Peter M.G. Munro

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Characterization of the Limbal Epithelial Stem Cell Niche: Novel Imaging Techniques Permit In Vivo Observation and Targeted Biopsy of Limbal Epithelial Stem Cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State‐of‐the‐art imaging techniques were used to construct a three‐dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63α and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies.

The Drusenlike Phenotype in Aging Ccl2-Knockout Mice Is Caused by an Accelerated Accumulation of Swollen Autofluorescent Subretinal Macrophages

PURPOSE Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD. METHODS The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry. RESULTS The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice. CONCLUSIONS These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Three-dimensional structure of the vertebrate muscle A-band: III. M-region structure and myosin filament symmetry☆

Ultrathin transverse sections of the body muscle of bony fish and the sartorius muscle of frog have been analysed in detail by optical diffraction and image averaging to reveal the ultrastructures of the myosin filaments both in the M-region and in the outer ends of the filament (the tip region). The evidence is unequivocal that the myosin filaments in both muscle types have 3-fold rotational symmetry in all of the regions where symmetry can be seen. Using the nomenclature of Sjostrom & Squire (1977a), the appearances of cross-sections in fish at different axial locations are as follows. (Note that fish myosin filaments are arranged in a simple hexagonal lattice.) 1. (1)At M1 (central M-bridge line) the myosin filament profile is almost circular, but does sometimes show a three-component structure. There are six M-bridges from each filament. 2. (2) At M4 the filament backbone comprises three kidney-shaped subunits related by a triad axis, together with six M-bridges. There are two types of M-bridge interaction site. 3. (3) At M6 there are three kidney-shaped subunits related by a triad. There are no M-bridges but there does seem to be extra protein here. 4. (4) In the bare region there are three nearly circular subunits related by a triad. 5. (5) At M9 or the start of the bridge region there is a Y-shaped filament profile from which three projections radiate towards three of the six actin filament positions (trigonal points). 6. (6) There is an apparent change of hand of the filament profiles across the M-band when viewed in the same section (i.e. from M4 to M4′ or M6 to M6′). This implies that the bipolar myosin filament has the symmetry of the dihedral point group 32. 7. (7) There is a change in orientation of the 3-fold myosin filament profile across the M-band so that the bare region profiles are about 40 ° apart. Less detail has been seen in frog sartorius muscle sections because of the presence of the superlattice (see paper II in this series, Luther & Squire, 1980). However, the observations are entirely compatible with a myosin filament with the same structure as in fish but arranged in a superlattice. The superlattice is apparent not only in the bare region in frog, but also at the M-bridge level M4. There are two classes of M-bridge interaction at M4 just as in fish. At the A-band edges of both muscles (outer D-zone) the filament profiles appear either triangular, Y-shaped or composed of three subunits. The lattices are insufficiently ordered for satisfactory image averaging to be carried out. As yet the main part of the bridge region shows little ultrastructural detail. However, the present results show clearly that the vertebrate myosin filament has 3-fold rotational symmetry; there can be little doubt that the symmetry of the crossbridge array approximates to that of a three-stranded helix (Squire, 1972). A model is proposed for the structure of the myosin filament in the vertebrate M-region. It explains the observed appearances without going into molecular detail. It is also suggested that it is the M-bridges at M4 that are primarily responsible for defining the two types of A-band structure (simple lattice and superlattice) and that the M1 bridges may have a secondary role.

Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups

Summary Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention.

Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy

We report mutations in the gene for topoisomerase I-binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families--one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain-containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy.

Real-Time In Vivo Imaging of Retinal Cell Apoptosis after Laser Exposure

PURPOSE To investigate whether the detection of apoptosing retinal cells (DARC) could detect cells undergoing apoptosis in a laser model of retinal damage. METHODS Laser lesions were placed, with the use of a frequency-doubled Nd:YAG laser, on the retina in 34 eyes of anesthetized Dark Agouti rats. Lesion size and laser-induced retinal elevation were analyzed using in vivo reflectance imaging. Development of retinal cell apoptosis was assessed using intravitreal fluorescence-labeled annexin 5 in vivo with DARC technology from baseline until 90 minutes after laser application. Histologic analysis of retinal flat mounts and cross-sections was performed. RESULTS The lateral and anteroposterior depth extension of the zone of laser damage was significantly larger for higher exposure settings. A strong diffuse signal, concentrated at the outer retina, was seen with DARC for low exposures (<300 ms and <300 mW). In comparison, higher exposures (>300 ms and >300 mW) resulted in detectable hyperfluorescent spots, mainly at the level of the inner retinal layers. Dose-dependent effects on spot density and positive correlation of spot density between lesion size (P < 0.0001) and retinal elevation (P < 0.0001) were demonstrated. Histology confirmed the presence of apoptosing retinal cells in the inner nuclear and the ganglion cell layers. CONCLUSIONS This is the first time that DARC has been used to determine apoptotic effects in the inner nuclear layer. The ability to monitor changes spatially and temporally in vivo promises to be a major advance in the real-time assessment of retinal diseases and treatment effects.

The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice.

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration.

Muscle ultrastructure in the teleost fish

Abstract The muscles of teleost fish are different in ultrastructure from those of most other kinds of vertebrate. A distinct difference is that they are much more highly ordered than the frog, rabbit or human muscles conventionally used as vertebrate muscle stereotypes. The improved order permits the full application of powerful techniques such as electron microscopy and image processing, and X-ray diffraction and model building. Here ultrastructural aspects of the red and white fibres in typical teleosts are compared through paired sets of electron micrographs, and ultrastructural details of teleost M-bands and Z-bands obtained from the application of three-dimensional reconstruction methods are described. The potential application of teleost muscle in production of “Muscle—The Movie”, which it is hoped will help to define the mechanism of force generation, is introduced.