Cdc7 kinase is required for postnatal brain development

The evolutionally conserved Cdc7 kinase plays crucial roles in initiation of DNA replication as well as in other chromosomal events. To examine the roles of Cdc7 in brain development, we have generated mice carrying Cdc7 knockout in neural stem cells by using Nestin-Cre. The Cdc7^Fl/Fl Nestin^Cre mice were born, but exhibited severe growth retardation and impaired postnatal brain development. These mice exhibited motor dysfunction within 9 days after birth and did not survive for more than 19 days. The cerebral cortical layer formation was impaired, although the cortical cell numbers were not altered in the mutant. In the cerebellum undergoing hypoplasia, granule cells (CGC) decreased in number in Cdc7^{Fl/F l}Nestin^Cre mice compared to the control at E15-18, suggesting that Cdc7 is required for DNA replication and cell proliferation of CGC at mid embryonic stage (before embryonic day 15). On the other hand, the Purkinje cell numbers were not altered but its layer formation was impaired in the mutant. These results indicate differential roles of Cdc7 in DNA replication/cell proliferation in brain. Furthermore, the defects of layer formation suggest a possibility that Cdc7 may play an additional role in cell migration during neural development.     

Emerging technologies from the Human Genome Project for understanding susceptibility and risk

The new technologies from the Human Genome Program provide exceptional opportunities for surveying and measuring human exposure, as well as determining susceptibility on an individual-by-individual basis. These new technologies will soon enable rapid screening of populations at risk, as well as the broader public, for a variety of genes known to be associated with increased risk. These include specific oncogenes, tumor suppressor genes and DNA repair enzymes. Use of these technologies also presents a number of ethical issues, both in screening and in use of the information about individuals. Overall, the use of rapid genotyping technologies will introduce a specificity and possible group identifiers that will present new challenges to the determination of risk within the EPA mandate.     

Manifestations of diabeters mellitus on mouse preimplantation developement: effect of elevated concentration of metabolic intemediated

The metabolic derangements of pregnancies complicated by diabetesmellitus, specifically hyperglycaemia and hyper-ketonaemia,are known to be teratogenic during the period of organogenesisin animals. We have shown previously that poorly controled diabetesmellitus impairs in-vivo and in-vitro mouse preimplantationembryo growth, and that culturing embryos in elevated glucoseconcentrations only partially recreated this developmental dealy.To extend this observation we examined the effect on mouse preimplantationembryo growth of elevated concentrations of other metabolicintermediates, which may be deranged in diabetes mellitys, namelylipids, lactate, glycerol, amino acids, and ketones. Two-cellembryos from ovulation-induced B₆C₃F₁ mice were cultured for72 h in the presence of added lipids (250 mg/dl), lactate (5mM), glycerol (160 µM) or mixed amino acids (8.5% travosol,7 mM) and showed no significant difference in growth over 72h verus their control groups. However, growth of preimplantationembryos in acetoacetate (10 mM) or in the racemic micture ofDL--hydroxybutyrate (16 and 32mM) revealed marked retardationversus controls when assessed either by distribution of developmentalstages over time (24, 48, 72 h, P <0.001) or by the differencein the average rank of sums indicating a delay in maturation(P<0.0001). We conclude that elevated ketone concentrationsadversely affect preimplantation embryo development. These findingsextend previous studies which correlate uncontrolled diabetesmellitus as well as hyperglycaemia with abnormal organogenesis,and demonstrate tht exposure to metabolic derangements may alsohinder reproductive performane at even earlier stages in gestation.     

Characterization and prediction of IVF cycles generating “slow-cleaving” embryos

Purpose To characterize and predict cycles generating slowcleaving embryos in in vitro fertilization, 86 cycles were retrospectively divided into two groups (slow, n=41, and fast, n=45 according to whether the number of blastomeres per embryo on day 3 was or > than the mean of the distribution, respectively.Results Cycles generating slowcleaving embryos were treated with luteinizing hormonereleasing hormone agonist before ovarian stimulation for a shorter period (12.1±0.5 versus 15.6±1.1 days; P0.01) and had higher immaturity grade of oocyte-corona-cumulus complexes which resulted in embryos (1.6±0.1 vs 1.3±0.1; P0.05) when compared to cycles producing fastcleaving embryos. Both variables entered in a logistic regression model applied in order to predict the probability of a cycle generating slowcleaving embryos (goodness-of-fit chisquare=180.0, degrees of freedom (df)=80, P=0.4786. This model predicted correctly 86.7% (13 of 15) of cycles generating slowcleaving embryos and 83.3% (10 of 12) of cycles producing fastcleaving embryos when the estimated probability of a cycle producing slowcleaving embryos was 0.7 or 0.3, respectively.Conclusion Shorter treatment with hormone-releasing hormone agonist before ovarian stimulation and higher immaturity grade of oocyte-corona-cumulus complexes which result in embryos are predictive characteristics of in vitro fertilization cycles generating slow-cleaving embryos.     

The function of presenilin-1 in amyloid β-peptide generation and brain development

Several mutations in genes that cause the familial form of Alzheimer’s Disease (FAD) have been identified. All mutations in the three FAD genes, i.e., amyloid precursor protein (APP), presenilin 1 (PS-1), and presenilin 2 (PS-2) cause an increased production of a longer, more amyloidogenic form of the amyloid peptide corroborating strongly the idea that abnormal processing of APP is central to the pathogenesis. In PS-1 deficient mice, 80% less amyloid peptide was produced. Instead, membrane associated carboxyterminal fragments generated by α- and β-secretase accumulated suggesting that PS-1 is involved in the gamma-secretase activity cleaving the transmembrane domain of APP after α- and β-secretase cleavage has occured. The clinical mutations in PS-1 which increase the production of βA4_1-42 therefore seem to cause a “selective” gain of its normal function. During cortical plate development in PS-1-deficient mice, neurons do not terminate their movement at the outer margin of the cortical plate, but enter the marginal zone and subarachnoid space. These focal heterotopias closely resemble those occuring, e.g., in human lissencephaly type II. The extracellular matrix of the cortical plate and marginal zone was altered as a consequence of a loss of Cajal-Retzius (CR) neurons from the marginal zone. The pathogenesis of this neuronal migration disorder is associated with a reduction and redistribution of notch-1 immunoreactivity in CR- and cortical plate neurons, a cell surface receptor operative in cell fate selection, which similar to APP is cleaved in its transmembrane domain during activation by a γ-secretase like protease.     

Hypoxia induces differential changes of dopamine metabolism in mature and immature mesencephalic and diencephalic cell cultures

Summary. Perinatal hypoxia is known as a high risk factor for the development of long-lasting abnormalities in dopaminergic system. The early developmental alterations of dopamine (DA) metabolism induced by hypoxia could contribute to these abnormalities. To understand the hypoxia-induced changes of intra- and extracellular dopamine levels and its main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in immature dopaminergic neurons, we compared these changes in rat mesencephalic and diencephalic cell cultures on day in vitro (DIV) 2 (immature cells), DIV 8 and DIV 13 (mature cells). Cell cultures were exposed to an oxygen-free gas mixture in a Billups chamber for 2–4 hours. Mature cell cultures responded to hypoxia with an increase of DA levels in the cells and in the medium during the first 45 min (by an average of 57 and 114% respectively). Thereafter, DA levels decreased, and returned to the baseline within the next 30 min. The cellular DA levels continued to decrease up to 15% of the baseline during 255 min hypoxia whereas the extracellular DA content stabilized at the prehypoxic levels. Immature cell cultures (DIV 2) in contrast to mature ones, were unable to maintain normal extracellular DA levels during hypoxia and showed a decrease of the cellular and extracellular levels to 50% of the prehypoxic levels. DOPAC and HVA changes mimick, however, at a lower level, the pattern of DA changes during the exposure to hypoxia. In principle, in the diencephalic cell culture similar effects of hypoxia exposure on the investigated parameters were found (studied during 0–120 min). The present study demonstrates that mature and immature dopaminergic cells differ in the regulation of the extra- and intracellular DA levels during hypoxia. In immature cells the low synthetic capacity of tyrosine hydroxylase and the deficient capacities of the transport and storage processes result in decreased extracellular DA levels. This could be an important factor for the long-term modulation of the expression of tyrosine hydroxylase and subsequent long-term behavioral and/or neurological abnormalities induced by perinatal hypoxia. Received June 8, 1998; accepted July 21, 1998     

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.     

Potentiation of glutamatergic agonist-induced inositol phosphate formation by basic fibroblast growth factor is related to developmental features in hippocampal cultures: neuronal survival and glial cell proliferation

We investigated the modulation by growth factors of phospholipase C (PLC)-linked glutamate receptors during in vitro development of hippocampal cultures. In defined medium, glial cells represent between 3 and 14% of total cell number. When we added basic fibroblast growth factor (bFGF) 2 h after plating, we found: (i) a neuroprotection from naturally occurring death for up to 5 days; (ii) a proliferation of glial cells from day 3; and (iii) a potentiation of quisqualate (QA)-induced inositol phosphate (IP) formation from 1 to 10 days in vitro (DIV) and 1S, 3R-amino-cyclopentane-1,3-dicarboxylate (ACPD) response from 3 to 10 DIV. The antimitotic cytosine-beta,D-arabinofuranoside (AraC) blocked glial cell proliferation induced by bFGF, but not neuroprotection. Under these conditions, the early potentiation of the QA response (1-3 DIV) was not changed, while the ACPD and late QA response potentiations were prevented (5-10 DIV). Epidermal growth factor was not neuroprotective but it induced both glial cell proliferation and late QA or ACPD potentiation. Surprisingly, the early bFGF-potentiated QA-induced IP response was blocked by 6, 7-dinitro-quinoxaline-2,3-dione (DNQX), suggesting the participation of ionotropic (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptors. The delayed bFGF-potentiated ACPD-induced IP response is inhibited by (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), indicating possible activation of glial metabotropic receptors. These results suggest that, in hippocampal cultures, bFGF modulates AMPA and metabotropic glutamate receptors linked to the IP cascade, possibly in relation to the regulation of neuronal survival and glial cell proliferation, respectively.     

Expression, distribution and ultrastructural localization of the synapse-organizing molecule agrin in the mature avian retina

At the vertebrate neuromuscular junction the extracellular matrix molecule agrin is responsible for the formation, maintenance and regeneration of most if not all postsynaptic specializations. Several agrin isoforms are generated by alternative splicing which differ in their function and which are all expressed in the CNS. To analyse the role of agrin in the CNS, we investigated the expression and ultrastructural localization of agrin in the posthatched chick retina. In situ hybridization revealed the presence of agrin mRNA in all cellular layers of the mature retina, indicating that most if not all major retinal cell types synthesize agrin. Pan-specific as well as isoform-specific antiagrin antisera stained the optic fibre layer and the outer plexiform layer. However, only the pan-specific antiserum additionally stained the inner limiting membrane. Immunoelectron microscopy showed that in the optic fibre layer agrin was associated with ganglion cell axons and that at least part of this agrin corresponds to a neuronal isoform of agrin. In the outer plexiform layer, agrin was localized in the cleft between the photoreceptor terminals and the invaginating horizontal and bipolar cell dendrites. In the synapse-containing inner plexiform layer both antisera revealed punctate immunoreactivity. This staining corresponded to agrin concentrated in the synaptic cleft of conventional synapses as determined by preembedding immunoelectron microscopy. Agrin is thus concentrated at mature interneuronal synapses as it is at the neuromuscular junction, consistent with a role of agrin during formation and/or maintenance of synapses in the CNS.