Neurodegenerative Diseases Associated with Mutations in <em>SLC25A46</em>

Neurodegenerative diseases present substantial clinical challenges. Their processes have been linked with various genetic causes, including mutations of genes encoding proteins associated with mitochondrial dynamics. Biallelic mutations in SLC25A46 have been identified as novel causes of a wide spectrum of neurological diseases with recessive inheritance, including optic atrophy, Charcot-Marie-Tooth neuropathy (CMT) type 2A neuropathy, Leigh syndrome, progressive myoclonic ataxia, and lethal congenital pontocerebellar hypoplasia. SLC25A46 (solute carrier family 25 member 46) is a membrane transit protein that is expressed in the mitochondrial outer membrane where it plays a major role in mitochondrial dynamics and cristae maintenance. This chapter presents recent findings on: (1) the clinical heterogeneity of SLC25A46-related neuropathies; (2) the SLC25A46 mutation spectrum and associated genotype-phenotype correlation; and (3) pathophysiological functions of SLC25A46 as characterized in cells and mouse models. A better understanding of the etiology of SLC25146-linked diseases will elucidate therapeutic perspectives

Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

OPA1 +/− -iPSCs (OL) are unfavorable to differentiate into neural rosette. Culture conditions were the same as those in Fig. 3. Panel A shows day 5 EBs, which were derived from control and OL-iPSCs, respectively. Morphologically, OL-EBs look similar to control EBs. Panel B shows neuron rosettes (NRs) after 5 days of EB attachment. Fewer and smaller neuron rosettes were derived from OL-EBs, compared with neuron rosettes from control EBs. Figure S2 OPA1 +/− -iPSCs (OL) failed to differentiate into RGCs with culture medium supplemented with 10 % FBS and DAPT. Differentiation method in Fig. 4 was followed. Putative RGCs were fixed on day 24 before fixation, followed by IF staining. Upper panel shows staining of TUJ1 (green) and BRN3a (red), while lower panel displays staining of TUJ1 (green) and ISLET1 (red). The scale bars equal 50 μM. Figure S3 IF analysis of RGCs derived from the control and VO-iPSCs on day 38. Neurospheres were plated onto PLO/L-coated plates and cultured in hESC medium containing 10 μM DAPT and 10 % FBS for 21 days before fixation with 4 % PFA on day 38. Cells were stained with TUJ1 (green), BRN3a (red) and ISLET-1 (red) antibodies. The scale bars equal 20 μM. Figure S4 NIM promoted OPA1 +/− -RGC (OL) generation detected by IF. Putative OL-RGCs derived from OL-iPSCs were cultured with hESC medium containing 10 μM DAPT, 10 % FBS, and 10 % neural induction medium (NIM) for 14 days before fixation on day 31. Upper panel shows TUJ1 (green) and BRN3a (red) staining. Lower panel shows staining of TUJ1 (green) and ISLET1 (red). The scale bars equal 50 μM. Figure S5 Quantification of RGC differentiation efficiency. The culture medium used for RGC differentiation contained 10 % neural induction medium. Samples were normalized to the control BRN3a/DAPI staining or the control ISLET-1/DAPI staining. The number of BRN3a or ISLET-1 signals versus the number of DAPI signals was calculated. A p-value of 0.084 was obtained for the BRN3a signal comparison between the control and VO, and a p-value of 0.076 was obtained for the ISLET-1 signal comparison between the control and VO. Student’s t-test was used to analyze differences between two groups. Figure S6 Noggin rescued OPA1 +/− -iPSC (OL) differentiation into RGCs confirmed by IF. Mature EBs were cultured with hES medium containing 10 % FBS and 100 ng/mL Noggin to obtain neuron rosettes, followed by culturing putative RGCs with hES medium containing 10 % FBS, DAPT and 100 ng/mL Noggin. Cells were fixed with 4 % PFA on day 31. Upper panel shows IF staining of TUJ-1 (green) and BRN3a (red), whereas lower panel shows staining of TUJ-1 (green) and ISLET1 (red). The scale bars equal 50 μM in both panels. Figure S7 Addition of 17β-estradiol in RGC differentiation medium promoted generation of OPA1 +/− -RGCs (OL). Putative RGCs were incubated with 100 nM 17β-estradiol in the cell culture medium during all of the differentiation stages, followed by fixation on day 31. Upper panel shows IF staining against TUJ1 (green) and BRN3a (red). Lower panel shows TUJ1 (green) and ISLET-1 (red) staining. The scale bars equal 50 μM. (PPTX 12904 kb

TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model

<p>Abstract</p> <p>Background</p> <p>The T-box transcription factor TBX3 is necessary for early embryonic development and for the normal development of the mammary gland. Homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. In humans, mutations that result in the haploinsufficiency of TBX3 causes Ulnar Mammary Syndrome (UMS) characterized by mammary gland hypoplasia as well as other congenital defects. In addition to its role in mammary gland development, various studies have also supported a role for Tbx3 in breast cancer development. TBX3 is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. Previous studies have suggested that TBX3 contributes to cancer development by its ability to bypass senescence by repressing the expression of p14^ARF-tumor suppressor. Although many studies have shown that a dysregulation of TBX3 expression may contribute to cancer progression, no direct evidence shows TBX3 causes breast cancer.</p> <p>Results</p> <p>In this study, we created doxycycline inducible double transgenic mice (MMTV-rtTA;tet-myc-TBX3-IRES-Luciferase) to test whether TBX3 over-expression can induce tumor formation within the mammary gland. Although over-expression of TBX3, alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. We also show that TBX3 directly binds to and represses <it>NFκBIB</it>, an inhibitor of the NF-κB pathway known to play a role in regulating cell proliferation. Lastly, we also show that the over-expression of TBX3 is associated with an increase in mammary stem-like cells.</p> <p>Conclusions</p> <p>Overall, our data suggests that over-expression of TBX3 may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the NF-κB pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.</p

The Molecular Mechanisms of OPA1-Mediated Optic Atrophy in Drosophila Model and Prospects for Antioxidant Treatment

Mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial protein, is the most common cause for autosomal dominant optic atrophy (DOA). The condition is characterized by gradual loss of vision, color vision defects, and temporal optic pallor. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, we analyzed phenotypes in the developing and adult Drosophila eyes produced by mutant dOpa1 (CG8479), a Drosophila ortholog of human OPA1. Heterozygous mutation of dOpa1 by a P-element or transposon insertions causes no discernable eye phenotype, whereas the homozygous mutation results in embryonic lethality. Using powerful Drosophila genetic techniques, we created eye-specific somatic clones. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning) and glossy (decreased lens and pigment deposition) eye phenotypes in adult flies; this phenotype was reversible by precise excision of the inserted P-element. Furthermore, we show the rough eye phenotype is caused by the loss of hexagonal lattice cells in developing eyes, suggesting an increase in lattice cell apoptosis. In adult flies, the dOpa1 mutation caused an increase in reactive oxygen species (ROS) production as well as mitochondrial fragmentation associated with loss and damage of the cone and pigment cells. We show that superoxide dismutase 1 (SOD1), Vitamin E, and genetically overexpressed human SOD1 (hSOD1) is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our results show dOpa1 mutations cause cell loss by two distinct pathogenic pathways. This study provides novel insights into the pathogenesis of optic atrophy and demonstrates the promise of antioxidants as therapeutic agents for this condition

Integrated analysis of the molecular pathogenesis of FDXR-associated disease.

The mitochondrial flavoprotein ferredoxin reductase (FDXR) is required for biogenesis of iron-sulfur clusters and for steroidogenesis. Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, and an increasing number of disorders are associated with disruptions in the synthesis of Fe-S clusters. Our previous studies have demonstrated that hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans and mice, attributed in part to reduced function of the electron transport chain (ETC) as well as elevated production of reactive oxygen species (ROS). Inflammation and peripheral neuropathy are also hallmarks of this disease. In this paper, we demonstrate that FDXR mutation leads to significant optic transport defects that are likely to underlie optic atrophy, a major clinical presentation in FDXR patients, as well as a neurodegenerative loss of cells in the central nervous system (CNS). Molecular analysis indicates that FDXR mutation also leads to mitochondrial iron overload and an associated depolarization of the mitochondrial membrane, further supporting the hypothesis that FDXR mutations cause neurodegeneration by affecting FDXR\u27s critical role in iron homeostasis

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among them recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, FRM patients often experience worsening or demise following stress associated with infections. We investigated two female FRM patients carrying the novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from three FRM patients' fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance

A novel RUNX2 missense mutation predicted to disrupt DNA binding causes cleidocranial dysplasia in a large Chinese family with hyperplastic nails

Background: Cleidocranial dysplasia (CCD) is a dominantly inherited disease characterized by hypoplastic or absent clavicles, large fontanels, dental dysplasia, and delayed skeletal development. The purpose of this study is to investigate the genetic basis of Chinese family with CCD. Methods: Here, a large Chinese family with CCD and hyperplastic nails was recruited. The clinical features displayed a significant intrafamilial variation. We sequenced the coding region of the RUNX2 gene for the mutation and phenotype analysis. Results: The family carries a c. T407C (p.L136P) mutation in the DNA- and CBF beta-binding Runt domain of RUNX2. Based on the crystal structure, we predict this novel missense mutation is likely to disrupt DNA binding by RUNX2, and at least locally affect the Runt domain structure. Conclusion: A novel missense mutation was identified in a large Chinese family with CCD with hyperplastic nails. This report further extends the mutation spectrum and clinical features of CCD. The identification of this mutation will facilitate prenatal diagnosis and preimplantation genetic diagnosis

Heterozygous Mutation of Drosophila Opa1 Causes the Development of Multiple Organ Abnormalities in an Age-Dependent and Organ-Specific Manner

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner

Heterozygous Mutation of Opa1 in Drosophila Shortens Lifespan Mediated through Increased Reactive Oxygen Species Production

Optic atrophy 1 (OPA1) is a dynamin-like GTPase located in the inner mitochondrial membrane and mutations in OPA1 are associated with autosomal dominant optic atrophy (DOA). OPA1 plays important roles in mitochondrial fusion, cristae remodeling and apoptosis. Our previous study showed that dOpa1 mutation caused elevated reactive oxygen species (ROS) production and resulted in damage and death of the cone and pigment cells in Drosophila eyes. Since ROS-induced oxidative damage to the cells is one of the primary causes of aging, in this study, we examined the effects of heterozygous dOpa1 mutation on the lifespan. We found that heterozygous dOpa1 mutation caused shortened lifespan, increased susceptibility to oxidative stress and elevated production of ROS in the whole Drosophila. Antioxidant treatment partially restored lifespan in the male dOpa1 mutants, but had no effects in the females. Heterozygous dOpa1 mutation caused an impairment of respiratory chain complex activities, especially complexes II and III, and reversible decreased aconitase activity. Heterozygous dOpa1 mutation is also associated with irregular and dysmorphic mitochondria in the muscle. Our results, for the first time, demonstrate the important role of OPA1 in aging and lifespan, which is most likely mediated through augmented ROS production