Caroline M. Connor

DNA methylation changes in schizophrenia and bipolar disorder

The etiology of the major psychotic disorders, including schizophrenia and bipolar disorder, remains poorly understood. Postmortem brain studies have revealed altered expression of multiple mRNAs, affecting neurotransmission, metabolism, myelination and other functions. Epigenetic mechanisms could be involved, because for a limited number of genes, the alterations on the mRNA level were linked to inverse DNA methylation changes at sites of the corresponding promoters. However, results from independent studies have been inconsistent and when expressed in quantitative terms, disease-related methylation changes appeared to be comparatively subtle. A recent study identified approximately 100 loci with altered CpG methylation in schizophrenia or bipolar disorder, the majority of which were gender-specific. Additional work will be necessary to clarify the origin and timing of these methylation changes in psychosis, and to determine the specific cell types affected in CNS.

SETDB1 HISTONE METHYLTRANSFERASE REGULATES MOOD-RELATED BEHAVIORS AND EXPRESSION OF THE NMDA RECEPTOR SUBUNIT NR2B

Histone methyltransferases specific for the histone H3-lysine 9 residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair, and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to <1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30 kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wild-type mice, systemic treatment with the NR2B antagonist, Ro25-6981 [R-(R,S)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propranol], and hippocampal small interfering RNA-mediated NR2B/Grin2b knockdown resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations.

White matter neuron alterations in schizophrenia and related disorders

Increased density and altered spatial distribution of subcortical white matter neurons (WMNs) represents one of the more well replicated cellular alterations found in schizophrenia and related disease. In many of the affected cases, the underlying genetic risk architecture for these WMN abnormalities remains unknown. Increased density of neurons immunoreactive for Microtubule‐Associated Protein 2 (MAP2) and Neuronal Nuclear Antigen (NeuN) have been reported by independent studies, though there are negative reports as well; additionally, group differences in some of the studies appear to be driven by a small subset of cases. Alterations in markers for inhibitory (GABAergic) neurons have also been described. For example, downregulation of neuropeptide Y (NPY) and nitric oxide synthase (NOS1) in inhibitory WMN positioned at the gray/white matter border, as well as altered spatial distribution, have been reported. While increased density of WMN has been suggested to reflect disturbance of neurodevelopmental processes, including neuronal migration, neurogenesis, and cell death, alternative hypotheses—such as an adaptive response to microglial activation in mature CNS, as has been described in multiple sclerosis –should also be considered. We argue that larger scale studies involving hundreds of postmortem specimens will be necessary in order to clearly establish the subset of subjects affected. Additionally, these larger cohorts could make it feasible to connect the cellular pathology to environmental and genetic factors implicated in schizophrenia, bipolar disorder, and autism. These could include the 22q11 deletion (Velocardiofacial/DiGeorge) syndrome, which in some cases is associated with neuronal ectopias in white matter.

Cingulate White Matter Neurons in Schizophrenia and Bipolar Disorder

BACKGROUND Increased neuronal density in prefrontal, parietal, and temporal white matter of schizophrenia subjects is thought to reflect disordered neurodevelopment; however, it is not known if this cellular alteration affects the cingulate cortex and whether similar changes exist in bipolar disorder. METHOD Eighty-two postmortem specimens (bipolar 15, schizophrenia 22, control 45) were included in this clinical study. Densities for two neuronal markers, neuron-specific nuclear protein (NeuN) and neuregulin 1 alpha (NRG), were determined in white matter up to 2.5 mm beneath the anterior cingulate cortex; density of NeuN immunopositive neurons (NeuN+) was also determined for a subset of cases in prefrontal cortex. Changes during normal development were monitored in a separate cohort of 14 brains. RESULTS Both the schizophrenia and bipolar cohorts demonstrated a twofold increase in NeuN+ density in cingulate white matter; this effect could be attributed to approximately 25% of cases that exceeded the second standard deviation from control subjects. Similar changes were observed in prefrontal cortex. In contrast density of NRG expressing neurons was unaltered. Cases with increased NeuN+ densities in two-dimensional (2-D) counts also showed a pronounced, > fivefold elevation in NeuN+ nuclei per cubic millimeter. Additionally, the developmental cohort demonstrated a 75% decline in NeuN+ neuronal density during the first postnatal year but was stable thereafter. CONCLUSIONS Increased neuronal density in white matter of cingulate cortex in schizophrenia provides further evidence that this alteration occurs in multiple cortical areas. Similar changes in some cases with bipolar illness suggest that the two disorders may share a common underlying defect in late prenatal or early postnatal neurodevelopment.

Amygdalar GABAergic-rich neural grafts attenuate anxiety-like behavior in rats

Transplantation experiments have shown that neurologic deficits may be reversed by engrafting fresh tissue or engineered cells within dysfunctional neural circuitry. In experimental and clinical settings, this approach has provided insights into the pathology and treatment of neurologic diseases, primarily movement disorders. The present experiments were designed to investigate whether a similar strategy is feasible as a method to investigate, and perhaps repair, circuitry integral to emotional disorders. We focused on the amygdala, a macrostructure known to be involved in the expression of anxiety- and fear-related behaviors. GABAergic cell-rich suspensions were prepared from E17 rat lateral ganglionic eminence and engrafted bilaterally into the lateral and basolateral amygdaloid nuclei of young adult rats. After 6 weeks, increased numbers of GABAergic neurons were identified in the vicinity of the graft sites, and electron microscopy provided evidence for functional integration of transplanted cells. Rats with these grafts spent more time in the open arms of the elevated-plus maze, consistent with an anxioloytic-like phenotype. These rats were also less sensitive to the unconditioned anxiogenic effects of light on the acoustic startle response, although fear-potentiated startle was not affected, suggesting that the grafts produced an attenuation of unlearned fear but did not affect acquisition of conditioned fear. Our results raise the possibility that distinct components of emotion can be modulated by strategic neural engraftment.

A simple method for improving the specificity of anti-methyl histone antibodies

Antibodies differentiating between the mono-, di- and trimethylated forms of specific histone lysine residues are a critical tool in epigenome research, but show variable specificity, potentially limiting comparisons across studies and between samples. Using trimethyl histone H3 lysine 4 (H3K4me3)—a mark enriched at transcription start sites (TSS) of active genes—as an example, we describe how simple co-incubation with synthetic peptide of the K4me2 modification leads to increased specificity for K4me3 and a much sharper peak distribution proximal to TSS following chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq).

Posttranslational Histone Modifications and the Neurobiology of Psychosis

Schizophrenia and related major psychiatric disease is typically defined by the conspicuous absence of a defining neuropathology and a lack of straightforward identifiable genetic factors in the majority of affected individuals. On the other hand, there is increasing evidence that a distinct set of RNAs, many of which encode proteins of critical importance for myelin regulation and oligodendrocyte function, or GABAergic inhibitory and glutamatergic excitatory neurotransmission are expressed at altered levels in diseased brain. This chapter explores the mechanisms by which epigenetic regulators of gene expression, including covalent histone modifications, could contribute to dysregulation of gene expression in schizophrenia. There is also discussion on the methodological and scientific limitations of histone-focused approaches, as it pertains to the human (postmortem) brain, as well as brief remarks on the topic of epigenetic heritability of chromatin structures potentially altered in schizophrenia. The authors predict that the study of histone modifications, both at defined candidate gene loci and genome-wide, will become an important tool in the investigation of gene expression abnormalities and potential epigenetic dysregulation in the brains of subjects on the psychosis spectrum.