Duska J. Sidjanin

Loss-of-Function Mutations in TBC1D20 Cause Cataracts and Male Infertility in blind sterile Mice and Warburg Micro Syndrome in Humans

blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology.

Blind sterile 2 (bs2), a hypomorphic mutation in Agps, results in cataracts and male sterility in mice

Blind sterile 2 (bs2) is a spontaneous autosomal recessive mouse mutation exhibiting cataracts and male sterility. Detailed clinical and histological evaluation revealed that bs2 mice have cataracts resulting from severely disrupted lens fiber cells. Analysis of bs2 testes revealed the absence of mature sperm and the presence of large multinucleate cells within the lumens of seminiferous tubules. Linkage analysis mapped the bs2 locus to mouse chromosome 2, approximately 45cM distal from the centromere. Fine mapping established a 3.1Mb bs2 critical region containing 19 candidate genes. Sequence analysis of alkylglycerone-phosphate synthase (Agps), a gene within the bs2 critical region, revealed a G to A substitution at the +5 position of intron 14. This mutation results in two abundantly expressed aberrantly spliced Agps transcripts: Agps(∆exon14) lacking exon 14 or Agps(exon∆13-14) lacking both exons 13 and 14 as well as full-length Agps transcript. Agps is a peroxisomal enzyme which catalyzes the formation of the ether bond during the synthesis of ether lipids. Both aberrantly spliced Agps(∆exon14) and Agps(exon∆13-14) transcripts led to a frame shift, premature stop and putative proteins lacking the enzymatic FAD domain. We present evidence that bs2 mice have significantly decreased levels of ether lipids. Human mutations in Agps result in rhizomelic chondrodysplasia punctata type 3 (RCDP3), a disease for which bs2 is the only genetic model. Thus, bs2 is a hypomorphic mutation in Agps, and represents a useful model for investigation of the tissue specificity of ether lipid requirements which will be particularly valuable for elucidating the mechanism of disease phenotypes resulting from ether lipid depletion.

Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation

RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary ‘RAB3GAP’ complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.

Waved with open eyelids 2 (woe2) is a novel spontaneous mouse mutation in the protein phosphatase 1, regulatory (inhibitor) subunit 13 like (Ppp1r13l) gene

BackgroundWaved with open eyelids 2 (woe2) is a novel autosomal recessive mouse mutation that arose spontaneously in our animal facility. Upon initial evaluation, mutant mice exhibited eyelids open at birth (EOB) and wavy fur phenotypes. The goals of this study were to phenotypically characterize the woe2 mice and to identify the gene harboring the mutation responsible for the woe2 phenotype.ResultsHistological analysis of woe2 embryos identified the failure of embryonic eyelid closure. Clinical and histological analysis of woe2 adult eyes identified severe corneal opacities, abnormalities of the anterior segment of the eye, and the absence of meibomian glands. Abnormalities in the fur texture and the absence of meibomian glands prompted us to evaluate other epidermal appendages: skin, teeth, and nails--as well as lacrimal, mammary, salivary, sebaceous and sweat glands. No obvious morphological differences between WT and woe2 mice were identified in these tissues. However, the analysis of woe2 identified cardiac abnormalities. Positional cloning of the woe2 locus identified a 1308 bp deletion in the Ppp1r13l gene. The deletion resulted in an aberrant Ppp1r13lΔexon9-11 transcript that lacks exons 9, 10 and 11 resulting in a premature stop and a loss of 223 amino acids from the C-terminal end of the putative mutant PPP1R13L protein. Immunohistological analysis during eye development identified expression of PPP1R13L in the palpebral epidermis, palpebral and bulbar conjunctiva, corneal epithelium and meibomian glands.ConclusionsThe woe2 mouse harbors a novel deletion within the Ppp1r13l gene, likely resulting in a complete loss of PPP1R13L function. Results from this study provide evidence that PPP1R13L has an essential role in embryonic eyelid closure as well in development of meibomian glands and the anterior segment of the eye. The woe2 mice are a useful model for investigation of the role of PPP1R13L, especially during ocular and eyelid development.

Targeted disruption of Tbc1d20 with zinc-finger nucleases causes cataracts and testicular abnormalities in mice.

BackgroundLoss-of-function mutations in TBC1D20 cause Warburg Micro syndrome 4 (WARBM4), which is an autosomal recessive syndromic disorder characterized by eye, brain, and genital abnormalities. Blind sterile (bs) mice carry a Tbc1d20-null mutation and exhibit cataracts and testicular phenotypes similar to those observed in WARBM4 patients. In addition to TBC1D20, mutations in RAB3GAP1, RAB3GAP2 and RAB18 cause WARBM1-3 respectively. However, regardless of which gene harbors the causative mutation, all individuals affected with WARBM exhibit indistinguishable clinical presentations. In contrast, bs, Rab3gap1-/-, and Rab18-/- mice exhibit distinct phenotypes; this phenotypic variability of WARBM mice was previously attributed to potential compensatory mechanisms. Rab3gap1-/- and Rab18-/- mice were genetically engineered using standard approaches, whereas the Tbc1d20 mutation in the bs mice arose spontaneously. There is the possibility that another unidentified mutation within the bs linkage disequilibrium may be contributing to the bs phenotypes and thus contributing to the phenotypic variability in WARBM mice. The goal of this study was to establish the phenotypic consequences in mice caused by the disruption of the Tbc1d20 gene.ResultsThe zinc finger nuclease (ZFN) mediated genomic editing generated a Tbc1d20 c.[418_426del] deletion encoding a putative TBC1D20-ZFN protein with an in-frame p.[H140_Y143del] deletion within the highly conserved TBC domain. The evaluation of Tbc1d20ZFN/ZFN eyes identified severe cataracts and thickened pupillary sphincter muscle. Tbc1d20ZFN/ZFN males are infertile and the analysis of the seminiferous tubules identified disrupted acrosomal development. The compound heterozygote Tbc1d20ZFN/bs mice, generated from an allelic bs/+ X Tbc1d20ZFN/+ cross, exhibited cataracts and aberrant acrosomal development indicating a failure to complement.ConclusionsOur findings show that the disruption of Tbc1d20 in mice results in cataracts and aberrant acrosomal formation, thus establishing bs and Tbc1d20ZFN/ZFN as allelic variants. Although the WARBM molecular disease etiology remains unclear, both the bs and Tbc1d20ZFN/ZFN mice are excellent model organisms for future studies to establish TBC1D20-mediated molecular and cellular functions.

Functional analysis of HSF4 mutations found in patients with autosomal recessive congenital cataracts.

PURPOSE The goal of this study was to functionally evaluate three previously uncharacterized heat shock factor protein 4 (HSF4) mutations (c.595_599delGGGCC, c.1213C>T, c.1327+4A>G) encoding mutant HSF4 proteins (G199EfsX15, R405X, and M419GfsX29) with missing C-terminal ends. These HSF4 mutations were previously identified in families with congenital autosomal recessive cataracts. METHODS FLAG-tagged recombinant wild type (WT) and mutant HSF4 proteins were analyzed using the protein stability assay, cellular immunofluorescence, Western blotting, electrophoretic mobility shift assay (EMSA), and reporter activation. RESULTS HSF4 mutant proteins did not differ in the protein turnover rate when compared with WT HSF4. Immunofluorescence revealed that WT and mutant HSF4 proteins were properly trafficked to the nucleus. EMSA analysis revealed that the G199EfsX15 and M419GfsX29 proteins exhibited decreased heat shock element (HSE)-mediated DNA binding, whereas the R405X mutant exhibited increased HSE-mediated DNA binding when compared with WT HSF4. All three HSF4 mutant proteins exhibited abolished HSE-mediated luciferase reporter activation. Detailed evaluation of the C-terminal region identified three novel domains: two activation domains and one repression domain. CONCLUSIONS The three HSF4 autosomal recessive mutations evaluated here result in a loss of HSF4 function due to a loss of regulatory domains present at the C-terminal end. These findings collectively indicate that the transcriptional activation of HSF4 is mediated by interactions between activator and repressor domains within the C-terminal end.

ADAM17 Transactivates EGFR Signaling during Embryonic Eyelid Closure

PURPOSE During mammalian embryonic eyelid closure ADAM17 has been proposed to play a role as a transactivator of epidermal growth factor receptor (EGFR) signaling by shedding membrane bound EGFR ligands. However, ADAM17 also sheds numerous other ligands, thus implicating ADAM17 in additional molecular pathways. The goal of this study was to experimentally establish the role of ADAM17 and determine ADAM17-mediated pathways essential for the embryonic eyelid closure. METHODS Wild-type (WT) and woe mice, carrying a hypomorphic mutation in Adam17, were evaluated using H&E and scanning electron microscopy. Expressions of ADAM17, EGFR, and the phosphorylated form EGFR-P were evaluated using immunohistochemistry. BrdU and TUNEL assays were used to evaluate cell proliferation and apoptosis, respectively. In vitro scratch assays of primary cultures were used to evaluate cell migration. Clinical and histologic analyses established if the hypermorphic Egfr(Dsk5) allele can rescue the woe embryonic eyelid closure. RESULTS woe mice exhibited a failure to develop the leading edge of the eyelid and consequently failure of the embryonic eyelid closure. Expression of ADAM17 was identified in the eyelid epithelium in the cells of the leading edge. ADAM17 is essential for epithelial cell migration, but does not play a role in proliferation and apoptosis. EGFR was expressed in both WT and woe eyelid epithelium, but the phosphorylated EGFR-P form was detected only in WT. The Egfr(Dsk5) allele rescued woe eyelid closure defects, but also rescued woe anterior segment defects and the absence of meibomian glands. CONCLUSIONS We provide in vivo genetic evidence that the role of ADAM17 during embryonic eyelid closure is to transactivate EGFR signaling.

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation

ABSTRACT In humans, loss of TBC1D20 (TBC1 domain family, member 20) protein function causes Warburg Micro syndrome 4 (WARBM4), an autosomal recessive disorder characterized by congenital eye, brain, and genital abnormalities. TBC1D20-deficient mice exhibit ocular abnormalities and male infertility. TBC1D20 is a ubiquitously expressed member of the family of GTPase-activating proteins (GAPs) that increase the intrinsically slow GTP-hydrolysis rate of small RAB-GTPases when bound to GTP. Biochemical studies have established TBC1D20 as a GAP for RAB1B and RAB2A. However, the cellular role of TBC1D20 still remains elusive, and there is little information about how the functional loss of TBC1D20 causes clinical manifestations in WARBM4-affected children. Here we evaluate the role of TBC1D20 in cells carrying a null mutant allele, as well as TBC1D20-deficient mice, which display eye and testicular abnormalities. We demonstrate that TBC1D20, via its RAB1B GAP function, is a key regulator of autophagosome maturation, a process required for maintenance of autophagic flux and degradation of autophagic cargo. Our results provide evidence that TBC1D20-mediated autophagosome maturation maintains lens transparency by mediating the removal of damaged proteins and organelles from lens fiber cells. Additionally, our results show that in the testes TBC1D20-mediated maturation of autophagosomes is required for autophagic flux, but is also required for the formation of acrosomes. Furthermore TBC1D20-deficient mice, while not mimicking severe developmental brain abnormalities identified in WARBM4 affected children, display disrupted neuronal autophagic flux resulting in adult-onset motor dysfunction. In summary, we show that TBC1D20 has an essential role in the maturation of autophagosomes and a defect in TBC1D20 function results in eye, testicular, and neuronal abnormalities in mice implicating disrupted autophagy as a mechanism that contributes to WARBM4 pathogenesis.

A Disintegrin and Metalloproteinase10 (ADAM10) Regulates NOTCH Signaling during Early Retinal Development

ADAM10 and ADAM17 are two closely related members of the ADAM (a disintegrin and metalloprotease) family of membrane-bound sheddases, which proteolytically cleave surface membrane proteins. Both ADAM10 and ADAM17 have been implicated in the proteolytic cleavage of NOTCH receptors and as such regulators of NOTCH signaling. During retinal development, NOTCH signaling facilitates retinal neurogenesis by maintaining progenitor cells in a proliferative state and by mediating retinal cell fates. However, the roles of ADAM10 and ADAM17 in the retina are not well defined. In this study, we set out to clarify the roles of ADAM10 and ADAM17 during early retinal development. The retinal phenotype of conditionally abated Adam17 retinae (Adam17 CKO) did not differ from the controls whereas conditionally ablated Adam10 retinae (Adam10 CKO) exhibited abnormal morphogenesis characterized by the formation of rosettes and a loss of retinal laminae phenotypically similar to morphological abnormalities identified in mice with retinal NOTCH signaling deficiency. Additionally, Adam10 CKO retinae exhibited abnormal neurogenesis characterized by fewer proliferating progenitor cells and greater differentiation of early photoreceptors and retinal ganglion cells. Moreover, constitutive activation of the NOTCH1-intracellular domain (N1-ICD) rescued Adam10 CKO abnormal neurogenesis, as well as abnormal retinal morphology by maintaining retinal cells in the progenitor state. Collectively these findings provide in vivo genetic evidence that ADAM10, and not ADAM17, is indispensable for proper retinal development as a regulator of NOTCH signaling.

Type I Interferon Counteracts Antiviral Effects of Statins in the Context of Gammaherpesvirus Infection

ABSTRACT The cholesterol synthesis pathway is a ubiquitous cellular biosynthetic pathway that is attenuated therapeutically by statins. Importantly, type I interferon (IFN), a major antiviral mediator, also depresses the cholesterol synthesis pathway. Here we demonstrate that attenuation of cholesterol synthesis decreases gammaherpesvirus replication in primary macrophages in vitro and reactivation from peritoneal exudate cells in vivo. Specifically, the reduced availability of the intermediates required for protein prenylation was responsible for decreased gammaherpesvirus replication in statin-treated primary macrophages. We also demonstrate that statin treatment of a chronically infected host attenuates gammaherpesvirus latency in a route-of-infection-specific manner. Unexpectedly, we found that the antiviral effects of statins are counteracted by type I IFN. Our studies suggest that type I IFN signaling counteracts the antiviral nature of the subdued cholesterol synthesis pathway and offer a novel insight into the utility of statins as antiviral agents. IMPORTANCE Statins are cholesterol synthesis inhibitors that are therapeutically administered to 12.5% of the U.S. population. Statins attenuate the replication of diverse viruses in culture; however, this attenuation is not always obvious in an intact animal model. Further, it is not clear whether statins alter parameters of highly prevalent chronic herpesvirus infections. We show that statin treatment attenuated gammaherpesvirus replication in primary immune cells and during chronic infection of an intact host. Further, we demonstrate that type I interferon signaling counteracts the antiviral effects of statins. Considering the fact that type I interferon decreases the activity of the cholesterol synthesis pathway, it is intriguing to speculate that gammaherpesviruses have evolved to usurp the type I interferon pathway to compensate for the decreased cholesterol synthesis activity.