decline are not clear. Studies are beginning to define the molecular and physiologic changesin the microenvironment of the aging subventricular zone niche. New revelations from aging studies will allow for a more thorough understanding of what reparative functions are lost in the aged brain, the progression of niche demise and the possibility for therauptic intervention to improve aging brain function.

Midbrain dopamine neurons associated with reward processing innervate the neurogenic subventricular zone.

Coordinated regulation of the adult neurogenic subventricular zone (SVZ) is accomplished by a myriad of intrinsic and extrinsic factors. The neurotransmitter dopamine is one regulatory molecule implicated in SVZ function. Nigrostriatal and ventral tegmental area (VTA) midbrain dopamine neurons innervate regions adjacent to the SVZ, and dopamine synapses are found on SVZ cells. Cell division within the SVZ is decreased in humans with Parkinson's disease and in animal models of Parkinson's disease following exposure to toxins that selectively remove nigrostriatal neurons, suggesting that dopamine is critical for SVZ function and nigrostriatal neurons are the main suppliers of SVZ dopamine. However, when we examined the aphakia mouse, which is deficient in nigrostriatal neurons, we found no detrimental effect to SVZ proliferation or organization. Instead, dopamine innervation of the SVZ tracked to neurons at the ventrolateral boundary of the VTA. This same dopaminergic neuron population also innervated the SVZ of control mice. Characterization of these neurons revealed expression of proteins indicative of VTA neurons. Furthermore, exposure to the neurotoxin MPTP depleted neurons in the ventrolateral VTA and resulted in decreased SVZ proliferation. Together, these results reveal that dopamine signaling in the SVZ originates from a population of midbrain neurons more typically associated with motivational and reward processing.

Aging of the subventricular zone neural stem cell niche.

The persistence of an active subventricular zone neural stem cell niche in the adult mammalian forebrain supports its continued role in the production of new neurons and in generating cells to function in repair through adulthood. Unfortunately, with increasing age the niche begins to deteriorate, compromising these functions. The reasons for this decline are not clear. Studies are beginning to define the molecular and physiologic changes in the microenvironment of the aging subventricular zone niche. New revelations from aging studies will allow for a more thorough understanding of which reparative functions are lost in the aged brain, the progression of niche demise and the possibility for therauptic intervention to improve aging brain function.

In-vitro analysis of Pitx3 in mesodiencephalic dopaminergic neuron maturation.

The transcription factor Pitx3 is expressed exclusively by mesodiencephalic dopaminergic neurons; however, ablation of Pitx3 results in selective degeneration of primarily dopaminergic neurons of the substantia nigra pars compacta, the neuronal population that is most vulnerable in Parkinson's disease. Although the exact molecular mechanisms of the action of Pitx3 are unclear, roles in both terminal maturation and/or survival of substantia nigra dopaminergic neurons have been suggested. To investigate the connection between Pitx3 and selective neurodegeneration, we generated embryonic stem cells from a Pitx3-deficient mouse (aphakia) for in-vitro differentiation to dopaminergic neurons. This 'loss of function'in-vitro system allowed us to examine characteristic features in dopaminergic neuron development and to assess the role that Pitx3 plays in the differentiation/maturation process. We found that aphakia embryonic stem cells generated 50% fewer tyrosine hydroxylase-positive/microtubule-associated protein (Map)2-positive mature neurons compared with control cultures. The expression of dopamine transport regulators and vesicle release proteins was reduced and dopamine release was unregulated in the Pitx3-deficient tyrosine hydroxylase-positive neurons generated. Treatment of aphakia embryonic stem cell cultures with retinoic acid resulted in a significant increase in mesodiencephalic tyrosine hydroxylase-positive neurons, providing further support for the role of Pitx3 in dopaminergic neuron specification through the retinoic acid pathway. Our study, using Pitx3-deficient embryonic stem cells in an in-vitro differentiation culture system, allowed us to assess the role of Pitx3 in the specification and final maturation of dopaminergic neurons.

Subventricular zone-mediated ependyma repair in the adult mammalian brain.

The subventricular zone (SVZ) of the adult mouse brain is a narrow stem cell niche that lies along the length of the lateral wall of the lateral ventricles. The SVZ supports neurogenesis throughout adulthood; however, with increasing age, the ventral SVZ deteriorates and only the dorsolateral SVZ remains neurogenic. Associated with the elderly dorsolateral SVZ, we reported previously an increased number of astrocytes interposed within the adjacent ependymal lining. Here, we show that astrocytes integrated within the ependyma are dividing, BrdU-labeled astrocytes that share cellular adherens with neighboring ependymal cells. By tracking BrdU-labeled astrocytes over time, we observed that, as they incorporated within the ependyma, they took on antigenic and morphologic characteristics of ependymal cells, suggesting a novel form of SVZ-supported "regenerative" repair in the aging brain. A similar form of SVZ-mediated ependyma repair was also observed in young mice after mild ependymal cell denudation with low dosages of neuraminidase. Together, this work identifies a novel non-neuronal mechanism of regenerative repair by the adult SVZ.

In vitro generation of dopaminergic neurons from adult subventricular zone neural progenitor cells.

The adult subventricular zone (SVZ) supports a population of cells that display the hallmarks of stem cells: they are self-renewing and multipotent-capable of generating neurons, oligodendrocytes, and astrocytes. In vivo, these adult neural stem cells (aNSCs) are fated primarily for a gamma-amino butyric acid (GABA)-ergic lineage of olfactory bulb interneurons, a small subpopulation of which is dopaminergic. Here, we investigate the plasticity of aNSCs in vitro, in particular, their ability to generate a specific neuronal lineage, midbrain dopamine neurons. Previous work using mouse embryonic stem (ES) cells showed that introduction of early developmental inductive cues, sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF-8), directed ES cell-derived neuroepithelial cells to generate midbrain dopaminergic neurons, those lost in Parkinson's disease. Placing aNSCs under similar culture conditions, immunocytochemistry and RT-PCR analysis revealed early dopaminergic neuron specification. However, aNSC-derived neurons remained morphologically immature, exhibiting concurrent nestin and tyrosine hydroxylase (TH) expression, with cell death occurring in the final differentiation stage. High-performance liquid chromatography (HPLC) analysis revealed that while aNSC-derived neurons released dopamine, release was not significantly increased following depolarization with K+. In contrast, ES cell-generated TH+ neurons expressed the mature markers MAP2 and NeuN and showed K+-evoked release of dopamine. Reduced culture time of aNSC-derived nestin+ progenitors in FGF-2-containing medium improved survival of TH+ neurons. However, these neurons exhibited characteristics of forebrain dopamine neurons and also expressed low levels of midbrain transcription factors. Together, our data indicate that when presented with in vitro conditions that promote midbrain-specific dopamine neuron specification, aNSCs instead generate forebrain-like dopamine neurons, demonstrating their restricted and prescribed nature.

The neural stem cell niche.

The neural stem cell niche defines a zone in which stem cells are retained after embryonic development for the production of new cells of the nervous system. This continual supply of new neurons and glia then provides the postnatal and adult brain with an added capacity for cellular plasticity, albeit one that is restricted to a few specific zones within the brain. Critical to the maintenance of the stem cell niche are microenvironmental cues and cell-cell interactions that act to balance stem cell quiescence with proliferation and to direct neurogenesis versus gliogenesis lineage decisions. Ultimately, based on the location of the niche, stem cells of the adult brain support regeneration in the dentate gyrus of the hippocampus and the olfactory bulb through neuron replacement. Here, we provide a summary of the current understanding of the organization and control mechanisms of the neural stem cell niche.

Neuroblast protuberances in the subventricular zone of the regenerative MRL/MpJ mouse.

The MRL mouse is unique in its capacity for regenerative healing of wounds. This regenerative ability includes complete closure, with little scarring, of wounds to the ear pinna and repair of cardiac muscle, without fibrosis, following cryoinjury. Here, we examine whether neurogenic zones within the MRL brain show enhanced regenerative capacity. The largest neurogenic zone in the adult brain, the subventricular zone (SVZ), lies adjacent to the lateral wall of the lateral ventricle and is responsible for replacement of interneuron populations within the olfactory bulb. Initial gross observation of the anterior forebrain in MRL mice revealed enlarged lateral ventricles; however, little neurodegeneration was detected within the SVZ or surrounding tissues. Instead, increased proliferation within the SVZ was observed, based on incorporation of the thymidine analogue bromodeoxyuridine. Closer examination using electron microscopy revealed that a significant number of SVZ astrocytes interpolated within the ependyma and established contact with the ventricle. In addition, subependymal, protuberant nests of cells, consisting primarily of neuroblasts, were found along the anterior SVZ of MRL mice. Whole mounts of the lateral wall of the lateral ventricle stained for the neuroblast marker doublecortin revealed normal formation of chains of migratory neuroblasts along the entire wall and introduction of enhanced green fluorescent protein-tagged retrovirus into the lateral ventricles confirmed that newly generated neuroblasts were able to track into the olfactory bulb.

The aging neurogenic subventricular zone.

In the adult mouse brain, the subventricular zone (SVZ) is a neurogenic stem cell niche only 4-5 cell diameters thick. Within this narrow zone, a unique microenvironment supports stem cell self-renewal, gliogenesis or neurogenesis lineage decisions and tangential migration of newly generated neurons out of the SVZ and into the olfactory bulb. However, with aging, SVZ neurogenesis declines. Here, we examine the dynamic interplay between SVZ cytoarchitecture and neurogenesis through aging. Assembly of high-resolution electron microscopy images of corresponding coronal sections from 2-, 10- and 22-month-old mice into photomontages reveal a thinning of the SVZ with age. Following a 2-h BrdU pulse, we detect a significant decrease in cell proliferation from 2 to 22 months. Neuroblast numbers decrease with age, as do transitory amplifying progenitor cells, while both SVZ astrocytes and adjacent ependymal cells remain relatively constant. At 22 months, only residual pockets of neurogenesis remain and neuroblasts become restricted to the anterior dorsolateral horn of the SVZ. Within this dorsolateral zone many key components of the younger neurogenic niche are maintained; however, in the aged SVZ, increased numbers of SVZ astrocytes are found interposed within the ependyma. These astrocytes co-label with markers to ependymal cells and astrocytes, form intercellular adherens junctions with neighboring ependymal cells, and some possess multiple basal bodies of cilia within their cytoplasm. Together, these data reveal an age-related, progressive restriction of SVZ neurogenesis to the dorsolateral aspect of the lateral ventricle with increased numbers of SVZ astrocytes interpolated within the ependyma.

Neural stem cells and the regulation of adult neurogenesis.

Presumably, the 'hard-wired' neuronal circuitry of the adult brain dissuades addition of new neurons, which could potentially disrupt existing circuits. This is borne out by the fact that, in general, new neurons are not produced in the mature brain. However, recent studies have established that the adult brain does maintain discrete regions of neurogenesis from which new neurons migrate and become incorporated into the functional circuitry of the brain. These neurogenic zones appear to be vestiges of the original developmental program that initiates brain formation. The largest of these germinal regions in the adult brain is the subventricular zone (SVZ), which lines the lateral walls of the lateral ventricles. Neural stem cells produce neuroblasts that migrate from the SVZ along a discrete pathway, the rostral migratory stream, into the olfactory bulb where they form mature neurons involved in the sense of smell. The subgranular layer (SGL) of the hippocampal dentate gyrus is another neurogenic region; new SGL neurons migrate only a short distance and differentiate into hippocampal granule cells. Here, we discuss the surprising finding of neural stem cells in the adult brain and the molecular mechanisms that regulate adult neurogenesis.