Objectives: To create a gene expression signature (GES) to represent the biological effects of a combination of known drugs for bipolar disorder (BD) on cultured human neuronal cells (NT2-N) and rat brains, which also has evidence of differential expression in individuals with BD. To use the GES to identify new drugs for BD using Connectivity Map (CMap).Methods: NT2-N (n = 20) cells and rats (n = 8) were treated with a BD drug combination (lithium, valproate, quetiapine and lamotrigine) or vehicle for 24 and 6 h, respectively. Following next-generation sequencing, the differential expression of genes was assessed using edgeR in R. The derived GES was compared to differentially expressed genes in post-mortem brains of individuals with BD. The GES was then used in CMap analysis to identify similarly acting drugs.Results: A total of 88 genes showed evidence of differential expression in response to the drug combination in both models, and therefore comprised the GES. Six of these genes showed evidence of differential expression in post-mortem brains of individuals with BD. CMap analysis identified 10 compounds (camptothecin, chlorambucil, flupenthixol, valdecoxib, rescinnamine, GW-8510, cinnarizine, lomustine, mifepristone and nimesulide) acting similarly to the BD drug combination.Conclusions: This study shows that GES and CMap can be used as tools to repurpose drugs for BD.

The drugs commonly used to treat bipolar disorder have limited efficacy and drug discovery is hampered by the paucity of knowledge of the pathophysiology of this disease. This study aims to explore the role of microRNAs in bipolar disorder and understand the molecular mechanisms of action of commonly used bipolar disorder drugs. The transcriptional effects of bipolar disorder drug combination (lithium, valproate, lamotrigine and quetiapine) in cultured human neuronal cells were studied using next generation sequencing. Differential expression of genes (n=20) and microRNAs (n=6) was assessed and the differentially expressed microRNAs were confirmed with TaqMan MicroRNA Assays. The expression of the differentially expressed microRNAs were inhibited to determine bipolar disorder drug effects on their target genes (n=8). Independent samples t-test was used for normally distributed data and Kruskal-Wallis/Mann-Whitney U test was used for data not distributed normally. Significance levels were set at p<0.05. We found that bipolar disorder drugs tended to increase the expression of miR-128 and miR-378 (p<0.05). Putative target genes of these microRNAs targeted pathways including those identified as “neuron projection development” and “axonogenesis”. Many of the target genes are inhibitors of neurite outgrowth and neurogenesis and were downregulated following bipolar disorder drug combination treatment (all p<0.05). The bipolar disorder drug combination tended to decrease the expression of the target genes (NOVA1, GRIN3A, and VIM), however this effect could be reversed by the application of microRNA inhibitors. We conclude that at a transcriptional level, bipolar disorder drugs affect several genes in concert that would increase neurite outgrowth and neurogenesis and hence neural plasticity, and that this effect is mediated (at least in part) by modulation of the expression of these two key microRNAs.

Objectives: To understand the therapeutic mechanisms of bipolar disorder (BD) drugs at molecular and cellular levels. Methods: Next generation sequencing was used to determine the transcriptional effects of a combination of four commonly prescribed BD drugs (lithium, valproate, lamotrigine and quetiapine) or vehicle (0.2% DMSO) in NT2-N (human neuronal) cells and rats. Differential expression of genes and pathway analysis were performed using edgeR in R and Gene Set Enrichment Analysis software respectively. Free cholesterol levels and neurite outgrowth were quantified in NT2-N cells following combination and individual BD drug treatments. Results: Pathway analysis showed up-regulation of many elements of the cholesterol biosynthesis pathway in NT2-N cells and oxidative phosphorylation in rat brains. Intracellular cholesterol transport genes were upregulated (NPC1, NPC2 and APOE), while the cholesterol efflux gene (ABCA1) was downregulated. BD drug combination tended to increase intracellular cholesterol levels and neurite outgrowth, but these effects were not seen for the drugs when used individually. Conclusions: These data suggest that BD drug combination is increasing cholesterol biosynthesis and the newly synthesised cholesterol is being utilised within the cells, possibly for synthesis of new membranes to facilitate neurite outgrowth. This mechanism possibly underpins clinical efficacy in individuals with BD treated with polypharmacy.

Objectives: To investigate the actions of lithium, valproate, lamotrigine and quetiapine on bioenergetic pathways in cultured NT2-N neuronal-like cells and C8-B4 microglial cells. Methods: NT2-N and C8-B4 cells were cultured and treated with lithium (2.5mM), valproate (0.5mM), quetiapine (0.05mM) or lamotrigine (0.05mM) for 24 hours. Gene expression and the mitochondrial bioenergetic profile were measured in both cell lines. Results: In NT2-N cells, valproate increased oxidative phosphorylation (OXPHOS) gene expression, mitochondrial uncoupling and maximal respiratory capacity, while quetiapine decreased OXPHOS gene expression and respiration linked to ATP turnover, as well as decreasing the expression of genes in the citric acid cycle. Lamotrigine decreased OXPHOS gene expression but had no effect on respiration, while lithium reduced the expression of genes in the citric acid cycle. In C8-B4 cells, valproate and lithium increased OXPHOS gene expression, and valproate increased basal respiratory rate and maximal and spare respiratory capacities. In contrast, quetiapine significantly reduced basal respiratory rate and maximal and spare respiratory capacities. Conclusions: Overall our data suggest that some drugs used to treat neuropsychiatric and affective disorders have actions on a range of cellular bioenergetic processes, which could impact their effects in patients.

Direct reprogramming of brain-resident glial progenitor cells (GPCs) into induced neurons (iN) shows great promise for future regenerative therapies for brain diseases. This type of cell conversion is facilitated by introducing lineage-specific transcription factors or small molecules to somatic cells, and can even be achieve in vivo , targeting resident glia cells in the brain for in vivo conversion. While this have shown promising results in animal models, a crucial step is to show the long-term survival and maturation of human reprogrammed cells in vivo. However, this remains largely unexplored partly due to the inaccessible cell source of GPC and difficulty in transplanting converted neurons. In this study, we assessed human GPCs derived iNs for their long-term structural and functional neuronal maturation after transplantation to the immunodeficient mouse brain. GPCs were transduced with established transcription factor cocktail and transplanted to the medial prefrontal cortex as three-dimensional cultures to improve survival and integration upon transplantation. Our results reveal survival of hiNs, for up to 10 months post-transplantation (MPT) and wide distribution across local and proximal functionally connected brain regions. Morphological analysis revealed a gradual neuronal maturation over time, characterized by increase in cellular complexity and expression of neuronal markers. Supporting these findings, electrophysiological recordings demonstrated progressive functional maturation up to 5 MPT with increase in sodium and potassium currents, and the ability to generate multiple evoked and spontaneous action potentials, validating a neuronal phenotype. Importantly, these changes were absent in control grafts of non-converted hGPCs that retained their glia identity. Our findings indicate that hGPCs derived iNs maintain a stable neuronal phenotype that persist in the mouse brain for extended periods. These cells exhibit survival and remarkable adaptability to the host environment, enabling both structural and functional maturation over time. These results highlight their potential as a promising strategy for neural replacement therapies aimed at restoring damaged circuits and promoting brain repair. Highlights: • Human neurons reprogrammed from glia progenitor cells survive long term upon transplantation in the immunodeficient mouse brain. • The human induced neurons migrate to local and distal connected brain regions within prefrontal cortex circuitry. • Grafted neurons gradually develop neuronal complex morphology and electrophysiological functional properties in the mouse brain.