Pocket proteins pRb and p107 are required for cortical lamination independent of apoptosis

Pocket proteins (pRb, p107 and p130) are well studied in their role of regulating cell cycle progression. Increasing evidence suggests that these proteins also control early differentiation and even later stages of cell maturation, such as migration. However, pocket proteins also regulate apoptosis, and many of the developmental defects in knock out models have been attributed to increased cell death. Here, we eliminate ectopic apoptosis in the developing brain through the deletion of Bax, and show that pocket proteins are required for radial migration independent of their role in cell death regulation. Following loss of pRb and p107, a population of cortical neurons fails to pass through the intermediate zone into the cortical plate. Importantly, these neurons are born at the appropriate time and this migration defect cannot be rescued by eliminating ectopic cell death. In addition, we show that pRb and p107 regulate radial migration through a cell autonomous mechanism since pRb/p107 deficient neurons fail to migrate to the correct cortical layer within a wild type brain. These results define a novel role of pocket proteins in regulating cortical lamination through a cell autonomous mechanism independent of their role in apoptosis.     

Quantitative analysis of the roles of IRM cell adhesion molecules in column formation in the fly brain

The Drosophila visual center shows columnar structures, basic structural and functional units of the brain, that are shared with the mammalian cerebral cortex. Visual information received in the ommatidia in the compound eye is transmitted to the columns in the brain. However, the developmental mechanisms of column formation are largely unknown. The Irre Cell Recognition Module (IRM) proteins are a family of immunoglobulin cell adhesion molecules. The four Drosophila IRM proteins are localized to the developing columns, the structure of which is affected in IRM mutants, suggesting that IRM proteins are essential for column formation. Since IRM proteins are cell adhesion molecules, they may regulate cell adhesion between columnar neurons. To test this possibility, we specifically knocked down IRM genes in columnar neurons and examined the defects in column formation. We developed a system that automatically extracts the individual column images and quantifies the column shape. Using this system, we demonstrated that IRM genes play critical roles in regulating column shape in a core columnar neuron, Mi1. We also show that their expression in the other columnar neurons, Mi4 and T4/5, is essential, suggesting that the interactions between IRM proteins and multiple neurons shape the columns in the fly brain.     

Mitosis in Neurons: Roughex and APC/C Maintain Cell Cycle Exit to Prevent Cytokinetic and Axonal Defects in Drosophila Photoreceptor Neurons

The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.     

Expression of the mouse PR domain protein Prdm8 in the developing central nervous system

It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord.     

Reelin induces EphB activation

The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the ''classical'' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.     

Mitochondrial Impairment in the Developing Brain After Hypoxia–Ischemia

The pattern of cell death in the immature brain differs from that seen in the adult CNS. During normal development, more than half of the neurons are removed through apoptosis, and mediators like caspase-3 are highly upregulated. The contribution of apoptotic mechanisms in cell death appears also to be substantial in the developing brain, with a marked activation of downstream caspases and signs of DNA fragmentation. Mitochondria are important regulators of cell death through their role in energy metabolism and calcium homeostasis, and their ability to release apoptogenic proteins and to produce reactive oxygen species. We find that secondary brain injury is preceded by impairment of mitochondrial respiration, signs of membrane permeability transition, intramitochondrial accumulation of calcium, changes in the Bcl-2 family proteins, release of proapoptotic proteins (cytochrome C, apoptosis inducing factor) and downstream activation of caspase-9 and caspase-3 after hypoxia-ischemia. These data support the involvement of mitochondria-related mechanisms in perinatal brain injury.     

An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development

We previously identified mNAT1 (murine N-terminal acetyltransferase 1) as an embryonic gene that is expressed in the developing brain and subsequently down-regulated, in part, by the onset of N-methyl-d-aspartate (NMDA) receptor function. By searching the data base we discovered a second closely related gene, mNAT2. mNAT1 and mNAT2 are highly homologous to yeast NAT1, a gene known to regulate entry into the G0 phase of the cell cycle. However, in the absence of further characterization, including evidence that mammalian homologues of NAT1 encode functional acetyltransferases, the significance of this relationship has been unclear. Here we focus on mNAT1. Biochemical analysis demonstrated that mNAT1 and its evolutionarily conserved co-subunit, mARD1, assemble to form a functional acetyltransferase. Transfection of mammalian cells with mNAT1 and mARD1 followed by immunofluorescent staining revealed that these proteins localize to the cytoplasm in both overlapping and separate compartments. In situ hybridization demonstrated that throughout brain development mNAT1 and mARD1 are highly expressed in areas of cell division and migration and are down-regulated as neurons differentiate. Finally, mNAT1 and mARD1 are expressed in proliferating mouse P19 embryonic carcinoma cells; treatment of these cells with retinoic acid initiates exit from the cell cycle, neuronal differentiation, and down-regulation of mNAT1 and mARD1 as the NMOA receptor 1 gene is induced. The results provide the first direct evidence that vertebrate homologues of NAT1 and ARD1 form an evolutionarily conserved N-terminal acetyltransferase and suggest that expression and down-regulation of this enzyme complex plays an important role in the generation and differentiation of neurons.     

Mitochondria and ischemic reperfusion damage in the adult and in the developing brain

The developing and the adult brain respond in similar ways to ischemia, but also display clear differences. For example, the relative contributions of necrosis and apoptosis to neuronal death may be different, such that apoptotic mechanisms would be more prevalent in the developing brain. During normal development, more than half of the neurons in some brain regions are removed through apoptosis, and effectors like caspase-3 are highly upregulated in the immature brain. Mitochondria are pivotal regulators of cell death through their role in energy production and calcium homeostasis, their capacity to release apoptogenic proteins and to produce reactive oxygen species. This review will summarize some of the current studies dealing with mitochondria-related mechanisms of ischemic brain damage, with special reference to developmental aspects.     

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.     

Characterization of an RNA granule from developing brain

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.     

Antidepressants and Cdk inhibitors: Releasing the brake on neurogenesis?

It is now clear that neurogenesis occurs in the brain of adult mammals. Many studies have attempted to establish relationships among neurogenesis, depression and the mechanism of action of antidepressant drugs. Therapeutic effects of antidepressants appear to be linked to increased neurogenesis in the hippocampus. Cdk inhibitors are expressed in multiple brain regions, presumably maintaining quiescence in differentiated neurons. Recently, the abundant expression of p21Cip1 was found in neuroblasts and in newly developing neurons in the subgranular zone of the hippocampus, a region where adult neurogenesis occurs. Chronic treatment with the tricyclic antidepressant imipramine markedly decreased p21Cip1 mRNA and protein levels and stimulated neurogenesis in this region. These results suggest that p21Cip1 restrains neurogenesis in the hippocampus, and antidepressant-induced stimulation of neurogenesis might be a consequence of decreased p21Cip1 expression, with the subsequent release of neuronal progenitor cells from the blockade of proliferation. These findings suggest the potential for new therapeutic strategies for the treatment of depression that target cell cycle proteins. However, there is a possibility that long-term stimulation of neurogenesis might exhaust the proliferation potentials of neuronal progenitors.     

Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex

The size of brain regions depends on the balance between proliferation and differentiation. During development of the mouse cerebral cortex, ventricular zone (VZ) progenitors, neuroepithelial and radial glial cells, enlarge the progenitor pool by proliferative divisions, while basal progenitors located in the subventricular zone (SVZ) mostly divide in a differentiative mode generating two neurons. These differences correlate to the existence of an apico-basal polarity in VZ, but not SVZ, progenitors. Only VZ progenitors possess an apical membrane domain at which proteins of the Par complex are strongly enriched. We describe a prominent decrease in the amount of Par-complex proteins at the apical surface during cortical development and examine the role of these proteins by gain- and loss-of-function experiments. Par3 (Pard3) loss-of-function led to premature cell cycle exit, reflected in reduced clone size in vitro and the restriction of the progeny to the lower cortical layers in vivo. By contrast, Par3 or Par6 (Pard6alpha) overexpression promoted the generation of Pax6+ self-renewing progenitors in vitro and in vivo and increased the clonal progeny of single progenitors in vitro. Time-lapse video microscopy revealed that a change in the mode of cell division, rather than an alteration of the cell cycle length, causes the Par-complex-mediated increase in progenitors. Taken together, our data demonstrate a key role for the apically located Par-complex proteins in promoting self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.     

Role of the Cell Cycle in the Pathobiology of Central Nervous System Trauma

Up-regulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce up-regulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.     

MALS-3 regulates polarity and early neurogenesis in the developing cerebral cortex

Apicobasal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian NPCs is poorly understood. Here, we characterize the function of the PDZ domain protein MALS-3 in the developing cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs. Mice lacking all three MALS genes fail to localize the polarity proteins PATJ and PALS1 apically in NPCs, whereas the formation and maintenance of adherens junctions appears normal. In the absence of MALS proteins, early NPCs progressed more slowly through the cell cycle, and their daughter cells were more likely to exit the cell cycle and differentiate into neurons. Interestingly, these effects were transient; NPCs recovered normal cell cycle properties during late neurogenesis. Experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of apicobasal polarity, loss of adherens junctions, and a slowing of the cell cycle. Our results suggest that MALS-3 plays a role in maintaining apicobasal polarity and is required for normal neurogenesis in the developing cortex.     

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.     

Synthesis and localization of plasma proteins in the developing human brain. Integrity of the fetal blood-brain barrier to endogenous proteins of hepatic origin

The distribution and possible origins of plasma proteins in the human embryonic and fetal brain at different stages of development have been investigated by a combination of isolation and translation of mRNAs and immunocytochemistry using specific antisera. As many as 23 plasma-like proteins have been identified using immunocytochemical methods at the light microscopical level. The presence of mRNAs for 13 of the immunocytochemically positive plasma proteins was demonstrated by in vitro and in ovo translation followed by crossed immunoelectrophoresis and autoradiography; this indicates in situ synthesis of these proteins (e.g., alpha-fetoprotein, alpha 1-antitrypsin, GC-globulin, alpha 2-macroglobulin, pseudocholinesterase, and transferrin) in some brain regions. The regional distribution of some proteins and the absence of some mRNAs suggest that the presence of certain plasma proteins in developing brain may be accounted for by uptake from csf or via nerve processes extending beyond the blood-brain barrier. In several cases, specific proteins appear to be associated with defined cell types, e.g., alpha-fetoprotein, GC-globulin, and ceruloplasmin with neurons, alpha 2-macroglobulin with endothelial cells, and ferritin with glial cells. Some proteins were associated with two or three cell types, e.g., alpha 1-antitrypsin with neurons and glia, and transferrin and alpha 2HS-glycoprotein with neurons, glia, and endothelial cells. Comparison of the expression of mRNAs from fetal brain and liver injected into Xenopus oocytes showed that a few proteins (transferrin and ceruloplasmin) were secreted when liver mRNA was injected, but not when brain mRNA was injected. This suggests that there may be an important difference in the structure and/or processing of these proteins in the brain which may reflect a function different from that associated with them when they originate from the liver. Staining was generally intracellular rather than extracellular; plasma proteins were not associated with the areas immediately around blood vessels although there was a strong immunoprecipitation for each protein within the lumen of cerebral blood vessels. These immunocytochemical findings together with the identification of mRNAs for a large number of plasma proteins in immature brain are discussed in relation to animal experimental work which suggests that the blood-brain barrier to protein is present even at very early stages of brain development.