Uncoupling Neogenin association with lipid rafts promotes neuronal survival and functional recovery after stroke.

The dependence receptor Neogenin and its ligand, the repulsive guidance molecule a (RGMa), regulate apoptosis and axonal growth in the developing and the adult central nervous system (CNS). Here, we show that this pathway has also a critical role in neuronal death following stroke, and that providing RGMa to neurons blocks Neogenin-induced death. Interestingly, the Neogenin pro-death function following ischemic insult depends on Neogenin association with lipid rafts. Thus, a peptide that prevents Neogenin association with lipid rafts increased neuronal survival in several in vitro stroke models. In rats, a pro-survival effect was also observed in a model of ocular ischemia, as well as after middle cerebral artery occlusion (MCAO). Treatments that prevented Neogenin association with lipid rafts improved neuronal survival and the complexity of the neuronal network following occlusion of the middle artery. Toward the development of a treatment for stroke, we developed a human anti-RGMa antibody that also prevents Neogenin association with lipid rafts. We show that this antibody also protected CNS tissue from ischemic damage and that its application resulted in a significant functional improvement even when administrated 6 h after artery occlusion. Thus, our results draw attention to the role of Neogenin and lipid rafts as potential targets following stroke.

Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations.

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Here, we describe the first characterization and neuronal differentiation of induced pluripotent stem (iPS) cells derived from Mecp2-deficient mice. Fully reprogrammed wild-type (WT) and heterozygous female iPS cells express endogenous pluripotency markers, reactivate the X-chromosome and differentiate into the three germ layers. We directed iPS cells to produce glutamatergic neurons, which generated action potentials and formed functional excitatory synapses. iPS cell-derived neurons from heterozygous Mecp2(308) mice showed defects in the generation of evoked action potentials and glutamatergic synaptic transmission, as previously reported in brain slices. Further, we examined electrophysiology features not yet studied with the RTT iPS cell system and discovered that MeCP2-deficient neurons fired fewer action potentials, and displayed decreased action potential amplitude, diminished peak inward currents and higher input resistance relative to WT iPS-derived neurons. Deficiencies in action potential firing and inward currents suggest that disturbed Na(+) channel function may contribute to the dysfunctional RTT neuronal network. These phenotypes were additionally confirmed in neurons derived from independent WT and hemizygous mutant iPS cell lines, indicating that these reproducible deficits are attributable to MeCP2 deficiency. Taken together, these results demonstrate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in primary neurons, and these identified phenotypes further illustrate the requirement of MeCP2 in neuronal development and/or in the maintenance of normal function. By validating the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, we identify deficiencies that can be targeted for in vitro translational screens.

Diminished prevalence but preserved synaptic distribution of N-methyl-D-aspartate receptor subunits in the methyl CpG binding protein 2(MeCP2)-null mouse brain.

In this study, we examined the prevalence and distribution of NMDA receptor subunits within crude synaptic membranes derived from the brains of mice lacking methyl CpG binding protein 2 (MeCP2). Our results show that the distribution of NMDA receptor subunits within the detergent soluble, detergent resistant, and postsynaptic density microdomains is preserved at MeCP2-null synapses. However, analysis of the NMDA receptor subunit expression revealed a decrease in the prevalence of the GluN1 and GluN2A subunits in MeCP2-null tissue. Collectively, these results indicate that synaptic membrane microdomains at synapses of the MeCP2-null brain develop normally, and that NMDA receptor subunits are properly targeted and distributed within them. The under-representation of the GluN1 and GluN2A subunits suggests that MeCP2-null synapses contain fewer mature NMDA receptor complexes, and raises the possibility that impaired NMDA receptor ontogeny could contribute to Rett syndrome pathophysiology.

Identification of a murine methyl cytosine phosphate guanine binding domain-containing factor 3 (MBD3) promoter segment sufficient for driving reporter gene expression in neurons in vitro and in vivo.

In this study, we characterize the functional properties of a segment of the murine methyl cytosine phosphate guanine binding domain-containing factor 3 (MBD3) promoter region. Transient transfection of a chimera consisting of a 1072 base pair region extending upstream from the MBD3 initiation codon fused to a luciferase complementary DNA (cDNA) confirmed the presence of a functional promoter unit. Primer extension analysis failed to identify a single predominant transcription initiation site, but rather detected multiple transcription initiation sites in both brain tissue and cultured neuroblastomaxglioma cell line (NG108-15) and rat pheochromocytoma cell line (PC12) cells. Reporter gene assays revealed that this 1072 base pair fragment efficiently drives expression in transfected NG108-15 cells, PC12 cells, cultured primary neurons, and in neurons of a transgenic mouse brain. Deletion analysis mapped the critical region for promoter activity to a segment of approximately 518 base pairs, located from positions -585 to -68 relative to the translational start codon. Taken together, these data indicate that a 1072 base pair fragment of the MBD3 promoter is sufficient to drive expression in cell lines and primary cultured neurons, and is able to direct transgene expression in the mouse brain in a pattern with spatial similarity to that of native MBD3.

Transient forebrain ischemia alters the mRNA expression of methyl DNA-binding factors in the adult rat hippocampus.

We have examined how transient cerebral ischemia affects the mRNA expression of a family of methyl CpG-binding domain (MBD)-containing factors in the rat hippocampus. Our results show that each member of this family is affected by cerebral ischemia challenge, but with differing patterns of responsiveness. At 3, 6 and 12 h following reperfusion, MeCP2 and MBD1 expression is maintained at control levels throughout the hippocampus. At 24 h, MeCP2 and MBD1 are induced in both the CA1 and CA3 subfields. This delayed pattern of induction is in contrast to the responses of MBD2 and MBD3. Both MBD2 and MBD3 display significant changes in expression at early times following reperfusion, although their changes are opposite in direction. MBD2 expression is induced throughout the hippocampal formation at 6 h, and remains elevated at 12 and 24 h. MBD3 expression decreases as early as 3 h following insult in the CA3 and dentate gyrus, and the decreased expression remains in the vulnerable CA1 subfield at 6, 12, and 24 h. Taken together, these results are the first to illustrate that the expression of methyl DNA-binding factors are affected by challenges to the brain, and they also illustrate that each methyl DNA-binding factor responds differently to cerebral ischemic challenge. As each of these family members is associated either directly or indirectly with the inhibition of gene transcription, our results suggest that following cerebral ischemia the normal pattern of transcriptional inhibition provided by these factors may be altered in the hippocampus.

Expression and function of A1 adenosine receptors in the rat hippocampus following transient forebrain ischemia.

We investigated how transient cerebral ischemia affects the gene expression, immunoreactive protein levels, and the function of the A1 subtype of adenosine receptor in the rat hippocampus at different times following reperfusion. A1 receptor mRNA levels were altered significantly in different hippocampal subfields as early as 6 h following insult. However, these changes in mRNA levels were not paralleled at the protein level, as western blotting with A1 receptor-specific antibodies revealed that hippocampal A1 adenosine receptor prevalence did not differ from sham control at either 6 or 24 h following insult. The lack of change in A1 receptor prevalence was consistent with functional examinations, as only marginal changes were observed in the ability of A1 receptors to attenuate excitatory post-synaptic potentials in the CA1 subfield at 24 h following reperfusion. These data illustrate that although the mRNA expression levels of the A1 adenosine receptor are altered by transient cerebral ischemia, the immunoreactive prevalence and function of this receptor are maintained in the post-ischemic hippocampus at times preceding the death of the vulnerable neurons.

Kindling induces the mRNA expression of methyl DNA-binding factors in the adult rat hippocampus.

We have investigated the gene expression responses of a family of methyl CpG-binding domain-containing factors (MeCP2, MBD1, MBD2, and MBD3) in the hippocampus of electrically kindled rats. Expression was examined in both amygdala- and partial perforant-pathway-kindled subjects, 24 h and 28 days following the final stimulation. In general, the responses of MBDs 2 and 3 paralleled each another, both temporally and spatially. The expression of both genes was significantly elevated in all hippocampal subfields at 24 h following either the fifth stage 5 seizure (amygdala kindling) or the 15th stimulation of the perforant pathway. This induced expression was transient, however, as the expression of both genes returned to control levels by 28 days. This pattern of response contrasted to that observed for MeCP2 and MBD1. MeCP2 displayed no change in expression either 24 h or 28 days after amygdala kindling, but did display a late-developing, significant increase in expression in the dentate gyrus at 28 days following perforant-pathway kindling. The expression of MBD1 was unchanged by partial perforant-pathway kindling, but was induced in the dentate gyrus 28 days after amygdala kindling. These results demonstrate that electrical kindling alters the hippocampal expression of methyl DNA-binding factors, but does not affect each factor equivalently. The responsive patterns observed suggest that this family of transcriptional regulators can be differentially altered in the hippocampus by seizure activity.

Somatostatin type 2 receptor expression in the rat hippocampus following cerebral ischemia.

We examined how transient cerebral ischemia affects the mRNA expression, and the immunoreactive distribution, of the somatostatin type 2 (sst2) receptor in the adult rat hippocampus. Following reperfusion, sst2 mRNA levels increased significantly in the CA1 region by 3 h, and were also increased in the CA3 and CA4/hilus subfields at 6 and 12 h. At 24 h, however, sst2 receptor mRNA levels returned to baseline throughout the hippocampus. At the protein level, we found the regional immunoreactivity of the sst2a receptor was maintained, or slightly elevated, throughout the hippocampus at 6 h, but not different from control at 24 h. These results suggest that sst2 receptors maintain their normal distribution and prevalence in the post-ischemic hippocampus before the deterioration of the vulnerable CA1 neurons. Thus, they represent attractive targets for neuroprotective interventions.

Perforant pathway kindling transiently induces the mRNA expression of GABA-B receptor subtypes R1A and R2 in the adult rat hippocampus.

We examined the gene expression responses of GABA-B R1A, R1B and R2 receptor subtypes in the hippocampus of perforant pathway-kindled rats at 24 h and 28 days after 15 consecutive daily stimulations. We found R1A expression, but not R1B expression, to be significantly induced in the dentate gyrus at 24 h. No change in the expression of R1A or R1B was observed at 28 days. R2 expression was induced throughout the hippocampus at 24 h, but also returned to control levels by 28 days. Thus, our results show that kindling induces a transient increase in GABA-B receptor mRNA in the hippocampus.

Cloning and tissue distribution of a novel isoform of the rat GABA(B)R1 receptor subunit.

We have identified a novel splice variant of the metabotropic GABA(B) receptor (R) subunit I, designated GABA(B)R1f, from a rat hippocampus cDNA library screen. GABA(B)R1f shares sequence homology with rat GABA(B)R1a, with the exception of an in-frame deletion of exon 4, resulting in a 21 bp deletion in the coding sequence of the N-terminal extracellular domain. In addition, GABA(B)R1f also contains a 93 bp in-frame insertion in a region of the sequence corresponding to the second extracellular loop and the fifth transmembrane domain, similar to that found in rat GABA(B)R1c. While being ubiquitously (but variably) expressed, reverse-transcription polymerase chain reaction analysis revealed the GABA(B)R1f isoform to be most prevalent in peripheral vs central tissues, suggesting a potential role for this novel isoform in either the mediation of inhibitory transmission in these various tissues, or in as yet defined actions unrelated to central synaptic regulatory mechanisms attributable to GABA(B)R.

Diazepam-potentiated [3H]phenytoin binding is associated with peripheral-type benzodiazepine receptors and not with voltage-dependent sodium channels.

In the presence of diazepam, [3H]phenytoin binds with high affinity to brain membranes. The present experiments examined whether this high affinity [3H]phenytoin-binding site co-localized with the standard [3H]phenytoin-binding site on the voltage-dependent sodium channel (VDSC). Veratridine, a pharmacological activator of the voltage-dependent sodium channel, that inhibits standard [3H]phenytoin binding, failed to affect the high affinity diazepam-potentiated [3H]phenytoin binding in brain membranes, suggesting that the potentiated binding interaction resides at a site distinct from the voltage-dependent sodium channel. This possibility was confirmed by anion exchange chromatography of digitonin-solubilized rat brain membranes, as diazepam-potentiated high affinity [3H]phenytoin binding eluted in column fractions that were distinct from [3H]saxitoxin-defined voltage-dependent sodium channels. To examine whether diazepam-potentiated [3H]phenytoin binding might be associated with other 'classic' benzodiazepine receptor sites, we tested whether specific ligands for benzodiazepine receptors would either produce or block potentiated [3H]phenytoin binding. Neither agonists, nor antagonists, of the high affinity central-type benzodiazepine receptor affected potentiated [3H]phenytoin binding, suggesting that the high affinity potentiated binding site is not likely associated with central benzodiazepine receptors. Peripheral-type benzodiazepine receptor agonists, however, did potentiate [3H]phenytoin binding, and a specific receptor antagonist (PK11195) attenuated the potentiation seen with diazepam. Overall, these data illustrate that [3H]phenytoin interacts with a novel site in brain membranes that is distinct from the voltage-dependent sodium channel and is allosterically revealed by peripheral-type, but not central-type, benzodiazepine receptor agonists.

Reversible attenuation of glutamatergic transmission in hippocampal CA1 neurons of rat brain slices following transient cerebral ischemia.

The present experiments were conducted to determine the time course of synaptic dysfunction in the vulnerable regions of the post-ischemia hippocampus. Following transient cerebral ischemia, neurons in the CA1 subfield of the hippocampus undergo a delayed degeneration that develops about 48 h after reperfusion. We have shown previously that CA1 glutamatergic transmission is decreased in the CA1 subfield well before any morphological deterioration of the CA1 cells is visible under the light microscope. However, it is unknown whether a time window exists after insult in which attenuated synaptic activity may be restored to normal levels. We show here that evoked CA1 somatic population spikes and dendritic field potential responses decline progressively after reperfusion in the CA1 subfield, such that by 72 h post-insult, the challenged neurons are unable to elicit evoked excitatory responses. This attenuation of synaptic transmission was confined to the vulnerable neurons of the hippocampus, however, as the evoked responses in the dentate gyrus displayed amplitudes that were not significantly diminished from sham control after challenge. In brain slices obtained from 24 h post-ischemia rats with significantly impaired CA1 somatic responses, the application of 5 or 50 microM of the potassium channel blocker 4-aminopyridine (4-AP) restored the magnitude of the evoked excitatory response to control values. At 36 h post-ischemia, the decreased CA1 evoked responses could be partially improved by 4-AP, but not to control levels. Based upon these results, we conclude that the decreased CA1 synaptic activity at 24 h post-ischemia is potentially reversible, and suggest that 4-AP improves the CA1 synaptic responses at least in part by improving transmitter release at post-ischemia glutamatergic synapses.

Increased calpain I-mediated proteolysis, and preferential loss of dephosphorylated NF200, following traumatic spinal cord injury.

We investigated the hypothesis that the Ca2+-activated protease calpain is involved in the pathophysiology of spinal cord injury, and is linked to the proteolytic degradation of cytoskeletal proteins. We report here that levels of calpain I (mu-calpain)-mediated spectrin breakdown products are increased by 15 min post-injury, with peak levels reached by 2 h post-injury. The dephosphorylated form of the neurofilament protein NF200 is substantially lost over the same time-period. A 35-g compressive injury was applied to the midthoracic rat spinal cord for 1 min, and animals were killed at 15 min, 1, 2, 4, 8, 16, and 24 h post-injury. Calpain I-mediated spectrin breakdown products accumulated post-injury, with peak levels reached at 2 h. Secondly, we have demonstrated a progressive loss of the 200,000 mol. wt neurofilament protein NF200, a cytoskeletal calpain substrate, which began within 1-2 h post-injury. Densitometric analyses confirmed that loss of NF200 is a substrate-specific phenomenon, since (i) dephosphorylated NF200 was preferentially lost while phosphorylated NF200 was relatively spared, and (ii) actin, which is not a substrate for calpain, was relatively spared following spinal cord injury. Finally, we demonstrated calpain I-mediated spectrin breakdown within NF200-positive neuronal processes post-injury. We conclude that the accumulation of spectrin breakdown products is temporally and spatially correlated with loss of dephosphorylated NF200 after spinal cord injury.

Chronic ethanol exposure alters MK-801 binding sites in the cerebral cortex of the near-term fetal guinea pig.

The mechanism of ethanol central nervous system (CNS) teratogenesis, resulting from chronic maternal ingestion of high-dose ethanol during pregnancy, is not clearly understood. One of the target sites for ethanol-induced damage in the developing brain is the cerebral cortex. It has been proposed that chronic prenatal ethanol exposure alters NMDA receptors in the developing cerebral cortex. To test this hypothesis, timed pregnant guinea pigs were administered one of the following oral treatments throughout gestation: 4 g ethanol/kg maternal body weight/day; isocaloric sucrose/pair-feeding; water; or no treatment (ad lib). Near-term fetuses were studied at gestational day (GD) 63 (term, about GD 68). This ethanol regimen produced a maternal blood ethanol concentration of 66+/-4 mM (304+/-19 mg/dl) at 1 h after the daily dose on GD 58. The chronic ethanol regimen decreased near-term fetal body weight (12-26% decrease), brain weight (23% decrease), and cerebral cortical weight (21% decrease), compared with the isocaloric sucrose/pair-feeding, and combined water/ad lib experimental groups. Saturation analysis of near-term fetal cerebral cortical membranes using a [3H]MK-801 radioligand binding assay demonstrated a decreased affinity and increased number of MK-801 binding sites for the chronic ethanol regimen compared with the control treatments. These data support the suggestion that upregulation of NMDA receptors in the cerebral cortex after chronic prenatal ethanol exposure could lead to NMDA receptor-mediated excitotoxicity in this brain region.

Altered expression levels of SEF-2 and p112 in the rat hippocampus after transient cerebral ischemia: identification by mRNA differential display.

The authors used mRNA differential display to identify genes whose expression levels are altered in the adult rat hippocampus 24 hours after global ischemia. At this time after challenge, the basic helix-loop-helix transcription factor, SEF-2, and the 26S proteasome complex subunit, p112, were identified as genes whose expression levels are decreased and increased, respectively, in the hippocampus. To determine the spatial and temporal patterns of expression change for each gene, the authors antisense in situ hybridization to paired brain sections of sham-operated and global ischemia-challenged rats at 6, 12, and 24 hours after reperfusion SEF-2 expression was not significantly altered from that of sham-operated controls in any hippocampal subfield at or before 12 hours after challenge. At 24 hours after ischemia, however, SEF-2 expression levels were significantly diminished in the vulnerable CA1 subfield, but not in the less vulnerable CA3 or dentate granule cell subfields. The proteasome p112 subunit gene displayed no change in expression levels at 6 hours after insult; however, an elevated expression was observed at 12 hours after challenge in the dentate granule cell subfield. By 24 hours after challenge, p112 expression was significantly elevated in both the CA1 and dentate granule cell subfields. These results demonstrate that a member of the basic helix-loop-helix family of transcription factors, SEF-2, and the major subunit of the 26S proteasome complex, p112, display altered gene expression in the hippocampus after transient cerebral ischemia.

Decreased hippocampal expression, but not functionality, of GABA(B) receptors after transient cerebral ischemia in rats.

We have examined the effects of transient global ischemia on both the gene expression levels and the functionality of GABA(B) receptors in rat brain, using antisense in situ hybridization and electrophysiological evaluations. At the level of gene expression, no significant change in GABA(B) receptor expression was observed in any hippocampal subfield at either 6 or 12 h after challenge. At 24 h postchallenge, however, a significant decrease in GABA(B) receptor expression was observed in both the CA1 and CA3 subfields, whereas no change was observed in the dentate granule cell layer. Although expression in both the vulnerable CA1 and less vulnerable CA3 subfields was diminished at this time postchallenge, there was no significant difference in the degree of the diminished expression between these subfields. At the functional level, the dose-dependent ability of baclofen (1-100 microM) to inhibit an evoked excitatory postsynaptic potential (f-EPSP) in the CA1 subfield was evaluated at 24 h postischemia, in comparison with the dose-response observed in sham-operated subjects. No significant differences were observed in the efficacy of GABA(B) receptor-mediated inhibition of the elicited f-EPSP at any of the baclofen concentrations examined. These data demonstrate that although the mRNA expression levels for the GABA(B) receptor are diminished in both vulnerable and less vulnerable neurons of Ammon's horn at 24 h following transient global ischemia, the functionality of the GABA(B) receptor system is maintained at this time postchallenge.

Amygdala-kindled and electroconvulsive seizures alter hippocampal expression of the m1 and m3 muscarinic cholinergic receptor genes.

Expression of m1 and m3 muscarinic cholinergic receptors mRNAs was examined in rat hippocampus following either: (1) kindling to five Stage 5 amygdala-kindled seizures; or (2) eight electroconvulsive shock (ECS) seizures. Twenty-four hours after the last seizure of either type, there was a significant decrease in both m1 and m3 mRNAs in CA1, CA3 and the dentate gyrus subfields of the hippocampus. Twenty-eight days after the last seizure of either type, there was a significant increase in m1 mRNAs in CA1, CA3, and the dentate gyrus; for m3 mRNAs, there was a significant increase in CA3 28 days after the last ECS seizure, and in CA1 and CA3 28 days after the last kindled seizure. These results suggest that seizures alter the cholinergic system in the hippocampus, and that some of the alterations are very long-lasting.

Attenuation of hypoxic current by intracellular applications of ATP regenerating agents in hippocampal CA1 neurons of rat brain slices.

Hypoxia-induced outward currents (hyperpolarization) were examined in hippocampal CA1 neurons of rat brain slices, using the whole-cell recording technique. Hypoxic episodes were induced by perfusing slices with an artificial cerebrospinal fluid aerated with 5% CO2/95% N2 rather than 5% CO2/95% O2, for about 3 min. The hypoxic current was consistently and reproducibly induced in CA1 neurons dialysed with an ATP-free patch pipette solution. This current manifested as an outward shift in the holding current in association with increased conductance, and it reversed at -78 +/- 2.5 mV, with a linear I-V relation in the range of -100 to -40 mV. To provide extra energy resources to individual neurons recorded, agents were added to the patch pipette solution, including MgATP alone, MgATP + phosphocreatine + creatine kinase, or MgATP + creatine. In CA1 neurons dialysed with patch solutions including these agents, hypoxia produced small outward currents in comparison with those observed in CA1 neurons dialysed with the ATP-free solution. Among the above agents examined, whole-cell dialysis with MgATP + creatine was the most effective at decreasing the hypoxic outward currents. We suggest that the hypoxic hyperpolarization is closely related to energy metabolism in individual CA1 neurons, and that the energy supply provided by phosphocreatine metabolism may play a critical role during transient metabolic stress.

Decreased expression and functionality of NMDA receptor complexes persist in the CA1, but not in the dentate gyrus after transient cerebral ischemia.

The authors investigated the gene expression of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptor and the functional electrophysiologic activity of NMDA receptor complexes in the vulnerable CA1 and less vulnerable dentate gyrus subfields of the rat hippocampus at different times after transient cerebral ischemia. Decreased expression for both subtypes was observed in both the CA1 subfield and dentate granule cell layer at early times after challenge; however, the decreased expression in the dentate granule cell layer was reversible because mRNA levels for both the NR2A and NR2B subtypes recovered to, or surpassed, sham-operated mRNA levels by 3 days postchallenge. No recovery of expression for either subtype was observed in the CA1 subfield. The functional activity of NMDA receptor complexes, as assessed by slow field excitatory postsynaptic potentiations (slow f-EPSP) in CA1 pyramidal neurons, was maintained at 6 hours postchallenge; however, this activity was diminished greatly by 24 hours postchallenge, and absent at 7 days postchallenge. A similar pattern was observed for the non-NMDA receptor-mediated fast f-EPSP. In dentate granule neurons, however, no significant change in NMDA receptor-mediated slow f-EPSP from sham control was observed at any time after insult. The non-NMDA receptor-generated fast f-EPSPs also were maintained at all times postinsult in the dentate gyrus. These results illustrate that the activity of NMDA receptors remains functional in dentate granule neurons, but not in the pyramidal neurons of the CA1 subfield, at early and intermediate times after transient cerebral ischemia, and suggest that there is a differential effect of ischemia on the glutamatergic transmission systems in these two hippocampal subfields.