Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities.

Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders.

Quantitative mass spectrometry for human melanocortin peptides in vitro and in vivo suggests prominent roles for ß-MSH and desacetyl α-MSH in energy homeostasis.

OBJECTIVE: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. METHODS: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. RESULTS: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of ß-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin. CONCLUSIONS: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and ß-MSH are likely to be the predominant physiological products acting on melanocortin receptors.

Generation and Characterization of Functional Human Hypothalamic Neurons.

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species-specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases. We and others have developed methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons and related cell types, such as astrocytes. This protocol builds on published studies by providing detailed step-by-step instructions for neuronal differentiation, quality control, long-term neuronal maintenance, and the functional interrogation of hypothalamic cells by calcium imaging. Together, these protocols should enable any group with appropriate facilities to generate and study human hypothalamic cells. © 2017 by John Wiley & Sons, Inc.

C57BL/6J mice as a polygenic developmental model of diet-induced obesity.

Susceptibility to obesity changes during the course of life. We utilized the C57BL/6J (B6) and 129S mouse as a genetic model for variation in diet-induced obesity to define the adiposity phenotypes from birth to maturity at 8 weeks-of-age. From birth to 8 weeks-of-age, both male and female 129S mice had significantly higher fat mass and adiposity index than B6 mice, although they were not obese. After 8 weeks-of-age, B6 had greater adiposity/obesity than 129S mice in response to a high fat (HF). We sought to determine the mechanism activating the fat accumulation in B6 mice at 8-weeks-of-age. We used microarray analysis of gene expression during development of inguinal fat to show that molecular networks of lipogenesis were maximally expressed at 8 weeks-of-age. In addition, the DNA methylation analysis of the Sfrp5 promoter and binding of acetylated histones to Sfrp5 and Acly promoter regions showed that major differences in the expression of genes of lipogenesis and chromatin structure occur during development. Differences in lipogenesis networks could account for the strain-dependent differences in adiposity up to 8 weeks-of-age; however, changes in the expression of genes in these networks were not associated with the susceptibility to DIO in B6 male mice beyond 8 weeks-of-age.

Obesity and related consequences to ageing.

Obesity has become a major public health problem. Given the current increase in life expectancy, the prevalence of obesity also raises steadily among older age groups. The increase in life expectancy is often accompanied with additional years of susceptibility to chronic ill health associated with obesity in the elderly. Both obesity and ageing are conditions leading to serious health problems and increased risk for disease and death. Ageing is associated with an increase in abdominal obesity, a major contributor to insulin resistance and the metabolic syndrome. Obesity in the elderly is thus a serious concern and comprehension of the key mechanisms of ageing and age-related diseases has become a necessary matter. Here, we aimed to identify similarities underlying mechanisms related to both obesity and ageing. We bring together evidence that age-related changes in body fat distribution and metabolism might be key factors of a vicious cycle that can accelerate the ageing process and onset of age-related diseases.

Mest and Sfrp5 are biomarkers for healthy adipose tissue.

Obesity depends on a close interplay between genetic and environmental factors. However, it is unknown how these factors interact to cause changes in the obese condition during the progression of obesity from the neonatal to the aged individual. We have utilized Mest and Sfrp5 genes, two genes highly correlated with adipose tissue expansion in diet-induced obesity, to characterize the obese condition during development of 2 genetic models of obesity. A model for the early onset of obesity was presented by leptin-deficient mice (ob/ob), whereas late onset of obesity was induced with high-fat diet (HFD) consumption in C57BL/6J mice with inherent risk of obesity (DIO). We correlated obese and diabetic phenotypes with Mest and Sfrp5 gene expression profiles in subcutaneous fat during pre-weaning, pre-adulthood and adulthood. A rapid development of obesity began in ob/ob mice immediately after weaning at 21 days of age, whereas the obesity of DIO mice was not evident until after 2 months of age. Even after 5 months of HFD treatment, the adiposity index of DIO mice was lower than in ob/ob mice at 2 months of age. In both obesity models, the expression of Mest and Sfrp5 genes increased in parallel with fat mass expansion; however, gene expression proceeded to decrease when the adiposity reached a plateau. The reduction in the expression of genes of caveolae structure and glucose metabolism were also suppressed in the aging adipose tissue. The analysis of fat mass and adipocyte size suggests that reduction in Mest and Sfrp5 is more sensitive to the age of the fat than its morphology. The balance of factors controlling fat deposition can be evaluated in part by the differential expression profiles of Mest and Sfrp5 genes with functions linked to fat deposition as long as there is an active accumulation of fat mass.