Global State Measures of the Dentate Gyrus Gene Expression System Predict Antidepressant-Sensitive Behaviors

Background Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are the most common form of medication treatment for major depression. However, approximately 50% of depressed patients fail to achieve an effective treatment response. Understanding how gene expression systems respond to treatments may be critical for understanding antidepressant resistance. Methods We take a novel approach to this problem by demonstrating that the gene expression system of the dentate gyrus responds to fluoxetine (FLX), a commonly used antidepressant medication, in a stereotyped-manner involving changes in the expression levels of thousands of genes. The aggregate behavior of this large-scale systemic response was quantified with principal components analysis (PCA) yielding a single quantitative measure of the global gene expression system state. Results Quantitative measures of system state were highly correlated with variability in levels of antidepressant-sensitive behaviors in a mouse model of depression treated with fluoxetine. Analysis of dorsal and ventral dentate samples in the same mice indicated that system state co-varied across these regions despite their reported functional differences. Aggregate measures of gene expression system state were very robust and remained unchanged when different microarray data processing algorithms were used and even when completely different sets of gene expression levels were used for their calculation. Conclusions System state measures provide a robust method to quantify and relate global gene expression system state variability to behavior and treatment. State variability also suggests that the diversity of reported changes in gene expression levels in response to treatments such as fluoxetine may represent different perspectives on unified but noisy global gene expression system state level responses. Studying regulation of gene expression systems at the state level may be useful in guiding new approaches to augmentation of traditional antidepressant treatments

Genomic Imprinting of the X-linked Gene Transketolase-like 1 in Mouse and Human

Genomic imprinting is defined as differential allele expression based on parental origin. Imprinting of X-linked genes has been hypothesized to contribute to the 4-fold male:female sex bias in autism. This hypothesis emerged from studies of Turner syndrome, where girls with a maternal X (45, X m) show greater propensity to social impairment and have a higher rate of autism compared to paternal X (45, Xp) females and the general population. Through the use of murine models, the X-linked Xlr3/4 locus and Rhox5 have been identified as imprinted. However, no imprinted orthologs of these genes have been found in humans. Therefore, the search was expanded to identify X-linked genes that are imprinted in both mouse and human. ^ Allele-specific quantitative real-time PCR was used to examine expression of candidate genes in developing brain. As a result, we have identified Transketolase-like 1 (Tktl1) as an X-linked imprinted gene. Tktl1 codes for a transketolase enzyme, which operates in the pentose phosphate pathway (PPP). One function of the PPP is maintaining glutathione in a reduced state by reduction of NADP to NADPH. Since aberrant glutathione levels have been found associated with autistic spectrum conditions, the effect of Tktl1 expression on the state of glutathione was studied in the developing brain. In addition, Tktl1 allows for another opportunity to determine the mechanism of X-linked genomic imprinting, which is of current interest to our laboratory.

Total Levels of Hippocampal Histone Acetylation Predict Normal Variability in Mouse Behavior

<div><p>Background</p><p>Genetic, pharmacological, and environmental interventions that alter total levels of histone acetylation in specific brain regions can modulate behaviors and treatment responses. Efforts have been made to identify specific genes that are affected by alterations in total histone acetylation and to propose that such gene specific modulation could explain the effects of total histone acetylation levels on behavior — the implication being that under naturalistic conditions variability in histone acetylation occurs primarily around the promoters of specific genes.</p><p>Methods/Results</p><p>Here we challenge this hypothesis by demonstrating with a novel flow cytometry based technique that normal variability in open field exploration, a hippocampus-related behavior, was associated with total levels of histone acetylation in the hippocampus but not in other brain regions.</p><p>Conclusions</p><p>Results suggest that modulation of total levels of histone acetylation may play a role in regulating biological processes. We speculate in the discussion that endogenous regulation of total levels of histone acetylation may be a mechanism through which organisms regulate cellular plasticity. Flow cytometry provides a useful approach to measure total levels of histone acetylation at the single cell level. Relating such information to behavioral measures and treatment responses could inform drug delivery strategies to target histone deacetylase inhibitors and other chromatin modulators to places where they may be of benefit while avoiding areas where correction is not needed and could be harmful.</p></div

Schematic of flow cytometry protocol.

<p>Specific brain regions are microdisected, enzymatically digested, and dissociated via trituration. Subsequently, dissociated cells are immunostained with anti-AcH3 antibody, labeled with diamidino-2-phenylindole (DAPI) dye to identify stained nuclei, and run on a flow cytometer. Levels of AcH3 are measured as the median fluorescence intensity of DAPI-positive events.</p

Possible interaction between AcH3 in the hippocampus and striatum.

<p>A group of four mice had high levels of AcH3 in the striatum and hippocampus (panel a). In contrast with mice that had high AcH3 only in the hippocampus, these mice were very hypoactive, and were even less active than low hippocampal AcH3 mice. Panel (b) shows a detailed time course for percent distance in the center on day 1 and demonstrates that in this measure, mice with high AcH3 in both regions were indistinguishable from low AcH3 mice (repeated measures ANOVA - main effect of group p = 0.01). Panel (c) shows a time course of total distance on day 1 and demonstrates that this group of mice was significantly less active than mice with high AcH3 only in the hippocampus (repeated measures ANOVA - main group effect p = 0.001) and mice with low levels of AcH3 in the hippocampus (repeated measures ANOVA - time x group interaction p = 0.004). Similar results were seen on other testing days (not shown).</p

Levels of AcH3 in the hippocampus predict levels of open-field exploration.

<p>Figure shows bivariate plots of percent distance travelled in the center on day 1 (x-axis) versus AcH3 levels in various brain regions. There was a significant correlation between percent center and AcH3 in the hippocampus (panel a – Spearman r = 0.47*) but not for other regions (panels b–d). Spearman r correlation values for other open-field measures are found in table S1.</p

Levels of AcH3 in the hippocampus predict levels of anxiety-like behavior.

<p>Mice were split at the median by hippocampal AcH3. Groups of mice separated by hippocampal AcH3 were significantly different in nearly all center measures (Percent distance in center (a), Time in center (b), Distance in center (c), Entries into center (d)) on all testing days. Differences decreased for total distance travelled (panel e), especially when testing was done in the dark. Panel (f) shows a more detailed time course for Total distance by 5-minute bins. Figure shows that differences between groups were most pronounced at the beginning of sessions in the light, whereas, differences decreased in the dark and towards the end of sessions.</p

SPATA5 mutations cause a distinct autosomal recessive phenotype of intellectual disability, hypotonia and hearing loss

We examined an extended, consanguineous family with seven individuals with severe intellectual disability and microcephaly. Further symptoms were hearing loss, vision impairment, gastrointestinal disturbances, and slow and asymmetric waves in the EEG. Linkage analysis followed by exome sequencing revealed a homozygous variant in SPATA5 (c.1822_1824del; p.Asp608del), which segregates with the phenotype in the family. Molecular modelling suggested a deleterious effect of the identified alterations on the protein function. In an unrelated family, we identified compound heterozygous variants in SPATA5 (c.[2081G > A];[989_991delCAA]; p.[Gly694Glu];[.Thr330del]) in a further individual with global developmental delay, infantile spasms, profound dystonia, and sensorineural hearing loss. Molecular modelling suggested an impairment of protein function in the presence of both variants. SPATA5 is a member of the ATPase associated with diverse activities (AAA) protein family and was very recently reported in one publication to be mutated in individuals with intellectual disability, epilepsy and hearing loss. Our results describe new, probably pathogenic variants in SPATA5 that were identified in individuals with a comparable phenotype. We thus independently confirm that bi-allelic pathogenic variants in SPATA5 cause a syndromic form of intellectual disability, and we delineate its clinical presentation

Principal components analysis generates a robust meaure (PCA1) of gene expression system state.

<p>Panel (a) plots the proportion of variance explained by principal components 1–4. Panel (b) is a bivariate plot of PCA1 values obtained from PCA done on all samples grouped together (x-axis) versus PCA1 from separate PCAs on samples grouped by region (y-axis) (r = 0.99, p<0.001). Panel (c) shows that the data processing algorithm used has no effect on values for PCA1 (r = 1.00, p<0.001). Panel (d) compares PCA1 values of dorsal and ventral dentate from the same mice (r = 0.97, p<0.001). Panels (e–f) are cross-correlation tables for PCA1 values obtained from PCA on independent groups of 2000 expression levels/group in dorsal (e) and ventral (f) dentate samples. Panel (g) plots gene connectivity (x-axis) against the correlation coefficient of genes with PCA1 demonstrating that more connected genes follow more closely with PCA1. Panel (h) plots PCA1 versus the Euclidean distance of transcriptomes from the transcriptome with the lowest value for PCA1 (open circle).</p

PCA1 is inversely related to levels of antidepressant-sensitive behaviors.

<p>Panels (a–b) plot immobility in the FST (y-axis) against PCA1 (x-axis) for the dorsal (a, Spearman r = −0.63, p = 0.004) and ventral (b, Spearman r = −0.63, p = 0.004) dentate gyrus samples. Panels (c–d) plot latency to eat in the NSF (y-axis) against PCA1 (x-axis) for the dorsal (c, Spearman r = −0.81, p = 0.004) and ventral (d, Spearman r = −0.79, p = 0.004) dentate gyrus samples.</p