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. Author manuscript; available in PMC: 2025 Sep 7.
Published in final edited form as: J Endocrinol. 2025 May 7;265(3):e240333. doi: 10.1530/JOE-24-0333

Single cell resolution of neurosteroidogenesis in the murine brain: Intermediary biosynthesis

Prasanthi P Koganti 1, Vimal Selvaraj 1,*
PMCID: PMC12413686  NIHMSID: NIHMS2077181  PMID: 40261706

Abstract

Neurosteroids synthesized within the central nervous system modulate neurotransmission, enhance neuroprotection, regulate immune responses, and influence cognitive and behavioral processes. Beyond de novo synthesis of pregnenolone (PREG) from cholesterol, the brain also engages in intermediary synthesis, converting local or circulating precursors into active neurosteroids. However, the specific cell types and brain regions involved remain poorly defined. In this study, we used single-cell transcriptomic data to map the expression of steroidogenic genes and identify cell populations in the murine brain responsible for intermediary neurosteroid biosynthesis. Our findings reveal that the synthesis of bioactive steroids downstream of PREG is not streamlined but selectively compartmentalized. Notably, cells involved in de novo neurosteroid biosynthesis are largely disjointed from intermediary steps indicating reliance on regional diffusion and/or systemic sources. Capacity for synthesis of androgens and corticosteroids anew are practically absent. While sterol sulfotransferases are expressed, sterol sulfatase required for desulfation is absent, indicating irreversible sulfonation of PREG and DHEA. Other enzymes involved in bioconversions of pregnanes and androstanes, when expressed, showed cell type- and region-specific ramifications. Although certain limitations exist in fully deciphering these results due to certain gaps in enzyme substrate specificity and isoform catalytic preferences, our study reveals valuable insights into the brain’s intermediary neurosteroid pathways. The distinct compartmentalization of these processes suggests precise control over steroid regulation, which could have far-reaching functional implications. By mapping the neurosteroidogenic potential in the murine brain and sex-associated variations, this study sets the stage for future investigations into the roles of neurosteroids in brain function and their therapeutic potential in neurological disorders.

Keywords: neuron, progesterone, estrogen, corticosterone, androgen

Introduction

The influence of steroids on brain function has long been recognized across various contexts (McEwen 1991). Neurosteroids, a term commonly used to describe steroids synthesized within the brain (Baulieu 1981), are not limited to products of de novo synthesis from cholesterol as a precursor, but are predominantly products of intermediary synthesis using different peripheral steroids from the gonads and adrenal glands, which reach the brain via systemic circulation. By modulating local autocrine and paracrine signaling processes (Robel & Baulieu 1994), neurosteroids have been described to influence a wide range of physiologic and pathophysiologic functions in the brain (Paul & Purdy 1992; Mellon & Griffin 2002; Lloyd-Evans & Waller-Evans 2020). However, as a clear departure from committed steroidogenic cells of the gonads and adrenal glands, intermediary synthesis of neurosteroids occurs in cell types with predefined dedicated neuronal and supportive brain functions (Do Rego et al. 2009). This principle not only affirms the functional evolution of distinct brain cells and regional neurosteroid-driven mechanisms in vivo, but also parallels the spatial and multiscale complexity of a variety of neuronal functions. Nevertheless, neurosteroid impacts on the broad domains of brain structure and function have been extremely difficult to delineate as knowledge of the cell types and regions involved in specific intermediary biosynthetic steps remain deficient.

Intermediary neurosteroid biosynthesis, distinct from de novo production (as described in ‘Single cell resolution of neurosteroidogenesis in the murine brain: de novo biosynthesis’ (Koganti & Selvaraj 2025)), involves single or sequential enzymatic conversions of pregnenolone and other steroid substrates by various cell types. Most steroid substrates are highly membrane permeable and brain cells that mediate specific enzymatic bioconversions can generate the next intermediary step or the terminal neurosteroid directly at their presumptive target sites within brain regions. Thus, local availability of steroid precursors defines the extent of terminal neurosteroid synthesis at each brain region and cell type. Among the key enzymatic steps, aromatization of circulating androstenedione to estrone and estradiol (Naftolin et al. 1971), 17α-hydroxylation of pregnenolone to 17-hydroxypregnenolone (Hojo et al. 2004), 5α-reduction of progesterone to 5α-dihydroprogesterone (5α-DH PROG) and 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) (Cheng & Karavolas 1973), hydroxysteroid sulfate conjugation of pregnenolone to pregnenolone sulfate and DHEA to DHEA sulfate (Corpéchot et al. 1981, 1983), and 7α-hydroxylation of DHEA and pregnenolone to 7α-hydroxy DHEA and 7α-hydroxy pregnenolone (Rose et al. 1997), have been reported to occur in brain slices or in in vitro cell cultures. While studies such as the in situ hybridization of Srd5a1 and Akr1c14 in the murine brain have localized certain neurosteroid biosynthetic capacity for products like 3α,5α-TH PROG and 3α,5α-TH DOC (Agís-Balboa et al. 2006), the absence of data on other isoforms of Srd5a and Akr1c limits our understanding of the distribution and cellular specificity of these processes. Such gaps in knowledge of the brain-wide capabilities of cell types and regions active in intermediary biosynthesis is particularly significant in the context of pathophysiology (Compagnone & Mellon 2000), where understanding abnormal neurosteroidogenesis and its functional basis in the complex etiology of many behavioral and neurological disorders have been difficult to dissect.

Clinical prospects of exogenous neuroactive steroids are increasingly being recognized in human and animal models of disease (Maguire & Mennerick 2024; Singhal et al. 2024), and several synthetic steroids are under development or have been approved as a new class of drugs that might address a variety of neurological, inflammatory and neuropsychiatric disorders (Bianchi & Baulieu 2012; Meltzer-Brody et al. 2018; Deligiannidis et al. 2021; Vaitkevicius et al. 2022; Balan et al. 2023; Haney et al. 2023). To address a key gap in knowledge about the sites of intermediary neurosteroid biosynthesis, we analyze brain single-cell transcriptomics data in this study to identify neurosteroid biosynthetic gene expression in classified cell populations in different brain regions and map specific cell types capable of producing different target intermediates and terminal neurosteroids.

Materials and Methods

Dataset

Used in this study is single cell datasets (Yao et al. 2023) generated by the BRAIN Initiative Cell Census Network, available through the Neuroscience Multiomic Data Archive (NeMO; https://data.nemoarchive.org/other/grant/aibs_internal/zeng/transcriptome/scell/). It includes single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) data obtained from 95 (v2) and 222 (v3) mice using next GEM technology (10xGenomics) representing mice at various developmental stages (postnatal P56-P120) and both sexes. The samples were from various brain regions that were dissected and dissociated to single cells for sequencing. To ensure comprehensive neuronal profiling, fluorescence-activated cell sorting (FACS) sorting or isolating single nuclei were also incorporated to enrich for neuronal cell populations to address challenges in isolating neuronal cell types. Sequenced data were processed using the CellRanger v8.0 pipeline with the mouse reference transcriptome GRCm38 and vM23. The protocol used by the authors (Yao et al. 2023), included stringent quality control parameters such as the detection levels of 62 housekeeping genes, a quality control score assessing the integrity of cytoplasmic RNA, and the removal of cells with a doublet score greater than 0.3 that filtered out of 43% of cells from the 10xv3 dataset and 29% from the 10xv2 dataset, ensuring that only high-quality cells were retained for subsequent combination and analyses. Clusters (32 cell types) identified based on the number and significance of conserved differentially expressed genes was available in the metadata files for both 10xv2 and 10xv3 datasets and was incorporated in our analysis.

Datamining

Hierarchical data formats (HDF) that stored both expression values (Log2 CPM), cell, region, and quality control as multidimensional arrays were analyzed using a Linux platform. Unzipped HDF files from the 10 regions that were sequenced in 10Xv2 and 13 regions in 10Xv3 were read using an R toolkit for single cell genomics Seurat v5 (Hao et al. 2021, 2024) in the R statistical computing platform (https://www.r-project.org). The Seurat objects created contained the count matrix which represents the expression of all the genes across all the cell types, which was subsequently extracted and converted to data matrices. The Ensembl ID for the genes (Table S1) were separated in data files to extract the expression levels across all the different regions. Parameters of the sequenced cells including the brain region it was obtained from, cell-type characterization, sex and age of the animal it was derived from, the X and Y coordinates for clustering cell-types were integrated from the metadata to ensure comprehensive profiling of transcriptomic variations. The different regions were subsequently concatenated into a master file that contained the expression and metadata information for all the sequenced cells. Data extraction, sorting and pivoting data were performed by using tidyr 1.3.1 (Wickham, H et al. 2024a) and dplyr 1.1.4 (Wickham, H et al. 2024b) packages in the R platform.

Cell type and regional nomenclature

Expression data were classified for both neuronal and non-neuronal cell types across different brain regions based on metadata from the single-cell transcriptomic atlas. Neuronal populations included GABAergic neurons from the cerebellum (CB), caudal ganglionic eminence (CGE), cerebral nuclei – Lateral Ganglionic Eminence (CNU-LGE), cerebral nuclei–hypothalamic area (CNU-HYa), hypothalamus (HY), lateral septal complex (LSX), midbrain (MB), medial ganglionic eminence (MGE), medulla or myelencephalon (MY), and pons (P), Olfactory bulb-immature (OB-IMN). Glutamatergic neurons were identified from the cerebellum (CB), cerebral nuclei–hypothalamic area (CNU-HYa), gonadotropin-releasing hormone–expressing neurons in the hypothalamus (HY Gnrh1), hypothalamus (HY), medial mammillary region of the hypothalamus (HY MM), intratelencephalic and extratelencephalic projections (IT-ET), midbrain (MB), medial habenula-Lateral Habenula (MH-LH), main olfactory bulb – Cajal-Retzius (MOB-CR), medulla (MY), cortical layer 6b projection neurons (NP-CT-L6b), pons (P), pineal gland, thalamus (TH), and the dentate gyrus of the olfactory bulb (MOB-DG). Dopaminergic neurons were derived from the midbrain (MB), and serotonergic neurons were classified from midbrain and hindbrain (MB-HB) regions. Non-neuronal cell types included astrocytes and ependymal cells (Astro-Epen), olfactory ensheathing cells (OEC), immune cells (including microglia and other myeloid-derived cells), oligodendrocyte precursor and mature oligodendrocytes (OPC-Oligo), and vascular cells comprising endothelial and perivascular populations.

Data filtering and calculation

For all single cell expression values, an exclusion cut-off of Log2 CPM<1 was applied to all datapoints analyzed. For overall expression in each cell type, the ratio of cell numbers that show expression to total number of cells sequenced in each cell type was obtained for representing the percentage of expressing cells. For total expression of genes involved, analysis comprised of assessment based on calculating the sum of expression for each classified cell type. Sex-specific differences in gene expression were analyzed by extracting donor sex and gene expression data from metadata for 2,381,293 male and 1,782,372 female cells. For each gene, the percentage of cells expressing was calculated for each cell type, and male-to-female ratios were computed.

Data visualization

The t-distributed stochastic neighbor embedding (t-SNE) plots were constructed to visualize the distribution of cells with specific gene expressions. For this plot, we used the X and Y coordinates generated for the combined 10xv2 and 10xv3 datasets through nonlinear dimensionality reduction available from metadata files (Yao et al. 2023). Initially, a scatter plot was created using the X and Y coordinates of all sequenced cells to distinguish cell type and region information consistent with and reproducing the original data package (Yao et al. 2023). Subsequently, cells expressing individual genes of interest, colored based on expression levels, were layered on the map of all sequenced cells. This permitted visualization of the spatial distribution of cells expressing the specific genes compared to the total population of sequenced cells. For graphing the overall expression of specific genes across different cell types, the ratios computed for each cell type were visualized as percentages using the ggplot2 package in R (Wickham 2016).

Results

Cell map classification of the murine brain

To investigate steroidogenic gene expression, we utilized a high-resolution transcriptomic and spatial cell-type atlas of the murine brain (Yao et al. 2023). This single-cell map reconstructs the various cell types and brain regions in mice (Figure 1A), facilitating the exploration and investigation of intermediary steroidogenic gene activity. This comprehensive map categorizes brain cells into 34 distinct functional classes, including neuronal subtypes—GABAergic, glutamatergic, dopaminergic, and serotonergic neurons—and non-neuronal subtypes—astrocytes, ependymal cells, oligodendrocytes, immune cells (includes microglia), olfactory ensheathing cells, and vascular cells (Figure 1A–B). Each cell type is linked to specific brain regions, identified by prefixes: CB (cerebellum), CNU (cerebral nuclei), CT (corticothalamic layer), CTX (cortex), ET (extratelencephalic), HB (hindbrain), HY (hypothalamus), IT (intratelencephalic), LH (lateral habenula), LSX (lateral septal complex), MB (midbrain), MH (medial habenula), MY (medulla), OB (olfactory bulb), P (pons) and TH (thalamus). This detailed classification serves as a foundation for examining region- and cell-type-specific capacities for intermediary neurosteroidogenesis based on gene expression (Figure 1B) for the different enzymatic bioconversions and substrate availability.

Figure 1. Cell and region map classification of the murine brain and intermediary neurosteroid biosynthetic enzymes.

Figure 1.

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(A) A t-distributed stochastic neighbor embedding (t-SNE) plot visualizing single cell gene expression data patterns from the murine brain color coded based on marker-defined cell types. Plot is a reconstruction from the cell atlas of the whole mouse brain. (B) Intermediary neurosteroid biosynthesis involves the conversion of the first steroid pregnenolone to pregnanes, androstanes, estranes, sulfated neurosteroids, and corticosteroids. Genes involved in the intermediary neurosteroid biosynthesis pathway are shown to indicate the range of enzymatic bioconversions that occur to generate intermediates and terminal steroids; genes that are not expressed in the murine brain based on our results are indicated (in grey).

SULT2 for biosynthesis of PREG-S and DHEA-S

Expression of Sult2a1 was found to be extremely low across all cell types in the different brain regions (Figure 2A), and Sult2b1 was found high in glutamatergic neurons of IT-ET followed by expression in NP-CT-L6b (Figure 2A). In addition, albeit at lower levels, a greater percentage of glutamatergic neurons in the pons, thalamus and pineal, and serotonergic neurons in MB-HB also expressed Sult2b1. With substrate preference for SULT2A1 sulfotransferase being DHEA and androsterone, and SULT2B1 being pregnenolone (Strott 2002), these findings indicated that PREG-S from pregnenolone bioconversion could occur at higher rates in the aforementioned neurons followed by androsterone sulfonation to Andro-S. Production of DHEA-S from DHEA might be scant as expression of Sult2a1 with known preference for this biosynthesis is not prominent the murine brain. Nevertheless, some DHEA-S synthesis might be possible from SULT2B1 activity. Both Sult2a1 and Sult2b1 were not prominent in non-neuronal cell populations with OPC-Oligo and immune cells showing modest levels of expression. This indicated that majority of the SULT2-based intermediary biosynthesis was associated with neurons. The co-expression analysis of Sult2 with Cyp11a1 indicated that there is some extent of cellular and regional coupling between de novo PREG biosynthesis and its sulfation in intermediary biosynthesis (Figure S1A) The sterol sulfatase, Sts, responsible for the hydrolysis of aryl and alkyl steroid sulfates was not detected in any neuronal or non-neuronal cell types in the brain (Figure 2B). Intermediary enzymatic bioconversions associated with SULT2 and STS are illustrated in Figure 2E.

Figure 2. SULT2 and HSD3B expression and associated intermediary steroid catalysis.

Figure 2.

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Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Sult2a1 and Sult2b1 (A), Sts (B), Hsd3b1, Hsd3b2 and Hsd3b3 (C), and Cyp17a1 (D) (colors indicate zero and positives as four quartile ranges). Intermediary enzymatic steroid bioconversions mediated by SULT2, STS, HSD3B and CYP17A enzymes are illustrated (E).

HSD3B for biosynthesis of PROG, 17OH-PROG, androstenedione and testosterone

Of the Hsd3b genes expressed in the murine brain, distinct patterns and expression levels were observed (Figure 2C; Figure S1). However, expression of the well-studied genes Hsd3b1, Hsd3b2 and Hsd3b3 that are known to mediate the bioconversion of PREG to PROG showed only extremely low to low expression (Figure 2C). Albeit low, Hsd3b2 expression showed minor enrichments in subpopulations of non-neuronal OPC-Oligo, and glutamatergic neurons of IT-ET and DG-IMN. Similarly, expression of Hsd3b3 was also low, but showed minor enrichments in glutamatergic neurons of IT-ET and DG-IMN. As hydroxysteroid dehydrogenases, HSD3B1, HSD3B2 and HSD3B3 are also known to bioconvert 17OH-PREG to 17OH-PROG, DHEA to androstenedione, androstenediol to testosterone, and 5α-Androstane-3β-17β-diol to DHT (Payne et al. 1997). Genes Hsd3b4 and Hsd3b5 that are 3-ketosteroid reductases involved in the bioconversion of dihydrotestosterone to 5α-Androstane-3β-17β-diol (Payne et al. 1997) showed little to no expression in the murine brain (Figure S1A). Similarly, Hsd3b6 with an undefined enzymatic activity, showed little to no expression in the murine brain. Among the relatively high expressed Hsd3b7 and Hsd3b8 genes, constitutive moderate levels across all cell types and regions were observed with specific enrichments for Hsd3b7 in non-neuronal cells and Hsd3b8 in glutamatergic neurons of IT-ET and HY-Gnrh1, and non-neuronal vascular cells (Figure S1A). However, HSD3B7 is involved in epimerization of the 3β-hydroxyl of cholesterol to its active 3α conformation, an essential step in the bile acid biosynthetic pathway (Shea et al. 2007), and not steroidogenesis. And substrate specificity and reactions mediated by HSD3B8 and HSD3B9 remains to be clearly defined. These knowledge gaps in enzymatic activities for the different Hsd3b genes do not allow direct assessment of the extent of coupling between de novo and intermediary steps in neurosteroidogenesis. If only the known HSD3B1, HSD3B2 and HSD3B3 are considered to direct intermediary biosynthesis of PREG to PROG, their coupling with cells/regions of de novo PREG synthesis is extremely low (Figure S1C). Cells expressing different Hsd3b genes could also indicate the ability to convert 17OH-PREG to 17OH-PROG, and DHEA to androstenedione. The extent of intermediary enzymatic steroid bioconversions associated with HSD3B are illustrated in Figure 2E.

CYP17A1 for biosynthesis of 17OH-PREG, DHEA, 17OH-PROG and androstenedione

Expression of Cyp17a1 showed an almost complete absence across all cell types in the different brain regions (Figure 2D). This indicated that intermediary biosynthesis of 17OH-PREG and DHEA from PREG, and 17OH-PROG and androstenedione from PROG are highly unlikely to occur in the murine brain. Intermediary enzymatic bioconversions associated with CYP17A1 are illustrated in Figure 2E.

CYP21A for biosynthesis of DOC and deoxycortisol

Expression of Cyp21a1, showed only extremely low to absent levels in brain cells (Figure 3A). Similarly, expression of Cyp21a2 was not detected in any of these datasets analyzed for the murine brain (Figure 3A). These findings indicated that intermediary biosynthesis of DOC from PROG, and 11-deoxycortisol from 17OH-PROG are highly unlikely to occur in the murine brain. Intermediary enzymatic bioconversions associated with CYP21A are illustrated in Figure 3D.

Figure 3. CYP21A, CYP11B and HSD11B expression and associated intermediary steroid catalysis.

Figure 3.

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Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Cyp21a1 and Cyp21a2 (A), Cyp11b1 and Cyp11b2 (B), and Hsd11b1 and Hsd11b2 (C) (colors indicate zero and positives as four quartile ranges). Intermediary enzymatic steroid bioconversions mediated by CYP21A, CYP11B and HSD11B enzymes are illustrated (D).

CYP11B for biosynthesis of corticosterone and cortisol

Expression of Cyp11b1 was found to be extremely low across all cell types in the different brain regions, and Cyp11b2 was low to absent in most brain regions (Figure 3B). These findings indicated that intermediary biosynthesis of cortisol from deoxycortisol and corticosterone from DOC are low to non-existent in the murine brain. Within the scope of corticosterone effects, HSD11B1 mediating the directional synthesis of systemically available cortisone to cortisol and 11-dehydrocorticosterone to corticosterone was observed in populations of glutamatergic neurons of IT-ET and CB, GABAergic neurons of the CTX-CGE and CB, non-neuronal cells of Astro-Epen and parts of OPC-Oligo. Expression in Astro-Epen was notably higher than in all other regions (also see Figure 8). However, Hsd11b2 that mediates the reverse directional conversion of cortisol to cortisone and corticosterone to 11-dehydrocorticosterone was extremely low across all brain regions (Figure 3C). Intermediary enzymatic bioconversions associated with CYP11B and HSD11B are illustrated in Figure 3D.

SRD5A and AKR1D for biosynthesis of DHP, DHDOC and DHT

Expression of Srd5a1 and Srd5a3 were prominent in different brain regions, whereas levels of Srd5a2 was low (Figure 4A); Akr1d1 that mediates reverse reactions was extremely low in all brain cells and regions (Figure 4B). High expression of Srd5a1 was observed in glutamatergic neurons of IT-ET, NP-CT-L6b, DG-IMN, CB, P and MY, GABAergic neurons of CTX-CGE and CB, dopaminergic neurons of MB, serotonergic neurons of MB-HB, and in non-neuronal cells with OPC-Oligo showing a high level of expression followed by Astro-Epen and vascular cells. For Srd5a3, high expression was observed in glutamatergic neurons of IT-ET, NP-CT-L6b, DG-IMN, CB, TH and HY-Gnrh1, and GABAergic neurons of CTX-CGE and CNU-LGE. Non-neuronal Srd5a3 expression was similar to Srd5a1, but also higher in vascular cells, Astro-Epen, and immune cells in addition to OPC-Oligo. This indicated that intermediary biosynthesis through SRD5A of 5α-DHP from PROG, 5α-DHDOC from DOC, and 5α-DHT from testosterone occurs in specific brain regions in the murine brain. The reverse 5β-reduction by AKR1D1 might be highly scant in the murine brain. Intermediary enzymatic bioconversions associated with SRD5A and AKR1D1 are illustrated in Figure 4C.

Figure 4. SRD5A and AKR1D expression and associated intermediary steroid catalysis.

Figure 4.

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Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Srd5a1, Srd5a2 and Srd5a3 (A), and Akr1d1 (B) (colors indicate zero and positives as four quartile ranges). Intermediary enzymatic steroid bioconversions mediated by SRD5A and AKR1D enzymes are illustrated (C).

AKR1C for biosynthesis of THP, THDOC, androstanediol and androstanedione

Of the 8 forms of Akr1c expressed in the murine brain, the expression levels were low but region-specific enrichments could be observed for some forms. Expression of Akr1c6 was extremely low for most cell types in the different brain regions (Figure 5A). Expression of Akr1c12 was also low but enriched in non-neuronal vascular cells. Expression of Akr1c13 was enriched in non-neuronal OPC-Oligo, Immune, Astro-Epen and vascular cells. Expression of Akr1c14 was highly enriched in vascular cells. Expression of Akr1c18 was enriched in GABAergic neurons of CTX-MGE and OB-IMN. Expression of Akr1c19 was observed at low levels across different neuronal populations and non-neuronal Astro-Epen population. Expression of Akr1c20 was also extremely low without any cell type or regional specificity. Expression of Akr1c21 was low across all cell populations with minor levels of enrichment in non-neuronal OPC-Oligo and Astro-Epen. This indicated that the intermediary biosynthesis of 3α,5α-THP (ALLO) from 5α-DHP, 5α,3α-THDOC from 5α-DHDOC, 5α-androstanediol from 5α-DHT, and androstanedione from androstenedione can be active at different levels across different murine brain regions. Intermediary enzymatic bioconversions associated with AKR1C are illustrated in Figure 5B.

Figure 5. AKR1C expression and associated intermediary steroid catalysis.

Figure 5.

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Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Akr1c6, Akr1c12, Akr1c13, Akr1c14, Akr1c18, Akr1c19, Akr1c20, and Akr1c21 (A) (colors indicate zero and positives as four quartile ranges). Intermediary enzymatic steroid bioconversions mediated by AKR1C enzymes are illustrated (B).

HSD17B for dehydrogenation of 17β-hydroxyl and reduction of 17-keto steroids

Of the Hsd17b genes expressed in the murine brain, distinct patterns and expression levels were observed, with some being among the highest levels of the enzymes under consideration for neurosteroid intermediary biosynthesis (Figure 6A). However, different Hsd17b genes have substrate specificities ranging from alcohols, bile acids, fatty acids and retinols (Moeller & Adamski 2009), and the nomenclature is also not consistent between species (Adamski & Jakob 2001). Therefore, it is difficult to attribute functions for the largely uncharacterized isoforms that need careful consideration. Among the highly expressed were Hsd17b4, Hsd17b7, Hsd17b8, Hsd17b10, Hsd17b11 and Hsd17b12, all of which showed almost constitutive expression across all cell types and regions of both neuronal and non-neuronal cells, but with some level of prominence in glutamatergic neurons of IT-ET and all non-neuronal cells. Of the low expressed alleles, Hsd17b1 expression showed minor enrichments in subpopulations of GABAergic neurons of CB, CTX-CGE, and CNU-LGE; Hsd17b2 also showed minor enrichments in subpopulations of GABAergic neurons of MB and MY, glutamatergic neurons of MB, MY and HY; Hsd17b3 showed minor enrichment in a subpopulation of non-neuronal Astro-Epen; Hsd17b13 and Hsd17b14 were low, but showed minor enrichment in glutamatergic neurons of IT-ET; Hsd17b6 was extremely low in most cells. Broadly interpreting known functions of Hsd17b genes, these data indicated that brain cells might be highly active in the intermediary biosynthesis via reversible reactions producing testosterone from androstenedione, estradiol from estrone, and androstenediol from DHEA. This is in addition to steps reversing effects of AKR1C in synthesis of DHP and DHDOC. Although many Hsd17b genes’ enzymatic activity have not been experimentally validated, homology-based predictions indicate additional possible substrates in the biosynthesis of androsterone from androstanediol, and PROG from 20α-dihydro-PROG. Intermediary enzymatic bioconversions associated with HSD17B are illustrated in Figure 6B.

Figure 6. HSD17B expression and associated intermediary steroid catalysis.

Figure 6.

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Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Hsd17b1, Hsd17b2, Hsd17b3, Hsd17b6, Hsd17b7, Hsd17b8, Hsd17b10, Hsd17b11, Hsd17b12, Hsd17b13, and Hsd17b14 (A) (colors indicate zero and positives as four quartile ranges). Intermediary enzymatic steroid bioconversions mediated by HSD17B enzymes are illustrated (B).

Aromatase/CYP19A1 for biosynthesis of estrone and estradiol

Expression of Cyp19a1 (aromatase) was extremely low for most cell types in the different brain regions (Figure 7A). Enrichments of positive cells were observed in subpopulations of GABAergic neurons of the CNU-HYa and glutamatergic neurons of the DG-IMN. Levels of Cyp19a1 in other brain regions were sparce. This indicated that intermediary biosynthesis of estrone from androstenedione and estradiol from testosterone were largely restricted to the aforementioned cells in the murine brain. Intermediary enzymatic bioconversions associated with CYP19A1 are illustrated in Figure 7C.

Figure 7. CYP19A1 and CYP7B expression and associated intermediary steroid catalysis.

Figure 7.

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Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Cyp19a1 (A), and Cyp7b1 (B) (colors indicate zero and positives as four quartile ranges). Intermediary enzymatic steroid bioconversions mediated by CYP19A1 and CYP7B1 enzymes are illustrated (C).

CYP7B for biosynthesis of 7α-OH-PREG and 7α-OH-DHEA

Expression of Cyp7b1 showed specific high levels of expression in glutamatergic neurons of the IT-ET, NP-CT-L6b, DG-IMN, CB and OB-CR, GABAergic neurons of the CB and CNU-LGE, and non-neuronal cells (OPC-Oligo, Astro-Epen, OEC and vascular cells) (Figure 7B).This indicated that high levels of cellular and regionally distributed intermediary biosynthesis of 7α-OH-PREG and 7α-OH-DHEA can occur in the murine brain. Intermediary enzymatic bioconversions associated with CYP7B are illustrated in Figure 7C.

Coupling of intermediary neurosteroid biosynthetic capabilities in the murine brain

Expression of Cyp11a1 indicating enrichment of de novo steroidogenes was observed in the glutamatergic neurons of IT-ET and NP-CT-L6b (Koganti, PP & Selvaraj, V Under Review). Albeit the possibility of PREG diffusion to different regions remains high, direct coupling between de novo and intermediary neurosteroidogenesis indicated by high expression of Sult2b1 (for conversion to PREG-S), and low to moderate expression of Hsd3b (for conversion to PROG) in the glutamatergic neurons of IT-ET and NP-CTL6b exist (Figure 8; Figure S1B and C). Moreover, expression of Srd5a1 and Srd5a3 that catalyzes sequential conversion of PROG to 5α-DHP was also relatively prominent in glutamatergic neurons of IT-ET and NP-CT-L6b (Figure 8). Most other intermediary biosynthetic capabilities were only single-step bioconversions and also not in continuum with de novo neurosteroid biosynthesis. Non-neuronal Astro-Epen was predominant in the expression of Hsd11b1 for bioconversions of 11-DHC to corticosterone and cortisone to cortisol; OPC-Oligo showed high expression of Srd5a1 and Srd5a3 for synthesis of 5α-DHP, 5α-DHDOC and/or 5α-DHT, similar to glutamatergic IT-ET. Of the Hsd17b isoforms, Hsd17b4, Hsd17b7, Hsd17b8, Hsd17b10, Hsd17b11 and Hsd17b12 were at relatively high yet variable levels in the different non-neuronal and neuronal cells (Figure 8). Although all the aforementioned HSD17B isoforms may not be involved in neurosteroid biosynthesis, their cell type and regional abundance measures provide a framework for future investigations. Similarly, although highly expressed, compartmentalization of the dual roles for CYP7B1 in bile acid and intermediary steroid biosynthesis in the brain remains unclear (Figure 8).

Figure 8. Intermediary neurosteroid biosynthetic map based on cell type and regional enzyme expression.

Figure 8.

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Heatmap showing total expression combined with a bubble chart showing the percentage of cells positive for the different enzymes involved in de novo and intermediary neurosteroid biosynthesis across the different cell types and regions in the murine brain. Patterns of expression indicate the presence of specific catalytic steps pinpointing the capacity for biosynthesis, coupling of different neurosteroid intermediates and target synthesis of terminal neurosteroid products across different cell types and brain regions. However, several enzymes (*) are of unknown function, specificity and/or do not steroidogenic bioconversions. Few enzymes were absent or were detected at extremely low levels in the murine brain (red #).

Receptors associated with intermediary neurosteroid signaling

Analysis of steroid receptor expression in the murine brain indicated target regions for specific neurosteroid-mediated signaling. Expression of GABAA receptor genes, chloride channels associated with different neurosteroid effects were broadly expressed in the murine brain (Figure S2). As additional targets for allosteric modulation, expression of AMPA (Gria) and kainite (Grik) receptor subunits were broad and abundant in most brain regions (Figures S3 and S4). Expression of NMDA receptor (Grin) showed both broad expression and cell-type specificity for some of the subunits (Figure S5). Expression of the different purinoceptors (P2rx) were variable with P2rx4 showing broad expression and other showing cell selective expression patterns (Figure S6). Expression of the cannabinoid receptor (Cnr1) and the sigma 1 receptor (Sigmar1) appeared generalized and abundant in the murine brain (Figure S7). For orthosteric effects based on systemic steroid receptors that mediate transcriptional responses and non-genomic signaling we identified expression of estrogen receptors that could mediate signaling through estradiol and estrone binding (Esr1, Esr2, and the membrane associated receptor Gper1) were at lower levels and only in defined populations (Figure S8A). Expression of progesterone receptors that could mediate signaling through PROG, 17OH-PROG and DHP (Pgr and the membrane associated receptors, Pgrmc1 and Pgrmc2) were also broadly expressed and abundant in the murine brain (Figure S8B). Expression of the androgen receptor (Ar) that could mediate signaling through 5α-dihydrotestosterone (DHT) and testosterone was also broadly expressed and abundant (Figure S8C). Expression of the glucocorticoid receptor (Nr3c1) that could mediate signaling through corticosterone, cortisol, DOC, and cortisone was also broadly expressed in the murine brain (Figure S8D).

Sex-associated variations in intermediary neurosteroidogenesis

Data revealed subtle but region-specific sex differences in the expression of key enzymes involved in intermediary neurosteroidogenesis. For Sult2b1, there were minimal sex differences across brain regions (Figure S9). For Hsd3b isoforms, subtle sex differences were observed in HY-GABA, HY-MM Glut, MH-LH Glut, LSX-GABA, OB-CR Glut, OB-DG-IMN and OEC (Figure S10); cells expressing Hsd3b7 in Pineal Glut were detected only in female brains. For Hsd11b1, expression was detected only in female OB-CR Glut, and a female bias was noted for HY-MM Glut (Figure S11). For Srd5a1, only HY-MM Glut showed a subtle bias towards females. For SRD5A3 expressing cells, a subtle female bias was noted for OEC and BY-MM Glut; Pineal Glut cells expressing Srd5a3 showed male only detection (Figure S12). Among Hsd17b isoforms, Hsd17b4 had a subtle female bias in HY-MM Glut (Figure S13). Cells expressing Hsd17b7 were found only in male Pineal Glut. For Hsd17b10, subtle female bias was detected in OEC and male bias detected in Pineal Glut (Figure S13). For Cyp7b1, no notable sex differences across neuronal and non-neuronal cell types were detected (Figure S14).

Discussion

Lack of delineation between de novo and intermediary steroid biosynthesis in the brain literature has often confounded mechanisms that rely on local or systemic steroid availability and its regulation. Through evaluation of the capacity for different brain cells to catalyze specific neurosteroid biosynthetic steps, the relevance of de novo vs peripheral sources in intermediary biosynthetic capabilities, and the sites of terminal bioactive neurosteroid biosynthesis, this study offers a reference map for locating regions engendering and possibly responsive to the functional impact of neurosteroidogenesis.

Different brain cells and regions capable of de novo biosynthesis of PREG from cholesterol are defined in an accompanying manuscript (Koganti, PP & Selvaraj, V Under Review). In examining intermediary conversions from PREG, we found that the synthesis of PREG-S and DHEA-S by SULT2 family members is unidirectional in the brain, as Sts is not expressed. This finding is significant, contrasting with previous reports that suggest that Sts expression and activity can be detected in the murine brain (Mortaud et al. 1996). Our observation is confirmed by other transcriptome databases of the murine brain that indicate failure to detect expression of Sts (Zhang et al. 2014; Apicco et al. 2019; Milenkovic et al. 2024). Public datasets reveal a clear species difference in that STS can be detected in human brain cells (Hawrylycz et al. 2012), but not in murine brain cells (comparisons in: https://brainrnaseq.org; https://www.proteinatlas.org). The irreversible nature of PREG and DHEA sulfation could have significant implications in that it would allow stable accumulation resulting in enhanced and prolonged effects. PREG-S is known to enhance excitatory neurotransmission, cognitive functions and neuroprotection, via potentiating NMDA receptor activity (Wu et al. 1991; Bowlby 1993), while antagonizing GABAA receptors (Majewska et al. 1988, 1989, 1990a). Similarly, DHEA-S can influence neuroplasticity and neuronal survival, via antagonizing GABAA receptors (Majewska et al. 1990b; Demirgören et al. 1991). The prolonged modulation of PREG-S and DHEA-S targets may physiologically support sustained neuronal signaling and plasticity, which could be critical for cognitive resilience (Vallée et al. 1997). This regulation through irreversible sulfation is a distinction from PREG-S and DHEA-S dispositions in the human brain.

Recognized for its pleiotropic effects, primarily mediated through classical progesterone receptors (Wnuk & Kajta 2017), PROG has been shown to promote neuroprotection after hypoxic brain injury, ishemic stroke and traumatic brain injury in therapeutic preclinical studies (Allitt et al. 2017; Dong et al. 2018; Tameh et al. 2018). Within the Hsd3b gene family that convert PREG to PROG, distinct expression patterns indicate potential for unique contributions. Different Hsd3b genes are associated with different functions; while HSD3B1, HSD3B2 and HSD3B3 function as dehydrogenases/isomerases, and HSD3B4 and HSD3B5 preferentially function as 3-ketosteroid reductases (Payne et al. 1997). In contrast to neurosteroidogenesis, bile acid synthesis from cholesterol relies on HSD3B7 (Gardner et al. 2024), highlighting both the functional diversity within the Hsd3b gene family. Therefore, pinpointing their activity in PREG biosynthesis remains a challenge. Moreover, physiological specificity for HSD3B6, HSD3B8 and HSD3B9 remain unclear, representing a significant gap in our understanding of their catalytic activity and substrate specificity, which is critical for accurate interpretation.

The absence of detectable Cyp17a1 mRNA expression in the murine brain presents a significant finding that capacity for local synthesis of key androgens, such as DHEA and androstenedione, from progestogens is deficient. While the absence of CYP17A1 has been reported in other rodents (Le Goascogne et al. 1991), some studies have presented contradictory findings, suggesting CYP17A1 expression and DHEA synthesis occurs in astrocytes and neurons within the cerebral cortex (Zwain & Yen 1999). In the neurosteroid literature, DHEA and androstenedione have been associated with modulating synaptic plasticity, providing neuroprotection, enhancing cognitive function, and regulating mood via a multi-faceted regulation (Prough et al. 2016). Our findings suggest that in the murine brain, DHEA and androstenedione from PREG and PROG are not produced locally but entirely sourced from systemic circulation, potentially affecting interpretations surrounding impacts on neural signaling involved in androgen-mediated behaviors.

Downstream of HSD3B bioconversions, CYP21A is the most common enzyme deficiency in congenital adrenal hyperplasia in human patients due to corticosteroid insufficiency (Hannah-Shmouni et al. 2017). In humans, local synthesis of corticosteroids through extra-adrenal 21-hydroxylase has been reported to occur in the hippocampus (Beyenburg et al. 2001). Transcripts for 21-hydroxylase has also been reported in the rat brain stem (Iwahashi et al. 1993; Strömstedt & Waterman 1995). However these findings remain controversial as other reports have indicated its complete absence of CYP21A in any extra-adrenal tissue (Mellon & Miller 1989), and the possible existence of another P450 21-hydroxylase has not been completely ruled out. While the 21-hydroxylated steroids DOC and deoxycortisol are considered critical in modulating neural activity in stress responses (Purdy et al. 1991), our findings imply that the murine brain largely relies on peripheral sources for these steroids rather than synthesizing them locally. Downstream of DOC and deoxycortisol, our findings also indicated that the synthesis of corticosterone and cortisone mediated by Cyp11b was also almost deficient. In contrast, Hsd11b1 that directionally prefers to mediate conversion of cortisone to cortisol or 11-dehydro corticosterone to corticosterone was prominent in specific brain cells and regions suggesting that active cortisol or corticosterone can undergo targeted synthesis towards amplifying regional responses. Although cortisol is considered the major glucocorticoid in humans, it is not the same for mice that have corticosterone as the predominant stress hormone (Joëls et al. 2018). Our findings indicate that there might be pockets of cortisol and/or corticosterone biosynthesis from systemic glucocorticoids in the murine brain creating distinctions from peripheral expression profiles. These findings are supported by direct glucocorticoid measurements that have been made in brain tissues, but indications of DOC synthesis remains inconsistent (Hamden et al. 2021). Nevertheless, our overall data patterns make sense in that a direct coordination of brain’s responses to systemic stress and metabolic challenges mediated by peripheral corticosteroids might be beneficial to direct, amplify and influence central regulatory dynamics and behavioral adaptations.

The 5α-reduction of PROG, DOC and corticosterone to produce DHP, DHDOC and DHT is unidirectional mediated by SRD5A1 (Andersson & Russell 1990) and SRD5A3 (Uemura et al. 2008; Fouad Mansour et al. 2016); 5β-reduction of these substrates by AKR1D1 is almost completely absent in the murine brain. High levels of locally synthesized DHP and DHDOC in brain regions have been reported in response to stress (Sze et al. 2018). Beyond being intermediary products subsequent steroid synthesis, knowledge of direct effects of DHP and DHDOC are scant, whereas DHT is a known high-potent mediator of androgen receptor targets in hormone regulated gene expression that surpasses that of testosterone (Grino et al. 1990). Albeit low, DHP has documented affinity for progesterone receptors (Rupprecht et al. 1993), and DHDOC is known to have direct effects on GABAA-activated inhibitory effects on neuronal transmission (Reddy & Rogawski 2002). Further 3α-reduction of 5α-DHP, 5α-DHDOC and 5α-DHT by AKR1C to generate ALLO, THDOC and Androstanediol, that all have higher potency at GABAA receptor effects compared to androgen, progesterone or corticosterone receptor effects (Purdy et al. 1991; Paul & Purdy 1992; Reddy & Rogawski 2002), they would have direct effects on the central nervous system inhibitory tone and resultant behavioral processes. Moreover, it is recognized that there is poor phylogenetic conservation between murine and human AKR1C enzymes as indicated by varied substrate preferences (Veliça et al. 2009), of relevance to interpreting neuronal functions and extrapolating models to human neurophysiology.

The family of Hsd17b genes are among the most abundant in intermediary neurosteroid biosynthesis in the murine brain, with expression spread across distinct populations of both neuronal and non-neuronal cells. Nevertheless, functional assignments for HSD17B and substrate specificity continues to be a topic of study with several members having multiple functionality in steroid bioconversions, fatty acid metabolism and bile acid synthesis (Moeller & Adamski 2009). For example, the highly expressed Hsd17b4, Hsd17b10 and Hsd17b12 coded enzymes have added substrate specificity for short chain fatty acids, branched fatty acids and bile acids (Möller et al. 1999; Shafqat et al. 2003; Moeller & Adamski 2009), while for others like Hsd17b13 the functional specificity remains unclear. Activity of HSD17B10 is additionally capable of biosynthesis of DHT via a non-classical androgen synthesis pathway (He et al. 2003). Specific to neuronal diseases, it must be noted that HSD17B10 has been associated with protein-protein interactions with amyloid beta (Du Yan et al. 1997), and mitochondrial toxicity in an Alzheimer’s disease mouse model (Lustbader et al. 2004). Among the other abundant isoforms, Hsd17b7 enzymatic activity has been shown to convert estrone to estradiol (Nokelainen et al. 1998); Hsd17B8 enzymatic activity has been shown to convert estradiol to estrone and lower extent testosterone to androstenedione (Fomitcheva et al. 1998); Hsd17b11 has been shown to convert 3α-Adiol to androsterone (Chai et al. 2003); Hsd17b14 has be shown to target estradiol and testosterone (Lukacik et al. 2007). Although the HSD17B influence through neurosteroids in neurologic pathologies have been difficult to delineate, it certainly highlights that HSD17B family members have broad and perhaps multiple functions. This is indicated in their tissue distributions with many isoforms detected in non-steroidogenic tissues such as the liver, heart, lung, small intestine, skeletal muscle, adipose tissue and kidney [summarized in (Moeller & Adamski 2009)]. Therefore, metabolic pathways mediated by HSD17Bs are not entirely dedicated to steroidogenesis, converting steroids at position 17, as the name indicates. Therefore, our interpretations need to be limited and require further delineation to study and interpret neurosteroidogenesis and functional effects in the brain.

Local aromatization of testosterone to produce estrogen by CYP19A1 has been long known in the realm of neurosteroidogenesis (Naftolin et al. 1975), and is considered a path to modulating neuronal function and providing neuroprotection (Boon et al. 2010). Restricted expression of Cyp19a1 to GABAergic neuronal circuitry in the hypothalamus might indicate potential direct effects for estrogen-regulated systemic behavioral responses (Gu et al. 1999; Kelly et al. 2005). Although production might be localized, distribution of estrogen receptor mediated effects can be widespread (Morissette et al. 2008), and key to interpreting any functional responses.

There are expanding roles for enzymes and products that add to intermediary synthesis products whose biological activities continue to be investigated. For example, CYP7B1, a key mediator of 7α-hydroxylation of cholesterol in the liver for biosynthesis of bile acids (Setchell et al. 1998), has also been demonstrated to act on 3β-hydroxy steroids such as PREG and DHEA (Rose et al. 1997). Although the role of 7α-OH-PREG and 7α-OH-DHEA remain to be fully defined, it is clear that it adds to the complex contribution of steroid biochemistry towards local and systemic influences of neuronal function.

In interpreting functions, receptor-mediated actions for different neurosteroids have overwhelmingly focused on acute allosteric effects on GABAA potentiation or inhibition relevant for therapeutic interventions (Bäckström et al. 2022; Thompson 2024). Nevertheless, it is known that effects of neurosteroids on GABAA are influenced by the receptors subunit composition (Hosie et al. 2006; Chen et al. 2019). Our data indicate that targeting synthesis would be a primary modality of region-specific effects because expression of different GABAA subunits almost seem ubiquitous in the murine brain. Similarly, allosteric effects mediated via AMPA receptors (Yaghoubi et al. 1998; Shirakawa et al. 2005), kainite receptors (Wu et al. 1998; Yaghoubi et al. 1998), NMDA receptors (Wu et al. 1991; Hrcka Krausova et al. 2020), purinergic receptors (Sivcev et al. 2023), cannabinoid receptors (Raux et al. 2022), and sigma 1 receptors (Su et al. 1988; Monnet & Maurice 2006), could be segregated via a combination of both targeted intermediary biosynthesis and receptor expression in the different neuronal cell types. Conversely, there is also evidence for regulation of intermediary neurosteroid biosynthesis by effects exerted by glutamate and GABA (Do Rego et al. 2012), highlighting composite regulation of various neurophysiological and behavioral processes. Although not directly linked to ionic currents, neurosteroid effects can also be mediated through directed actions towards nuclear or membrane-associated steroid hormone receptors targeted by gonadal steroids (Cyr et al. 2001; Smith & McMahon 2005; Srivastava et al. 2008). Although we find abundant expression of receptors for estrogen, progesterone, testosterone and corticosterone in the murine brain, effects mediated via such coupled genomic and non-genomic steroid signaling largely remain to be functionally elucidated in the brain (Mahfouz et al. 2016).

Sex differences in localized neurosteroid availability may contribute to previously observed variations in synaptic plasticity, stress resilience, neuroendocrine regulation and pathologies (Meffre et al. 2007; Caruso et al. 2013; Mendell & MacLusky 2018; Giatti et al. 2020). While some intermediary steroidogenic enzymes showed regionally conserved mRNA expression across sexes, others exhibited sex-exclusive or sex-biased transcript patterns. Intermediary neurosteroidogenesis in the pineal gland, a central regulator of circadian rhythms and melatonin synthesis, exhibited the highest degree of sex-exclusive expression in Pineal Glut. Hsd3b7-expressing cells were detected only in female Pineal Glut, and conversely, Hsd17b7 and Srd5a3-expressing cells were detected only in male Pineal Glut. This suggests that local progesterone synthesis is higher in females, and dihydrotestosterone (DHT) and androgen biosynthesis were higher in males. This finding aligns with prior evidence that pineal-derived neurosteroids interact with melatonin pathways to influence circadian rhythmicity and associated physiologies (Haraguchi & Tsutsui 2020). The hypothalamus, a key center for hormonal regulation, stress response, and reproductive behavior exhibited a female bias in Srd5a1 and Srd5a3 suggesting that 5α-reduced neurosteroids such as allopregnanolone and DHT may be preferentially synthesized in females within the medial mammillary hypothalamus. Additionally, Hsd3b2, Hsd3b8, and Hsd17b4 exhibited a female bias in various hypothalamic populations, suggesting a more active progesterone metabolism in these regions. These differences could contribute to sex differences in hypothalamus regulated stress, and reproductive physiology (Oyola & Handa 2017). Hsd11b1-expressing cells were detected only in female OB-CR Glut, suggesting female-specific glucocorticoid metabolism impacting olfactory circuits. Additionally, Hsd3b3-expressing cells exhibited sex differences in OB-CR Glut and OB-DG-IMN, while Srd5s3 showed a female bias in OEC. These findings corroborate the molecular basis for sex impacts on sensory processing and social behaviors (Abaffy et al. 2023). In the limbic system, Hsd3b8 showed a female bias in MH-LH Glut and HY-MM Glut, which may contribute to sex differences in progesterone-mediated anxiolysis and depression susceptibility (Kundakovic & Rocks 2022). Hsd3b2 expression differences in LSX-GABA and HY-GABA may further indicate sex-dependent modulation of stress and behavioral responses (Wirth 2011). These findings highlight how sex-linked intermediary neurosteroid biosynthesis may shape region-localized neural functions and influence related neurophysiological processes.

In critically evaluating the dataset used in this analysis (Yao et al. 2023), it is important to acknowledge the inherent limitations common to large-scale studies. Cell dissociation from dissected brain regions can introduce bias, as mature neuronal cell bodies are often more challenging to isolate, leading to disproportionate losses and variability in RNA yields. This process can also influence the representation of certain cell types across different brain regions. Additionally, sequencing depth can vary across both cell types and within individual populations, potentially affecting the resolution of some findings. Despite these challenges, the dataset’s remarkable scale encompassing 4,041,289 brain cells analyzed across diverse cell populations provides a robust foundation for our overall conclusions. Moreover, although transcriptome-based analyses do not directly confirm the presence of protein or bioactivity of enzymes, they compellingly indicate the enzymatic potential for specific structural steroid bioconversions. Ultimately, while predicting the precise substrates and terminal steroids produced for each enzyme might remain complex, this work represents a significant step forward by delineating a cellular framework that defines the potential or absence of intermediary neurosteroidogenic capability.

In summary, our results reveal a compartmentalized approach to neurosteroidogenesis, with distinct pathways being favored or restricted based on cell type, regional distribution, and substrate availability. These insights not only enhance our understanding of the physiological roles of neurosteroids but also lay the groundwork for future studies exploring therapeutic interventions targeting neurosteroid pathways in neurological diseases. Understanding these cell-specific and region-specific pathways will be crucial for leveraging neurosteroidogenic mechanisms in therapeutic contexts, especially for neurodegenerative diseases, stress-related disorders, and cognitive impairments.

Supplementary Material

01

Figure S1. Coupling of de novo and intermediary neurosteroid biosynthesis. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Hsd3b4, Hsd3b5, Hsd3b6, Hsd3b7, Hsd3b8, and Hsd3b9 (A) (colors indicate zero and positives as four quartile ranges), co-expression of Cyp11a1 and combined Sult2a1+Sult2b1 (B), and co-expression of Cyp11a1 and combined Hsd3b1+Hsd3b2+Hsd3b3 (C).

Figure S2. Expression of GABAA receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Gabra1, Gabra2, Gabra3, Gabra4, Gabra5, Gabrb1, Gabrb2, Gabrb3, Gabrd, and Gabrg1, (colors indicate zero and positives as four quartile ranges).

Figure S3. Expression of AMPA receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Gria1, Gria2, Gria3 and Gria4, (colors indicate zero and positives as four quartile ranges).

Figure S4. Expression of kainate receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Grik1, Grik2, Grik3, Grik4 and Grik5, (colors indicate zero and positives as four quartile ranges).

Figure S5. Expression of NMDA receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Grin1, Grin2a, Grin2b, Grin2c, Grin2d, Grin3a, Grin3b, and Grina, (colors indicate zero and positives as four quartile ranges).

Figure S6. Expression of purinergic receptor subtypes. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of P2rx1, P2rx2, P2rx3, P2rx4, P2rx5, P2rx6 and P2rx7, (colors indicate zero and positives as four quartile ranges).

Figure S7. Expression of cannabinoid receptor and sigma-1 receptor. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of (A) Cnr1 and (B) Sigmar1, (colors indicate zero and positives as four quartile ranges).

Figure S8. Expression of steroid hormone receptors. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in co-expression of estrogen receptors (Esr1, Esr2, and Gper1) (A), progesterone receptors (Pgr, Pgrmc1, and Pgrmc2) (B), androgen receptor (Ar) (C), and glucocorticoid receptor (Nr3c1) (D).

Figure S9. Sex-associated variations in SULT2B1 expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Sult2b1. No brain regions exhibited exclusive expression in one sex. Only subtle differences between sexes were observed across few regions.

Figure S10. Sex-associated variations in HSD3B isoform expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Hsd3b2, Hsd3b3, Hsd3b7, and Hsd3b8. In Pineal Glut, cells expressing Hsd3b7 were detected only in females (circled star). Notable sex differences were detected in HY-GABA, HY-MM Glut and MH-LH Glut for Hsd3b2, LSX-GABA, OB-CR Glut and OB-DG IMN for Hsd3b3, OEC for Hsd3b7, HY-MM Glut and MH-LH Glut for Hsd3b8 (circled dots).

Figure S11. Sex-associated variations in HSD11B1 expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Hsd11b1. In OB-CR Glut, cells expressing Hsd11b1 were detected only in females (circled star). Notable sex differences were also detected in HY-MM Glut (circled dot).

Figure S12. Sex-associated variations in SRD5A1 and SRD5A3 expression. t-SNE plots (left) and the ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Srd5a1 (top) and Srd5a3 (bottom) expression across different brain cell types. In Pineal Glut, cells expressing Srd5a3 were detected only in males (circled star). Notable sex differences were detected in HY-MM Glut for Srd5a1, OEC and HY-MM Glut for Srd5a3 (circled dots).

Figure S13. Sex-associated variations in HSD17B isoform expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Hsd17b4, Hsd17b7, Hsd17b8, Hsd17b10, Hsd17b11, and Hsd17b12. In Pineal Glut, cells expressing Hsd17b7 were detected only in males (circled star). Notable sex differences were detected in HY-MM Glut for Hsd17b4, OEC and Pineal Glut for Hsd17b10 (circled dots).

Figure S14. Sex-associated variations in CYP7B1 expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Cyp7b1. No brain regions exhibited exclusive expression in one sex. No notable sex differences were detected across neuronal and non-neuronal cell types.

02

Table S1. Genes names and Ensembl IDs for data extracted for mapping.

Acknowledgements

The authors would like to thank the Allen Institute for Brain Science and all its contributors for generating different datasets available for public, non-profit use.

Funding

Funding for this study was from the National Institutes of Health (Grant Number DK110059) to VS.

Footnotes

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Data availability statement

All raw data analyzed in this study are available as part of the Allen Brain Map available for public, non-profit use (https://portal.brain-map.org/). All processed data presented in this study are available within the article and its Supplementary Materials.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Figure S1. Coupling of de novo and intermediary neurosteroid biosynthesis. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Hsd3b4, Hsd3b5, Hsd3b6, Hsd3b7, Hsd3b8, and Hsd3b9 (A) (colors indicate zero and positives as four quartile ranges), co-expression of Cyp11a1 and combined Sult2a1+Sult2b1 (B), and co-expression of Cyp11a1 and combined Hsd3b1+Hsd3b2+Hsd3b3 (C).

Figure S2. Expression of GABAA receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Gabra1, Gabra2, Gabra3, Gabra4, Gabra5, Gabrb1, Gabrb2, Gabrb3, Gabrd, and Gabrg1, (colors indicate zero and positives as four quartile ranges).

Figure S3. Expression of AMPA receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Gria1, Gria2, Gria3 and Gria4, (colors indicate zero and positives as four quartile ranges).

Figure S4. Expression of kainate receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Grik1, Grik2, Grik3, Grik4 and Grik5, (colors indicate zero and positives as four quartile ranges).

Figure S5. Expression of NMDA receptor subunits. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of Grin1, Grin2a, Grin2b, Grin2c, Grin2d, Grin3a, Grin3b, and Grina, (colors indicate zero and positives as four quartile ranges).

Figure S6. Expression of purinergic receptor subtypes. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of P2rx1, P2rx2, P2rx3, P2rx4, P2rx5, P2rx6 and P2rx7, (colors indicate zero and positives as four quartile ranges).

Figure S7. Expression of cannabinoid receptor and sigma-1 receptor. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in expression of (A) Cnr1 and (B) Sigmar1, (colors indicate zero and positives as four quartile ranges).

Figure S8. Expression of steroid hormone receptors. Plots showing t-SNE data visualizing single cell levels of gene expression in the murine brain to identify cell type and regional specificity in co-expression of estrogen receptors (Esr1, Esr2, and Gper1) (A), progesterone receptors (Pgr, Pgrmc1, and Pgrmc2) (B), androgen receptor (Ar) (C), and glucocorticoid receptor (Nr3c1) (D).

Figure S9. Sex-associated variations in SULT2B1 expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Sult2b1. No brain regions exhibited exclusive expression in one sex. Only subtle differences between sexes were observed across few regions.

Figure S10. Sex-associated variations in HSD3B isoform expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Hsd3b2, Hsd3b3, Hsd3b7, and Hsd3b8. In Pineal Glut, cells expressing Hsd3b7 were detected only in females (circled star). Notable sex differences were detected in HY-GABA, HY-MM Glut and MH-LH Glut for Hsd3b2, LSX-GABA, OB-CR Glut and OB-DG IMN for Hsd3b3, OEC for Hsd3b7, HY-MM Glut and MH-LH Glut for Hsd3b8 (circled dots).

Figure S11. Sex-associated variations in HSD11B1 expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Hsd11b1. In OB-CR Glut, cells expressing Hsd11b1 were detected only in females (circled star). Notable sex differences were also detected in HY-MM Glut (circled dot).

Figure S12. Sex-associated variations in SRD5A1 and SRD5A3 expression. t-SNE plots (left) and the ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Srd5a1 (top) and Srd5a3 (bottom) expression across different brain cell types. In Pineal Glut, cells expressing Srd5a3 were detected only in males (circled star). Notable sex differences were detected in HY-MM Glut for Srd5a1, OEC and HY-MM Glut for Srd5a3 (circled dots).

Figure S13. Sex-associated variations in HSD17B isoform expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Hsd17b4, Hsd17b7, Hsd17b8, Hsd17b10, Hsd17b11, and Hsd17b12. In Pineal Glut, cells expressing Hsd17b7 were detected only in males (circled star). Notable sex differences were detected in HY-MM Glut for Hsd17b4, OEC and Pineal Glut for Hsd17b10 (circled dots).

Figure S14. Sex-associated variations in CYP7B1 expression. t-SNE plots (left) and the sex ratio of positive expression in cells across various brain regions (right) illustrate sex differences in Cyp7b1. No brain regions exhibited exclusive expression in one sex. No notable sex differences were detected across neuronal and non-neuronal cell types.

NIHMS2077181-supplement-01.pdf (5MB, pdf)
02

Table S1. Genes names and Ensembl IDs for data extracted for mapping.

NIHMS2077181-supplement-02.pdf (78.6KB, pdf)

Data Availability Statement

All raw data analyzed in this study are available as part of the Allen Brain Map available for public, non-profit use (https://portal.brain-map.org/). All processed data presented in this study are available within the article and its Supplementary Materials.

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