Deep transcriptome profiling of human hypothalamic agouti-related protein and proopiomelanocortin neurons regulating energy homeostasis
1Laboratory of Reproductive Neurobiology, Hun-Ren Institute of Experimental Medicine, Budapest, 1083 Hungary;
2Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, 4032 Hungary;
3Department of Pathology and Experimental Cancer Research, Semmelweis University, 1083 Budapest, Hungary
**Correspondence: takacs.szabolcs@koki.hun-ren.hu*Correspondence: hrabovszky.erik@koki.hun-ren.huSummary
We have developed and validated a pioneering ‘IHC/LCM-Seq’ method for transcriptome profiling of spatially defined neuronal cell types detected with immunohistochemistry in sections of formaldehyde-fixed human brains. IHC/LCM-Seq provided unprecedented insight, with 14,000-16,000 transcripts identified, into the gene expression landscape of human agouti-related peptide (AgRP) neurons, which drive appetite and energy storage, and proopiomelanocortin (POMC) neurons, which suppress feeding and promote energy expenditure. These cells differed from each other, and from fertility-regulating kisspeptin neurons, in their distinct enrichments of co-transmitters, transcription factors, and receptors. The AgRP neuron transcriptome was rich in receptors for proinflammatory cytokines, metabolic hormones and growth hormone, whereas POMC neurons expressed reproductive hormone-, glucagon-like peptide– and endocannabinoid receptors. IHC/LCM-Seq, a versatile spatial transcriptomic approach for characterizing cell types in postmortem brains, opened a new window onto molecular mechanisms regulating energy homeostasis in the human hypothalamus and highlighted possible pharmacological targets for weight management.
Article notes
Competing Interest Statement
The authors have declared no competing interest.
Introduction
The hypothalamic arcuate (aka infundibular) nucleus plays a crucial role in energy homeostasis, primarily through the actions of two functionally antagonistic neuronal populations: Agouti-related protein (AgRP) neurons, which stimulate appetite and promote energy conservation, and proopiomelanocortin (POMC) neurons, which suppress food intake and enhance energy expenditure.1 AgRP and POMC neurons are highly responsive to peripheral metabolic cues – including leptin, insulin, and ghrelin – and their dysfunction is strongly linked to disorders of energy homeostasis, such as obesity, anorexia, and diabetes.1,2 Defining the molecular signatures of AgRP and POMC neurons is fundamental for identifying therapeutic strategies against metabolic disorders. In particular, detailed characterization of their receptor profiles may uncover novel mechanisms regulating energy homeostasis and guide targeted approaches to address the obesity pandemic. For example, expression of GLP1R in anorexigenic POMC neurons likely contributes to the anti-obesity effects of semaglutide, whereas calcitonin receptor (CALCR) expression in these neurons may underlie the efficacy of recent combination therapies pairing semaglutide with the amylin analogue cagrilintide, which can act on CALCR.3
Current knowledge of the regulatory mechanisms within hypothalamic neuronal circuits targeted by obesity and diabetes treatments has largely been derived from animal studies, limiting the translational potential for developing effective therapies in humans. Recent advances in single-nucleus RNA sequencing (snRNA-seq) have enabled the creation of a spatio-cellular transcriptional map (‘HYPOMAP’) of the human hypothalamus.4 The authors identified approximately 2,000 transcripts per cell and integrated their snRNA-seq data with a hypothalamic expression matrix derived from publicly available whole-brain human datasets.5 Through multi-level hierarchical unsupervised clustering, they delineated 291 hypothalamic neuronal clusters, including a single AgRP neuron cluster defined by five signature genes, and three distinct POMC neuron clusters characterized by eight marker genes.4
The small quantity of input RNA severely limits the sensitivity of various single-cell sequencing techniques. By contrast, bulk sequencing of neuronal pools isolated via fluorescence-activated cell sorting (FACS) or laser capture microdissection (LCM) enables the detection of rare transcripts and produces highly detailed transcriptomic profiles that are otherwise difficult to obtain. Our laboratory recently achieved a methodological breakthrough in bulk sequencing by integrating innovative RNA-preserving strategies during immunohistochemistry (IHC) with the isolation of labeled neurons using LCM, followed by RNA-Seq.6 In the present study, we adapted the ‘IHC/LCM-Seq’ approach to human postmortem brains fixed by immersion in 4% formaldehyde (FA). This adaptation enabled transcriptomic profiling of AgRP and POMC neurons in the arcuate/infundibular nucleus, as well as fertility-regulating kisspeptin (KP) neurons7 of the same region, which we selected for comparison with a third cell type. The IHC/LCM-Seq methodology yielded unprecedented transcriptome coverage – approximately 14,000 to 16,000 transcripts per cell type. These included rare transcription factors, regulatory RNAs, and, notably, receptors, some of which may become potential targets for future weight-management strategies.
Results
Immersion-fixation of postmortem brain samples preserves tissue RNA integrity
Perfusion fixation of the brain with 4% FA constituted the initial step of the IHC/LCM-Seq protocol we developed for laboratory rodents.6 Because this fixation protocol is not applicable to autopsy material, we first assessed how postmortem delay and immersion fixation jointly affect tissue RNA integrity. Neocortical blocks (∼4 mm in each dimension) were dissected from the brain of a 53-year-old woman ∼20 h after death and immersion-fixed in cold (4 °C) 4% FA for 24, 48, or 72 h, then infiltrated with 20% sucrose for 24 h. Twenty-µm-thick free-floating sections were prepared using a freezing microtome and stored at – 20 °C in a cryoprotectant solution formulated for long-term RNA preservation6 (Fig. 1A). As we proposed recently6, all solutions included the RNase inhibitor aluminon8 (aurintricarboxylic acid, ammonium salt; ATA; 0.05%). Bioanalyzer measurements of total RNA extracted from test sections revealed consistently high RNA integrity (RIN > 7.0) and comparable yields (∼2 ng/mm2), regardless of fixation duration (Fig. 1B, Table S1).
A modified immunohistochemistry protocol preserves tissue RNAs
Encouraged by the high RIN values of immersion fixed samples, we next adapted the IHC/LCM-Seq workflow to human tissue sections. The modified IHC protocol began with epitope retrieval in 0.1 M citrate buffer (pH 6.0, 80 °C, 30 min), supplemented with either 0.05% ATA or polyvinyl sulfonate (PVSA) at concentrations of 1%, 3%, or 10% (Fig. 1C). In the absence of RNase inhibitors, RIN values dropped sharply from 7.8 ± 0.3 to 3.1 ± 0.2. In contrast, inclusion of 0.05% ATA or 3-10% PVSA effectively preserved RNA integrity (Fig. 1D, Table S1). As a final validation of our RNA-preserving IHC protocol, we visualized cortical parvalbumin neurons immunohistochemically (Fig. 1E), followed by RNA quality analysis. Sections immunostained without RNase inhibitors exhibited substantial RNA degradation, with RIN values of 2.4 ± 0.1 at 4 °C and 2.1 ± 0.2 at RT. In contrast, supplementing all antibody and buffer solutions with 0.05% ATA or 2% PVSA effectively preserved RNA, yielding RIN values of 7.7 ± 0.5 (ATA) and 6.8 ± 0.0 (PVSA) at 4 °C, and 7.1 ± 0.8 (ATA) and 6.6 ± 0.2 (PVSA) at RT (Fig. 1D, Table S1).
Of note, ATA tended to compromise specific immunostaining and increase nonspecific background (Fig. 1E), consistent with our previous observations.6 Therefore, the finalized IHC/LCM-Seq protocol for profiling hypothalamic AgRP, POMC, and KP neurons incorporated 2% PVSA in all buffer and antibody solutions (Fig. 2A).
Optimized tissue processing enables high-resolution transcriptome profiling of hypothalamic AgRP, POMC and KP neurons
The optimized IHC/LCM-Seq protocol (Fig. 2A) enabled robust immunohistochemical detection of AgRP, POMC and KP neurons in the human hypothalamus, facilitating their capture by LCM (∼850 labeled cells per cell type per subject). This was followed by RNA isolation, cDNA library preparation, and Illumina-based RNA sequencing (Fig. 2B). Pre-IHC RINs from hypothalamic test sections of seven male subjects ranged from 3.1 to 6.7 (Fig. 2C). Sequencing data have been deposited in BioProject under accession numbers PRJNA1305226 (AgRP and POMC neurons) and PRJNA1305334 (KP neurons).
Sequencing of eight libraries – comprising 3 AgRP, 2 POMC, and 3 KP samples – generated a total of 211.8 million raw reads (17.6-33 M per sample). Following trimming, adapter removal, alignment to the human reference genome (Ensembl release 111) using STAR (v2.7.11b), and gene assignment with FeatureCounts (subread v2.0.6), an average of 2.2 ± 0.6 million reads per sample (17.6 million total) were uniquely mapped and assigned to genes. This enabled identification of approximately 18,000 ± 1200 transcripts with ≥ 1 read, and 15,000 ± 800 transcripts with ≥ 5 reads (Fig. 2D, Table S2). Principal component analysis (PCA) verified three distinct transcriptomic profiles, each corresponding to a specific cell type (Fig. 2E). The absence of marker neuropeptide transcripts (AGRP, POMC, and KISS1) outside their respective neuronal populations, together with asymmetric expression of established neuronal phenotype markers, confirmed that there was minimal – if any – cross-contamination between transcriptomes (Fig. 2F).
Differential gene expression analysis reveals cell type-specific transcript enrichment, including 50 transcription factors
A total of 1,210 transcripts were significantly enriched in a cell type-specific manner among AgRP, POMC, and KP neurons (adjusted p-value /p.adj./ < 0.05, DESeq2; Table S2). Differentially expressed genes (DEGs) are visualized through heat maps (Fig. 3A), numerical expression data (Fig. 3B), and volcano plot markers across the three pairwise comparisons (Figs. 3C-E). We further examined 902 transcription factor (TF) transcripts annotated in the KEGG BRITE database to explore their potential roles in shaping the distinct transcriptomic profiles of AgRP, POMC, and KP neurons (Table S3). Of the 40 most abundant TFs selected per cell type (63 in total), 13 were significantly differentially expressed, indicating cell type-specific transcriptional regulation (Fig. 3F, Table S3). Expanding the analysis to all expressed TFs revealed 50 that differed between at least two neuronal populations, underscoring broader cell type-specific transcriptional regulation (p.adj. < 0.05, DESeq2; Fig. 3G, Table S3). Many of the shared transcription factors could reflect similar developmental programs, humoral signals, and afferent inputs. Transcription factors with significant cell type-specific enrichment (p.adj. < 0.05, fold change > 1.5 vs. other cell types) included NR3C1, OTP, XBP1, KLF9, FOXO1, ETV5, CREM, ZBTB16, and ST18 for AgRP neurons; ZFHX4, ONECUT1, and LEF1 for POMC neurons; and BCL6, ISL1, and PROX1 for both AgRP and POMC neurons. Unlike AgRP neurons, POMC cells also shared expression for several transcription factors with KP neurons (PGR, AR, ESR1, NHLH2, and SOX1). SOX14, STAT5A, and PLAGL1 were enriched in KP neurons (Fig. 3G; Table S3).
AgRP, POMC and KP neurons exhibit distinct neuropeptide co-transmitter profiles
Neuropeptide and granin expression profiles, based on annotations from the Neuropeptide Database9 (www.neuropeptides.nl), showed marked differences among the three neuronal populations. Normalized transcript abundances, expressed in TPM, are presented in Fig. 4A and Table S4. AgRP neurons were enriched for NPY, AGRP, SST, and UTS2, and exhibited higher expression of TAC1, GAL, VGF, and CRH compared to POMC and KP neurons. POMC neurons uniquely expressed POMC and VIP, and had higher levels of ADCYAP1 relative to KP neurons. KP neurons selectively expressed TAC3 and KISS1, and showed elevated expression of CBLN1, NXPH1, PENK, CBLN4, PDYN, and NMU compared to AgRP and POMC neurons (Figs. 4A, B).
A subset of receptors expressed in AgRP, POMC, and/or KP neurons was implicated in genetic disorders affecting energy homeostasis
To identify transcripts associated with metabolic disorders, we selected fourteen metabolism-related traits from the GWAS Catalog10 (Fig. 5A, Table S5). These traits included 2,493 genes that were also expressed in AgRP, POMC, and/or KP neurons, applying cutoffs of mean TPM ≥ 10 and at least 5 reads in each sample of the respective cell type (Fig. 5B, Tables S6-S7). Among these, we identified 52 receptor genes (Fig. 5C, Table S8), several of which showed cell type-specific enrichment. Receptor expression profiles were further examined to identify potential therapeutic targets for the treatment of metabolic disorders.
Receptor transcripts reveal distinct humoral and neuronal regulatory mechanisms
A total of 205 receptor transcripts detectable in AgRP, POMC, and/or KP neurons (cutoffs: mean TPM ≥ 10 and reads ≥ 5 in each sample of the same cell type) are illustrated in Fig. 6 and listed in Table S8. This dataset includes new candidate receptors for targeting hypothalamic metabolic and/or reproductive pathways. Of these, 112 showed at least twofold enrichment, and 62 exhibited significant differential expression in pairwise comparisons between cell types. ACVR1C encoding an activin receptor was the most abundantly expressed receptor, occurring exclusively in AgRP neurons. These neurons also showed selective enrichment for receptors of proinflammatory cytokines (IL18R1, IL1R1), circulating hormones (GHR, GHSR, NR3C1), and various neuropeptides related to energy homeostasis (RXFP1, NPY2R, AVPR1A, MC3R, AGTR1, and CALCRL). POMC neurons showed enrichment for several receptors involved in metabolic hormone and neuropeptide signaling, including NPY1R, CALCR, SSTR1, HCRTR2, QRFPR, GLP1R, CCKAR, PROKR1, and GRPR), as well as the type-1 endocannabinoid-(CNR1) and the H1 histamin receptor (HRH1). The leptin receptor (LEPR) was co-enriched in AgRP and POMC cells. POMC and KP neurons both expressed reproductive hormone receptors (PRLR, PGR, AR, ESR1), with higher levels in the latter, whereas opioid receptors (OPRM1, OPRL1, OPRK1) were expressed more prominently in POMC than in KP neurons. (Fig. 6; Table S8). IL6ST, ADCYAP1R1, and the insulin receptor (INSR) were among the most highly expressed receptors in all three cell types.
Discussion
Methodological considerations
The protocols used in this study are based on our previously established ‘LCM-Seq’ method, which was originally developed for spatial transcriptomic profiling of fluorescently labeled neurons in transgenic mice.11 To eliminate the reliance on genetic tagging, we subsequently developed the more versatile ‘IHC/LCM-Seq’ method, in which we replaced transgenic labeling with RNA-preserving immunohistochemistry. This change enabled deep transcriptomic profiling of neuronal populations across species.6 In the present study, we successfully adapted the IHC/LCM-Seq technique for use in postmortem human brain tissue. This transition introduced unique challenges inherent to autopsy-derived material, necessitating the implementation of rigorous quality control measures throughout the workflow. We found that RNA integrity was well preserved in samples collected within 24 hours postmortem and immersion-fixed in cold 4% FA for 24-72 hours. Variations within this fixation window had minimal – if any – impact on RNA quality, offering practical flexibility for tissue processing. Importantly, transcript detection was also largely independent of the initial RNA Integrity Number (RIN), which ranged from 3.1 to 6.7 across samples. We attributed this robustness to the use of random primers in the TruSeq library preparation protocol, which can accommodate highly fragmented RNA.12 Post-fixation handling – comprising cold 20% sucrose infiltration, sectioning at 20 µm, and section storage at –20 °C in cryoprotectant solution with 0.05% ATA – followed protocols we had previously optimized for mouse and rat brains.6 When working with immersion-fixed human brains, successful immunolabeling often requires an epitope retrieval step (30 min at 80 °C in 0.1 M citrate buffer, pH 6.0). While this pretreatment initially compromised RNA quality, the addition of 10% PVSA to the retrieval buffer effectively mitigated degradation. Subsequent immunostaining in the presence of either 0.05% ATA or 2% PVSA efficiently preserved RNA integrity at both 4 °C and RT. However, ATA caused increased nonspecific background staining and also compromised the specific signal, making PVSA the preferred RNase inhibitor for routine IHC/LCM-Seq applications. Overall, our methodological results confirmed that IHC/LCM-Seq is a robust, highly sensitive, and species-independent technique. Adapting this method to postmortem human brains in this study has substantial translational and clinical relevance.
As we discussed previously6, IHC/LCM-Seq offers several advantages over single-cell approaches such as scRNA-Seq and snRNA-Seq, which rely on enzymatic dissociation of brain tissue. i) LCM preserves the spatial context of neurons, maintaining their native anatomical architecture and microenvironment. ii) Tissue fixation prior to RNA extraction stabilizes the transcriptome and reduces artifacts introduced during dissociation and cell isolation processes. iii) IHC/LCM-Seq provides considerably enhanced sensitivity for detecting low-abundance transcripts, including long non-coding RNAs, transcription factors, and rare receptor mRNAs. However, we note that achieving unparalleled sensitivity involves a trade-off in cellular resolution. Unlike single-cell methods, bulk RNA sequencing cannot resolve cellular heterogeneity or distinguish subpopulations within a given neuronal type. For instance, recent snRNA-Seq studies have revealed three transcriptionally distinct subclusters of human POMC neurons – variability that remained undetectable in our bulk datasets. Thus, IHC/LCM-Seq and single-cell approaches should be viewed as complementary methodologies, each offering unique advantages depending on the biological question.
Functional implications of transcriptomic findings
Our study provides unprecedented insight into the molecular architecture of orexigenic AgRP and anorexigenic POMC neurons playing central roles in the regulation of energy homeostasis. The inclusion of KP neurons as a third cell type offers a valuable comparative perspective; while localized similarly in the arcuate nucleus, these neurons play a primary role in regulating the reproductive axis.7 With 14,000-16,000 transcripts detected per neuronal cell type, our dataset offers a comprehensive resource for dissecting the transcriptional programs that underlie the mediobasal hypothalamic control of energy balance and reproduction.
TFs reflecting shared and unique regulatory features and developmental origins of arcuate neuron phenotypes
Despite their distinct functional roles, AgRP, POMC, and KP neurons exhibited substantial overlaps in their TF profiles, similarities potentially reflecting shared developmental origins13 and/or common microenvironmental influences; among the 902 detected TFs, only 50 were differentially expressed across cell types.
TFs typical of AgRP neurons included OTP, FOXO1, and the glucocorticoid-responsive TFs NR3C114, KLF915, and ZBTB1616. Most TFs detected in POMC cells were not unique, as many were also expressed in AgRP or KP neurons. The presence of sex steroid receptors (PGR, AR, ESR1) provides a potential mechanistic link between gonadal hormone signaling and the hypothalamic regulation of metabolic functions17,18; not surprisingly, these receptors are expressed at the highest levels in KP neurons, consistent with the crucial involvement of this cell type in negative sex steroid feedback to the reproductive axis.7
Distinct transcriptomic signatures of AgRP, POMC, and KP neurons
Using differential expression analysis, we detected 1,210 transcripts exhibiting significant differences in expression across the three neuronal populations. CARTPT was expressed in AgRP neurons but not in POMC cells, representing a notable species difference from rodents that has already been reported previously.19 High levels of this mRNA were also detected in KP neurons, consistent with our laboratory’s earlier immunohistochemical observations.20 Additional neuropeptide mRNAs with enriched expression in AgRP neurons included NPY, TAC1, SST, GHRH, GAL, CRH, VGF and UTS2. Some of the encoded peptides may play thus far underappreciated roles in human orexigenic circuitry. ADCYAP1 and VIP represented two neuropeptides expressed predominantly in POMC neurons and only at relatively low levels. Further transcripts typical of the POMC cell type – including PENK, CBLN1, CBLN4, NXPH1, and PDYN – were even more prominent in KP neurons, whereas TAC3, encoding neurokinin B, was exclusively expressed in KP neurons.
Supporting information
Acknowledgments
This work was supported by grants to Project no. RRF-2.3.1-21-2022-00011, titled National Laboratory of Translational Neuroscience implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Széchenyi Plan Plus, the National Research, Development and Innovation Office (K138137 to E.H. and PD134837 to K.S). We would like to thank Drs. C. Fekete and W.S. Dhillo for kind donation of the POMC and KP antibodies, respectively.
Declaration of interests
The authors declare no competing interests.
Supplemental information titles and legends
Table S1: RIN and RNA yield values for Figure 1B, RIN values and ANOVA statistics for Figure 1D
Table S2: Whole transcriptome profile of hypothalamic AgRP, POMC and KP neurons including differential expression data
Table S3: Gene expression profile of transcription factors
Table S4: Gene expression profile of neuropeptides and granins
Table S5: Selected, metabolism-related mapped traits from the GWAS Catalog
Table S6: Transcripts filtered for GWAS association by mean TPM ≥ 10 and at least 5 reads in each sample of the same neuronal phenotype
Table S7: Transcripts of ‘MAPPED_GENEs’ expressed in AgRP, POMC or KP neurons that were associated with metabolic disorders in the GWAS Catalog
Table S8: Receptor expression profiles of AgRP, POMC and KP neurons including GWAS trait associations
Methods
Experimental model and subject details
Human subjects
Histological samples from adult human brains (N = 8) were obtained during autopsies conducted at the Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary. The specimens were derived from one 53-year-old female and seven male individuals aged 28, 33, 38, 46, 52, 53, and 56 years, all with no documented neurological disorders. Tissue collection was performed 12 to 24 hours postmortem. Ethical approval was granted by the Regional and Institutional Committee of Science and Research Ethics of Semmelweis University (SE-TUKEB 251/2016), in compliance with the Declaration of Helsinki and relevant Hungarian legislation (Act CLIV of 1997 and Decree 18/1998 [XII.27.] of the Ministry of Health), which does not mandate prior written consent from the deceased.
Method details
Experimental procedures
For all experiments, reagents were of molecular biology grade. MQ water and PBS were pretreated overnight with diethylpyrocarbonate (DEPC; Thermo Scientific; 1 mL/L) and subsequently autoclaved. All other solutions were prepared using DEPC-treated and autoclaved MQ water. Reagents and buffers were maintained under RNase-free, sterile conditions with or without the additional use of the RNase inhibitors. Work surfaces were cleaned with RNaseZAP (Merck KGaA, Darmstadt, Germany).
Optimization of the IHC/LCM-Seq method for human brain tissues
Immersion fixation and section preparation
To optimize the IHC/LCM-Seq protocol for brain samples gained from autopsies, test experiments were performed using 4-mm-thick tissue blocks (n = 9) dissected 20 h postmortem from the frontal cortex of a 53-year-old female subject. Samples were immersion-fixed for 24, 48, or 72 hours (n = 3 per fixation time) in freshly prepared ice-cold 4% formaldehyde (FA) in 0.1 M phosphate-buffered saline (PBS; pH 7.4). Following fixation, tissues were infiltrated with ice-cold sterile 20% sucrose in PBS for 24 hours, snap-frozen on powdered dry ice, and stored at –80 °C until sectioning. The fixative and sucrose solutions were supplemented with 0.05% aluminon (triammonium salt of aurintricarboxylic acid, ATA; Merck) to enhance RNA preservation.6,8 Prior to sectioning, the white matter was trimmed from each tissue block. Free-floating 20-µm-thick sections were prepared using a Leica freezing microtome (Leica Biosystems, Wetzlar, Germany) (Fig. 1A).
Assessing the impact of immersion fixation on RNA quality and recovery
RNA was extracted from individual cortical sections fixed for 24, 48, or 72 hours using the PureLink FFPE RNA Isolation Kit (Thermo Scientific, Waltham, MA, USA). For each fixation time (n = 3), RNA yield – normalized to the section area (mm2) – and RNA Integrity Number (RIN) were determined using the Agilent 2100 Bioanalyzer system with Eukaryotic Total RNA Pico Chips and the 2100 Expert software (Agilent, Santa Clara, CA, USA). Results were presented as mean ± SEM from triplicate measurements (Table S1).
Tissue archiving and optimization of RNA preservation during antigen retrieval
Most cortical sections were stored for one month at –20 °C in a modified cryoprotectant solution (30% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer; pH 7.4) supplemented with 0.05% aluminon (ATA). We have previously demonstrated that this storage method preserves RNA integrity for three years or longer.6 Optimization of section pretreatments and the IHC protocol used the three tissue blocks that were immersion-fixed for 72 hours. Sections retrieved from the antifreeze medium were rinsed in sterile PBS and subjected to a 30-minute heat-induced epitope retrieval step in 0.1 M citrate buffer (pH 6.0, 80 °C), either without additives or in the presence of the RNase inhibitors ATA (0.05%) or polyvinyl sulfonic acid (PVSA)45 used at 1%, 3%, or 10%. Following brief PBS washes, total RNA was extracted, and RNA integrity was assessed using the Agilent 2100 Bioanalyzer system. Results were presented as mean ± SEM (Table S1) of three measurements on independent samples. Differences between treatment groups – with and without antigen retrieval – were assessed by one-way ANOVA followed by Tukey’s post hoc tests.
Optimization of the RNA-preserving immunohistochemistry (IHC) protocol
Antigen retrieval was performed in the presence of 10% polyvinyl sulfonic acid (PVSA) which efficiently protected RNA in the above experiment. Subsequently, sections were processed for immunohistochemical detection of cortical parvalbumin-expressing neurons at either RT or 4 °C, with or without the use of RNase inhibitors. To inhibit RNases, all antibody and buffer solutions (except the peroxidase developer) contained either 0.05% aluminon (ATA) or 2% PVSA. Sections were pretreated with 1% H2O2 and 0.5% Triton X-100 in PBS for 20 minutes, followed by incubation with a commercially available affinity-purified goat polyclonal antibody against human parvalbumin (#EB06776; Everest Biotech; 1:30,000) for either 40 hours at 4 °C or overnight at RT. The primary antibody was reacted with Biotin-SP-AffiniPure anti-goat IgG secondary antibody (Jackson ImmunoResearch; 1:500; overnight at 4 °C or 2 hours at RT), followed by the ABC Elite reagent (Vector Laboratories, Burlingame, CA, USA; 1:1,000; 1 hour at RT) in PBS. The peroxidase reaction was visualized at RT using a developer containing 10 mg diaminobenzidine, 30 mg nickel-ammonium sulfate, and 0.003% H2O2 in 20 ml of 0.05 M Tris-HCl buffer (pH 8.0). RNA was extracted from pre-IHC sections (n = 3) and from post-IHC sections (n = 3 per treatment group), and RNA integrity was assessed using the Agilent 2100 Bioanalyzer System (Table S1). Differences in RIN values among the post-IHC groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test.
For qualitative analysis of the immunohistochemical labeling, the immunostained sections were mounted onto microscope slides from Elvanol, air-dried for 30 minutes, then sequentially dehydrated in 70%, 95%, and 100% ethanol (5 minutes each), followed by clearing in xylene (2 × 5 minutes). Coverslipping was performed using DPX mounting medium (Merck, Darmstadt, Germany) for light microscopic analysis and imaging. Digital images were adjusted for brightness and contrast using Adobe Photoshop (Adobe Systems), and final composite figures were assembled in Adobe Illustrator (Adobe Systems).
IHC/LCM-Seq studies of hypothalamic AgRP, POMC and KP neurons
Immersion fixation of hypothalamic samples
Four-mm-thick hypothalamic slices from seven male individuals (Fig. 2C) were immersion-fixed in ice-cold 4% FA in 0.1 M phosphate-buffered saline (PBS; pH 7.4) for 72 hours, followed by infiltration in 20% sucrose (24 h). Both solutions were supplemented with 0.05% aluminon (ATA). Free-floating 20-µm-thick coronal sections were prepared from the mediobasal hypothalamus (atlas plates 24-27)46,47 using a freezing microtome and stored in modified cryoprotectant solution containing 0.05% ATA, as described for cortical samples.
RNA quality testing
Total RNA was extracted from a hypothalamic section per case using the PureLink FFPE RNA Isolation Kit (Thermo Scientific) its integrity was assessed with the Agilent 2100 Bioanalyzer system. Samples used in subsequent IHC/LCM-Seq experiments exhibited RNA Integrity Number (RIN) values ranging from 3.1 to 6.7 (Fig. 2C).
Immunohistochemical detection of AgRP, POMC and KP neurons
The RNA-preserving IHC protocol optimized for cortical parvalbumin labeling was adapted to detect hypothalamic AgRP, POMC and KP neurons. All antibody and buffer solutions were supplemented with 2% PVSA, except the epitope retrieval solution, which contained 10% PVSA, and the peroxidase developer, which was free of RNase inhibitors.
Mediobasal hypothalamic sections (every 12th) were removed from the cryoprotectant solution, rinsed in ice-cold PBS (3 × 5 min), subjected to antigen retrieval in 0.1 M citrate buffer (pH 6.0; 80 °C; 30 min), then incubated at 4 °C for 20 minutes in PBS containing 1% H2O2 and 0.5% Triton X-100. The following primary antibodies were applied for 40 hours at 4 °C: Goat anti-AgRP (EB06725; Everest Biotech; 1:10,000); Sheep anti-POMC (#41; gift from Dr. Csaba Fekete; 1:30,000)48; Sheep anti-KP (GQ2; gift from Dr. Waljit S. Dhillo; 1:50,000)49. After PBS washes, sections were incubated for 2 hours at RT with species-specific biotinylated secondary antibodies raised in donkeys (Jackson ImmunoResear ch; 1:500), followed by a 1-hour incubation with the ABC Elite reagent (Vector Laboratories, Burlingame, CA, USA; 1:1,000) in PBS. Immunoreactivity was visualized using Ni-DAB chromogen as described for cortical parvalbumin labeling.
Selection and exclusion criteria of immunostained sections for RNA-Seq studies
Due to variability in staining quality, not all seven hypothalamic specimens were included in all downstream analyses. Transcriptome profiling was conducted using: 3 samples for AgRP neurons, 2 samples for POMC neurons and 3 samples for KP neurons
Laser capture microdissection of immunostained neurons
Immunolabeled sections were mounted onto PEN membrane glass slides (Membrane Slide 1.0 PEN, Carl Zeiss) from Elvanol containing 2% PVSA, air-dried, and processed for LCM via sequential dehydration: 70% ethanol (60 s), 96% ethanol (60 s), 100% ethanol (2 × 60 s), and xylene (60 s). Slides were stored at –80 °C in slide mailers with silica gel desiccants unless processed immediately for LCM. Using a PALM Microbeam system (Carl Zeiss) equipped with a 40× objective lens and RoboSoftware, immunolabeled neurons were individually selected from the arcuate/infundibular nucleus and pressure-catapulted into adhesive caps (Adhesive Cap 500, Carl Zeiss). Approximately 850 neurons were collected per sample and stored at –80 °C in LCM caps until RNA extraction.
RNA extraction
Total RNA was isolated from pooled neurons using the Arcturus Paradise PLUS FFPE RNA Isolation Kit (Applied Biosystems, Waltham, MA, USA), according to the manufacturer’s protocol.
Library preparation and RNA Sequencing
Sequencing libraries were generated with the TruSeq Stranded Total RNA Library Preparation Gold kit (Illumina, San Diego, CA, USA), incorporating 16 PCR cycles for fragment enrichment.6,50 A pooled library mix (1 nM; 120 µl total volume) containing eight indexed samples was sequenced on an Illumina NextSeq 500 platform using the NextSeq 500/550 High Output v2.5 kit (75-cycle configuration).11
Bioinformatics
Low-quality reads and adapter sequences were removed using Trimmomatic (v0.39; parameters: LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:36)52 and Cutadapt (v4.6)51, respectively. Cleaned reads were aligned to the Homo sapiens GRCh38 Ensembl reference genome (release hs111) using the STAR aligner (v2.7.11b)53 with default parameters. Gene-level quantification was carried out using featureCounts (Subread v2.0.6).54 Transcriptome coverage was assessed based on the number of unique transcripts detected at two read count thresholds: ≥1 and ≥5 reads. Metrics were calculated and reported as the mean ± standard error of the mean (SEM) across biological replicates. Subsequent analysis was conducted in R (v4.5.0).55 TPM values were computed from read counts normalized to gene lengths obtained via FeatureCounts, using gene models defined in the corresponding Ensembl GTF annotation file. For differential expression analysis, raw gene counts were normalized and analyzed using the DESeq2 package (v1.44.0).56 The following R packages were utilized for data transformation, analysis, and visualization: tidyverse (v2.0.0)57, openxlsx (v4.2.8)58, ggplot2 (v3.5.2)59, ggfortify (v0.4.17)60, cluster (v2.1.8.1)61, pheatmap (v1.0.12)62, RColorBrewer (v1.1-3)63, ggrepel (v0.9.6)64, EnhancedVolcano (v1.22.0)65, gridExtra (v2.3)66, ggpubr (v0.6.0)67, eulerr (v7.0.2)68.
Functional classification
Transcripts were assigned to functional categories using the KEGG BRITE database69 (https://www.genome.jp/kegg/brite.html). Transcriptional regulators were identified based on the KEGG ‘Transcription Factors’ category (ko03000) (see Table S3). Receptor classes included G protein-coupled receptors (ko04030), cytokine receptors (ko04050), pattern recognition receptors (ko04054), and nuclear receptors (ko03310). This functional category was extended to include ACVR1C and INSR from the receptor serine/threonine kinases and receptor tyrosine kinases categories (ko01001+K08873), respectively. Receptor genes were considered expressed in a given neuronal phenotype if their mean TPM value was ≥10 and they were supported by ≥5 reads in all biological replicates of that group (Table S8). Neuropeptides were classified based on the neuropeptides.nl database9 (Table S4). A comprehensive list of functional classifications is provided in Tables S3, S4, S8.
Populational enrichment of gene expression
Gene expression was considered enriched in a specific neuronal population if the transcript exhibited at least a twofold increase in normalized read counts (log2 fold change > 1) relative to the other populations. When two neuronal types each showed ≥2-fold higher expression compared to the third, the gene was classified as co-enriched in those two populations.
GWAS associations
Trait– and disease-associated SNPs were retrieved from the GWAS Catalog10 (https://www.ebi.ac.uk/gwas/). Traits and diseases relevant to malnutrition were manually curated to match the 14 mapped GWAS traits presented in Figure 5 and Table S5. Trait categories included all descendant terms, except for ‘Body weight’, where child terms were excluded to maintain specificity. For comparative analysis, only intragenic SNPs reaching genome-wide significance (p < 5 × 10-8) were retained. The resulting gene list was then cross-referenced with neuronal population-specific enrichment data (Tables S6, S7).
Data availability
RNA-Seq files for AgRP and POMC neurons are available in BioProject with the accession number PRJNA1305226. (Release date 01/01/2026). KP sequencing data have been deposited under PRJNA1305334 (Release date 01/01/2026).
Code availability
Scripts are available at https://github.com/goczbalazs/PRJNA1305226_1305334.
Quantification and statistical analysis
Statistical analyses for Figure 1D were conducted using one-way ANOVA followed by Tukey’s HSD post hoc tests, implemented via the aov() and TukeyHSD() functions in R. Normality was evaluated with the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene’s test (Brown–Forsythe; median-centered) – via the shapiro.test() and leveneTest() functions. Significance was set at p < 0.05. Full statistical results are presented in Table S1.
Differential expression analysis for RNA-Seq data was performed with the DESeq2 package. Genes with Benjamini-Hochberg adjusted p-values < 0.05 were considered differentially expressed. Detailed results are provided in Table S2.
Significant SNP-trait associations from GWAS were defined by p < 5×10-8.70,71 The annotation and filtering of SNPs by intragenic localization and trait mapping are described above under GWAS Associations.
Codes are available at https://github.com/goczbalazs/PRJNA1305226_1305334
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Erik Hrabovszky (hrabovszky.erik@koki.hun-ren.hu).
Materials availability
This study did not generate new unique reagents.