Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development.

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.

LPA receptors: subtypes and biological actions.

Lysophosphatidic acid (LPA) is a small, ubiquitous phospholipid that acts as an extracellular signaling molecule by binding to and activating at least five known G protein-coupled receptors (GPCRs): LPA(1)-LPA(5). They are encoded by distinct genes named LPAR1-LPAR5 in humans and Lpar1-Lpar5 in mice. The biological roles of LPA are diverse and include developmental, physiological, and pathophysiological effects. This diversity is mediated by broad and overlapping expression patterns and multiple downstream signaling pathways activated by cognate LPA receptors. Studies using cloned receptors and genetic knockout mice have been instrumental in uncovering the significance of this signaling system, notably involving basic cellular processes as well as multiple organ systems such as the nervous system. This has further provided valuable proof-of-concept data to support LPA receptors and LPA metabolic enzymes as targets for the treatment of medically important diseases that include neuropsychiatric disorders, neuropathic pain, infertility, cardiovascular disease, inflammation, fibrosis, and cancer.

Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1α-notch signal interaction in the developing brain.

Oxygen levels in tissues including the embryonic brain are lower than those in the atmosphere. We reported previously that Notch signal activation induces demethylation of astrocytic genes, conferring astrocyte differentiation ability on midgestational neural precursor cells (mgNPCs). Here, we show that the oxygen sensor hypoxia-inducible factor 1α (HIF1α) plays a critical role in astrocytic gene demethylation in mgNPCs by cooperating with the Notch signaling pathway. Expression of constitutively active HIF1α and a hyperoxic environment, respectively, promoted and impeded astrocyte differentiation in the developing brain. Our findings suggest that hypoxia contributes to the appropriate scheduling of mgNPC fate determination.

Lysophosphatidic acid (LPA) and its receptors.

Lysophosphatidic acid (LPA), a bioactive phospholipid, and its family of cognate G protein-coupled receptors have demonstrated roles in many biological functions in the nervous system. To date, five LPA receptors have been identified, and additional receptors may exist. Most of these receptors have been genetically deleted in mice toward identifying biological and medically relevant roles. In addition, small molecule agonists and antagonists have been reported. Here we review recent data on the nervous system functions of LPA signaling, and summarize data on reported agonists and antagonists of LPA receptors.

Lysophospholipid activation of G protein-coupled receptors.

One of the major lipid biology discoveries in last decade was the broad range of physiological activities of lysophospholipids that have been attributed to the actions of lysophospholipid receptors. The most well characterized lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Documented cellular effects of these lipid mediators include growth-factor-like effects on cells, such as proliferation, survival, migration, adhesion, and differentiation. The mechanisms for these actions are attributed to a growing family of 7-transmembrane, G protein-coupled receptors (GPCRs). Their pathophysiological actions include immune modulation, neuropathic pain modulation, platelet aggregation, wound healing, vasopressor activity, and angiogenesis. Here we provide a brief introduction to receptor-mediated lysophospholipid signaling and physiology, and then discuss potential therapeutic roles in human diseases.

Np95/Uhrf1 regulates tumor suppressor gene expression of neural stem/precursor cells, contributing to neurogenesis in the adult mouse brain.

Adult neurogenesis is a process of generating new neurons from neural stem/precursor cells (NS/PCs) in restricted adult brain regions throughout life. It is now generally known that adult neurogenesis in the hippocampal dentate gyrus (DG) and subventricular zone participates in various higher brain functions, such as learning and memory formation, olfactory discrimination and repair after brain injury. However, the mechanisms underlying adult neurogenesis remain to be fully understood. Here, we show that Nuclear protein 95 KDa (Np95, also known as UHRF1 or ICBP90), which is an essential protein for maintaining DNA methylation during cell division, is involved in multiple processes of adult neurogenesis. Specific ablation of Np95 in adult NS/PCs (aNS/PCs) led to a decrease in their proliferation and an impairment of neuronal differentiation and to suppression of neuronal maturation associated with the impairment of dendritic formation in the hippocampal DG. We also found that deficiency of Np95 in NS/PCs increased the expression of tumor suppressor genes p16 and p53, and confirmed that expression of these genes in NS/PCs recapitulates the phenotype of Np95-deficient NS/PCs. Taken together, our findings suggest that Np95 plays an essential role in proliferation and differentiation of aNS/PCs through the regulation of tumor suppressor gene expression in adult neurogenesis.

Insights into the pharmacological relevance of lysophospholipid receptors.

The discovery of lysophospholipid (LP) 7-transmembrane, G protein-coupled receptors (GPCRs) that began in the 1990s, together with research into the functional roles of the major LPs known as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), have opened new research avenues into their biological processes and mechanisms. Major examples of LP signalling effects include embryogenesis, nervous system development, vascular development, uterine implantation, immune cell trafficking, and inflammatory reactions. LP signalling also influences the pathophysiology of many diseases including cancer, autoimmune and inflammatory diseases, which indicate that LP receptors may be attractive targets for pharmacological therapies. A key example of such a therapeutic agent is the S1P receptor modulator FTY720, which upon phosphorylation and continued drug exposure, acts as an S1P receptor functional antagonist. This compound (also known as fingolimod or Gilenya) has recently been approved by the FDA for the treatment of relapsing forms of multiple sclerosis. Continued basic and translational research on LP signalling should provide novel insights into both basic biological mechanisms, as well as novel therapeutic approaches to combat a range of human diseases.

Lysophosphatidic acid signaling may initiate fetal hydrocephalus.

Fetal hydrocephalus (FH), characterized by the accumulation of cerebrospinal fluid, an enlarged head, and neurological dysfunction, is one of the most common neurological disorders of newborns. Although the etiology of FH remains unclear, it is associated with intracranial hemorrhage. Here, we report that lysophosphatidic acid (LPA), a blood-borne lipid that activates signaling through heterotrimeric guanosine 5'-triphosphate-binding protein (G protein)-coupled receptors, provides a molecular explanation for FH associated with hemorrhage. A mouse model of intracranial hemorrhage in which the brains of mouse embryos were exposed to blood or LPA resulted in development of FH. FH development was dependent on the expression of the LPA(1) receptor by neural progenitor cells. Administration of an LPA(1) receptor antagonist blocked development of FH. These findings implicate the LPA signaling pathway in the etiology of FH and suggest new potential targets for developing new treatments for FH.

Aneuploid mosaicism in the developing and adult cerebellar cortex.

Neuroprogenitor cells (NPCs) in several telencephalic proliferative regions of the mammalian brain, including the embryonic cerebral cortex and postnatal subventricular zone (SVZ), display cell division "defects" in normal cells that result in aneuploid adult progeny. Here, we identify the developing cerebellum as a major, nontelencephalic proliferative region of the vertebrate central nervous system (CNS) that also produces aneuploid NPCs and nonmitotic cells. Mitotic NPCs assessed by metaphase chromosome analyses revealed that 15.3% and 20.8% of cerebellar NPCs are aneuploid at P0 and P7, respectively. By using immunofluorescent analysis of cerebellar NPCs, we show that chromosome segregation defects contribute to the generation of cells with an aneuploid genomic complement. Nonmitotic cells were assessed by fluorescence-activated cell sorting (FACS) coupled with fluorescence in situ hybridization (FISH), which revealed neuronal and nonneuronal aneuploid populations in both the adult mouse and human cerebellum. Taken together, these results demonstrate that the prevalence of neural aneuploidy includes nontelencephalic portions of the neuraxis and suggest that the generation and maintenance of aneuploid cells is a widespread, if not universal, property of central nervous system development and organization.

Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex.

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.

Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1.

Cadherin-related neuronal receptor (CNR) proteins are a diverse set of synaptic protocadherins, but little is known about its adhesive properties. We found that overexpressed CNR1 protein localized on the cell surface of HEK293T cells and increased the calcium-dependent cell aggregation potential. However, we could not detect the strong homophilic binding activity of CNR1 EC-Fc fusion protein in vitro. Parental HEK293T cells adhered to Arg-Gly-Asp (RGD) motif of EC1 domain of CNR1-Fc fusion protein. The fusion protein that the Asp73 of EC1 point-mutated to Glu (RGE-Fc) lost the adhesive activity. The adhesion activity of HEK293T cells to CNR1 EC-Fc fusion protein was completely blocked by inhibitors of integrins, including RGDS peptide and anti-beta1 integrin antibodies. The increased cell-aggregative property of CNR1 transfectants was also blocked by RGDS peptides. At cell-cell junctions of the CNR1 transfectants, co-localization between CNR1 and HEK293T endogenous beta1 integrin was observed. Furthermore, the spatiotemporal expression patterns of CNR and beta1 integrin nearly overlapped in the molecular layer of the developing mouse cerebellum in the main stage of synaptogenesis. These results indicate that CNR1 has a heterophilic, calcium-dependent cell adhesion activity with the beta1 integrin subfamily, and raise the possibility of CNR-beta1 integrin association in synaptogenesis.

Interaction with protocadherin-gamma regulates the cell surface expression of protocadherin-alpha.

The protocadherin-alpha (CNR/Pcdhalpha) and protocadherin-gamma (Pcdhgamma) proteins, members of the cadherin superfamily, are putative cell recognition/adhesion molecules in the brain. Overexpressed cadherins are generally expressed on the cell surface and elicit cell adhesion activity in several cell lines, although hardly any overexpressed CNR/Pcdhalpha proteins are expressed on the cell surface, except on HEK293T cells, which show low expression. We analyzed the expression of CNR/Pcdhalpha and Pcdhgamma in HEK293T cells and found that they formed a protein complex and that Pcdhgamma enhanced the surface expression of CNR/Pcdhalpha. This enhanced surface expression was confirmed by flow cytometry analysis and by marking cell surface proteins with biotin. The enhancement was observed using different combinations of CNR/Pcdhalpha and Pcdhgamma proteins. The surface expression activity was enhanced by the extracellular domains of the proteins, which could bind each other. Their cytoplasmic domains also had binding activity and influenced their localization. Their protein-protein interaction was also detected in extracts of mouse brain and two neuroblastoma cell lines. Thus, interactions between CNR/Pcdhalpha and Pcdhgamma regulate their surface expression and contribute to the combinatorial diversity of cell recognition proteins in the brain.