Dysregulation of circadian clock gene expression patterns in a treatment-resistant animal model of depression.

Circadian rhythm (CR) disturbances are among the most commonly observed symptoms during major depressive disorder, mostly in the form of disrupted sleeping patterns. However, several other measurable parameters, such as plasma hormone rhythms and differential expression of circadian clock genes (ccgs), are also present, often referred to as circadian phase markers. In the recent years, CR disturbances have been recognized as an essential aspect of depression; however, most of the known animal models of depression have yet to be evaluated for their eligibility to model CR disturbances. In this study, we investigate the potential of adrenocorticotropic hormone (ACTH)-treated animals as a disease model for research in CR disturbances in treatment-resistant depression. For this purpose, we evaluate the changes in several circadian phase markers, including plasma concentrations of corticosterone, ACTH, and melatonin, as well as gene expression patterns of 13 selected ccgs at 3 different time points, in both peripheral and central tissues. We observed no impact on plasma corticosterone and melatonin concentrations in the ACTH rats compared to vehicle. However, the expression pattern of several ccgs was affected in the ACTH rats compared to vehicle. In the hippocampus, 10 ccgs were affected by ACTH treatment, whereas in the adrenal glands, 5 ccgs were affected and in the prefrontal cortex, hypothalamus and liver 4 ccgs were regulated. In the blood, only 1 gene was affected. Individual tissues showed changes in different ccgs, but the expression of Bmal1, Per1, and Per2 were most generally affected. Collectively, the results presented here indicate that the ACTH animal model displays dysregulation of a number of phase markers suggesting the model may be appropriate for future studies into CR disturbances.

Novelty-induced memory consolidation is accompanied by increased Agap3 transcription: a cross-species study.

Novelty-induced memory consolidation is a well-established phenomenon that depends on the activation of a locus coeruleus-hippocampal circuit. It is associated with the expression of activity-dependent genes that may mediate initial or cellular memory consolidation. Several genes have been identified to date, however, to fully understand the mechanisms of memory consolidation, additional candidates must be identified. In this cross-species study, we used a contextual novelty-exploration paradigm to identify changes in gene expression in the dorsal hippocampus of both mice and rats. We found that changes in gene expression following contextual novelty varied between the two species, with 9 genes being upregulated in mice and 3 genes in rats. Comparison across species revealed that ArfGAP with a GTPase domain, an ankyrin repeat and PH domain 3 (Agap3) was the only gene being upregulated in both, suggesting a potentially conserved role for Agap3. AGAP3 is known to regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor trafficking in the synapse, which suggests that increased transcription of Agap3 may be involved in maintaining functional plasticity. While we identified several genes affected by contextual novelty exploration, we were unable to fully reverse these changes using SCH 23390, a dopamine D1/D5 receptor antagonist. Further research on the role of AGAP3 in novelty-induced memory consolidation could lead to better understanding of this process and guide future research.

Dysregulation of miR-185, miR-193a, and miR-450a in the skin are linked to the depressive phenotype.

BACKGROUND: Dysregulated microRNAs (miRNAs) in dermal fibroblasts of depressive subjects, indicate biomarker potential and can possibly aid clinical diagnostics. To overcome methodological challenges related to human experiments and fibroblast cultures, we here validate 38 miRNAs previously observed to be dysregulated in human fibroblasts from depressed subjects, in the skin of four distinct rat models of depression. METHODS: In the presented study male rats from the adrenocorticotropic hormone (ACTH) model (n = 10/group), the chronic mild stress model (n = 10/group), Wistar Kyoto/Wistar Hannover rats (n = 10/group), and Flinders Resistant/Flinders Sensitive Line rats (n = 8/group) were included. Real-time qPCR was utilized to investigate miRNA alterations in flash-frozen skin-biopsies from the ear and fibroblast cultures. RESULTS: In the ACTH rat model of depression, we identified nine dysregulated miRNAs in the skin and three in the fibroblasts. As the skin presented three times the amount of dysregulated miRNAs compared to the fibroblasts, skin instead of fibroblasts were continuously used for studies with the other rat models. In the skin from the four rat models of depression, 15 out of 38 miRNAs re-exhibited significant dysregulation in at least one of the rat models of depression and 67% were regulated in the same direction as in the human study. miR-450a and miR-193a presented dysregulation across rat models and miR-193a and miR-185 exhibited very strong dysregulation (30-fold and 50-fold, respectively). Lastly, an Ingenuity Pathway Analysis indicated functional overlap between dysregulated miRNAs, and common regulated pathways. CONCLUSION: Flash-frozen skin is a valid alternative to fibroblast cultures as the skin appear to retain more of the miRNA dysregulation present in vivo. A sub-population of 15 miRNAs appear to be specific for the depressive phenotype, as they are dysregulated in both human depressed patients and distinct rat models of depression. We propose miR-450a, miR-185, and miR-193a as biomarker candidates of particular interest.

Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds.

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.