Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

Food perception promotes phosphorylation of MFFS131 and mitochondrial fragmentation in liver.

Liver mitochondria play a central role in metabolic adaptations to changing nutritional states, yet their dynamic regulation upon anticipated changes in nutrient availability has remained unaddressed. Here, we found that sensory food perception rapidly induced mitochondrial fragmentation in the liver through protein kinase B/AKT (AKT)-dependent phosphorylation of serine 131 of the mitochondrial fission factor (MFFS131). This response was mediated by activation of hypothalamic pro-opiomelanocortin (POMC)-expressing neurons. A nonphosphorylatable MFFS131G knock-in mutation abrogated AKT-induced mitochondrial fragmentation in vitro. In vivo, MFFS131G knock-in mice displayed altered liver mitochondrial dynamics and impaired insulin-stimulated suppression of hepatic glucose production. Thus, rapid activation of a hypothalamus-liver axis can adapt mitochondrial function to anticipated changes of nutritional state in control of hepatic glucose metabolism.

Reciprocal activity of AgRP and POMC neurons governs coordinated control of feeding and metabolism.

Agouti-related peptide (AgRP)-expressing and proopiomelanocortin (POMC)-expressing neurons reciprocally regulate food intake. Here, we combine non-interacting recombinases to simultaneously express functionally opposing chemogenetic receptors in AgRP and POMC neurons for comparing metabolic responses in male and female mice with simultaneous activation of AgRP and inhibition of POMC neurons with isolated activation of AgRP neurons or isolated inhibition of POMC neurons. We show that food intake is regulated by the additive effect of AgRP neuron activation and POMC neuron inhibition, while systemic insulin sensitivity and gluconeogenesis are differentially modulated by isolated-versus-simultaneous regulation of AgRP and POMC neurons. We identify a neurocircuit engaging Npy1R-expressing neurons in the paraventricular nucleus of the hypothalamus, where activated AgRP neurons and inhibited POMC neurons cooperate to promote food consumption and activate Th+ neurons in the nucleus tractus solitarii. Collectively, these results unveil how food intake is precisely regulated by the simultaneous bidirectional interplay between AgRP and POMC neurocircuits.

CerS6-dependent ceramide synthesis in hypothalamic neurons promotes ER/mitochondrial stress and impairs glucose homeostasis in obese mice.

Dysregulation of hypothalamic ceramides has been associated with disrupted neuronal pathways in control of energy and glucose homeostasis. However, the specific ceramide species promoting neuronal lipotoxicity in obesity have remained obscure. Here, we find increased expression of the C16:0 ceramide-producing ceramide synthase (CerS)6 in cultured hypothalamic neurons exposed to palmitate in vitro and in the hypothalamus of obese mice. Conditional deletion of CerS6 in hypothalamic neurons attenuates high-fat diet (HFD)-dependent weight gain and improves glucose metabolism. Specifically, CerS6 deficiency in neurons expressing pro-opiomelanocortin (POMC) or steroidogenic factor 1 (SF-1) alters feeding behavior and alleviates the adverse metabolic effects of HFD feeding on insulin sensitivity and glucose tolerance. POMC-expressing cell-selective deletion of CerS6 prevents the diet-induced alterations of mitochondrial morphology and improves cellular leptin sensitivity. Our experiments reveal functions of CerS6-derived ceramides in hypothalamic lipotoxicity, altered mitochondrial dynamics, and ER/mitochondrial stress in the deregulation of food intake and glucose metabolism in obesity.

Development of a genetically-encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.

Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration.

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.

HypoMap-a unified single-cell gene expression atlas of the murine hypothalamus.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders.

Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone's effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders.

Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition.

Neddylation is a ubiquitylation-like pathway that controls cell cycle and proliferation by covalently conjugating Nedd8 to specific targets. However, its role in neurons, nonreplicating postmitotic cells, remains unexplored. Here we report that Nedd8 conjugation increased during postnatal brain development and is active in mature synapses, where many proteins are neddylated. We show that neddylation controls spine development during neuronal maturation and spine stability in mature neurons. We found that neddylated PSD-95 was present in spines and that neddylation on Lys202 of PSD-95 is required for the proactive role of the scaffolding protein in spine maturation and synaptic transmission. Finally, we developed Nae1(CamKIIα-CreERT2) mice, in which neddylation is conditionally ablated in adult excitatory forebrain neurons. These mice showed synaptic loss, impaired neurotransmission and severe cognitive deficits. In summary, our results establish neddylation as an active post-translational modification in the synapse regulating the maturation, stability and function of dendritic spines.

Proteomic analysis identifies dysfunction in cellular transport, energy, and protein metabolism in different brain regions of atypical frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is an umbrella term for a heterogeneous group of young-onset dementias of uncertain prevalence and incidence worldwide. Atypical cases of FTLD with fused in sarcoma inclusions (aFTLD-U) have been described recently, but their molecular characterization is still due. Using shotgun mass spectrometry, we identified a total of 107 differentially expressed proteins in the prefrontal cortex, cerebellum and occipital lobe from aFTLD-U patients compared to controls. These proteins are involved in a range of biological pathways such as cellular transport in the prefrontal cortex, energy metabolism in the cerebellum, and protein metabolism in the occipital lobe. In addition, they were validated by selective reaction monitoring (SRM). Comparison of the aFTLD-U proteomic findings with similar studies of Alzheimer's disease and schizophrenia led to identification of proteins that may be related to dementias and psychoses, respectively. Further studies of aFTLD-U and other FTLD subtypes are warranted, although this will require intensive biobanking efforts.

Characterization of the human primary visual cortex and cerebellum proteomes using shotgun mass spectrometry-data-independent analyses.

We present the first characterization of the human occipital lobe (primary visual cortex) and cerebellum proteomes. Proteins were identified using a combination of gel electrophoresis and data-independent nanoflow liquid chromatography mass spectrometry (nLC-MS(E) ). The resulting data sets comprised 391 and 330 unique proteins in occipital lobe and cerebellum, respectively, present in at least 75% of the analyzed samples with 297 proteins found in common. These proteins have been associated previously with conditions, such as neurological disorder, progressive motor neuropathy, Parkinson's disease and schizophrenia. The unique proteins identified in the occipital lobe included the interesting finding of growth hormone and several members of the Ca²âº-dependent calmodulin kinase and serine/threonine protein phosphatase families. The complete mapping of these and other brain proteomes may help in the elucidation of neurological processes and identify potential targets for therapeutic strategies.