Proteome Analysis of Thyroid Hormone Transporter Mct8/Oatp1c1-Deficient Mice Reveals Novel Dysregulated Target Molecules Involved in Locomotor Function

Abstract

Thyroid hormone (TH) transporter MCT8 deficiency causes severe locomotor disabilities likely due to insufficient TH transport across brain barriers and, consequently, compromised neural TH action. As an established animal model for this disease, Mct8/Oatp1c1 double knockout (DKO) mice exhibit strong central TH deprivation, locomotor impairments and similar histo-morphological features as seen in MCT8 patients. The pathways that cause these neuro-motor symptoms are poorly understood. In this paper, we performed proteome analysis of brain sections comprising cortical and striatal areas of 21-day-old WT and DKO mice. We detected over 2900 proteins by liquid chromatography mass spectrometry, 67 of which were significantly different between the genotypes. The comparison of the proteomic and published RNA-sequencing data showed a significant overlap between alterations in both datasets. In line with previous observations, DKO animals exhibited decreased myelin-associated protein expression and altered protein levels of well-established neuronal TH-regulated targets. As one intriguing new candidate, we unraveled and confirmed the reduced protein and mRNA expression of Pde10a, a striatal enzyme critically involved in dopamine receptor signaling, in DKO mice. As altered PDE10A activities are linked to dystonia, reduced basal ganglia PDE10A expression may represent a key pathogenic pathway underlying human MCT8 deficiency.

Keywords: Allan–Herndon–Dudley Syndrome; CNS; Mct8; Oatp1c1; Slc16a2; Slco1c1; T3; T4; basal ganglia; myelination.

Figure 1

Proteomic profiling of brain sections by LC-MS/MS. (A) Brain sections of WT and DKO mice at P21 were analyzed by LC-MS/MS. (B) Volcano plot displaying the regulation of proteins indicated by −log₁₀ q-value and log₂FC. Proteins with q < 0.01 were colored red and blue to indicate significant up- and down-regulation, respectively, in DKO versus WT animals. Grey dots indicate proteins that were not found to be differentially expressed. (C) Heatmap of the 67 regulated proteins with a q < 0.01 sorted by descending fold change. (D) Spider plot of the top 4 non-redundant, enriched pathways and their annotated genes from the over-representation analysis with the mouse-specific MSigDB category M5-GO “Gene Ontology gene sets” using the significantly regulated proteins. SNR: signal-to-noise ratio. n = 10 (WT) and n = 8 (DKO).

Figure 2

Comparison of proteomic and transcriptomic alterations. (A) Venn diagram indicating overlapping molecules between our P21 DKO proteome data and a published transcriptomic dataset [16] on age-matched DKO striatum. (B) Regression scatter plot of the 713 overlapping molecules based on the interception of the Venn diagrams with their respective proteomic and transcriptomic log₂FC. Molecules that are down-regulated on the transcript and protein level are depicted in blue, while those that are up-regulated in tandem are shown in red. Black dots indicate molecules that are differentially expressed on the transcript and protein level with opposite directions. Factors that are not concomitantly regulated in both datasets are displayed in grey. The grey band along the regression line indicates the regression’s 95% confidence interval. (C) StringDB analysis of co-regulated molecules with a log₂FC for transcriptome and proteome data >0.5. The molecules labelled in grey are not annotated to any of the highlighted terms and were therefore collectively grouped in the term “miscellaneous”. (D) The top 50 molecules of the term dystonia, according to PubMed datamining, were compared to the proteomic and transcriptomic regulations in DKO versus WT mice, revealing Pde10a and Pvalb as key affected molecules.

Figure 3

Reduced Pde10a mRNA and protein levels in the striatum of DKO mice. (A) Illustration of the analyzed brain areas for FISH and IHC (striatum; black box), and Western blot (whole forebrain hemisphere; green box). Icons were created with BioRender.com (accessed on 2 September 2023). (B,C) Fluorescence in situ hybridization analysis of striatal Pde10a mRNA (in green) expression revealed significantly reduced signal intensities in DKO mice at P21 ((B) WT: 3 females and 1 male; DKO: 2 females and 2 males) and P120 ((C) all males). (D) Immunofluorescence staining for Pde10a protein (in green) demonstrating reduced signal intensities in DKO mice at P21. (E) Western blot analysis of forebrain homogenates confirmed reduced Pde10a protein levels in female DKO mice at P21. As a housekeeping protein for the normalization of signal intensities, Gadph was used. Blue color in (C,D) indicates Hoechst33258-counterstained cell nuclei. Scale bar: 50 μm. n = 3–5. ** p < 0.01; *** p < 0.001.