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Supplemental material, Supplementary_Information_JOP_02_07_19 for Drugs used to treat bipolar disorder act via microRNAs to regulate expression of genes involved in neurite outgrowth by Srisaiyini Kidnapillai, Ben Wade, Chiara C Bortolasci, Bruna Panizzutti, Briana Spolding, Timothy Connor, Tamsyn Crowley, Stéphane Jamain, Laura Gray, Marion Leboyer, Michael Berk and Ken Walder in Journal of Psychopharmacology

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.

Genetic analyses have revolutionised our understanding of avian mating systems. However, the majority of such studies to date have focused on passerine species. Despite this taxonomic bias, extra-pair paternity (EPP) and conspecific brood parasitism (CBP) are usually concluded to be less common in taxa with pronounced longevity, socially monogamous mating systems, and bi-parental care. The order Psittaciformes, with around 350 species worldwide, has long-lived species that are relatively under-studied with respect to parentage and pair fidelity. Although studies have revealed a variety of mating systems in Psittaciformes, there is little work testing whether EPP or CBP occur in Psittaciformes. To test for genetic and social pair fidelity we studied an Australian parrot, the Crimson Rosella (Platycercus elegans), over 8 years at three sites in south-eastern Australia. Using nine microsatellite markers in 42 pairs and their offspring, we found no cases of EPP. However, we found one case of CBP, suggesting that P. elegans females do adopt alternative breeding strategies, albeit at low levels. We also show that over the 8 years of study 32% of recaptured individuals paired with more than one partner in different years. Our results are consistent with assumption of low EPP in parrots, but challenge the notion that this is associated with long-term pair bonds.

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.

Additional file 2: Figure S2. Determination of T1, T2 and CT contrast of the synthesized MRI/CT contrast agent. A slight enhancement in T1 contrast (A) was observed in Hsp-70 Lf-PEG-ZF nanoparticles when compared to commercial ferrite nanoparticles. (B) A significant increase in the T2 contrast was observed in Hsp-70 Lf-PEG-ZF nanoparticles when compared to commercial ferrite nanoparticles. (C) A slight enhancement in CT contrast was observed in Hsp-70 Lf-PEG-ZF nanoparticles when compared to commercial ferrite nanoparticles.

Additional file 1: Figure S1. Determination of serum stability of the synthesized MRI/CT contrast agents. The serum stability was conducted in with 1 mg/ml solution of Hsp-70 Lf-PEG-ZF, Hsp-70 Lf-ZF and Hsp-70 Ch-Lf-ZF nanoparticles in 10 % BSA for a period of 7 days. Analysis was performed using DLS.

Additional file 5: Figure S5. Detailed images from Fig. 3b. More images of ex vivo hart and aorta T2 MRI sagittal scans revealed a higher accumulation of Hsp-70 Lf-PEG-ZF nanoparticles in adult mice (16 months) when compared to juvenile mice (6 months old).

Additional file 4: Figure S4. Heat map images of MRI scans and determination of enhancement in T2 contrast. (A) The heat map images of representative images from Fig. 3 are represented with encircled region of interests (ROI). (B) The enhancement in contrast intensity was calculated from 5 different images of each treatment using imgae J software and is represented in a graph.

Additional file 6: Figure S6. Site specific localisation of Hsp-70 Lf-PEG-ZF nanoparticles. The Hsp-70 Lf-PEG-ZF nanoparticles were found to be specifically bound to the region around the plaque in the aorta in adult mice.

Additional file 8: Figure S8. Diagram schematically depicting the formation and steps involved in Hsp-70 Lf-PEG-ZF nanocomplex preparation.

Additional file 7: Figure S7. Diagram schematically representing the steps involved in the chemical synthesis of Hsp-70 Lf-PEG-ZF nanocomplex.

Additional file 3: Figure S3. Concentration-dependent lactate dehydrogenase (LDH) release cell cytotoxicity and CyQUANT cell proliferation assays with THP-1 and Jurkat cells. The cells were incubated with Hsp-70 Lf-PEG-ZF (A), Hsp-70 Lf-ZF (B) and Hsp-70 Ch-Lf-ZF (C) nanocomplexes.