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.

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.

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 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.

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.

The waved with open eyelids (woe) locus is a hypomorphic mouse mutation in Adam17.

The waved with open eyes (woe) locus is a spontaneous recessive mouse mutation that exhibits wavy fur, eyelids open at birth, and enlarged heart and esophagus. In this study, we confirmed the previously identified woe phenotypes and additionally identified anterior eye segment defects, absence of the meibomian glands, and defects in the semilunar cardiac valves. Positional cloning identified a C794T substitution in the Adam17 gene that ablates a putative exonic splicing enhancer (ESE) sequence in exon 7 resulting in aberrant Adam17 splicing. The predominant woe transcript, Adam17(Delta)(exon7), lacks exon 7 resulting in an in-frame deletion of 90 bp and a putative Adam17(Delta252-281) protein lacking residues 252-281 from the metalloprotease domain. Western blot analysis in woe identified only the precursor form of Adam17(Delta252-281) protein. Absence of cleavage of the prodomain renders Adam17(Delta252-281) functionally inactive; however, constitutive and stimulated shedding of Adam17 substrates was detected in woe at significantly reduced levels. This residual Adam17 shedding activity in woe most likely originates from full-length Adam17(T265M) encoded by the Adam17(C794T) transcript identified expressed at severely reduced levels. These results show that even small amounts of functional Adam17 allow woe mice to survive into adulthood. In contrast to Adam17(-/-) mice that die at birth, the viability of woe mice provides an excellent opportunity for studying the role of Adam17 throughout postnatal development and homeostasis.

Genetic and clinical evaluation of juvenile retinoschisis.

Juvenile retinoschisis is a rare retinal dystrophy caused by RS1 gene mutations.(1) Clinical examinations and molecular testing definitively diagnosed juvenile retinoschisis in 2 male infants, one of whom had a novel mutation not previously reported in the United States. Genetic testing may be the simplest way to confirm this diagnosis in infants.

Identification of two novel mutations in families with X-linked ocular albinism.

PURPOSE: Our goal was to evaluate the OA1 gene, also known as G-protein coupled receptor 143 (GPR143), in two United States families, one from the mid-west and one from the mid-south, who had clinical features of X-linked ocular albinism. Both families had previously tested negative for mutations. METHODS: Selected family members underwent a detailed ophthalmologic evaluation. Blood samples were obtained, and genomic DNA isolated. Mutational analysis by direct sequencing was used to evaluate OA1 exons and intron/exon junction. RESULTS: Ophthalmic features in the evaluated family members were consistent with X-linked ocular albinism. Mutation screening and sequence analysis of the OA1 gene in the mid-west family identified a novel 190delC deletion. The 190delC mutation was predicted to result in a frameshift following Ser63, an addition of 16 novel amino acids and a premature stop. In the mid-south family, a 346T>G substitution was identified in exon 2. The 346T>G mutation was predicted to result in a substitution of the highly conserved Cys116 to Gly and disruption of the disulfide bridge essential for the normal structure and function of the OA1 protein. CONCLUSIONS: Two novel mutations in the OA1 gene were identified in two families with ocular albinism. The identified mutations are likely loss-of-function mutations. These findings confirm that mutations in the OA1 gene are associated with the majority of X-linked ocular albinism cases.

Early transposable element insertion in intron 9 of the Hsf4 gene results in autosomal recessive cataracts in lop11 and ldis1 mice.

Lens opacity 11 (lop11) is an autosomal recessive mouse cataract mutation that arose spontaneously in the RIIIS/J strain. At 3 weeks of age mice exhibit total cataracts with vacuoles. The lop11 locus was mapped to mouse chromosome 8. Analysis of the mouse genome for the lop11 critical region identified Hsf4 as a candidate gene. Molecular evaluation of Hsf4 revealed an early transposable element (ETn) in intron 9 inserted 61 bp upstream of the intron/exon junction. The same mutation was also identified in a previously mapped cataract mutant, ldis1. The ETn insertion altered splicing and expression of the Hsf4 gene, resulting in the truncated Hsf4 protein. In humans, mutations in HSF4 have been associated with both autosomal dominant and recessive cataracts. The lop11 mouse is an excellent resource for evaluating the role of Hsf4 in transparency of the lens.

Radiation hybrid map, physical map, and low-pass genomic sequence of the canine prcd region on CFA9 and comparative mapping with the syntenic region on human chromosome 17.

Progressive rod-cone degeneration (prcd) is a canine retinal disease that maps to the centromeric end of CFA9 in a region of synteny with the distal part of HSA17q. As such, prcd has been postulated as the only animal model of RP17, a human retinitis pigmentosa locus that maps to 17q22. In an effort to establish more detailed regions of synteny between dog CFA9 and the HSA17q-ter region, we created a robust gene-enriched CFA9-RH08(3000) map with 34 gene-based markers and 12 microsatellites, with the highest resolution and number of markers for the centromeric end of CFA9. Furthermore, we built an approximately 1.5-Mb physical map containing both GRB2 and GALK1, genes so far identified by meiotic linkage analysis as being closest to the prcd locus, and generated about 1.2 Mb low-pass (3.2x) canine sequence. Canine to human comparative sequence analysis identified 49 transcripts that had been previously mapped to the HSA17q25 region. The generated low-pass canine sequence was annotated with a working draft of human sequence from HSA17q25, and we used this scaffold to order and orient the canine sequence against human. This order and orientation are preliminary, as high-throughput genomic sequencing of HSA17q-ter has not been fully completed.

A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice.

Hfi is a dominant cataract mutation where heterozygotes show hydropic lens fibers and homozygotes show total lens opacity. The Hfi locus was mapped to the distal part of mouse chromosome 10 close to the major intrinsic protein (Mip), which is expressed only in cell membranes of lens fibers. Molecular analysis of Mip revealed a 76-bp deletion that resulted in exon 2 skipping in Mip mRNA. In Hfi/Hfi this deletion resulted in a complete absence of the wildtype Mip. In contrast, Hfi/+ animals had the same amount of wildtype Mip as +/+. Results from pulse-chase expression studies excluded hetero-oligomerization of wildtype and mutant Mip as a possible mechanism for cataract formation in the Hfi/+. We propose that the cataract phenotype in the Hfi heterozygote mutant is due to a detrimental gain of function by the mutant Mip resulting in either cytotoxicity or disruption in processing of other proteins important for the lens. Cataract formation in the Hfi/Hfi mouse is probably a combined result of both the complete loss of wildtype Mip and a gain of function of the mutant Mip.

Mapping of the autosomal dominant cataract mutation (Coc) on mouse chromosome 16.

PURPOSE: To characterize the mouse cataract mutation Coc. METHODS: Coc is an X-radiation-induced autosomal dominant cataract mutation maintained on a murine C3H inbred strain. The affected heterozygotes were outcrossed to C57BL/6, and (C3H Coc/+ x C57BL/6) mice that were Coc/+ were then backcrossed to C57BL/6 to generate a panel of 103 progeny for mapping. For linkage analysis, microsatellites from each autosome were selected. The maximum distance between markers was 30 centimorgans (cM). RESULTS: The initial genome-wide screen of 14 backcrossed progeny indicated that the Coc locus resides on chromosome 16. Further mapping with additional markers from chromosome 16 for all 103 backcrossed progeny positioned Coc between markers D16Mit134 and D16Mit63. This region is syntenic to human chromosome 3. CONCLUSIONS: Mapping of the Coc locus to mouse chromosome 16 provides the positional information necessary to identify the candidate gene responsible for the Coc phenotype. The molecular characterization of the gene disrupted in the Coc mutation will provide insight into the mechanisms involved in cataract formation.