Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

Xing, Mingming; Li, Yang; Zhang, Yuqi; Zhou, Juemou; Ma, Danting; Zhang, Mengqi; Tang, Minglei; Ouyang, Ting; Zhang, Fumiao; Shi, Xiaofeng; Sun, Jianyuan; Chen, Zuxin; Zhang, Weiping J.; Zhang, Shuli; Xie, Xiangyang

doi:10.1038/s41467-024-53258-6

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Published: 16 October 2024

Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits

Mingming Xing¹^na1,
Yang Li^2,3^na1,
Yuqi Zhang¹^na1,
Juemou Zhou¹^na1,
Danting Ma¹,
Mengqi Zhang¹,
Minglei Tang¹,
Ting Ouyang¹,
Fumiao Zhang¹,
Xiaofeng Shi¹,
Jianyuan Sun^3,4,
Zuxin Chen ORCID: orcid.org/0000-0002-5412-3140⁵,
Weiping J. Zhang¹,
Shuli Zhang ORCID: orcid.org/0000-0001-8006-1134^2,3 &
…
Xiangyang Xie ORCID: orcid.org/0000-0003-4317-7327¹

Nature Communications volume 15, Article number: 8939 (2024) Cite this article

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Abstract

The paraventricular hypothalamus (PVH) is crucial for food intake control, yet the presynaptic mechanisms underlying PVH neurons remain unclear. Here, we show that RUVBL2 in the PVH is significantly reduced during energy deficit, and knockout (KO) of PVH RUVBL2 results in hyperphagic obesity in mice. RUVBL2-expressing neurons in the PVH (PVH^RUVBL2) exert the anorexigenic effect by projecting to the arcuate hypothalamus, the dorsomedial hypothalamus, and the parabrachial complex. We further demonstrate that PVH^RUVBL2 neurons form the synaptic connections with POMC and AgRP neurons in the ARC. PVH RUVBL2 KO impairs the excitatory synaptic transmission by reducing presynaptic boutons and synaptic vesicles near active zone. Finally, RUVBL2 overexpression in the PVH suppresses food intake and protects against diet induced obesity. Together, this study demonstrates an essential role for PVH RUVBL2 in food intake control, and suggests that modulation of synaptic plasticity could be an effective way to curb appetite and obesity.

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Introduction

The brain maintains energy homeostasis by regulating food intake and energy expenditure after integrating both peripheral and central signals^1,2,3. This regulation relies on a complex neural network composed of neurons within and outside of the hypothalamus^4,5,6. Numerous studies has established the regulatory role for several hypothalamic regions in energy homeostasis including the arcuate hypothalamus (ARC), the paraventricular hypothalamus (PVH), the dorsomedial hypothalamus (DMH), the ventromedial hypothalamus (VMH), and the lateral hypothalamus (LH)^7,8,9.

The PVH is a heterogeneous region in the hypothalamus and has been shown to regulate energy homeostasis through a variety of cell types expressing various markers^{10,11,12,13,14,15,16,17}. Of interest, the vast majority of PVH neurons have been demonstrated to promote satiety, supporting an indispensable role for the PVH in satiety control¹⁸. The neural circuits underlying the anorexigenic effects of PVH neurons have also been extensively studied^15,19,20,21. Some downstream target sites receiving projections from the PVH have been defined including the parabrachial complex (PB)¹⁵ and the VMH¹³. Nevertheless, given the prominence of the PVH in energy homeostasis, additional markers and/or downstream sites projected by PVH neurons need to be identified to better understand the regulation mechanisms underlying PVH control of satiety.

Synaptic plasticity is defined as a reshape of the strength and efficacy of synaptic transmission among neurons, which could last for milliseconds, hours, days, or even longer^22,23. A growing body of evidence has indicated a close relationship between synaptic plasticity and energy homeostasis^24,25,26. The initial evidence for a potential role of synaptic plasticity in energy homeostasis came from the observations that leptin treatment in ob/ob mice rapidly restored synaptic inputs to POMC-expressing neurons (ARC^POMC) and AgRP-expressing neurons (ARC^AgRP) in the ARC before the occurrence of leptin’s anorexigenic effect²⁷. Although an intimate relationship between synaptic plasticity and energy homeostasis has been further suggested by later studies over the past two decades^{28,29,30,31,32,33,34,35,36,37}, whether modulating synaptic plasticity regulates appetite and body weight remains unclear.

RuvB-like 2 (RUVBL2) is a highly conserved AAA+ (ATPases associated with diverse cellular activities) ATPase that has been shown to participate in a variety of cellular processes^38,39 including chromatin remodeling³⁹, transcriptional regulation⁴⁰, glucose transport⁴¹, and assembly of macromolecular complexes^39,42. Interestingly, one recent study conducting exome-sequencing of type 2 diabetes (T2D) patients and controls indicates that RUVBL2 together with the melanocortin-4 receptor (MC4R) and others are the most significant T2D-associated genes according to gene-level analyses⁴³, suggesting a potential role of RUVBL2 in metabolic control. Although RUVBL2 has been reported to be ubiquitously expressed in the brain⁴⁴, and we observed its abundant expression in the hypothalamus, particularly in the PVH, the functional role of RUVBL2 in energy homeostasis is unknown.

In this study, we found that RUVBL2 expression in the PVH is significantly reduced during energy deficit. Using different mouse models combined with stereotaxic injection, chemo-/opto-genetics, viral tracing and circuit-specific chemo-/opto-genetics, we show that knockout (KO) of PVH RUVBL2 leads to hyperphagic obesity, and RUVBL2-expressing neurons in the PVH (PVH^RUVBL2) exert the anorexigenic effect by projecting to the ARC, the DMH, and the PB. Interestingly, PVH^RUVBL2 neurons project to both ARC^POMC and ARC^AgRP neurons. Through electrophysiology, transmission electron microscopy, immunohistochemistry, and chromatin immunoprecipitation sequencing (ChIP-Seq) combined with RNA sequencing (RNA-Seq), we further reveal that RUVBL2 KO impairs the excitatory synaptic transmission by reducing presynaptic boutons and synaptic vesicles near the active zone (AZ), which is owing to RUVBL2 transcription control of genes related to neuron axonogenesis and synaptic vesicle. Finally, RUVBL2 overexpression increases synaptic vesicles near the AZ and the excitatory synaptic transmission, consequently suppressing food intake and protecting against diet-induced obesity, suggesting that modulation of synaptic plasticity could be a feasible way to regulate food intake and body weight.

Results

RUVBL2 KO in the adult PVH leads to massive obesity owing to hyperphagia

Using a monoclonal antibody against RUVBL2, we showed that RUVBL2 is broadly expressed in the brain, including the hypothalamus (Supplementary Fig. 1a, b). The PVH is an indispensable hypothalamic site regulating energy homeostasis¹⁸. We observed that RUVBL2 is expressed throughout the PVH, ranging from the anterior, the medial, to the posterior part (Supplementary Fig. 1c). Interestingly, fasting significantly reduces the expression of RUVBL2 in the PVH at both mRNA and protein levels (Fig. 1a,b), suggesting a potential role for RUVBL2 in control of energy homeostasis.

To study the role of PVH RUVBL2 in energy homeostasis, we generated the floxed Ruvbl2 mouse strain (Ruvbl2^fl/fl)⁴² (Supplementary Fig. 2a), and bilaterally injected adeno-associated virus (AAV) (AAV2-GFP or AAV2-Cre-GFP) into the PVH of Ruvbl2^fl/fl mice (Fig. 1c, d and Supplementary Fig. 2b). Of interest, one recent study reported that injection of some batches of AAV-Cre in the mouse PVH resulted in obesity due to their neuronal toxicity⁴⁵. Hence, we also examined the toxicity of AAV2-Cre by injecting AAV2-Cre-GFP or AAV2-GFP as control into the PVH of 8 week-old male C57BL/6 J mice and monitored their weekly body weight. It turned out that there was no difference in body weight of AAV2-GFP- and AAV2-Cre-GFP-injected mice (Supplementary Fig. 2c), excluding the possible neuronal toxicity of AAV2-Cre-GFP.

As shown by immunohistochemical (IHC) staining, AAV2-Cre-GFP infection drastically reduced the expression of RUVBL2 in the PVH (Fig. 1e). The mice injected with AAV2-Cre-GFP became massively obese 12 weeks post virus injection compared with those injected with AAV2-GFP (Fig. 1f, g and Supplementary Fig. 2d, e). Daily food intake of mice with AAV2-Cre-GFP injection was significantly increased (Fig. 1h, i). Using comprehensive lab animal monitoring system (CLAMS), we showed that mice injected with AAV2-Cre-GFP exhibited similar energy expenditure (metabolic rate) (Fig. 1j and Supplementary Fig. 2f), locomotor activity (Fig. 1k and Supplementary Fig. 2g), and respiratory exchange ratio (RER) (Supplementary Fig. 2h, i) in both light and dark cycles, compared with the mice injected with AAV2-GFP.

Note that our metabolic cages measurements were carried out in mice showing different body weights after 12 weeks of AAV injection. One study has suggested that analysis of covariance (ANCOVA) should be performed to evaluate the metabolic rate in animals with different body weights⁴⁶. Hence, we performed ANCOVA using body weight as covariate, indicating similar metabolic rate between two groups (Fig. 1l and Supplementary Fig. 2j). In addition, mice with AAV2-Cre-GFP injection showed significantly reduced blood glucose control and insulin sensitivity as demonstrated by the intraperitoneal glucose tolerance tests (IPGTT) and the intraperitoneal insulin tolerance tests (IPITT), respectively (Fig. 1m, n and Supplementary Fig. 2k, l). Taken together, these data demonstrate that PVH RUVBL2 KO leads to hyperphagic obesity.

PVH^RUVBL2 neurons constitute the majority of PVH neurons

We next characterized PVH^RUVBL2 neurons by examining the co-localization of RUVBL2 with other known markers. To investigate the co-localization between RUVBL2 and SIM1, an essential molecule for PVH development⁴⁷, Sim1-Cre transgenic mice were crossed with Ai14 (Rosa26-CAG-LSL-tdTomato) reporter mice⁴⁸ to generate Sim1-Cre;Ai14 mice. As shown, the majority (79.2%) of PVH^SIM1 neurons expresses RUVBL2 (Supplementary Fig. 3a), suggesting that PVH^RUVBL2 neurons constitute a large part of PVH neurons.

MC4R-expressing neurons (PVH^MC4R) and prodynorphin-expressing neurons (PVH^PDYN) in the PVH are two well-characterized cell types that have been suggested to account for the whole satiety control by the PVH¹⁵. Therefore, we analyzed the co-localization of RUVBL2 with MC4R or PDYN in the PVH. Through Mc4r-2a-Cre mice⁴⁹ and Ai14 mice, we showed that ~32.7% of PVH^MC4R neurons co-express RUVBL2, while ~16.8% of PVH^RUVBL2 neurons co-expresses MC4R (Supplementary Fig. 3b). On the other hand, the vast majority (~93%) of PVH^PDYN neurons co-expresses RUVBL2, while ~55.8% of PVH^RUVBL2 neurons co-expresses PDYN (Supplementary Fig. 3c). We also analyzed the co-expression of RUVBL2 with several other markers in the PVH including Oxytocin, CRH, TH, and TRH. As shown, most of neurons investigated co-expresses RUVBL2, while only a small part of PVH^RUVBL2 neurons co-expresses these markers (Supplementary Fig. 3d–g). Together, these data indicate that PVH^RUVBL2 neurons constitute the majority of PVH neurons, and also suggest that PVH^RUVBL2 neurons may exert an anorexigenic effect, given an established role for the PVH in satiety control¹⁸.

Manipulation of PVH^RUVBL2 neuron activity alters food intake

To explore the role of PVH^RUVBL2 neurons in satiety control, designer receptors exclusively activated by designer drugs (DREADDs) and their specific ligand clozapine N-oxide (CNO)⁵⁰ were applied to manipulate PVH^RUVBL2 neuron activity. We first generated Ruvbl2-Cre knock-in mice by placing Cre sequence after the coding sequence of Ruvbl2 gene using CRISPR-Cas9 (Supplementary Fig. 4a), and bilaterally delivered the Cre-dependent AAV9-DIO-hM3D(Gq)-mCherry (stimulatory) or control AAV9-DIO-mCherry into the PVH of Ruvbl2-Cre mice (Fig. 2a). Administration of CNO robustly increased the PVH^RUVBL2 neuron activity as revealed by c-Fos immunoreactivity (Fig. 2b). Chemogenetic activation of PVH^RUVBL2 neurons significantly reduced food intake of fasted mice in both light and dark cycles (Fig. 2c, d and Supplementary Fig. 4b, c). Note that chemogenetic activation of PVH^RUVBL2 neurons did not influence the energy expenditure of mice in both cycles as demonstrated by metabolic rate or oxygen consumption rate (VO₂) (Supplementary Fig. 4d–g).

**Fig. 2: Modulation of PVH^RUVBL2 neuron activity alters food intake.**

To investigate whether inhibition of PVH^RUVBL2 neuron activity affects food intake, we bilaterally injected the Cre-dependent AAV9-DIO-hM4D(Gi)-mCherry (inhibitory) or control AAV9-DIO-mCherry into the PVH of Ruvbl2-Cre mice (Fig. 2e, f). Chemogenetic inhibition of PVH^RUVBL2 neurons significantly increased food intake within 4 h after CNO administration (Fig. 2g, h and Supplementary Fig. 4h, i), without any effect on the energy expenditure of mice (Supplementary Fig. 4j–m).

We also performed optogenetics⁵¹ by injecting Cre-dependent AAV9-DIO-hChannelrhodopsin-2 (hChR2)-mCherry or control AAV9-DIO-mCherry into the PVH of male Ruvbl2-Cre mice that were simultaneously implanted with an optic fiber above the PVH (Fig. 2i). After recovery and acclimation, optogenetics was performed using a 465-nm blue laser with 10 ms pulse duration at 20 Hz for 10 min (Fig. 2j). Optogenetic stimulation significantly reduced food intake of overnight-starved mice injected with AAV9-ChR2 compared with AAV9-mCherry, while no change of food intake between these two groups was observed either before or after stimulation (Fig. 2k, l and Supplementary Movie 1), supporting that activation of PVH^RUVBL2 neurons acutely suppresses food intake. Together, these chemogenetics and optogenetics studies have demonstrated the anorexigenic effect of PVH^RUVBL2 neurons.

PVH^RUVBL2 neurons project to several brain regions

To identify the brain regions receiving axonal projections from PVH^RUVBL2 neurons, we carried out anterograde monosynaptic tracing⁵². The helper virus AAV8-DIO- EGFP-T2A-TK was first unilaterally injected into the PVH of 8 week-old male Ruvbl2-Cre mice that received the unilateral injection of the herpes simplex virus (HSV) (HSV-ΔTK-LSL-tdTomato) into the same site after 3 weeks of virus injection (Fig. 3a).

We observed the unilateral infection of both helper virus and HSV in the PVH (Fig. 3b), and that seven brain regions throughout the brain clearly showed tdTomato fluorescent signals, suggesting direct projections from PVH^RUVBL2 neurons (Fig. 3c). These brain regions included the ventral lateral septum nucleus (LSv) (Fig. 3c1), the preoptic area (POA) (Fig. 3c2), the DMH (Fig. 3c3), the ARC (Fig. 3c4), the medial amygdala (MeA) (Fig. 3c5), the PB including the parabrachial nucleus (PBN) (Fig. 3c6) and the pre-locus coeruleus (pLC) (Fig. 3c7), and the nucleus tractus solitarius (NTS) (Fig. 3c8). Consistently, previous studies have indicated that several cell types in the PVH including PVH^MC4R, PVH^PDYN, PVH^TrkB, and PVH^TRH neurons regulate energy homeostasis by projecting to the PB (LPBN or pLC)^15,49 or the ARC^17,35.

We next performed retrograde tracing⁵³ to demonstrate the synaptic connections between PVH^RUVBL2 neurons and the above-mentioned seven brain regions (Supplementary Fig. 5a–h). These data together demonstrate the direct projections from PVH^RUVBL2 neurons to the brain regions identified by anterograde monosynaptic tracing.

ARC^POMC and ARC^AgRP neurons get involved in regulation of appetite as an important component of central melanocortin system¹⁸. Thus, we asked whether PVH^RUVBL2 neurons regulate appetite by projecting to ARC^POMC and/or ARC^AgRP neurons. To address this, Ruvbl2-Cre mice were crossed with Pomc-EGFP mice⁵⁴ to generate Ruvbl2-Cre;Pomc-EGFP mice that were used for anterograde monosynaptic tracing (Supplementary Fig. 6a). We observed that many anterograde tdTomato signals from PVH^RUVBL2 neurons are localized in ARC^POMC neurons (Supplementary Fig. 6b,c), indicating the monosynaptic projection from PVH^RUVBL2 neurons to ARC^POMC neurons. To validate this synaptic connection, we performed retrograde monosynaptic tracing⁵⁵ in Pomc-Cre mice⁵⁶ (Fig. 3d), showing that almost all of the retrograde DsRed signals from ARC^POMC neurons are localized in PVH^RUVBL2 neurons (Fig. 3e,f). Interestingly, PVH^RUVBL2 neurons also form synaptic connections with ARC^AgRP neurons as revealed by retrograde monosynaptic tracing in Agrp-Cre mice⁵⁷ (Fig. 3g-i). Note that we observed that most of PVH^RUVBL2 neurons projecting to ARC^POMC and ARC^AgRP neurons are glutamatergic (Supplementary Fig. 6d,e). Taken together, these results demonstrate PVH^RUVBL2 → ARC^POMC and PVH^RUVBL2 → ARC^AgRP neuronal circuits.

PVH^RUVBL2 neurons exert their anorexigenic effect through PVH^RUVBL2 → ARC, PVH^RUVBL2 → DMH, and PVH^RUVBL2 → PB neural circuits

To investigate which neural circuit (s) mediates the anorexigenic effect of PVH^RUVBL2 neurons, we performed projection-specific chemogenetics. Control AAV5-Con/Fon-EGFP or Cre- and FLP-dependent AAV5-Con/Fon-hM4D(Gi)-EGFP were bilaterally injected into the PVH of Ruvbl2-Cre mice that simultaneously received bilateral injection of retroAAV-FLP into PVH^RUVBL2-projected regions as identified above. We first investigated whether PVH^RUVBL2 → ARC regulated food intake, given a well-established role for the ARC in energy homeostasis³. The injection sites of PVH and ARC were verified by histological analysis (Fig. 4a, b). Chemogenetic inhibition of ARC-projecting PVH^RUVBL2 neurons significantly increased food intake in both light and dark cycles (Fig. 4c, d).

**Fig. 4: PVH^RUVBL2 neurons exert the anorexigenic effect through PVH^RUVBL2 → ARC, PVH^RUVBL2 → DMH, and PVH^RUVBL2 → PB neural circuits.**

We next reasoned whether PVH^RUVBL2 → DMH regulates food intake, considering that DMH is another hypothalamic region that is known to regulate energy homeostasis⁶. Likewise, we verified the injection sites of PVH and DMH (Fig. 4e, f). Chemogenetic inhibition of DMH-projecting PVH^RUVBL2 neurons significantly increased food intake (Fig. 4g, h).

In addition, we tested the other PVH^RUVBL2-projected structures including the PB, the LSv, the POA, the MeA, and the NTS. Chemogenetic inhibition of PB-projecting PVH^RUVBL2 neuron activity significantly increased food intake in both light and dark cycles (Supplementary Fig. 7a–c). These data are consistent with previous studies demonstrating the critical role for the PB in mediating the anorexgenic effects of several known cell types in the PVH^13,15,49. In comparison, chemogenetic inhibition of PVH^RUVBL2 neurons projecting to other regions (LSv, MeA, POA, and NTS) had no effect on food intake (Supplementary Fig. 7d–o). Note that CNO alone has no effect on mouse food intake (Supplementary Fig. 7p, q). Taken together, these results indicate that PVH^RUVBL2 neurons suppresses food intake through their projections to the ARC, the DMH, and the PB.

We also performed projection-specific optogenetics to examine the anorexigenic effect of PVH^RUVBL2 → ARC and PVH^RUVBL2 → DMH neural circuits. To examine the anorexigenic effect of PVH^RUVBL2 → ARC neural circuit, control AAV9-DIO-mCherry or Cre-dependent AAV9-DIO-hChR2-mCherry were bilaterally injected into the PVH of Ruvbl2-Cre mice that simultaneously were implanted with an optic fiber above the ARC for neuron terminal activation (Fig. 4i,j). Optogenetic activation of ARC-projecting PVH^RUVBL2 neurons triggered a marked decrease of food intake in overnight-fasted mice (Fig. 4k and Supplementary Movie 2).

To examine the anorexigenic effect of PVH^RUVBL2 → DMH neural circuit, control AAV9-DIO-mCherry or Cre-dependent AAV9-DIO-hChR2-mCherry were bilaterally injected into the PVH of Ruvbl2-Cre mice that simultaneously received implantation of an optic fiber above the DMH for neuron terminal activation (Fig. 4l,m). Optogenetic activation of DMH-projecting PVH^RUVBL2 neurons triggered a significant decrease of food intake in overnight-fasted mice (Fig. 4n). These data collectively demonstrate that PVH^RUVBL2 → ARC, PVH^RUVBL2 → DMH, and PVH^RUVBL2 → PB neural circuits mediate the anorexigenic effect of PVH^RUVBL2 neurons.

RUVBL2 KO in the PVH impairs the excitatory synaptic transmission

We next aimed to study the synaptic mechanisms underlying the anorexigenic effect of PVH RUVBL2 and PVH^RUVBL2 neurons. First, we examined whether PVH^RUVBL2 neurons are excitatory or inhibitory by analyzing the co-localization of RUVBL2 with VGLUT2 or VGAT as the excitatory or inhibitory neuron marker, respectively^12,58. Accordingly, Vglut2-ires-Cre⁵⁸ or Vgat-ires-Cre⁵⁸ mice were crossed with Ai14 reporter mice to generate Vglut2-ires-Cre;Ai14 or Vgat-ires-Cre;Ai14 mice. We observed that ~49% of PVH^RUVBL2 neurons are VGLUT2⁺ excitatory neurons, while ~8% of PVH^RUVBL2 neurons are VGAT⁺ inhibitory neurons (Supplementary Fig. 8a, b), suggesting that most PVH^RUVBL2 neurons are excitatory.

We then determined the valence of functional monosynaptic connections between PVH^RUVBL2 neurons and the ARC or the DMH using ChR2-assisted circuit mapping (CRACM)⁵⁹. To achieve this, we injected Cre-dependent AAV9-EF1a-DIO-hChR2-mCherry expressing the ChR2 into the PVH of Ruvbl2-Cre mice, and recorded the excitatory post-synaptic currents (EPSCs) and the inhibitory post-synaptic currents (IPSCs) in ARC or DMH neurons by optogenetically stimulating PVH^RUVBL2 neuron terminals in the ARC or the DMH, respectively (Supplementary Fig. 8c, d). We recorded the excitatory postsynaptic currents (EPSCs) in 50% (6 out of 12 neurons) of ARC neurons, and the inhibitory post-synaptic currents (IPSCs) in 9.5% (2 out of 21 neurons) of ARC neurons (Supplementary Fig. 8e, f). In addition, 38.5% (5 out of 13 neurons) and 14.3% (3 out of 21 neurons) of DMH neurons displayed the EPSCs and the IPSCs, respectively (Supplementary Fig. 8g, h). These data demonstrate that PVH^RUVBL2 neurons send both excitatory and inhibitory projections to the ARC and the DMH.

Considering that abnormalities in feeding behavior may arise from the disorder in synaptic transmission (excitatory or inhibitory)^30,60, we reasoned that RUVBL2 KO in the PVH may impair synaptic transmission. To test this, we bilaterally injected AAV2-Cre-GFP into the PVH of 8 week-old Ruvbl2^fl/fl mice to knock out RUVBL2. After 6 weeks of AAV infection, we first performed the voltage-clamp recordings in the brain slices to investigate the spontaneous EPSCs (sEPSCs) in ARC neurons (Fig. 5a). Both amplitude and frequency of sEPSC were significantly decreased in ARC neurons (Fig. 5b–d), suggesting a presynaptic effect of RUVBL2 KO on the excitatory synaptic transmission. Next, we investigated whether RUVBL2 KO affects the evoked EPSCs (eEPSCs) by comparing eEPSCs in ARC neurons under afferent fiber stimulation. Interestingly, the peak amplitude of eEPSCs was also significantly decreased after RUVBL2 KO (Fig. 5e, f), while the pair-pulse ratio (PPR) (interstimulus interval (ISI), 50 ms) reflecting the evoked vesicle release probability was significantly increased (Fig. 5g). Together, these results indicate a presynaptic role of RUVBL2 in regulating the excitatory synaptic transmission in PVH^RUVBL2 → ARC circuit.

**Fig. 5: RUVBL2 KO in the PVH impairs the excitatory synaptic transmission.**

We also examined the potential effect of RUVBL2 KO on the inhibitory synaptic transmission in ARC neurons, but observed no differences in the spontaneous IPSCs (sIPSCs) (amplitude and frequency) between WT and RUVBL2 KO mice (Fig. 5h–j). Taken together, these results reveal that RUVBL2 KO impairs the excitatory synaptic transmission in PVH^RUVBL2 → ARC circuit.

We next investigated whether PVH RUVBL2 KO affected synaptic transmission in PVH^RUVBL2 → DMH circuit (Fig. 5k). RUVBL2 KO significantly decreased both amplitude and frequency of sEPSCs (Fig. 5l–n), and the amplitude of eEPSCs in DMH neurons (Fig. 5o, p). In addition, the pair-pulse ratio (PPR) (ISI, 50 ms) in DMH neurons was significantly increased after RUVBL2 KO (Fig. 5q). We further demonstrate that there were no changes in either amplitude or frequency of sIPSCs in DMH neurons after RUVBL2 KO (Fig. 5r–t). Collectively, these results demonstrate that PVH RUVBL2 KO impairs the excitatory synaptic transmission in both PVH^RUVBL2 → ARC and PVH^RUVBL2 → DMH circuits.

RUVBL2 KO impairs the excitatory synaptic transmission in PVH^RUVBL2 → ARC^POMC and PVH^RUVBL2 → ARC^NPY circuits

Considering that PVH RUVBL2 KO impairs the excitatory synaptic transmission to ARC neurons (Fig. 5a–j), and PVH^RUVBL2 neurons send projections to ARC^POMC and ARC^AgRP neurons (Fig. 3d–i), we investigated whether PVH RUVBL2 KO impairs excitatory synaptic transmission to ARC^POMC or ARC^AgRP neurons. To this end, we first generated Ruvbl2^fl/fl;Pomc-EGFP mice by crossing Ruvbl2^fl/fl mice with Pomc-EGFP mice, bilaterally injected AAV2-Cre-mCherry into the PVH of 8 week-old Ruvbl2^fl/fl;Pomc-EGFP mice to knock out RUVBL2, and performed electrophysiological recordings in the brain slices containing ARC^POMC neurons after 6 weeks of AAV injection (Fig. 6a). As shown (Fig. 6b–d), both amplitude and frequency of sEPSCs in ARC^POMC neurons were significantly decreased after PVH RUVBL2 KO in the PVH. In addition, the peak amplitude of eEPSCs was also significantly decreased (Fig. 6e, f), while the PPR was significantly increased after PVH RUVBL2 KO (Fig. 6g). These results demonstrate a presynaptic role of RUVBL2 in the excitatory synaptic transmission to ARC^POMC neurons.

**Fig. 6: RUVBL2 KO impairs the excitatory synaptic transmission to ARC^POMC or ARC^NPY neurons.**

To examine whether PVH RUVBL2 KO impacted the synaptic transmission to ARC^AgRP neurons, we generated Ruvbl2^fl/fl;Npy-GFP mice by crossing Ruvbl2^fl/fl mice with Npy-GFP mice²⁷ (Co-expression of Npy and AgRP in the ARC²⁰), bilaterally injected AAV2-Cre-mCherry into the PVH of 8 week-old Ruvbl2^fl/fl;Npy-GFP mice to knock out RUVBL2, and performed electrophysiological recordings in the brain slices containing ARC^NPY neurons (Fig. 6h). PVH RUVBL2 KO did not change the amplitude of sEPSCs (Fig. 6i, j), but significantly decreased the frequency of sEPSCs in ARC^NPY neurons (Fig. 6k). In addition, PVH RUVBL2 KO significantly decreased the peak amplitude of eEPSCs in ARC^NPY neurons (Fig. 6l, m), but did not alter PPR (ISI, 50 ms) (Fig. 6n). Collectively, these data indicate that PVH RUVBL2 is required for excitatory synaptic transmission between PVH^RUVBL2 neurons and ARC^POMC or ARC^NPY neurons.

As ARC^POMC and ARC^AgRP neurons exert an opposing role in feeding, and our projection-specific chemogenetics and optogenetics have demonstrated the anorexigenic effect of PVH^RUVBL2 → ARC, we postulated that the anorexigenic effect of PVH^RUVBL2 → ARC^POMC and other ARC neurons could outweigh the orexigenic effect of PVH^RUVBL2 → ARC^AgRP. To test this, we bilaterally injected Cre-dependent AAV9-DIO-hChR2-mCherry into the PVH of 8 week-old Ruvbl2-Cre mice that were implanted with an optic fiber above the ARC and also received bilateral injection of AAV9-Npy-FLP and FLP-dependent AAV9-fDIO-hM3D(Gq)-EGFP in the ARC (Fig. 6o). In this way, we were able to simultaneously activate ARC-projecting PVH^RUVBL2 and ARC^NPY neurons. As shown, activation of both ARC-projecting PVH^RUVBL2 and ARC^NPY neurons conferred a marked anorexigenic effect (Fig. 6p), supporting that the anorexigenic effect of PVH^RUVBL2 → ARC outweighs the orexigenic effect of PVH^RUVBL2 → ARC^AgRP.

To demonstrate that activation of PVH^RUVBL2 → ARC^POMC neural circuit partially, if not completely, mediates the anorexigenic effects by activation of PVH^RUVBL2 → ARC projections, we bilaterally injected Cre-dependent AAV9-DIO-hChR2-mCherry into the PVH of 8 week-old Ruvbl2-Cre mice implanted with an optic fiber above the ARC. The mice were pre-treated with SHU9119, a potent antagonist of MC3/4R⁶¹, prior to optical activation of PVH^RUVBL2 → ARC projections for 6 h. We observed that SHU9119 pretreatment significantly attenuated the anorexigenic effects induced by activation of PVH^RUVBL2 → ARC projections (Fig. 6q), supporting the important contribution of the melanocortin pathway to anorexia by activation of PVH^RUVBL2 → ARC.

RUVBL2 KO in the PVH reduces presynaptic boutons and alters synaptic vesicle distribution

Impairment in synaptic transmission could arise from aberrations of presynaptic inputs and/or synaptic vesicle distribution, which have been suggested to have direct impacts on synaptic plasticity^22,62. Thus, we reasoned that RUVBL2 KO might impair presynaptic inputs and/or presynaptic vesicle distribution, consequently causing abnormalities in synaptic transmission. To address this, we bilaterally injected AAV2-GFP or AAV2-Cre-GFP into the PVH of WT or Ruvbl2^fl/fl mice to knock out RUVBL2, and then analyzed presynaptic boutons as reflected by VGluT2-labeled puncta. As shown (Fig. 7a–d), VGluT2-labeled puncta in the ARC or DMH are significantly reduced after PVH RUVBL2 KO.

Next, we applied transmission electron microscopy (TEM) to examine the distribution of synaptic vesicles. Although RUVBL2 KO does not alter the length of active zone (AZ), it significantly reduces synaptic vesicles within 200 nm from the AZ in PVH^RUVBL2 neuron terminals in the ARC or DMH (Fig. 7e-l). Given that PVH RUVBL2 is the only variable in these experiments, we suppose that the altered presynaptic boutons and synaptic vesicle distribution are ascribed to RUVBL2 KO in the PVH. Note that these results are also consistent with our electrophysiology analysis. Collectively, these data suggest that RUVBL2 KO-caused impairment in the excitatory synaptic transmission underlies food intake increase.

We next investigated how RUVBL2 KO reduces presynaptic boutons and alters synaptic vesicle distribution. As RUVBL2 has been shown to participate in transcriptional control by interacting with different transcription factors or forming various chromatin-modifying complexes⁴⁰, we performed ChIP-Seq of the hypothalamus isolated from 8 week-old male WT mice. Our ChIP-Seq data show that RUVBL2 binds to the promoters of numerous genes that get involved in regulation of synaptic plasticity, in particular synaptic vesicle or neuron axonogenesis (Fig. 7m–o).

To further demonstrate the transcription control of genes related to synaptic vesicle or neuron axonognenesis by RUVBL2, we carried out RNA-Seq of the PVH from Ruvbl2^fl/fl mice that received bilateral injection of AAV2-GFP or AAV2-Cre-GFP into the PVH to knock out RUVBL2. Our combined analyses of RNA-Seq and the above-described ChIP-Seq reveal the down-regulation of a number of genes related to synaptic vesicle or neuron axonognenesis after RUVBL2 KO in the PVH (Fig. 7p, q). Together, these data are in support of our findings that PVH RUVBL2 KO reduces presynaptic boutons and alters synaptic vesicle distribution.

RUVBL2 overexpression enhances the excitatory synaptic transmission and suppresses food intake

We finally asked whether increased expression of RUVBL2 in the PVH enhances the excitatory synaptic transmission and suppresses food intake. To address this, we made a Cre-dependent RUVBL2 overexpression mouse model by inserting mouse Ruvbl2 coding sequence into the Hipp11 (H11) locus⁶³ (Ruvbl2^KI/+) (Supplementary Fig. 8a), and crossed Ruvbl2^KI/+ mice with Sim1-Cre transgenic mice¹⁰ to generate Ruvbl2^KI/KI;Sim1-Cre mice showing RUVBL2 overexpression in the PVH (Supplementary Fig. 8b, c).

Using Ruvbl2^KI/KI;Sim1-Cre mice, we examined the electrophysiological characteristics of ARC and DMH neurons receiving projections from PVH^RUVBL2 neurons. We found that the amplitude of sEPSCs was significantly increased in ARC or DMH neurons of Ruvbl2^KI/KI;Sim1-Cre mice compared with WT mice (Fig. 8a–d), although there was no difference in the frequency of sEPSCs (Fig. 8e, f). In addition, the amplitude of eEPSCs was significantly increased in ARC or DMH neurons of Ruvbl2^KI/KI;Sim1-Cre mice (Fig. 8g–j), while no difference in PPR (ISI 50 ms) was observed (Fig. 8k, l). Note that no changes were observed in either amplitude or frequency of sIPSCs in ARC or DMH neurons after RUVBL2 overexpression (Supplementary Fig. 8d–i). Together, these results demonstrate that RUVBL2 overexpression through the Sim1-Cre transgene enhances the excitatory synaptic transmission in PVH^RUVBL2 → ARC and PVH^RUVBL2 → DMH circuits.

**Fig. 8: RUVBL2 overexpression enhances the excitatory synaptic transmission and suppresses food intake.**

Our electrophysiological results implicate that RUVBL2 overexpression may influence the distribution of synaptic vesicles. Through TEM, we observed that synaptic vesicles within 10 nm from the AZ are significantly increased in PVH^RUVBL2 neuron terminals in the ARC or DMH of Ruvbl2^KI/KI;Sim1-Cre mice (Fig. 8m–t), suggesting that RUVBL2 overexpression alters the distribution of synaptic vesicles, thus enhancing the excitatory synaptic transmission.

When fed a regular chow diet, Ruvbl2^KI/KI;Sim1-Cre mice showed significantly decreased body weight (Fig. 8u, v). The decrease of body weight in Ruvbl2^KI/KI;Sim1-Cre mice is attributed to significantly decreased food intake (Fig. 8w), as there is comparable energy expenditure between these two genotypes as indicated by ANOVA (Supplementary Fig. 8j, k). Together, these data demonstrate that RUVBL2 overexpression suppresses food intake and this effect could be ascribed to an increase in the excitatory transmission.

To evaluate whether RUVBL2 overexpression confers any protection against diet-induced obesity (DIO), WT and Ruvbl2^KI/KI;Sim1-Cre mice started HFD feeding at the age of 8 weeks, and their weekly body weight was monitored. Ruvbl2^KI/KI;Sim1-Cre mice showed robust resistance to DIO (Fig. 8x, y), which is owing to the significantly reduced HFD intake (Fig. 8z), as no difference of energy expenditure was observed between these two genotypes as demonstrated by ANOVA (Supplementary Fig. 8l, m). We also observed significant improvement in blood glucose control and insulin sensitivity in Ruvbl2^KI/KI;Sim1-Cre mice compared with WT mice after HFD (Supplementary Fig. 8n–q). These data collectively demonstrate that RUVBL2 overexpression protects mice against DIO by suppressing food intake, and indicate that PVH RUVBL2 controls food intake by modulating synaptic plasticity (synaptic vesicle distribution) and synaptic transmission

Given that Sim1 is expressed in multiple regions other than the PVH¹³, we sought to further confirm the functional role of PVH RUVBL2 in energy homeostasis by bilaterally injecting Cre-dependent AAV2-DIO-RUVBL2 into the PVH of 4 week-old Sim1-Cre mice (Supplementary Fig. 10a, b). As shown, RUVBL2 overexpression conferred resistance to DIO (Supplementary Fig. 10c, d), which was primarily attributed to decreased food intake (Supplementary Fig. 10e, f). RUVBL2 overexpression mice showed significantly improved glucose tolerance and insulin sensitivity (Supplementary Fig. 10g, h). Together, these results support that PVH RUVBL2 inhibits food intake and protects against body weight gain and DIO.

Discussion

In this study, we reveal that RUVBL2 in the PVH suppresses food intake by modulating synaptic plasticity, and demonstrate that PVH^RUVBL2 neurons exert their anorexigenic effect through projecting to three brain regions. The elucidation of neural circuits between PVH^RUVBL2 neurons and these brain regions should greatly advance our understanding of PVH control of satiety.

We found that RUVBL2 is abundantly expressed in the PVH and PVH^RUVBL2 neurons constitute the majority of PVH^SIM1 neurons. Previous studies have established the PVH as a crucial satiety control site in the brain^1,18,20. Consistently, either KO of RUVBL2 in the PVH or chemogenetic inhibition of PVH^RUVBL2 neurons increase food intake, whereas chemogenetic or optogenetic activation of PVH^RUVBL2 neurons suppress food intake. Note that neither altering PVH RUVBL2 expression nor manipulating PVH^RUVBL2 neuron activity caused any observed changes on energy expenditure, suggesting that RUVBL2 and PVH^RUVBL2 neurons control energy balance primarily by regulating food intake. Nevertheless, it could be hard to exclude a potential effect of RUVBL2 on energy expenditure, given its abundant expression in the PVH and the reported role of PVH^BDNF and PVH^Oxytocin neurons in energy expenditure in previous studies^14,64.

Our study suggests three neural circuits in total mediating the anorexigenic effect of PVH^RUVBL2 neurons: PVH^RUVBL2 → PB, PVH^RUVBL2 → ARC, and PVH^RUVBL2 → DMH. We found that chemogenetic inhibition of PB-projecting PVH^RUVBL2 neurons significantly increases food intake. Consistently, previous studies have shown that several types of PVH neurons including PVH^MC4R, PVH^PDYN, and PVH^TrkB neurons exert their anorexigenic effects by projecting to the PB^13,15,49. These studies together indicate that the PB is an important downstream brain structure mediating the anorexigenic effects of PVH neurons.

The anorexigenic effect of PVH^RUVBL2 neurons could also be ascribed to their projections to the ARC that senses nutritional state and controls energy homeostasis⁵. On one hand, ARC^POMC and ARC^AgRP neurons project to distinct downstream brain sites as two well-studied cell types and essential components of central melanocortin system⁸. One well-established neural pathway is composed of the projections from ARC^POMC and ARC^AgRP neurons to the PVH, resulting in activation and inhibition of PVH^MC4R neuron, respectively⁵. On the other hand, ARC^POMC or ARC^AgRP neuron activity is also regulated by either peripheral signals²¹, or presynaptic inputs from a number of brain regions such as the brain stem⁶⁵, the DMH³⁴, the VMH²⁸, and the PVH³⁵. Interestingly, one study has suggested that leptin exerts it anorexigenic effects mainly through reducing the GABAergic inhibitory tone to POMC neurons⁵⁸. In the present study, we show that ARC^POMC neurons receive dense projections from glutamatergic PVH^RUVBL2 neurons, and PVH RUVBL2 KO impaired the excitatory synaptic transmission to ARC^POMC neurons, suggesting that PVH^RUVBL2 → ARC^POMC circuit mediates the anorexigenic activity of PVH^RUVBL2 neurons. Consistently, one study reported the synaptic connection between the PVH and ARC^POMC neurons, without elucidating the function⁶⁶.

One previous study showed that activation of ARC^POMC neurons did not suppress food intake instantly, but significantly reduced food intake within 24 hr⁶⁷, while our study showed that optogenetic activation of PVH^RUVBL2 → ARC circuit instantly suppressed food intake. These suggest that projections of PVH^RUVBL2 neurons to some non-POMC ARC neurons could exert an instant appetite-suppressing effect; yet the identities of these non-POMC ARC neurons remain to be defined in future studies. In addition, this study suggests a potential positive-feedback loop between the ARC and the PVH, thereby promoting a maximal or sustained satiety. Given the prominent role of ARC^POMC → PVH^MC4R neural pathway in suppressing food intake and a part of PVH^MC4R neurons (~33%) are RUVBL2-positive, we would suppose that ARC^POMC neurons and MC4R-positive PVH^RUVBL2 neurons could form reciprocal circuits; or within the PVH there may exist a neuronal circuit between RUVBL2-negative PVH^MC4R neurons and MC4R-negative PVH^RUVBL2 neurons. These possible neural circuits connect the PVH with the ARC, forming an enclosed circuit that functions as a positive-feedback loop and thus promotes substantial satiety.

PVH^RUVBL2 → DMH is another neural circuit mediating the anorexigenic effect of PVH^RUVBL2 neurons. The DMH controls energy homeostasis through regulating food intake and energy expenditure^68,69. Accumulative studies have demonstrated the stimulatory role for DMH neurons in adaptive thermogenesis^{70,71,72,73,74}. Interestingly, however, DMH neurons have been shown to exert different roles in regulation of food intake depending on cell types^75,76,77,78. In the present study, we found that alteration of PVH RUVBL2 expression or manipulation of PVH^RUVBL2 neuron activity only affect food intake, but do not influence energy expenditure. Therefore, the identity of DMH neurons receiving axonal terminals from PVH^RUVBL2 neurons remains to be defined.

Multiple factors are involved in modulating synaptic plasticity including quantity or activity of postsynaptic receptors, presynaptic input, and quantity or distribution of synaptic vesicles^79,80,81,82. To date, most studies on synaptic plasticity mainly focused on postsynaptic mechanisms including the number or property of postsynaptic receptor; in contrast, the presynaptic mechanisms (e.g., presynaptic inputs, and quantity or distribution of synaptic vesicles) have been relatively underestimated. The presynaptic alterations including structural remodeling and reorganization of presynapses tend to last long, thereby underlying a long-term behavioral change^22,83. Interestingly, one recent study showed that the increased axonal terminals in PVH^TRH → ARC^AgRP circuit promoted appetite and body weight (re)gain in the setting of weight loss, suggesting a role for structural synaptic plasticity in regulation of food intake³⁵. Consistently, we demonstrated that PVH RUVBL2 KO decreases the presynaptic boutons of ARC-projecting PVH^RUVBL2 neurons, impairs synaptic transmission, and increases food intake, implicating a causal role of structural synaptic plasticity in food intake control.

The quantity and distribution of synaptic vesicle could also affect synaptic plasticity in either a short-term (minutes to hours) or a long-term (days or more) manner, thereby altering synaptic strength and ultimately changing behaviors⁷⁹. In agreement with this, we observed that RUVBL2 KO significantly reduces synaptic vesicles, whereas RUVBL2 overexpression significantly increases synaptic vesicles.

RUVBL2 has been shown to control transcription by forming different transcription-associated protein complexes⁴⁰, or regulating the clustering of RNA polymerase II with different transcription factors⁸⁴, which is in agreement with its abundant distribution in the nucleus⁴¹. Our data indicate that RUVBL2 modulates synaptic plasticity and synaptic transmission by regulating the expression of genes related to axonogenesis and synaptic vesicle, consistent with the role for RUVBL2 in transcription control⁴⁰. In support of the role for PVH RUVBL2 in modulating synaptic plasticity, overexpression of RUVBL2 through the Sim1-Cre transgene increases synaptic vesicles within 10 nm near the AZ in PVH^RUVBL2 neuron terminals in the ARC or DMH. In addition, we observed that PVH RUVBL2 is significantly reduced during energy deficit, suggesting that RUVBL2 regulates synaptic plasticity in a dynamic manner in response to changing energy state. Of interest, however, whether RUVBL2 modulates synaptic plasticity and controls food intake dependent of its ATPase activity remains unknown, given that hypothalamic AMP-activated protein kinase (AMPK), a cellular energy sensor, has been shown to regulate food intake through diverse mechanisms^85,86,87,88.

In summary, this study reveals two neural circuits of PVH^RUVBL2 → ARC^POMC and PVH^RUVBL2 → DMH that mediate the anorexigenic effect of the PVH. Moreover, this study demonstrates an essential role for PVH RUVBL2 in food intake control by modulating synaptic plasticity, suggesting that modulation of synaptic plasticity could be an effective way to regulate food intake and body weight.

Methods

Animals

All animal care and use procedures were approved by the Animal Care and Use Committee (ACUC) at Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University (DXBYY-IACUC-2020036). The mice were euthanized with CO₂, followed by cervical dislocation. Both male and female mice were used in the study.

All mice were maintained on C57BL/6 J background. Mice were housed at the animal facility harboring a stable temperature (~22 °C), humidity and 12/12 h light/dark cycle, and gained free access to food and water. C57BL/6 WT mice were purchased from GemPharmatech (Nanjing, China). Ai14 (Stock #:007914), Sim1-Cre (Stock #:006395), Mc4r-2a-Cre (Stock #:030759), Pomc-EGFP (Stock #:009593), Agrp-Cre (Stock #012899), Npy-GFP (Stock #008321), Vglut2-ires-cre (Stock #:016963), Vgat-ires-cre (Stock #:028862), and Pomc-Cre (Stock #:005965) mouse strains were from the Jackson Laboratory (Bar harbor, ME, USA).

Loxp-flanked Ruvbl2 (Ruvbl2^fl/+) mice were generated by BIOCYTOGEN (Beijing, China). To generate Ruvbl2^fl/+ mice, two loxp sequences were inserted to flank the exons E3-E6 of Ruvbl2 via CRISPR-Cas9⁴².

Both Ruvbl2-Cre and Cre-dependent Ruvbl2 overexpression knock-in (Ruvbl2^KI/+) mice were generated by GemPharmatech (Nanjing, China). To generate Ruvbl2-Cre mice, P2A-Cre sequence was inserted after the coding sequence of the Ruvbl2 gene via CRISPR-Cas9 system. To generate Ruvbl2^KI/+ knock-in mice, a donor vector harboring the LoxP-Stop-LoxP (LSL) cassette and the mouse RUVBL2 coding sequence (CDS) that is in fusion with an HA tag was inserted into the ubiquitous H11 locus via CRISPR–Cas9 system.

Eight-week-old WT and Ruvbl2^KI/KI;Sim1-Cre were fed a high-fat diet (HFD) and their weekly body weight was recorded. After 8 weeks of HFD feeding, analyses of daily food intake and metabolic cages, IPGTT and IPITT were carried out.

Stereotaxic Injection of AAV

Mice were deeply anesthetized with 1.2% avertin and placed in a stereotaxic apparatus. Body temperature was maintained at 37 °C using a heating pad, and AAV was injected into the PVH of mice using a 5 μl Hamilton syringe with a 33-gauge needle that was attached to a stereotaxic arm. Infusion of each virus (0.2 μl, 1.8 μl/h, 10¹² viral particles/ml) was accomplished by a microsyringe pump into the PVH region using the following coordinates: AP, −0.65 mm; ML, ±0.28 mm; and DV,−5.05 mm relative to the bregma. After infusion, the needle was left in the brain for another 10 min to reduce backflow of viral injection along the needle track. The mice were recovered in a 37 °C incubator before returning to home cage. Mice with missed viral injections or unilateral injections were excluded from analysis (e.g., body weight and food intake measurement) after examining the virus-associated fluorescent protein expression.

The corresponding coordinates of other target brain regions in this study are as follows (calculated from the bregma): the ARC (AP, −1.85 mm; ML, ±0.27 mm; DV, −5.85 mm), the DMH (AP, −1.95 mm; ML, ±0.26 mm; DV, −5.10 mm), the POA (AP, −0.00 mm; ML, ±0.260 mm; DV, −5.15 mm), the MeA (AP,−1.46 mm; ML, ±1.95 mm; DV, −5.50 mm), the LSv (AP, 0.20 mm; ML, ±0.5 mm; DV, −3.50 mm), the NTS (AP, −7.0 mm; ML, ±0.7 mm; DV, −4.7 mm), and the PB (AP, −4.96; ML, ±1.4 mm; DV, −5.5 mm).

The following AAV were used: AAV2-GFP (HANBIO, Cat#57070645), AAV2-Cre-GFP (HANBIO, Cat#57070646), AAV8-EF1α-DIO-EGFP-T2A-TK (BrainVTA, Cat#PT-0087), HSV-ΔTK-LSL-tdTomato (BrainVTA, Cat#H03002), AAV2/retro-DIO-EGFP (OBIO, Cat#H6363), AAV8-EF1α-DIO-oRVG-WPRE (BrainVTA, #PT-0023), AAV8-EF1α-DIO-H2B-EGFP-T2A-TVA-WPRE (BrainVTA, #PT-0021), RV-ENVA-ΔG-DsRed (BrainVTA, Cat#R01002), AAV9-EF1a-DIO-hM3D(Gq)-mCherry (BrainVTA, Cat#PT-0042), AAV9-EF1a-DIO-hM4D(Gi)-mCherry (BrainVTA, Cat#PT-0043), AAV9-EF1a-DIO-mCherry (BrainVTA, Cat#PT-0013), AAV5-hSyn-Con/Fon-hM4D(Gi)-EYFP (BrainVTA, Cat#PT-1572), AAV2/retro-Flp (BrainVTA, Cat#PT-0341), AAV9-EF1a-DIO-hChR2-mCherry (BrainVTA, Cat#PT-0002), AAV9-Npy-FLP (BrainVTA, Cat#PT-2297), AAV9-fDIO-hM3D(Gq)-EGFP (BrainVTA, Cat#PT-1837), AAV2-mCherry (BrainVTA, Cat#PT-0407), AAV2-Cre-mCherry (BrainVTA, Cat#PT-0100), AAV2-DIO-GFP (OBIO, Cat#H15469), AAV2-DIO-RUVBL2-GFP (OBIO, Cat#H29352).

Isolation of the PVH

Mice were euthanized with CO₂, followed by cervical dislocation. The whole mouse brain was rapidly dissected and placed in a brain mold (Reinwald, model: 68707). Slices at the thickness of 1 mm (bregma ~−0.4- ~ −1.4 mm) were obtained using a blade, and then placed under a dissecting microscope (Leica EZ4) to isolate the PVH region carefully using fine scissors and forceps. The collected samples were stored in Trizol reagent (Invitrogen, Cat# 15596018) at −80 °C for subsequent RNA extraction, cDNA synthesis and quantitative PCR (qPCR).

Quantitative real-time PCR (qPCR)

The extraction of RNA and qPCR were performed as previously described⁸⁹. In brief, total RNA was extracted from the hypothalamic paraventricular nucleus (PVH) using Trizol reagent (Invitrogen, Cat# 15596018). Reverse transcription PCR was carried out using a RevertAid RT kit (thermoscientific, Cat#K1691), and quantitative PCR was conducted using SGExcel FastSYBR Mixture (Sangon Biotech, Cat# B532955) through QuantStudio 3 Real-Time PCR System (Applied Biosystems) following the manufacturer’s instructions. Hprt1 (forward: 5’-CAGTCCCAGCGTCGTGATTA-3’, reverse: 5’-TGGCCTCCCATCTCCTTCAT-3’), Ruvbl2 (forward: 5’-TTGGTGTCCGGATCAAGGAG-3’, reverse: 5’-CTTGAGGGTCAGTTTGCCCA-3’). The relative expression of target genes was calculated based on 2^-ΔΔCt method with Hprt1 as the reference gene.

Western blotting (WB)

Eight week-old male C57BL/6 J mice fed a regular chow diet ad libitum or fasted for 48 h were euthanized with CO₂, followed by cervical dislocation. The PVH was isolated and homogenized in 1 × lysis buffer containing 1% deoxycholic acid, 10 mM Na₄P₂O₇, 1% Triton 100, 100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, and 0.1% SDS. Protein concentrations were determined using a BCA protein assay kit (Biosharp, Cat# BL521). 20 μg of total protein was loaded in SDS-PAGE and transferred to PVDF membranes, followed by 1 h of blocking with 5% nonfat milk and incubation with primary antibodies against RUVBL2 (1:10000; proteintech #67851-1-Ig) or HSP90 (1:10000; proteintech #13171-1-AP) at 4 °C overnight. Finally, ECL detection systems were applied to develop signals. Densitometric analysis of WB bands was performed using ImageJ software.

Physiological measurements

Body weight was measured weekly after virus injection, or before and after HFD; daily food intake was measured after 12 weeks of virus injection or after 8 weeks of HFD as described previously⁹⁰. Briefly, mice were singly housed for 1 week to minimize the stress before their daily food intake was measured. After measurement of weekly body weight and daily food intake, energy expenditure (metabolic rate or oxygen consumption rate VO₂) was monitored using Promethion High-Definition Multiplexed Respirometry System (Sable Systems, North Las Vegas, NV, USA).

Immunohistochemistry

Mice were anesthetized with 1.2% avertin and perfused transcardially with ice-cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. Brain tissues were postfixed overnight at 4 °C and then immersed in 30% sucrose (in PBS) for 3 days at 4 °C before being sectioned at 30 μm thicknesses using a sliding microtome (Leica, SM2010R). Brain sections were rinsed once with PBS, permeabilized with 1% Triton X-100 in PBS for 10 min and washed with PBS for 5 min, then incubated with blocking buffer (0.2% Triton X-100, 5% goat serum or 1% bovine serum albumin and 10% goat serum in PBS) for 1 h at room temperature, and subsequently incubated with primary antibodies diluted in the blocking buffer overnight at 4 °C. The following primary antibodies were used: mouse anti-RUVBL2 (1:800; proteintech #67851-1-Ig), rabbit anti-RUVBL2 (1:400; abcam #ab36569), rabbit anti-prodynorphin (1:100; ABclonal #A5830), mouse anti-Oxytocin (1:1000; Millipore #MAB5296), rabbit anti-CRH (1:200; proteintech # 10944-1-AP), rabbit anti-tyrosine hydroxylase (1:1000; Sigma #AB152), rabbit anti-TRH (1:500; ThermoFisher #PA5-57331), rabbit anti-c-Fos (1:300; proteintech # 66590-1-Ig), rabbit anti-HA-Tag (1:1000; Cell Signaling Technology # 3724), rabbit anti-vGlut2 (1:1000, abcam #ab216463), rabbit anti-glutamate (1:500, Sigma #G6642). After three washes in PBST (PBS containing 0.2% Triton X-100) for 20 min each, sections were incubated with appropriate fluorescent secondary antibodies for 1 h at room temperature. Sections were washed in PBST for three times (20 min per time and a last wash in PBS for 10 min), incubated with 4′,6-diamidino-2- phenylindole (DAPI) for counter staining for 2 min, and then mounted onto slides for confocal image capture using a ZEISS confocal microscope (LSM 800).

An antibody against VGluT2 was used for IHC staining of the excitatory synapses in the ARC and DMH. AAV2-Cre-GFP or control AAV2-GFP was bilaterally injected into the PVH of 8 week-old Ruvbl2^fl/fl mice that were euthanized for IHC staining 6 weeks later. To quantify vGlut2⁺ puncta, images were saved as CZI files and analyzed with NIH Image software. For each image, the quantity of VGlut2⁺ puncta in a selected area was counted.

To investigate the co-expression of RUVBL2 with PDYN, Oxytocin, or CRH, mice were treated with 20 μg of colchicine (Sigma, C3915) in 1 μL of H₂O through intracerebraventricular injection with a microinjector at a slow rate (50 nl min⁻¹) with customized controllers (Coordinates: 1.0 mm lateral, 0.4 mm back from the bregma, and 2.5 mm below the skull). Injection was begun 5 min after tissue penetration, and the microinjector was pulled out 10 min after injection. After suturing, mice were recovered in a warm bracket before they were transferred to housing cages for 48 h before perfusion. The IHC staining images were captured using a ZEISS confocal microscope (LSM 800).

Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)

IPGTT and IPITT were performed as previously described⁹⁰. Briefly, as to IPGTT, mice were fasted overnight for 16 h with free access to water. The blood glucose levels were measured at baseline (0 min) from the tail vein using a glucometer before mice were intraperitoneally injected with glucose (1.5 g/kg body weight). After glucose injection, blood glucose levels were measured every 30 min until 2 h post injection. As to IPITT, mice were fasted for 6 h with free access to water. The fasting glucose levels were measured at baseline (0 min) before an intraperitoneal injection of insulin (0.5 U/kg body weight). Blood glucose levels were measured every 30 min until 2 h post injection of insulin. A portable glucometer (OneTouch Ultra) was used to measure blood glucose levels.

Anterograde monosynaptic tracing

The helper AAV8-EF1α-DIO-EGFP-T2A-TK and HSV-ΔTK-LSL-tdTomato were used to carry out anterograde monosynaptic tracing⁵². In the absence of the thymidine kinase (TK), the HSV-ΔTK does not replicates or spreads in neurons, but is capable of transmitting to the postsynaptic neurons in the presence of complementary TK expressed by the helper AAV8-EF1α-DIO-EGFP-T2A-TK, thereby labeling the postsynaptic neurons by tdTomato⁵². 8 week-old male Ruvbl2-Cre mice were deeply anesthetized with avertin and unilaterally injected with AAV8-DIO-EGFP-T2A-TK into the PVH. After infusion, the needle was left in the brain for another 10 min to reduce backflow of viral injection along the needle track before it was removed. The mice were recovered in a 37 °C incubator before returning to home cage. Three weeks later, HSV-ΔTK-LSL-tdTomato was injected into the same site of these mice as the previous helper AAV. Three days after HSV injection, mice were perfused and their brains were collected and sectioned for subsequent IHC analysis.

Retrograde tracing

Eight-week-old Ruvbl2-Cre mice were deeply anesthetized with 1.2% avertin and stereotaxically injected with AAV2/retro-DIO-EGFP into different brain regions identified by anterograde monosynaptic tracing studies including the LSv, the POA, the MeA, the DMH, the ARC, the PBN, and the NTS. After infusion, the needle was left in the brain for another 10 min to reduce backflow of the viral injection along the needle track. The wound was closed with sutures, and the mice were recovered in a 37 °C incubator before returning to a home cage. 2–3 weeks after viral injection, animals were perfused and the brains were harvested for IHC analysis.

Retrograde monosynaptic tracing

8 week-old Pomc-Cre and AgRP-Cre mice were deeply anesthetized with 1.2% avertin and unilaterally injected with Cre-dependent AAV8-EF1α-DIO-oRVG-WPRE and AAV8-EF1α-DIO-H2B-EGFP-T2A-TVA-WPRE into the ARC. After infusion, the needle was left in the brain for another 10 min to reduce the backflow of injected virus along the needle track. Three weeks later, these mice received a second injection of RV-ENVA-ΔG-DsRed into the same injection site. Seven days after viral injection, animals were deeply anesthetized and perfused.

Chemogenetics and projection-specific chemogenetics

For chemogenetics, 200 nL of AAV9-EF1a-DIO-hM3D(Gq)-mCherry, AAV9-EF1a-DIO-hM4D(Gi)-mCherry or AAV9-EF1a-DIO-mCherry were injected bilaterally into the PVH (AP, −0.65; ML, ±0.28: DV, −5.05) of 8 week-old Ruvbl2-Cre mice. For projection-specific chemogenetics, 200 nL AAV5-hSyn-Con/Fon-hM4D(Gi)-EYFP were injected bilaterally into the PVH of 8 week-old Ruvbl2-Cre mice that simultaneously received injection of 200 nL of AAV2/retro-Flp (into the various brain regions projected by PVH^RUVBL2 neurons. These brain regions include the LSv, the POA, the MeA, the DMH, the ARC, the PB, and the NTS. After surgery, the mice were allowed to recover for at least two weeks and received daily handling to reduce stress prior to experiment.

To measure food intake after chemogenetic activation, mice were fasted overnight (for food intake during day time) or 12 h (for food intake during night time), and then injected with vehicle or CNO (1 mg/kg BW, Tocris, Cat#4936). Hourly food intake was measured within 4 h after CNO treatment. To measure food intake of mice after chemogenetic inhibition, mice were injected with vehicle or CNO (1 mg/kg BW) and hourly food intake was measured within 4 h after CNO administration.

Optogenetics and projection-specific optogenetics

For optogenetics, AAV9-EF1a-DIO-hChR2-mCherry or AAV9-EF1a-DIO-mCherry was bilaterally injected into the PVH (AP = −0.65, ML = ± 0.28, DV = −5.05) of Ruvbl2-Cre mice. A 200 μm diameter optic fiber was implanted into the target regions, including the PVH (AP, −0.65; ML, 0.28; DV, −4.95), the DMH (AP, −1.95; ML, 0.26; DV, −5.10), and the ARC (AP, −1.85; ML, 0.27; DV, −5.85), to activate RUVBL2 neurons in the PVH, or the axon terminals of PVH^RUVBL2 neurons innervated the DMH or the ARC. Animals were singly housed for at least 2 weeks with daily handling prior to experiment.

Before optogenetics, mice were fasted overnight. For optogenetic activation, a 465 nm blue laser light with 20-ms pulse duration at 20 Hz for 10 min (constant 2 s on and 2 s interval alternating) was applied to activate the cell bodies or the axon terminals of PVH^RUVBL2 neurons. Cumulative food intake during 10 min of laser stimulation, 10 min before or after laser stimulation was measured, respectively.

To simultaneously activate ARC-projecting PVH^RUVBL2 neurons and ARC^NPY neurons, Cre-dependent AAV9-DIO-ChR2-mCherry was injected into the PVH of Ruvbl2-Cre mice that received implantation of an optic fiber above the ARC and injection of mixture of AAV9-Npy-FLP and FLP-dependent AAV9-fDIO-hM3D(Gq)-EGFP (1:1 ratio) into the ARC. For simultaneous activation of ARC-projecting PVH^RUVBL2 neurons and ARC^NPY neurons, mice were injected with vehicle or CNO (2 mg/kg BW), followed by optical activation of the axon terminals of PVH^RUVBL2 neurons in the ARC for 1 h using a 465-nm blue laser light. Cumulative food intake of mice within 1 h was measured.

To determine the contribution of the melanocortin pathways to PVH^RUVBL2 → ARC projection-induced anorexia, Cre-dependent AAV9-DIO-ChR2-mCherry was bilaterally injected into the PVH of 8 week-old male Ruvbl2-Cre mice that received implantation of an optic fiber above the ARC. At night time, mice were first intraperitoneally injected with SHU9119 (0.5 mg/kg, MCE, HY-P0227), followed by optical activation of the axon terminals of PVH^RUVBL2 neurons in the ARC using a 465-nm blue laser light for 6 h when the laser light was switched on every other 30 min. Cumulative food intake of mice within 6 h was measured.

Brain slice preparation

For electrophysiology recording, brain slices containing the PVH, the DMH, and the ARC were prepared from Ruvbl2^fl/fl mice injected with AAV2-GFP or AAV2-Cre-GFP, or from Ruvbl2^fl/fl;Pomc-EGFP and Ruvbl2^fl/fl;Npy-GFP mice injected with AAV2-mCherry or AAV2-Cre-mCherry into the PVH for 8 weeks. For ChR2-assisted circuit mapping (CRACM), brain slices containing the PVH, the DMH, and the ARC were prepared from Ruvbl2-Cre mice injected with AAV9-EF1a-DIO-hChR2-mCherry for 4 weeks. Precise viral injection sites were confirmed by fluorescence in the PVH. Brain slices containing the DMH and the ARC were prepared from WT and Ruvbl2^KI/KI;Sim1-Cre mice for electrophysiology recording of neurons after RUVBL2 overexpression.

Mice were euthanized with CO₂, followed by cervical dislocation. Coronal brain slices (200 μm thickness) were cut in ice-cold cutting solution containing (mM): 72 sucrose, 83 NaCl, 26 NaHCO₃, 22 glucose, 2.5 KCl, 1 NaH₂PO₄, 5 MgCl₂ and 1 CaCl₂ on a Leica vibratome (Leica). Brain slices were transferred to artificial cerebrospinal fluid (aCSF) containing (mM): 126 NaCl, 21.4 NaHCO₃, 10 glucose, 2.5 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂ and 2.4 CaCl₂ and incubated at 34 °C for 45 min. Brain slices in aCSF were kept at room temperature (24–26 °C) for at least 30 min before electrophysiological recording.

Electrophysiology

Visually guided (infrared DIC videomicroscopy and water-immersion X40 objective) whole-cell recordings were obtained with patch pipettes (2.5-4.5 MΩ) pulled from borosilicate capillary glass with a Sutter P97 puller (Sutter Instrument, Novato, CA, USA). All recordings were obtained with a Multiclamp 700B amplifier, Digidata 1550B converter and pClamp 10.7 software (Axon Instruments), sampled at 20 or 50 kHz, and filtered at 2 kHz. All data were analyzed with Igor Pro-6.2 (WaveMetrics). During recordings, the chamber contained brain slice was continuously perfused with aCSF solution bubbled with 95% O₂ and 5% CO₂ at room temperature.

Whole-cell voltage-clamp recordings were made from DMH and ARC neurons with membrane potential clamped at −70 mV. sEPSCs and evoked EPSCs were recorded with an internal solution containing (in mM): 140 Cs-gluconate, 15 HEPES, 0.5 EGTA, 2 TEA-Cl, 2 MgATP, 0.3 NaGTP, 10 phosphocreatine, 2 QX 314-Cl, pH was adjusted to 7.2 with CsOH. 20 μM picotoxin (PTX) were applied in aCSF to block GABAergic synaptic transmission. sIPSCs and evoked IPSCs were recorded with an internal solution containing (in mM): 140 CsCl, 1 BAPTA, 10 HEPES, 5 MgCl₂, 5 Mg-ATP, 0.3 Na₃GTP, 10 QX 314-Cl, pH was adjusted to 7.2 with CsOH. 50 μM D-(-)−2-amino-5-phosphonopentanoic acid (D-APV) and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were applied in aCSF to block AMPAR- and NMDAR-mediated synaptic transmission. A pair of electrical stimulations (0.1 ms, 20–40 V) with 50 ms intervals was applied via a bipolar electrode positioned near the recorded cells. Electrical output was controlled by a programmable pulse stimulator (Master-8, A.M.P.I.) and pClamp 10.7 software.

ChR2-assisted circuit mapping (CRACM)

ChR2-assited circuit mapping (CARCM) was conducted as previously described⁵⁹. In brief, Cre-dependent AAV9-EF1a-DIO-hChR2-mCherry was injected into the PVH of 8 week-old male Ruvbl2-Cre mice. Four weeks after surgery for recovery, the mice were ready to use. To photo-stimulate ChR2-expressing fibers, a blue light (473 nm wavelength, 5 ms, maximum) was applied to the field around the recorded cells in the ARC or the DMH via an optic fiber. The light output was controlled by a programmable pulse stimulator and pClamp 10.7 software.

Transmission electron microscopy (TEM)

Mice were euthanized with CO₂, followed by cervical dislocation. Coronal brain sections (200 μm thickness) containing the ARC were prepared and transferred to freshly prepared solution containing 2.5% glutaraldehyde and 2% paraformaldehyde for at least 1 h at room temperature. The fixed sections were stored in 4 °C refrigerator overnight. On the next day, the sections were trimmed into ~ 0.2 mm³ cube containing the ARC under a stereoscope. The sections were post-fixed in 1% osmium tetroxide for 1.5 h, dehydrated in an ascending series of ethanol [30, 50, 70, 85, 95, and 100% (vol/vol)], and stored in epoxy (EPON 812) overnight. The sections were then embedded in epoxy resin at 60 °C for 48 h of polymerization. The semithin sections (500 nm thickness) were stained with Toluidine blue and the ARC was confirmed by bright-field microscope. Then the resin was further trimmed until only the ARC was included. The ultrathin sections (50 nm thickness) were cut by Leica EM UC6 tramicrotome and mounted on Formvar-coated slot grids. After poststaining with 2% (wt/vol) uranyl acetate and 8% (wt/vol) lead citrate, the ultrathin sections were examined on a Tecnai Spirit TEM (FEI) at 120 kV. Data were analyzed with IMOD 4.11 (University of Colorado).

Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

ChIP-Seq library preparation

Eight-week-old male C57BL/6 J mice were euthanized with CO₂, followed by cervical dislocation. The hypothalamus was isolated, cross-linked with 1% formaldehyde for 10 min at room temperature, and quenched with 125 mM glycine. The chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-RUVBL2 antibody (ABclonal, #A1905). After reverse crosslinking, CHIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adapter ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA libraries were amplified for 15 cycles and sequenced using Illumina NovaSeq 6000 platform with paired-end 2 × 150 as the sequencing mode.

Data analysis

Raw reads were filtered to obtain high-quality clean reads by removing sequencing adapters, short reads (length < 35 bp) and low quality reads using Cutadapt v1.18 (https://doi.org/10.1093/bioinformatics/btu170) (non-default parameters: --max-n 0 --minimum-length 35) and Trimmomatic v0.38 (https://doi.org/10.1089/cmb.2017.0096) (non-default parameters: SLIDINGWINDOW:4:15 LEADING:10 TRAILING:10 MINLEN:35). Then FastQC (https://f1000research.com/articles/8-1874/v2) (with default parameters) was used to ensure high reads quality. The clean reads were mapped to the mouse genome (assembly GRCm38) using the Bowtie2 v2.3.4.1 (https://www.nature.com/articles/nmeth.1923) (with default parameters) software. Duplicate reads were then removed using picard MarkDuplicates. Peak detection was performed using the MACS v2.1.2 (https://doi.org/10.1186/gb-2008-9-9-r137) (non-default parameters: -f BAMPE -g hs/mm -p 0.01) peak finding algorithm with 0.01 set as the p-value cutoff. Annotation of peak sites to gene features was performed using the ChIPseeker R package (https://doi.org/10.1093/bioinformatics/btv145).

RNA sequencing (RNA-Seq)

AAV2-GFP and AAV2-Cre-GFP were injected into the PVH of 8 week-old male Ruvbl2^fl/fl mice. 6 weeks after virus injection, mice were euthanized with CO₂, followed by cervical dislocation. The PVH was isolated for total RNA extraction using Trizol reagent (Invitrogen), followed by RNA-Seq and data analysis completed by LC-Bio Technology CO. Ltd. (Hangzhou, China).

Quantitation and statistical analysis

All data are presented as means ± SEM. ImageJ was used to quantify WB images and VGluT2⁺ puncta of IHC images. Statistical analyses were performed using two-tailed unpaired or paired Student’s t-test (Excel 2011), or two-way ANOVA followed by Bonferroni’s test for multiple comparisons (GraphPad Prism 9). ANCOVA was performed using SPSS Version 26 (IBM SPSS Statistics, Chicago, IL, USA). p < 0.05 was considered statistically significant. Electrophysiology data were off-line analyzed with Igor 6.2 software (WaveMetrics, Portland, OR, USA). Prism 9 and Adobe Illustrator CS6 were used to generate and prepare all the figures.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All the data generated or analyzed in this study are included in this paper or the Supplementary Information. The CHIP-Seq and RNA-Seq data generated in the study have been deposited in Gene Expression Omnibus (GEO) under accession code GSE249791 and GSE252308, respectively. Source data are provided with this paper.

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Acknowledgements

We thank Profs. Dandan Wang (Fudan University, Shanghai, China), Cheng Zhan and Ji Liu (University of Science and Technology of China, Hefei, China), Juxue Li (Nanjing Medical University, Nanjing, China), and Yuanqing Gao (Nanjing Medical University, Nanjing, China) for sharing Ai14, Agrp-Cre and Npy-GFP, Mc4r-2a-Cre, Pomc-Cre, and Pomc-EGFP mouse models, respectively. We are grateful to Xueke Tan, Zhongshuang Lv, Xixia Li, and Qing Bian at the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Sciences, for help in sample preparation for electron microscopy and image capture. We thank Prof. Zhiping Pang (Rutgers University Robert Wood Johnson Medical School, USA) and Prof. Baoji Xu (UF Scripps Biomedical Research, University of Florida, USA) for critical reading of the manuscript. This work was supported by the National Key Research and Development Program of China (2019YFA0802500 to X.X. and W.J.Z.), the National Natural Science Foundation of China-Guangdong Joint Fund (U20A6005 to J.S.), ST2030-Majr Projects (2021ZD0202500 to J.S.), the National Natural Science Foundation of China (31971076 and 32271202 to X.X.), the Tianjin Municipal Science and Technology Commission (20JCJQJC00240 to X.X.), the R&D Program of Guangzhou National Laboratory (GZNL2023A02012 to X.X. and W.J.Z.), the Instrument Developing Project of the Chinese Academy of Sciences (YJKYYQ20180028 to J.S.), and the Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-032A).

Author information

These authors contributed equally: Mingming Xing, Yang Li, Yuqi Zhang, Juemou Zhou.

Authors and Affiliations

NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, The province and ministry co-sponsored collaborative innovation center for medical epigenetics, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300134, China
Mingming Xing, Yuqi Zhang, Juemou Zhou, Danting Ma, Mengqi Zhang, Minglei Tang, Ting Ouyang, Fumiao Zhang, Xiaofeng Shi, Weiping J. Zhang & Xiangyang Xie
State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
Yang Li & Shuli Zhang
University of Chinese Academy of Sciences, Beijing, 100049, China
Yang Li, Jianyuan Sun & Shuli Zhang
The Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China
Jianyuan Sun
Shenzhen Key Laboratory of Drug Addiction, Shenzhen Neher Neural Plasticity Laboratory, Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, 518055, China
Zuxin Chen

Authors

Mingming Xing
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Yang Li
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Yuqi Zhang
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Juemou Zhou
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Danting Ma
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Mengqi Zhang
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Minglei Tang
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Ting Ouyang
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Fumiao Zhang
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Xiaofeng Shi
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Jianyuan Sun
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Zuxin Chen
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Weiping J. Zhang
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Shuli Zhang
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Xiangyang Xie
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Contributions

Conceptualization and methodology, X.X., S.Z., and Z.C.; Investigation, M.X., Y.L., Y.Z., J.Z., D.M., M.Z., M.T., T.O., and F.Z.; Supervision, X.X., S.Z., X.S., J.S., and W.J.Z.; Writing-Original Draft, X.X., S.Z., M.X., Y.L., Y.Z., and J.Z.; Resources, X.X., S.Z., and W.J.Z.

Corresponding authors

Correspondence to Shuli Zhang or Xiangyang Xie.

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Nature Communications thanks Qingchun Tong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Xing, M., Li, Y., Zhang, Y. et al. Paraventricular hypothalamic RUVBL2 neurons suppress appetite by enhancing excitatory synaptic transmission in distinct neurocircuits. Nat Commun 15, 8939 (2024). https://doi.org/10.1038/s41467-024-53258-6

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Received: 08 March 2024
Accepted: 03 October 2024
Published: 16 October 2024
DOI: https://doi.org/10.1038/s41467-024-53258-6