Epigenetic Determinants of Altered Gene Expression in Schizophrenia: a Dissertation

Schizophrenia is a neurodevelopmental disorder affecting 1% of the general population. Dysfunction of the prefrontal cortex (PFC) is associated with the etiology of schizophrenia. Moreover, a substantial deficit of GAD1mRNA in schizophrenic PFC has been reported by different groups. However, the underlying molecular mechanisms are still unclear. Interestingly, epigenetic factors such as histone modifications and DNA methylation could be involved in the pathogenesis of schizophrenia during the maturation of the PFC. In my work, I identified potential epigenetic changes in schizophrenic PFC and developmental changes of epigenetic marks in normal human PFC. Furthermore, mouse and neuronal precursor cell models were used to confirm and investigate the molecular mechanisms of the epigenetic changes in human PFC. My initial work examined whether chromatin immunoprecipitation can be applied to human postmortem brain. I used micrococcal nuclease (MNase)-digested chromatin instead of cross-linked and sonicated chromatin for further immunoprecipitation with specific anti-methyl histone antibodies. Surprisingly, the integrity of mono-nucleosomes was still maintained at least 30 hrs after death. Moreover, differences of histone methylation at different genomic loci were detectable and were preserved within a wide range of autolysis times and tissue pH values. Interestingly, MNase-treated chromatin is more efficient for subsequent immunoprecipitation than crosslinked and sonicated chromatin. During the second part of my dissertation work, I profiled histone methylation at GABAergic gene loci during human prefrontal development. Moreover, a microarray analysis was used to screen which histone methyltransferase (HMT) could be involved in histone methylation during human prefrontal development. Mixed-lineage leukemia 1 (MLL1), an HMT for methylation at histone H3 lysine 4 (H3K4), appears to be the best candidate after interpreting microarray results. Indeed, decreased methylation of histone H3 lysine 4 at a subset of GABAergic gene loci occurred in Mll1 mutant mice. Interestingly, clozapine, but not haloperidol, increased levels of trimethyl H3K4 (H3K4me3) and Mll1 occupancy at the GAD1 promoter. I profiled histone methylation and gene expression for GAD1 in schizophrenics and their matched controls. Interestingly, there are deficits of GAD1 mRNA levels and GAD1 H3K4me3 in female schizophrenics. Furthermore, I was also interested in whether the changes of GAD1 chromatin structure could contribute to cortical pathology in schizophrenics with GAD1 SNPs. Remarkably, homozygous risk alleles for schizophrenia at the 5’ end of the GAD1 gene are associated with a deficit of GAD1 mRNA levels together with decreased GAD1 H3K4me3 and increased GAD1H3K27me3 in schizophrenics. Finally, I shifted focus on whether DNA methylation at the GAD1 promoter could contribute to a deficit of GAD1 mRNA in schizophrenia. However, no reproducible techniques are available for extracting genomic DNA specifically from GABAergic neurons in human brain. Therefore, I used an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of active and silenced gene expression. The methylation frequencies of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined from two chromatin fractions (H3K4me3 and H3K27me3) separately. Consistently, the proximal promoter region of GAD1 is more resistant to methylation in comparison to intron 2 of GAD1 in either open or repressive chromatin fractions. Interestingly, overall higher levels of DNA methylation were seen in repressive chromatin than in open chromatin. Surprisingly, schizophrenic subjects showed a significant decrease of DNA methylation at the GAD1proximal promoter from repressive chromatin. Taken together, my work has advanced our knowledge of epigenetic mechanisms in human prefrontal development and the potential link to the etiology of schizophrenia. It could eventually provide a new approach for the treatment of schizophrenia, especially in the regulation of methylation at histone H3 lysine 4

Isolation of neuronal chromatin from brain tissue

<p>Abstract</p> <p>Background</p> <p>DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. However, chromatin assays typically lack single cell resolution, and therefore little is known about chromatin regulation of differentiated neuronal nuclei that reside in brain parenchyma intermingled with various types of non-neuronal cells.</p> <p>Results</p> <p>Here, we describe a protocol to selectively tag neuronal nuclei from adult brain – either by (anti-NeuN) immunolabeling or transgene-derived histone H2B-GFP fusion protein – for subsequent fluorescence-activated sorting and chromatin immunoprecipitation (ChIP). To illustrate an example, we compared histone H3 lysine 4 and 9 methylation marks at select gene promoters in neuronal, non-neuronal and unsorted chromatin from mouse forebrain and human cerebral cortex, and provide evidence for neuron-specific histone methylation signatures.</p> <p>Conclusion</p> <p>With the modifications detailed in this protocol, the method can be used to collect nuclei from specific subtypes of neurons from any brain region for subsequent ChIP with native/un-fixed or crosslinked chromatin preparations. Starting with the harvest of brain tissue, ChIP-ready neuronal nuclei can be obtained within one day.</p

Exploring Public Library Practices for Promoting Family Engagement from the Perspective of Librarians

This study aims to explore the promotion of family engagement practices in public libraries from librarians’ perspectives. This is a qualitative study, involving 18 librarians working in township libraries in Taiwan. Data were gathered through interviews, organized and analyzed employing a thematic analysis approach. Research findings show that public librarians’ perception of family engagement predominantly focuses on family members’ company and parent-child shared reading. Libraries promote family engagement primarily through close attention to reading needs of minority groups, and active in-depth community-based resident services. In addition, libraries provide diverse reading activities based on the needs and interests of various families. At the same time, libraries take a proactive approach in partnership working to expand the impact of family engagement. Nevertheless, libraries have encountered obstruction in the process. Based on the findings, this study puts forward suggestions for research and practice regarding promotion of family engagement in public libraries. (Article content in Chinese with English extended abstract

Advances in understanding visual cortex plasticity

Visual cortical plasticity can be either rapid, occurring in response to abrupt changes in neural activity, or slow, occurring over days as a homeostatic process for adapting neuronal responsiveness. Recent advances have shown that the magnitude and polarity of rapid synaptic modifications are regulated by neuromodulators, while homeostatic modifications can occur through regulation of cytokine actions or NMDA receptor subunit composition. Synaptic and homeostatic plasticity together produce the normal physiological response to monocular impairments. In vivo studies have now overturned the dogma that robust plasticity is limited to an early critical period. Indeed, rapid physiological plasticity in the adult can be enabled by prior, experience-driven anatomical rearrangements or through pharmacological manipulations of the epigenome

‘福芳’甜椒嫁接植株之田間生育情形

In autumn crop, the graft success rate of 'Lucky Fragrance' sweet pepper were 100％, except grafted on 'M210101', 'Passion' and 194 rootstocks were 61.1％, 77％ and 77％, respectively. Flowering of these combinations were early then grafted on 'Miles Flavor', 'Passion' and 'Born Fire' rootstocks. The early fruit yield and total yield of plants grafted on 'Passion' and 'Born Fire' rootstockswere increased. The yields of plants grafted on 'Miles Flavor' and 'M210101' rootstocks were decreased. In spring crop, the field survival rate of 'Lucky Fragrance' sweet pepper grafted on 'M210101' and 'Born Fire' rootstocks were 88.9％ and 94.4％, respectively. Plants grafted on 'Miles Flavor' , 'Passion' and 'Born Fire' rootstocks enhanced anthesis. The plant height, stem node and fresh weight were increased by grafted on 'Passion' and '194' rootstocks. The plants had early fruit yield, fruit number and total yield in the combination of grafted on 'Passion' rootstock, but they were decreased when plants grafted on '31346','M210101' rootstocks and control.秋作以'福芳'接穗嫁接於M210101之成活率為61.1%,'百香'及'生生4號'砧者為77%，其他砧木者為100%。始花期以'百里香'、'百香'及'艷茹'砧顯著較早，其他地上部植株性狀於各穗砧組合無顯著差異。嫁接苗單株果實初期產量及總產量，以'百香'及'艷茹'砧較高，'百里香'及M2l0l0l玷之產童為最低。'福芳'春作嫁接於M210101砧之成活率為79.3%，定植30天之田間存活率除'艷茹'砧之94.4%及M210101砧之88.9%，其他組合皆為100%。始花期以'百里香'、'百香''及'艷茹'砧 顯著較早，嫁接植林之株高、節數及地上部鮮乾重以'百香'及'生生4號'砧顯 著最佳，31346砧及共砧之生育較差。'百香'砧嫁接苗單株初期產量，總產量果數為最高，31346、M210101砧及'福芳'對照植株為顯著最低

Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters

Alterations in GABAergic mRNA expression play a key role for prefrontal dysfunction in schizophrenia and other neurodevelopmental disease. Here, we show that histone H3-lysine 4 methylation, a chromatin mark associated with the transcriptional process, progressively increased at GAD1 and other GABAergic gene promoters (GAD2, NPY, SST) in human prefrontal cortex (PFC) from prenatal to peripubertal ages and throughout adulthood. Alterations in schizophrenia included decreased GAD1 expression and H3K4-trimethylation, predominantly in females and in conjunction with a risk haplotype at the 5\u27 end of GAD1. Heterozygosity for a truncated, lacZ knock-in allele of mixed-lineage leukemia 1 (Mll1), a histone methyltransferase expressed in GABAergic and other cortical neurons, resulted in decreased H3K4 methylation at GABAergic gene promoters. In contrast, Gad1 H3K4 (tri)methylation and Mll1 occupancy was increased in cerebral cortex of mice after treatment with the atypical antipsychotic, clozapine. These effects were not mimicked by haloperidol or genetic ablation of dopamine D2 and D3 receptors, suggesting that blockade of D2-like signaling is not sufficient for clozapine-induced histone methylation. Therefore, chromatin remodeling mechanisms at GABAergic gene promoters, including MLL1-mediated histone methylation, operate throughout an extended period of normal human PFC development and play a role in the neurobiology of schizophrenia

Antinociceptive Activities and the Mechanisms of Anti-Inflammation of Asiatic Acid in Mice

Asiatic acid (AA), a pentacyclic triterpene compound in the medicinal plant Centella asiatica, was evaluated for antinociceptive and anti-inflammatory effects. Treatment of male ICR mice with AA significantly inhibited the numbers of acetic acid-induced writhing responses and the formalin-induced pain in the late phase. In the anti-inflammatory test, AA decreased the paw edema at the 4th and 5th h after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. AA decreased the nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels on serum level at the 5th h after Carr injection. Western blotting revealed that AA decreased Carr-induced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and nuclear factor-κB (NF-κB) expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with AA also diminished neutrophil infiltration into sites of inflammation as did indomethacin (Indo). The anti-inflammatory mechanisms of AA might be related to the decrease in the level of MDA, iNOS, COX-2, and NF-κB in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver

Behavioral deficits in an Angelman syndrome model: Effects of genetic background and age

Angelman syndrome (AS) is a severe neurodevelopmental disorder associated with disruption of maternally inherited UBE3A (ubiquitin protein ligase E3A) expression. At the present time, there is no effective treatment for AS. Mouse lines with loss of maternal Ube3a (Ube3am–/p+) recapitulate multiple aspects of the clinical AS profile, including impaired motor coordination, learning deficits, and seizures. Thus, these genetic mouse models could serve as behavioral screens for preclinical efficacy testing, a critical component of drug discovery for AS intervention. However, the severity and consistency of abnormal phenotypes reported in Ube3am–/p+ mice can vary, dependent upon age and background strain, which is problematic for the detection of beneficial drug effects. As part of an ongoing AS drug discovery initiative, we characterized Ube3am–/p+ mice on either a 129S7/SvEvBrd-Hprtb-m2 (129) or C57BL/6J (B6) background across a range of functional domains and ages to identify reproducible and sufficiently large phenotypes suitable for screening therapeutic compounds. The results from the study showed that Ube3am–/p+ mice have significant deficits in acquisition and reversal learning in the Morris water maze. The findings also demonstrated that Ube3am–/p+ mice exhibit motor impairment in a rotarod task, hypoactivity, reduced rearing and marble-burying, and deficient fear conditioning. Overall, these profiles of abnormal phenotypes can provide behavioral targets for evaluating effects of novel therapeutic strategies relevant to AS

Follistatin mediates learning and synaptic plasticity via regulation of Asic4 expression in the hippocampus.

The biological mechanisms underpinning learning are unclear. Mounting evidence has suggested that adult hippocampal neurogenesis is involved although a causal relationship has not been well defined. Here, using high-resolution genetic mapping of adult neurogenesis, combined with sequencing information, we identify follistatin (Fst) and demonstrate its involvement in learning and adult neurogenesis. We confirmed that brain-specific Fst knockout (KO) mice exhibited decreased hippocampal neurogenesis and demonstrated that FST is critical for learning. Fst KO mice exhibit deficits in spatial learning, working memory, and long-term potentiation (LTP). In contrast, hippocampal overexpression of Fst in KO mice reversed these impairments. By utilizing RNA sequencing and chromatin immunoprecipitation, we identified Asic4 as a target gene regulated by FST and show that Asic4 plays a critical role in learning deficits caused by Fst deletion. Long-term overexpression of hippocampal Fst in C57BL/6 wild-type mice alleviates age-related decline in cognition, neurogenesis, and LTP. Collectively, our study reveals the functions for FST in adult neurogenesis and learning behaviors