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.

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

BACKGROUND: Loss-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. RESULTS: The 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 Tbc1d20 (ZFN/ZFN) eyes identified severe cataracts and thickened pupillary sphincter muscle. Tbc1d20 (ZFN/ZFN) males are infertile and the analysis of the seminiferous tubules identified disrupted acrosomal development. The compound heterozygote Tbc1d20 (ZFN/bs) mice, generated from an allelic bs/+ X Tbc1d20 (ZFN/+) cross, exhibited cataracts and aberrant acrosomal development indicating a failure to complement. CONCLUSIONS: Our findings show that the disruption of Tbc1d20 in mice results in cataracts and aberrant acrosomal formation, thus establishing bs and Tbc1d20 (ZFN/ZFN) as allelic variants. Although the WARBM molecular disease etiology remains unclear, both the bs and Tbc1d20 (ZFN/ZFN) mice are excellent model organisms for future studies to establish TBC1D20-mediated molecular and cellular functions.

Functional analysis of the Hsf4(lop11) allele responsible for cataracts in lop11 mice.

PURPOSE: Lens opacity 11 (lop11) is a spontaneous autosomal recessive mouse mutation resulting in cataracts. Insertion of an early transposable element (ETn) in intron 9 of heat shock factor 4 (Hsf4) was previously identified as responsible for lop11 cataracts. Although molecular analysis showed that the ETn insertion resulted in an aberrant Hsf4 transcript encoding a truncated mutant HSF4(lop11) protein, the function of the mutant HSF4(lop11) protein was not investigated. The goal of this study was to functionally evaluate the mutant HSF4(lop11) protein and to establish the onset and progression of cataracts in lop11 lenses. METHODS: HSF4 is expressed as two alternatively transcribed isoforms Hsf4a and Hsf4b. Given that only Hsf4b is expressed in the lens we pursued evaluation of the mutant Hsf4b isoform only. Recombinant wild type HSF4b and mutant HSF4b(lop11) proteins were analyzed using elecrophoretic mobility shift, reporter transactivation, western blotting and protein half-life assays in HEK293 cells. Prenatal and postnatal wild type and lop11 lenses were evaluated using a combination of clinical, histological, and immunohistological analyses. RESULTS: HSF4b(lop11) stability and nuclear translocation of did not differ from wild type HSF4b. However, HSF4b(lop11) exhibited abolished HSE-mediated DNA binding and transactivation. Further investigation identified that HSF4b(lop11) fails to form trimers. Histological analysis of lop11 lenses indicated the persistence of nuclei in lens fiber cells as early as postnatal day 0.5 (P0.5). No differences were observed between wild type and lop11 in lens epithelial cell proliferation and spatio-temporal differentiation to fiber cells. However, by P10-12, lop11 lenses develop severely vacuolated cataracts commonly accompanied by rupture of the lens capsule and release of the lenticular material in the vitreous cavity. Clinically, lop11 vacuolated cataracts were visible upon eyelid opening between P12-14. CONCLUSIONS: The ETn insertion in lop11 mice results in abolished HSF4b function. Loss of 132 amino acids from the COOH-terminus in HSF4b(lop11) results in the failure of trimer formation and subsequent failure of HSE-mediated DNA binding and transactivation. These findings highlight the importance of the COOH-terminal region for normal function. The persistence of nuclei in postnatal lop11 lens fiber cells was identified as the initial lens abnormality, thus confirming a previously identified role of HSF4b in denucleation of lens fiber cells. By P14 lop11 lenses develop severe fiber cell vacuoles although how the loss of HSF4b function results in this process remains unknown. Collectively, these findings further our understanding of the mechanism of HSF4 loss of function as well as the resulting implications on lop11 cataractogenesis.

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.

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation.

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.

Alkylglycerone phosphate synthase (AGPS) deficient mice: models for rhizomelic chondrodysplasia punctate type 3 (RCDP3) malformation syndrome.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous autosomal recessive syndrome characterized by congenital cataracts, shortening of the proximal limbs, neurological abnormalities, seizures, growth delays, and severe intellectual disability. Most RCDP children die in the first decade of life due to respiratory complications. Mutations in alkylglycerone phosphate synthase (AGPS) cause RCDP type 3 (RCDP3). We've previously established that cataracts and male infertility in blind sterile 2 (bs2) mice are caused by a spontaneous hypomorphic mutation in Agps. As a part of this study, we set out to further explore the bs2 phenotypes and how they correlate to the clinical presentations of RCDP3 patients. Our results show that ∼50% bs2 mice die embryonically and surviving bs2 mice exhibit growth delays that they overcome by adulthood. The X-ray analysis of adult bs2 mice revealed significant humeral, but not femoral shortening. Clinical and histological eye evaluations revealed that bs2 lenses undergo normal development with first opacities developing at P21 that by P28 rapidly progress to mature cataracts. Evaluation of testes determined that infertility in bs2 mice is due to the aberrant formation of multicellular cellular clusters that undergo apoptosis. Given that the bs2 locus is a hypomorphic Agps mutation, we set out to generate Agps knockout mice utilizing Knockout Mouse Project (KOMP) resource. Our results showed that ∼85% of Agps knock-out mice die embryonically whereas surviving adult Agps knock-out mice phenotypically exhibit cataracts and testicular abnormalities similar to those observed in bs2 mice. Given that the majority of Agps knock-out mice die embryonically presented a challenge for further analyses of Agps deficiency in mouse models. Although not done as a part of this study, Agps-KOMP mice or ES cells can be further modified with FLP recombinase to generate mice suitable for subsequent matings with a transgenic Cre strain of choice, thereby providing an opportunity to study conditional Agps deficiency in a specific tissue or desired developmental time points without Agps deficiency-mediated embryonic lethality.

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.