Rodent models for the analysis of tissue clock function in metabolic rhythms research

The circadian timing system consists on a distributed network of cellular clocks that together coordinate 24-h rhythms of physiology and behavior. Clock function and metabolism are tightly coupled, from the cellular to the organismal level. Genetic and non-genetic approaches in rodents have been employed to study circadian clock function in the living organism. Due to the ubiquitous expression of clock genes and the intricate interaction between the circadian system and energy metabolism, genetic approaches targeting specific tissue clocks have been used to assess their contribution in systemic metabolic processes. However, special requirements regarding specificity and efficiency have to be met to allow for valid conclusions from such studies. In this review, we provide a brief summary of different approaches developed for dissecting tissue clock function in the metabolic context in rodents, compare their strengths and weaknesses, and suggest new strategies in assessing tissue clock output and the consequences of circadian clock disruption in vivo.Fil: Tsang, Anthony H.. University of Lübeck; Alemania. University of Cambridge; Reino UnidoFil: Astiz, Mariana. University of Lübeck; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leinweber, Brinja. University of Lübeck; AlemaniaFil: Oster, Henrik. University of Lübeck; Alemani

Nutrient sensing in the nucleus of the solitary tract mediates non-aversive suppression of feeding via inhibition of AgRP neurons.

UNLABELLED: The nucleus of the solitary tract (NTS) is emerging as a major site of action for the appetite-suppressive effects of leading pharmacotherapies currently investigated to treat obesity. However, our understanding of how NTS neurons regulate appetite remains incomplete. OBJECTIVES: In this study, we used NTS nutrient sensing as an entry point to characterize stimulus-defined neuronal ensembles engaged by the NTS to produce physiological satiety. METHODS: We combined histological analysis, neuroanatomical assessment using inducible viral tracing tools, and functional tests to characterize hindbrain-forebrain circuits engaged by NTS leucine sensing to suppress hunger. RESULTS: We found that NTS detection of leucine engages NTS prolactin-releasing peptide (PrRP) neurons to inhibit AgRP neurons via a population of leptin receptor-expressing neurons in the dorsomedial hypothalamus. This circuit is necessary for the anorectic response to NTS leucine, the appetite-suppressive effect of high-protein diets, and the long-term control of energy balance. CONCLUSIONS: These results extend the integrative capability of AgRP neurons to include brainstem nutrient sensing inputs

Rodent models for the analysis of tissue clock function in metabolic rhythms research

The circadian timing system consists on a distributed network of cellular clocks that together coordinate 24-h rhythms of physiology and behavior. Clock function and metabolism are tightly coupled, from the cellular to the organismal level. Genetic and non-genetic approaches in rodents have been employed to study circadian clock function in the living organism. Due to the ubiquitous expression of clock genes and the intricate interaction between the circadian system and energy metabolism, genetic approaches targeting specific tissue clocks have been used to assess their contribution in systemic metabolic processes. However, special requirements regarding specificity and efficiency have to be met to allow for valid conclusions from such studies. In this review, we provide a brief summary of different approaches developed for dissecting tissue clock function in the metabolic context in rodents, compare their strengths and weaknesses, and suggest new strategies in assessing tissue clock output and the consequences of circadian clock disruption in vivo.Instituto de Investigaciones Bioquímicas de La Plat

Characterization of Retinal Structure in ATF6-Associated Achromatopsia.

PurposeMutations in six genes have been associated with achromatopsia (ACHM): CNGA3, CNGB3, PDE6H, PDE6C, GNAT2, and ATF6. ATF6 is the most recent gene to be identified, though thorough phenotyping of this genetic subtype is lacking. Here, we sought to test the hypothesis that ATF6-associated ACHM is a structurally distinct form of congenital ACHM.MethodsSeven genetically confirmed subjects from five nonconsanguineous families were recruited. Foveal hypoplasia and the integrity of the ellipsoid zone (EZ) band (a.k.a., IS/OS) were graded from optical coherence tomography (OCT) images. Images of the photoreceptor mosaic were acquired using confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Parafoveal cone and rod density values were calculated and compared to published normative data as well as data from two subjects harboring CNGA3 or CNGB3 mutations who were recruited for comparative purposes. Additionally, nonconfocal dark-field AOSLO images of the retinal pigment epithelium were obtained, with quantitative analysis performed in one subject with ATF6-ACHM.ResultsFoveal hypoplasia was observed in all subjects with ATF6 mutations. Absence of the EZ band within the foveal region (grade 3) or appearance of a hyporeflective zone (grade 4) was seen in all subjects with ATF6 using OCT. There was no evidence of remnant foveal cone structure using confocal AOSLO, although sporadic cone-like structures were seen in nonconfocal split-detection AOSLO. There was a lack of cone structure in the parafovea, in direct contrast to previous reports.ConclusionsOur data demonstrate a near absence of cone structure in subjects harboring ATF6 mutations. This implicates ATF6 as having a major role in cone development and suggests that at least a subset of subjects with ATF6-ACHM have markedly fewer cellular targets for cone-directed gene therapies than do subjects with CNGA3- or CNGB3-ACHM

RBP3-retinopathy - inherited high myopia and retinal dystrophy: Genetic Characterization, Natural History, and Deep Phenotyping

Objective: To examine the genetic and clinical features and the natural history of RBP3-associated retinopathy. // Design: Multi-center international, retrospective, case series. // Setting: Three tertiary referral centers. // Participants: Adults and children, with molecularly confirmed RBP3-associated retinopathy. // Main Outcomes and Measures: Multi-center, international, retrospective, consecutive observational study in three tertiary referral centers of adults and children, with molecularly confirmed RBP3-retinopathy. The genetic, clinical and retinal imaging findings, including optical coherence tomography (OCT) and fundus autofluorescence (FAF), were investigated both cross-sectionally and longitudinally. The results of International standard full-field and pattern electroretinography (ERG; PERG) were reviewed. // Results: We ascertained 12 patients (5 females), from 10 families, with four patients previously reported. Eight novel disease-causing RBP3 variants were identified. Ten patients were homozygous. The mean age (±SD, range) of the group was 21.4 years (±19.1, 2.9-60.5 years) at baseline evaluation. All 12 patients were highly myopic with a mean spherical equivalent of -16.0D (range; -7.0D to -33.0D). Visual acuity was not significantly different between eyes and no significant anisometropia was observed. Mean best corrected visual acuity (BCVA) was 0.48 LogMAR (range; 0.2-1.35, SD; ± 0.29 LogMAR) at baseline. Eleven patients had longitudinal BCVA assessment, with a mean BCVA of 0.46 LogMAR after a mean follow-up of 12.6 years. All patients were symptomatic with reduced VA and myopia by the age of 7 years. All patients had myopic fundi and features in keeping with high myopia on OCT, including choroidal thinning. The 4 youngest patients had no fundus pigmentary changes, with the rest presenting with a variable degree of mid-peripheral pigmentation and macular changes. FAF showed variable phenotypes, ranging from areas of increased signal to advanced atrophy in older patients. OCT showed cystoid macular edema at presentation in three patients, which persisted during follow-up in two patients and resolved to atrophy for the third patient. The ERGs were abnormal in 9 of 9 cases, revealing variable relative involvement of rod and cone photoreceptors with additional milder dysfunction post-phototransduction in some. All but one had PERG evidence of macular dysfunction, severe in most. // Conclusions: This study details the clinical and functional phenotype of RBP3-retinopathy in the largest cohort reported to date. RBP3-retinopathy is a disease characterized by early onset, slow progression over decades, and high myopia. The phenotypic spectrum and natural history as described herein has prognostic and counselling implications. RBP3-related disease should be considered in children with high myopia and retinal dystrophy

Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies

Calcitonin Receptor Neurons in the Mouse Nucleus Tractus Solitarius Control Energy Balance via the Non-aversive Suppression of Feeding.

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating CalcrNTS neurons decreased food intake and body weight but (unlike neighboring CckNTS cells) failed to promote aversion, revealing that CalcrNTS neurons mediate a non-aversive suppression of food intake. While both CalcrNTS and CckNTS neurons decreased feeding via projections to the PBN, CckNTS cells activated aversive CGRPPBN cells while CalcrNTS cells activated distinct non-CGRP PBN cells. Hence, CalcrNTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing CalcrNTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, CalcrNTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells

Multiexon deletion alleles of ATF6 linked to achromatopsia

Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease