The C57BL/6J Mouse Strain Background Modifies the Effect of a Mutation in Bcl2l2

Bcl2l2 encodes BCL-W, an antiapoptotic member of the BCL-2 family of proteins. Intercross of Bcl2l2 +/− mice on a mixed C57BL/6J, 129S5 background produces Bcl2l2 −/− animals with the expected frequency. In contrast, intercross of Bcl2l2 +/− mice on a congenic C57BL/6J background produces relatively few live-born Bcl2l2 −/− animals. Genetic modifiers alter the effect of a mutation. C57BL/6J mice (Mus musculus) have a mutant allele of nicotinamide nucleotide transhydrogenase (Nnt) that can act as a modifier. Loss of NNT decreases the concentration of reduced nicotinamide adenine dinucleotide phosphate within the mitochondrial matrix. Nicotinamide adenine dinucleotide phosphate is a cofactor for glutathione reductase, which regenerates reduced glutathione, an important antioxidant. Thus, loss of NNT activity is associated with increased mitochondrial oxidative damage and cellular stress. To determine whether loss of Bcl2l2 −/− mice on the C57BL/6J background was mediated by the Nnt mutation, we outcrossed Bcl2l2 congenic C57BL/6J (Nnt −/−) mice with the closely related C57BL/6JEiJ (Nnt +/+) strain to produce Bcl2l2 +/− ; Nnt +/+ and Bcl2l2 +/− ; Nnt −/− animals. Intercross of Bcl2l2 +/− ; Nnt +/+ mice produced Bcl2l2 −/− with the expected frequency, whereas intercross of Bcl2l2 +/− ; Nnt −/− animals did not. This finding indicates the C57BL/6J strain background, and possibly the Nnt mutation, modifies the Bcl2l2 mutant phenotype. This and previous reports highlight the importance of knowing the genetic composition of mouse strains used in research studies as well as the accurate reporting of mouse strains in the scientific literature

Carrier Screening for Spinal Muscular Atrophy (SMA) in 107,611 Pregnant Women during the Period 2005–2009: A Prospective Population-Based Cohort Study

BACKGROUND: Spinal muscular atrophy (SMA) is the most common neuromuscular autosomal recessive disorder. The American College of Medical Genetics has recently recommended routine carrier screening for SMA because of the high carrier frequency (1 in 25-50) as well as the severity of that genetic disease. Large studies are needed to determine the feasibility, benefits, and costs of such a program. METHODS AND FINDINGS: This is a prospective population-based cohort study of 107,611 pregnant women from 25 counties in Taiwan conducted during the period January 2005 to June 2009. A three-stage screening program was used: (1) pregnant women were tested for SMA heterozygosity; (2) if the mother was determined to be heterozygous for SMA (carrier status), the paternal partner was then tested; (3) if both partners were SMA carriers, prenatal diagnostic testing was performed. During the study period, a total of 2,262 SMA carriers with one copy of the SMN1 gene were identified among the 107,611 pregnant women that were screened. The carrier rate was approximately 1 in 48 (2.10%). The negative predictive value of DHPLC coupled with MLPA was 99.87%. The combined method could detect approximately 94% of carriers because most of the cases resulted from a common single deletion event. In addition, 2,038 spouses were determined to be SMA carriers. Among those individuals, 47 couples were determined to be at high risk for having offspring with SMA. Prenatal diagnostic testing was performed in 43 pregnant women (91.49%) and SMA was diagnosed in 12 (27.91%) fetuses. The prevalence of SMA in our population was 1 in 8,968. CONCLUSION: The main benefit of SMA carrier screening is to reduce the burden associated with giving birth to an affected child. In this study, we determined the carrier frequency and genetic risk and provided carrier couples with genetic services, knowledge, and genetic counseling

Mitochondrial Function Is Required for Secretion of DAF-28/Insulin in C. elegans

While insulin signaling has been extensively studied in Caenorhabditis elegans in the context of ageing and stress response, less is known about the factors underlying the secretion of insulin ligands upstream of the insulin receptor. Activation of the receptor governs the decision whether to progress through the reproductive lifecycle or to arrest growth and enter hibernation. We find that animals with reduced levels of the mitochondrial outer membrane translocase homologue TOMM-40 arrest growth as larvae and have decreased insulin signaling strength. TOMM-40 acts as a mitochondrial translocase in C. elegans and in its absence animals fail to import a mitochondrial protein reporter across the mitochondrial membrane(s). Inactivation of TOMM-40 evokes the mitochondrial unfolded protein response and causes a collapse of the proton gradient across the inner mitochondrial membrane. Consequently these broadly dysfunctional mitochondria render an inability to couple food abundance to secretion of DAF-28/insulin. The secretion defect is not general in nature since two other neuropeptides, ANF::GFP and INS-22::VENUS, are secreted normally. RNAi against two other putative members of the TOMM complex give similar phenotypes, implying that DAF-28 secretion is sensitive to mitochondrial dysfunction in general. We conclude that mitochondrial function is required for C. elegans to secrete DAF-28/insulin when food is abundant. This modulation of secretion likely represents an additional level of control over DAF-28/insulin function

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

Genes and Physiological Role of the Eukaryotic Nicotinamide Nucleotide Transhydrogenase

Nicotinamide nucleotide transhydrogenase (NNT, EC 1.6.1.1) is a proton pump which catalyses the reversible reduction of NADP+ by NADH linked to proton translocation across the membrane, according to the reaction: H+out + NADP+ + NADH H+in + NAD+ + NADPH In higher eukaryotes the enzyme is located in the inner mitochondrial membrane and is thus a possible provider of mitochondrial NADPH. NADPH is, among other things, important in the mitochondrial defence against oxidative damage. Reactive oxygen species and the damage they do to biological systems have been implicated in neurodegenerative diseases and ageing. In the present work the NNT genes from mouse, man and a green algae have been cloned and characterised, showing that the mitochondrial NNT genes have the same structure. The expression pattern in mouse tissues and subsections of human brain has been studied, as well as the expression pattern of Green Fluorescent Protein under control of the NNT promoter in the nematode Caenorhabditis elegans. The results show a tissue and cell-specific expression and, interestingly, a high expression in certain nematode neurons. A C. elegans mutant, lacking functional NNT enzyme, has been isolated and shown to grow slower than wild-type worms in the presence of the superoxide anion generator methyl viologen, providing evidence for the importance of the NNT enzyme as a supplier of mitochondrial NADPH for detoxification, e.g. of peroxides

Genes and Physiological Role of the Eukaryotic Nicotinamide Nucleotide Transhydrogenase

Nicotinamide nucleotide transhydrogenase (NNT, EC 1.6.1.1) is a proton pump which catalyses the reversible reduction of NADP+ by NADH linked to proton translocation across the membrane, according to the reaction: <p />H⁺_out + NADP⁺ + NADH <-> H⁺_in + NAD⁺ + NADPH <p />In higher eukaryotes the enzyme is located in the inner mitochondrial membrane and is thus a possible provider of mitochondrial NADPH. NADPH is, among other things, important in the mitochondrial defence against oxidative damage. Reactive oxygen species and the damage they do to biological systems have been implicated in neurodegenerative diseases and ageing. <p />In the present work the NNT genes from mouse, man and a green algae have been cloned and characterised, showing that the mitochondrial NNT genes have the same structure. The expression pattern in mouse tissues and subsections of human brain has been studied, as well as the expression pattern of Green Fluorescent Protein under control of the NNT promoter in the nematode <I>Caenorhabditis elegans</I>. The results show a tissue and cell-specific expression and, interestingly, a high expression in certain nematode neurons. A C. elegans mutant, lacking functional NNT enzyme, has been isolated and shown to grow slower than wild-type worms in the presence of the superoxide anion generator methyl viologen, providing evidence for the importance of the NNT enzyme as a supplier of mitochondrial NADPH for detoxification, e.g. of peroxides