The CBP KIX domain regulates long-term memory and circadian activity.

BACKGROUND: CREB-dependent transcription necessary for long-term memory is driven by interactions with CREB-binding protein (CBP), a multi-domain protein that binds numerous transcription factors potentially affecting expression of thousands of genes. Identifying specific domain functions for multi-domain proteins is essential to understand processes such as cognitive function and circadian clocks. We investigated the function of the CBP KIX domain in hippocampal memory and gene expression using CBPKIX/KIX mice with mutations that prevent phospho-CREB (Ser133) binding. RESULTS: We found that CBPKIX/KIX mice were impaired in long-term memory, but not learning acquisition or short-term memory for the Morris water maze. Using an unbiased analysis of gene expression in the dorsal hippocampus after training in the Morris water maze or contextual fear conditioning, we discovered dysregulation of CREB, CLOCK, and BMAL1 target genes and downregulation of circadian genes in CBPKIX/KIX mice. Given our finding that the CBP KIX domain was important for transcription of circadian genes, we profiled circadian activity and phase resetting in CBPKIX/KIX mice. CBPKIX/KIX mice exhibited delayed activity peaks after light offset and longer free-running periods in constant dark. Interestingly, CBPKIX/KIX mice displayed phase delays and advances in response to photic stimulation comparable to wildtype littermates. Thus, this work delineates site-specific regulation of the circadian clock by a multi-domain protein. CONCLUSIONS: These studies provide insight into the significance of the CBP KIX domain by defining targets of CBP transcriptional co-activation in memory and the role of the CBP KIX domain in vivo on circadian rhythms.

Transcriptional corepressor SIN3A regulates hippocampal synaptic plasticity via Homer1/mGluR5 signaling.

Long-term memory depends on the control of activity-dependent neuronal gene expression, which is regulated by epigenetic modifications. The epigenetic modification of histones is orchestrated by the opposing activities of 2 classes of regulatory complexes: permissive coactivators and silencing corepressors. Much work has focused on coactivator complexes, but little is known about the corepressor complexes that suppress the expression of plasticity-related genes. Here, we define a critical role for the corepressor SIN3A in memory and synaptic plasticity, showing that postnatal neuronal deletion of Sin3a enhances hippocampal long-term potentiation and long-term contextual fear memory. SIN3A regulates the expression of genes encoding proteins in the postsynaptic density. Loss of SIN3A increases expression of the synaptic scaffold Homer1, alters the metabotropic glutamate receptor 1α (mGluR1α) and mGluR5 dependence of long-term potentiation, and increases activation of ERK in the hippocampus after learning. Our studies define a critical role for corepressors in modulating neural plasticity and memory consolidation and reveal that Homer1/mGluR signaling pathways may be central molecular mechanisms for memory enhancement.

Outcome Trajectories after Primary Perinatal Hemorrhagic Stroke.

BACKGROUND: Perinatal hemorrhagic stroke in late preterm and term neonates is understudied. We describe two-month and two-year neurological outcomes in a prospective cohort. METHODS: Neonates ≥36 weeks' gestation with spontaneous hemorrhagic stroke (parenchymal and intraventricular) presenting at age ≤28 days were enrolled between March 2007 and May 2015 at three tertiary pediatric centers. Hemorrhagic transformation of arterial ischemic stroke or cerebral sinovenous thrombosis was excluded. The Pediatric Stroke Outcome Measure (PSOM) assessed outcomes. Wilcoxon signed-rank tests evaluated change over time. RESULTS: Twenty-six neonates were included (median age: 1 day, interquartile range [IQR] 0 to 16; median gestational age: 38.3 weeks, IQR 37.0 to 39.0). Hemorrhage was isolated intraventricular in seven (27%), isolated intraparenchymal in six (23%), and a combination in 10 (39%). Three neonates (12%) died during hospitalization; one died later due to cardiac disease. Among 22 survivors, outcomes were assessed at a median of 2.1 months (IQR 1.7 to 3.3) in 96% and 1.9 years (IQR 1.3 to 2.0) in 73%. Median PSOM scores were 0.0 (IQR 0.0 to 1.0) and 0.25 (IQR 0.0 to 1.3), respectively. At two years, 45% of patients had no or nonimpairing deficits (PSOM <1.0), 30% had mild deficits (PSOM 1.0 to 2.0), and 5% had moderate deficits (PSOM 2.5 to 4.5). Over time, 31% worsened and 6% improved. Although total PSOM scores did not change significantly (P = 0.08), language subscores worsened (P = 0.009). No child developed epilepsy. CONCLUSIONS: Perinatal hemorrhagic stroke survivors had favorable outcomes in early childhood; at two years moderate to severe deficits occurred in 5%. Language deficits may emerge over time, warranting close follow-up.

Neurologic Outcome Predictors in Pediatric Intracerebral Hemorrhage: A Prospective Study.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage is a considerable source of morbidity and mortality. This 3-center study describes outcomes of pediatric intracerebral hemorrhage and identifies 2-year neurological outcome predictors. METHODS: Children 29 days to 18 years of age presenting with intracerebral hemorrhage from March 2007 to May 2015 were enrolled prospectively. Exclusion criteria included trauma; intracranial tumor; hemorrhagic transformation of arterial ischemic stroke or cerebral sinovenous thrombosis; isolated subdural, epidural, or subarachnoid hemorrhage; and abnormal baseline neurological function. Intracerebral hemorrhage and total brain volumes were measured on neuroimaging. The Pediatric Stroke Outcome Measure assessed outcomes. RESULTS: Sixty-nine children were included (median age: 9.7 years; interquartile range: 2.2-14). Six children (9%) died during hospitalization. Outcomes in survivors were assessed at early follow-up in 98% (median 3.1 months; interquartile range: 3.1-3.8) and at later follow-up in 94% (median: 2.1 years; interquartile range: 1.3-2.8). Over a third had a significant disability at 2 years (Pediatric Stroke Outcome Measure >2). Total Pediatric Stroke Outcome Measure score improved over time (P=0.0003), paralleling improvements in the sensorimotor subscore (P=0.0004). Altered mental status (odds ratio, 13; 95% confidence interval, 3.9-46; P<0.001), hemorrhage volume ≥4% of total brain volume (odds ratio, 17; 95% confidence interval, 1.9-156; P=0.01), and intensive care unit length of stay (odds ratio, 1.1; 95% confidence interval, 1.0-1.2; P=0.002) were significantly associated with poor 2-year outcome. CONCLUSIONS: Over one third of children experienced significant disability at 2 years. Improvements in outcomes were driven by recovery of sensorimotor function. Altered mental status, hemorrhage volume ≥4% of total brain volume, and intensive care unit length of stay were independent predictors of significant disability at 2 years.

Efficacy of artisanal preparations of cannabidiol for the treatment of epilepsy: Practical experiences in a tertiary medical center.

Medically refractory epilepsy continues to be a challenge worldwide, and despite an increasing number of medical therapies, approximately 1 in 3 patients continues to have seizures. Cannabidiol (CBD), one of many constituents of the Cannabis sativa or marijuana plant, has received renewed interest in the treatment of epilepsy. While highly purified CBD awaits Food and Drug Administration (FDA) approval, artisanal formulations of CBD are readily available and are seeing increased use in our patient population. Although randomized controlled trials of CBD are ongoing and promising, data regarding artisanal formulations of CBD are minimal and largely anecdotal. Here, we report a retrospective study to define the efficacy of artisanal CBD preparations in children with epilepsy. Given the known interaction between CBD and clobazam, we also conducted a subgroup comparison to determine if clobazam use was related to any beneficial effects of CBD. Additionally, we compared response rates with CBD and with clobazam alone within an overlapping patient cohort. A pediatric cohort with epilepsy of 108 patients was identified through a medical record search for patients using CBD oil. The addition of CBD resulted in 39% of patients having a >50% reduction in seizures, with 10% becoming seizure-free. The responder rate for clobazam was similar. No patients achieved CBD monotherapy, although the weaning of other antiepileptic drugs (AEDs) became possible in 22% of patients. A comparable proportion had AED additions during CBD therapy. With concomitant use of clobazam, 44% of patients had a 50% reduction in seizures upon addition of CBD compared with 33% in the population not taking clobazam; this difference was not statistically significant. The most common reported side effect of CBD was sedation in less than 4% of patients, all of whom were also taking clobazam. Increased alertness and improved verbal interactions were reported in 14% of patients in the CBD group and 8% of patients in the CBD and clobazam group. Benefits were more marked in the CBD alone group, in contrast to the CBD and clobazam group, but this difference was not statistically significant. In summary, these findings support efficacy of artisanal CBD preparations in seizure reduction with few significant side effects. The response to CBD was independent of concurrent clobazam use, although clobazam may contribute to the sedation seen with concurrent CBD use.

Learning-dependent chromatin remodeling highlights noncoding regulatory regions linked to autism.

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder that is associated with genetic risk factors. Most human disease-associated single-nucleotide polymorphisms (SNPs) are not located in genes but rather are in regulatory regions that control gene expression. The function of regulatory regions is determined through epigenetic mechanisms. Parallels between the cellular basis of development and the formation of long-term memory have long been recognized, particularly the role of epigenetic mechanisms in both processes. We analyzed how learning alters chromatin accessibility in the mouse hippocampus using a new high-throughput sequencing bioinformatics strategy we call DEScan (differential enrichment scan). DEScan, which enabled the analysis of data from epigenomic experiments containing multiple replicates, revealed changes in chromatin accessibility at 2365 regulatory regions-most of which were promoters. Learning-regulated promoters were active during forebrain development in mice and were enriched in epigenetic modifications indicative of bivalent promoters. These promoters were disproportionally intronic, showed a complex relationship with gene expression and alternative splicing during memory consolidation and retrieval, and were enriched in the data set relative to known ASD risk genes. Genotyping in a clinical cohort within one of these promoters (SHANK3 promoter 6) revealed that the SNP rs6010065 was associated with ASD. Our data support the idea that learning recapitulates development at the epigenetic level and demonstrate that behaviorally induced epigenetic changes in mice can highlight regulatory regions relevant to brain disorders in patients.

Contextual fear conditioning induces differential alternative splicing.

The process of memory consolidation requires transcription and translation to form long-term memories. Significant effort has been dedicated to understanding changes in hippocampal gene expression after contextual fear conditioning. However, alternative splicing by differential transcript regulation during this time period has received less attention. Here, we use RNA-seq to determine exon-level changes in expression after contextual fear conditioning and retrieval. Our work reveals that a short variant of Homer1, Ania-3, is regulated by contextual fear conditioning. The ribosome biogenesis regulator Las1l, small nucleolar RNA Snord14e, and the RNA-binding protein Rbm3 also change specific transcript usage after fear conditioning. The changes in Ania-3 and Las1l are specific to either the new context or the context-shock association, while the changes in Rbm3 occur after context or shock only. Our analysis revealed novel transcript regulation of previously undetected changes after learning, revealing the importance of high throughput sequencing approaches in the study of gene expression changes after learning.