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Extracellular vesicles, including exosomes/microvesicles (EMVs), have been described as sensitive biomarkers that represent disease states and response to therapies. In light of recent reports of disease-mirroring EMV molecular signatures, the present study profiled two EMVs from different Parkinson's disease (PD) tissue sources: (a) neural progenitor cells derived from an endogenous adult stem/progenitor cell, called adult human neural progenitor (AHNP) cells, that we found to be pathological when isolated from postmortem PD patients' substantia nigra; and (b) leucine-rich repeat kinase 2 (LRRK2) gene identified patient induced pluripotent stem cells (iPSCs), which were used to isolate EMVs and begin to characterize their cargoes. Initial characterization of EMVs derived from idiopathic patients (AHNPs) and mutant LRRK2 patients showed differences between both phenotypes and when compared with a sibling control in EMV size and release based on Nanosight analysis. Furthermore, molecular profiling disclosed that neurodegenerative-related gene pathways altered in PD can be reversed using gene-editing approaches. In fact, the EMV cargo genes exhibited normal expression patterns after gene editing. This study shows that EMVs have the potential to serve as sensitive biomarkers of disease state in both idiopathic and gene-identified PD patients and that following gene-editing, EMVs reflect a corrected state. This is relevant for both prodromal and symptomatic patient populations where potential responses to therapies can be monitored via non-invasive liquid biopsies and EMV characterizations., (© 2019 Wiley Periodicals, Inc.)

Malignant glioma is among of the most devastating, and least curable, types of cancer. Since the re-emergence of the cancer stem cell hypothesis, much progress has been made towards elucidating the cellular origin of these tumors. The hypothesis that tumors are hierarchically organized, with a cancer stem cell at the top that shares defining features with somatic stem cells and provides therapeutic refractoriness properties, has put adult stem cells into the limelight as prime suspect for malignant glioma. Much confusion still exists, though, as to the particular cell type and processes that lead to oncogenic transformation. In this review, we will discuss recent developments and novel hypotheses regarding the origin of malignant gliomas, especially glioblastoma. In particular, we argue that glioblastoma is the result of different pathways originating in multiple sources that all ultimately converge in the same disease. Further attention is devoted to potential scenarios leading to transformation of different stem/progenitor cell types of the brain, and the probability and relevance of these scenarios for malignant tumorigenesis., (Copyright © 2011 Wiley-Liss, Inc.)

Residual tumor cells remain beyond the margins of every glioblastoma (GBM) resection. Their resistance to postsurgical therapy is considered a major driving force of mortality, but their biology remains largely uncharacterized. In this study, residual tumor cells were derived via experimental biopsy of the resection margin after standard neurosurgery for direct comparison with samples from the routinely resected tumor tissue. In vitro analysis of proliferation, invasion, stem cell qualities, GBM-typical antigens, genotypes, and in vitro drug and irradiation challenge studies revealed these cells as unique entities. Our findings suggest a need for characterization of residual tumor cells to optimize diagnosis and treatment of GBM.

Microglia, the resident immune cells of the brain, have recently been hypothesized to play a role both in neuronal diseases and age-related neurogenic decline, and are theorized to be modulators of adult neurogenesis. Current methods for the isolation of microglia from cultured primary brain tissue result in relatively poor yield, requiring a large tissue sample or multiple specimens to obtain a sufficient number of microglia for cell and molecular analysis. We report here a method for the repetitive isolation of microglia from established glial monolayer cultures from which it is possible to expand the initial population of microglia roughly 10,000-fold. The expanded population expresses appropriate microglial morphology and phenotype markers, and demonstrates functionally normal phagocytosis, thus providing a high-yield assay for the investigation and analysis of microglia from a single initial dissection of primary tissue. Furthermore, this massive expansion is limited to microglia derived from the subventricular zone as the fold expansion of isolatable microglia was found to be up to 20 times greater than cultures from other brain regions, indicating unique properties for this persistently neurogenic region., (Copyright 2008 Wiley-Liss, Inc.)

Microglia are increasingly implicated as a source of non-neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self-renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia-conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self-renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain., ((c) 2006 Wiley-Liss, Inc.)

To the extent that their fate choice and differentiation processes can be understood and manipulated, neural stem cells represent a promising therapeutic tool for a variety of neuropathologies. We have previously shown that mature astrocytes possess neural stem cell attributes, and can give rise to neurons through the formation of multipotent neurosphere clones. Here we show that relatively mature neurons generated from neurospheres derived from postnatal subependymal zone or cerebellar cortex undergo a phenotypic transformation into astrocytes that coincides with the appearance of a nonfused, hybrid cell type that shares the morphology, antigenicity, and physiology of both neurons and astrocytes. We refer to this astrocyte/neuron hybrid as an "asteron," and hypothesize that it represents an intermediate step in the trans- or dedifferentiation of neurons into astrocytes. The present finding suggests that seemingly terminally differentiated neural cells may in fact represent points along a bidirectionally fluid continuum of differentiation, with intermediate points represented by "hybrid" cells coexpressing phenotypic markers of more than one lineage.

Recently discovered multipotent astrocytic stem cells are discussed in light of current nomenclature for glial precursor and lineage-associated cells in the developing, postnatal, and adult mammalian brain. Defining the phenotype of any immature cell in the nervous system is a challenge, and a position is stated that includes the need for categorizing cells within a continuum of differentiation potential. The possibility for dedifferentiating glial cells into clonogenic stem-like cells offers numerous possibilities for translating knowledge and technology from this subfield of stem cell biology to regenerative medicine. Along with the need for developing a new lexicon for defining the cellular players that contribute to the generation of glia and neurons in the developing and mature central nervous system, the relationships also need to be established among potency, repopulation attempts, and tumorigenesis of cells meeting the criteria of glial stem cells. Finally, it is possible that understanding the normal differentiation, de- and transdifferentiation potential of glial stem-like cells in the mature central nervous system will provide insights into the possible use of these cells, or biogenic factors associated with their growth and differentiation, in therapeutic approaches for a variety of neurological disorders., (Copyright 2003 Wiley-Liss, Inc.)

Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem-like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage-specific proteins. The high frequencies of clone-forming cells (about 0.1-10 x 10(-3)) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT-PCR analyses of populations of normal brain- and tumor-derived sister clones revealed transcripts for nestin, neuron-specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor-derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch-signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma-derived clones contain cells immunopositive separately for GFAP or neuronal beta-III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors., (Copyright 2002 Wiley-Liss, Inc.)

A transgenic mouse containing the first exon of the human Huntington's disease (HD) gene has revealed a variety of behavioral and pathophysiological anomalies reminiscent of certain aspects of human Huntington's disease (HD). The present study has found that expression of the extracellular matrix glycoprotein tenascin-C appears to be unaffected in astroglial cells in wild-type and R6/2 transgenic mice that express the mutant huntingtin protein but that it is conspicuously absent in two neuronal populations within the cerebral cortex and thalamus of the R6/2 mice. Loss of tenascin-C expression begins between the fourth and eighth postnatal weeks, coincidental with the onset of abnormal behavioral phenotype and the appearance of intranuclear inclusion bodies and neuropil aggregates. By 12 weeks, R6/2 mice exhibit a complete absence of tenascin-C neuronal immunolabeling, a disappearance of cRNA probe-positive neurons across discrete cytoarchitectonic regions of the dorsal thalamus (e.g., the ventromedial, parafascicular, lateral posterior, and posterior thalamic groups) and frontal cortex, and an accompanying thalamic astrogliosis. The loss of neuronal tenascin-C expression includes structures that are known to send converging excitatory axonal projections to the caudate-putamen, the structure that is most at risk for neurodegeneration in HD. Altered neuronal expression of tenascin-C in R6/2 mice implicates altered transcriptional activities of the mutant huntingtin protein. The abnormal biochemistry and possibly abnormal activity of thalamostriate and corticostriate projection neurons may also affect abnormal neuronal activities in their primary connectional target, the neostriatum, which is severely compromised in HD., (Copyright 2001 Wiley-Liss, Inc.)

Using a novel suspension culture approach, previously undescribed populations of neural precursor cells have been isolated from the adult mouse brain. Recent studies have shown that neuronal and glial precursor cells proliferate within the subependymal zone of the lateral ventricle throughout life, and a persistent expression of developmentally regulated surface and extracellular matrix molecules implicates cell-cell and cell-substrate interactions in the proliferation, migration, and differentiation of these cells. By using reagents that may affect cell-cell interactions, dissociated adult brain yields two types of cell aggregates, type I and type II spheres. Both sphere types are proliferative, and type I spheres evolve into type II spheres. Neurons and glia arise from presumptive stem cells of type II spheres, and they can survive transplantation to the adult brain.

We have addressed the issue of whether or not peripherally expressed nerve growth factor (NGF) influences the formation of whisker-specific patterns in the brain by regulating the survival of sensory neurons. Transgenic mice that overexpress an NGF cDNA in the skin were examined. In these animals, excess NGF expression is controlled by promoter and enhancer sequences of a keratin gene, thus restricting the higher levels of NGF expression to basal keratinocytes of the epidermis. Twice the number of trigeminal sensory neurons survive in transgenic mice as in normal animals, and a corresponding hyperinnervation of the whisker pad is noted, both around the vibrissa follicles and along the intervibrissal epidermis. However, the increased survival of sensory neurons and the enhanced peripheral projections do not interfere with the development of whisker-specific patterns in the trigeminal brainstem, in the ventrobasal thalamic complex or in the face-representation region of the primary somatosensory (SI) cortex. These results demonstrate that vibrissa-related central patterns are able to form in the virtual absence of trigeminal ganglion cell death and suggest that mechanisms other than a selective elimination of sensory neurons control the development of whisker-specific neural patterns in the brain.

The subependymal zone (SEZ) of the lateral ventricle of adult rodents has long been known to be mitotically active. There has been increased interest in the SEZ, since it has been demonstrated that neuroepithelial stem cells residing there generate neurons in addition to glia in vitro. In the present study, we have examined parasagittal sections of the adult mouse brain using immunocytochemistry for extracellular matrix (ECM) molecules (tenascin and chondroitin sulfate-containing proteoglycans), glial fibrillary acidic protein (GFAP, a cytoskeletal protein prominently expressed by immature and reactive astrocytes), RC-2 (a radial glial and immature astrocyte cytoskeletal marker), TuJ1 (a class III beta-tubulin isoform expressed solely by postmitotic and adult neurons), nestin (a cytoskeletal protein associated with stem cells), neuron-specific enolase, and bromodeoxyuridine (BrdU, which is taken up by dividing cells). Our results demonstrate that a population of young neurons reside within an ECM-rich, GFAP-positive astrocyte pathway from the rostral SEZ all the way into the olfactory bulb. Furthermore, BrdU labeling studies indicate that there is a high level of cell division along the entire length of this path, and double-labeling studies indicate that neurons committed to a neuronal lineage (i.e., TuJ1+) take up BrdU (suggesting they are in the DNA synthesis phase of the cell cycle), again along the entire length of the SEZ "migratory pathway." Thus, the SEZ appears to retain the ability to produce neurons and glia throughout the life of the animal, functioning as a type of "brain marrow." The implications of these findings are discussed in relation to the role that such a glial/ ECM-rich boundary (as seen in the embryonic cortical subplate and other developing areas) may play in: confining the migratory populations and maintaining them in a persistent state of immaturity; facilitating their migration to the olfactory bulb, where they are incorporated into established adult circuitries; and potentially altering SEZ cell cycle dynamics that eventually lead to cell death.

The subventricular zone (SVZ) of the lateral ventricle remains mitotically active in the adult mammalian central nervous system (CNS). Recent studies have suggested that this region may contain neuronal precursors (neural stem cells) in adult rodents. A variety of neuronal and glial markers as well as three extracellular matrix (ECM) markers were examined with the hope of understanding factors that may affect the growth and migration of neurons from this region throughout development and in the adult. This study has characterized the subventricular zone of late embryonic, postnatal, and adult mice using several neuronal markers [TuJ1, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), neuron-specific enolase (NSE)], glial markers [RC-2, vimentin, glial fibrillary acidic protein (GFAP), galactocerebroside (Gal-C)], ECM markers [tenascin-C (TN-C), chondroitin sulfate, a chondroitin sulfate proteoglycan termed dermatan sulfate-dependent proteoglycan-1 (DSD-1-PG)], stem-cell marker (nestin), and proliferation-specific marker [bromodeoxyuridine (BrdU)]. TuJ1+ and nestin+ cells (neurons and stem cells, respectively) persist in the region into adulthood, although the numbers of these cells become more sparse as the animal develops, and they appear to be immature compared to the cells in surrounding forebrain structures (e.g., not expressing NSE and having few, if any, processes). Likewise, NADPH-d+ cells are found in and around the SVZ during early postnatal development but become more sparse in the proliferative zone through maturity, and, by adulthood, only a few labeled cells can be found at the border between the SVZ and surrounding forebrain structures (e.g., the striatum), and even smaller numbers of positive cells can be found within the adult SVZ proper. BrdU labeling also seems to decrease significantly after the first postnatal week, but it still persists in the SVZ of adult animals. The disappearance of RC-2+ (radial) glia during postnatal development and the persistence of glial-derived ECM molecules such as tenascin and chondroitin sulfate proteoglycans (as well as other "boundary" molecules) in the adult SVZ may be associated with a persistence of immaturity, cell death, and a lack of cell emigration from the SVZ in the adult.

Plant lectins were used to examine the disposition of glycosylated molecules in vibratome sections through the barrel subfield of mouse somatosensory cortex at selected times during postnatal development. The peroxidase conjugates of peanut agglutinin (PNA, specific for N-acetylgalactosamine), concanavalin A (specific for mannose), and wheat germ agglutinin (specific for N-acetylglucosamine and N-acetylneuraminic acid) were used to study lectin binding in aldehyde-fixed tissue sections of cortex. Following peroxidase cytochemistry and light microscopy, it was found that all three lectins bound in the region of the barrel subfield as early as postnatal day 3 (day of birth = postnatal day 1). The lectins bound to the prospective sides and/or septae of individual barrels in preference to the prospective hollows. This lectin demarcation of the barrel field occurred prior to the detection of this region with cresyl violet staining and was still demonstrable on postnatal day 6, when the individual barrels became discernible with cresyl violet. This suggests that the lectin binding material is present before the barrel field becomes a fully formed and organized region. A decrease in lectin affinity for binding sites in these tissue sections occurs during postnatal development (Cooper and Steindler: Soc. Neurosci. (Abstr.) 10: 43a, '84) and this study demonstrates that lectins do not delineate the barrel field of more mature animals (2-3 months old), whereas barrels can be detected with cresyl violet at this time. A preliminary electron microscope analysis of the postnatal day 6 somatosensory cortex demonstrates that the lectin PNA binds to elements of the forming neuropil and also to Golgi apparatus intermediate saccules in neuronal cells. The prospective barrel field can be detected with lectins during a critical period in development in which alterations can occur in the barrel field in response to peripheral deprivation (Jeanmonod et al: Neuroscience 6:1503-35, '81) and therefore we suggest that the glycans visualized with lectin-peroxidase conjugates denote possible candidates for molecules involved in shaping barrel structure.

Double retrograde axonal tracing experiments were carried out in order to reveal potential patterns of divergence in axonal projections from the two major sensory nuclei of the mouse brainstem trigeminal complex: the principal sensory and spinal trigeminal nuclei (oralis, interpolaris, and caudalis divisions). The tracers wheat germ agglutinin, N-[acetyl-3H] and horseradish peroxidase were used in paired injection strategies within portions of the cerebellum, superior colliculus, and thalamic ventrobasal complex and/or posterior group of adult ICR white mice. Trigeminal neurons with projections to tactile areas of the cerebellar cortex or underlying deep cerebellar nuclei were found scattered throughout the principal sensory nucleus and interpolaris division, and mainly in dorsal regions of the oralis division of the spinal trigeminal nucleus. Injections of either tracer which involved lateral portions of the rostral half of the superior colliculus labeled trigeminotectal neurons mainly in the contralateral interpolaris division, ventral half of the oralis division, and a ventral region of the principal sensory nucleus near the oralis border. Fewer trigeminotectal neurons were found scattered throughout the principal sensory nucleus and the magnocellular layer of the caudalis divisions, although an occasional labeled neuron wa also found in the marginal layer. Contralaterally projecting trigeminothalamic neurons were observed throughout the principal sensory nucleus, interpolaris division, and within the marginal and magnocellular layers of caudalis. Double-labeled neurons were observed only after paired injections of the tracers in the thalamus and ipsilateral superior colliculus, and they were found within the caudoventral portion of the principal sensory nucleus near the oralis border, throughout the interpolaris division, within the magnocellular layer of caudalis, and only a few double-labeled neurons were also found within the marginal layer. After such injections, 50% of the labeled tectum-projecting neurons in the principal sensory nucleus, 64% in the interpolaris division, and 57% in the caudalis division are branched neurons which have collateralized projections to both the superior colliculus and thalamus. These projections, which have not been described before, appear to arise from more than one class of projection neuron which is differentially distributed within different regions of the trigeminus.

The dispositions of galactosyl-containing glycoconjugates were studied during postnatal development of the caudate putamen in mice. The binding of the lectin peanut agglutinin, which has an affinity for galactosyl B-1,3 N-acetylgalactosamine residues, was compared to acetylcholinesterase staining and tyrosine hydroxylase immunoreactivity in the immature and adult neostriatum. The binding of peanut agglutinin conjugated to horseradish peroxidase, in sections that were processed for peroxidase histochemistry, was extremely pronounced in the neostriatum through the first postnatal week and constituted ringlike or polygonally shaped structures, which, overall, produced a variegated mosaic. These structures consist of outer rims of dense lectin-associated reaction product surrounding lightly labeled centers. Lectin delineations of the neostriatal mosaic are no longer visible in the second postnatal week. When adjacent sections were processed for lectin binding or acetylcholinesterase histochemistry, the dense lectin binding sites represented borders of acetylcholinesterase-rich and -poor zones. The distribution of dense patches of tyrosine hydroxylase immunoreactive fibers and terminals also coincides with the acetylcholinesterase-rich zones during the same times, and thus the glycoconjugate-delineated boundaries can also be directly compared with the distribution of nigrostriatal dopaminergic projections. The findings presented here represent the first demonstration of a probe that recognizes apparent borders of neostriatal compartments during a limited period of development. They are consistent with previous observations made on transient glycoconjugate "hidden boundaries" during development of other central nervous system structures, including the somatosensory cortical barrel field, and thalamic and brainstem nuclei (Cooper and Steindler, '86a,b; Steindler and Cooper, in press). In those studies, glia were shown to be the major source of glycoconjugate-associated patterns, and thus, glia and glycoconjugates that they synthesize during pattern formation events may be involved in the formation and stabilization of neurochemically distinct components of the neostriatal mosaic.

The intranuclear organization of divergently projecting neurons of the midbrain raphe in the rat was studied by using double retrograde axonal tracing. Paired injections of the tracers N-[acetyl-3H] WGA and horseradish peroxidase were made within known projection targets of the midbrain raphe (caudate-putamen, amygdala, hippocampus, substantia nigra, and locus coeruleus). After injections of either tracer in the aforementioned targets, retrograde labeled neurons were found mainly ipsilaterally and within midline portions of the dorsal raphe nucleus, its caudal B6 portion, and within the linear and superior central nuclei of the median raphe complex. There are discrete intranuclear distributions of raphe neurons that project to these forebrain and brainstem sites, and there is an overall rostrocaudal topographic order within the raphe with neurons projecting to the neostriatum, amygdala, and substantia nigra residing most rostrally and neurons projecting to the hippocampus and/or locus coeruleus occupying caudal portions of the B6 and superior central nuclei. Such distributions of projection neurons suggest the existence of an "encephalotopic" intranuclear organization within the raphe; that is, each central nervous system structure that receives midbrain raphe projections has its own unique representation within a topographically distinct portion of one or more of the raphe subgroups. These findings suggest an overall functional organization within the midbrain raphe nuclear complex whereby rostral portions are associated with the basal ganglia and related nuclei, and caudal portions relate to the limbic system. An intermediate representation of amygdala-projecting raphe neurons functionally conjoins the two. Collateralized neurons are found within complex zones of overlap in the topographically organized distributions of raphe neurons projecting to functionally related structures.

The binding properties of derivatized wheat germ agglutinin (WGA) have been examined in fixed tissue sections from the adult mouse cerebellum and also in axonal tracing paradigms following cerebellar injections. The aim of these studies is to begin to distinguish the roles different binding sites may play in generating diverse biological activities which lead to neuronal uptake and axonal transport of lectins or glycoconjugates. Vibratome sections from aldehyde-fixed cerebellum were incubated in N-[acetyl-3H] WGA or WGA conjugated to horseradish peroxidase (WGA-HRP). Sections from this in vitro binding paradigm and those from cerebellar pressure injection cases using those tracers (in vivo binding paradigm) were processed for light microscopic autoradiography, histochemistry, and electron microscopy. Blocking experiments were also performed with various sugar haptens to confirm the binding specificity of these lectin preparations. Light microscopy of lectin binding patterns within the cerebellar cortex has revealed that both derivatized WGA preparations bind most intensely to the molecular layer. Within the deep cerebellar nuclei, binding is unique and produces a punctate delineation of cell bodies and dendrites. Electron microscopy revealed that these binding sites are associated with glial processes which abut the plasma membrane of deep nuclei cells. Cerebellar WGA-HRP injection sites contain labeled profiles involved in uptake and axonal transport of the labeled lectin (e.g., multivesicular and dense bodies) in addition to label associated with synapses, glia, undetermined components of the extracellular space, and neuronal plasma membranes. These sites are therefore presumed to possess a high affinity or capacity for binding derivatized WGA. Binding studies performed here thus reveal, for the first time, the existence of discrete glial sites that display an extraordinary attraction for lectins such as WGA. The roles such glial glycoconjugates play in diverse biological activities including neuronal uptake and transport of macromolecules need further study.