Deletion of POMT2 in Zebrafish Causes Degeneration of Photoreceptors.

Mutations in the extracellular matrix protein eyes shut homolog (EYS) are a common cause of retinitis pigmentosa, a blinding disease characterized by photoreceptor degeneration. EYS binds to matriglycan, a carbohydrate modification on O-mannosyl glycan substitutions of the cell-surface glycoprotein α-dystroglycan. Patients with mutations in enzymes required for the biosynthesis of matriglycan exhibit syndromic retinal atrophy, along with brain malformations and congenital muscular dystrophy. Protein O-mannosyltransferase 2 (POMT2) is an enzyme required for the synthesis of O-mannosyl glycans. To evaluate the roles of O-mannosyl glycans in photoreceptor health, we generated protein O-mannosyltransferase 2 (pomt2) mutant zebrafish by CRISPR. pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to α-dystroglycan. Mutant zebrafish presented with hydrocephalus and hypoplasia of the cerebellum, as well as muscular dystrophy. EYS protein was enriched near photoreceptor connecting cilia in the wild-type, but its presence and proper localization was significantly reduced in mutant animals. The mutant retina exhibited mis-localization of opsins and increased apoptosis in both rod and cone photoreceptors. Immunofluorescence intensity of G protein subunit alpha transducin 2 (GNAT2) antibody (a general cone marker) and 1D4 antibody (a long double cone marker) in mutant retinas did not differ from wild-type retinas at 1-month post fertilization, but was reduced at 6 months post fertilization, indicating significant cone degeneration. These data suggest that POMT2-mediated O-mannosyl glycosylation is required for EYS protein localization to the connecting cilium region and photoreceptor survival.

Interactions between C8orf37 and FAM161A, Two Ciliary Proteins Essential for Photoreceptor Survival.

Mutations in C8orf37 cause Bardet-Biedl syndrome (BBS), retinitis pigmentosa (RP), and cone-rod dystrophy (CRD), all manifest in photoreceptor degeneration. Little is known about which proteins C8orf37 interacts with to contribute to photoreceptor survival. To determine the proteins that potentially interact with C8orf37, we carried out a yeast two-hybrid (Y2H) screen using C8orf37 as a bait. FAM161A, a microtubule-binding protein localized at the photoreceptor cilium required for photoreceptor survival, was identified as one of the preys. Double immunofluorescence staining and proximity ligation assay (PLA) of marmoset retinal sections showed that C8orf37 was enriched and was co-localized with FAM161A at the ciliary base of photoreceptors. Epitope-tagged C8orf37 and FAM161A, expressed in HEK293 cells, were also found to be co-localized by double immunofluorescence staining and PLA. Furthermore, interaction domain mapping assays identified that the N-terminal region of C8orf37 and amino acid residues 341-517 within the PFAM UPF0564 domain of FAM161A were critical for C8orf37-FAM161A interaction. These data suggest that the two photoreceptor survival proteins, C8orf37 and FAM161A, interact with each other which may contribute to photoreceptor health.

Ba2B5O8(OH)2(NO3)·3H2O: the design of an alkaline earth metal borate-nitrate optimized from a hydroxylic borate.

The first alkaline earth metal borate-nitrate, namely Ba2B5O8(OH)2(NO3)·3H2O (BBNOH), has been synthesized by the hydrothermal method. BBNOH crystallizes in the space group of P21/c and shows two-dimensional (2D) 2∞[B5O8(OH)2]3- borate anion layers, and the hydrated barium cations and the [NO3]- anions are located between the layers. The process of optimizing the structure of Ba2B5O8(OH)2OH to BBNOH has been discussed. The first principles calculation has been used to calculate the birefringence of Ba2B5O8(OH)2(NO3)·3H2O, and the value is 0.033@1064 nm, which is mainly originated from the borate anions and the π conjugated [NO3]- anions.

TMEM216 Deletion Causes Mislocalization of Cone Opsin and Rhodopsin and Photoreceptor Degeneration in Zebrafish.

Purpose: Mutations in TMEM216, a ciliary transition zone tetraspan transmembrane protein, are linked to Joubert syndrome and Meckel syndrome. Photoreceptor degeneration is a prominent phenotype in Joubert syndrome. How TMEM216 contributes to photoreceptor health is poorly understood. Methods: We have generated tmem216 knockout zebrafish by CRISPR genome editing. The impact of TMEM216 deletion on photoreceptors was evaluated by immunofluorescence staining and electron microscopy. Results: Homozygous tmem216 knockout zebrafish died before 21 days after fertilization. Their retina exhibited reduced immunoreactivity to rod photoreceptor outer segment marker 4D2 and cone photoreceptor outer segment marker G protein subunit α transducin 2 (GNAT2). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed an increase in TUNEL-positive nuclei in the knockout retina, indicating photoreceptor degeneration. The tmem216 mutation resulted in shortened photoreceptor ciliary axoneme, as revealed by acetylated α-tubulin immunostaining. Photoreceptors in knockout zebrafish exhibited mislocalization of outer segment proteins such as rhodopsin, GNAT2, and red opsin to the inner segment and cell bodies. Additionally, electron microscopy revealed that the mutant photoreceptors elaborated outer segment with abnormal disc morphology such as shortened discs and vesicles/vacuoles within the outer segment. Conclusion: Our results indicate that TMEM216 is essential for normal genesis of outer segment disc structures, transport of outer segment materials, and survival of photoreceptors in zebrafish. These tmem216 knockout zebrafish will be useful in studying how transition zone proteins regulate photoreceptor outer segment formation and maintenance.

Eyes shut homolog (EYS) interacts with matriglycan of O-mannosyl glycans whose deficiency results in EYS mislocalization and degeneration of photoreceptors.

Mutations in eyes shut homolog (EYS), a secreted extracellular matrix protein containing multiple laminin globular (LG) domains, and in protein O-mannose ß1, 2-N-acetylglucosaminyl transferase 1 (POMGnT1), an enzyme involved in O-mannosyl glycosylation, cause retinitis pigmentosa (RP), RP25 and RP76, respectively. How EYS and POMGnT1 regulate photoreceptor survival is poorly understood. Since some LG domain-containing proteins function by binding to the matriglycan moiety of O-mannosyl glycans, we hypothesized that EYS interacted with matriglycans as well. To test this hypothesis, we performed EYS Far-Western blotting assay and generated pomgnt1 mutant zebrafish. The results showed that EYS bound to matriglycans. Pomgnt1 mutation in zebrafish resulted in a loss of matriglycan, retention of synaptotagmin-1-positive EYS secretory vesicles within the outer nuclear layer, and diminished EYS protein near the connecting cilia. Photoreceptor density in 2-month old pomgnt1 mutant retina was similar to the wild-type animals but was significantly reduced at 6-months. These results indicate that EYS protein localization to the connecting cilia requires interaction with the matriglycan and that O-mannosyl glycosylation is required for photoreceptor survival in zebrafish. This study identified a novel interaction between EYS and matriglycan demonstrating that RP25 and RP76 are mechanistically linked in that O-mannosyl glycosylation controls targeting of EYS protein.

Laminin-dystroglycan signaling regulates retinal arteriogenesis.

Proper arteriovenous morphogenesis is crucial for maintaining normal tissue perfusion. However, our understanding of how arterial morphogenesis is regulated in the CNS is incomplete. In this study, we asked whether vascular basement membrane (BM) laminins, specifically the γ3-containing isoforms, regulate retinal arterial morphogenesis. We provide evidence that Laminin-γ3 is deposited at both arterial and venous BMs during arteriogenesis. Vascular BM Laminin-γ3 bound dystroglycan (DG), a laminin receptor preferentially expressed by arterial endothelial cells (ECs) during arteriogenesis. Blockade of laminin-DG binding in vitro led to decreased Delta-like ligand (DLL)-4 expression in ECs. Moreover, genetic deletion of the Laminin-γ3- and EC-specific deletion of DG led to similar defects in retinal arteriogenesis, including reduced Dll4 expression, hyperbranching and reduced smooth muscle coverage. These results implicate a newly identified Laminin-γ3-DG signaling cascade that regulates arterial Dll4/Notch signaling to specify and stabilize retinal arteries.-Biswas, S., Watters, J., Bachay, G., Varshney, S., Hunter, D. D., Hu, H., Brunken, W. J. Laminin-dystroglycan signaling regulates retinal arteriogenesis.

Postnatal Gene Therapy Improves Spatial Learning Despite the Presence of Neuronal Ectopia in a Model of Neuronal Migration Disorder.

Patients with type II lissencephaly, a neuronal migration disorder with ectopic neurons, suffer from severe mental retardation, including learning deficits. There is no effective therapy to prevent or correct the formation of neuronal ectopia, which is presumed to cause cognitive deficits. We hypothesized that learning deficits were not solely caused by neuronal ectopia and that postnatal gene therapy could improve learning without correcting the neuronal ectopia formed during fetal development. To test this hypothesis, we evaluated spatial learning of cerebral cortex-specific protein O-mannosyltransferase 2 (POMT2, an enzyme required for O-mannosyl glycosylation) knockout mice and compared to the knockout mice that were injected with an adeno-associated viral vector (AAV) encoding POMT2 into the postnatal brains with Barnes maze. The data showed that the knockout mice exhibited reduced glycosylation in the cerebral cortex, reduced dendritic spine density on CA1 neurons, and increased latency to the target hole in the Barnes maze, indicating learning deficits. Postnatal gene therapy restored functional glycosylation, rescued dendritic spine defects, and improved performance on the Barnes maze by the knockout mice even though neuronal ectopia was not corrected. These results indicate that postnatal gene therapy improves spatial learning despite the presence of neuronal ectopia.

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates.

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.

Eyes shut homolog is required for maintaining the ciliary pocket and survival of photoreceptors in zebrafish.

Mutations in the extracellular matrix protein eyes shut homolog (EYS) cause photoreceptor degeneration in patients with retinitis pigmentosa 25 (RP25). Functions of EYS remain poorly understood, due in part to the lack of an EYS gene in mouse. We investigated the localization of vertebrate EYS proteins and engineered loss-of-function alleles in zebrafish. Immunostaining indicated that EYS localized near the connecting cilium/transition zone in photoreceptors. EYS also strongly localized to the cone outer segments and weakly to the rod outer segments and cone terminals in primate retinas. Analysis of mutant EYS zebrafish revealed disruption of the ciliary pocket in cone photoreceptors, indicating that EYS is required for maintaining the integrity of the ciliary pocket lumen. Mutant zebrafish exhibited progressive loss of cone and rod photoreceptors. Our results indicate that EYS protein localization is species-dependent and that EYS is required for maintaining ciliary pocket morphology and survival of photoreceptors in zebrafish.

New concepts in basement membrane biology.

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.

Biochemical and biophysical changes underlie the mechanisms of basement membrane disruptions in a mouse model of dystroglycanopathy.

Mutations in glycosyltransferases, such as protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1), causes disruptions of basement membranes (BMs) that results in neuronal ectopias and muscular dystrophy. While the mutations diminish dystroglycan-mediated cell-ECM interactions, the cause and mechanism of BM disruptions remain unclear. In this study, we established an in vitro model to measure BM assembly on the surface of neural stem cells. Compared to control cells, the rate of BM assembly on POMGnT1 knockout neural stem cells was significantly reduced. Further, immunofluorescence staining and quantitative proteomic analysis of the inner limiting membrane (ILM), a BM of the retina, revealed that laminin-111 and nidogen-1 were reduced in POMGnT1 knockout mice. Finally, atomic force microscopy showed that the ILM from POMGnT1 knockout mice was thinner with an altered surface topography. The results combined demonstrate that reduced levels of key BM components cause physical changes that weaken the BM in POMGnT1 knockout mice. These changes are caused by a reduced rate of BM assembly during the developmental expansion of the neural tissue.

Adeno-associated viral-mediated LARGE gene therapy rescues the muscular dystrophic phenotype in mouse models of dystroglycanopathy.

Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, and fukutin, creating the possibilities of a one-for-all gene therapy. To determine the feasibility of LARGE gene therapy, a serotype 9 adeno-associated viral vector for overexpressing LARGE (AAV9-LARGE) was injected intracardially into newborns of two mouse models of CMD: the natural LARGE mutant Large(myd) mice and protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice. AAV9-LARGE virus treatment yielded partial restoration of α-DG glycosylation and ligand-binding activity. The muscular dystrophy phenotype in skeletal muscles was ameliorated as revealed by significantly reduced fibrosis, necrosis, and numbers of centrally located nuclei with improved motor function. These results indicate that LARGE overexpression in vivo by AAV9-mediated gene therapy is effective at restoring functional glycosylation of α-DG and rescuing the muscular dystrophy phenotype in deficiency of not only LARGE but also POMGnT1, providing evidence that in vivo LARGE gene therapy may be broadly useful in dystroglycanopathies.

Differential glycosylation of α-dystroglycan and proteins other than α-dystroglycan by like-glycosyltransferase.

Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of α-dystroglycan (α-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of α-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and α-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than α-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of α-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of α-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on α-DG but also N-glycans on proteins other than α-DG.

Breaches of the pial basement membrane are associated with defective dentate gyrus development in mouse models of congenital muscular dystrophies.

A subset of congenital muscular dystrophies (CMDs) has central nervous system manifestations. There are good mouse models for these CMDs that include POMGnT1 knockout, POMT2 knockout and Large(myd) mice with all exhibiting defects in dentate gyrus. It is not known how the abnormal dentate gyrus is formed during the development. In this study, we conducted a detailed morphological examination of the dentate gyrus in adult and newborn POMGnT1 knockout, POMT2 knockout, and Large(myd) mice by immunofluorescence staining and electron microscopic analyses. We observed that the pial basement membrane overlying the dentate gyrus was disrupted and there was ectopia of granule cell precursors through the breached pial basement membrane. Besides these, the knockout dentate gyrus exhibited reactive gliosis in these mouse models. Thus, breaches in the pial basement membrane are associated with defective dentate gyrus development in mouse models of congenital muscular dystrophies.

LARGE expression augments the glycosylation of glycoproteins in addition to α-dystroglycan conferring laminin binding.

Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity.

Conditional knockout of protein O-mannosyltransferase 2 reveals tissue-specific roles of O-mannosyl glycosylation in brain development.

The meninges produce essential signaling molecules and major protein components of the pial basement membrane during normal brain development. Disruptions in the pial basement membrane underlie neural ectopia seen in those congenital muscular dystrophies (CMDs) caused by mutations in genes involved in O-mannosyl glycosylation. In mammals, biosynthesis of O-mannosyl glycans is initiated by a complex of mutually indispensable protein O-mannosyltransferases 1 and 2 (POMT1 and 2). To study the roles of O-mannosylation in brain development we generated a conditional allele of POMT2. POMT2 nulllizygosity resulted in embryonic lethality because of a defective Reichert's membrane. Brain-specific deletion of POMT2 resulted in hypoglycosylation of α-dystroglycan (DG) and abolished laminin binding activity. The effect of POMT2 deletion on brain development was dependent on timing, as earlier deletion resulted in more severe phenotypes. Multiple brain malformations including overmigration of neocortical neurons and migration failure of granule cells in the cerebellum were observed. Immunofluorescence staining and transmission electron microscopy revealed that these migration defects were closely associated with disruptions in the pial basement membrane. Interestingly, POMT2 deletion in the meninges (and blood vessels) did not disrupt the development of the neocortex. Thus, normal brain development requires protein O-mannosylation activity in neural tissue but not the meninges. These results suggest that gene therapy should be directed to the neural tissue instead of the meninges.

Glycomic analyses of mouse models of congenital muscular dystrophy.

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1(-/-)). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with ß1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical ß1,2-elongation and ß1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

Pikachurin interaction with dystroglycan is diminished by defective O-mannosyl glycosylation in congenital muscular dystrophy models and rescued by LARGE overexpression.

Congenital muscular dystrophies (CMD) such as muscle-eye-brain disease caused by defective glycosylation of α-dystroglycan (α-DG) exhibit defective photoreceptor synaptic function. Mouse knockouts of dystroglycan and its extracellular matrix binding partner pikachurin recapitulate this phenotype. In this study, pikachurin-α-dystroglycan interactions in several mouse models of CMD were examined by pikachurin overlay experiments. The results show that hypoglycosylation of α-dystroglycan resulted in markedly reduced pikachurin-α-dystroglycan interactions. Expression of pikachurin is abolished at the outer plexiform layer of two mouse models, protein O-mannose N-acetylglucosaminyl transferase 1 (POMGnT1) knockout and Large(myd) mice. Overexpressing LARGE restored this interaction in POMGnT1 knockout cells. These results indicate that pikachurin interactions with α-dystroglycan and its localization at the photoreceptor ribbon synapse require normal glycosylation of α-dystroglycan.

Cellular and molecular characterization of abnormal brain development in protein o-mannose N-acetylglucosaminyltransferase 1 knockout mice.

Protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that catalyzes the transfer of N-acetylglucosamine to O-mannose of glycoproteins. It is involved in posttranslational modification of alpha-dystroglycan (alpha-DG). POMGnT1-null mice were generated by gene trapping with a retroviral vector inserted into exon 2 of the POMGnT1 gene. Expression of POMGnT1 was completely disrupted as evidenced by absence of its mRNA expression. POMGnT1 knockout mice were viable but with reduced fertility and variable lifespan. The functional glycosylated form of alpha-DG was markedly reduced in POMGnT1 knockout mice along with impaired alpha-DG-laminin binding activity. Multiple developmental defects in muscle, brain, and eye were observed. In addition, the knockout mice exhibited extensive abnormalities in the neocortex, including changed neuron distribution, presence of ectopic fibroblasts, and GFAP-positive reactive astrocytes. Analysis of POMGnT1 knockout neocortex at several developmental stages revealed that these defects were secondary to disruptions of the pial basement membrane.

Retinal ectopias and mechanically weakened basement membrane in a mouse model of muscle-eye-brain (MEB) disease congenital muscular dystrophy.

PURPOSE: Some forms of congenital muscular dystrophy are associated with cortical and retinal dysplasias. Protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice, one of the mouse models of muscular dystrophy, exhibit a thinner retina with reduced density of retinal ganglion cells. This study is aimed to further characterize the knockout retina, with special emphasis on the inner limiting membrane, the basement membrane of the retina. METHODS: Immunofluorescence staining and transmission electron microscopy were used to analyze the retinas. Atomic force microscopy was performed on the inner limiting membrane preparations to examine their mechanical properties. RESULTS: The inner limiting membrane of the knockout mice exhibited frequent breaks with protrusions of the Müller glial processes and ectopic placement of retinal ganglion cells into the vitreous humor. Disruptions in inner limiting membrane integrity developmentally precede the cellular abnormalities. Regions of disrupted inner limiting membrane were also associated with molecular abnormalities of Müller glia that included diminished presence of the integral membrane proteins Kir4.1 (an inwardly rectifying potassium channel) and aquaporin-4. When measured with atomic force microscopy, the POMGnT1 knockout mouse inner limiting membrane (ILM) exhibited significantly reduced Young's modulus and is therefore mechanically weaker than the ILM from controls. CONCLUSIONS: Deficiency of POMGnT1-mediated glycosylation of dystroglycan is implicated in reduced stiffness of the ILM. The weakened ILM results in the disruption of the membrane and subsequent reduction in retinal integrity.