Identification and Characterization of Leptin Responsive Neurons Involved in the Metabolic Control of Reproduction

Reproductive function is an adaptive system that is critical for the survival of all species. In vertebrates, fertility (the ability to reproduce) is regulated by the hypothalamic-pituitary-gonadal (HPG) axis. This axis is centrally driven by the release of gonadotropin releasing hormone (GnRH) from the hypothalamus. There are many endogenous and exogenous factors that can influence the HPG axis; here I focus on metabolic regulation of fertility. The adipokine leptin, primarily produced by adipose tissue, circulates throughout the body in concentrations relative to the amount of energy stored in the adipose tissue. Leptin plays a critical role in the hypothalamic control of energy balance, but also has profound effects on central regulation of reproduction. The effect of leptin on the HPG axis is indirect, as GnRH neurons do not express leptin receptor (Quennell et al., 2009). Therefore it is assumed that intermediate leptin responsive neurons must exist that provides leptin-to-GnRH signaling. Trying to elucidate this neuronal pathway has been a challenge to many researchers in the field over the past years, as Lepr neurons exhibit a widespread and heterogynous distribution throughout the brain. Here, I investigated different neuronal pathways and neurotransmitters that might relay the signal of leptin onto the HPG axis. I used transgenic mouse models to visualize Lepr neurons, and combined that with retrograde tract tracing to identify leptin responsive inputs to the region of GnRH neurons. Additionally, conditional deletion of the receptors for leptin from neuronal nitric oxide synthase (nNOS), glutamate, and γ-aminobutyric acid (GABA) neuron populations was used to assess the effects on reproductive function. With these experiments I investigated the role of these neurons and signaling molecules in mediating leptin-to-GnRH signaling. From these studies I found that Lepr neurons, particularly from the arcuate nucleus of the hypothalamus (Arc), provide neuronal input to the region of the GnRH neurons. Recent literature shows that leptin signaling in nNOS neurons might play a role in the metabolic regulation of fertility, however my attempt to conditionally delete Lepr from nNOS neurons was unsuccessful, presumably due to an incompatibility of the Cre and LoxP genes used. I found that leptin signaling in glutamate neurons is not critical for reproductive functioning. Most interestingly I found that GABA neuron specific Lepr knockout caused significant disruptions in reproductive functioning. Both females and males displayed delayed puberty onset. Observations of various adult fertility parameters revealed that these knockout animals have decreased fecundity and females had disordered estrous cycles. These experiments showed that leptin signaling in GABAergic neurons plays a critical role in the metabolic regulation of fertility. These exciting results were followed up by measuring changes in hypothalamic gene expression between control and GABA specific Lepr knockout females. The GABA specific Lepr knockout females showed a significant increase of Agrp gene expression in the Arc, and Npy gene expression in the LHA. These results will focus future research (including Lepr knockout or rescue experiments) to GABAergic leptin receptor-expressing AgRP neurons in Arc. The experiments presented in this thesis form an important step in explaining the mechanisms by which central leptin signaling modulates the reproductive system. This work helps to narrow down the location and possible identity of mediators of leptin-to-GnRH signaling. Future research now, should be more focused on the GABAergic Lepr cells

Identification and Characterization of Leptin Responsive Neurons Involved in the Metabolic Control of Reproduction

Reproductive function is an adaptive system that is critical for the survival of all species. In vertebrates, fertility (the ability to reproduce) is regulated by the hypothalamic-pituitary-gonadal (HPG) axis. This axis is centrally driven by the release of gonadotropin releasing hormone (GnRH) from the hypothalamus. There are many endogenous and exogenous factors that can influence the HPG axis; here I focus on metabolic regulation of fertility. The adipokine leptin, primarily produced by adipose tissue, circulates throughout the body in concentrations relative to the amount of energy stored in the adipose tissue. Leptin plays a critical role in the hypothalamic control of energy balance, but also has profound effects on central regulation of reproduction. The effect of leptin on the HPG axis is indirect, as GnRH neurons do not express leptin receptor (Quennell et al., 2009). Therefore it is assumed that intermediate leptin responsive neurons must exist that provides leptin-to-GnRH signaling. Trying to elucidate this neuronal pathway has been a challenge to many researchers in the field over the past years, as Lepr neurons exhibit a widespread and heterogynous distribution throughout the brain. Here, I investigated different neuronal pathways and neurotransmitters that might relay the signal of leptin onto the HPG axis. I used transgenic mouse models to visualize Lepr neurons, and combined that with retrograde tract tracing to identify leptin responsive inputs to the region of GnRH neurons. Additionally, conditional deletion of the receptors for leptin from neuronal nitric oxide synthase (nNOS), glutamate, and γ-aminobutyric acid (GABA) neuron populations was used to assess the effects on reproductive function. With these experiments I investigated the role of these neurons and signaling molecules in mediating leptin-to-GnRH signaling. From these studies I found that Lepr neurons, particularly from the arcuate nucleus of the hypothalamus (Arc), provide neuronal input to the region of the GnRH neurons. Recent literature shows that leptin signaling in nNOS neurons might play a role in the metabolic regulation of fertility, however my attempt to conditionally delete Lepr from nNOS neurons was unsuccessful, presumably due to an incompatibility of the Cre and LoxP genes used. I found that leptin signaling in glutamate neurons is not critical for reproductive functioning. Most interestingly I found that GABA neuron specific Lepr knockout caused significant disruptions in reproductive functioning. Both females and males displayed delayed puberty onset. Observations of various adult fertility parameters revealed that these knockout animals have decreased fecundity and females had disordered estrous cycles. These experiments showed that leptin signaling in GABAergic neurons plays a critical role in the metabolic regulation of fertility. These exciting results were followed up by measuring changes in hypothalamic gene expression between control and GABA specific Lepr knockout females. The GABA specific Lepr knockout females showed a significant increase of Agrp gene expression in the Arc, and Npy gene expression in the LHA. These results will focus future research (including Lepr knockout or rescue experiments) to GABAergic leptin receptor-expressing AgRP neurons in Arc. The experiments presented in this thesis form an important step in explaining the mechanisms by which central leptin signaling modulates the reproductive system. This work helps to narrow down the location and possible identity of mediators of leptin-to-GnRH signaling. Future research now, should be more focused on the GABAergic Lepr cells

Molecular Signature of Neuroinflammation Induced in Cytokine-Stimulated Human Cortical Spheroids

Crucial in the pathogenesis of neurodegenerative diseases is the process of neuroinflammation that is often linked to the pro-inflammatory cytokines Tumor necrosis factor alpha (TNFα) and Interleukin-1beta (IL-1β). Human cortical spheroids (hCSs) constitute a valuable tool to study the molecular mechanisms underlying neurological diseases in a complex three-dimensional context. We recently designed a protocol to generate hCSs comprising all major brain cell types. Here we stimulate these hCSs for three time periods with TNFα and with IL-1β. Transcriptomic analysis reveals that the main process induced in the TNFα- as well as in the IL-1β-stimulated hCSs is neuroinflammation. Central in the neuroinflammatory response are endothelial cells, microglia and astrocytes, and dysregulated genes encoding cytokines, chemokines and their receptors, and downstream NFκB- and STAT-pathway components. Furthermore, we observe sets of neuroinflammation-related genes that are specifically modulated in the TNFα-stimulated and in the IL-1β-stimulated hCSs. Together, our results help to molecularly understand human neuroinflammation and thus a key mechanism of neurodegeneration

Key role for lipids in cognitive symptoms of schizophrenia

International audienceSchizophrenia (SZ) is a psychiatric disorder with a convoluted etiology that includes cognitive symptoms, which arise from among others a dysfunctional dorsolateral prefrontal cortex (dlPFC). In our search for the molecular underpinnings of the cognitive deficits in SZ, we here performed RNA sequencing of gray matter from the dlPFC of SZ patients and controls. We found that the differentially expressed RNAs were enriched for mRNAs involved in the Liver X Receptor/Retinoid X Receptor (LXR/RXR) lipid metabolism pathway. Components of the LXR/RXR pathway were upregulated in gray matter but not in white matter of SZ dlPFC. Intriguingly, an analysis for shared genetic etiology, using two SZ genome-wide association studies (GWASs) and GWAS data for 514 metabolites, revealed genetic overlap between SZ and acylcarnitines, VLDL lipids, and fatty acid metabolites, which are all linked to the LXR/RXR signaling pathway. Furthermore, analysis of structural T1-weighted magnetic resonance imaging in combination with cognitive behavioral data showed that the lipid content of dlPFC gray matter is lower in SZ patients than in controls and correlates with a tendency towards reduced accuracy in the dlPFC-dependent task-switching test. We conclude that aberrations in LXR/RXR-regulated lipid metabolism lead to a decreased lipid content in SZ dlPFC that correlates with reduced cognitive performance

Cannabinoid Modulation of Midbrain Urocortin 1 Neurones During Acute and Chronic Stress

Neurones in the centrally projecting Edinger-Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour. © 2012 The Authors. Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe