Integrative multi-omics landscape of fluoxetine action across 27 brain regions reveals global increase in energy metabolism and region-specific chromatin remodelling.

Depression and anxiety are major global health burdens. Although SSRIs targeting the serotonergic system are prescribed over 200 million times annually, they have variable therapeutic efficacy and side effects, and mechanisms of action remain incompletely understood. Here, we comprehensively characterise the molecular landscape of gene regulatory changes associated with fluoxetine, a widely-used SSRI. We performed multimodal analysis of SSRI response in 27 mammalian brain regions using 310 bulk RNA-seq and H3K27ac ChIP-seq datasets, followed by in-depth characterisation of two hippocampal regions using single-cell RNA-seq (20 datasets). Remarkably, fluoxetine induced profound region-specific shifts in gene expression and chromatin state, including in the nucleus accumbens shell, locus coeruleus and septal areas, as well as in more well-studied regions such as the raphe and hippocampal dentate gyrus. Expression changes were strongly enriched at GWAS loci for depression and antidepressant drug response, stressing the relevance to human phenotypes. We observed differential expression at dozens of signalling receptors and pathways, many of which are previously unknown. Single-cell analysis revealed stark differences in fluoxetine response between the dorsal and ventral hippocampal dentate gyri, particularly in oligodendrocytes, mossy cells and inhibitory neurons. Across diverse brain regions, integrative omics analysis consistently suggested increased energy metabolism via oxidative phosphorylation and mitochondrial changes, which we corroborated in vitro; this may thus constitute a shared mechanism of action of fluoxetine. Similarly, we observed pervasive chromatin remodelling signatures across the brain. Our study reveals unexpected regional and cell type-specific heterogeneity in SSRI action, highlights under-studied brain regions that may play a major role in antidepressant response, and provides a rich resource of candidate cell types, genes, gene regulatory elements and pathways for mechanistic analysis and identifying new therapeutic targets for depression and anxiety.

Transposable elements that have recently been mobile in the human genome.

BACKGROUND: Transposable elements (TE) comprise nearly half of the human genome and their insertions have profound effects to human genetic diversification and as well as disease. Despite their abovementioned significance, there is no consensus on the TE subfamilies that remain active in the human genome. In this study, we therefore developed a novel statistical test for recently mobile subfamilies (RMSs), based on patterns of overlap with > 100,000 polymorphic indels. RESULTS: Our analysis produced a catalogue of 20 high-confidence RMSs, which excludes many false positives in public databases. Intriguingly though, it includes HERV-K, an LTR subfamily previously thought to be extinct. The RMS catalogue is strongly enriched for contributions to germline genetic disorders (P = 1.1e-10), and thus constitutes a valuable resource for diagnosing disorders of unknown aetiology using targeted TE-insertion screens. Remarkably, RMSs are also highly enriched for somatic insertions in diverse cancers (P = 2.8e-17), thus indicating strong correlations between germline and somatic TE mobility. Using CRISPR/Cas9 deletion, we show that an RMS-derived polymorphic TE insertion increased the expression of RPL17, a gene associated with lower survival in liver cancer. More broadly, polymorphic TE insertions from RMSs were enriched near genes with allele-specific expression, suggesting widespread effects on gene regulation. CONCLUSIONS: By using a novel statistical test we have defined a catalogue of 20 recently mobile transposable element subfamilies. We illustrate the gene regulatory potential of RMS-derived polymorphic TE insertions, using CRISPR/Cas9 deletion in vitro on a specific candidate, as well as by genome wide analysis of allele-specific expression. Our study presents novel insights into TE mobility and regulatory potential and provides a key resource for human disease genetics and population history studies.

Cleavable cellulosic sponge for functional hepatic cell culture and retrieval.

Interconnected macroporous hydrogel is hydrophilic; it exhibits soft tissue-like mechanical property and aqueous-stable macroporosity for 3D spheroid culture. There is an unmet need to develop cleavable macroporous hydrogel, for the ease of retrieving functional spheroids for further in vitro and in vivo applications. We have developed and comprehensively characterized a hydroxypropyl-cellulose-disulfide sponge by systematically identifying strategies and synthesis schemes to confer cleavability to the sponge under cell-friendly conditions. It preserved the essential advantages of the macroporous hydrogel to support 3D spheroid formation and maintenance of sensitive hepatocytes while allowing rapid cleavage and retrieval of functional spheroids. By culturing HepaRG as spheroids in the cleavable sponge, we have accelerated HepaRG differentiation to 9 days compared to 28 days in 2D culture. Cytochrome P450 basal activity reached significantly higher level, while albumin secretion and fluorescein diacetate staining indicated the same at day 5. The purity of albumin+ hepatocytes reached 92.9% versus 7.1% of CK19+ cholangiocytes at day 9, a much stronger preference for hepatocytes than the 60% albumin+ hepatocytes purity in 2D culture. HepaRG differentiated hepatocytes were retrieved by cleaving the sponge with 10 mM tris-(2-carboxyethyl)-phosphine (TCEP) within 30 min preserving viability, plateability and positive albumin staining of the hepatocyte spheroids. This cleavable macroporous hydrogel sponge will support the rapid development of various 3D spheroid- or organoid-based applications in basic research and drug testing.

Hepatic spheroids used as an in vitro model to study malaria relapse.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.