A Novel cdc2-Related Protein Kinase Expressed in the Nervous System

Abstract: We report the cloning and characterization of a cDNA encoding a cdc2-related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34^cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of ～58–60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34^cdc2 and cdk5.     

Cloning and expression of mars, a novel member of the guanylate kinase associated protein family in Drosophila

The Membrane-Associated Guanylate Kinase (MAGUK) family of anchor proteins are involved in organising a range of molecules such as cell adhesion molecules, receptors, and intracellular signalling molecules at cell junctions. In mammals, the PSD-95/SAP-90/hDlg class of MAGUK proteins bind to a family of Guanylate Kinase Associated Proteins (GKAPs) that have been found at presumptive synaptic sites in neurons. Here we describe the identification of Mars, a novel Drosophila protein belonging to the GKAP family. RT-PCR analysis reveals that Drosophila mars mRNA and protein are predominantly expressed in embryos and in the adult germline. In embryos, mars is expressed in central nervous system and brain, as determined by RNA in situ hybridisation. In testes, mars is strongly expressed in pre-meiotic germ cells, but is not found in somatic or post-meiotic cells, indicating that in addition to their role in neuronal cells, GKAP proteins are also likely to play a role in germline development.     

A unified hypothesis on the lineage of neural stem cells

For many years, it was assumed that neurons and glia in the central nervous system were produced from two distinct precursor pools that diverged early during embryonic development. This theory was partially based on the idea that neurogenesis and gliogenesis occurred during different periods of development, and that neurogenesis ceased perinatally. However, there is now abundant evidence that neural stem cells persist in the adult brain and support ongoing neurogenesis in restricted regions of the central nervous system. Surprisingly, these stem cells have the characteristics of fully differentiated glia. Neuroepithelial stem cells in the embryonic neural tube do not show glial characteristics, raising questions about the putative lineage from embryonic to adult stem cells. In the developing brain, radial glia have long been known to produce cortical astrocytes, but recent data indicate that radial glia might also divide asymmetrically to produce cortical neurons. Here we review these new developments and propose that the stem cells in the central nervous system are contained within the neuroepithelial --> radial glia --> astrocyte lineage.     

The Expression Patterns of MHC Class I Molecules in the Developmental Human Visual System

It has been considered that healthy neurons in central nervous system (CNS) do not express major histocompatibility complex (MHC) class I molecules. However, recent studies clearly demonstrated the expression of functional MHC class I in the mammalian embryonic, neonatal and adult brain. Until now, it is still unknown whether MHC I molecules are expressed in the development of human brain. We collected nine human brain tissues from fetuses aged from 21 to 31 gestational weeks (GW), one newborn of postnatal 55 days and one adult. The expression of MHC class I molecules was detected during the development of visual system in human brain by immunohistochemistry and immunofluorescence. MHC class I proteins were located at lateral geniculate nucleus (LGN) and the expression was gradually increased from 21 GW to 31 GW and reached high levels at 30–31 GW when fine-scale refinement phase was mediated by neural electric activity. However, there was no expression of MHC class I molecules in the visual cortical cortex during all the developmental stages examined. We also concluded that MHC class I molecules were mainly expressed in neurons but not in astrocytes at LGN. In the developing visual system, the expression of β2M protein on neurons was not found in our study.     

Bcl-2 family members in the development and degenerative pathologies of the nervous system

Neuronal death is an essential feature in the normal development of the nervous system and in neurodegenerative states of the adult or ageing brain. Bcl-2 is the prototype of a growing family of proteins which control cell death. Many of these proteins are expressed in the nervous system during development and in the adult. Numerous observations have suggested that this family of proteins plays a central role in the control of naturally occurring and pathological neuronal death. In this review, I will discuss the possible mechanisms of action of these proteins as well as their potential use in treating neurodegenerative diseases.     

Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain

APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.     

Temporal and spatial patterns of expression of p35, a regulatory subunit of cyclin-dependent kinase 5, in the nervous system of the mouse

The protein p35 is a regulatory subunit of cyclin-dependent kinase 5. It has no recognized homology to cyclins but binds to and activates cyclin-dependent kinase 5 directly in the absence of other protein molecules. Cyclin-dependent kinase 5 was initially isolated by homology to the key cell cycle regulator cdc2 kinase and later identified as a neuronal kinase that phosphorylates histone H1, tau or neurofilaments. This kinase is localized in axons of the developing and mature nervous system. To understand the role of p35 as a regulator of cyclin-dependent kinase 5 activity in the CNS, we examined the pattern of expression of p35 mRNA in the nervous system of embryonic, early postnatal and adult mice. In separate experiments, we also examined the spatial distribution of cyclin-dependent kinase 5 mRNA and the activity of cyclin-dependent kinase 5/p35 kinase complex. Postmitotic cells express p35 mRNA immediately after they leave the zones of cell proliferation. It is also expressed in developing axonal tracts in the brain. Cyclin-dependent kinase 5 mRNA is present in postmitotic and in proliferative cells throughout the embryonic central nervous system. During early postnatal period signal for p35 mRNA declines while that for cyclin-dependent kinase 5 mRNA increases throughout the brain. In the adult brain although both p35 and cyclin-dependent kinase 5 mRNAs are expressed at relatively high levels in certain structures associated with the limbic system, considerable differences exist in the patterns of their distribution in other parts of the brain. These data suggest that the p35/cyclin-dependent kinase 5 complex may be associated with early events of neuronal development such as neuronal migration and axonal growth while in the limbic system of the mature brain it may be associated with the maintenance of neuronal plasticity.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

A Critical role for Bim in retinal ganglion cell death

Optic nerve transection results in the death of retinal ganglion cells (RGCs) by apoptosis. Apoptosis is regulated by the Bcl-2 family of proteins, of which the Bcl-2 homology (BH3) -only proteins forms a subset. As BH3-only proteins have been shown to play a significant role in regulating cell death in the central nervous system, we wished to investigate the role of Bcl-2 interacting mediator of cell death (Bim), a prominent member of this protein family in the regulation of cell death in the RGC layer using in vitro retinal explants. In this study, we use an innovative retinal shaving procedure to isolate the cells of the ganglion cell layer to use for western blotting. Members of the BH3-only protein family are down-regulated during retinal development and are not normally expressed in the adult retina. Using this procedure, we demonstrate that Bim is re-expressed and its expression is increased over time following axotomy. Expression of Bad and Bik decreases over the same time course, whereas there is no indication that Bid and Puma are re-expressed. We show that explants from Bim knockout mice are resistant to axotomy-induced death when compared with their wild-type counterparts. Genetic deletion of Bim also prevents caspase 3 cleavage. The activity of Bim can be negatively regulated by phosphorylation. We show that the decrease of Bim phosphorylation correlates with a decrease in expression of survival kinases such as pAkt and pERK over the same time course. These results implicate Bim re-expression as being essential for axotomy-induced death of RGCs and that phosphorylation of Bim negatively regulates its activity in RGCs.     

The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat.

We have used immunocytochemical methods to localize the homeodomain LIM protein Isl-1 in the adult rat. Isl-1 immunoreactivity is expressed in polypeptide hormone-producing cells of the endocrine system, in neurons of the peripheral nervous system, and in a subset of brain nuclei. Isl-1 is also expressed in a subset of motoneurons in the spinal cord and brain stem, but not in regions of the central nervous system involved in sensory function or in neocortical areas. The pattern of expression of Isl-1 suggests that this gene may be involved in the specification and maintenance of differentiated phenotypical properties of these cells.     

Neural stem cells in the brain

Stem cells in the central nervous system were usually considered as relevant for evaluation only in embryonic time. Recent advances in molecular cloning and immunological identification of the different cell types prove the presence of neurogenesis of the new neurons in adult mammals brains. New neurons are born in two areas of the mammal and human brain--sybventricular zone and subgranular zone of dentate gyrus. New born granular neurons of dentate gyrus have a great importance for memory and learning. New neurons originate from precursors which in culture and in situ could also transform into astrocytes and oligodendrocytes, thus fulfill criteria of neural stem cells. In culture, mitotic activity of these stem sells depends on fibroblast growth factor 2 and epidermal growth factor. Depletion of cultural medium of these factors and addition of serum, other growth factors (Platelet-derived growth factor and ciliary neurotrophic factor) leads to generation of neurons and astrocytes. Isolation and clonal analysis of stem cells is based on immunological markers such as nestin, beta-tubulin III, some types of membrane glicoproteids. Identification and visualization of stem cells in brain revealed two populations of cells which have properties of stem cells. In embryonic time, radial glia cells could give origin to neurons, in mature brain cells expressing glial fibrillar acidic protein typical marker of astrocytes fulfill criteria for stem cells. Neural stem cells could transform not only into mature neurons and glial cells but also into blood cells, thus revealing broad spectrum of progenitors from different embryonic tissues. Further progress in this field of neurobiology could give prosperity in the cell therapy of many brain diseases.     

p73 is an essential regulator of neural stem cell maintenance in embryonal and adult CNS neurogenesis

The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.     

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve,brain, and spinal cord during development

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.     

Glutathione binding to the Bcl-2 homology-3 domain groove: a molecular basis for Bcl-2 antioxidant function at mitochondria

Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4). Given the opposing effects of Bcl-2 and Bax/BH3-only proteins on the redox state of mitochondria, we hypothesized that the antioxidant function of Bcl-2 is antagonized by its interaction with the BH3 domains of pro-apoptotic family members. Here, we show that BH3 mimetics that bind to a hydrophobic surface (the BH3 groove) of Bcl-2 induce GSH-sensitive mitochondrial dysfunction and apoptosis in cerebellar granule neurons. BH3 mimetics displace a discrete mitochondrial GSH pool in neurons and suppress GSH transport into isolated rat brain mitochondria. Moreover, BH3 mimetics and the BH3-only protein, Bim, inhibit a novel interaction between Bcl-2 and GSH in vitro. These results suggest that Bcl-2 regulates an essential pool of mitochondrial GSH and that this regulation may depend upon Bcl-2 directly interacting with GSH via the BH3 groove. We conclude that this novel GSH binding property of Bcl-2 likely plays a central role in its antioxidant function at mitochondria.     

Neuronal Apoptosis: BH3-Only Proteins the Real Killers?

At present there is a poor understanding of the events that lead up to neuronal apoptosis that occurs in neurodegenerative diseases and following acute ischemic episodes. Apoptosis is critical for the elimination of unwanted neurons within the developing nervous system. The Bcl-2 family of proteins contains pro- and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis. There is increasing interest in a subfamily of the Bcl-2 family, the BH3-only proteins, and their pro-apoptotic effects within neurons. Recently ischemic and seizure-induced neuronal injury has been shown to result in the activation of the BH3-only protein, Bid. This protein is cleaved and the truncated protein (tBid) translocates to the mitochondria. The translocation of tBid to the mitochondria is associated with the activation of outer mitochondrial membrane proteins Bax/Bak and the release of cytochrome C from the mitochondria. ER stress also has been implicated as a factor for the induction of apoptosis in ischemic neuronal injury. The induction of ER stress in hippocampal neurons has been shown to activate expression of bb3/PUMA, a member of the BH3-only gene family. Activation of PUMA is associated with the activation and clustering of the pro-apoptotic Bcl-2 family member Bax and the loss of cytochrome C from the mitochondria.     

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.     

Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death

Mouse embryos genetically null for all alphav integrins develop intracerebral hemorrhage owing to defective interactions between blood vessels and brain parenchymal cells. Here, we have used conditional knockout technology to address whether the cerebral hemorrhage is due to primary defects in vascular or neural cell types. We show that ablating alphav expression in the vascular endothelium has no detectable effect on cerebral blood vessel development, whereas deletion of alphav expression in central nervous system glial cells leads to embryonic and neonatal cerebral hemorrhage. Conditional deletion of alphav integrin in both central nervous system glia and neurons also leads to cerebral hemorrhage, but additionally to severe neurological defects. Approximately 30% of these mutants develop seizures and die by 4 weeks of age. The remaining mutants survive for several months, but develop axonal deterioration in the spinal cord and cerebellum, leading to ataxia and loss of hindlimb coordination. Collectively, these data provide evidence that alphav integrins on embryonic central nervous system neural cells, particularly glia, are necessary for proper cerebral blood vessel development, and also reveal a novel function for alphav integrins expressed on axons in the postnatal central nervous system.