Temperature thresholds and degree-day model for Marmara gulosa (Lepidoptera: Gracillariidae).

The developmental thresholds for Marmara gulosa Guillén & Davis (Lepidoptera: Gracillariidae) were investigated in the laboratory by using 17, 21, 25, 29, and 33 degrees C. The lowest mortality occurred in cohorts exposed to 25 and 29 degrees C. Other temperatures caused >10% mortality primarily in egg and first and second instar sap-feeding larvae. Linear regression analysis approximated the lower developmental threshold at 12.2 degrees C. High mortality and slow developmental rate at 33 degrees C indicate the upper developmental threshold is near this temperature. The degree-day (DD) model indicated that a generation requires an accumulation of 322 DD for development from egg to adult emergence. Average daily temperatures in the San Joaquin Valley could produce up to seven generations of M. gulosa per year. Field studies documented two, five, and three overlapping generations of M. gulosa in walnuts (Juglans regia L.; Juglandaceae), pummelos (Citrus maxima (Burm.) Merr.; Rutaceae), and oranges (Citrus sinensis (L.) Osbeck; Rutaceae), for a total of seven observed peelminer generations. Degree-day units between generations averaged 375 DD for larvae infesting walnut twigs; however, availability of green wood probably affected timing of infestations. Degree-day units between larval generations averaged 322 for pummelos and 309 for oranges, confirming the laboratory estimation. First infestation of citrus occurred in June in pummelo fruit and August in orange fruit when fruit neared 60 mm in diameter. Fruit size and degree-day units could be used as management tools to more precisely time insecticide treatments to target the egg stage and prevent rind damage to citrus. Degree-day units also could be used to more precisely time natural enemy releases to target larval instars that are preferred for oviposition.

Nitric oxide release is induced by dopamine during illumination of the carp retina: serial neurochemical control of light adaptation.

Several lines of indirect evidence have suggested that nitric oxide may play an important role during light adaptation of the vertebrate retina. We aimed to verify directly the effect of light on nitric oxide release in the isolated carp retina and to investigate the relationship between nitric oxide and dopamine, an established neuromodulator of retinal light adaptation. Using a biochemical nitric oxide assay, we found that steady or flicker light stimulation enhanced retinal nitric oxide production from a basal level. The metabotropic glutamate receptor agonist L-amino-4-phosphonobutyric acid, inhibited the light adaptation-induced nitric oxide production suggesting that the underlying cellular pathway involved centre-depolarizing bipolar cell activity. Application of exogenous dopamine to retinas in the dark significantly enhanced the basal production of nitric oxide and importantly, inhibition of endogenous dopaminergic activity completely suppressed the light-evoked nitric oxide release. The effect of dopamine was mediated through the D1 receptor subtype. Imaging of the nitric oxide-sensitive fluorescent indicator 4,5-diaminofluorescein di-acetate in retinal slices revealed that activation of D1 receptors resulted in nitric oxide production from two main spatial sources corresponding to the photoreceptor inner segment region and the inner nuclear layer. The results taken together would suggest that during the progression of retinal light adaptation there is a switch from dopaminergic to nitrergic control, probably to induce further neuromodulatory effects at higher levels of illumination and to enable more efficient spreading of the light adaptive signal.

Diversion of water flow from a floodplain wetland stream: an analysis of geomorphological setting and hydrological and ecological consequences.

Diversion of water has been ongoing in the Mkuze Wetland for several decades. Two canals form the focus of this study; the Mpempe-Demazane Canal and the Tshanetshe Canal. The former involved an ambitious excavation over a distance of 13.5 km in the lower part of the wetland, while the latter was a minor excavation over a distance of approximately 100 m in the upper part of the wetland. Although ambitious and costly, the Mpempe-Demazane Canal resulted in little downward or headward erosion, and there was minor diversion of flow. However, the minor excavation of the Tshanetshe Canal resulted in erosion downstream of the excavation (the Tshanetshe Stream), downward and lateral erosion of the excavated section, and headward erosion that has propagated almost 4 km upstream along the Mkuze River. Most of the flow of the Mkuze River has been captured by the Tshanetshe Canal and Stream. The impact of canalisation on floodplain wetlands is thus more dependent on the location than the scale of activity. The avulsion of the Mkuze River into the Tshanetshe Canal and Stream is due to a large difference in elevation between the Mkuze River and floodplain into which it was diverted, and the fact that in this region the river typically has high discharges. This avulsion may have been inevitable as a result of natural processes of sedimentation. In contrast, the difference in elevation between the Mkuze River and the basin into which it was diverted via the Mpempe Canal was small as is discharge of the Mkuze River in this part of the wetland. Thus, the diversion was unsuccessful. The presence of hippos that create hydraulically efficient pathways that are oriented parallel to the regional hydraulic slope, may accelerate avulsion in large African wetlands. Overall, it is argued that the environmental consequences of excavation need to be viewed against the background that wetlands are dynamic features within the landscape.

Characterization of cell death pathways in murine retinal neurodegeneration implicates cytochrome c release, caspase activation, and bid cleavage.

Apoptosis is considered to be the final common pathway of photoreceptor cell death in different inherited retinal diseases. However, apoptosis encompasses diverse pathways of molecular interactions culminating in cellular demise. To begin dissecting these interactions, we have investigated key participants in the rd (retinal degeneration) model of retinal neurodegeneration. By Western blot analysis and immunocytochemistry, we found that cytochrome c release occurs in rd retinas concurrently with the activation of the proapoptotic protein Bid. Active forms of caspase-8 and the mitogen-activated protein kinase p38, both of which are capable of cleaving Bid, were detected in rd retinas at the peak time of photoreceptor death. In addition, the activated form of the cell death effector caspase-3 was detectable particularly at the photoreceptors in parallel with this peak degenerative phase. These data suggest that activation of both major apoptotic pathways occurs during photoreceptor degeneration in the rd mouse model of inherited blindness.

Activation of nicotinic receptors on GABAergic amacrine cells in the rabbit retina indirectly stimulates dopamine release.

The retina possesses subpopulations of amacrine cells, which utilize different transmitters, including acetylcholine (ACh), GABA, and dopamine. We have examined interactions between these neurones by studying the effects of nicotinic agonists on GABA and dopamine release. Isolated rabbit retinas were incubated with [3H]dopamine and then superfused. Fractions of the superfusate (2 min) were collected and the [3H]dopamine in each sample was measured. Endogenous GABA release was examined by incubating retinas in a small chamber. At 5-min intervals, the medium was changed and the GABA measured by high-pressure liquid chromatography (HPLC). Exposure of the retina to nicotine, epibatidine, and other nicotinic agonists increased the release of both GABA and dopamine. The effects of nicotine and epibatidine were blocked by mecamylamine, confirming an action on nicotinic receptors. The action of epibatidine on dopamine release was unaffected by glutamate antagonists but was blocked by picrotoxin and gabazine. These results suggested that nicotine might increase dopamine release indirectly by stimulating the release of GABA, which in turn inhibited the release of an inhibitory transmitter acting tonically on the dopaminergic amacrines. Exposure of the retina to GABA caused a small increase in dopamine release. This hypothetical inhibitory transmitter was not GABA, an opioid, adenosine, glycine, nociceptin, a cannabinoid, or nitric oxide because appropriate antagonists did not affect the resting release of dopamine. However, metergoline, a 5HT1/5HT2 receptor antagonist, and ketanserin, a 5HT2A receptor antagonist, but not the 5HT1A antagonist WAY100635, increased the resting release of dopamine and blocked the effects of nicotine. The 5HT1A/5HT7 agonist 8-hydroxy DPAT inhibited both the nicotine and GABA-evoked release of dopamine. We conclude that nicotinic agonists directly stimulate the release of GABA, but the evoked release of dopamine is indirect, and arises from GABA inhibiting the input of an inhibitory transmitter, which we tentatively identify as serotonin.

Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells.

PURPOSE: Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice. METHODS: The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography. RESULTS: Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed. CONCLUSIONS: Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.

Evidence for a complex influence of nicotinic acetylcholine receptors on hippocampal serotonin release.

The effects of nicotine on 5-hydroxytryptamine (5-HT) release from serotonergic nerve endings in rat dorsal hippocampal slices were studied. Nicotine (50-500 microM:) caused a concentration-dependent increase in 5-HT release. This effect was antagonised by mecamylamine (0.5 microM:), indicating an action at nicotinic receptors. Nicotine-evoked 5-HT release was not affected by tetrodotoxin (3 microM:), cadmium chloride (0.1 mM:), or the absence of Ca(2+) or Na(+) in the superfusion medium. Unexpectedly, higher concentrations of mecamylamine alone (1-50 microM:) increased 5-HT release. This suggested the presence of inhibitory input to 5-HT neurones and that these inhibitory neurones possess tonically active nicotinic receptors. The effect of mecamylamine (50 microM:) on 5-HT release was reduced by the muscarinic M(1) receptor agonist, McN-A-343 (100 microM:), but pirenzepine (0.005-1 microM:), which blocks M(1) receptors, alone increased 5-HT release. Hippocampal serotonergic neurones are known to possess both excitatory nicotinic receptors and inhibitory M(1) receptors. Although there may be several explanations for our results, one possible explanation is that nicotine stimulates 5-HT release by activating nicotinic heteroreceptors on 5-HT terminals. Mecamylamine (0.5 microM:) antagonises this effect, but higher concentrations increase 5-HT release indirectly by blocking the action of endogenous acetylcholine on nicotinic receptors situated on cholinergic neurones that provide muscarinic inhibitory input to 5-HT neurones.

Altered expression of secreted frizzled-related protein-2 in retinitis pigmentosa retinas.

PURPOSE: Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive death of the photoreceptors due to apoptosis. To identify changes in gene expression associated with the degenerative state in RP retinas, expression profiling of apoptosis-related genes was performed using a gridded array technique. METHODS: Total RNAs from RP and control retinas were used to generate radiolabeled cDNA probes to screen gridded membrane arrays of 205 apoptosis-related genes. Reverse transcription-polymerase chain reaction was used to generate probes corresponding to differentially expressed genes for Northern blot analysis and for mRNA in situ hybridization studies of retinal cryosections. Fluorescence immunocytochemistry was performed on retinal sections using available antibodies. RESULTS: By expression profiling, we identified upregulated expression of the mRNA for secreted Frizzled-related protein-2 (SFRP2) in RP retina in comparison with control. By Northern blot analysis, SFRP2 mRNA levels were 2- to 20-fold higher in RP samples than in controls. The localization of SFRP2 mRNA by in situ hybridization varied according to the degree of degeneration, from stratified in relatively well-preserved retinas to diffuse in the highly degenerative state. By immunofluorescence, SFRP2 protein in RP retinas was found mainly to colocalize with the cell adhesion and signal transducing protein beta-catenin. CONCLUSIONS: SFRPs can regulate apoptosis in vitro and appear to interact with the Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. Increased SFRP2 expression in RP retinas suggests that an altered pattern of Wnt signal transduction may be a step in the degenerative process linking causal mutations with eventual photoreceptor demise.

Modulated expression of secreted frizzled-related proteins in human retinal degeneration.

Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive loss of photoreceptors, apparently by apoptosis, and our recent report of increased secreted Frizzled-related protein-2 (SFRP2) in RP retinas suggests altered Wnt signalling may be a component of the degenerative process. The present study shows that levels of SFRPI, SFRP3 and SFRP5 mRNAs are also abnormal in RP, giving rise to idiosyncratic expression patterns. In highly degenerative retinas, the SFRP proteins localize mainly to the inner limiting membrane, but in a well-preserved retina SFRPI and SFRP5 are notably localized to the surviving photoreceptors. Together with increased c-jun mRNA expression in all cases examined, these results support the notion that disruptions of Wnt network signalling are involved retinal neurodegeneration.

Identification by array screening of altered nm23-M2/PuF mRNA expression in mouse retinal degeneration.

In the rd/rd mouse model of inherited retinal degeneration, the majority of photoreceptors die apoptotically between postnatal age (P)10 and 20 days, during which period the inner retina appears morphologically unaffected. To examine mRNA changes associated with the degeneration, we performed differential screening of 588 arrayed murine cDNAs using probes reverse-transcribed from P8 predegenerative and control mouse retinal RNAs. We detected altered expression of the gene encoding nm23-M2, a member of the family of nucleoside diphosphate kinases implicated in diverse processes including metastasis suppression and transcriptional regulation. Retinal nm23 mRNA levels increased during degeneration while control levels decreased over age-matched time-points. In situ hybridization showed the high level of expression at P20 in rd/rd was concentrated in the retinal ganglion cells. Previous studies have indicated upregulation of the stress-response related gene alphaB-crystallin in the rd/rd inner retina, and increased nm23 levels may be a component of this response to photoreceptor loss and altered retinal architecture.

Release of endogenous ascorbic acid preserves extracellular dopamine in the mammalian retina.

PURPOSE: To investigate whether the inhibitory effect of nitric oxide (NO) on dopamine release from the retina is due to chemical oxidation of dopamine in the extracellular medium rather than to an inhibitory effect on dopamine release from retinal neurons. METHODS: Dopamine was incubated in Krebs bicarbonate medium and its rate of chemical degradation measured by high-performance liquid chromatography (HPLC). The effects of NO donors and antioxidants on dopamine were assessed by comparing dopamine degradation in the presence and absence of drug. The effects of NO donors on the K-evoked release of [3H]dopamine were measured from isolated superfused rabbit retinas. The release of ascorbic acid from the isolated rat retina and from an eyecup preparation in anesthetized rabbits was measured by HPLC. RESULTS: After 10 minutes' incubation in Krebs bicarbonate medium, the dopamine concentration decreased by 20%. This decline increased to 80% in the presence of S-nitroso-N-acetyl-DL-penicillamine (SNAP) or sodium nitroprusside (SNP). The increased rate of dopamine degradation was abolished if retina was incubated in the medium and then removed before the incubation of dopamine. The protective effect of preincubation with tissue was lost in the presence of ascorbate oxidase suggesting the release of ascorbic acid. HPLC analysis confirmed a substantial release of ascorbic acid from both rabbit and rat retinas. The K-evoked release of [3H]dopamine from the rabbit retina was inhibited by SNP. CONCLUSIONS: NO can rapidly, oxidize dopamine in physiological medium, but in the presence of retina, sufficient endogenous antioxidants (mainly ascorbate) are released to prevent this chemical reaction. Thus, the inhibitory action of NO on dopamine release results from an action on retinal neurons. Ascorbate release in the retina may have an important physiological role in prolonging the life of dopamine, which often has to diffuse long distances from axons in the inner plexiform layer to receptors in other retinal layers.

Retinal expression of gamma-crystallins in the mouse.

PURPOSE: High levels of expression of a form of gamma-crystallin mRNA in mouse retina have been identified. Because the six murine gamma-crystallins have generally been regarded as specific to the lens, the expression of these crystallins at the mRNA and protein levels in the retina were evaluated in more detail. METHODS: Expression of gammaE/F-crystallin mRNA was examined by northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis applied to murine retinal and lens total RNAs. For gammaA-D-crystallin mRNAs, a multiplex RT-PCR was used on total cDNAs. The detection of total gamma-crystallin protein in the retina was performed using an antibody to bovine lens gamma-crystallins, applied to protein extracts in immunoblot analysis and to cryostat sections of ocular tissues in immunofluorescence studies. RESULTS: By RT-PCR, we confirmed expression of both gammaE-and gammaF-crystallin as well as all four (gammaA-gammaD) remaining crystallins at the mRNA level in the mouse retina. Gamma-crystallin proteins were also detectable in murine retina by immunoblot analysis, although at a lower level than in the lens. By immunocytochemistry, gamma-crystallins were localized particularly to the inner retina, outer plexiform layer, and the photoreceptors during postnatal development. CONCLUSIONS: Our findings of gamma-crystallin mRNA and protein expression in the retina indicate that none of the major crystallin classes is uniquely expressed in the lens. The expression of gamma-crystallins in the developing murine retina suggests a role analogous to the anti-stress properties established for the small heat-shock protein alphaB-crystallin, perhaps in response to varying exposure to light.

Effect of targeted expression of clusterin in photoreceptor cells on retinal development and differentiation.

Clusterin expression is increased in tissues undergoing apoptosis, including neurodegenerative retina, but the causal relationships remain to be clarified. To test the hypothesis that overexpression of clusterin could induce apoptosis in neurons, transgenic mice were generated in which rat clusterin transgene was expressed in photoreceptor cells under the transcriptional control of the human interphotoreceptor retinoid-binding protein (IRBP) promoter. Photoreceptor cell death in the resulting transgenic mice was examined by histology and TUNEL techniques. The expression of the clusterin transgene was confirmed by in situ hybridization in the photoreceptor cells, and results in a complex pattern of clusterin protein distribution in the retina. A reduction in apoptotic staining in the transgenic retinas was observed from birth to postnatal day 15. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor cell death, but may relate to cytoprotective functions.

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration.

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.

Expression patterns of neurturin and its receptor components in developing and degenerative mouse retina.

PURPOSE: Neurturin (NTN) and its receptor components (GFRalpha2 and Ret) play an important role in the survival of different populations of neurons in the central and peripheral nervous systems. To gain insight into their possible functions throughout normal retinal development and during retinal neuronal apoptosis, the retinal distribution of expression of NTN and GFRalpha2 mRNAs and Ret protein were compared in control and retinal degeneration (rd) mice. METHODS: Eyes from control and rd animals were fixed in paraformaldehyde before sectioning. For in situ hybridization, retinal sections were hybridized with 35S-radiolabeled sense and antisense riboprobes for murine NTN and GFRalpha2 and were autoradiographed. Ret localization was detected by immunofluorescence. RESULTS: Neurturin mRNA expression was modulated through normal postnatal retinal development and was localized primarily to the inner retina and photoreceptor outer segments. GFRalpha2 mRNA displayed a diffuse developmental pattern of expression, but in the mature normal retina, NTN and GFRalpha2 mRNAs were more closely colocalized. Ret protein was localized particularly at the outer segments of photoreceptors, inner retina, and ganglion cell layers, but there were no prominent differences among genotypes. Increased NTN mRNA expression was detected in the retinal pigment epithelium and neural retina in concert with photoreceptor degeneration in rd mouse. In contrast, the level of GFRalpha2 mRNA was lower in rd compared with that in normal retina. CONCLUSIONS: These results suggest that NTN and its receptor are involved in retinal postnatal development and maintenance and that alterations in their transcription patterns are associated with inherited retinal degeneration.

Expression of Pax-6 mRNA in the retinal degeneration (rd) mouse.

The homeobox gene Pax-6 is expressed during eye development in both the retina and lens, and Pax-6 mutations cause ocular abnormalities including retinal defects. We investigated the pattern of Pax-6 gene expression in the rd/rd mouse model of inherited retinal degeneration in comparison with nondegenerative controls, using Northern blot, reverse-transcription (RT)-PCR and in situ hybridization analysis. We observed an increased level of Pax-6 mRNA expression in the degenerative state, which appeared to affect equally the major Pax-6 exon 5a transcriptional splice variants as detected by RT-PCR. By in situ hybridization, Pax-6 mRNA was localized to the inner nuclear and ganglion cell layers of nondegenerative retina, but showed a more diffuse signal pattern in the rd/rd retina. This modulation of Pax-6 mRNA levels and localization is suggestive of activation of expression in retinal glial cells and may reflect reorganization of cellular interactions in response to the degenerative processes.

Modulation of extracellular GABA levels in the retina by activation of glial P2X-purinoceptors.

1. In the rat retina, gamma-aminobutyric acid (GABA) released as a transmitter is inactivated by uptake mainly into glial cells (Müller cells). Activation of P2-purinoceptors in Müller cells increases [Ca2+]i and the present study was undertaken to see whether this action affected the glial release of [3H]-GABA from the superfused rat isolated retina. 2. Adenosine 5'-triphosphate (ATP) and the P2X-purinoceptor agonists, alpha,beta-methylene-ATP (alpha,beta-meATP) and beta,gamma-methyleneATP (beta,gamma-meATP) significantly increased the KCl-evoked release of [3H]-GABA from the retina. 3. Adenosine and the P2Y-purinoceptor agonist, 2-chloroATP, had no effect on the KCl-evoked release of [3H]-GABA from the retina. However, 2-methylthioATP (2-Me-S-ATP) significantly enhanced the evoked release of [3H]-GABA. 4. The effect of ATP on the glial release of [3H]-GABA was abolished by the P2-antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. When the superfused retina was exposed to the GABA uptake inhibitor, SKF89976A, the enhancing effect of alpha,beta-meATP on the KCl-evoked release of GABA was abolished. 6. The KCl-evoked release of [3H]-GABA from the frog retina and rat cerebrocortical slices, which take up GABA mainly into neurones, was not affected by ATP or alpha,beta-meATP. 7. We concluded that the glial Müller cells in the rat retina possess P2-receptors, activation of which increases the 'release' of preloaded [3H]-GABA apparently by reducing uptake. On balance, the results suggest the involvement of P2X-purinoceptors, although we cannot exclude the possibility that P2Y-purinoceptors may be involved. Our results suggest that ATP, as well as being a conventional transmitter in the retina, may be involved in neuronal-glial signalling and modulate the extracellular concentration of GABA.

Expression of alphaB-crystallin in a mouse model of inherited retinal degeneration.

AlphaB-crystallin, which is abundantly expressed in the lens but also in a diversity of other tissues, functions as a stress-inducible molecular chaperone and is increased in brain neurodegenerative diseases. We compared retinal alphaB-crystallin expression in a model of inherited retinal degeneration, the rd mouse, and controls. Northern and in situ hybridization analysis showed alphaB-crystallin mRNA to have an altered spatio-temporal pattern with increased levels localized to glial cells in the degenerative state. Immunocytochemistry confirmed increased expression at Müller cells and astrocytes, together with transiently increased localization to the degenerating photoreceptors. These findings suggest that increased alphaB-crystallin expression is associated with glial cell reaction to neuronal damage in the retina, and may comprise part of the retina's overall defensive response to the stress of apoptotic photoreceptor cell death.

Localization of tissue inhibitor of metalloproteinases-3 in neurodegenerative retinal disease.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is one of a family of genes whose products are implicated in the regulation of remodelling of the extracellular matrix. The level of mRNA coding for TIMP-3 is increased in retinas affected by the photoreceptor degenerative disease, simplex retinitis pigmentosa (RP), and mutations in TIMP-3 are associated with an inherited form of macular dystrophy. Here we compare TIMP-3 protein expression in normal retina and in those affected by RP and by age-related macular degeneration. Immunoreactive TIMP-3 is present in normal retinal pigment epithelium, and in degenerative retinas particularly at Bruch's membrane and additionally in photoreceptor-retaining regions in simplex RP. The pattern suggests a role for TIMP-3 in normal retinal homeostasis, and, in the disease state, in the modulation of extracellular matrix metabolism and neovascularization.

Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration.

Knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. Towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (AAV) vector to deliver and express the correct form of the cGMP phosphodiesterase-beta (PDE-beta) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessive mutation in the endogenous gene. A truncated murine opsin promoter was used to drive expression of the PDE-beta cDNA. Following intraocular injection of AAV. PDE-beta, increased retinal expression of immunoreactive PDE protein was observed, including within photoreceptor cell bodies. Compared with age-matched controls, treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light as measured by in vitro electroretinography. These findings provide evidence that rescue of functional photoreceptor neurons can be achieved by somatic gene therapy.