Decreased glutamate receptor 2 expression and enhanced epileptogenesis in immature rat hippocampus after perinatal hypoxia-induced seizures.

Hypoxic encephalopathy is the most common cause of neonatal seizures and can lead to chronic epilepsy. In rats at postnatal days 10-12 (P10-12), global hypoxia induces spontaneous seizures and chronically decreases seizure threshold, thus mimicking clinical aspects of neonatal hypoxia. We have shown previously that the acute and chronic epileptogenic effects of hypoxia are age-dependent and require AMPA receptor activation. In this study, we aimed to determine whether hypoxia-induced seizures and epileptogenesis are associated with maturational and seizure-induced changes in AMPA receptor composition and function. Northern and Western blots indicated that glutamate receptor 2 (GluR2) mRNA and protein expression were significantly lower in neocortex and hippocampus at P10-12 compared with adult. After hypoxia-induced seizures at P10, GluR2 mRNA was significantly decreased within 48 hr, and GluR2 protein was significantly decreased within 96 hr. AMPA-induced Co(2+) uptake by neurons in hippocampal slices indicated higher expression of Ca(2+)-permeable AMPA receptors in immature pyramidal neurons compared with adult. In slices obtained 96 hr after hypoxia-induced seizures, AMPA-induced Co(2+) uptake was significantly increased compared with age-matched controls, and field recordings revealed increased tetanus-induced afterdischarges that could be kindled in the absence of NMDA receptor activation. In situ end labeling showed no acute or delayed cell death after hypoxia-induced seizures. Our results indicate that susceptibility to hypoxia-induced seizures occurs during a developmental stage in which the expression of Ca(2+)-permeable AMPA receptors is relatively high. Furthermore, perinatal hypoxia-induced seizures induce increased expression of Ca(2+)-permeable AMPA receptors and an increased capacity for AMPA receptor-mediated epileptogenesis without inducing cell death.

Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein.

X-linked lissencephaly and "double cortex" are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. We identified a novel 10 kb brain-specific cDNA interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration.

Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.

Long-range, directed migration is particularly dramatic in the cerebral cortex, where postmitotic neurons generated deep in the brain migrate to form layers with distinct form and function. In the X-linked dominant human disorder periventricular heterotopia (PH), many neurons fail to migrate and persist as nodules lining the ventricular surface. Females with PH present with epilepsy and other signs, including patent ductus arteriosus and coagulopathy, while hemizygous males die embryonically. We have identified the PH gene as filamin 1 (FLN1), which encodes an actin-cross-linking phosphoprotein that transduces ligand-receptor binding into actin reorganization, and which is required for locomotion of many cell types. FLN1 shows previously unrecognized, high-level expression in the developing cortex, is required for neuronal migration to the cortex, and is essential for embryogenesis.

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Quantitative analysis of the expression of a VIP transgene.

We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

Corruption of genomic databases with anomalous sequence.

We describe evidence that DNA sequences from vectors used for cloning and sequencing have been incorporated accidentally into eukaryotic entries in the GenBank database. These incorporations were not restricted to one type of vector or to a single mechanism. Many minor instances may have been the result of simple editing errors, but some entries contained large blocks of vector sequence that had been incorporated by contamination or other accidents during cloning. Some cases involved unusual rearrangements and areas of vector distant from the normal insertion sites. Matches to vector were found in 0.23% of 20,000 sequences analyzed in GenBank Release 63. Although the possibility of anomalous sequence incorporation has been recognized since the inception of GenBank and should be easy to avoid, recent evidence suggests that this problem is increasing more quickly than the database itself. The presence of anomalous sequence may have serious consequences for the interpretation and use of database entries, and will have an impact on issues of database management. The incorporated vector fragments described here may also be useful for a crude estimate of the fidelity of sequence information in the database. In alignments with well-defined ends, the matching sequences showed 96.8% identity to vector; when poorer matches with arbitrary limits were included, the aggregate identity to vector sequence was 94.8%.

Characterization of the gene and messages for vasoactive intestinal polypeptide (VIP) in rat and mouse.

We have studied the structure and expression of the gene for vasoactive intestinal polypeptide (VIP) in rodents. We used a human cDNA to identify and clone a fragment of the rat VIP gene. This genomic fragment contained two separate exons, one encoding VIP itself and the other encoding a closely related neuropeptide, peptide histidine-isoleucine (PHI-27). Probes containing either exon, or both, hybridized to two messages: a prominent 1700-base (b) mRNA and a rare 1000-b species. These messages are expressed together in a tissue-specific manner, with highest levels in polyadenylated RNA from cerebral cortex and from small intestine, paralleling the reported levels of the neuropeptides themselves in these tissues. Using the rat genomic fragment as a probe, we isolated the mouse VIP gene in its entirety. The mouse gene is similar in organization to its human counterpart, with a total of 7 exons spanning 8 kilobases (kb). The 7th and largest exon, which is transcribed into the bulk of the 3' untranslated region of the messages, bears two potential polyadenylation sites 700 basepairs (bp) apart. S-1 nuclease protection with a fragment of this exon indicated that the two identifiable VIP messages differ in the extent of their 3' untranslated regions. Conversely, we found no evidence for differential splicing to produce messages encoding only one of the neuropeptides. Instead, specific oligonucleotide-directed digestion with RNase H demonstrated that all of the detectable mRNA from this gene contains both VIP and PHI coding sequences.

Optimizing the northern blot procedure.

We describe methods for preparing formaldehyde-agarose gels for use in Northern blotting which yield consistent high quality results. Using these methods, we tested seven different commercially available membranes in Northern blots. Each membrane was handled as specified by the manufacturer in a course of hybridization, stripping and rehybridization. Filter background was low in all cases, but the intensity of the signal generated by specific hybridization varied markedly between filters after both the first and second hybridization.

Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases.

A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined. The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease. The insert is truncated at different locations. The vector DNA is also partially digested. The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection. The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA. This method is simpler and thus may be more reliable than established subcloning schemes.

Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease.

Amyloid B-protein/amyloid A4 is a peptide present in the neuritic plaques, neurofibrillary tangles and cerebrovascular deposits in patients with Alzheimer's disease and Down's syndrome (trisomy 21) and may be involved in the pathogenesis of Alzheimer's disease. Recent molecular genetic studies have indicated that amyloid protein is encoded as part of a larger protein by a gene on human chromosome 21 (refs 6-9). The amyloid protein precursor (APP) gene is expressed in brain and in several peripheral tissues, but the specific biochemical events leading to deposition of amyloid are not known. We have now screened complementary DNA libraries constructed from peripheral tissues to determine whether the messenger RNA encoding APP in these tissues is identical to that expressed in brain, and we identify a second APP mRNA that encodes an additional internal domain with a sequence characteristic of a Kunitz-type serine protease inhibitor. The alternative APP mRNA is present in both brain and peripheral tissues of normal individuals and those with Alzheimer's disease, but its pattern of expression differs from that of the previously reported APP mRNA.

Myelination of jp,jpmsd, and qk axons by normal glia in vitro: ultrastructural and autoradiographic evidence.

Normal optic nerve glia were 'injected' into hypomyelinated mutant jp,jpmsd, and qk cerebellum by co-culturing explants in direct physical contact. Quantitative light microscopic studies demonstrated that such glial injection significantly increased the number of myelin profiles counted in cultures, suggesting that axons in all 3 mutants can accept myelination from competent glia when they are made available. In each mutant, the observed increase in myelination was independent of the ages of donor optic nerves and recipient cultures, but absolutely required positioning of the optic nerve so that direct contact occurred with the mutant cerebellar explants. The additional myelin found near the zone of fusion with the optic nerve morphologically resembled normal, not mutant myelin. Autoradiographs made after [3H]thymidine-labeled normal optic nerve was injected into jpmsd cultures showed that labeled cells had colonized the nearby mutant tissue. Labeled cells identified as oligodendrocytes by ultrastructural criteria were found adjacent to myelin segments near the fusion zone, but direct continuity between processes of these oligodendrocytes and myelin sheaths was not demonstrated. The astrocytes and phagocytic cells which were also labeled had no obvious relationship to myelinated axons. These results provide experimental evidence that the primary abnormalities produced by the three mutations jp,jpmsd, and qk are inherent in their glial cells, probably although not definitely in the oligodendrocytes.