Npy transcription is regulated by noncanonical STAT3 signaling in hypothalamic neurons: Implication with lipotoxicity and obesity.

Neuropeptide Y (Npy) is an abundant neuropeptide expressed in the central and peripheral nervous systems. NPY-secreting neurons in the hypothalamic arcuate nucleus regulate energy homeostasis, and Npy mRNA expression is regulated by peripheral nutrient and hormonal signals like leptin, interleukin-6 (IL-6), and fatty acids. This study demonstrates that IL-6, which phosphorylates tyrosine 705 (Y705) of STAT3, decreased Npy mRNA in arcuate immortalized hypothalamic neurons. In parallel, inhibitors of STAT3-Y705 phosphorylation, stattic and cucurbitacin I, robustly upregulated Npy mRNA. Chromatin-immunoprecipitation showed high baseline total STAT3 binding to multiple regulatory regions of the Npy gene, which are decreased by IL-6 exposure. The STAT3-Npy interaction was further examined in obesity-related pathologies. Notably, in four different hypothalamic neuronal models where palmitate potently stimulated Npy mRNA, Socs3, a specific STAT3 activity marker, was downregulated and was negatively correlated with Npy mRNA levels (R2 = 0.40, p < 0.001), suggesting that disrupted STAT3 signaling is involved in lipotoxicity-mediated dysregulation of Npy. Finally, human NPY SNPs that map to human obesity or body mass index were investigated for potential STAT3 binding sites. Although none of the SNPs were linked to direct STAT3 binding, analysis show that rs17149106 (-602 G > T) is located on an upstream enhancer element of NPY, where the variant is predicted to disrupt validated binding of KLF4, a known inhibitory cofactor of STAT3 and downstream effector of leptin signaling. Collectively, this study demonstrates that STAT3 signaling negatively regulates Npy transcription, and that disruption of this interaction may contribute to metabolic disorders.

Oleate restores altered autophagic flux to rescue palmitate lipotoxicity in hypothalamic neurons.

Accumulation of excess lipids in non-adipose tissues, such as the hypothalamus, is termed lipotoxicity and causative of free fatty acid-mediated pathology in metabolic disease. This study aimed to elucidate the molecular mechanisms behind oleate (OA)- and palmitate (PA)-mediated changes in hypothalamic neurons. Using the well-characterized hypothalamic neuronal cell model, mHypoE-46, we assessed gene changes through qRT-PCR, cell death with quantitative imaging, PA metabolism using stable isotope labeling, and cellular mechanisms using pharmacological modulation of lipid metabolism and autophagic flux. Palmitate (PA) disrupts gene expression, including Npy, Grp78, and Il-6 mRNA in mHypoE-46 hypothalamic neurons. Blocking PA metabolism using triacsin-C prevented the increase of these genes, implying that these changes depend on PA intracellular metabolism. Co-incubation with oleate (OA) is also potently protective and prevents cell death induced by increasing concentrations of PA. However, OA does not decrease U-13C-PA incorporation into diacylglycerol and phospholipids. Remarkably, OA can reverse PA toxicity even after significant PA metabolism and cellular impairment. OA can restore PA-mediated impairment of autophagy to prevent or reverse the accumulation of PA metabolites through lysosomal degradation, and not through other reported mechanisms. The autophagic flux inhibitor chloroquine (CQ) mimics PA toxicity by upregulating autophagy-related genes, Npy, Grp78, and Il-6, an effect partially reversed by OA. CQ also prevented the OA defense against PA toxicity, whereas the autophagy inducer rapamycin provided some protection. Thus, PA impairment of autophagic flux significantly contributes to its lipotoxicity, and OA-mediated protection requires functional autophagy. Overall, our results suggest that impairment of autophagy contributes to hypothalamic lipotoxicity.

scREMOTE: Using multimodal single cell data to predict regulatory gene relationships and to build a computational cell reprogramming model.

Cell reprogramming offers a potential treatment to many diseases, by regenerating specialized somatic cells. Despite decades of research, discovering the transcription factors that promote cell reprogramming has largely been accomplished through trial and error, a time-consuming and costly method. A computational model for cell reprogramming, however, could guide the hypothesis formulation and experimental validation, to efficiently utilize time and resources. Current methods often cannot account for the heterogeneity observed in cell reprogramming, or they only make short-term predictions, without modelling the entire reprogramming process. Here, we present scREMOTE, a novel computational model for cell reprogramming that leverages single cell multiomics data, enabling a more holistic view of the regulatory mechanisms at cellular resolution. This is achieved by first identifying the regulatory potential of each transcription factor and gene to uncover regulatory relationships, then a regression model is built to estimate the effect of transcription factor perturbations. We show that scREMOTE successfully predicts the long-term effect of overexpressing two key transcription factors in hair follicle development by capturing higher-order gene regulations. Together, this demonstrates that integrating the multimodal processes governing gene regulation creates a more accurate model for cell reprogramming with significant potential to accelerate research in regenerative medicine.

Spexin: Its role, regulation, and therapeutic potential in the hypothalamus.

Spexin is the most recently discovered member of the galanin/kisspeptin/spexin family of peptides. This 14-amino acid peptide is highly conserved and is implicated in homeostatic functions including, but not limited to, metabolism, energy homeostasis, and reproduction. Spexin is expressed by neurons in the hypothalamus, which coordinate energy homeostasis and reproduction. Critically, levels of spexin appear to be altered in disorders related to energy homeostasis and reproduction, such as obesity, diabetes, and polycystic ovarian syndrome. In this review, we discuss the evidence for the involvement of spexin in the hypothalamic control of energy homeostasis and reproduction. The anorexigenic properties of spexin have been attributed to its effects on the energy-regulating neuropeptide Y/agouti-related peptide neurons and proopiomelanocortin neurons. While the role of spexin in reproduction remains unclear, there is evidence that gonadotropin-releasing hormone expressing neurons may produce and respond to spexin. Furthermore, we discuss the disorders and concomitant treatments, which have been reported to alter spexin expression, as well as the underlying signaling mechanisms that may be involved. Finally, we discuss the biochemical basis of spexin, its interaction with its cognate receptors, and how this information can be adapted to develop therapeutics for disorders related to the alteration of energy homeostasis and reproduction.

Palmitate differentially regulates Spexin, and its receptors Galr2 and Galr3, in GnRH neurons through mechanisms involving PKC, MAPKs, and TLR4.

The function of the gonadotropin-releasing hormone (GnRH) neuron is critical to maintain reproductive function and a significant decrease in GnRH can lead to disorders affecting fertility, including hypogonadotropic hypogonadism. Spexin (SPX) is a novel hypothalamic neuropeptide that exerts inhibitory effects on reproduction and feeding by acting through galanin receptor 2 (GALR2) and galanin receptor 3 (GALR3). Fatty acids can act as nutritional signals that regulate the hypothalamic-pituitary-gonadal (HPG) axis, and elevated levels of circulating saturated fatty acids associated with high fat diet (HFD)-feeding have been shown to induce neuroinflammation, endoplasmic reticulum stress and hormonal resistance in the hypothalamus, as well as alter neuropeptide expression. We previously demonstrated that palmitate, the most common saturated fatty acid in a HFD, elevates the expression of Spx, Galr2 and Galr3 mRNA in a model of appetite-regulating neuropeptide Y hypothalamic neurons. Here, we found that Spx, Galr2 and Galr3 mRNA were also significantly induced by palmitate in a model of reproductive GnRH neurons, mHypoA-GnRH/GFP. As a follow-up to our previous report, we examined the molecular pathways by which Spx and galanin receptor mRNA was regulated in this cell line. Furthermore, we performed inhibitor studies, which revealed that the effect of palmitate on Spx and Galr3 mRNA involved activation of the innate immune receptor TLR4, and we detected differential regulation of the three genes by the protein kinases PKC, JNK, ERK, and p38. However, the intracellular metabolism of palmitate to ceramide did not appear to be involved in the palmitate-mediated gene regulation. Overall, this suggests that SPX may play a role in reproduction at the level of the hypothalamus and the pathways by which Spx, Galr2 and Galr3 are altered by fatty acids could provide insight into the mechanisms underlying reproductive dysfunction in obesity.

NAMPT and BMAL1 Are Independently Involved in the Palmitate-Mediated Induction of Neuroinflammation in Hypothalamic Neurons.

Obesity is a prominent metabolic disease that predisposes individuals to multiple comorbidities, including type 2 diabetes mellitus, cardiovascular diseases, and cancer. Elevated circulating levels of fatty acids contribute to the development of obesity, in part, by targeting the hypothalamus. Palmitate, the most abundant circulating saturated fatty acid, has been demonstrated to dysregulate NAMPT and circadian clock proteins, as well as induce neuroinflammation. These effects ultimately result in hypothalamic dysregulation of feeding behavior and energy homeostasis. NAMPT is the rate-limiting enzyme of the NAD+ salvage pathway and its expression is under the control of the circadian clock. NAD+ produced from NAMPT can modulate the circadian clock, demonstrating bidirectional interactions between circadian and metabolic pathways. Using NPY/AgRP-expressing mHypoE-46 neurons as well as the novel mHypoA-BMAL1-WT/F and mHypoA-BMAL1-KO/F cell lines, we studied whether there were any interactions between NAMPT and the core circadian clock protein BMAL1 in the palmitate-mediated induction of neuroinflammation. We report that palmitate altered Nampt, Bmal1, Per2 and the inflammatory genes Nf-κb, IκBα, Il-6, and Tlr4. Contrary to studies performed with peripheral tissues, the palmitate-mediated induction in Nampt was independent of BMAL1, and basal Nampt levels did not appear to exhibit rhythmic expression. Palmitate-induced downregulation of Bmal1 and Per2 was independent of NAMPT. However, NAMPT and BMAL1 were both involved in the regulation of Nf-κb, IκBα, Il-6, and Tlr4, as NAMPT inhibition resulted in the repression of basal Nf-κb and IκBα and normalized palmitate-mediated increases in Il-6, and Tlr4. On the other hand, BMAL1 deletion repressed basal Nf-κb, but increased basal Il-6. We conclude that NAMPT and BMAL1 do not interact at the transcriptional level in hypothalamic neurons, but are independently involved in the expression of inflammatory genes.

Palmitate and Nitric Oxide Regulate the Expression of Spexin and Galanin Receptors 2 and 3 in Hypothalamic Neurons.

Spexin (SPX) is a novel satiety factor that putatively binds the galanin receptors R2 and R3 (GalR2/R3). SPX reduces body weight, and circulating SPX is decreased in obesity. It is unknown how SPX and its receptors are regulated in the hypothalamus, critical for energy homeostasis. We therefore examined the regulation of hypothalamic Spx, GalR2 and GalR3 gene expression in mouse primary and immortalized hypothalamic neurons. We report that Spx, GalR2 and GalR3 mRNA levels were regulated by acute treatments of palmitate, a dietary saturated fatty acid, as well as the nitric oxide (NO) donor sodium nitroprusside (SNP), but through a pathway independent of cyclic GMP and protein kinase G. Additionally, the palmitate- and NO-mediated induction of Spx and galanin receptors was blocked with the PKC inhibitor k252c. Furthermore, palmitate induced mRNA levels of endoplasmic reticulum (ER) stress markers, including Chop, Grp78 and Bax/Bcl2, as well as C/ebp-ß, whereas SNP induced Bax/Bcl2 and C/ebp-ß. Transcriptional changes in Spx, GalR2, GalR3, C/ebp-ß and ER stress marker mRNAs were blocked by pre-treatment with at least one of the chemical chaperones PBA or TUDCA. We also describe the presence of OCT-1 and C/EBP-ß response elements in the 5' regulatory region of Spx and demonstrate that SNP increases binding of C/EBP-ß to this region, but not Oct-1 mRNA nor OCT-1 binding. Our findings suggest an acute modulation of anorexigenic SPX signaling by palmitate and NO. Furthermore, ER stress and C/EBP-ß appear to mediate the changes in Spx, GalR2 and GalR3 in hypothalamic neurons.

Immunization With Mycobacterium tuberculosis Antigens Encapsulated in Phosphatidylserine Liposomes Improves Protection Afforded by BCG.

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol Mycobacterium tuberculosis, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.