Subset Of Hypothalamic Neurons Research Articles

Structural insights into the mechanism of leptin receptor activation

Leptin is an adipocyte-derived protein hormone that promotes satiety and energy homeostasis by activating the leptin receptor (LepR)–STAT3 signaling axis in a subset of hypothalamic neurons. Leptin signaling is dysregulated in obesity, however, where appetite remains elevated despite high levels of circulating leptin. To gain insight into the mechanism of leptin receptor activation, here we determine the structure of a stabilized leptin-bound LepR signaling complex using single particle cryo-EM. The structure reveals an asymmetric architecture in which a single leptin induces LepR dimerization via two distinct receptor-binding sites. Analysis of the leptin–LepR binding interfaces reveals the molecular basis for human obesity-associated mutations. Structure-based design of leptin variants that destabilize the asymmetric LepR dimer yield both partial and biased agonists that partially suppress STAT3 activation in the presence of wild-type leptin and decouple activation of STAT3 from LepR negative regulators. Together, these results reveal the structural basis for LepR activation and provide insights into the differential plasticity of signaling pathways downstream of LepR.

Neurobiology: To care or not to care.

The behaviour of adult mice towards pups varies depending on gender and sexual experience. The activity of a population of neurons in the hypothalamus of the brain has now been found to regulate these differing responses. See Article p.325 Sexual experience brings radical change in how male mice behave with pups — virgin males attack them whereas experienced fathers display parental care. Now Catherine Dulac and colleagues show that virgin males with impaired pheromone sensing do not attack pups. Even more spectacularly, the authors identify a subset of hypothalamic neurons expressing the neuropeptide galanin whose activation in virgin males suppresses aggression and induces pup grooming. Genetic ablation of galanin-expressing neurons results in dramatic impairment of both maternal and paternal responses to pups. The molecular identification of a neuronal population controlling parental behaviour in both males and females is likely to attract wide interest from both neuroscientists and ethologists.

Understanding the Direct Synaptic Effects of Estradiol

More than 35 years ago, Kelly and coworkers (1) demonstrated that iontophoretic application of an estradiol ester altered the firing rates of a subset of hypothalamic neurons in a matter of milliseconds, an effect that appeared to be stereospecific in that responses were observed to esters of 17 -estradiol, but not its 17 isomer. These observations accompanied the almost simultaneous demonstration by Pietras and Szego (2) that endometrial cells had estradiol recognition sites on their plasma membranes. Kelly et al (1) proposed that neurons might also express stereospecific membrane receptor sites, capable of mediating rapid electrophysiological responses to changes in ovarian estradiol secretion. Progress in characterizing these putative membrane receptors was initially slow, because most work continued to focus on estrogen receptor (ER) actions in the cell nucleus and the regulation of transcriptional response mechanisms. This situation changed dramatically in the early 1990s with the demonstration of rapid effects of estradiol on a number of intracellular signaling pathways (3, 4) as well as direct evidence of ER-like immunoreactivity in dendrites, axon terminals, and postsynaptic densities in the brain (5–7) (reviewed in Blaustein, Ref. 8). Over the ensuing 20 years, multiple potential receptor systems for estradiol have been identified in the central nervous system, signaling via a number of different intracellular pathways (reviewed in Micevych and Christensen, Ref. 9). Studies in cells transfected with vectors expressing ER or ER have shown that whereas newly synthesized receptors concentrate in the cell nucleus, they also incorporate into the plasma membrane (10) where they can initiate estradiol-dependent changes in ERK phosphorylation (11), suggesting that plasma membrane and nuclear ER or ER may be derived from a common source. The demonstration that ER immunoreactivity is present in multiple neuronal cellular compartments raises the question of how the distribution of extranuclear ER is controlled. Are ERs associated with specific cellular organelles or do synaptic and dendritic ERs represent receptors en passant to the plasma membrane? Studies in nonneuronal systems have demonstrated that that ER is trafficked to the plasma membrane via a caveolin-dependent process (12) where it can be anchored in place by palmitoylation (13). Is the same true for ER in neurons? Is the density of ER in the plasma membrane regulated independently of nuclear receptor concentrations? Is ER distributed evenly throughout neuronal cell membranes or is it concentrated specifically in target sites where it can both respond to locally synthesized steroids and exert rapid effects on neurotransmission? The paper by Tabatadze et al (14) in this issue of Endocrinology begins to answer some of these questions. Using tissue fractionationtechniques, theauthorsdemonstrate that ER is concentrated in synaptosomes, particularly in association with synaptic vesicles and neuropeptide-containing large dense-core vesicles. Immunoisolation experiments showthatER is associatedwithbothgammaaminobutryic acid (GABA)and glutamate-containing vesicles. On Western blots, synaptosomal ER immunoreactivity migrates as two bands at approximately 66 and 70 kDa, the ratio of the higher to lower molecular mass forms being greatly decreased by phosphatase treatment, suggesting that the 70kDa band represents a phosphorylated form of the receptor. Finally, treatment of hippocampal slices in vitro was found to decrease the quantity of ER associated with synaptosomal membranes, via a depalmitoylation-dependent mechanism. Overall, the results suggest that ER associates with synaptosomal membranes via a palmitoyla-

Cross-talk between metabolism and reproduction: the role of POMC and SF1 neurons

Energy homeostasis and reproduction require tight coordination, but the mechanisms underlying their interaction are not fully understood. Two sets of hypothalamic neurons, namely pro-opiomelanocortin (POMC) neurons in the arcuate nucleus and steroidogenic factor-1 (SF1) neurons in the ventromedial hypothalamic nucleus, are emerging as critical nodes where metabolic and reproductive signals communicate. This view is supported by recent genetic studies showing that disruption of metabolic signals (e.g., leptin and insulin) or reproductive signals (e.g., estradiol) in these neurons leads to impaired regulation of both energy homeostasis and fertility. In this review, we will examine the potential mechanisms of neuronal communication between POMC, SF1, and gonadotropin-releasing hormone neurons in the regulation of metabolism and reproduction.

Embryonic GnRH Signaling Is Necessary for Maturation of the Male Reproductive Axis.

Gonadotropin-releasing hormone (GnRH) signaling regulates reproductive physiology in mammals. GnRH is released by a subset of hypothalamic neurons and binds to its receptor (GnRHR) on gonadotropes in the anterior pituitary gland to control production and secretion of gonadotropins that in turn regulate the activity of the gonads. Central control of reproduction is well understood in adult animals, however GnRH signaling has also been implicated in the development of the reproductive axis. To investigate the role of GnRH signaling during development, we selectively ablated GnRHR expressing cells in mice. This genetic strategy permitted us to identify an essential stage in male reproductive axis development, which depends on embryonic GnRH signaling. Our experiments revealed a striking dichotomy in the gonadotrope population of the fetal anterior pituitary gland. We show, that only luteinizing hormone (LH)- but not follicle-stimulating hormone (FSH)-expressing gonadotropes express the GnRHR at embryonic day 16.75. Furthermore, we demonstrate that an embryonic increase in LH secretion is needed to promote development of FSH-expressing gonadotropes, which might be mediated by paracrine interactions within the pituitary. Moreover, migration of GnRH neurons into the hypothalamus appeared normal with appropriate axonal connections to the median eminence, providing genetic evidence against autocrine regulation of GnRH neurons. Surprisingly, genetic ablation of GnRHR expressing cells significantly increased the number of GnRH neurons in the anterior hypothalamus, suggesting an unexpected role of GnRH signaling in establishing the size of the GnRH neuronal population. Our experiments define a functional role of embryonic GnRH signaling for the first time. (platform)

Immunohistochemical localization of chemokine CXCL14 in rat hypothalamic neurons

We immunohistochemically investigated the distribution of CXCL14, also called BRAK protein in the rat hypothalamus using anti-human CXCL14 serum. CXCL14-immunoreactive somata were localized in the periventricular area and paraventricular and supraoptic hypothalamic nuclei. In the former, immunoreactive neuronal somata, confirmed by double staining with a neuronal marker, NeuN, contained diffuse CXCL14-like immunoreactivity in their perikarya. In contrast, immunoreactive somata in the latter contained immunoreactive puncta within their perikarya. Very dense immunoreactive fibers and puncta were seen in the median eminence. Dense immunoreactive fibers were seen in the arcuate nucleus and ventromedial hypothalamic nucleus. Other hypothalamic areas contained a few immunoreactive fibers and puncta. These results demonstrated for the first time that CXCL14 protein is present in a subset of hypothalamic neurons and suggest that CXCL14 participates in hypothalamic functions such as control of autonomic nervous systems and/or participates in immune cell recruitment via the median eminence.

Embryonic gonadotropin-releasing hormone signaling is necessary for maturation of the male reproductive axis

Gonadotropin-releasing hormone (GnRH) signaling regulates reproductive physiology in mammals. GnRH is released by a subset of hypothalamic neurons and binds to GnRH receptor (GnRHR) on gonadotropes in the anterior pituitary gland to control production and secretion of gonadotropins that in turn regulate the activity of the gonads. Central control of reproduction is well understood in adult animals, but GnRH signaling has also been implicated in the development of the reproductive axis. To investigate the role of GnRH signaling during development, we selectively ablated GnRHR-expressing cells in mice. This genetic strategy permitted us to identify an essential stage in male reproductive axis development, which depends on embryonic GnRH signaling. Our experiments revealed a striking dichotomy in the gonadotrope population of the fetal anterior pituitary gland. We show that luteinizing hormone-expressing gonadotropes, but not follicle-stimulating hormone-expressing gonadotropes, express the GnRHR at embryonic day 16.75. Furthermore, we demonstrate that an embryonic increase in luteinizing hormone secretion is needed to promote development of follicle-stimulating hormone-expressing gonadotropes, which might be mediated by paracrine interactions within the pituitary. Moreover, migration of GnRH neurons into the hypothalamus appeared normal with appropriate axonal connections to the median eminence, providing genetic evidence against autocrine regulation of GnRH neurons. Surprisingly, genetic ablation of GnRHR expressing cells significantly increased the number of GnRH neurons in the anterior hypothalamus, suggesting an unexpected role of GnRH signaling in establishing the size of the GnRH neuronal population. Our experiments define a functional role of embryonic GnRH signaling.

Increased E4 Activity in Mice Leads to Ubiquitin-containing Aggregates and Degeneration of Hypothalamic Neurons Resulting in Obesity

Obesity has become a serious worldwide public health problem. Although neural degeneration in specific brain regions has been suggested to contribute to obesity phenotype in humans, a causal relationship between these two conditions has not been demonstrated experimentally. We now show that E4B (also known as UFD2a), a mammalian ubiquitin chain elongation factor (E4), induces the formation of intracellular aggregates positive for ubiquitin and the adaptor protein p62 when overexpressed in cultured cells or the brain. Mice transgenic for E4B manifested neural degeneration in association with aggregate formation, and they exhibited functional impairment specifically in a subset of hypothalamic neurons that regulate food intake and energy expenditure, resulting in development of hyperphagic obesity and related metabolic abnormalities. The neural pathology of E4B transgenic mice was similar to that of human neurodegenerative diseases associated with the formation of intracellular ubiquitin-positive deposits, indicating the existence of a link between such diseases and obesity and related metabolic disorders. Our findings thus provide experimental evidence for a role of hypothalamic neurodegeneration in obesity, and the E4B transgenic mouse should prove to be a useful animal model for studies of the relationship between neurodegenerative diseases and obesity.

Huntingtin-associated Protein-1 Deficiency in Orexin-producing Neurons Impairs Neuronal Process Extension and Leads to Abnormal Behavior in Mice

Huntingtin-associated protein-1 (Hap1) is a neuronal protein that associates with huntingtin, the Huntington disease protein. Although Hap1 and huntingtin are known to be involved in intracellular trafficking, whether and how the impairment of Hap1-associated trafficking leads to neurological pathology and symptoms remain to be seen. As Hap1 is enriched in neuronal cells in the brain, addressing this issue is important in defining the role of defective intracellular trafficking in the selective neuropathology associated with Hap1 dysfunction. Here, we find that Hap1 is abundantly expressed in orexin (hypocretin)-producing neurons (orexin neurons), which are distinctly distributed in the hypothalamus and play an important role in the regulation of feeding and behavior. We created conditional Hap1 knock-out mice to selectively deplete Hap1 in orexin neurons via the Cre-loxP system. These mice show process fragmentation of orexin neurons and reductions in food intake, body weight, and locomotor activity. Sucrose density gradient fractionation reveals that loss of Hap1 in the mouse brain also reduces the distribution of trafficking protein complexes and cargo proteins in the fractions that are enriched in synaptosomes. These results suggest that Hap1 is critical for the transport of multiple proteins to the nerve terminals to maintain the integrity of neuronal processes and that selective disruption of the processes of orexin neurons can cause abnormal feeding and locomotor activity.

SOX3 is required during the formation of the hypothalamo-pituitary axis.

The pituitary develops from the interaction of the infundibulum, a region of the ventral diencephalon, and Rathke's pouch, a derivative of oral ectoderm. Postnatally, its secretory functions are controlled by hypothalamic neurons, which also derive from the ventral diencephalon. In humans, mutations affecting the X-linked transcription factor SOX3 are associated with hypopituitarism and mental retardation, but nothing is known of their etiology. We find that deletion of Sox3 in mice leads to defects of pituitary function and of specific central nervous system (CNS) midline structures. Cells in the ventral diencephalon, where Sox3 is usually highly expressed, have altered properties in mutant embryos, leading to abnormal development of Rathke's pouch, which does not express the gene. Pituitary and hypothalamic defects persist postnatally, and SOX3 may also function in a subset of hypothalamic neurons. This study shows how sensitive the pituitary is to subtle developmental defects and how one gene can act at several levels in the hypothalamic-pituitary axis.

Obese and sleepy without orexin

Using novel genetic techniques to cause degeneration of a subset of hypothalamic neurons in mice, researchers from the University of Tsukuba in Japan and the University of Texas at Dallas have shown that orexin-containing cells regulate sleep/wake states and energy metabolism. Orexins, or hypocretins, are neuropeptides expressed in the lateral hypothalamic area implicated in both feeding behavior and sleep dysfunction. A toxic Machado–Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch was linked to an orexin-specific promotor to cause postnatal degeneration of orexinergic neurons in transgenic mice. These mice exhibited a phenotype similar to human narcolepsy, including premature onset of REM sleep, cataplexic arrests, and fragmented wake and sleep episodes. Furthermore, these mice also developed late-onset obesity, despite eating less. This study suggests these neurons regulate wakefulness, energy and metabolism in complex ways. The full paper is in the May issue of Neuron. LO

Viral mapping of feeding circuits

Using a modified pseudorabies virus combined with Cre recombinase technology, researchers from Rockefeller and Princeton Universities have mapped brain circuits involved in feeding and satiety. The virus, which was engineered so that it would encode green fluorescent protein (GFP), was injected into the arcuate nucleus of mice engineered to express Cre in neuropeptide Y or leptin receptor cells. Only virus taken up into these neurons replicates and moves retrogradely through synaptic contacts, so researchers were able to visualize the upstream neural inputs into the neuropeptide Y or leptin receptor cells – a specific subset of hypothalamic neurons involved in feeding. Higher cortical centers, as well as the hypothalamus, amygdala and brainstem were labeled in this study, indicating the anatomic basis of the complex factors controlling decisions about whether or not to eat. The paper is published in the March 30 issue of Science. LO

Estrogen and Tamoxifen Differentially Regulate Beta-Endorphin and cFos Expression and Neuronal Colocalization in the Arcuate Nucleus of the Rat

Estrogen regulates hypothalamic gene expression, synthesis and release of the endogenous opioid peptide β-endorphin (βEND), although a consensus estrogen response element sequence has not been identified in the rat proopiomelanocortin (POMC) gene. POMC gene expression is also regulated by the activation of AP-1 promoter elements, which are known to be estrogen sensitive. The present studies examine whether estrogen modulates the hypothalamic POMC system through a non-classical mechanism involving AP-1 binding proteins such as cFos. Immunohistochemical double-labeling for βEND and cFos was used and immunoreactive (-ir) populations were quantified in the arcuate nucleus and periarcuate area across time using unbiased stereological methods. Ovariectomized rats were injected with 50 µg estradiol (E₂), 500 µg tamoxifen citrate (TAM) or both (E₂+TAM) and were perfused 1, 2, 4 or 48 h later. E₂ rapidly increased numbers of cFos-ir, βEND-ir and doubly-labeled cells after 4 h, and the number of βEND-ir cells remained high 48 h later, suggesting that the stimulatory effects of cFos on POMC in the hypothalamus persist after the cFos signal decays. Treatment with TAM alone did not affect the numbers of immunoreactive cells, although E₂+TAM blocked the E₂-mediated induction in all immunoreactive populations. Similar effects were seen at the transcriptional level. E₂ increased hypothalamic POMC mRNA after 4 h, while TAM treatment or coadministration of E₂+TAM did not significantly change the levels of POMC mRNA. Cellular colocalization of βEND-ir and cFos-ir supports a possible intracellular co-regulation of these peptides by an estrogen-dependent mechanism within a subset of hypothalamic neurons. It does not, however, appear that E₂ acts directly through an AP-1 site within the POMC gene.

Bovine Oxytocin Transgenes in Mice: HYPOTHALAMIC EXPRESSION, PHYSIOLOGICAL REGULATION, AND INTERACTIONS WITH THE VASOPRESSIN GENE

To gain insights into the molecular mechanisms that restrict the expression of the oxytocin gene to anatomically defined groups of neurons in the hypothalamus, we generated transgenic mice bearing bovine oxytocin genomic fragments. Appropriate neuron-specific and physiological regulation was observed in mice bearing transgene bOT3.5, which consists of the oxytocin structural gene flanked by 0.6 kilobase pair (kbp) of upstream and 1.9 kbp of downstream sequences. bOT3.5 is expressed in oxytocin magnocellular neurons in the mouse supraoptic nucleus and paraventricular nucleus, but transgene RNAs are excluded from vasopressin neurons. Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increased the abundance of bovine oxytocin transcripts in the supraoptic nucleus, but not in the paraventricular nucleus, in parallel with the endogenous mouse oxytocin RNA. Surprisingly, mimicry of the endogenous oxytocin gene expression pattern was lost with larger transgenes. Addition of 0.7 kbp of contiguous downstream sequences (transgene bOT) or linkage to the bovine vasopressin gene (transgene VP-B/bOT3.5) repressed hypothalamic expression. No mice were derived bearing transgene bOT6.4, which consists of the oxytocin structural gene flanked by 3 kbp of upstream and 2.6 kbp of downstream sequences, suggesting that the presence of this DNA is detrimental to normal embryonic development. These data suggest that while bOT3.5 contains sufficient cis-acting sequences to mediate expression to particular subsets of hypothalamic neurons, the overall regulation of the oxytocin gene is governed by multiple interacting enhancers and repressors.

Non‐NMDA glutamate receptors are present throughout the primate hypothalamus

To determine the distributions of glutamate receptors throughout the macaque hypothalamus, we utilized highly specific antipeptide antibodies to visualize alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits (GluR1, GluR2 and GluR3 [designated as GluR2/3], and GluR4); kainate receptor subunits (GluR6 and GluR7, [designated as GluR6/7]), and a metabotropic receptor (mGluR1 alpha). The results indicate that these glutamate receptors are distributed differentially throughout the monkey hypothalamus. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors are the dominant non-N-methyl-D-aspartate glutamate receptors within the monkey hypothalamus, and the GluR2 subunit is most abundant. GluR1-immunoreactive neurons and neuropil are observed predominantly in the tuberal and mammillary nuclei. GluR2/3-immunoreactive neurons and neuropil have a broader distribution within preoptic, anterior, tuberal, and caudal regions. Separate (but partially overlapping) distributions of GluR1- and GluR2/3-immunoreactive neurons were found, suggesting that the GluR1, GluR2, and/or GluR3 subunits may be coexpressed in subsets of hypothalamic neurons. In contrast, GluR4 immunoreactivity was expressed minimally within monkey hypothalamus. GluR6/7 immunoreactivity was enriched selectively within the suprachiasmatic nucleus. mGluR1 alpha immunoreactivity was present in the mammillary complex. The localization of non-N-methyl-D-aspartate glutamate receptor subunits to neurons throughout the macaque hypothalamus provides further evidence for the glutamatergic regulation of neuroendocrine, autonomic, and limbic circuits. Differential distributions of glutamate receptor subunits may increase the dynamic range of the effects of presynaptic glutamate, allowing for the regulation of several distinct functions subserved by hypothalamic neurons.