Smith-Magenis syndrome protein Rai1 regulates body weight homeostasis through hypothalamic Bdnf-producing neurons and TrkB signalling

Reviewer #1 (Public Review):

Summary:
Rai1 encodes the transcription factor retinoic acid-induced 1 (RAI1), which regulates expression of factors involved in neuronal development and synaptic transmission. Rai1 haploinsufficiency leads to the monogenic disorder Smith-Magenis syndrome (SMS), which is associated with excessive feeding, obesity and intellectual disability. Consistent with findings in human subjects, Rai1+/- mice and mice with conditional deletion of Rai1 in Sim+ neurons, which are abundant in the paraventricular nucleus (PVN), exhibit hyperphagia, obesity and increased adiposity. Furthermore, RAI1-deficient mice exhibit reduced expression of brain-derived neurotrophic factor (BDNF), a satiety factor essential for the central control of energy balance. Notably, overexpression of BDNF in PVN of RAI1-deficient mice mitigated their obesity, implicating this neurotrophin in the metabolic dysfunction these animals exhibit. In this follow up study, Javed et al. interrogated the necessity of RAI1 in BDNF+ neurons promoting metabolic health.

Consistent with previous reports, the authors observed reduced BDNF expression in the hypothalamus of Rai1+/- mice. Moreover, proteomics analysis indicated impairment in neurotrophin signaling in the mutants. Selective deletion of Rai1 in BDNF+ neurons in the brain during development resulted in increased body weight, fat mass and reduced locomotor activity and energy expenditure without changes in food intake. There was also a robust effect on glycemic control, with mutants exhibiting glucose intolerance. Selective depletion of RAI1 in BDNF+ neurons in PVN in adult mice also resulted in increased body weight, reduced locomotor activity, and glucose intolerance without affecting food intake. Blunting RAI1 activity also leads to increases and decreases in the inhibitory tone and intrinsic excitability, respectively, of BDNF+ neurons in the PVN.

Strengths:
Overall, the experiments are well designed and multidisciplinary approaches are employed to demonstrate that RAI1 deficits in BDNF+ neurons diminish hypothalamic BDNF signaling and produce metabolic dysfunction. The most significant advance relative to previous reports is the finding from electrophysiological studies showing that blunting RAI1 activity leads to increases and decreases the inhibitory tone and intrinsic excitability, respectively, of BDNF+ neurons in the PVN. Furthermore, that intact RAI1 function is required in BDNF+ neurons for the regulation of glucose homeostasis.

Weaknesses:
Some of the data need to be reconciled with previous findings by others. For example, the authors report that more than 50% of BDNF+ neurons in PVN also express pTrkB whereas about 20% of pTrkB+ cells contain BDNF, raising the possibility that autocrine mechanisms might be at play. This is in conflict with a previous study by An et al, (2015) showing that these cell populations are largely non-overlapping in the PVN.

Another issue that deserves more in depth discussion is that diminished BDNF function appears to play a minor part driving deficits in energy balance regulation. Accordingly, both global central depletion of Rai1 in BDNF+ neurons during development and deletion of Rai1 in BDNF+ neurons in the adult PVN elicited modest effects on body weight (less than 18% increase) and did not affect food intake. This contrasts with mice with selective Bdnf deletion in the adult PVN, which are hyperphagic and dramatically obese (90% heavier than controls). Therefore, the results suggest that deficits in RAI1 in PVN or the whole brain only moderately affect BDNF actions influencing energy homeostasis and that other signaling cascades and neuronal populations play a more prominent role driving the phenotypes observed in Rai1+/- mice, which are hyperphagic and 95% heavier than controls. The results from the proteomic analysis of hypothalamic tissue of Rai1 mutant mice and controls could be useful in generating alternative hypotheses.

Depleting RAI1 in BDNF+ neurons had a robust effect compromising glycemic control. However, as the approach does not necessarily impact BDNF exclusively, there should be a larger discussion of alternative mechanisms.

Reviewer #2 (Public Review):

Understanding disease conditions often yields valuable insights into the physiological regulation of biological functions, as well as potential therapeutic approaches. In previous investigations, the author's research group identified abnormal expression of brain-derived neurotrophic factor (BDNF) in the hypothalamus of a mouse model exhibiting Smith-Magenis syndrome (SMS), which is caused by heterozygous mutations of the Rai1 gene. Human SMS is associated with distinct facial characteristics, sleep disturbances, behavioral issues, and intellectual disabilities, often accompanied by obesity. Conditional knockout (cKO) of the Bdnf gene from the paraventricular hypothalamus (PVH) in mice led to hyperphagic obesity, while overexpression of the Bdnf gene in the PVH of Rai1 heterozygous mice restored the SMS-like obese phenotype. Based on these preceding findings, the authors of the present study discovered that homozygous Rai1 cKO restricted to Bdnf-expressing cells, or Rai1 gene knockdown solely in Bdnf-positive neurons in the PVH, induced obesity along with intricate alterations in adipose tissue composition, energy expenditure, locomotion, feeding patterns, and glucose tolerance, some of which varied between sexes. Additionally, the authors demonstrated that a brain-penetrating drug capable of activating the TrkB pathway, a downstream signaling pathway of BDNF, partially alleviated the SMS-like obesity phenotype in female mice with Rai1 heterozygous mutations. Although the specific (neural) cell type responsible for this TrkB signaling remains an open question, the present study unequivocally highlights the importance of Rai1 gene function in PVH Bdnf neurons for the obesity phenotype, providing valuable insights into potential therapeutic strategies for managing obesity associated with SMS.

In the proteomic analysis (Fig. 1), the authors elucidated that multiple phospho-protein signaling pathways, including Akt and mTOR pathways, exhibited significant attenuation in the SMS model mice. Of significance, the manifestation of haploinsufficiency of the Rai1 gene exclusively within the BDNF+ cells demonstrated negligible impact on body weight (Fig. 2-supple 3D), despite observing a reduction in BDNF levels in the heterozygous Rai1 mutant (Fig. 1A). Conversely, the homozygous Rai1 cKO in the BDNF+ cells prominently displayed an obesity phenotype, suggesting substantial dissimilarities in the gene expression profiles between Rai1 heterozygous and homozygous conditions within the BDNF+ cell population. It would be advantageous to precisely identify the responsible differentially expressed genes, possibly including Bdnf itself, in the homozygous cKO model. The observed reduction in the excitability of PVH BDNF+ cells (Fig. 3) is presumably attributed to aberrant gene expression other than Bdnf itself, which may serve as a prospective target for gene expression analysis. Notably, the Rai1 homozygous cKO mice in BDNF+ cells exhibited some sexual dimorphisms in feeding and energy expenditures, as evidenced by Fig. 2 and related figures. Exploring the potential relevance of these sexual differences to human SMS cases and investigating the underlying cellular/molecular mechanisms in the future would provide valuable insights.

Although the CRISPR-mediated knockdown of the Rai1 gene (Fig. 4) appears to be highly effective, given the broad transduction of AAV serotype 9, it may be helpful to exclude the possibility of other brain regions adjacent to the PVH, such as the DMH or VMH, being affected by this viral procedure. If the PVH-specificity is established, the majority of Rai1 cKO effects in Bdnf+ cells are primarily attributed to PVH-Bdnf+ cells based on the similarity of phenotypes observed. With regards to the apparent rescue of the body weight phenotype in Rai1 heterozygous mutants using a selective TrkB activator, the specific biological processes, and neurons responsible for this effect remain unclear to this reviewer. Elucidating these aspects would be significant when considering potential applications to human SMS cases.

Overall, the present study represents a valuable addition to the authors' series of high-quality molecular genetic investigations into the in vivo functions of the Rai1 gene. This reviewer particularly commends their diligent efforts to enhance our comprehension of SMS and contribute to the future development of more effective therapies for this syndrome.

Reviewer #3 (Public Review):

Summary:
Smith-Magenis syndrome (SMS) is associated with obesity and is caused by deletion or mutations in one copy of the Rai1 gene which encodes a transcriptional regulator. Previous studies have shown that Bdnf gene expression is reduced in the hypothalamus of Rai1 heterozygous mice. This manuscript by Javed et al. further links SMS-associated obesity with reduced Bdnf gene expression in the PVH.

Strengths:
The authors show that deletion of the Rai1 gene in all BDNF-expressing cells or just in the PVH BDNF neurons postnatally caused obesity. Interestingly, mutant mice displayed sexual dimorphism in the cause for the obesity phenotype. Overall, the data are well presented and convincing except the data from LM22A-4.

Weaknesses:
1. The most serious concern is about data from LM22A-4 administration experiments (Figure 5 and associated supplemental figures). A rigorous study has demonstrated that LM22A-4 does not activate TrkB (Boltaev et al., Science Signaling, 2017), which is consistent with unpublished results from many labs in the neurotrophin field. It is tricky to interpret body weight data from pharmacological studies because compounds always have some side effects, which can reduce body weight non-specifically.

2. The resolution of all figures are poor, and thus I could not judge the quality of the micrographs.

3. Citation of the literature is not precise. The study by An et al. (2015) shows that deletion of the Bdnf gene in the PVH leads to obesity due to increased food intake and reduced energy expenditure (not just hyperphagic obesity; Line 72). Furthermore, the study by Unger et al. (2017) carried out Bdnf deletion in the VMH and DMH using AAV-Cre and did not discuss SF1 neurons at all (Line 354). The two studies by Yang et al. (Mol Endocrinol, 2016) and Kamitakahara et al. (Mol Metab, 2015) did use SF1-Cre to delete the Bdnf gene and did not observe any obesity phenotype.

4. Animal number is not described in many figure legends.

Author Response

We express our sincere gratitude to the editors and reviewers for their invaluable input. To further improve our manuscript, we have devised a plan to perform additional histological experiments of Bdnf and TrkB expression. Specifically, we will replace the phospho-TrkB antibody with an anti-TrkB antibody to quantify Bdnf/TrkB co-expression. Moreover, we acknowledge the concern raised by the reviewers regarding the clarity of some explanations and the potential influence of alternative mechanisms influencing the defects observed in Bdnf neurons. We aim to provide a clearer explanation and discussion. We also intend to provide a more comprehensive discussion of the limitations of our LM22A-4 drug treatment experiment. By addressing these points, we wish to ensure that our research is informative to the eLife readership.