The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin–melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.

Background: The local availability of thyroid hormones (TH) depends on transporters like monocarboxylate transporter 8 (MCT8). While its importance in fetal brain development is established, a comprehensive regional characterization of MCT8 in the adult human brain remains lacking. This knowledge is essential for understanding MCT8's role in normal physiology, aging, and neurological disease.Methods: We investigated MCT8 distribution in the adult and elderly human brain across multiple structural levels, encompassing distinct cell types, the cellular microenvironment, and cytoarchitecture. Comparing 8 brain regions from 14 donors, we employed immunostaining, whole-slide image analysis, fluorescence in situ hybridization (RNAscope), and stimulated emission depletion (STED) microscopy to assess protein and transcriptome expression. Braak staging was applied to assess MCT8 expression in relation to Alzheimer's disease (AD) neurofibrillary pathology.Findings: MCT8 protein and SLC16A2 mRNA were robustly expressed in glial cells, neurons, and brain barriers across all examined regions. RNAscope confirmed strong concordance between transcript presence and immunoreactivity. STED microscopy localized MCT8 signal to plasma membranes, perinuclear regions, and axonal/dendritic compartments. Braak staging revealed no significant difference in MCT8 expression between donors with high versus low neurofibrillary pathology.Interpretation: MCT8 is broadly expressed throughout the adult and elderly human brain at both macro- and microenvironmental levels. Its distinct localization suggests ongoing functional roles in inter- and intracellular TH transport, and its expression appears to remain stable regardless of AD progression.

The local availability of thyroid hormones (TH) depends on TH transporters such as monocarboxylate transporter 8 (MCT8, encoded by SLC16A2 ). Mutations of SLC16A2 cause Allan-Herndon-Dudley syndrome (AHDS), a disorder characterized by severe neurodevelopmental and motor deficits. While MCT8’s importance in foetal brain development is well-established, its precise topographical, cellular, and subcellular distribution in the adult human brain has remained largely uncharacterized, limiting our understanding of which structures depend on MCT8-mediated TH transport throughout life and whether neurodegenerative processes such as Alzheimer’s disease (AD) affect local transporter availability. In this study, we systematically mapped MCT8 expression across eight functionally distinct brain regions from 14 aged human body donors. Our multiscale approach combined immunostaining, automated whole-slide image analysis of over 13 million cells, multiplex RNAscope in situ hybridization, stimulated emission depletion (STED) microscopy, and single-nucleus RNA sequencing data analysis. We additionally applied standardized Braak staging to investigate whether AD progression correlates with alterations in MCT8 expression. We demonstrate robust MCT8 expression across brain barriers, glial cells, and in distinct neuronal populations. RNAscope confirmed a strong concordance between SLC16A2 mRNA presence and MCT8 immunoreactivity. STED and confocal microscopy localized MCT8 to plasma membranes, perinuclear regions, and axonal/dendritic compartments, indicating roles in both trans- and intracellular TH transport. MCT8 expression was preserved across Braak stages, with no significant difference between donors with varying degrees of neurofibrillary pathology. Our findings establish a comprehensive cellular and regional framework for MCT8-mediated transport in the human brain, mapping its specific anatomical distribution across regions crucial for motor control and cognitive processing. Stable MCT8 expression across Braak stages suggests that cerebral TH dysregulation in AD likely reflects altered hormone metabolism rather than transporter loss.