Endocrine Abstracts

Background: Resistance to thyroid hormone alpha (RTHα), caused by mutations in the T3-receptor alpha 1 (TRα1) isoform, includes growth retardation, intellectual disability, and abnormal thyroid function tests. The current paradigm entails that disease features arise from decreased T3 action in TRα1-expressing tissues. However, also for patients that carry mutations that completely abolish T3-stimulated activity, neurological features vary strongly, ranging from mild motor impairments to severe intellectual disability and absence of speech. This implies that other mechanisms besides impaired T3-binding affect the neurocognitive phenotype, such as altered recruitment of regulatory cofactors. Our aim was to elucidate the molecular mechanisms underlying the large differences in neurocognitive features among RTHα patients.

Methods: Human SHSY5Y neuroblastoma cells were stably transduced with FLAG-HA-tagged (FH) wild type (WT), C380fsx387-TRα1 (severe phenotype) or F397fsx406-TRα1 (mild phenotype), or empty vector as control. Cells were stimulated for 4 hrs with vehicle or 10 nM T3 and transcriptomes were analyzed by RNAseq. To characterize the TRα1-interactome, WT FHTRα1 was tandem-affinity purified from nuclear extract from and co-purifying proteins identified by LC-MS/MS. Selected cofactors were further tested for co-immunoprecipitation with WT and mutant FHTRα1 in SHSY5Y cells, or FLAGTRα1 in transiently transfected HepG2 cells. Direct interactions between receptors and the co-repressor NCoR1 were further tested using a mammalian two-hybrid system.

Results: In contrast to WT, cells expressing the frameshift mutants C380fsx387-FHTRα1 and F397fsx406-FHTRα1 lacked any T3-stimulated gene expression. Unstimulated gene expression also differed for the mutants, particularly for the more severe C380fsx387-TRα1 mutant, with many dysregulated genes involved in neuronal development and migration. Compared to WT and F397fsx406-TRα1, C380fsx387-TRα1 showed a reduced interaction with the corepressor NCoR1 in co-immunoprecipitation and in the 2-hybrid assays, and an increased association with CHD4, ADNP and HP1γ, three proteins that form a repressive complex (ChAHP) that is important for neuronal lineage specification. The nonsense-mutant C380X-TRα1 also displayed decreased NCoR1 and increased ChAHP recruitment, indicating that the location of the mutation, rather than the altered amino acid sequence caused by the frameshift causes the changes in cofactor recruitment.

Conclusion: C380fsx387-TRα1 and F397fsx406-TRα1 both lack T3-binding but show differential effects on gene expression in human neuronal cells, which likely causes the neurological phenotypic differences in RTHα. C380fsx387-TRα1 has increased recruitment of the repressive ChAHP complex, which likely contributes to the severe clinical phenotype in patients with this mutation. Major rearrangement in cofactor recruitment is a disease mechanism for nuclear receptors that has not been previously described.