Proteomic Analysis After Status Epilepticus Identifies UCHL1 as Protective Against Hippocampal Injury.

Brief, non-harmful seizures (preconditioning) can temporarily protect the brain against prolonged, otherwise injurious seizures. Following focal-onset status epilepticus (SE) in preconditioned (tolerance) and sham-preconditioned (injury) mice, we screened for protein changes using a proteomic approach and identified several putative candidates of epileptic tolerance. Among SE-induced changes to both proteomic screens, proteins clustered in key regulatory pathways, including protein trafficking and cytoskeletal regulation. Downregulation of one such protein, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), was unique to injury and not evident in tolerance. UCHL1 inhibition decreased hippocampal ubiquitin, disrupted UPS function, interfered with seizure termination and exacerbated seizure-induced cell death. Though UCHL1 transcription was maintained after SE, we observed downregulation of the pro-translational antisense Uchl1 (AsUchl1) and confirmed that both AsUchl1 and rapamycin can increase UCHL1 expression in vivo. These data indicate that the post-transcriptional loss of UCHL1 following SE is deleterious to neuronal survival and may contribute to hyperexcitability, and are suggestive of a novel modality of rapamycin therapy.

Transcriptional Response of Polycomb Group Genes to Status Epilepticus in Mice is Modified by Prior Exposure to Epileptic Preconditioning.

Exposure of the brain to brief, non-harmful seizures can activate protective mechanisms that temporarily generate a damage-refractory state. This process, termed epileptic tolerance, is associated with large-scale down-regulation of gene expression. Polycomb group (PcG) proteins are master controllers of gene silencing during development that are re-activated by injury to the brain. Here, we explored the transcriptional response of genes associated with polycomb repressive complex (PRC) 1 (Ring1A, Ring1B, and Bmi1) and PRC2 (Ezh1, Ezh2, and Suz12), as well as additional transcriptional regulators Sirt1, Yy1, and Yy2, in a mouse model of status epilepticus (SE). Findings were contrasted to changes after SE in mice previously given brief seizures to evoke tolerance. Real-time quantitative PCR showed SE prompted an early (1 h) increase in expression of several genes in PRC1 and PRC2 in the hippocampus, followed by down-regulation of many of the same genes at later times points (4, 8, and 24 h). Spatio-temporal differences were found among PRC2 genes in epileptic tolerance, including increased expression of Ezh2, Suz12, and Yy2 relative to the normal injury response to SE. In contrast, PRC1 complex genes including Ring 1B and Bmi1 displayed differential down-regulation in epileptic tolerance. The present study characterizes PcG gene expression following SE and shows prior seizure exposure produces select changes to PRC1 and PRC2 composition that may influence differential gene expression in epileptic tolerance.

Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy.

Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain.

Investigating gene promoter methylation in a mouse model of status epilepticus.

Epigenetic modification of DNA by methylation of the cytosine present in CG dinucleotides constitutes a key regulatory mechanism in the control of gene expression in neurological diseases. In this chapter, we describe an in-depth methodology of methylated DNA immunoprecipitation used in combination with tiling microarrays (MeDIP-chip) in order to analyze genome-wide gene promoter methylation in the hippocampus of mice following status epilepticus (prolonged seizure). While a specific mouse model and array format are described, the method can be applied to DNA from many tissues to analyze the methylation status of promoter regions across whole genomes, using a wide range of available array formats (both custom designed and commercially catalogued). We conclude the chapter with the description of bisulfite sequencing validation of MeDIP-chip results.

Spatio-temporally restricted blood-brain barrier disruption after intra-amygdala kainic acid-induced status epilepticus in mice.

Mesial temporal lobe epilepsy (MTLE) is the most common, intractable seizure disorder in adults. Blood-brain barrier (BBB) disruption, including interruption of endothelial tight cell junctions and serum protein and immunoglobulin G (IgG) extravasation into brain parenchyma, has been reported in experimental and human MTLE and implicated in disease pathogenesis. Triggering status epilepticus in mice by intra-amygdala microinjection of kainic acid produces damage mainly within the CA3 subfield of the ipsilateral hippocampus, and recurrent spontaneous seizures emerge during the following week. To investigate whether BBB impairment is a feature of this model, we characterized endothelial tight cell junction proteins and IgG and albumin in the hippocampus up to three weeks after status epilepticus. Hippocampal microvessels displayed a reduction in continuous staining for zonula occludens 1 (ZO-1), the main tight junction protein, after status epilepticus and in epileptic mice, although western blotting found ZO-1 protein levels in the hippocampal subfields were not different from controls at any time. Increased IgG and albumin were detected in damaged and non-damaged ipsilateral hippocampal subfields, mainly 4-24h after status epilepticus, although increased serum protein extravasation was also found in the CA3 subfield in epileptic mice. Thus, BBB opening or damage occurs mainly in the period shortly after status epilepticus but may also persist within the CA3 subfield as a feature of the pathophysiology of chronic epilepsy in this model.

Differential DNA methylation patterns define status epilepticus and epileptic tolerance.

Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.

Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis.

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 µM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 µM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 µM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.

Critical role for Syk in responses to vascular injury.

Although current antiplatelet therapies provide potent antithrombotic effects, their efficacy is limited by a heightened risk of bleeding and failure to affect vascular remodeling after injury. New lines of research suggest that thrombosis and hemorrhage may be uncoupled at the interface of pathways controlling thrombosis and inflammation. Here, as one remarkable example, studies using a novel and highly selective pharmacologic inhibitor of the spleen tyrosine kinase Syk [PRT060318; 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide] coupled with genetic experiments, demonstrate that Syk inhibition ameliorates both the acute and chronic responses to vascular injury without affecting hemostasis. Specifically, lack of Syk (murine radiation chimeras) attenuated shear-induced thrombus formation ex vivo, and PRT060318 strongly inhibited arterial thrombosis in vivo in multiple animal species while having minimal impact on bleeding. Furthermore, leukocyte-platelet-dependent responses to vascular injury, including inflammatory cell recruitment and neointima formation, were markedly inhibited by PRT060318. Thus, Syk controls acute and long-term responses to arterial vascular injury. The therapeutic potential of Syk may be exemplary of a new class of antiatherothrombotic agents that target the interface between thrombosis and inflammation.

Plxdc2 is a mitogen for neural progenitors.

The development of different brain regions involves the coordinated control of proliferation and cell fate specification along and across the neuraxis. Here, we identify Plxdc2 as a novel regulator of these processes, using in ovo electroporation and in vitro cultures of mammalian cells. Plxdc2 is a type I transmembrane protein with some homology to nidogen and to plexins. It is expressed in a highly discrete and dynamic pattern in the developing nervous system, with prominent expression in various patterning centres. In the chick neural tube, where Plxdc2 expression parallels that seen in the mouse, misexpression of Plxdc2 increases proliferation and alters patterns of neurogenesis, resulting in neural tube thickening at early stages. Expression of the Plxdc2 extracellular domain alone, which can be cleaved and shed in vivo, is sufficient for this activity, demonstrating a cell non-autonomous function. Induction of proliferation is also observed in cultured embryonic neuroepithelial cells (ENCs) derived from E9.5 mouse neural tube, which express a Plxdc2-binding activity. These experiments uncover a direct molecular activity of Plxdc2 in the control of proliferation, of relevance in understanding the role of this protein in various cancers, where its expression has been shown to be altered. They also implicate Plxdc2 as a novel component of the network of signalling molecules known to coordinate proliferation and differentiation in the developing nervous system.

Measured visual field extent varies with peripheral stimulus flicker rate in very young children.

PURPOSE: The purpose of this article is to describe measured visual field extent in very young children in response to variation in peripheral stimulus flicker rate. METHODS: Binocular visual field extent was measured using a black, double-arc perimeter and an LED static perimetry procedure in 120 11-month-old, 120 17-month-old, and 120 30-month-old children and 40 adults. Each subject was tested with one of four flicker rates: 1 Hz, 10 Hz, 20 Hz, or 40 Hz. An interpolated estimate of the eccentricity at which 50% of subjects detected the peripheral stimulus and the mean of the farthest eccentricity at which subjects detected the peripheral stimulus were calculated for each flicker rate for each age group. RESULTS: In 11-, 17-, and 30-month-old children, but not in adults, measured visual field extent (eccentricity at which the stimulus was detected) varied significantly with rate of stimulus flicker. The largest measured visual field extent was produced by a 10-Hz stimulus and the smallest was produced by 1-Hz and 40-Hz stimuli. Measured visual field extent in children was similar to that of adults for 10-Hz flicker, but smaller than that of adults for 1-Hz, 20-Hz, and 40-Hz flicker. CONCLUSIONS: These results underscore the importance of standardizing stimulus parameters when developing tests for clinical assessment of visual fields in children. Furthermore, for longitudinal assessment of young patients, use of a 10-Hz flicker rate, in combination with the other parameters used in the present study, would help to avoid difficulties in interpretation that could arise from an interaction between age-related and disease-related changes that might occur if other stimulus flicker rates were used.

The effect of flicker rate on nasal and temporal measured visual field extent in infants.

PURPOSE: To explore the effect of peripheral stimulus flicker rate on measured visual field extent (MVFE) in young infants. METHODS: Three hundred sixty infants (180 each at 3.5 and 7 months of age) were tested monocularly with a light-emitting diode static perimetry procedure using a double-arc perimeter with arms at 45 degrees , 135 degrees , 225 degrees , and 315 degrees . Each subject was tested with one of six flicker conditions: no flicker, 1 Hz, 3 Hz, 10 Hz, 20 Hz, and 40 Hz. An interpolated estimate of the location at which 50% of subjects detected the peripheral stimulus (corrected for spontaneous eye movements) and the mean location of the farthest spot seen were calculated across subjects for each perimeter arm for each flicker condition for each age group. RESULTS: Nasally, MVFE was larger for 7-month-old than for 3.5-month-old infants. Across both ages, infants showed larger MVFE for 10-Hz stimuli than for nonflickering stimuli, but MVFE did not differ between any other flicker conditions. Temporally, response to flicker varied with age. For 3.5-month-old infants, MVFE was smaller for the no flicker condition than for the 3-Hz, 10-Hz, and 20-Hz conditions, but there were no other differences across flicker conditions. For 7-month-old infants, MVFE was larger for 3-Hz stimuli than for the no flicker, 1-Hz, 20-Hz, and 40-Hz conditions, but there were no other differences across flicker conditions. Additional analyses showed that the effect of flicker rate on the percentage of subjects looking at a peripheral stimulus at a single eccentricity (29 degrees ) was similar to the effect of stimulus flicker across eccentricities, as reflected in MVFE. CONCLUSIONS: Peripheral stimulus flicker can enhance the MVFE in 3.5- and 7-month-old infants. However, the effect depends on flicker rate and is consistent with previous data indicating that 10 Hz and perhaps 3 Hz are especially effective in enhancing MVFE in older infants and young children.

Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis.

BACKGROUND: This study was designed to determine whether (1) P2Y12 antagonism synergizes with other antithrombotics and (2) anticoagulants (thrombin inhibitors) affect the antithrombotic activity elicited by P2Y12 antagonism. METHODS AND RESULTS: Thrombosis was achieved by perfusion of human and murine blood through type III collagen-coated capillaries at arterial shear rate. CT50547, a direct-acting P2Y12 antagonist, inhibited thrombosis in PPACK- but not heparin-anticoagulated human blood. In contrast, CT50547 inhibited thrombosis in aspirin-treated individuals independently of the anticoagulant. Thrombin and TXA2 also synergized with P2Y12 in the absence of anticoagulation, because combined treatment of aspirin or C921-78 (a factor Xa inhibitor) with CT50547 or 2-MeSAMP (a P2Y12 antagonist) inhibited the thrombotic process, whereas all treatments failed to inhibit thrombosis when used individually. Synergism was also observed ex vivo when P2Y12-deficient (P2Y12-/-) mice were administered aspirin or coagulation inhibitors (C921-78 and bivalirudin). Finally, using intravital microscopy, we found that both C921-78 and bivalirudin abrogated the thrombotic process in P2Y12+/- mice, whereas each showed only partial efficacy in P2Y12+/+ animals. CONCLUSIONS: Our study indicates that (1) thrombin inhibitors and aspirin have a demonstrable synergy of antithrombotic activity with P2Y12 antagonism and (2) the in vitro analysis of the antithrombotic activity of P2Y12 antagonists is affected by the anticoagulant used for blood collection. This suggests that the antithrombotic potential of P2Y12 antagonists in vitro may be overestimated in anticoagulated samples of blood and best achieved in vivo by the inclusion of aspirin and/or a thrombin inhibitor.

The influence of stimulus size on measured visual field extent in infants.

PURPOSE: To compare measured visual field extent for a 6 degrees stimulus (typical size used in studies of infants) with a 1.5 degrees stimulus (similar to the largest size used in Goldmann perimetry) in young infants. METHODS: A total of 120 infants (60 each at 3.5 months and 7 months of age) and 24 adults were tested monocularly with a kinetic perimetry procedure using a black double-arc perimeter. Each subject was tested with either a 6 degrees or 1.5 degrees white sphere, which was mounted on a black wand and moved smoothly toward the intersection of the perimeter arms at 3.4 degrees /s. Visual field extent along each perimeter arm was defined as the median of 2 to 3 measurements of the position of the leading edge of the stimulus when the subject made an eye movement toward the stimulus. RESULTS: The 6 degrees stimulus produced larger measured visual field extent than the 1.5 degrees stimulus in 3.5-month olds (temporal field only) and in 7-month olds (nasal and temporal field), but not in adults. CONCLUSIONS: Using the testing conditions of the present study, increasing stimulus size beyond the largest used in a Goldmann perimeter (approximately 2 degrees) increases measured visual field extent in young infants, but not in adults. This may relate to differences in peripheral summation areas or to differences in attentional factors between infants and adults.

Scientific and therapeutic insights into the role of the platelet P2Y12 receptor in thrombosis.

Platelets are important mediators of thrombosis in both healthy and diseased vessels. When platelets become activated by various soluble agonists or by adhesion to subendothelium under high shear, they release adenosine-5'-diphosphate that acts in a positive feedback mechanism on two different G-protein coupled receptors (P2Y(12), P2Y(1)) on platelets. This released adenosine-5'-diphosphate, acting through P2Y(12), is critical for sustained aggregation and stabilization of thrombi. P2Y(12) is the target of antithrombotic drugs (ticlopidine, clopidogrel), whereas the role of P2Y(1) in thrombosis remains to be fully established. Recent studies using either inhibitors of key components of signaling pathways or genetically engineered mice have contributed to our understanding of the signaling mechanisms in platelets mediated by adenosine-5'-diphosphate through the P2Y(12) receptor. Studies of patients with defective adenosine-5'-diphosphate mediated aggregation, as well as P2Y(12)-null mice, have revealed the importance of this receptor in mediating platelet activation and aggregation. Recent clinical trials using approved P2Y(12) blockers have extended the use of these drugs to additional patient populations. Recent data demonstrating the role of P2Y(12) in mediating platelet adhesion to thrombogenic surfaces (collagen, von Willebrand factor) provide further rationale as to the clinical efficacy of P2Y(12) blockers. P2Y(12) antagonists in combination with anticoagulants (thrombin inhibitors, factor Xa inhibitors) act synergistically in inhibiting thrombus formation (similar to aspirin) ex vivo. These findings suggest the potential for combination therapies (P2Y(12) antagonists with inhibitors of GPIIb-IIIa, thrombin or Factor Xa, etc.) to provide additional clinical benefit to patients with various cardiovascular diseases, especially those who may be aspirin-resistant.

P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40-50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12-/- mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12-/- platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12-/- mice on collagen were less dense and less activated than their WT counterparts. P2Y12-/- platelet activation was also reduced in response to ADP or a PAR-4-activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.