Analysis of dehydrogenase-independent functions of HSD17B10 in humans and animal models

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with an atypical progressive neurodegenerative disease course (Zschocke et al, 2000). The symptoms in HSD10 deficiency patients are not correlated with residual dehydrogenase activity of mutated HSD10. LOF and rescue experiments in Xenopus embryos showed that a knock-down of HSD10 caused apoptosis. The dehydrogenase activity of HSD10 was not required for cell survival suggesting that HSD10 has additional functions. The pathogenetic basis of HSD10 deficiency has so far remained elusive but the symptoms observed in patients are likely related to defects in general mitochondrial function. Therefore, the effect of HSD10 LOF on mitochondrial structural and functional integrity was investigated. Embryonic Xenopus cells displayed severe disruption of mitochondrial morphology and function when translation of HSD10 mRNA was blocked. Similar effects on mitochondria were observed in cells derived from conditional HSD10 knock-out mice and in fibroblasts from patients with a severe clinical phenotype. In Xenopus overexpression of two HSD10 mutations, R130C and D86G, associated with severe disease in humans, strongly induced apoptosis in a dominant-negative manner which was not due to the unfolded protein response that is occasionally triggered by overexpression of (mutated) proteins. In contrast, wildtype HSD10 and the Q165H mutation had little effect on apoptosis. Expression analysis of apoptosis-associated genes in HSD10 depleted cells or cells carrying different HSD10 mutations demonstrated that no specific apoptotic pathway was activated. This indicated that intrinsic as well as extrinsic apoptosis signals contribute to cell death when HSD10 function is perturbed. Symptoms in patients usually develop after metabolic stress situations. Therefore the stress response behaviour of fibroblasts carrying HSD10 mutations was studied under different stress conditions. In contrast to control cells, fibroblasts from patients were not able to stimulate tRNA transcription upon oxidative stress. Interestingly cells with the R130C mutation could cope with stress just like the Q165H mutation. This was unexpected, since the R130C mutation causes a severe clinical phenotype whereas the Q165H mutation has been found in neurologically normal boys. In order to understand the mechanisms behind the physiological function of HSD10 a search for its binding partners was performed. In a homology based BLAST in yeast as well as in an IMAC approach putative HSD10-interacting proteins were identified. Unfortunately none of these candidates were directly connected to mitochondrial function or apoptosis. In a previously performed yeast-2-hybrid screen several HSD10 interaction partners had been identified. One of those binding proteins, UXT, was tested for a functional interaction with HSD10 in Xenopus embryos. Co-expression of HSD10 and UXT enhanced the induction of apoptosis. The mechanism of this functional interaction remains to be investigated. HSD10 is a component of the RNAseP complex which is essential for the 5’ processing of mitochondrial tRNAs. Therefore I tested the function of RNaseP in HSD10 deficiency in patient fibroblast and in a rescue experiment in HSD10 depleted cells. The rescue experiment, in which wildtype HSD10 function was substituted by the mutations R130C, D86G and Q165H indicated that all mutations still have RNaseP activity. This held also true for patient fibroblasts where no tRNA precursor accumulation or inhibition of mitochondrial translation was detected. Experiments with cells that are depleted of mitochondrial DNA and hence do not require mitochondrial transcription or mtRNaseP function revealed that these cells still become apoptotic upon HSD10 knock-down. Therefore, the apoptosis phenotype upon HSD10 LOF is not dependent on RNaseP function. Taken together, these experiments indicate that clinical symptoms in HSD10 deficiency are not fully explained by an impairment of RNaseP function

A non-enzymatic function of 17 beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required

A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required