Search for: All records

Abstract BackgroundFolate is an essential B-group vitamin and a key methyl donor with important biological functions including DNA methylation regulation. Normal neurodevelopment and physiology are sensitive to the cellular folate levels. Either deficiency or excess of folate may lead to neurological disorders. Recently, folate has been linked to tRNA cytosine-5 methylation (m⁵C) and translation in mammalian mitochondria. However, the influence of folate intake on neuronal mRNA m⁵C modification and translation remains largely unknown. Here, we provide transcriptome-wide landscapes of m⁵C modification in poly(A)-enriched RNAs together with mRNA transcription and translation profiles for mouse neural stem cells (NSCs) cultured in three different concentrations of folate. ResultsNSCs cultured in three different concentrations of folate showed distinct mRNA methylation profiles. Despite uncovering only a few differentially expressed genes, hundreds of differentially translated genes were identified in NSCs with folate deficiency or supplementation. The differentially translated genes induced by low folate are associated with cytoplasmic translation and mitochondrial function, while the differentially translated genes induced by high folate are associated with increased neural stem cell proliferation. Interestingly, compared to total mRNAs, polysome mRNAs contained high levels of m⁵C. Furthermore, an integrative analysis indicated a transcript-specific relationship between RNA m⁵C methylation and mRNA translation efficiency. ConclusionsAltogether, our study reports a transcriptome-wide influence of folate on mRNA m⁵C methylation and translation in NSCs and reveals a potential link between mRNA m⁵C methylation and mRNA translation. 

Background/Objectives: Transient Receptor Potential Melastatin 8 (TRPM8) is a non-selective, Ca2+-permeable cation channel involved in thermoregulation and other physiological processes, such as basal tear secretion, cell differentiation, and insulin homeostasis. The activation and deactivation of TRPM8 occur through genetic modifications, channel interactions, and signaling cascades. Recent evidence suggests a significant role of TRPM8 in the hypothalamus and amygdala related to pain sensation and sexual behavior. Notably, TRPM8 has been implicated in neuropathic pain, migraines, and neurodegenerative diseases such as Parkinson’s disease. Our laboratory has identified testosterone as a high-affinity ligand of TRPM8. TRPM8 deficiency appears to influence behavioral traits in mice, like increased aggression and deficits in sexual satiety. Here, we aim to explore the pathways altered in brain tissues of TRPM8-deficient mice using the expression and methylation profiles of messenger RNA (mRNA) and long non-coding RNA (lncRNA). Specifically, we focused on brain regions integral to behavioral and hormonal control, including the olfactory bulb, hypothalamus, amygdala, and insula. Methods: RNA was isolated and purified for microarray analysis collected from male wild-type and TRPM8 knockout mice. Results: We identified various differentially expressed genes tied to multiple signaling pathways. Among them, the androgen–estrogen receptor (AR-ER) pathway, steroidogenesis pathway, sexual reward pathway, and cocaine reward pathway are particularly worth noting. Conclusions: These results should bridge the existing gaps in the knowledge regarding TRPM8 and inform potential targets for future studies to elucidate its role in the behavior changes and pathology of the diseases associated with TRPM8 activity. 

Background: The Transient Receptor Potential Melastatin 8 (TRPM8) is a cold/pain-sensitive Ca2+ channel. Testosterone is a high-affinity agonist for TRPM8, and TRPM8 -/- male mice exhibit disrupted sexual behavior: indiscriminate approach, increased mounting, and delayed satiety, possibly due to decreased ventral tegmental area dopamine (DA) neuron activity. DA plays a critical role in motivated behaviors, including behavioral activation, detection of reward-relevant stimuli, and reinforcement learning. Hypothesis: It is hypothesized that TRPM8 KO mice will exhibit disruptions across a range of motivationally-relevant behaviors, including spontaneous locomotor activation, detection of novel stimuli, sucrose preference, and sensitivity to the psychomotor stimulant amphetamine. Methods: Adult mice (Jackson Laboratory) were individually housed and locomotor activity was assessed for 48 hours. To assess detection of novel stimuli, a novel object recognition task was performed. Mice were habituated to two identical objects for two hours. A novel object was introduced and interaction with the novel vs familiar object was recorded. Sucrose (0.1%) preference was assessed using a two-bottle choice procedure. Tests for amphetamine sensitization (1.0 mg/kg i.p.) are in progress. Results: Female mice were more active compared to male mice (F (1,26) = 7.14, p < 0.05). Time course analysis of the nocturnal activity of males revealed a statistically significant decrease (F (1,12) = 23.41, p < 0.001) in activity among TRPM8 -/- compared to wildtype mice. In contrast, the TRPM8 deletion had no effect on the activity of female mice (F (1,12) = 0.32, n.s.). Preliminary analysis of the novel object recognition task revealed a trend towards increased exploration of the novel object and decreased time with the familiar object among male TRPM8 -/- mice compared to wildtype (Cohen’s d > 0.58). Finally, male TRPM8 -/- mice exhibited a robust preference for sucrose compared to wildtype mice. Additional data collection is in progress. Conclusion: TRPM8-/- mice were less active during the active phase of the day/night cycle compared to wildtype mice. However, TRPM8-/- mice exhibited increased interest in a novel object and a robust preference for sucrose, indicating increased sensitivity to motivationally-relevant stimuli. These behavioral data suggest that TRPM8 -/- mice are likely to exhibit decreased basal DA levels in reward-relevant brain areas, but that motivationally relevant stimuli likely elicit robust increases in DA. 

Testosterone exerts high affinity for the Transient Receptor Potential Melastatin 8 (TRPM8) Ca2+ channel. TRPM8 -/- male mice exhibit disrupted sexual behavior (e.g., indiscriminate approach, delayed satiety), possibly due to decreased ventral tegmental area dopamine neuron activity. It is hypothesized that TRPM8 null mutant mice (Jackson Laboratories) will exhibit disruptions across a range of motivationally-relevant behaviors, including spontaneous locomotor activation, detection of novel stimuli, sucrose preference, and sensitivity to the psychomotor stimulant amphetamine. Initial findings indicate that male TRPM8 mutant mice (n=6) exhibit decreased nocturnal locomotor activity (F(1,12)=23.41, p<0.001), increased behavioral anxiety in the light/dark task (t(10)=2.44, p<0.05; d=1.4), and behavioral despair in the forced swim task (t(10)=3.70, p<0.005; d=2.1). In contrast, these mice tended to prefer a low concentration (0.1%) of sucrose compared to wildtype males (n=6; t(10)=1.35, p=0.09; d=0.83). Tests for sensitivity to amphetamine are in progress. These data suggest a pivotal role for TRPM8 in motivated behavior. 

Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner. 

Post-transcriptional RNA modifications have been recognized as key regulators of neuronal differentiation and synapse development in the mammalian brain. While distinct sets of 5-methylcytosine (m5C) modified mRNAs have been detected in neuronal cells and brain tissues, no study has been performed to characterize methylated mRNA profiles in the developing brain. Here, together with regular RNA-seq, we performed transcriptome-wide bisulfite sequencing to compare RNA cytosine methylation patterns in neural stem cells (NSCs), cortical neuronal cultures, and brain tissues at three postnatal stages. Among 501 m5C sites identified, approximately 6% are consistently methylated across all five conditions. Compared to m5C sites identified in NSCs, 96% of them were hypermethylated in neurons and enriched for genes involved in positive transcriptional regulation and axon extension. In addition, brains at the early postnatal stage demonstrated substantial changes in both RNA cytosine methylation and gene expression of RNA cytosine methylation readers, writers, and erasers. Furthermore, differentially methylated transcripts were significantly enriched for genes regulating synaptic plasticity. Altogether, this study provides a brain epitranscriptomic dataset as a new resource and lays the foundation for further investigations into the role of RNA cytosine methylation during brain development.