eIF2A-knockout mice reveal decreased life span and metabolic syndrome.

Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.

Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction.

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

Mutations in the mitochondrial ribosomal protein MRPS22 lead to primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is characterized by amenorrhea and loss or dysfunction of ovarian follicles prior to the age of 40. POI has been associated with autosomal recessive mutations in genes involving hormonal signaling and folliculogenesis, however, the genetic etiology of POI most often remains unknown. Here we report MRPS22 homozygous missense variants c.404G>A (p.R135Q) and c.605G>A (p.R202H) identified in four females from two independent consanguineous families as a novel genetic cause of POI in adolescents. Both missense mutations identified in MRPS22 are rare, occurred in highly evolutionarily conserved residues, and are predicted to be deleterious to protein function. In contrast to prior reports of mutations in MRPS22 associated with severe mitochondrial disease, the POI phenotype is far less severe. Consistent with this genotype-phenotype correlation, mitochondrial defects in oxidative phosphorylation or rRNA levels were not detected in fibroblasts derived from the POI patients, suggesting a non-bioenergetic or tissue-specific mitochondrial defect. Furthermore, we demonstrate in a Drosophila model that mRpS22 deficiency specifically in somatic cells of the ovary had no effect on fertility, whereas flies with mRpS22 deficiency specifically in germ cells were infertile and agametic, demonstrating a cell autonomous requirement for mRpS22 in germ cell development. These findings collectively identify that MRPS22, a component of the small mitochondrial ribosome subunit, is critical for ovarian development and may therefore provide insight into the pathophysiology and treatment of ovarian dysfunction.

Widespread epistasis regulates glucose homeostasis and gene expression.

The relative contributions of additive versus non-additive interactions in the regulation of complex traits remains controversial. This may be in part because large-scale epistasis has traditionally been difficult to detect in complex, multi-cellular organisms. We hypothesized that it would be easier to detect interactions using mouse chromosome substitution strains that simultaneously incorporate allelic variation in many genes on a controlled genetic background. Analyzing metabolic traits and gene expression levels in the offspring of a series of crosses between mouse chromosome substitution strains demonstrated that inter-chromosomal epistasis was a dominant feature of these complex traits. Epistasis typically accounted for a larger proportion of the heritable effects than those due solely to additive effects. These epistatic interactions typically resulted in trait values returning to the levels of the parental CSS host strain. Due to the large epistatic effects, analyses that did not account for interactions consistently underestimated the true effect sizes due to allelic variation or failed to detect the loci controlling trait variation. These studies demonstrate that epistatic interactions are a common feature of complex traits and thus identifying these interactions is key to understanding their genetic regulation.

Coordinated transcriptional control of adipocyte triglyceride lipase (Atgl) by transcription factors Sp1 and peroxisome proliferator-activated receptor γ (PPARγ) during adipocyte differentiation.

The breakdown of stored fat deposits into its components is a highly regulated process that maintains plasma levels of free fatty acids to supply energy to cells. Insulin-mediated transcription of Atgl, the enzyme that mediates the rate-limiting step in lipolysis, is a key point of this regulation. Under conditions such as obesity or insulin resistance, Atgl transcription is often misregulated, which can contribute to overall disease progression. The mechanisms by which Atgl is induced during adipogenesis are not fully understood. We utilized computational approaches to identify putative transcriptional regulatory elements in Atgl and then tested the effect of these elements and the transcription factors that bind to them in cultured preadipocytes and mature adipocytes. Here we report that Atgl is down-regulated by the basal transcription factor Sp1 in preadipocytes and that the magnitude of down-regulation depends on interactions between Sp1 and peroxisome proliferator-activated receptor γ (PPARγ). In mature adipocytes, when PPARγ is abundant, PPARγ abrogated transcriptional repression by Sp1 at the Atgl promoter and up-regulated Atgl mRNA expression. Targeting the PPARγ-Sp1 interaction could be a potential therapeutic strategy to restore insulin sensitivity by modulating Atgl levels in adipocytes.

Contrasting genetic architectures in different mouse reference populations used for studying complex traits.

Quantitative trait loci (QTLs) are being used to study genetic networks, protein functions, and systems properties that underlie phenotypic variation and disease risk in humans, model organisms, agricultural species, and natural populations. The challenges are many, beginning with the seemingly simple tasks of mapping QTLs and identifying their underlying genetic determinants. Various specialized resources have been developed to study complex traits in many model organisms. In the mouse, remarkably different pictures of genetic architectures are emerging. Chromosome Substitution Strains (CSSs) reveal many QTLs, large phenotypic effects, pervasive epistasis, and readily identified genetic variants. In contrast, other resources as well as genome-wide association studies (GWAS) in humans and other species reveal genetic architectures dominated with a relatively modest number of QTLs that have small individual and combined phenotypic effects. These contrasting architectures are the result of intrinsic differences in the study designs underlying different resources. The CSSs examine context-dependent phenotypic effects independently among individual genotypes, whereas with GWAS and other mouse resources, the average effect of each QTL is assessed among many individuals with heterogeneous genetic backgrounds. We argue that variation of genetic architectures among individuals is as important as population averages. Each of these important resources has particular merits and specific applications for these individual and population perspectives. Collectively, these resources together with high-throughput genotyping, sequencing and genetic engineering technologies, and information repositories highlight the power of the mouse for genetic, functional, and systems studies of complex traits and disease models.

Transgenerational inheritance of metabolic disease.

Metabolic disease encompasses several disorders including obesity, type 2 diabetes, and dyslipidemia. Recently, the incidence of metabolic disease has drastically increased, driven primarily by a worldwide obesity epidemic. Transgenerational inheritance remains controversial, but has been proposed to contribute to human metabolic disease risk based on a growing number of proof-of-principle studies in model organisms ranging from Caenorhabditis elegans to Mus musculus to Sus scrofa. Collectively, these studies demonstrate that heritable risk is epigenetically transmitted from parent to offspring over multiple generations in the absence of a continued exposure to the triggering stimuli. A diverse assortment of initial triggers can induce transgenerational inheritance including high-fat or high-sugar diets, low-protein diets, various toxins, and ancestral genetic variants. Although the mechanistic basis underlying the transgenerational inheritance of disease risk remains largely unknown, putative molecules mediating transmission include small RNAs, histone modifications, and DNA methylation. Due to the considerable impact of metabolic disease on human health, it is critical to better understand the role of transgenerational inheritance of metabolic disease risk to open new avenues for therapeutic intervention and improve upon the current methods for clinical diagnoses and treatment.

Zinc finger protein 407 (ZFP407) regulates insulin-stimulated glucose uptake and glucose transporter 4 (Glut4) mRNA.

The glucose transporter GLUT4 facilitates insulin-stimulated glucose uptake in peripheral tissues including adipose, muscle, and heart. GLUT4 function is impaired in obesity and type 2 diabetes leading to hyperglycemia and an increased risk of cardiovascular disease and neuropathy. To better understand the regulation of GLUT4 function, a targeted siRNA screen was performed and led to the discovery that ZFP407 regulates insulin-stimulated glucose uptake in adipocytes. The decrease in insulin-stimulated glucose uptake due to ZFP407 deficiency was attributed to a reduction in GLUT4 mRNA and protein levels. The decrease in GLUT4 was due to both decreased transcription of Glut4 mRNA and decreased efficiency of Glut4 pre-mRNA splicing. Interestingly, ZFP407 coordinately regulated this decrease in transcription with an increase in the stability of Glut4 mRNA, resulting in opposing effects on steady-state Glut4 mRNA levels. More broadly, transcriptome analysis revealed that ZFP407 regulates many peroxisome proliferator-activated receptor (PPAR) γ target genes beyond Glut4. ZFP407 was required for the PPARγ agonist rosiglitazone to increase Glut4 expression, but was not sufficient to increase expression of a PPARγ target gene reporter construct. However, ZFP407 and PPARγ co-overexpression synergistically activated a PPARγ reporter construct beyond the level of PPARγ alone. Thus, ZFP407 may represent a new modulator of the PPARγ signaling pathway.

Zinc finger protein 407 overexpression upregulates PPAR target gene expression and improves glucose homeostasis in mice.

The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors is central to the pathophysiology and treatment of metabolic disease through the receptors' ability to regulate the expression of genes involved in glucose homeostasis, adipogenesis, and lipid metabolism. However, the mechanism by which PPAR is regulated remains incompletely understood. We generated a transgenic mouse strain (ZFP-TG) that overexpressed Zfp407 primarily in muscle and heart. Transcriptome analysis by RNA-Seq identified 1,300 differentially expressed genes in the muscle of ZFP-TG mice, among which PPAR target genes were significantly enriched. Among the physiologically important PPARγ target genes, Glucose transporter (Glut)-4 mRNA and protein levels were increased in heart and muscle. The increase in Glut4 and other transcriptional effects of Zfp407 overexpression together decreased body weight and lowered plasma glucose, insulin, and HOMA-IR scores relative to control littermates. When placed on high-fat diet, ZFP-TG mice remained more glucose tolerant than their wild-type counterparts. Cell-based assays demonstrated that Zfp407 synergistically increased the transcriptional activity of all PPAR subtypes, PPARα, PPARγ, and PPARδ. The increased PPAR activity was not associated with increased PPAR mRNA or protein levels, suggesting that Zfp407 posttranslationally regulates PPAR activity. Collectively, these results demonstrate that Zfp407 overexpression improved glucose homeostasis. Thus, Zfp407 represents a new drug target for treating metabolic disease.

Deep congenic analysis identifies many strong, context-dependent QTLs, one of which, Slc35b4, regulates obesity and glucose homeostasis.

Although central to many studies of phenotypic variation and disease susceptibility, characterizing the genetic architecture of complex traits has been unexpectedly difficult. For example, most of the susceptibility genes that contribute to highly heritable conditions such as obesity and type 2 diabetes (T2D) remain to be identified despite intensive study. We took advantage of mouse models of diet-induced metabolic disease in chromosome substitution strains (CSSs) both to characterize the genetic architecture of diet-induced obesity and glucose homeostasis and to test the feasibility of gene discovery. Beginning with a survey of CSSs, followed with genetic and phenotypic analysis of congenic, subcongenic, and subsubcongenic strains, we identified a remarkable number of closely linked, phenotypically heterogeneous quantitative trait loci (QTLs) on mouse chromosome 6 that have unexpectedly large phenotypic effects. Although fine-mapping reduced the genomic intervals and gene content of these QTLs over 3000-fold, the average phenotypic effect on body weight was reduced less than threefold, highlighting the "fractal" nature of genetic architecture in mice. Despite this genetic complexity, we found evidence for 14 QTLs in only 32 recombination events in less than 3000 mice, and with an average of four genes located within the three body weight QTLs in the subsubcongenic strains. For Obrq2a1, genetic and functional studies collectively identified the solute receptor Slc35b4 as a regulator of obesity, insulin resistance, and gluconeogenesis. This work demonstrated the unique power of CSSs as a platform for studying complex genetic traits and identifying QTLs.

Cell-state dependent regulation of PPAR γ signaling by ZBTB9 in adipocytes.

Adipocytes play a critical role in metabolic homeostasis. Peroxisome proliferator-activated receptor- γ (PPAR γ ) is a nuclear hormone receptor that is a master regulator of adipocyte differentiation and function. ZBTB9 was predicted to interact with PPAR γ based on large-scale protein interaction experiments. In addition, GWAS studies in the type 2 diabetes (T2D) Knowledge Portal revealed associations between Z btb9 and both BMI and T2D risk. Here we show that ZBTB9 positively regulates PPAR γ activity in mature adipocytes. Surprisingly Z btb9 knockdown (KD) also increased adipogenesis in 3T3-L1 cells and human preadipocytes. E2F activity was increased and E2F downstream target genes were upregulated in Zbtb9 -KD preadipocytes. Accordingly, RB phosphorylation, which regulates E2F activity, was enhanced in Zbtb9 -KD preadipocytes. Critically, an E2F1 inhibitor blocked the effects of Zbtb9 deficiency on adipogenic gene expression and lipid accumulation. Collectively, these results demonstrate that Zbtb9 inhibits adipogenesis as a negative regulator of Pparg expression via altered RB-E2F1 signaling. Our findings reveal complex cell-state dependent roles of ZBTB9 in adipocytes, identifying a new molecule that regulates adipogenesis and adipocyte biology as both a positive and negative regulator of PPAR γ signaling depending on the cellular context, and thus may be important in the pathogenesis and treatment of obesity and T2D.

Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407.

Cofactors interacting with PPARγ can regulate adipogenesis and adipocyte metabolism by modulating the transcriptional activity and selectivity of PPARγ signaling. ZFP407 was previously demonstrated to regulate PPARγ target genes such as GLUT4, and its overexpression improved glucose homeostasis in mice. Here, using a series of molecular assays, including protein-interaction studies, mutagenesis, and ChIP-seq, ZFP407 was found to interact with the PPARγ/RXRα protein complex in the nucleus of adipocytes. Consistent with this observation, ZFP407 ChIP-seq peaks significantly overlapped with PPARγ ChIP-seq peaks, with more than half of ZFP407 peaks overlapping with PPARγ peaks. Transcription factor binding motifs enriched in these overlapping sites included CTCF, RARα/RXRγ, TP73, and ELK1, which regulate cellular development and function within adipocytes. Site-directed mutagenesis of frequent PPARγ phosphorylation or SUMOylation sites did not prevent its regulation by ZFP407, while mutagenesis of ZFP407 domains potentially necessary for RXR and PPARγ binding abrogated any impact of ZFP407 on PPARγ activity. These data suggest that ZFP407 controls the activity of PPARγ, but does so independently of post-translational modifications, likely by direct binding, establishing ZFP407 as a newly identified PPARγ cofactor. In addition, ZFP407 ChIP-seq analyses identified regions that did not overlap with PPARγ peaks. These non-overlapping peaks were significantly enriched for the transcription factor binding motifs of TBX19, PAX8, HSF4, and ZKSCAN3, which may contribute to the PPARγ-independent functions of ZFP407 in adipocytes and other cell types.

Ancestral paternal genotype controls body weight and food intake for multiple generations.

Current treatments have largely failed to slow the rapidly increasing world-wide prevalence of obesity and its co-morbidities. Despite a strong genetic contribution to obesity (40-70%), only a small percentage of heritability is explained with current knowledge of monogenic abnormalities, common sequence variants and conventional modes of inheritance. Epigenetic effects are rarely tested in humans because of difficulties arranging studies that distinguish conventional and transgenerational inheritance while simultaneously controlling environmental factors and learned behaviors. However, growing evidence from model organisms implicates genetic and environmental factors in one generation that affect phenotypes in subsequent generations. In this report, we provide the first evidence for paternal transgenerational genetic effects on body weight and food intake. This test focused on the obesity-resistant 6C2d congenic strain, which carries the Obrq2a(A/J) allele on an otherwise C57BL/6J background. Various crosses between 6C2d and the control C57BL/6J strain showed that the Obrq2a(A/J) allele in the paternal or grandpaternal generation was sufficient to inhibit diet-induced obesity and reduce food intake in the normally obesity-susceptible, high food intake C57BL/6J strain. These obesity-resistant and reduced food intake phenotypes were transmitted through the paternal lineage but not the maternal lineage with equal strength for at least two generations. Eliminating social interaction between the father and both his offspring and the pregnant dam did not significantly affect food intake levels, demonstrating that the phenotype is transmitted through the male germline rather than through social interactions. Persistence of these phenotypes across multiple generations raises the possibility that transgenerational genetic effects contribute to current metabolic conditions.

The juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity.

Despite considerable effort, the identification of genes that regulate complex multigenic traits such as obesity has proven difficult with conventional methodologies. The use of a chromosome substitution strain-based mapping strategy based on deep congenic analysis overcame many of the difficulties associated with gene discovery and led to the finding that the juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity. The effects of a mild Cntnap2 mutation on body weight were highly dependent on genetic background, as both obesity-promoting and obesity-resistant effects of Cntnap2 were observed on different genetic backgrounds. The more severe effect of complete TAG1 deficiency, by decreasing food intake, completely prevented the weight gain normally associated with high-fat-diet feeding. Together, these studies implicate two novel proteins in the regulation of diet-induced obesity. Moreover, as juxtaparanodal proteins have previously been implicated in various neurological disorders, our results suggest a potential genetic and molecular link between obesity and diseases such as autism and epilepsy.

Low-Dose Anti-HIV Drug Efavirenz Mitigates Retinal Vascular Lesions in a Mouse Model of Alzheimer's Disease.

A small dose of the anti-HIV drug efavirenz (EFV) was previously discovered to activate CYP46A1, a cholesterol-eliminating enzyme in the brain, and mitigate some of the manifestation of Alzheimer's disease in 5XFAD mice. Herein, we investigated the retina of these animals, which were found to have genetically determined retinal vascular lesions associated with deposits within the retinal pigment epithelium and subretinal space. We established that EFV treatment activated CYP46A1 in the retina, enhanced retinal cholesterol turnover, and diminished the lesion frequency >5-fold. In addition, the treatment mitigated fluorescein leakage from the aberrant blood vessels, deposit size, activation of retinal macrophages/microglia, and focal accumulations of amyloid ß plaques, unesterified cholesterol, and Oil Red O-positive lipids. Studies of retinal transcriptomics and proteomics identified biological processes enriched with differentially expressed genes and proteins. We discuss the mechanisms of the beneficial EFV effects on the retinal phenotype of 5XFAD mice. As EFV is an FDA-approved drug, and we already tested the safety of small-dose EFV in patients with Alzheimer's disease, our data support further clinical investigation of this drug in subjects with retinal vascular lesions or neovascular age-related macular degeneration.

Diet-induced hepatocellular carcinoma in genetically predisposed mice.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with approximately 70% of cases resulting from hepatitis B and C viral infections, aflatoxin exposure, chronic alcohol use or genetic liver diseases. The remaining approximately 30% of cases are associated with obesity, type 2 diabetes and related metabolic diseases, although a direct link between these pathologies and HCCs has not been established. We tested the long-term effects of high-fat and low-fat diets on males of two inbred strains of mice and discovered that C57BL/6J but not A/J males were susceptible to non-alcoholic steatohepatitis (NASH) and HCC on a high-fat but not low-fat diet. This strain-diet interaction represents an important model for genetically controlled, diet-induced HCC. Susceptible mice showed morphological characteristics of NASH (steatosis, hepatitis, fibrosis and cirrhosis), dysplasia and HCC. mRNA profiles of HCCs versus tumor-free liver showed involvement of two signaling networks, one centered on Myc and the other on NFkappaB, similar to signaling described for the two major classes of HCC in humans. miRNA profiles revealed dramatically increased expression of a cluster of miRNAs on the X chromosome without amplification of the chromosomal segment. A switch from high-fat to low-fat diet reversed these outcomes, with switched C57BL/6J males being lean rather than obese and without evidence for NASH or HCCs at the end of the study. A similar diet modification may have important implications for prevention of HCCs in humans.

Adipocyte-specific deletion of zinc finger protein 407 results in lipodystrophy and insulin resistance in mice.

PPARγ deficiency in humans and model organisms impairs the transcriptional control of adipogenesis and mature adipocyte function resulting in lipodystrophy and insulin resistance. Zinc finger protein 407 (ZFP407) positively regulates PPARγ target gene expression and insulin-stimulated glucose uptake in cultured adipocytes. The in vivo physiological role of ZFP407 in mature adipocytes, however, remains to be elucidated. Here we generated adipocyte-specific ZFP407 knockout (AZKO) mice and discovered a partial lipodystrophic phenotype with reduced fat mass, hypertrophic adipocytes in inguinal and brown adipose tissue, and reduced adipogenic gene expression. The lipodystrophy was further exacerbated in AZKO mice fed a high-fat diet. Glucose and insulin tolerance tests revealed decreased insulin sensitivity in AZKO mice compared to control littermates. Cell-based assays demonstrated that ZFP407 is also required for adipogenesis, which may also contribute to the lipodystrophic phenotype. These results demonstrate an essential in vivo role of ZFP407 in brown and white adipose tissue formation and organismal insulin sensitivity.

Engineered nasal cartilage for the repair of osteoarthritic knee cartilage defects.

Osteoarthritis (OA) is the most prevalent joint disorder, causing pain and disability predominantly in the aging population but also affecting young individuals. Current treatments are limited to use of anti-inflammatory drugs to alleviate symptoms or degenerated joint replacement by a prosthetic implant at the end stage of the disease. We hypothesized that degenerative cartilage defects can be treated using nasal chondrocyte­based tissue-engineered cartilage (N-TEC). We demonstrate that N-TEC maintained cartilaginous properties when exposed in vitro to inflammatory stimuli found in osteoarthritic joints and favorably altered the inflammatory profile of cells from osteoarthritic joints. These effects were at least partially mediated by down-regulation of the WNT (wingless/integrated) signaling pathway through sFRP1 (secreted frizzled-related protein-1). We further report that N-TEC survive and engraft in vivo in ectopic mouse models reproducing a human osteochondral OA tissue environment, as well as in sheep articular cartilage defects that mimic degenerative settings. Last, we tested the safety of autologous N-TEC for the treatment of osteoarthritic cartilage defects in the knees of two patients with advanced OA (Kellgren and Lawrence grades 3 and 4) who were otherwise considered for unicondylar knee arthroplasty. No adverse reactions were recorded, and patients reported reduced pain as well as improved joint function and life quality 14 months after surgery. Together, our findings indicate that N-TEC can directly contribute to cartilage repair in osteoarthritic joints. A suitably powered clinical trial is now required to assess its efficacy in the treatment of patients with OA.

PI(3,4)P2-mediated cytokinetic abscission prevents early senescence and cataract formation.

Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)­dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2­binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell type­specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.

The ataxia3 mutation in the N-terminal cytoplasmic domain of sodium channel Na(v)1.6 disrupts intracellular trafficking.

The ENU-induced neurological mutant ataxia3 was mapped to distal mouse chromosome 15. Sequencing of the positional candidate gene Scn8a encoding the sodium channel Na(v)1.6 identified a T>C transition in exon 1 resulting in the amino acid substitution p.S21P near the N terminus of the channel. The cytoplasmic N-terminal region is evolutionarily conserved but its function has not been well characterized. ataxia3 homozygotes exhibit a severe disorder that includes ataxia, tremor, and juvenile lethality. Unlike Scn8a null mice, they retain partial hindlimb function. The mutant transcript is stable but protein abundance is reduced and the mutant channel is not detected in its usual site of concentration at nodes of Ranvier. In whole-cell patch-clamp studies of transfected ND7/23 cells that were maintained at 37 degrees C, the mutant channel did not produce sodium current, and function was not restored by coexpression of beta1 and beta2 subunits. However, when transfected cells were maintained at 30 degrees C, the mutant channel generated voltage-dependent inward sodium currents with an average peak current density comparable with wild type, demonstrating recovery of channel activity. Immunohistochemistry of primary cerebellar granule cells from ataxia3 mice demonstrated that the mutant protein is retained in the cis-Golgi. This trafficking defect can account for the low level of Na(v)1.6-S21P at nodes of Ranvier in vivo and at the surface of transfected cells. The data demonstrate that the cytoplasmic N-terminal domain of the sodium channel is required for anterograde transport from the Golgi complex to the plasma membrane.