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Unravelling neuronal heterogeneity in energy homeostasis regulation

Periodic Reporting for period 1 - ARC-NeuroHet (Unravelling neuronal heterogeneity in energy homeostasis regulation)

Reporting period: 2017-04-01 to 2019-03-31

Obesity is one of the greatest public health challenges worldwide. In the European Union, its prevalence has been steadily increased during the last four decades. It causes a wide range of physical disabilities and psychological problems and drastically increases a person’s risk of developing a number of noncommunicable diseases (NCDs), including cardiovascular disease, cancer and diabetes. Obesity results from the deregulation of energy homeostasis, and recent research using experimental rodent models has expanded our understanding of energy balance regulation. Global energy balance in the organism is controlled by the central nervous system (CNS), which monitors the energy availability and regulates food intake, energy expenditure and substrate use by the peripheral organs. Neurons in the Arcuate nucleus of the hypothalamus (ARC) constantly monitor the energy levels of the organism through sensing metabolic (glucose, fatty acids, etc.) and hormonal (insulin, leptin, etc.) cues carried in the blood. ARC neuronal populations integrate this information and coordinate global metabolism through the regulation of activity of downstream neurons. Inside the ARC, AgRP and POMC neurons have opposing effects on the regulation of energy homeostasis. Moreover, both populations have a high degree of heterogeneity, being composed by several neuronal subpopulations that react to different and specific stimuli. We require brand new experimental approaches to investigate in detail those subpopulations and how they affect the regulation of energy balance. The newly available Dre/rox recombinase technology offer great opportunity to combine with existing technologies (optogenetics, DREADDS, etc.) to produce improved transgenic mouse models. Novel approaches in the design of murine mouse models are indispensable to study with further detail how certain neuronal subpopulations affect energy balance. Additionally, new druggable targets can be discover trough cell sequencing techniques. The knowledge obtained from these studies will help to the scientific and medical community to understand the complex regulation of energy homeostasis, and the possible causes for its deregulation that results in obesity. In the long term, this knowledge will be the basis for new health policies aimed to curb the obesity epidemic.
In the ARC NeuroHet project, I planned to combine the new Dre/rox recombinase technology with the well-established Cre/loxP technology to study the heterogeneity of the AgRP population. I planned to use the novel AgRP-Dre mouse line, engineered in the host laboratory “Neuronal Control of Metabolism” (NCM) group, at the Max Planck Institute for Metabolism Research (MPI-MR) in Cologne, Germany. By breeding AgRP-Dre mouse with the P2Y6-Cre mouse line, I can directly express a specific transgene (fluorescent reporter, Ca2+ reporter, etc.) only in neurons that express AgRP and P2Y6 markers. By doing this, I will delineate the AgRP, P2Y6 subpopulation localization, axonal projections and their response to physiological activators to unravel its specific role in energy homeostasis. Additionally, I will obtain their transcriptional profile and investigate for possible new druggable targets that affect this subpopulation exclusively. Unexpectedly, the validation of the AgRP-Dre mouse line revealed that Dre expression only reached 25-30% of the total AgRP population. This low expression level made the mouse model not suitable for the proposed experiments. Evaluation of the risk assessment, which was based in successful preliminary data obtained from the POMC-Dre transgenic line created at the host laboratory, confirmed that we underestimate an important critical risk.
Then it was decided to start an alternative research project based in the same premises as the original project: combine the Dre/rox and Cre/LoxP technologies to further explore the regulation of energy homeostasis by AgRP and POMC neurons. For this alternative project, instead of dissect neuronal subpopulations, I focused into the study of the combined net effects of AgRP and POMC activated neurons into the overall energy homeostasis. AgRP and POMC are considered to have antagonic effect in the regulation of energy homeostasis. Nevertheless, they project their axonal projections to the same areas, suggesting they can connect to similar downstream neurons. For the new project, I combine the POMC-Dre line with the well establish AgRP-ires-Cre line (obtained from B. Lowell lab) and with already available DREADDs technology using a carefully breeding strategy. By this, I will simultaneously activate and inhibited AgRP and POMC neurons, respectively. Using different experimental paradigms, I will investigate the total net effect over the regulation of food intake, glucose homeostasis and goal-oriented behaviors (Figure 1). Additionally, I will collect whole hypothalamus to analyze the transcriptional profiling of downstream neuronal networks affected by AgRP and POMC neurons. This analysis will revealed new markers of downstream neurons that participate in the regulation of body weight and glucose homeostasis. Results obtained had discover an unexpected interaction between AgRP and POMC neurons in the regulation of overall insulin sensitivity. In addition, preliminary results suggest that peripheral organs modify their sensitivity to insulin depending of the net effect composed by AgRP and POMC activated states.
Further planned experiments will analyze carefully the interaction between these neuronal populations and their net effect in the energy homeostasis regulation. Lastly, the combination of Cre and Dre recombinase technologies represents a new promising experimental approach to tackle the study of neuroscience questions directed to investigate the causes of obesity or other brain-related diseases, as depression, addiction or neurodegenerative diseases.