Ganglioside deficiency in hypothalamic POMC neurons promotes body weight gain.

BACKGROUND: Glucosylceramide synthase (GCS; gene: UDP-glucose:ceramide glucosyltransferase (Ugcg))-derived gangliosides comprise a specific class of lipids in the plasma membrane that modulate the activity of transmembrane receptors. GCS deletion in hypothalamic arcuate nucleus (Arc) neurons leads to prominent obesity. However, it has not yet been studied how ganglioside depletion affects individual Arc neuronal subpopulations. The current study investigates the effects of GCS deletion specifically in anorexigenic pro-opiomelanocortin (POMC) neurons. Additionally, we investigate insulin receptor (IR) signaling and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) binding to ATP-dependent K+ (KATP) channels of GCS-deficient POMC neurons. MATERIALS AND METHODS: We generated Ugcgf/f-Pomc-Cre mice with ganglioside deficiency in POMC neurons. Moreover, the CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 technology was used to inhibit GCS-dependent ganglioside biosynthesis in cultured mouse POMC neurons, yielding UgcgΔ-mHypoA-POMC cells that were used to study mechanistic aspects in further detail. Proximity ligation assays (PLAs) visualized interactions between gangliosides, IR, and KATP channel subunit sulfonylurea receptor-1 (SUR-1), as well as intracellular IR substrate 2 (IRS-2) phosphorylation and PIP3. RESULTS: Chow-fed Ugcgf/f-Pomc-Cre mice showed a moderate but significant increase in body weight gain and they failed to display an increase of anorexigenic neuropeptide expression during the fasting-to-re-feeding transition. IR, IRS-2, p85, and overall insulin-evoked IR and IRS-2 phosphorylation were elevated in ganglioside-depleted UgcgΔ-mHypoA-POMC neurons. A PLA demonstrated that more insulin-evoked complex formation occurred between PIP3 and SUR-1 in ganglioside-deficient POMC neurons in vitro and in vivo. CONCLUSION: Our work suggests that GCS deletion in POMC neurons promotes body weight gain. Gangliosides are required for an appropriate adaptation of anorexigenic neuropeptide expression in the Arc during the fasting-to-re-feeding transition. Moreover, gangliosides might modulate KATP channel activity by restraining PIP3 binding to the KATP channel subunit SUR-1. Increased PIP3/SUR-1 interactions in ganglioside-deficient neurons could in turn potentially lead to electrical silencing. This work highlights that gangliosides in POMC neurons of the hypothalamic Arc are important regulators of body weight.

Locked nucleic acid-based in situ hybridisation reveals miR-7a as a hypothalamus-enriched microRNA with a distinct expression pattern.

MicroRNAs (miRNAs) are short (∼22 nucleotides) noncoding RNA molecules that post-transcriptionally repress the expression of protein-coding genes by binding to 3'-untranslated regions of the target mRNAs. To identify miRNAs selectively expressed within the hypothalamus, a part of the brain that controls vital bodily functions, we employed locked nucleic acid (LNA)-fluorescent in situ hybridisation (FISH). The expression pattern of the mature miRNAs miR-7a, miR-7b, miR-137 and miR-153 in mouse brain tissue sections was investigated. Although all studied miRNAs were present in the hypothalamus, miR-7a, was the only miRNA found to be enriched in the hypothalamus, with low or no expression in other parts of the central nervous system (CNS). Within the hypothalamus, strong miR-7a expression was distinct and restricted to some hypothalamic nuclei and adjacent areas. miR-7a expression was particularly prominent in the subfornical organ, as well as the suprachiasmatic, paraventricular, periventricular, supraoptic, dorsomedial and arcuate nuclei. Identical expression patterns for miR-7a were seen in mouse and rat hypothalamus. By combining LNA-FISH with immunohistochemistry, it was shown that miR-7a was preferentially present in small orexigenic neuropeptide Y/agouti-related protein-containing-neurones located in the ventromedial aspect of the arcuate nucleus but not in large pro-opiomelanocortin/cocaine- and amphetamine-regulated transcript-containing anorexigenic neurones of the ventrolateral part of the arcuate nucleus. The limited and distinct expression of miR-7a in the CNS suggests that miR-7a has a role in post-transcriptional regulation in hypothalamic neurones. Particularly strong expression of miR-7a in neurones located in the ventromedial division of the arcuate nucleus, a subregion with a weak blood-brain barrier, raises the possibility that miR-7a is influenced by circulating hormones and is a regulator of the genes involved in body weight control.