Single cell resolution of neurosteroidogenesis in the murine brain: de novo biosynthesis

Abstract

Neurosteroids synthesized within the central nervous system play essential roles in modulating neurotransmission, providing neuroprotection, regulating immune responses, influencing behavior and cognition and mediating stress physiology. Despite their broad significance, the specific brain cell types capable of de novo steroid synthesis from cholesterol remain poorly defined. In this study, we analyzed single-cell transcriptomic data to map steroidogenic gene expression across cell populations in the murine brain, focusing on the de novo production of the neurosteroid pregnenolone. Our findings reveal that de novo steroidogenesis, as marked by Cyp11a1 expression, is predominantly confined to specific neuronal subtypes, particularly glutamatergic neurons of the intra- and extra-telencephalic regions and the corticothalamic layer. In contrast, Star expression, which is essential for mitochondrial cholesterol import, was more broadly distributed, occurring in both neuronal and non-neuronal cells (including oligodendrocytes, astrocytes, immune cells and vascular cells). In these non-neuronal populations, Star was notably co-expressed with mitochondrial Cyp27a1, indicative of bile acid synthesis rather than neurosteroidogenesis. This distinction highlights that Star expression alone is not a reliable marker of de novo neurosteroidogenic capacity in the brain, as its functional significance depends on the broader enzymatic context in which it occurs. The resulting single-cell map of de novo neurosteroid biosynthetic capacity across brain regions, including modest sex-associated differences, provides a foundational framework for understanding neurosteroid signaling in distinct cell types and its relevance to brain physiology and pathophysiology.

Keywords: astrocyte; bile; microglia; neuron; oligodendrocyte; pregnenolone.

Figure 1.. Single cell map classification of the murine brain.

(A) A t-distributed stochastic neighbor embedding (t-SNE) plot visualizing single cell distribution patterns from the murine brain color coded based on marker-defined regional cell types. Plot is a reconstruction from the cell atlas of the whole mouse brain (Yao et al. 2023). (B) De novo steroidogenesis involves the synthesis of pregnenolone from precursor cholesterol. The process involves two key steps that are movement of cholesterol across the mitochondrial intermembrane space (IMS) mediated by the steroidogenic acute regulatory protein (STAR), and activity of the cytochrome P450 monooxygenase (CYP11A1) that catalyzes the side-chain hydroxylation and cleavage of cholesterol to pregnenolone.

Figure 2.. Neurons are the major contributors to de novo neurosteroid biosynthesis.

(A) Expression of Cyp11a1 from single cells visualized in a cell type defined t-SNE plot with colors that indicate zero and positives (as four quartile ranges). Valuation of regional and cell type specific Cyp11a1 expression (heatmap with total expression log₂CPM) and percentage of cell type-specific expression as indicators of pregnenolone production indicates that majority of de novo neurosteroidogenesis occurs in different types of glutamatergic and GABAergic neurons. The glutamatergic IT-ET neurons showed the highest levels of Cyp11a1 expression, followed by NP-CT-L6b neurons. (B) Expression of Star from single cells visualized in a cell type defined t-SNE plot (color range indicates quartiles) and heatmap. Valuation of regional and cell type specific Star as an essential step in steroidogenesis indicates a pattern inconsistent with Cyp11a1, encompassing both neuronal and non-neuronal cells. (C) A t-SNE plot visualizing co-expression of Cyp11a1 and Star (Red), highlights the frequency of co-expression correlated to the different neuronal cell populations.

Figure 3.. Non-neuronal glial cells appear active in bile acid synthesis.

(A) Expression of Cyp27a1 from single cells visualized in a cell type defined t-SNE plot with colors that indicate zero and positives (as four quartile ranges). Valuation of Cyp27a1 expression (heatmap with total expression log₂CPM) and percentage of cell type-specific expression as indicators of the acidic pathway of bile acid production shows that majority of bile acid synthesis occurs in different non-neuronal glial cells: OPC-oligo>Astro-Epen>Immune>Vascular cells. (B) A t-SNE plot visualizing co-expression of Cyp27a1 and STAR (Red), indicates substantial co-expression correlated to glial cell populations.

Figure 4.. Non-neuronal cells are scarce contributors to de novo neurosteroid biosynthesis.

(A). Cropped t-SNE label plots showing OPC-Oligo, Immune, Astro-Epen and Vascular and t-SNE expression plots for Cyp11a1, Cyp27a1, Pdgfra, Mog, Gfap, C1qa, Cd93 and Pdgfrb (color range indicates quartiles). (B) Co-expression of Cyp11a1 and Cyp27a1 with different non-neuronal cells (based on markers) are presented as a percentage of the total population of non-neuronal cells. Evaluating Cyp11a1 and Cyp27a1 expression together with co-expression of different non-neuronal cell lineages indicated a higher degree of bile acid biosynthetic capacity and diminutive de novo neurosteroidogenic capacity in these cells. (C) Distribution of Cyp27a1 expressing non-neuronal cells were primarily associated with OPC-Oligo with minor populations of Astro-Epen, Immune and Vascular cells.

Figure 5.. Extent of involvement by brain cells towards de novo neurosteroid biosynthesis.

Of the total 4,041,289 single cell data points processed for this study, 6.74% were positive for Cyp11a1. These Cyp11a1 positive cells were >99% neuronal (6.68%) with little representation of <1% in non-neuronal cells (0.06%). Within neurons, Cyp11a1 positive glutamatergic neurons were much higher (5.81%) than GABAergic neurons (0.86%), with much lower representation from dopaminergic and serotonergic neurons (0.01% for each). Among non-neuronal cells, oligodendrocytes and their progenitors showed highest expression (0.03%), followed by astrocytes and ependymal cells (0.016%), vascular cells (0.11%) and immune cells (0.005%). These findings indicate that de novo neurosteroid biosynthesis is predominantly from glutamatergic neurons within neuronal populations of the murine brain.