Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway.

Ataxia oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease caused by mutations in APTX, which encodes the DNA strand-break repair protein aprataxin (APTX). CoQ10 deficiency has been identified in fibroblasts and muscle of AOA1 patients carrying the common W279X mutation, and aprataxin has been localized to mitochondria in neuroblastoma cells, where it enhances preservation of mitochondrial function. In this study, we show that aprataxin deficiency impairs mitochondrial function, independent of its role in mitochondrial DNA repair. The bioenergetics defect in AOA1-mutant fibroblasts and APTX-depleted Hela cells is caused by decreased expression of SDHA and genes encoding CoQ biosynthetic enzymes, in association with reductions of APE1, NRF1 and NRF2. The biochemical and molecular abnormalities in APTX-depleted cells are recapitulated by knockdown of APE1 in Hela cells and are rescued by overexpression of NRF1/2. Importantly, pharmacological upregulation of NRF1 alone by 5-aminoimidazone-4-carboxamide ribonucleotide does not rescue the phenotype, which, in contrast, is reversed by the upregulation of NRF2 by rosiglitazone. Accordingly, we propose that the lack of aprataxin causes reduction of the pathway APE1/NRF1/NRF2 and their target genes. Our findings demonstrate a critical role of APTX in transcription regulation of mitochondrial function and the pathogenesis of AOA1 via a novel pathomechanistic pathway, which may be relevant to other neurodegenerative diseases.

ANO10 mutations cause ataxia and coenzyme Q10 deficiency.

Inherited ataxias are heterogeneous disorders affecting both children and adults, with over 40 different causative genes, making molecular genetic diagnosis challenging. Although recent advances in next-generation sequencing have significantly improved mutation detection, few treatments exist for patients with inherited ataxia. In two patients with adult-onset cerebellar ataxia and coenzyme Q10 (CoQ10) deficiency in muscle, whole exome sequencing revealed mutations in ANO10, which encodes anoctamin 10, a member of a family of putative calcium-activated chloride channels, and the causative gene for autosomal recessive spinocerebellar ataxia-10 (SCAR10). Both patients presented with slowly progressive ataxia and dysarthria leading to severe disability in the sixth decade. Epilepsy and learning difficulties were also present in one patient, while retinal degeneration and cataract were present in the other. The detection of mutations in ANO10 in our patients indicate that ANO10 defects cause secondary low CoQ10 and SCAR10 patients may benefit from CoQ10 supplementation.

Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis.

Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

Progressive myoclonus epilepsy with nephropathy C1q due to SCARB2/LIMP-2 deficiency: clinical report of two siblings.

Action myoclonus-renal failure syndrome (AMRF) is considered a rare form of progressive myoclonus epilepsy (PME) associated with renal failure. A mutation on the gene encoding the lysosomal integral membrane protein type 2-LIMP-2 (SCARB2), the receptor responsible for targeting glucocerebrosidase to the lysosomes, was recently described, allowing a better understanding of its etiopathogenesis. We describe clinically two sisters with AMRF that resulted from a mutation in the SCARB2 gene. The renal involvement was due to nephropathy C1q. When substrate-reduction therapy, to correct the possible glucocerebroside storage in the cells with glucocerebrosidase deficiency, was administered to one of the siblings, a significant improvement was observed. This report points out a rational for a therapeutical approach to this new lysossomopathy.

Uncoupling between CD1d upregulation induced by retinoic acid and conduritol-B-epoxide and iNKT cell responsiveness.

Gaucher disease (GD) is associated with upregulation of CD1d and MHC-class II expression by monocytes. While the physiological impact of CD1d upregulation remains uncertain, it has been proposed that MHC-class II upregulation is associated with inflammation. Hereby, we show that the decrease in MHC-class II expression seen in GD patients under therapy correlates positively with chitotriosidase activity, a marker of inflamed macrophages. We also show that retinoic acid (RA) and the beta-glucocerebrosidase inhibitor conduritol-B-epoxide (CBE) lead to upregulation of CD1d expression by THP-1 cells, which correlated with an increase in mRNA expression. In vitro co-culture experiments showed that RA treated THP-1 cells were more stimulatory for CD4(+) than for CD8(+) T cells, as determined by CFSE loss, in comparison to untreated THP-1 cells. Interestingly, even though addition of exogenous isoglobotrihexosylceramide (iGb3), a physiological CD1d ligand, augmented the percentage of dividing CD4(+) T cells, we could not detect a significant expansion of CD4(+)Valpha24(+) invariant Natural Killer T (iNKT) cells. In contrast, addition of alpha-galactosylceramide (alpha-GC) induced expansion of Valpha24(+) iNKT cells as determined by using alpha-GC-loaded human CD1d dimers. These results strengthen the existence of a cross-talk between monocyte lipid accumulation, inflammation and changes in cell surface CD1d and MHC-class II in monocytes, which may result in inappropriate recognition events by immune cells and perpetuate chronic inflammation.

A nonsense mutation in the LIMP-2 gene associated with progressive myoclonic epilepsy and nephrotic syndrome.

The main clinical features of two siblings from a consanguineous marriage were progressive myoclonic epilepsy without intellectual impairment and a nephrotic syndrome with a strong accumulation of C1q in capillary loops and mesangium of kidney. The biochemical analysis of one of the patients revealed a normal beta-glucocerebrosidase activity in leukocytes, but a severe enzymatic deficiency in cultured skin fibroblasts. This deficiency suggested a defect in the intracellular sorting pathway of this enzyme. The sequence analysis of the gene encoding LIMP-2 (SCARB2), the sorting receptor for beta-glucocerebrosidase, confirmed this hypothesis. A homozygous nonsense mutation in codon 178 of SCARB2 was found in the patient, whereas her healthy parents were heterozygous for the mutation. Besides lacking immunodetectable LIMP-2, patient fibroblasts also had decreased amounts of beta-glucocerebrosidase, which was mainly located in the endoplasmic reticulum, as assessed by its sensitivity to Endo H. This is the first report of a mutation in the SCARB2 gene associated with a human disease, which, contrary to earlier proposals, shares no features with Charcot-Marie-Tooth disease both at the clinical and neurophysiological levels.

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model.

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase-deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol-B-epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid-binding molecule CD1d. GluCerase-deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)-class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC-class I. However, CD1d and MHC-class II mRNA levels were not increased. GluCerase-deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC-class II and imbalances in the percentage of CD4+, CD8+, and Valpha24+ T cells. Interestingly, follow-up studies revealed that enzyme replacement therapy induced a decrease in MHC-class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.