siCtr indicates the negative control siRNA. Those genes with a p value of <0.05 and those with an expression change of more than 50% in the circTshz2-2-depleted cells compared to negative control are denoted by a red dot. Transcriptional response to circTshz2-2 dysregulationĪ The genes experiencing significant differential expression in response to circTshz2-2 knockdown in differentiated (Diff.) Neuro-2A cells at day 5. The data in E were evaluated using an ordinary two-way ANOVA test. Statistical significance for B–F was determined using an unpaired two-tailed t-test with Welch’s correction. Structural data for B–E represents the findings from at least three independent experiments (n = 3) with more than ten neurons analyzed per replicate. Data are represented as the mean ± SEM (n = 3). F Changes in SYP and PSD-95 protein levels following circTshz2-2 knockdown in PCN at DIV 7. S9E and results are described as the mean (solid line) ± SEM (dot line). The number of intersections was analyzed using the Sholl analysis method as described in Fig. E Changes in neural complexity following circTshz2-2 knockdown in PCN at DIV 7. The whiskers in graphs B–D represent the minimum and maximum values while the box line represents the first quartile, median, and third quartile, respectively. D Changes in the number of neurites from each soma following circTshz2-2 knockdown in PCN at DIV 7. C Changes in neural structures and total neurite length following circTshz2-2 knockdown in mouse primary cortical neurons (PCN) after 7 days in vitro (DIV 7). siCtr denotes the negative control siRNA. B Changes in neural structures and neurite length following circTshz2-2 knockdown in differentiated (Diff.) Neuro-2A cells at day 5. Back-splice junctions of circRNAs identified by Sanger sequencing are indicated as a silver triangle. Genomic information and PhastCons data were obtained from the USCS Genome Browser. Regulation of neuronal structures by circTshz2-2Ī Genomic information and conservation of circTshz2-2 in mouse Tshz2 locus (mm10). Our data have revealed potential regulatory roles of obesity-related circTshz2-2 on the neuronal cell cycle and memory function providing a novel link between metabolic syndromes and cognitive deficits. Finally, we showed that circTshz2-2 affects spatial memory in wild-type and obese mice. Inversely, overexpression of circTshz2-2 decreased BDNF expression, induced cell cycle proteins, and shortened the neurite length, indicating that circTshz2-2 regulates neuronal cell cycle and structure. Suppression of circTshz2-2 increased BDNF expression and reduced G2/M checkpoint proteins such as Cyclin B2 and CDK1 through BDNF/TrkB signaling pathway, resulting in cell cycle arrest and neurite elongation. We found that circTshz2-2 bound to the YY1 transcriptional complex and suppressed Bdnf transcription. Transcriptomic and cellular analysis indicated that circTshz2-2 dysregulation altered the expression of cell division-related genes and induced cell cycle arrest at the G2/M phase of the neuron. In this study, we scrutinized a conserved and neuron-specific circRNA, circTshz2-2, which affects neuronal cell cycle and spatial memory in the brain. Recently, we identified obesity-linked circular RNAs (circRNA) by analyzing the brain cortices of high-fat-fed obese mice. Only a few studies explored the contribution of non-coding genes in these pathophysiologies. Metabolic syndromes, including obesity, cause neuropathophysiological changes in the brain, resulting in cognitive deficits.
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