CE14 - Physiologie et physiopathologie

Role of Tbx3 in directing the functional identity of POMC neurons in obesity – neuroIDobese

Role of Tbx3 in directing the functional identity of POMC neurons in obesity

Chronic consumption of hypercaloric diets may trigger a ‘neuronal identity crisis’ in certain subpopulations of hypothalamic neurons due to the impaired action of the T-box gene 3 (Tbx3), a transcription factor that maintains hypothalamic neurons identity during postnatal life. Such a loss of identity may undermine the working machinery of POMC neurons, ultimately leading to obesity and its associated metabolic complications, albeit this hypothesis has yet to be investigated.

The main goal of this project is to investigate the role of Tbx3 in directing the functional identity of POMC neurons in obesity.

While executing the experiments mentioned in Aim 1, we encountered a few technical issues that we have been able to overcome by successfully introducing backup strategies. In the original plan, we proposed the use of single-cell mRNA sequencing from ARC micro punches and/or FACS sorting to achieve the single-cell transcriptome of POMC-traced neurons. However, both approaches were technically challenging regarding our ability to maintain good quality mRNAs and avoid potential off-target molecular effects driven by the cell dissociation procedure. To overcome these obstacles, we have set up the Patch-seq approach, where brain slices are analyzed ex-vivo without needing any cell dissociation procedure. This allows obtaining good quality and viable single neuronal cells (as verified by the analyses of the electric profile of the neurons) and analyzing these cells both in terms of electric and molecular profile. The latter is a critical advantage that might increase the quality of the information compared to the initial plan proposed (Aim1). Therefore, to achieve deliverable 1.1, we plan to use this alternative Patch-seq approach, which will not affect the main content of the proposal. We do not foresee that this change in the experimental strategy will lead to any significant delay in the project or any necessary adjustment in the budget.

Viral-based gain of Tbx3 function in POMC neurons has been achieved thanks to the use of a surgical stereotaxic procedure. We used a series of approaches to analyse the metabolic state of our mice after viral-based overexpression of Tbx3 in POMC neurons, including measurement of body weight, food intake, analysis of glucose metabolism thanks to the use of glucose tolerance tests, insulin tolerance tests, and quantification of plasma insulin levels. We have also employed whole-cell patch-clamp recording of hypothalamic ARC POMC neurons in brain slices from our POMC-tracing mice. This analysis was followed by single-cell QRT-PCR for a series of markers of cell identity and function. Finally, we employed immunohistochemistry and in-situ hybridization to analyze the expression of several markers of cell identity and function in ex-vivo hypothalamic slices obtained from our POMC-tracing mice. We then developed and used novel imaging tools to quantify the expression of these different markers in animals exposed to control or hypercaloric diets at a single cell resolution.

The work carried out so far has allowed demonstrating that, as initially hypothesized, changes in Tbx3 expression induced by dietary cues in POMC neurons play a crucial role in the pathophysiology of obesity-linked glucose intolerance. We have observed that viral-based overexpression of Tbx3 in POMC neurons protects the animals from alterations in systemic glucose handling induced by exposure to a hypercaloric diet (deliverable 3.1). These changes are notably independent of modifications in the body composition or food intake induced by the viral approach. Hence, our data reveal that Tbx3 action in POMC neurons affects systemic glucose metabolism during diet-induced metabolic stress, likely via direct mechanisms that link brain neuronal circuits with the control of peripheral metabolic organs. More experiments are currently ongoing to explore whether these same phenotypic changes are also observed in animals where Tbx3 is overexpressed in POMC neurons after the establishment of obesity (deliverable 3.2). Once these experiments are completed, we plan to proceed with the planned exploration of Tbx3-linked molecular factors possibly involved in the observed phenotype (deliverables 2.1 and 2.2).
A second important milestone achieved is the functional exploration of POMC neurons in our reporter (‘POMC-tracing’) mice (deliverable 1.2) at the single cell level, using whole-cell patch-clamp recordings combined with single-cell qPCR. Thanks to this approach, we have observed that diet-induced stress alters POMC neuronal activity in specific clusters of cells that are defined by a series of markers of neuronal identity, including Tbx3, but also additional markers such as CART, Leptin receptor, GLP-1 receptor, Insulin receptor.

Our results suggest that Tbx3-mediated mechanisms of identity plasticity are likely the core of POMC neuronal dysfunction in obesity. Moreover, we have identified a previously unknown subpopulation of POMC neurons, and observed that this population is particularly susceptible to dysfunction in obesity.
Obesity represents an important social and economic burden, but there are limited pharmacological tools to safely and effectively counteract this disease, reflecting the insufficient knowledge of the cellular and molecular mechanisms underlying its etiology, and therefore the scarce number of clinically relevant therapeutic targets. Our findings suggest that changes in the functional identity of hypothalamic POMC neurons may represent a novel mechanism implicated in the pathophysiology of type-2 diabetes development in obesity.

Research articles

1) Quarta C et al (2022). GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice. Nature Metabolism 4(8):1071-1083.

2) Castellanos-Jankiewicz A, ..Quarta C et al (2021). Hypothalamic bile acid-TGR5 signaling protects from obesity. Cell Metabolism S1550-4131(21)00175-3

3) Saucisse N., ..Quarta C‚ et al (2021). POMC neurons functional heterogeneity relies on mTORC1 signaling. Cell Reports 2021 Oct 12;37(2):109800.

Review and Perspectives

4) Allard C, Zizzari P, Quarta C, Cota D. (2022). Food intake and body weight in rodent studies: the devil is in the details. Nature Metabolism, in press.

5) Quarta C et al (2021). POMC neuronal heterogeneity in energy balance and beyond: an integrated view. Nature Metabolism 3(3):299-308.

6) Lee TH..Quarta C*. Yin-Yang control of energy balance by lipids in the hypothalamus: The endocannabinoids vs bile acids case. Biochimie 19:S0300-9084(22)00182-1. *corr. author.

7) Leon S.. Quarta C* (2021). Microglia–Neuron Crosstalk in Obesity: Melodious Interaction or Kiss of Death? International Journal of Molecular Sciences. 22, 5243. *corr. author.

Invited talks Dr Quarta

1) International Workshop Well-Aging and the Tanycytic Control of Health (WATCH). Title: Single-cell mapping of POMC neurons in obesity reveals novel subtypes with atypical identity. Prato, Italy (26-28 June 2022).

2) International virtual meeting French and British Societies for Neuroendocrinology (SNE/BSN), 2021. Title: Impact of hypercaloric diets on POMC neuronal plasticity. Bordeaux, France (22-24 September 2021)

3) Neurocentre Magendie’s Symposium. Title: Single-cell mapping of POMC neurons in obesity reveals novel subtypes with ‘atypical identity’.(Bordeaux, France 16/04/22).

4) Journées Francophones de Nutrition. Title: Contrôle moléculaire de l'identité des neurones hypothalamiques dans l'obésité.(online, congress, 2021).

5) NeuroFrance International congress 2021. Title: Molecular control of hypothalamic neurons identity (online, 20/05/21).

Oral presentations Leon Stéphane (thesis Director Dr. Quarta)

1) Single-cell molecular and functional mapping of POMC neurons in obesity: a multimodal approach. EMBO Workshop (03-06 October 2022, Malaga, Spain).

2) Single-cell mapping of POMC neurons in obesity reveals novel subtypes with ‘atypical identity’. 24th European Congress of Endocrinology (Milan, International congress, 24/05/22).

3)Lineage tracing of POMC neurons reveals heterogeneity in response to metabolic stress. 23rd European Congress of Endocrinology (e-ECE, online, International congress, 26/05/21).

4) Single-cell mapping of POMC neurons in obesity reveals novel subtypes with ‘atypical identity’. 19th Synape and Network day (Bordeaux, Scientific Day, 13/09/22).

5) Contrôle moléculaire de l’identité des neurones hypothalamiques dans l’obésité. Journées Francophones de Nutrition (online, National congress, 2021).

Hypercaloric diets may cause permanent alterations in systemic energy homeostasis by disrupting the differentiation state and therefore the functional identity of hypothalamic neurons, i.e. their cell-specific working machinery. This hypothesis still requires direct demonstration, due to our limited knowledge of the intrinsic neuronal factors involved. I recently uncovered the pivotal role played by the transcription factor Tbx3 in directing the functional identity of hypothalamic POMC neurons. Notably, diet-induced obesity inhibits hypothalamic Tbx3 expression in both murine models and humans. Thus, we will explore whether hypercaloric diets disrupt the functional and molecular identity of POMC neurons via intrinsic changes in Tbx3 expression and whether this mechanism contributes to the development of diet-induced obesity. Our studies might shed new light into the main molecular signals linking hypothalamic POMC neurons dysfunction with the etiology of obesity.

Project coordination

CARMELO QUARTA (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - UMR1215)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM - UMR1215 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - UMR1215

Help of the ANR 290,304 euros
Beginning and duration of the scientific project: March 2021 - 42 Months

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