Progressive Retinal Degeneration and Glial Activation in the CLN6nclf Mouse Model of Neuronal Ceroid Lipofuscinosis: A Beneficial Effect of DHA and Curcumin Supplementation

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6^nclf mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6^nclf retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6^nclf retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6^nclf mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6^nclf retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6^nclf retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.     

Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats

Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats.S.-P.H. and P.-K.L. contributed equally to this work.     

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.     

Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6 ^nclf/nclf cerebellar cells and compared them to wild-type and CbCln3 ^{Δex7/8/Δex7/8} cerebellar cells. CbCln6 ^nclf/nclf cells and CbCln3 ^{Δex7/8/Δex7/8} cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6 ^nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6 ^nclf/nclf and CbCln3 ^{Δex7/8/Δex7/8} cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3 ^Δex7/8 and Cln6 ^nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.     

A Murine Model of Variant Late Infantile Ceroid Lipofuscinosis Recapitulates Behavioral and Pathological Phenotypes of Human Disease

Neuronal ceroid lipofuscinoses (NCLs; also known collectively as Batten Disease) are a family of autosomal recessive lysosomal storage disorders. Mutations in as many as 13 genes give rise to ∼10 variants of NCL, all with overlapping clinical symptomatology including visual impairment, motor and cognitive dysfunction, seizures, and premature death. Mutations in CLN6 result in both a variant late infantile onset neuronal ceroid lipofuscinosis (vLINCL) as well as an adult-onset form of the disease called Type A Kufs. CLN6 is a non-glycosylated membrane protein of unknown function localized to the endoplasmic reticulum (ER). In this study, we perform a detailed characterization of a naturally occurring Cln6 mutant (Cln6^nclf) mouse line to validate its utility for translational research. We demonstrate that this Cln6^nclf mutation leads to deficits in motor coordination, vision, memory, and learning. Pathologically, we demonstrate loss of neurons within specific subregions and lamina of the cortex that correlate to behavioral phenotypes. As in other NCL models, this model displays selective loss of GABAergic interneuron sub-populations in the cortex and the hippocampus with profound, early-onset glial activation. Finally, we demonstrate a novel deficit in memory and learning, including a dramatic reduction in dendritic spine density in the cerebral cortex, which suggests a reduction in synaptic strength following disruption in CLN6. Together, these findings highlight the behavioral and pathological similarities between the Cln6^nclf mouse model and human NCL patients, validating this model as a reliable format for screening potential therapeutics.     

Pre-induced adult human peripheral blood mononuclear cells migrate widely into the degenerative retinas of rd1 mice

Background aimsRecent advances in stem cell research have raised the possibility of stem cells repairing or replacing retinal photoreceptor cells that are either dysfunctional or lost in many retinal diseases. Various types of stem cells have been used to replace retinal photoreceptor cells. Recently, peripheral blood stem cells, a small proportion of pluripotent stem cells, have been reported to mainly exist in the peripheral blood mononuclear cells (PBMCs).MethodsIn this study, the effects of pre-induced adult human PBMCs (hPBMCs) on the degenerative retinas of rd1 mice were investigated. Freshly isolated adult hPBMCs were pre-induced with the use of the conditioned medium of rat retinas for 4 days and were then labeled with chloromethyl-benzamidodialkylcarbocyanine (CM-DiI) and then transplanted into the subretinal space of the right eye of rd1 mice through a trans-scleral approach. The right eyes were collected 30 days after transplantation. The survival and migration of the transplanted cells in host retinas were investigated by whole-mount retinas, retinal frozen sections and immunofluorescent staining.ResultsAfter subretinal transplantation, pre-induced hPBMCs were able to survive and widely migrate into the retinas of rd1 mice. A few CM-DiI–labeled cells migrated into the inner nuclear layer and the retinal ganglion cell layer. Some transplanted cells in the subretinal space of rd1 host mice expressed the human photoreceptor–specific marker rhodopsin.ConclusionsThis study suggests that pre-induced hPBMCs may be a potential cell source of cell replacement therapy for retinal degenerative diseases.     

CNTF Induces Regeneration of Cone Outer Segments in a Rat Model of Retinal Degeneration

Background

Cone photoreceptors are responsible for color and central vision. In the late stage of retinitis pigmentosa and in geographic atrophy associated with age-related macular degeneration, cone degeneration eventually causes loss of central vision. In the present work, we investigated cone degeneration secondary to rod loss in the S334ter-3 transgenic rats carrying the rhodopsin mutation S334ter.

Methodology/Principal Findings

Recombinant human ciliary neurotrophic factor (CNTF) was delivered by intravitreal injection to the left eye of an animal, and vehicle to the right eye. Eyes were harvested 10 days after injection. Cone outer segments (COS), and cell bodies were identified by staining with peanut agglutinin and cone arrestin antibodies in whole-mount retinas. For long-term treatment with CNTF, CNTF secreting microdevices were implanted into the left eyes at postnatal day (PD) 20 and control devices into the right eyes. Cone ERG was recorded at PD 160 from implanted animals. Our results demonstrate that an early sign of cone degeneration is the loss of COS, which concentrated in many small areas throughout the retina and is progressive with age. Treatment with CNTF induces regeneration of COS and thus reverses the degeneration process in early stages of cone degeneration. Sustained delivery of CNTF prevents cones from degeneration and helps them to maintain COS and light-sensing function.

Conclusions/Significance

Loss of COS is an early sign of secondary cone degeneration whereas cell death occurs much later. At early stages, degenerating cones are capable of regenerating outer segments, indicating the reversal of the degenerative process. Sustained delivery of CNTF preserves cone cells and their function. Long-term treatment with CNTF starting at early stages of degeneration could be a viable strategy for preservation of central vision for patients with retinal degenerations.     

Oncostatin M protects rod and cone photoreceptors and promotes regeneration of cone outer segment in a rat model of retinal degeneration

Retinitis pigmentosa (RP) is a group of photoreceptor degenerative disorders that lead to loss of vision. Typically, rod photoreceptors degenerate first, resulting in loss of night and peripheral vision. Secondary cone degeneration eventually affects central vision, leading to total blindness. Previous studies have shown that photoreceptors could be protected from degeneration by exogenous neurotrophic factors, including ciliary neurotrophic factor (CNTF), a member of the IL-6 family of cytokines. Using a transgenic rat model of retinal degeneration (the S334-ter rat), we investigated the effects of Oncostatin M (OSM), another member of the IL-6 family of cytokines, on photoreceptor protection. We found that exogenous OSM protects both rod and cone photoreceptors. In addition, OSM promotes regeneration of cone outer segments in early stages of cone degeneration. Further investigation showed that OSM treatment induces STAT3 phosphorylation in Müller cells but not in photoreceptors, suggesting that OSM not directly acts on photoreceptors and that the protective effects of OSM on photoreceptors are mediated by Müller cells. These findings support the therapeutic strategy using members of IL-6 family of cytokines for retinal degenerative disorders. They also provide evidence that activation of the STAT3 pathway in Müller cells promotes photoreceptor survival. Our work highlights the importance of Müller cell-photoreceptor interaction in the retina, which may serve as a model of glia-neuron interaction in general.     

All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Ciliary neurotrophic factor: a survival and differentiation inducer in human retinal progenitors

Retinitis pigmentosa, age-related macular degeneration, and Parkinson’s disease remain major problems in the field of medicine. Some of the strategies being explored for treatment include replacement of damaged tissue by transplantation of healthy tissues or progenitor cells and delivery of neurotrophins to rescue degenerating tissue. One of the neurotrophins with promise is the ciliary neurotrophic factor (CNTF). In this study, we report the role played by CNTF in retinal cell differentiation and survival in retinal progenitors. We found that CNTF is a survival factor for multipotential human retinal cells and increased cell survival by 50%, over a 7-d period, under serum-free conditions, as determined by apoptotic assays (immunohistochemistry and flow cytometry). This effect is dose dependent with a maximum survival at a CNTF concentration of 20 ng/ml. We also report that CNTF might be a cell commitment factor, directing the differentiation mainly toward large multipolar cells with ganglionic and amacrine phenotype. These cells express tyrosine hydroxylase (amacrine cells) as well as, thy 1.1 and neuron-specific enolase (ganglionic cells). Additionally, there was also an increase in protein kinase C alpha, a protein expressed in rod and cone bipolars as well as cone photoreceptors and calbindin, a protein expressed in cone photoreceptors and horizontal cells. In our studies, CNTF doubled the number of cells with ganglionic phenotypes, and basic fibroblast growth factor doubled the number of cells with photoreceptor phenotype. Additionally, CNTF induced a subset of progenitors to undergo multiple rounds of cell division before acquiring the large multipolar ganglionic phenotype. Our conclusion is that CNTF could be an agent that has therapeutic potential and possibly induces differentiation of large multipolar ganglionic phenotype in a subset of progenitors.     

Müller Cell Reactivity in Response to Photoreceptor Degeneration in Rats with Defective Polycystin-2

Background

Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2.

Methods

Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution.

Findings

Müller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators.

Interpretation

Cellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.     

Ciliary neurotrophic factor blocks rod photoreceptor differentiation from postmitotic precursor cells in vitro

The development of photoreceptors in the mammalian retina is thought to be controlled by extrinsic signals. We have shown previously that ciliary neurotrophic factor (CNTF) potently inhibits photoreceptor differentiation in cultures of rat retina. The present study analyzes which developmental processes are affected by CNTF. Rod differentiation as determined by opsin and recoverin immunocytochemistry was effectively blocked by CNTF and leukemia inhibitory factor, but not by other neurotrophic agents tested. CNTF did not influence proliferation, cell death, or survival, and had no effect on the downregulation of nestin immunoreactivity in progenitor cells. Opsin-positive rods could be reverted to an opsin-negative state initially, but became unresponsive to CNTF later. No compensatory increase in the number of other cell types was observed. Application of neutralizing antibodies against CNTF revealed that rod development was partially blocked by an endogenous CNTF-like molecule in control cultures. Our results suggest that CNTF can act as a specific negative regulator of rod differentiation. Its action on photoreceptor precursor cells could serve to synchronize the maturation of photoreceptors, which are born over an extended period of time. Together with other stimulatory signals, CNTF may thus control the temporally and numerically correct integration of photoreceptors into the retinal network.     

Genetic Expression of Cyclic GMP Phosphodiesterase Activity Defines Abnormal Photoreceptor Differentiation in Neurological Mutants of Inherited Retinal Degeneration

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy‐induced apoptosis via modulation of retinal glia in vivo

Adenoviral‐mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy‐induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRα) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral‐mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter‐1 (GLAST‐1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRα, pSTAT₃, GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Palmitoylation-induced Aggregation of Cysteine-string Protein Mutants That Cause Neuronal Ceroid Lipofuscinosis

Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families.     

Rescue from Photoreceptor Degeneration in the rd Mouse by Human Immunodeficiency Virus Vector-Mediated Gene Transfer

Retinitis pigmentosa (RP) is the most common inherited retinal disease, in which photoreceptor cells degenerate, leading to blindness. Mutations in the rod photoreceptor cGMP phosphodiesterase beta subunit (PDEbeta) gene are found in patients with autosomal recessive RP as well as in the rd mouse. We have recently shown that lentivirus vectors based on human immunodeficiency virus (HIV) type 1 achieve stable and efficient gene transfer into retinal cells. In this study, we evaluated the potential of HIV vector-mediated gene therapy for RP in the rd mouse. HIV vectors containing a gene encoding a hemagglutinin (HA)-tagged PDEbeta were injected into the subretinal spaces of newborn rd mouse eyes. One to three rows of photoreceptor nuclei were observed in the eyes for at least 24 weeks postinjection, whereas no photoreceptor cells remained in the eyes of control animals at 6 weeks postinjection. Expression of HA-tagged PDEbeta in the rescued photoreceptor cells was confirmed by two-color confocal immunofluorescence analysis using anti-HA and anti-opsin antibodies. HIV vector-mediated gene therapy appears to be a promising means for the treatment of recessive forms of inherited retinal degeneration.     

Rhythmic Ganglion Cell Activity in Bleached and Blind Adult Mouse Retinas

In retinitis pigmentosa – a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor’s dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the understanding of the degeneration process and may guide future rescue strategies.     

Increased Expression of Retinal TIMP3 mRNA in Simplex Retinitis Pigmentosa Is Localized to Photoreceptor-Retaining Regions

Abstract: The human tissue inhibitor of metalloproteinases-3 (TIMP3) gene is the most recently characterized member of a family of genes whose products are implicated in extracellular matrix (ECM) remodelling. We previously described an increase in expression of TIMP3 mRNA in retinas affected by the progressive photoreceptor degenerative disease, simplex retinitis pigmentosa (RP). To gain further insight into the association between TIMP3 overexpression and retinal degeneration, we have analyzed the cellular localization of TIMP3 mRNA in control and simplex RP retinas using in situ hybridization. No TIMP3 mRNA expression was detectable in control neural retina. In RP-affected retinas, overexpression of TIMP3 mRNA was observed in photoreceptor inner segments and in the ganglion cell layer only in those regions retaining relatively nondystrophic retinal architecture. Modulation of TIMP3 expression in these regions, possibly in association with matrix metalloproteinases, may reflect remodelling of the retinal ECM and concomitant reorganization of neuronal connectivity.