Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability
Bengani, H;Grozeva, D;Moyon, L;Bhatia, S;Louros, SR;Hope, J;Jackson, A;Prendergast, JG;Owen, LJ;Naville, M;Rainger, J;Grimes, G;Halachev, M;Murphy, LC;Spasic-Boskovic, O;van Heyningen, V;Kind, P;Abbott, CM;Osterweil, E;Raymond, FL;Roest Crollius, H;FitzPatrick, DR;
PMID: 34388204 | DOI: 10.1371/journal.pone.0256181
Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use.
Kouvaros, S;Bizup, B;Solis, O;Kumar, M;Ventriglia, E;Curry, FP;Michaelides, M;Tzounopoulos, T;
PMID: 37294760 | DOI: 10.1126/sciadv.adf3525
Synaptic zinc is a neuromodulator that shapes synaptic transmission and sensory processing. The maintenance of synaptic zinc is dependent on the vesicular zinc transporter, ZnT3. Hence, the ZnT3 knockout mouse has been a key tool for studying the mechanisms and functions of synaptic zinc. However, the use of this constitutive knockout mouse has notable limitations, including developmental, compensatory, and brain and cell type specificity issues. To overcome these limitations, we developed and characterized a dual recombinase transgenic mouse, which combines the Cre and Dre recombinase systems. This mouse allows for tamoxifen-inducible Cre-dependent expression of exogenous genes or knockout of floxed genes in ZnT3-expressing neurons and DreO-dependent region and cell type-specific conditional ZnT3 knockout in adult mice. Using this system, we reveal a neuromodulatory mechanism whereby zinc release from thalamic neurons modulates N-methyl-d-aspartate receptor activity in layer 5 pyramidal tract neurons, unmasking previously unknown features of cortical neuromodulation.
Santana-Varela, S;Bogdanov, Y;Gossage, S;Okorokov, A;Li, S;de Clauser, L;Alves-Simoes, M;Sexton, J;Iseppon, F;Luiz, A;Zhao, J;Wood, J;Cox, J;
| DOI: 10.12688/wellcomeopenres.17090.1
Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.
Barry DM, Liu XT, Liu B, Liu XY, Gao F, Zeng X, Liu J, Yang Q, Wilhelm S, Yin J, Tao A, Chen ZF
PMID: 32170060 | DOI: 10.1038/s41467-020-15230-y
Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission
Maynard KR, Kardian A, Hill JL, Mai Y, Barry B, Hallock HL, Jaffe AE, Martinowich K
PMID: 31941661 | DOI: 10.1523/ENEURO.0310-19.2019
Brain-derived neurotrophic factor (BDNF) signals through its cognate receptor tropomyosin receptor kinase B (TrkB) to promote the function of several classes of inhibitory interneurons. We previously reported that loss of BDNF-TrkB signaling in cortistatin (Cort)-expressing interneurons leads to behavioral hyperactivity and spontaneous seizures in mice. We performed bulk RNA sequencing (RNA-seq) from the cortex of mice with disruption of BDNF-TrkB signaling in cortistatin interneurons, and identified differential expression of genes important for excitatory neuron function. Using translating ribosome affinity purification and RNA-seq, we define a molecular profile for Cort-expressing inhibitory neurons and subsequently compare the translatome of normal and TrkB-depleted Cort neurons, revealing alterations in calcium signaling and axon development. Several of the genes enriched in Cort neurons and differentially expressed in TrkB-depleted neurons are also implicated in autism and epilepsy. Our findings highlight TrkB-dependent molecular pathways as critical for the maturation of inhibitory interneurons and support the hypothesis that loss of BDNF signaling in Cort interneurons leads to altered excitatory/inhibitory balance
Dietschi, Q;Tuberosa, J;Fodoulian, L;Boillat, M;Kan, C;Codourey, J;Pauli, V;Feinstein, P;Carleton, A;Rodriguez, I;
PMID: 36383665 | DOI: 10.1126/sciadv.abn7450
Rodents perceive pheromones via vomeronasal receptors encoded by highly evolutionarily dynamic Vr and Fpr gene superfamilies. We report here that high numbers of V1r pseudogenes are scattered in mammalian genomes, contrasting with the clustered organization of functional V1r and Fpr genes. We also found that V1r pseudogenes are more likely to be expressed when located in a functional V1r gene cluster than when isolated. To explore the potential regulatory role played by the association of functional vomeronasal receptor genes with their clusters, we dissociated the mouse Fpr-rs3 from its native cluster via transgenesis. Singular and specific transgenic Fpr-rs3 transcription was observed in young vomeronasal neurons but was only transient. Our study of natural and artificial dispersed gene duplications uncovers the existence of transcription-stabilizing elements not coupled to vomeronasal gene units but rather associated with vomeronasal gene clusters and thus explains the evolutionary conserved clustered organization of functional vomeronasal genes.
Ito, N;Takatsu, A;Ito, H;Koike, Y;Yoshioka, K;Kamei, Y;Imai, SI;
PMID: 35905718 | DOI: 10.1016/j.celrep.2022.111131
Sarcopenia and frailty are urgent socio-economic problems worldwide. Here we demonstrate a functional connection between the lateral hypothalamus (LH) and skeletal muscle through Slc12a8, a recently identified nicotinamide mononucleotide transporter, and its relationship to sarcopenia and frailty. Slc12a8-expressing cells are mainly localized in the LH. LH-specific knockdown of Slc12a8 in young mice decreases activity-dependent energy and carbohydrate expenditure and skeletal muscle functions, including muscle mass, muscle force, intramuscular glycolysis, and protein synthesis. LH-specific Slc12a8 knockdown also decreases sympathetic nerve signals at neuromuscular junctions and β2-adrenergic receptors in skeletal muscle, indicating the importance of the LH-sympathetic nerve-β2-adrenergic receptor axis. LH-specific overexpression of Slc12a8 in aged mice significantly ameliorates age-associated decreases in energy expenditure and skeletal muscle functions. Our results highlight an important role of Slc12a8 in the LH for regulation of whole-body metabolism and skeletal muscle functions and provide insights into the pathogenesis of sarcopenia and frailty during aging.
Coverdell, TC;Abraham-Fan, RJ;Wu, C;Abbott, SBG;Campbell, JN;
PMID: 35705034 | DOI: 10.1016/j.celrep.2022.110962
Motor control of the striated esophagus originates in the nucleus ambiguus (nAmb), a vagal motor nucleus that also contains upper airway motor neurons and parasympathetic preganglionic neurons for the heart and lungs. We disambiguate nAmb neurons based on their genome-wide expression profiles, efferent circuitry, and ability to control esophageal muscles. Our single-cell RNA sequencing analysis predicts three molecularly distinct nAmb neuron subtypes and annotates them by subtype-specific marker genes: Crhr2, Vipr2, and Adcyap1. Mapping the axon projections of the nAmb neuron subtypes reveals that Crhr2nAmb neurons innervate the esophagus, raising the possibility that they control esophageal muscle function. Accordingly, focal optogenetic stimulation of cholinergic Crhr2+ fibers in the esophagus results in contractions. Activating Crhr2nAmb neurons has no effect on heart rate, a key parasympathetic function of the nAmb, whereas activating all of the nAmb neurons robustly suppresses heart rate. Together, these results reveal a genetically defined circuit for motor control of the esophagus.
Ventral pallidum DRD3 potentiates a pallido-habenular circuit driving accumbal dopamine release and cocaine seeking
Pribiag, H;Shin, S;Wang, EH;Sun, F;Datta, P;Okamoto, A;Guss, H;Jain, A;Wang, XY;De Freitas, B;Honma, P;Pate, S;Lilascharoen, V;Li, Y;Lim, BK;
PMID: 34048697 | DOI: 10.1016/j.neuron.2021.05.002
Drugs of abuse induce persistent remodeling of reward circuit function, a process thought to underlie the emergence of drug craving and relapse to drug use. However, how circuit-specific, drug-induced molecular and cellular plasticity can have distributed effects on the mesolimbic dopamine reward system to facilitate relapse to drug use is not fully elucidated. Here, we demonstrate that dopamine receptor D3 (DRD3)-dependent plasticity in the ventral pallidum (VP) drives potentiation of dopamine release in the nucleus accumbens during relapse to cocaine seeking after abstinence. We show that two distinct VP DRD3+ neuronal populations projecting to either the lateral habenula (LHb) or the ventral tegmental area (VTA) display different patterns of activity during drug seeking following abstinence from cocaine self-administration and that selective suppression of elevated activity or DRD3 signaling in the LHb-projecting population reduces drug seeking. Together, our results uncover how circuit-specific DRD3-mediated plasticity contributes to the process of drug relapse.
Naganuma F, Bandaru SS, Absi G, Chee MJ, Vetrivelan R.
PMID: 30284033 | DOI: 10.1007/s00429-018-1766-2
Neurons containing melanin-concentrating hormone (MCH) in the posterior lateral hypothalamus play an integral role in rapid eye movement sleep (REMs) regulation. As MCH neurons also contain a variety of other neuropeptides [e.g., cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1] and neurotransmitters (e.g., glutamate), the specific neurotransmitter responsible for REMs regulation is not known. We hypothesized that glutamate, the primary fast-acting neurotransmitter in MCH neurons, is necessary for REMs regulation. To test this hypothesis, we deleted vesicular glutamate transporter (Vglut2; necessary for synaptic release of glutamate) specifically from MCH neurons by crossing MCH-Cre mice (expressing Cre recombinase in MCH neurons) with Vglut2flox/flox mice (expressing LoxP-modified alleles of Vglut2), and studied the amounts, architecture and diurnal variation of sleep-wake states during baseline conditions. We then activated the MCH neurons lacking glutamate neurotransmission using chemogenetic methods and tested whether these MCH neurons still promoted REMs. Our results indicate that glutamate in MCH neurons contributes to normal diurnal variability of REMs by regulating the levels of REMs during the dark period, but MCH neurons can promote REMs even in the absence of glutamate.
Quillet, R;Dickie, AC;Polgár, E;Gutierrez-Mecinas, M;Bell, AM;Goffin, L;Watanabe, M;Todd, AJ;
PMID: 37041197 | DOI: 10.1038/s41598-023-32720-3
Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75-80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.
Zhou, B;Claflin, KE;Flippo, KH;Sullivan, AI;Asghari, A;Tadinada, SM;Jensen-Cody, SO;Abel, T;Potthoff, MJ;
PMID: 36001982 | DOI: 10.1016/j.celrep.2022.111239
Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine hormone that functions to regulate energy homeostasis and macronutrient intake. Recently, FGF21 was reported to be produced and secreted from hypothalamic tanycytes, to regulate peripheral lipid metabolism; however, rigorous investigation of FGF21 expression in the brain has yet to be accomplished. Using a mouse model that drives CRE recombinase in FGF21-expressing cells, we demonstrate that FGF21 is not expressed in the hypothalamus, but instead is produced from the retrosplenial cortex (RSC), an essential brain region for spatial learning and memory. Furthermore, we find that central FGF21 produced in the RSC enhances spatial memory but does not regulate energy homeostasis or sugar intake. Finally, our data demonstrate that administration of FGF21 prolongs the duration of long-term potentiation in the hippocampus and enhances activation of hippocampal neurons. Thus, endogenous and pharmacological FGF21 appear to function in the hippocampus to enhance spatial memory.
