CRTC1 nuclear localization in the hippocampus of the pilocarpine-induced status epilepticus model of temporal lobe epilepsy.

cAMP response-element binding protein (CREB)-dependent genes are differentially expressed in brains of temporal lobe epilepsy (TLE) patients and also in animal models of TLE. Previous studies have demonstrated the importance of CREB regulated transcription in TLE. However, the role of the key regulator of CREB activity, CREB-regulated transcription coactivator 1 (CRTC1), has not been explored in epilepsy. In the present study the pilocarpine-induced status epilepticus (SE) model of TLE was used to study the regulation of CRTC1 during and following SE. Nuclear translocation of CRTC1 is critical for its transcriptional activity, and dephosphorylation at serine 151 residue via calcineurin phosphatase regulates cytoplasmic to nuclear transit of CRTC1. Here, we examined the localization and phosphorylation (Ser151) of CRTC1 in SE-induced rat hippocampus at two different time points after SE onset. One hour after SE onset, we found that CRTC1 translocates to the nucleus of CA1 neurons but not CA3 or dentate granule neurons. We further found that this CRTC1 nuclear localization is independent of Ser151 dephosphorylation since we did not detect any difference in dephosphorylation of Ser151 between control and SE animals at this time point. In contrast, 48 h after SE CRTC1 shows increased nuclear localization in the dentate gyrus (DG) of the SE-induced rats. At 48 h after SE, FK506 treatment blocked CRTC1 nuclear localization and dephosphorylation of Ser151. Our results provide evidence that CREB cofactor CRTC1 translocates into the nucleus of a distinct subset of hippocampal neurons during and following SE and this translocalization is regulated by calcineurin at a later time point following SE. Nuclear CRTC1 can bind to CREB possibly altering transcription during epileptogenesis.

Increased sensitivity to kindling in mice lacking TSP1.

The development of a hyperexcitable neuronal network is thought to be a critical event in epilepsy. Thrombospondins (TSPs) regulate synaptogenesis by binding the neuronal α2δ subunit of the voltage-gated calcium channel. TSPs regulate synapse formation during development and in the mature brain following injury. It is unclear if TSPs are involved in hyperexcitability that contributes to the development of epilepsy. Here we explore the development of epilepsy using a pentylenetetrazole (PTZ) kindling model in mice lacking TSP1 and TSP2. Unexpectedly, we found increased sensitivity to PTZ kindling in mice lacking TSP1, while mice lacking TSP2 kindled similar to wild-type. We found that the increased seizure susceptibility in the TSP1 knockout (KO) mice was not due to a compensatory increase in TSP2 mRNA as TSP1/2 KO mice were sensitive to PTZ, similar to the TSP1 KO mice. Furthermore, there were similar levels of TGF-B signal activation during kindling in the TSP1 KO mice compared to wild-type. We observed decreased expression of voltage-dependent calcium channel subunit CACNA2D1 mRNA in TSP1, TSP2, and TSP1/2 KO mice. Decreased CACNA2D2 mRNA was only detected in mice that lacked TSP1 and α2δ-1/2 protein levels in the cortex were lower in the TSP 1/2 KO mice. CACNA2D2 knockout mice have spontaneous seizures and increased PTZ seizure susceptibility. Here we report similar findings, TSP1, and TSP1/2 KO mice have low levels of CACNA2D2 mRNA expression and α2δ-1/2 receptor level in the cortex, and are more susceptible to seizures. CACNA2D2 mutations in mice and humans can cause epilepsy. Our data suggest TSP1 in particular may control CACNA2D2 levels and could be a modifier of seizure susceptibility.

The role of transcription factors cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER) in epileptogenesis.

Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing approximately threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy.

Dystrophic neuritic processes in epileptic cortex.

Cortical dysplasia is a frequent finding in cortical resections from children with refractory epilepsy. Diagnostic criteria and a classification scheme for cortical dysplasia has been proposed, though the relationship between specific cortical dysplasia features and their causal relationship with epilepsy is poorly understood. We reviewed 28 surgical resections from children and identified a common and easily recognized feature of cortical dysplasia: maloriented, misshapen and occasionally coarse neurofilament stained process forming a dystrophic neuritic background. The dystrophic neuritic background was associated with other features of cortical dysplasia in all 28 patients with cortical dysplasia, 26 with refractory epilepsy and 2 patients with other neurologic diagnoses. In seven children with refractory epilepsy due to other pathologic diagnosis such as vascular or glial lesions, the dystrophic neuritic background was only found in one patient with a ganglioglioma and other features suggestive of an associated cortical dysplasia. Our data indicate that a dystrophic neuritic background is a common and relatively specific neuropathologic finding in cortical dysplasia.

Dysplasia: a common finding in intractable pediatric temporal lobe epilepsy.

BACKGROUND: Risk factors for temporal lobe epilepsy (TLE) include history of CNS infection, family history of epilepsy, and history of febrile convulsions (FC). Pre-existing cortical dysplasia (CD) may also predispose to refractory TLE, independent of other risk factors for epilepsy. METHODS: The authors reviewed the neuropathologic features of surgical tissue from temporal lobectomies of 33 pediatric patients with refractory TLE, with and without a history of epilepsy risk factors. RESULTS: CD was found in 64% (21/33) of all patients with refractory TLE, including 73% (11/15) patients with a history of FC, 66% (2/3) patients with CNS infections, and 83% (5/6) patients with a family history of epilepsy. Disrupted cortical lamination, dystrophic and maloriented neurons, and balloon cells characterized the CD found in the temporal neocortex. CONCLUSION: CD was seen in 21 of 33 surgical specimens from children with refractory TLE, including those with and without other epilepsy risk factors.

Interferon beta in multiple sclerosis: experience in a British specialist multiple sclerosis centre.

BACKGROUND: The efficacy of interferon beta (IFN beta) is well established in relapsing-remitting multiple sclerosis (MS). However, the use of this drug in clinical practice is complex, especially because it is only partially effective, its long term efficacy and side effects are unknown, its efficacy may be abrogated by the development of neutralising antibodies, compliance is variable, and its cost effectiveness is controversial. OBJECTIVES AND METHODS: Analysis of a prospectively followed up series of 101 MS patients treated with IFN beta was undertaken to: (1) monitor the outcome of IFN beta treatment in clinical practice; (2) compare the immunogenicity of the three commercial IFN beta preparations available; (3) assess the proportion of patients fulfilling the current guidelines of the Association of British Neurologists for stopping IFN beta therapy. RESULTS: During a median treatment period of 26 months (range 2-85), the relapse rate decreased by 41%. Although the reduction in the relapse rate was similar for all three commercial products, none of the Avonex treated patients were relapse free, compared with 19% of the Betaferon treated and 27% of the Rebif treated patients (p=0.02). Neutralising antibodies were not detected in Avonex treated patients (0 of 18), compared with 12 of 32 (38%) Betaferon treated and 10 of 23 (44%) Rebif treated patients (p=0.02). Forty of 101 (40%) patients satisfied the current (2001) Association of British Neurologists criteria for stopping IFN beta treatment at some stage during their treatment. CONCLUSION: IFN beta is effective in reducing the relapse rate in patients with relapsing-remitting MS in routine clinical practice. However, after a median treatment duration of 26 months, 40% of initially relapsing-remitting MS patients seem to have ongoing disease activity, presenting as disabling relapses or insidious progression.

A nationwide survey of self-reported red light running: measuring prevalence, predictors, and perceived consequences.

A United States probability sample of 880 licensed drivers participated in a telephone survey of red light running perceptions and behaviors. Despite most drivers believing red light running was problematic and dangerous, approximately one in five respondents reported running one or more red lights when entering the last ten signalized intersections. Among several demographic and attitude variables, only age group predicted recent red light running. Specifically, younger respondents were more likely to be violators. Drivers also reported being more likely to run red lights when alone, and were typically in a hurry when speeding up to be beat red lights. Contrary to expectations, frustration was not as important for predicting red light running as it was for other driving behaviors, such as speeding, tailgating, weaving, and gesturing angrily at others. Additionally, drivers perceived and received few consequences for running red lights. Less than 6% had received a traffic ticket for red light running and most believed that police would catch less than 20% of violators. Slightly more than one in ten had been involved in a red light running crash. Respondents most commonly suggested legal initiatives to reduce red light running. Accordingly, we recommend traffic safety experts pursue interventions that apply immediate and consistent negative consequences to violators to change the public's red light running perceptions and behavior.

Distinct structures and functions of related pre- and postsynaptic carbohydrates at the mammalian neuromuscular junction.

Carbohydrates that terminate in beta-linked N-acetylgalactosamine (betaGalNAc) residues are concentrated in the postsynaptic apparatus of the skeletal neuromuscular junction and have been implicated in the differentiation of the postsynaptic membrane. We now report that distinct synapse-specific betaGalNAc-containing carbohydrates are associated with motor nerve terminals. Two monoclonal antibodies that recognize distinct betaGalNAc-containing epitopes, CT1 and CT2, both stain synaptic sites on skeletal muscle fibers. However, CT1 selectively stains nerve terminal, whereas CT2 selectively stains the postsynaptic apparatus. Likewise, CT1 and CT2 selectively stain motoneuron-like and muscle cell lines, respectively. Using the cell lines, we identify distinct CT1- and CT2-reactive glycolipids and glycoproteins. Finally, we show that GalNAc modulates the adhesion of motoneuron-like cells to recombinant fragments of a synaptic cleft component, laminin beta2. Together, these results show that pre- as well as postsynaptic membranes bear and are affected by distinct but related synapse-specific carbohydrates.

Distinct adhesive properties of ciliary and choroid neurons from the avian ciliary ganglion.

The avian ciliary ganglion (CG) contains two populations of neurons: ciliary neurons, which innervate striated muscle, and choroid neurons, which innervate vascular smooth muscle. We used cell size (ciliary cells are larger) and somatostatin immunoreactivity (which is restricted to choroid cells) as markers to compare the adhesive properties of these two neuronal types. Similar numbers of freshly dissociated embryonic chick ciliary and choroid neurons adhered to laminin (laminin 1) and polylysine, consistent with the fact that each population comprises about half of the ganglionic neurons. In contrast, severalfold more ciliary neurons than choroid neurons adhered to a recombinant fragment of a synapsespecific basal lamina protein, s-laminin/laminin beta 2. Moreover, severalfold more ciliary neurons than choroid neurons adhered to a plastic surface when assayed by the method of Needels et al. in serum-free medium. Adhesion to s-laminin and plastic appears to be mediated by different cell surface components, as adhesion to recombinant s-laminin is inhibited by the tripeptide, LRE, and by Ca2+ ions, but not by heparin, whereas adhesion to plastic is LRE and Ca2+ insensitive but heparin sensitive. Both adhesive differences are apparent at embryonic day 8, soon after the ciliary and choroid neurons have begun to form synapses. Thus, two sets of neurons in the CG that send axons through different nerves and innervate different targets also show distinct adhesive behaviors.

A motoneuron-selective stop signal in the synaptic protein S-laminin.

Motor axons preferentially reinnervate original synaptic sites on denervated muscle fibers. We have shown that components of synaptic basal lamina direct this selectivity, and we identified a protein, s-laminin, that is concentrated in synaptic basal lamina. Here, we report that a recombinant s-laminin fragment inhibits neurite outgrowth promoted by laminin. A tripeptide sequence in this fragment, Leu-Arg-Glu (LRE), contributes to this inhibition and is itself sufficient to inhibit outgrowth. LRE-mediated inhibition is selective for motoneuron-like cells and is observed in mixtures with several, but not all, outgrowth-promoting substrates. Growth cones extending on laminin stop for up to several hours upon contacting deposits of the s-laminin fragment. Thus, LRE may serve as a cell type-selective and context-dependent target-derived signal that plays a role in synapse formation.

Gated migration: neurons migrate on but not onto substrates containing S-laminin.

Components of the extracellular matrix influence migration of diverse cell types. Some, such as laminin, promote neuronal migration, whereas others are nonpermissive or inhibitory. Here, we demonstrate that a recombinant fragment of s-laminin, a homologue of the laminin B1 chain, is a barrier to neuronal migration. NSC-34 (motoneuron-like) and ciliary ganglion cells were plated on substrates coated with alternating stripes of laminin and a mixture of laminin plus s-laminin. On these patterned substrates, cells seldom crossed from s-laminin-free to s-laminin-containing regions. Mutation of the tripeptide LRE, an adhesive site in s-laminin, abolished s-laminin's ability to block border crossing. However, overall rates of migration were similar on the two substrates. This behavior contrasts with that of previously reported barrier molecules, which decreases rates of cell migration when mixed with permissive substrates. Instead, s-laminin appears to block cell migration through a "gating" mechanism that acts primarily at borders.

S-laminin: mapping to mouse chromosome 9 and expression in the linked mutants tippy and ducky.

S-Laminin, a homologue of the laminin B1 chain, is present in a subset of basal laminae, including those of the skeletal neuromuscular junction and the renal glomerulus. Here, we show that the distribution and apparent size of murine S-laminin are similar to those documented previously for rat and human. We then use interspecific backcross analysis to map the S-laminin (Lams) gene to mouse chromosome 9. Thus, it is unlinked to genes for the laminin A, B1, and B2 chains. Finally, because the Lams gene mapped near two mutations that affect neuromuscular function, ducky (du) and tippy (tip), we assayed S-laminin by Southern blotting, immunoblotting, and immunohistochemistry in these mutants. No abnormality of the S-laminin gene or protein was detectable in either mutant.

Renal artery bullet embolism.

Bullet embolism is a well-recognized phenomenon. While bullet embolism is not common, renal artery bullet embolism appears to be very rare. We report three cases of bullet embolism to a renal artery. Autopsy findings and bullet types are presented. Such cases are of particular interest to both the medical examiner and the clinician. Failure to consider this phenomenon clinically could lead to infarction of a kidney. A consideration of this phenomenon at autopsy could shorten the dissection time required to retrieve the bullet.

Primary sequence of a motor neuron-selective adhesive site in the synaptic basal lamina protein S-laminin.

S-laminin, a novel homolog of laminin, is concentrated in a subset of basal laminae including the basal lamina that passes between motor nerve terminals and muscle fibers at the neuromuscular junction. Here we used recombinant fragments to localize a neuronal attachment site to the C-terminal 10% of s-laminin. We then used synthetic peptides spanning the active fragment to identify the primary sequence of the adhesive site as Leu-Arg-Glu (LRE): neurons attach to an immobilized LRE-containing peptide, and soluble LRE blocks attachment of neurons to the s-laminin fragment. Whereas ciliary ganglion neurons (which normally innervate muscle fibers) adhered well both to laminin and to an s-laminin fragment, sensory and central neurons and several neuronal cell lines all adhered well to laminin but poorly to the s-laminin fragment. Together, these results define a motor neuron-selective attachment site on s-laminin.

Restoration of cell volume and the reversal of carbohydrate transport and growth inhibition of osmotically upshocked Escherichia coli.

Resumption of growth in osmotically upshocked Escherichia coli was effected only by an external stimulus (betaine treatment) in severe upshock, but was spontaneous in less severe upshock. In either case, growth resumption was preceded by a reversal of glucose transport inhibition, and that reversal was preceded by a recovery of cell volume. We hypothesize that deformation of the membrane by osmotic stress results in conversion of a membrane component of the transport system to a less functional conformation, which results in the inhibition of transport and the consequent inhibition of growth. Relief of the deformation would then allow recovery to a more functional conformation, reversal of transport inhibition, and then resumption of growth.