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als mutations  als  asmcs  function  glut  homeostasis  igd  mitochondrial  mutations  prokr variants  prokr  toxicity  ubqln  variants  vitro 
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Preview: Human Molecular Genetics - Advance Access

Human Molecular Genetics Advance Access

Published: Fri, 17 Nov 2017 00:00:00 GMT

Last Build Date: Fri, 17 Nov 2017 06:44:29 GMT


Mutation-dependent aggregation and toxicity in a Drosophila model for UBQLN2-associated ALS


Members of the conserved ubiquilin (UBQLN) family of ubiquitin (Ub) chaperones harbor an antipodal UBL (Ub-like)-UBA (Ub-associated) domain arrangement and participate in proteasome and autophagosome-mediated protein degradation. Mutations in a proline-rich-repeat region (PRR) of UBQLN2 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD); however, neither the normal functions of the PRR nor impacts of ALS-associated mutations within it are well understood. In this study we show that ALS mutations perturb UBQLN2 solubility and folding in a mutation-specific manner. Biochemical impacts of ALS mutations were additive, transferrable to UBQLN1, and resulted in enhanced Ub association. A Drosophila melanogaster model for UBQLN2-associated ALS revealed that both wild-type and ALS-mutant UBQLN2 alleles disrupted Ub homeostasis; however, UBQLN2ALS mutants exhibited age-dependent aggregation and caused toxicity phenotypes beyond that seen for wild-type UBQLN2. Although UBQLN2 toxicity was not correlated with aggregation in the compound eye, aggregation-prone UBQLN2 mutants elicited climbing defects and NMJ abnormalities when expressed in neurons. An UBA domain mutation that abolished Ub binding also diminished UBQLN2 toxicity, implicating Ub binding in the underlying pathomechanism. We propose that ALS-associated mutations in UBQLN2 disrupt folding and that both aggregated species and soluble oligomers instigate neuron autonomous toxicity through interference with Ub homeostasis.

Modeling Mutant/Wild-type Interactions to Ascertain Pathogenicity of PROKR2 Missense Variants in Patients with Isolated GnRH Deficiency


A major challenge in human genetics is the validation of pathogenicity of heterozygous missense variants. This problem is well-illustrated by PROKR2 variants associated with Isolated GnRH Deficiency (IGD). Homozygous, loss of function variants in PROKR2 were initially implicated in autosomal recessive IGD; however, most IGD-associated PROKR2 variants are heterozygous. Moreover, while IGD patient cohorts are enriched for PROKR2 missense variants similar rare variants are also found in normal individuals. To elucidate the pathogenic mechanisms distinguishing IGD-associated PROKR2 variants from rare variants in controls, we assessed 59 variants using three approaches: (i) in silico prediction, (ii) traditional in vitro functional assays across 3 signaling pathways with mutant-alone transfections, and (iii) modified in vitro assays with mutant and wild-type expression constructs co-transfected to model in vivo heterozygosity. We found that neither in silico analyses nor traditional in vitro assessments of mutants transfected alone could distinguish IGD variants from control variants. However, in vitro co-transfections revealed that 15/34 IGD variants caused loss-of-function (LoF), including 3 novel dominant-negatives, while only 4/25 control variants caused LoF. Surprisingly, 19 IGD-associated variants were benign or exhibited LoF that could be rescued by WT co-transfection. Overall, variants that were LoF in ≥ 2 signaling assays under co-transfection conditions were more likely to be disease associated than benign or ‘rescuable’ variants. Our findings suggest that in vitro modeling of WT/Mutant interactions increases the resolution for identifying causal variants, uncovers novel dominant negative mutations, and provides new insights into the pathogenic mechanisms underlying heterozygous PROKR2 variants.

GLUT10 Maintains the Integrity of Major Arteries through Regulation of Redox Homeostasis and Mitochondrial Function


Glucose transporter 10 (GLUT10) is a member of the GLUT family of membrane transporters, and mutations in this gene cause arterial tortuosity syndrome (ATS). However, the physiological role and regulation of GLUT10 in arteries remains unclear. To further understand its physiological roles in major arteries, we examined the regulatory mechanisms of GLUT10 in ASMCs and aortic tissues. Interestingly, we find that targeting of GLUT10 to mitochondria is increased in ASMCs under both stress and aging conditions, which enhances dehydroascorbic acid (DHA) uptake and maintains intracellular ascorbic acid (AA) levels. We further demonstrate that the targeting of GLUT10 to mitochondria is important to maintain redox homeostasis, mitochondrial structure and mitochondrial function in ASMCs. A missense mutation of GLUT10 (Glut10G128E) impairs mitochondrial targeting in ASMCs. Consequently, ASMCs isolated from Glut10G128E mice exhibit increased reactive oxygen species (ROS) levels, fragmented mitochondria and impaired mitochondrial function, as well as enhanced cell proliferation and migration. In vivo, mitochondrial structure is altered, and ROS levels are heightened in aortic tissues of Glut10G128E mice. Furthermore, increased number and disorganization of ASMCs, along with progressive arterial wall remodeling were observed in aortic tissues of Glut10G128E mice. These defects were coincident with elevated systolic blood pressure in aged Glut10G128E animals. Our results describe a novel mechanism that GLUT10 targeting to mitochondria under stress and aging condition has a critical role in maintaining AA levels, redox homeostasis and mitochondrial structure and function in ASMCs, which is likely to contribute to the maintenance of healthy vascular tissue.