Biallelic EPCAM deletions induce tissue-specific DNA repair deficiency and cancer predisposition.

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies, we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAMdel iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression, increased mutational burden, MMRD signatures and MS-indel accumulation, the hallmarks of MMRD. In contrast, maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes, preserving MMR proficiency. The combined use of iPSC, organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD, which affects the clinical management of these patients.

First identification of ITM2B interactome in the human retina.

Integral Membrane Protein 2 B (ITM2B) is a type II ubiquitous transmembrane protein which role remains unclear. ITM2B mutations have been associated with different disorders: mutations leading to longer mutant proteins have been reported in two distinct Alzheimer-like autosomal dominant disorders with early-onset progressive dementia and cerebellar ataxia. Both disorders share neurological features including severe cerebral amyloid angiopathy, non-neuritic plaques, and fibrillary tangles as in Alzheimer disease. Our group reported a missense mutation in ITM2B, in an unusual retinal dystrophy with no dementia. This finding suggests a specific role of ITM2B in the retina. As the identification of retinal-specific ITM2B partners could bring new insights into the cellular functions of ITM2B, we performed quantitative proteomics of ITM2B interactome of the human retina. Overall, 457 ITM2B partners were identified with 8 of them involved in visual transduction. In addition, bulk Gene Ontology analyses showed that many ITM2B partners are involved in several other biological functions, such as microtubule organization, protein translation and interestingly, mitochondrial homeostasis. These data represent the first report of the ITM2B interactome in the human retina and may serve as a valuable inventory of new potential ITM2B partners for future investigations of ITM2B physiological functions and dysfunctions.

Glycine receptors in a population of adult mammalian cones.

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of alpha-beta-heteromeric glycine receptors. Glycine receptors were also activated by taurine and beta-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the beta subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (approximately 13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor beta subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.

Advanced glycation end products can induce glial reaction and neuronal degeneration in retinal explants.

BACKGROUND/AIMS: Neuronal degeneration has been reported to occur in diabetic retinopathy before the onset of detectable microvascular abnormalities. To investigate whether advanced glycation end products (AGE) could be directly responsible for retinal neurodegeneration, retinal explants were incubated with glycated bovine serum albumin (BSA). METHODS: Retinal explants obtained from non-diabetic adult rats were incubated 4 days with or without 200 mug/ml glycated BSA. Neural apoptosis was quantified by terminal dUTP nick end labelling (TUNEL) binding and immunostaining with anti-cleaved caspase-3 antibody. Expression of glial fibrillary acidic protein (GFAP) was localised by immunofluorescence. RESULTS: TUNEL and cleaved caspase-3 positive cells increased significantly by 2.2-fold and 2.5-fold in retinal explants incubated in glycated BSA (p<0.05), respectively. The ganglion cell layer was the most sensitive retinal layer to the glycated BSA. Neuronal degeneration was confirmed by the increased GFAP labelling in Müller glial cells from retinal explants treated with glycated BSA. CONCLUSION: These results suggest that AGE could induce retinal neurodegeneration in the absence of blood perfusion. Cells in the ganglion cell layer appeared to be the most sensitive as in diabetic retinopathy and its animal models. AGE toxicity could therefore contribute to the early pathological mechanisms of diabetic retinopathy.

Expression of Dp71 in Müller glial cells: a comparison with utrophin- and dystrophin-associated proteins.

PURPOSE: The abnormal retinal electrophysiology observed in patients with Duchenne muscular dystrophy (DMD) has been attributed to an altered expression of C-terminal products of the dystrophin gene. It has been shown that Dp260 is expressed by photoreceptor cells, whereas Dp71 is present in glial cells. The present study was intended to identify all known members of the dystrophin superfamily and their associated proteins expressed in Müller glial cells (MGC). METHODS: The expression of the proteins and of their messengers was studied in MGC cultures from 2-week-old rats, by polymerase chain reaction amplification, Western blot analysis, and immunocytochemistry. An immunocytochemical localization of the proteins was also performed on enzymatically dissociated Müller cells from adult rat retinas. RESULTS: MGCs expressed a spliced isoform of Dp71 called Dp71f, as well as utrophin, beta-dystroglycan, delta and gamma-sarcoglycans, and alpha1-syntrophin. In morphologically preserved differentiated Müller cells, Dp71f was localized in clusters, utrophin was diffusely distributed in the cytoplasm, and dystrophin-associated proteins (DAPs) were membrane-bound. Most of these proteins were preferentially expressed in the vitread portion of the cells. Dp71f and utrophin expression was restricted to MGCs, whereas all DAPs were also present in other retinal cell types. CONCLUSIONS: The exclusive localization of Dp71f and utrophin in MGCs suggests that these proteins, together with DAPs, play a specific role in these cells. Further knowledge of possible interactions of these proteins within a functional complex may provide new insights into the molecular basis of the electroretinogram phenotype in DMD.

Adult human retinal neurons in culture: Physiology of horizontal cells.

PURPOSE: Adult postmortem human retinal neurons in long-term monolayer cultures were recorded to characterize the voltage- and transmitter-gated currents in putative human horizontal cells (HCs). METHODS: Enzymatically and mechanically dissociated human retinal cells were seeded on polylysine and laminin- coated coverslips. Cells were identified by immunocytochemistry with cell type-specific antibodies and recorded with the patch-clamp technique. RESULTS: Immunostaining and responses to voltage steps confirmed the survival of various retinal cell types. Horizontal cells were identified by their specific glutamate-modulated anomalous rectifier K+ current conductance. This identification was further confirmed by subsequent immunolabeling of dye-labeled recorded cells with an anti-parvalbumin antibody that selectively stained HCs in frozen human retinal sections. Horizontal cells generated voltage-gated currents classically observed in HCs from fish to mammals: a transient outward K+ current, a sustained outward K+ current, and an L-type (Ca2+ current. Na+ currents were observed in only a few HCs. As in other species, glutamate, gamma-aminobutyric acid (GABA), and glycine generated responses mediated by the activation of kainate/(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), GABA(A), and glycine receptors, respectively. CONCLUSIONS: Various human retinal cell populations survive in vitro as indicated by immunolabeling with specific cell markers and by the diversity of responses to voltage steps. Human HCs exhibited extensive physiological similarities to HCs from other vertebrate species and a maintained expression of parvalbumin. These results constitute a comprehensive analysis of voltage- and transmitter-gated currents in a primate retinal neuron and validate the use of long-term monolayer culture of adult human neurons as a novel in vitro model for the study of human vision.

GABAA and GABAC receptors in adult porcine cones: evidence from a photoreceptor-glia co-culture model.

1. Edge contrast enhancement is an integrated visual function based on the complex centre-surround organization of the cone photoreceptor light response. While centre responses result from direct light activation, surround responses are thought to result from lateral inhibition mediated by horizontal cells. This feedback signal has been attributed to GABAA receptors which have been found in lower vertebrate cones. 2. In order to study the GABA response of adult mammalian photoreceptors, we designed a culture system consisting of isolated photoreceptors seeded on a layer of retinal glial cells. Mature rods and cones required the presence of Muller glial cells to survive and develop neurites; they degenerated in the absence of glial cells. 3. Cone photoreceptors generated large GABA responses whereas rod photoreceptors did not respond to GABA applications. 4. Cone GABA responses consisted of two distinct components, one suppressed by the GABAA receptor blockers bicuculline and SR95531, and the second by the GABAC receptor antagonists TPMPA and imidazole-4-acetic acid (I4AA). Pentobarbital greatly increased the GABAA receptor component whereas it did not affect, or even reduced, the GABAC receptor component. During long GABA applications, GABAA receptor currents desensitized by 78%, contrasting with the sustained GABAC response. 5. Expression of GABAC receptors in cone photoreceptors was confirmed by anti-rho-subunit immunolabelling of porcine retinal sections. 6. These results indicate that both GABAA and GABAC receptors may participate in the feedback synapse from horizontal cells to cone photoreceptors in the mammalian retina.

Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse.

The role of cellular interactions in the mechanism of secondary cone photoreceptor degeneration in inherited retinal degenerations in which the mutation specifically affects rod photoreceptors was studied. We developed an organ culture model of whole retinas from 5-week-old mice carrying the retinal degeneration mutation, which at this age contain few remaining rods and numerous surviving cones cocultured with primary cultures of mixed cells from postnatal day 8 normal-sighted mice (C57BL/6) retinas or retinal explants from normal (C57BL/6) or dystrophic (C3H/He) 5-week-old mice. After 7 days, the numbers of residual cone photoreceptors were quantified after specific peanut lectin or anti-arrestin antibody labeling by using an unbiased stereological approach. Examination of organ cultured retinas revealed significantly greater numbers of surviving cones (15-20%) if cultured in the presence of retinas containing normal rods as compared with controls or cocultures with rod-deprived retinas. These data indicate the existence of a diffusible trophic factor released from retinas containing rod cells and acting on retinas in which only cones are present. Because cones are responsible for high acuity and color vision, such data could have important implications not only for eventual therapeutic approaches to human retinal degenerations but also to define interactions between retinal photoreceptor types.

Ganglioside and neurotrophic growth factor interactions in retinal neuronal and glial cells.

Ganglioside (GG) and neurotrophic growth factor (GF) interactions in retinal neuronal and glial cells have been very little studied. Rat retinas were mechanically separated into outer (photoreceptor or PR) and inner (other neurons, IR) halves by planar vibratome sectioning and retinal Müller glial (RMG) cells were isolated and cultured according to previously published methods. The distribution on a percent molar basis of individual GG was different between the two halves: PR were dominated by GD3 (48% total GG) and contained only trace amounts (< 4%) of complex species (GT1b, GQ); IR was more typical of mature brain tissue, exhibiting substantial amounts (approximately 25%) of more complex GG. The GG profile of RMG cells was also simple, dominated by GM3 (60%) and GD1a (20%). A single addition to the medium of 500 pM bFGF or EGF for 48 hr to cultured RMG cells led to significant increases in total GG levels of 30-40%. Such treatments by both growth factors induced increases in GM3, whereas longer exposure (96 hr) of confluent RMG to these factors additionally stimulated synthesis of more complex GG. Incubations of RMG with [3H]-glucosamine showed that GG synthesis was 2-fold stimulated by growth factors. We also tested the effect of GM3 on one of the bFGF receptor transduction pathways, namely PI-3 kinase activation. To our knowledge these data constitute the first demonstration of neurotrophic factor stimulation of GG levels in cells of CNS in vitro. Such complex interactions may have particularly important consequences for neural physiopathology.

Survival and regeneration of adult human and other mammalian photoreceptors in culture.

PURPOSE: Fully mature neurons of central nervous system origin generally are considered unable to survive for extended periods of time in simple culture conditions. The authors report that adult and aged human, porcine, and rodent retinal neurons, including rod and cone photoreceptors, constitute an exception to this idea. METHODS: Cells were dissociated from human postmortem retinas, adult mammalian retinas, and selected brain regions and were seeded into tissue culture plates and left to develop as monolayer cultures for up to 2 months. A battery of antibody markers was used to identify the nature and morphology of the cells in vitro. RESULTS: Photoreceptor cell survival of rods and cones was observed routinely when the delay between the time of death until culture preparation was 50 hours or less, compatible with current eye bank practice. Two-week-old cultures were formed of rod photoreceptors, representing approximately 50% of neuronal cell types; cone photoreceptors, representing 5% to 30% of neuronal cell types; other retinal neurons (especially amacrine cells approximately 20%); and retinal glial cells, present in variable numbers. Glial cells were essential for long-term photoreceptor survival and neurite outgrowth. Adult mammalian brain neurons isolated under the same conditions did not survive. CONCLUSIONS: Fully adult human and other mammalian retinal neurons, including photoreceptors, exhibit remarkable plasticity in vitro, and such monolayer models may have applications in physiological, pharmacologic, and toxicologic studies of human and other mammalian retina.

New members of the 3 beta-hydroxysteroid dehydrogenase gene family.

Several bands of hydridization are detected when southern blots of human genomic DNA are proved with cDNA of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3 beta-HSD gene family. Firstly, primer designed to amplify 3 beta-HSD type I and II genes were found on occasion to amplify DNA products of appropriate length but which were resolved as distinct sequences by denaturing gradient gel electrophoresis (DGGE). Five of these novel bands were cloned and their sequences were found to be closely related to 3 beta-HSD types I and II. Secondly, 57 genomic clones were selected from two lambda genomic libraries by hybridization with exonic probes of 3 beta -HSD type I. These were screened for novel members of the gene family by pcr amplification using various combinations of PCR primers to the type I and II genes, particularly those primers that previously amplified novel PCR products from genomic DNA. Amplification products from (lambda) clones were screened for novel sequences by DGGE. As a result of these approaches, at least five new members of the 3 beta-HSD gene family were found, one of which locates to the 3 beta -HSD type I and II gene cluster on 1p13. The existence of additional closely related but distinct members of the gene family should be recognized as a potential complication when screening PCR fragments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from (lambda) clones to search for novel members of the gene family.

DNA replication in vitro by recombinant DNA-polymerase-alpha-primase.

DNA-polymerase-alpha--primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-alpha--primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-polymerase-alpha--primase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-alpha--primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-alpha--primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.

Survival and regeneration of adult human photoreceptors in vitro.

Adult human retina from enucleated ocular tissue was enzymatically dissociated and plated into plastic dishes for cell culture in serum-supplemented medium. Within a few days following seeding, islands of glial-like cells with rounded neurons growing on top of them were visible. Immunocytochemical labelling of these cultures revealed that virtually all the surviving neurons (> 98%) were rod photoreceptors, and that they extended long neurites across the glial cell surface. Hence, adult human photoreceptors retain a remarkable capacity for survival and regrowth, and such preparations may be of value for physiopathological and retinal grafting studies.

Exogenous gangliosides modulate calcium fluxes in cultured neuronal cells.

Previous work from this and other laboratories has shown that the neuritogenic effect due to exogenous gangliosides on primary neurons in culture is accompanied by several morphological and biochemical modifications. The present results indicate that the treatment of these neurons with gangliosides, under the experimental conditions which are known to produce a sprouting effect, inhibited the influx of 45Ca2+ and increased the release of 45Ca2+ from the cells. No significant differences were noted using concentrations of gangliosides (10(-8)-10(-5) M) either below or above the critical micellar concentrations. No apparent specificity was observed among various species of individual sialocompounds (GM1, GD1a). Moreover the presence or absence of fetal calf serum in the culture medium influenced the levels of 45Ca2+ fluxes. This study confirms the hypothesis that gangliosides may be considered as Ca2+ flux modulators in neuronal cells.

On the action of hydroxylamine, hydrazine and their derivatives on the water-oxidizing complex.

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S0 → S1, S1 → S2, S2 → S3, S3 → (S4) → S0. State S1 is most stable in the dark. Oxygen is released during S3 → (S4) → S0. Hydroxylamine and hydrazine interact with S1. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S1 → S2 : S2 → S3 : S3 → (S4) → S0 : S0 → S1 :... in the absence of the agents, to S1 (*) → S0 : S0 → S1 : S1 → S2 : S2 → S3 :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH(+)(S1 → S2) : 1H(+)(S2 → S3) : 2H(S3 → S0) : 1H(+)(S0 → S1) :... to 2H(+)(S1 (*) → S0) : 1H(+)(S0 → S1) : OH(+)(S1 → S2) : 1H(+)(S2 → S3) : 2H(+)(S3 → S0) :... The 2H(+) were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53-57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n≥3 molecules of hydroxylamine and of n≥2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl(-) or OH(-), which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S1.

Antianginal drug trapidil (Rocornal) inhibits spreading of platelets and formation of surface-bound thrombi-like aggregates.

Effects of an antianginal drug trapidil (Rocornal) on platelet interaction with a surface coated with fibrillar calf skin collagen (CSC) have been studied by scanning electron microscopy. Gel filtered human platelets were incubated with the CSC substrate in the absence or in the presence of soluble inducers of platelet activity: arachidonic acid (AA), stable analogue of prostaglandin (PG) endoperoxides, U46619, and thrombin. In the absence of soluble inducers, trapidil does not alter the total number of adherent platelets. At the same time, trapidil inhibits the shape change of adherent platelets, induced by the CSC substrate, increasing the percentage of discoid and decreasing the percentage of spread platelets. It was demonstrated earlier (1) and in the present study that soluble inducers of platelet activity stimulate massive spreading of platelets and formation of surface-bound thrombi-like aggregates on the CSC substrate. Trapidil completely prevents the effects of the exogenous AA and U46619 on platelet-substrate interactions, but inhibits the AA-stimulated synthesis of thromboxane A2 (TXA2) in platelets by 40-50% only. Trapidil also blocks platelet aggregation in suspension, spreading and formation of surface-bound aggregates induced by low, but not high, concentrations of thrombin. Possible sites of trapidil action are discussed.