Responsiveness of sphingosine phosphate lyase insufficiency syndrome to vitamin B6 cofactor supplementation.

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation.

Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney.

A small molecule screening to detect potential therapeutic targets in human podocytes.

WIDMEIER E, TAN W, AIRIK M, HILDEBRANDT F: A small molecule screening to detect potential therapeutic targets in human podocytes. Am J Physiol Renal Physiol 312: F157-F171, 2017. First published October 19, 2016; doi:10.1152/ajprenal.00386.2016. Steroid-resistant nephrotic syndrome (SRNS) inevitably progresses to end-stage kidney disease, requiring dialysis or transplantation for survival. However, treatment modalities and drug discovery remain limited. Mutations in over 30 genes have been discovered as monogenic causes of SRNS. Most of these genes are predominantly expressed in the glomerular epithelial cell, the podocyte, placing it at the center of the pathogenesis of SRNS. Podocyte migration rate (PMR) represents a relevant intermediate phenotype of disease in monogenic causes of SRNS. We therefore adapted PMR in a high-throughput manner to screen small molecules as potential therapeutic targets for SRNS. We performed a high-throughput drug screening of a National Institutes of Health Clinical Collection (NCC) library (n = 725 compounds) measuring PMR by videomicroscopy. We used the Woundmaker to perform individual 96-well scratch wounds and screened compounds using a quantitative kinetic live cell imaging migration assay using IncuCyte ZOOM technology. Using a normal distribution for the average PMR in wild-type podocytes with a vehicle control (DMSO), we applied a 90% confidence interval to define "distinct" compounds (5% faster/slower PMR) and found that 12 of 725 compounds (at 10 µM) reduced PMR. Clusters of drugs that alter PMR included actin/tubulin modulators such as the azole class of antifungals and antineoplastic vinca-alkaloids. We hereby identify compounds that alter PMR. The PMR assay provides a new avenue to test therapeutics for nephrotic syndrome. Positive results may reveal novel pathways in the study of glomerular diseases such as SRNS.

Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q10 deficiency.

Human COQ6 encodes a monooxygenase which is responsible for the C5-hydroxylation of the quinone ring of coenzyme Q (CoQ). Mutations in COQ6 cause primary CoQ deficiency, a condition responsive to oral CoQ10 supplementation. Treatment is however still problematic given the poor bioavailability of CoQ10. We employed S. cerevisiae lacking the orthologous gene to characterize the two different human COQ6 isoforms and the mutations found in patients. COQ6 isoform a can partially complement the defective yeast, while isoform b, which lacks part of the FAD-binding domain, is inactive but partially stable, and could have a regulatory/inhibitory function in CoQ10 biosynthesis. Most mutations identified in patients, including the frameshift Q461fs478X mutation, retain residual enzymatic activity, and all patients carry at least one hypomorphic allele, confirming that the complete block of CoQ biosynthesis is lethal. These mutants are also partially stable and allow the assembly of the CoQ biosynthetic complex. In fact treatment with two hydroxylated analogues of 4-hydroxybenzoic acid, namely, vanillic acid or 3-4-hydroxybenzoic acid, restored the respiratory growth of yeast Δcoq6 cells expressing the mutant huCOQ6-isoa proteins. These compounds, and particularly vanillic acid, could therefore represent an interesting therapeutic option for COQ6 patients.

Molecular cloning and expression of phospholipase C epsilon 1 in zebrafish.

Phospholipase C (PLC) is a key enzyme that generates inositol 1,4,5-triphosphate (IP(3)) and diacylglycerol (DAG), two second messengers in signal transduction. Phospholipase Cepsilon (PLCE1) is a unique member of the PLC family in that it carries both RasGEF and Ras-associating (RA) domains and may serve as an activator and an effector of small GTPases. Recently, mutations in PLCE1 have been associated with early-onset nephrotic syndrome. We have identified the zebrafish ortholog of PLCE1 and characterized its expression pattern in zebrafish embryos by in situ hybridization. Zebrafish plce1 gene encodes a protein of 2248 amino acids that shares 56% identity with mammalian PLCepsilon. During zebrafish embryonic development, plce1 is expressed in a dynamic pattern in a variety of organs, such as brain, muscle, liver, and pronephric glomeruli. Our results suggest that zebrafish may be used as a model organism to address the function of PLCepsilon during the development of these organs.