The A30P α-synuclein mutation decreases subventricular zone proliferation.

Parkinson's disease (PD) is associated with olfactory defects in addition to dopaminergic degeneration. Dopaminergic signalling is necessary for subventricular zone (SVZ) proliferation and olfactory bulb (OB) neurogenesis. Alpha-synuclein (α-syn or Snca) modulates dopaminergic neurotransmission, and SNCA mutations cause familial PD, but how α-syn and its mutations affect adult neurogenesis is unclear. To address this, we studied a bacterial artificial chromosome transgenic mouse expressing the A30P SNCA familial PD point mutation on an Snca-/- background. We confirmed that the SNCA-A30P transgene recapitulates endogenous α-syn expression patterns and levels by immunohistochemical detection of endogenous α-syn in a wild-type mouse and transgenic SNCA-A30P α-syn protein in the forebrain. The number of SVZ stem cells (BrdU+GFAP+) was decreased in SNCA-A30P mice, whereas proliferating (phospho-histone 3+) cells were decreased in Snca-/- and even more so in SNCA-A30P mice. Similarly, SNCA-A30P mice had fewer Mash1+ transit-amplifying SVZ progenitor cells but Snca-/- mice did not. These data suggest the A30P mutation aggravates the effect of Snca loss in the SVZ. Interestingly, calbindin+ and calretinin (CalR)+ periglomerular neurons were decreased in both Snca-/-, and SNCA-A30P mice but tyrosine hydroxylase+ periglomerular OB neurons were only decreased in Snca-/- mice. Cell death decreased in the OB granule layer of Snca-/- and SNCA-A30P mice. In the same region, CalR+ numbers increased in Snca-/- and SNCA-A30P mice. Thus, α-syn loss and human A30P SNCA decrease SVZ proliferation, cell death in the OB and differentially alter interneuron numbers. Similar disruptions in human neurogenesis may contribute to the olfactory deficits, which are observed in PD.

Regional differences in human ependymal and subventricular zone cytoarchitecture are unchanged in neuropsychiatric disease.

Much work has focused on the possible contribution of adult hippocampal neurogenesis to neuropsychiatric diseases. The hippocampal subgranular zone and the other stem cell-containing neurogenic niche, the subventricular zone (SVZ), share several cytological features and are regulated by some of the same molecular mechanisms. However, very little is known about the SVZ in neuropsychiatric disorders. This is important since it surrounds the lateral ventricles and in schizophrenia ventricular enlargement frequently follows forebrain nuclei shrinkage. Also, adult neurogenesis has been implicated in pharmacotherapy for affective disorders and many of the molecules associated with neuropsychiatric disorders affect SVZ biology. To assess the neurogenic niche, we examined material from 60 humans (Stanley Collection) and characterized the cytoarchitecture of the SVZ and ependymal layer in age-, sex- and post mortem interval-matched controls, and patients diagnosed with schizophrenia, bipolar illness, and depression (n = 15 each). There is a paucity of post mortem brains available for study in these diseases, so to maximize the number of possible parameters examined here, we quantified individual sections rather than a large series. Previous work showed that multiple sclerosis is associated with increased width of the hypocellular gap, a cell-sparse region that typifies the human SVZ. Statistically there were no differences between disease groups and controls in the width of the hypocellular gap or in the density of cells in the hypocellular gap. Because ventricular enlargement in schizophrenia may disrupt ependymal cells, we quantified them, but observed no difference between diagnostic groups and controls. There are significant differences in the prevalence of neuropsychiatric illness between the sexes. Therefore, we looked for male versus female differences, but did not observe any in the parameters quantified. We next turned to a finer spatial resolution and asked if there were differences amongst the disease groups in dorsal ventral subdivisions of the SVZ. Similar to when we treated the SVZ as a whole, we did not find such differences. However, compared to the dorsal SVZ, the ventral SVZ had a wider hypocellular gap and more ependymal cells in all four groups. In contrast, cell density was similar in dorsal ventral subregions of the SVZ hypocellular gap. These results show that though there are regional differences in the SVZ in humans, neuropsychiatric disorders do not seem to alter several fundamental histological features of this adult neurogenic zone.

Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.

The adult brain subventricular zone (SVZ) produces neuroblasts that migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) in a specialized niche. Galectin-3 (Gal-3) regulates proliferation and migration in cancer and is expressed by activated macrophages after brain injury. The function of Gal-3 in the normal brain is unknown, but we serendipitously found that it was expressed by ependymal cells and SVZ astrocytes in uninjured mice. Ependymal cilia establish chemotactic gradients and astrocytes form glial tubes, which combine to aid neuroblast migration. Whole-mount preparations and electron microscopy revealed that both ependymal cilia and SVZ astrocytes were disrupted in Gal3(-/-) mice. Interestingly, far fewer new BrdU(+) neurons were found in the OB of Gal3(-/-) mice, than in wild-type mice 2 weeks after labeling. However, SVZ proliferation and cell death, as well as OB differentiation rates were unaltered. This suggested that decreased migration in vivo was sufficient to decrease the number of new OB neurons. Two-photon time-lapse microscopy in forebrain slices confirmed decreased migration; cells were slower and more exploratory in Gal3(-/-) mice. Gal-3 blocking antibodies decreased migration and dissociated neuroblast cell-cell contacts, whereas recombinant Gal-3 increased migration from explants. Finally, we showed that expression of phosphorylated epidermal growth factor receptor (EGFR) was increased in Gal3(-/-) mice. These results suggest that Gal-3 is important in SVZ neuroblast migration, possibly through an EGFR-based mechanism, and reveals a role for this lectin in the uninjured brain.

Dopamine stimulation of postnatal murine subventricular zone neurogenesis via the D3 receptor.

We investigated the expression and role of the dopamine receptor 3 (D3R) in postnatal mouse subventricular zone (SVZ). In situ hybridization detected selective D3R mRNA expression in the SVZ. Fluorescence activated cell sorting (FACS) of adult SVZ subtypes using hGFAP-GFP and Dcx-GFP mice showed that transit amplifying progenitor cells and niche astrocytes expressed D3R whereas stem cell-like astrocytes and neuroblasts did not. To determine D3R's role in SVZ neurogenesis, we administered U-99194A, a D3R preferential antagonist, and bromodeoxyuridine in postnatal mice. In vivo D3R antagonism decreased the numbers of newborn neurons reaching the core and the periglomerular layer of the olfactory bulb. Moreover, it decreased progenitor cell proliferation but did not change the number of label-retaining (stem) cells, commensurate with its expression on transit amplifying progenitor cells but not SVZ stem cell-like astrocytes. Collectively, this study suggests that dopaminergic stimulation of D3R drives proliferation via rapidly amplifying progenitor cells to promote murine SVZ neurogenesis.

Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration.

BACKGROUND: The adult subventricular zone (SVZ) contains stem and progenitor cells that generate neuroblasts throughout life. Although it is well accepted that SVZ neuroblasts are migratory, recent evidence suggests their progenitor cells may also exhibit motility. Since stem and progenitor cells are proliferative and multipotential, if they were also able to move would have important implications for SVZ neurogenesis and its potential for repair. METHODOLOGY/PRINCIPAL FINDINGS: We studied whether SVZ stem and/or progenitor cells are motile in transgenic GFP+ slices with two photon time lapse microscopy and post hoc immunohistochemistry. We found that stem and progenitor cells; mGFAP-GFP+ cells, bright nestin-GFP+ cells and Mash1+ cells were stationary in the SVZ and rostral migratory stream (RMS). In our search for motile progenitor cells, we uncovered a population of motile betaIII-tubulin+ neuroblasts that expressed low levels of epidermal growth factor receptor (EGFr). This was intriguing since EGFr drives proliferation in the SVZ and affects migration in other systems. Thus we examined the potential role of EGFr in modulating SVZ migration. Interestingly, EGFr(low) neuroblasts moved slower and in more tortuous patterns than EGFr-negative neuroblasts. We next questioned whether EGFr stimulation affects SVZ cell migration by imaging Gad65-GFP+ neuroblasts in the presence of transforming growth factor alpha (TGF-alpha), an EGFr-selective agonist. Indeed, acute exposure to TGF-alpha decreased the percentage of motile cells by approximately 40%. CONCLUSIONS/SIGNIFICANCE: In summary, the present study directly shows that SVZ stem and progenitor cells are static, that EGFr is retained on some neuroblasts, and that EGFr stimulation negatively regulates migration. This result suggests an additional role for EGFr signaling in the SVZ.

Techniques and strategies to analyze neural progenitor cell migration.

One of the most surprising aspects of neural development is that cells do not remain in their birthplace but actively migrate along a variety of routes to their final destinations. This review traces past, present, and future techniques used to analyze progenitor cell migration in the brain, and also discusses their relevant strengths and weaknesses. The large majority of information regarding cell migration is from studies where migratory cells have been labeled, but in which the actual movements are not observed, ie., from static experiments. More recently, dynamic imaging of cell migration in living slices and, even in vivo, has provided a glimpse of how complex these phenomena truly are. A variety of new techniques, such as 2-photon videomicroscopy, are emerging that will continue to add to our body of knowledge concerning the migration of cells in the central nervous system.

Developmental changes of keratin expression in chick embryo olfactory epithelium in relation to cellular differentiation and neurogenesis in vivo and in vitro.

Olfactory embryogenesis was studied using an anti-chick keratin antibody on chick embryo sections as well as in vitro. Olfactory placodes form at embryonic day 3 (ED3) in the anterior facial ectoderm and invaginate to form the nasal pits. At ED5, the epidermal ectoderm and respiratory epithelium show the same dense cytokeratin immunoreaction. In contrast, absence of keratin expression in the basal part of olfactory epithelial primordium, in the deeper nasal pit area, coincides with one of the critical first steps of olfactory neurogenesis. However, beginning with periphero-central olfactory synaptogenesis at ED8, a new basal cell population starts to express keratin in the olfactory epithelium. Keratin positive cells appear to correspond, by their epithelial localisation and morphology, to sustentacular and basal cells. This interpretation was confirmed in vitro with ED14 chick primary olfactory cultures where TrKA immunoreactivity was used as a marker of horizontal basal cells (HBCs). After ED15, late keratin expression was detected in forming Bowman's glands. The density of keratin expressing basal cells was measured between ED10 and ED20, and appeared highest in the median part of the olfactory epithelium, the area of most active olfactory neurogenesis and neuronal maturation. Thus, keratin expression corresponds to a specialisation of horizontal basal cells as active neuronal stem cells.

Combination of 3' and 5' IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model.

To ensure the B cell differentiation stage specificity of the intronic Emu element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a V(H) promoter-GFP reporter gene linked to the 3'LCR region and the Emu element. By flow cytometry, GFP(+) lymphocytes were observed amongst pro-B cells (B220(+)CD43(+)CD117(+)) and at all stages of differentiation up to mature B cells (B220(+)IgM(+)IgD(+)). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP(+)Thy1,2(+) cells (T lymphocytes) and GFP(+)B220(-)CD117(+)CD43(+) cells (uncommitted lymphohematopoietic progenitors). Therefore, the Emu-GFP-3'LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3' and 5' IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.