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Blood Sugar Balance (BSB) is an accessible web-based game, created as an extension of our federally funded type 2 diabetes curriculum for high school biology classrooms. Modeling of complex systems and diseases, like metabolism and type 2 diabetes (T2D), is especially difficult and deeply impactful when executed in an engaging way. Blood Sugar Balance integrates environmental factors, biological factors, and personal choices to model glucose metabolism and understand the impact and risk factors for type 2 diabetes. Players earn points during gameplay by ensuring their game character maintains healthy blood glucose levels throughout the play period by regulating them. Players must make choices about food, exercise, and the release of hormones from the pancreas to manage blood glucose levels. Game settings can model the stages of type 2 diabetes as well as environmental factors that limit access to food, exercise, and health care options. Gameplay is fast and engaging, allowing exploration of factors that impact the final score. For example, how might accessibility to insulin impact the final score while playing at the type 2 diabetes setting? Here we describe the development of Blood Sugar Balance and the integration of data analysis into the accompanying NGSS-aligned lesson plan.

Access to professional development opportunities promotes equity for both teachers and students. We identified time, distance, and childcare needs as limiting factors for teachers seeking to attend our conference workshops and in-person professional development opportunities. Therefore, just months before the COVID-19 pandemic and the rapid shift to online learning, the Genome Sciences Education Outreach (GSEO) program transitioned to online professional development for the NIGMS sponsored SEPA grant Genes Environment and Me Network (GEMNet) program. Our program was well positioned to support teachers in our network by providing online PD that included technology support and rapid access to our online curriculum for use for distance learning. Here we share the process of creating online PD as well as converting our materials to the digital environment. Additionally, we provide survey data and lessons learned from eight online PD trainings from January 2020 to January 2021 for both Health/Family and Consumer Science Teachers (n=20) and Biology Teachers (n=23). Notably, Teachers provided feedback affirming our initial reasons for providing online PD, and appreciated being supported at an overwhelmingly difficult time to be an educator. Our findings support the need for both online and in-person professional development opportunities for future programs.

Serotonin is a key mediator of stress, anxiety, and depression, and novel therapeutic targets within serotonin neurons are needed to combat these disorders. To determine how stress alters the translational profile of serotonin neurons, we sequenced ribosome associated RNA from these neurons after repeated stress in male and female mice. We identified numerous sex- and stress-regulated genes. In particular, Fkbp5 mRNA, which codes for the glucocorticoid receptor co-chaperone protein FKBP51, was consistently upregulated in male and female mice following stress. Pretreatment with a selective FKBP51 inhibitor into the dorsal raphe prior to repeated forced swim stress decreased resulting stress-induced anhedonia. Our results support previous findings linking FKBP51 to stress-related disorders and provide the first evidence suggesting that FKBP51 function may be an important regulatory node integrating circulating stress hormones and serotonergic regulation of stress responses.

Background Microglia have recently been implicated in opioid dependence and withdrawal. Mu Opioid (MOR) receptors are expressed in microglia, and microglia form intimate connections with nearby neurons. Accordingly, opioids have both direct (MOR mediated) and indirect (neuron-interaction mediated) effects on microglia function. Methods To investigate this directly, we used RNA sequencing of ribosome-associated RNAs from striatal microglia (RiboTag-Seq) after the induction of morphine tolerance and followed by naloxone precipitated withdrawal (n=16). We validated the RNA-Seq data by combining fluorescent in-situ hybridization with immunohistochemistry for microglia (n=18). Finally, we expressed and activated the Gi/o-coupled hM4Di DREADD receptor in CX3CR1-expressing cells during morphine withdrawal (n=18). Results We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal. WGCNA analysis revealed an intriguing network of cAMP-associated genes that are known to be involved in microglial motility, morphology, and interactions with neurons that were downregulated with morphine tolerance and upregulated rapidly by withdrawal. Three-dimensional histological reconstruction of microglia allowed for volumetric, visual colocalization of mRNA within individual microglia that validated our bioinformatics results. Direct activation of Gi/o-coupled DREADD receptors in CX3CR1-expressing cells exacerbated signs of opioid withdrawal rather than mimicking the effects of morphine. Conclusions These results indicate that Gi-signaling and cAMP-associated gene networks are inversely engaged during opioid tolerance and early withdrawal, perhaps revealing a role of microglia in mitigating the consequences of opioids.

5-HT1B receptors modulate synaptic serotonin (5-HT) levels and play a significant role in the regulation of emotional behaviors. These receptors are Gαi/o-coupled and inhibit adenylyl cyclase but have also been reported to activate MAP kinases; however, the details of signaling cascades downstream of 5-HT1B receptor activation remain unclear, particularly in neuronal cells. We generated a stable 5-HT1B receptor-expressing Neuro2A (N2A-1B) neuronal cell line and demonstrate that activation of these receptors by the selective 5-HT1B agonist CP-94253 results in activation of ERK1/2 but not of other closely related MAP kinases. Phosphoproteomics revealed four novel phosphorylation sites on the third intracellular loop of the 5-HT1B receptor, and mutations of serine-256 and serine-291 to alanine led to reduced levels of ERK1/2 phosphorylation following receptor activation. Inhibition of Gαi/o signaling with pertussis toxin, as well as MEK1/2 inhibition with U0126, also reduced 5-HT1B-mediated ERK1/2 phosphorylation. Finally, we found that knockout of either β-arrestin 1 or β-arrestin 2 prevented 5-HT1B-mediated phosphorylation of ERK1/2. Taken together, these results show that 5-HT1B receptor activation selectively induces ERK1/2 activation through both the Gαi subunit and β-arrestin proteins. This work elucidates the signal transduction pathway of 5-HT1B receptors, as well as key phosphorylation sites within the receptor that modulate ERK1/2 activation, and further characterizes the intracellular mechanisms that underlie 5-HT1B receptor function.

Repeated cycles of alcohol intoxication and withdrawal both induce profound changes in gene expression that can contribute to the physiological and behavioral consequences of ethanol. Since neuroinflammation is an important consequence of these changes, we used a novel strategy to investigate the impact of repeated cycles of chronic intermittent ethanol vapor and withdrawal on the RNAs actively undergoing translation in striatal microglia. RiboTag was selectively expressed in the microglia of transgenic mice and was used to immunopurify the RNA “translatome” from striatal microglia, yielding a snapshot of RNA translation during alcohol intoxication and after 8 hours of withdrawal. We obtained highly enriched microglial RNAs and analyzed these in individual animals by deep sequencing. We found a dramatic shift in gene expression during acute intoxication compared to air-exposed controls, with increases in genes and pathways associated with cytokine signaling, indicating increased neuroinflammation and microglial activation. After 8 hours of ethanol withdrawal, many inflammatory pathways remained upregulated but phagocytotic and proapoptotic pathways were increased. Using an unbiased bioinformatic method, weighted gene coexpression network analysis, multiple differentially expressed gene modules were identified. One in particular was differentially expressed in ethanol intoxicated vs. withdrawing animals, and there was a strong correlation between the centrality of the genes to this gene network and their individual statistical significance in differential expression. The unfolded protein response was over-represented in this network after withdrawal. The induction of this pathway in microglia is important since this cellular stress response can either lead towards restoration of normal function or apoptosis.

The lateral habenula (LHb) integrates critical information regarding aversive stimuli that shapes decision making and behavioral responses. The three major LHb outputs innervate dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTg). LHb neurons that project to these targets are segregated and nonoverlapping, and this led us to consider whether they have distinct molecular phenotypes and adaptations to stress exposure. In order to capture a time-locked profile of gene expression after repeated forced swim stress, we used intersectional expression of RiboTag in rat LHb neurons and next-gen RNA sequencing to interrogate the RNAs actively undergoing translation from each of these pathways. The “translatome” in the neurons comprising these pathways was similar at baseline, but diverged after stress, especially in the neurons projecting to the RMTg. Using weighted gene co-expression network analysis, we found one module comprising genes downregulated after stress in the RMTg-projecting LHb neurons; there was an overrepresentation of genes associated with phosphoinositide 3 kinase (PI3K) signaling in this module. Reduced PI3K signaling in RMTg-projecting LHb neurons may be a compensatory adaptation that alters the functional balance of LHb outputs to GABAergic vs. monoaminergic neurons following repeated stress exposure.

Microglia have recently been implicated in dependence to opioids. To investigate this directly, we used RNA sequencing of ribosome associated RNAs from striatal microglia (RiboTag-Seq) after the induction of morphine tolerance and then the precipitation of withdrawal by naloxone. We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal, and bioinformatics analysis revealed an intriguing upregulation of cAMP-associated genes that are involved in microglial motility, morphology, and interactions with neurons. Three-dimensional histological reconstruction of microglia revealed changes in process branching and termination following opioid tolerance that were rapidly reversed during withdrawal and were consistent with cAMP effects on microglia morphology. Direct activation of Gi-coupled DREADD receptors in microglia, rather than mimicking the effects of morphine, exacerbated opioid withdrawal. Together these indicate that microglial response to cAMP signaling can mitigate the rapid manifestations of opioid withdrawal.

5-HT(6) (serotonin) receptors are promising targets for a variety of neuropsychiatric disorders and have been linked to several cellular signaling cascades. Endogenous 5-HT(6) receptors are restricted to the primary neuronal cilium, a small sensory organelle stemming from the cell body that receives numerous extrasynaptic signals. Inhibition of 5-HT(6) receptors decreases cilia length in primary neuronal cultures, but the signaling mechanisms involved are still unclear. Intense overexpression of exogenous 5-HT(6) receptors increases the probability for receptors to localize outside the primary cilium and have been associated with changes in cilia morphology and dendritic outgrowth. In the present study, we explore the role of 5-HT(6)R rescue on neuronal morphology in primary neuronal cultures from 5-HT(6)R-KO mice, at the same time maintaining a more physiologic level of expression, wherein the receptor localizes to cilia in 80%–90% of neurons (similar to endogenous 5-HT(6)R localization). We found that rescue of 5-HT(6)R expression is sufficient to increase cilia length and dendritic outgrowth, but primarily in neurons in which the receptor is located exclusively in the primary cilia. Additionally, we found that expression of 5-HT(6)R mutants deficient in agonist-stimulated cAMP or without the predicted Fyn kinase binding domain maintained constitutive activity for stimulating cAMP and still increased the length of cilia, and that the proposed Fyn kinase domain was required for stimulating dendritic outgrowth. These findings highlight the complexity of 5-HT(6)R function and localization, particularly with the use of exogenous overexpression, and provide greater understanding and potential mechanisms for 5-HT(6)R drug therapies.

5-HT(1B) receptors modulate synaptic serotonin (5-HT) levels and play a significant role in the regulation of emotional behaviors. These receptors are Gα(i/o)-coupled and inhibit adenylyl cyclase but have also been reported to activate MAP kinases; however, the details of signaling cascades downstream of 5-HT(1B) receptor activation remain unclear, particularly in neuronal cells. We generated a stable 5-HT(1B) receptor-expressing Neuro2A (N2A-1B) neuronal cell line and demonstrate that activation of these receptors by the selective 5-HT(1B) agonist CP-94253 results in activation of ERK1/2 but not of other closely related MAP kinases. Phosphoproteomics revealed four novel phosphorylation sites on the third intracellular loop of the 5-HT(1B) receptor, and mutations of serine-256 and serine-291 to alanine led to reduced levels of ERK1/2 phosphorylation following receptor activation. Inhibition of Gα(i/o) signaling with pertussis toxin, as well as MEK1/2 inhibition with U0126, also reduced 5-HT(1B)-mediated ERK1/2 phosphorylation. Finally, we found that knockout of either β-arrestin 1 or β-arrestin 2 prevented 5-HT(1B) -mediated phosphorylation of ERK1/2. Taken together, these results show that 5-HT(1B) receptor activation selectively induces ERK1/2 activation through both the Gα(i) subunit and β-arrestin proteins. This work elucidates the signal transduction pathway of 5-HT(1B) receptors, as well as key phosphorylation sites within the receptor that modulate ERK1/2 activation, and further characterizes the intracellular mechanisms that underlie 5-HT(1B) receptor function.

AML is not a single disease but rather a collection of diseases caused by a variety of mutations. About 30% of AML cases are characterized by a TCTG insertion in the exon 12 of Nucleophosmin 1 (NPM1). Due to this mutation, mutant NPM1 (NPM1c) is mislocalized and maintained predominantly in the cytoplasm of the cell. Previously it had been reported that NPM1 binds to CCCTC binding factor (CTCF). Based on this observation we hypothesized that NPM1c would mislocalize CTCF into the cytoplasm, preventing CTCF's access to binding sites in the nucleus, and allowing for epigenetic misregulation of gene expression. To demonstrate that NPM1c could mislocalize CTCF to the cytoplasm we used confocal microscopy. While CTCF was completely nuclear in cells expressing NPM1, CTCF was present in the nucleus and the cytoplasm of cells expressing NPM1c. The cytoplasmic interaction of NPM1c and CTCF was confirmed by immunoprecipitation and western blotting. Using fluorescently tagged sub-fragments of NPM1c and CTCF we found that a.a. 1-264 of CTCF interacted with a.a. 244-298 of NPM1c. CTCF has multiple effects on gene expression related to the level of CTCF in the cell/nucleus; overexpression of CTCF leads to cell senescence while reduced expression can lead to immortalization. Such effects are likely mediated by the effects of CTCF on gene expression and alternate splicing. Increased expression of HOXA9 is a characteristic of NPM1c AML and has been shown to be affected by CTCF binding between HOXA9 and HOXA7 in IMR90 cells. To determine if NPM1c would affect HOXA9 expression in IMR90 cells, we infected the cells with NPM1c or NPM1. Similar to CTCF knockdown in IMR90 cells, there was increased expression of HOXA9 in the NPM1c, but not NPM1 modified cells. Moreover, the IMR90 cells expressing NPM1c became immortal. Using chromatin immunoprecipitation we found that there was reduced binding of CTCF to the CTCF binding site 2kb downstream of HOXA9 in the IMR90 cells expressing NPM1c. Reduced CTCF binding to this site was observed in the NPM1c mutant human cell line OCI-AML-3 but not NB4 cells, an AML cell line, that lacks the mutation. Loss of CTCF binding in NPM1c expressing cells was further demonstrated by changes in CTCF mediated alternate splicing and enhancer blocking activity using reporter assays. Finally, interrogation of the TCGA AML data set showed increased methylation of CTCF binding sites in NPM1c AML. This is in keeping with de novo methylation that occurs when CTCF no longer binds to its cognate site. Based on these studies we propose that the transforming effect of NPM1c is due, in large part, to its ability to mislocalize CTCF and suggests that targeting the interaction between these two proteins could be of potential therapeutic benefit. Citation Format: Atom Wang, Youqi Han, Peikun Chen, Nanyang Jia, Mark D. Minden. Cytoplasmic mislocalization of CTCF by NPM1c in acute myeloid leukemia resulting in inhibited CTCF regulatory functions generating aberrant genetic and epigenetic profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2991.