PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo.

Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5(+) TPCs in vitro, while pharmacological inhibition of PAR 1 similarly slowed both the growth and migration of A2B5(+) TPCs in culture. In addition, PAR1 silencing potently suppressed tumor expansion in vivo, and significantly prolonged the survival of mice following intracranial transplantation of human TPCs. These data strongly suggest the importance of PAR1 to the self-renewal and tumorigenicity of A2B5-defined glioma TPCs; as such, the abrogation of PAR1-dependent signaling pathways may prove a promising strategy for gliomas.

Testosterone modulation of angiogenesis and neurogenesis in the adult songbird brain.

Throughout life, new neurons arise from the ventricular zone of the adult songbird brain and are recruited to the song control nucleus higher vocal center (HVC), from which they extend projections to its target, nucleus robustus of the arcopallium (RA). This process of ongoing parenchymal neuronal addition and circuit integration is both triggered and modulated by seasonal surges in systemic testosterone. Brain aromatase converts circulating testosterone to estradiol, so that HVC is concurrently exposed to both androgenic and estrogenic stimulation. These two signals cooperate to trigger HVC endothelial cell division and angiogenesis, by inducing the regionally-restricted expression of vascular endothelial growth factor (VEGF), its matrix-releasing protease MMP9, and its endothelial receptor VEGFR2. The expanded HVC microvascular network then secretes the neurotrophic factor BDNF, which in turn supports the recruitment of newly generated neurons. This process is striking for its spatial restriction and hence functional specificity. While androgen receptors are broadly expressed by the nuclei of the vocal control system, estrogen receptor (ERα) expression is largely restricted to HVC and its adjacent mediocaudal neopallium. The geographic overlap of these receptor phenotypes in HVC provides the basis for a regionally-defined set of paracrine interactions between the vascular bed and neuronal progenitor pool that both characterize and distinguish this nucleus. These interactions culminate in the focal attraction of new neurons to the adult HVC, the integration of those neurons into the extant vocal control circuits, and ultimately the acquisition and elaboration of song.

Sustained induction of neuronal addition to the adult rat neostriatum by AAV4-delivered noggin and BDNF.

Intraventricular ependymal infection by adenoviruses expressing brain-derived neurotrophic factor (BDNF) and noggin is sufficient to induce the heterotopic recruitment of new medium spiny neurons to the adult neostriatum, from endogenous subependymal neural progenitor cells. This approach was found to slow disease progression and extend survival in an R6/2 mouse model of Huntington's disease (HD). However, the practical therapeutic value of this strategy is limited by the transient expression and immunogenicity of adenoviral vectors. In addition, it has been unclear whether sustained overexpression of BDNF and noggin would yield similarly sustained neuronal production and striatal recruitment, or whether progenitor depletion or tachyphylaxis might supervene to limit the therapeutic potential of this approach. To address these issues, we used adeno-associated virus serotype 4 (AAV4), an ependymotrophic vector that is neither immunogenic nor neurotoxic, to achieve sustained BDNF and noggin expression. Using AAV4, we found that BDNF and noggin achieved levels sufficient to initiate and maintain, for at least 4 months, ongoing neuronal addition to the neostriatum and olfactory bulb. Over this period, we noted no diminution of treatment-associated neuronal recruitment from resident progenitors. AAV4:BDNF and noggin-induced neuronal addition may thus provide a means to provide longlasting and persistent striatal neuronal replacement in conditions of striatal neuronal loss, such as HD.

Disease targets and strategies for the therapeutic modulation of endogenous neural stem and progenitor cells.

Neural stem cells, able to self-renew and give rise to both neurons and glia, line the cerebral ventricles of the adult human brain. Humans also harbor lineage-restricted neuronal progenitors in the hippocampus and glial progenitor cells in both the gray and white matter of the forebrain. These various stem and progenitor cell types may provide targets for pharmacotherapy for a variety of disorders of the central nervous system. Each resident progenitor phenotype may be mobilized and induced to differentiate in vivo by the actions of both exogenous growth factors and small molecule modulators of progenitor-selective signaling pathways. This strategy may be particularly efficacious in neurodegenerations such as Huntington's disease, in which lost neurons may be replenished through the directed induction of progenitor cells lining the ventricular wall of the affected striatum. Similarly, the mobilization of glial progenitor cells may permit the introduction of new oligodendrocytes to demyelinated regions of adult white matter. Our rapidly increasing understanding of the molecular control of progenitor cell mobilization and differentiation should provide a wealth of new opportunities for recruiting endogenous progenitors as a means of treating neurological disease.

Progenitor cell-based myelination as a model for cell-based therapy of the central nervous system.

Diseases of the brain and spinal cord are especially daunting challenges for cell-based strategies of repair, given the multiplicity of cell types within the adult central nervous system, and the precision with which they must interact in both space and time. Nonetheless, a number of diseases are especially appropriate for cell-based therapy, in particular those in which single phenotypes are lost. Foremost among these are the disorders of myelin, in which oligodendrocytes are the specific and often sole victims of the underlying disease process. These include not only the vascular, traumatic, and inflammatory demyelinations of adulthood, but also the congenital and childhood dysmyelinating syndromes of the pediatric leukodystrophies. These congenital disorders of myelin formation and maintenance may present especially compelling targets for cell-based neurological therapy.

Identification and characterization of neuronal precursors and their progeny from human fetal tissue.

We have examined primary human neuronal precursors (HNPs) from 18-22-week-old fetuses. We showed that E-NCAM/MAP2/beta-III tubulin-immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament-associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage-gated ion channels, and ligand-gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Talpha1 tubulin promoter was E-NCAM/beta-III tubulin-immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population.

High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain.

Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.

Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters.

Many tissues arise from pluripotent stem cells through cell-type specification and maturation. In the bone marrow, primitive stem cells generate all the different types of blood cells via the sequential differentiation of increasingly committed progenitor cells. Cell-surface markers that clearly distinguish stem cells, restricted progenitors, and differentiated progeny have enabled researchers to isolate these cells and to study the regulatory mechanisms of hematopoiesis. Neuronal differentiation appears to involve similar mechanisms. However, neural progenitor cells that are restricted to a neuronal fate have not been characterized in vivo, because specific cell-surface markers are not available. We have developed an alternative strategy to identify and isolate neuronal progenitor cells based on dual-color fluorescent proteins. To identify and isolate directly progenitor cells from brain tissue without the need for either transfection or intervening cell culture, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanced yellow fluorescent protein (EYFP) in neuronal progenitor cells and neurons under the control of the Talpha1 alpha-tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cells under the control of the enhancer/promoter elements of the nestin gene. By crossing these lines we obtained mice expressing both transgenes. To isolate neuronal progenitor cells directly from the developing brain, we used flow cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study the mechanisms of neurogenesis and to develop cell-based therapies for neurological disorders.

Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain.

Neural progenitor cells persist throughout the adult forebrain subependyma, and neurons generated from them respond to brain-derived neurotrophic factor (BDNF) with enhanced maturation and survival. To induce neurogenesis from endogenous progenitors, we overexpressed BDNF in the adult ventricular zone by transducing the forebrain ependyma to constitutively express BDNF. We constructed a bicistronic adenovirus bearing BDNF under cytomegalovirus (CMV) control, and humanized green fluorescent protein (hGFP) under internal ribosomal entry site (IRES) control. This AdCMV:BDNF:IRES:hGFP (AdBDNF) was injected into the lateral ventricles of adult rats, who were treated for 18 d thereafter with the mitotic marker bromodeoxyuridine (BrdU). Three weeks after injection, BDNF averaged 1 microg/gm in the CSF of AdBDNF-injected animals but was undetectable in control CSF. In situ hybridization demonstrated BDNF and GFP mRNA expression restricted to the ventricular wall. In AdBDNF-injected rats, the olfactory bulb exhibited a >2.4-fold increase in the number of BrdU(+)-betaIII-tubulin(+) neurons, confirmed by confocal imaging, relative to AdNull (AdCMV:hGFP) controls. Importantly, AdBDNF-associated neuronal recruitment to the neostriatum was also noted, with the treatment-induced addition of BrdU(+)-NeuN(+)-betaIII-tubulin(+) neurons to the caudate putamen. Many of these cells also expressed glutamic acid decarboxylase, cabindin-D28, and DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa), markers of medium spiny neurons of the neostriatum. These newly generated neurons survived at least 5-8 weeks after viral induction. Thus, a single injection of adenoviral BDNF substantially augmented the recruitment of new neurons into both neurogenic and non-neurogenic sites in the adult rat brain. The intraventricular delivery of, and ependymal infection by, viral vectors encoding neurotrophic agents may be a feasible strategy for inducing neurogenesis from resident progenitor cells in the adult brain.

Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene.

Mesencephalic precursor cells may one day provide dopaminergic neurons for the treatment of Parkinson's disease. However, the generation of dopaminergic neurons from mesencephalic precursors has been difficult to follow, partly because an appropriate means for recognizing mesencephalic ventricular zone precursors has not been available. To visualize and isolate mesencephalic precursor cells from a mixed population, we used transgenic mice and rats carrying green fluorescent protein (GFP) cDNA under the control of the nestin enhancer. nestin-driven GFP was detected in the mesencephalic ventricular zone, and it colocalized with specific markers for neural precursor cells. In addition, data from flow-cytometry indicated that Prominin/CD133, a cell-surface marker for ventricular zone cells, was expressed specifically in these GFP-positive (GFP(+)) cells. After sorting by fluorescence-activated cell sorting, the GFP(+) cells proliferated in vitro and expressed precursor cell markers but not neuronal markers. Using clonogenic sphere formation assays, we showed that this sorted population was enriched in multipotent precursor cells that could differentiate into both neurons and glia. Importantly, many neurons generated from nestin-GFP-sorted mesencephalic precursors developed a dopaminergic phenotype in vitro. Finally, nestin-GFP(+) cells were transplanted into the striatum of a rat model of Parkinson's disease. Bromodeoxyuridine-tyrosine hydroxylase double-labeling revealed that the transplanted cells generated new dopaminergic neurons within the host striatum. The implanted cells were able to restore dopaminergic function in the host striatum, as assessed by a behavioral measure: recovery from amphetamine-induced rotation. Together, these findings indicate that precursor cells harvested from the embryonic ventral mesencephalon can generate dopaminergic neurons able to restore function to the chemically denervated adult striatum.

Creating a stable tympanic membrane perforation using mitomycin C.

OBJECTIVE: To determine the ability of topically applied mitomycin C to create a stable tympanic membrane perforation. STUDY DESIGN AND SETTING: Twenty-four rats underwent subtotal removal of the tympanic membranes bilaterally. Forty ears received 0.2 mg/ml of mitomycin C. The remaining 8 received phosphate-buffered saline solution (control). Photographs taken every 3 to 5 days for 44 days were digitally scanned and computer analyzed to calculate the percentage of residual perforation. Application of solutions, photography, and data analysis were performed in a blinded fashion. RESULTS: The mitomycin C treated ears had delayed closure time and healing rate (from day 0 to 25) compared to the control group. All controls healed by day 14. By day 44, 92.5% of the mitomycin C treated ears healed. CONCLUSION: Mitomycin C prolongs the closure and healing rate of myringotomies in rat tympanic membranes. SIGNIFICANCE: Myringotomy with concurrent mitomycin C application may be useful for creating an animal model for chronic tympanic membrane perforation and should be tested in human beings as a method to maintain myringotomy patency for long-term ventilation.

Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells.

We generated transgenic mice carrying enhanced green fluorescent protein (EGFP) under the control of the nestin second-intronic enhancer (E/nestin:EGFP). Flow cytometry followed by in vitro assays revealed that in situ EGFP expression in the embryonic brain correlated with the mitotic index, the cogeneration of both neurons and glia, and the frequency of neurosphere formation in vitro. High-level EGFP expressors derived from embryos included a distinct subpopulation of cells that were self-renewable and multipotent, criteria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich for NSCs using EGFP expression level. In adults, although E/nestin:EGFP-positive cells included the NSC population, the frequency of neurosphere formation did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Promoter-based isolation and fluorescence-activated sorting of mitotic neuronal progenitor cells from the adult mammalian ependymal/subependymal zone.

Neuronal precursor cells are widespread in the subependyma of the forebrain ventricular lining, and may provide a cellular substrate for brain repair. We have previously identified and isolated them from fetal brain, by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein (hGFP), driven by the early neuronal promoter for Talpha1 tubulin (P/Talpha1). Fetal neuronal precursors were thereby identified and harvested with both a high degree of enrichment, and a virtual abolition of glial contaminants. We have now extended this approach to include the isolation and purification of neuronal progenitors from the adult brain. Dissociates of the lateral ventricular wall, that included the combined ependymal/subependymal zone, were obtained from 3-month-old adult rats. These cells were cultured and transfected with P/Talpha1:hGFP plasmid DNA. Two days later, the cells were redissociated, sorted on the basis of Talpha1-driven GFP expression, and replated. The majority of these cells expressed the early neuronal proteins Hu and TuJ1/betaIII-tubulin upon FACS; within the week thereafter, most matured as morphologically-evident neurons, that coexpressed betaIII-tubulin and MAP-2. Fewer than 5% expressed astrocytic markers, compared to over half of the cells in matched samples that were either not sorted, or sorted after transfection with a plasmid bearing the nonfluorescent lacZ gene under the control of P/Talpha1 tubulin. Thus, the use of a fluorescent transgene under the control of an early neuron-selective promoter permits the enrichment of neuronal progenitor cells from the adult rat brain, in a form that may allow their heterologous implantation.

Promoter-targeted selection and isolation of neural progenitor cells from the adult human ventricular zone.

Adult humans, like their nonhuman mammalian counterparts, harbor persistent neural progenitor cells in the forebrain ventricular lining. In the absence of adequate surface markers, however, these cells have proven difficult to isolate for study. We have previously identified and selected neural progenitor cells from both the fetal and adult rodent ventricular zone (VZ), by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein driven by the early neuronal promoter for Talpha1 tubulin (P/Talpha1:hGFP). We have now extended this approach by purifying both P/Talpha1:hGFP tubulin-defined neuronal progenitors, as well as potentially less committed E/nestin:hGFP-defined neural progenitor cells, from the adult human VZ. The ventricular wall of the temporal horn of the lateral ventricle was dissected from temporal lobes obtained from four adult patients undergoing therapeutic lobectomy. These samples were dissociated, and the cultured cells transduced with either P/Talpha1:hGFP or E/nestin:EGFP plasmid DNA. A week later, the cells were redissociated, selected via fluorescence-activated cell sorting (FACS) on the basis of neural promoter-driven GFP expression, and replated. The majority of these cells expressed the early neuronal protein betaIII-tubulin upon FACS; within the week thereafter, most matured as morphologically evident neurons that coexpressed betaIII-tubulin and microtubule-associated protein (MAP)-2. Many of these neurons had incorporated bromodeoxyuridine in vitro in the days before FACS, indicating their mitogenesis in vitro. Thus, the use of fluorescent transgenes under the control of early neural promoters permits the enrichment of neuronal progenitor cells from the adult human ventricular zone. The specific acquisition, in both purity and number, of residual neural progenitor cells from the adult human brain may now permit hitherto unfeasible studies of both their biology and practical application.

Meningeal cells can communicate with astrocytes by calcium signaling.

Mechanical stimulation of adult human and rat pia-arachnoid cell cultures (loaded with calcium indicator dye) produced an increase in calcium in the stimulated cell. This change then propagated rapidly among neighboring cells, producing a calcium wave with a maximum distance of propagation and velocity resembling calcium waves in astrocytes. The pia-arachnoid waves were blocked by either octanol or apyrase, suggesting that propagation might occur either by gap junction communication or extracellular movement of ATP. Calcium waves in pia-arachnoid cells could invade contiguous astrocytes, and vice versa. Gap junction coupling between pia-arachnoid cells and astrocytes was shown by dye transfer experiments, in conjunction with immunostaining for connexin43. We infer that calcium signals from cells in the cortical parenchyma may be transmitted to the pia-arachnoid and might then serve in the induction of neurovascular changes, including those postulated to be responsible for the pain of migraine headache.

Use of a mail-out continuing education article to teach health professionals about drug-induced disease.

A U.S. Food and Drug Administration (FDA)/Georgetown University Medical Center conference was the basis for "Clinical Therapeutics and the Recognition of Drug-Induced Disease," the first MEDWATCH continuing education (CE) mail-out article. Developed as a major component of FDA MEDWATCH post-marketing surveillance outreach, the article used a clinical therapeutic approach to discuss topics including adverse drug events (ADEs) pharmacology and ADE reporting. Distributed nationwide through the MEDWATCH Partners, health professionals applied for CE credit by completing a self-assessment examination. With the overall response rate slightly more than 2%, 15,260 health professionals (55% physicians and 37% pharmacists) received CE credit. Evaluation of the initial approximately two-thirds (N = 10,021) of successfully completed exams found 99% agreement that stated learning objectives were met, and the article relevant to their clinical practice; spontaneous comments/letters were also very positive. The highest percentage responding specialists were internists (28%) and psychiatrists (17%), with notable differences found among specialties for response rate versus relative article distribution (such as relatively low response rates among surgeons and radiology/radiation physics specialists). The number of health professionals receiving CE credit, coupled with examination performance and overall response, indicates that "Clinical Therapeutics and the Recognition of Drug-Induced Disease" was well received and fulfilled learning objectives. The results provide encouragement for this continuing educational approach.

Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter.

Previous studies have suggested the persistence of oligodendrocyte progenitor cells in the adult mammalian subcortical white matter. To identify oligodendrocyte progenitors in the adult human subcortical white matter, we transfected dissociates of capsular white matter with plasmid DNA bearing the gene for green fluorescence protein (hGFP), placed under the control of the human early promoter (P2) for the oligodendrocytic protein cyclic nucleotide phosphodiesterase (P/hCNP2). Within 4 d after transfection with P/hCNP2:hGFP, a discrete population of small, bipolar cells were noted to express GFP. These cells were A2B5-positive (A2B5(+)), incorporated bromodeoxyuridine in vitro, and constituted <0.5% of all cells. Using fluorescence-activated cell sorting (FACS), the P/hCNP2-driven GFP(+) cells were then isolated and enriched to near-purity. In the weeks after FACS, most P/hCNP2:hGFP-sorted cells matured as morphologically and antigenically characteristic oligodendrocytes. Thus, the human subcortical white matter harbors mitotically competent progenitor cells, which give rise primarily to oligodendrocytes in vitro. By using fluorescent transgenes of GFP expressed under the control of an early oligodendrocytic promoter, these oligodendrocyte progenitor cells may be extracted and purified from adult human white matter in sufficient numbers for implantation and cell-based therapy.

Endothelial trophic support of neuronal production and recruitment from the adult mammalian subependyma.

Vascular endothelial cells are among the first cells that ventricular zone neuroblasts encounter during early development. The ventricular zone cells promote angiogenesis by the invading vasculature, with the release of endothelial mitogens. Yet the feedback support of young neurons by endothelial cells (ECs) has not hitherto been explored. We therefore asked whether ECs might participate in neuronal recruitment, by providing neurotrophic support to newly generated neurons. We used the neurogenic subependymal zone (SZ) of the adult rat forebrain as a model system, because of its well-characterized and relatively homogeneous population of neuronal precursor cells. We found that explants of the adult rat SZ raised on ECs generated more neurons, which survived longer, than explants raised on astrocytes, fibroblasts, or laminin. This endothelial trophic effect was humoral, in that it was also noted in SZ explants raised in noncontiguous coculture with ECs grown on porous inserts. RT-PCR for neurotrophin family members revealed that cultures of both human brain- and umbilical cord-derived ECs produced brain-derived neurotrophic factor (BDNF) mRNA, but no detectable NGF, NT-3, or NT-4 mRNA. ELISA revealed that BDNF protein was secreted by ECs into the medium at >1 ng/ml. The neurotrophic effect of ECs could be replaced by added BDNF, and was blocked by addition of 5 microg/ml trkB-Fc to endothelial-SZ cocultures. Thus, endothelial cells can act as sources of secreted BDNF, through which the capillary microvasculature may act to support neuronal recruitment and survival in the CNS.