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Long-read transcriptome profiling of the rat hippocampal neurons in primary culture at different time points after activation using Nanopore sequencing

ABSTRACT: Long-read transcriptome sequencing provides us with a convenient tool for thorough study of biological processes such as neuronal plasticity. In this study, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome limitations of short-read RNA-Seq approaches we performed a Oxford Nanopore Technologies (ONT)-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation (30 min; 1 h and 5 h). Overall, we found 23652 novel transcrits in comparison to reference annotations, out of which ~6k were entirely novel and mostly transposable element-derived loci. Analysis of differentially expressed genes (DEG) showed that a total of 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points respectively. Most notably, multiple genes encoding ribosomal proteins, with high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms, observed in this study, could help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, the result of differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE218694 | GEO | 2022/11/25

REPOSITORIES: GEO

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Project description:Exercise improves brain function enhancing neuronal plasticity and cognitive enhancement. For voluntary resistance wheel running (RWR) exercise, with a load of 30% of body weight, we reported enhancement of neurogenesis and spatial memory associated with hippocampal brain-derived neurotrophic factor (BDNF) signaling compared to wheel running (WR) without a load (Lee et al., 2012; Lee et al., 2013). Despite these new evidences, mechanisms for RWR-induced improvement of hippocampal function remained to be clarified. In the present study, we have utilized the high-throughput DNA microarray approach to gain deep insight into molecular mechanisms underlying these changes that could be novel targets of RWR-induced hippocampal plasticity. To do so, whole genome (4x44K) high-density oligonucleotide microarrays were used to monitor the expression level of gene transcripts in the hippocampus of rats voluntary running for 4 weeks in comparison with sedentary animals. These rats showed a significant decrease in average running distance although average work levels immensely increased 12-fold in the RWR group, resulting in muscular adaptation for the fast-twitch plantaris muscle. DNA microarray analysis revealed that 122 (sedentary x WR) and 157 (sedentary x RWR) genes were up-regulated (> 1.5-fold change) as compared with 97 (sedentary x WR) and 467 (sedentary x RWR) down-regulated genes (< 0.75-fold change). Functional categorization using the Ingenuity Pathway Analysis (IPA) revealed expression pattern changes in the major categories of disease and disorders, molecular functions and physiological system development and function. Among these, RWR up-regulated genes such as NFATc1, AVPR1A and FGFR4, which are crucial role for neuronal development and its functions. The down-regulated inflammatory cytokines (IL1B, IL10, IL2RA, TNF) and chemokines (CXCL1, CXCL9, CXCL10, CCL2, CCL13, CCR4) might be important to neuronal dysfunction and vulnerability. Taken together, these gene candidates are suggested to play a critical role in hippocampal plasticity. This is a first study presenting not only new information into the voluntary RWR influenced transcriptome in the rat hippocampus, but also provides a hypothesis for the RWR influenced enhancement in brain function.

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Long-read transcriptome profiling of the rat hippocampal neurons in primary culture at different time points after activation using Nanopore sequencing

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