Ocular cell transfection with the human basic fibroblast growth factor gene delays photoreceptor cell degeneration in RCS rats.

Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and 19%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic fibroblast growth factor isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to 17% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.

Quantitative analysis of subretinal injections in the rat.

BACKGROUND: Experimental therapeutic approaches to retinal degenerations often require the subretinal injection of a therapeutic agent. The injected volume and the age of the animal can influence the proportion of the retinal surface affected by the subretinal injection. We have investigated the quantitative effect of a single injection in the subretinal space. METHODS: Normal and Royal College of Surgeons rats aged 1 week, 3 weeks or 2 months received subretinal transscleral injections of 1, 3, 5 or 10 microl China ink. After 24 h, animals were killed, injected eyes were enucleated and fixated, and the retinas flattened. An image analyzing program was used to measure the total retinal surface and the retinal surface affected by the dye. RESULTS: The mean retinal surface affected by the injection ranged from 5.24+/-2.76 mm2 to 14.8+/-2.3 mm2, depending on animal age and injected volume. The injection affected 8.79+/-0.89 to 36.9+/-8.13% of total retinal surface. There was no statistically significant difference between normal and Royal College of Surgeons rats. Intravitreal leakage of the dye was more frequent with increasing injection volumes. CONCLUSION: The retinal surface affected by a single subretinal injection increases with the injected volume, but this increase is not proportional. Higher volumes lead to a loss of injected solution, either in the vitreous body or through the sclerotomy. In 2-month-old rats, a 3-microl subretinal injection appears to have the best reproducibility, with 20-30% of retinal surface covered by the injected dye.

Intravitreous transplantation of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delays photoreceptor cell degeneration in Royal College of Surgeons rats.

We developed an experimental approach with genetically engineered and encapsulated mouse NIH 3T3 fibroblasts to delay the progressive degeneration of photoreceptor cells in dark-eyed Royal College of Surgeons rats. These xenogeneic fibroblasts can survive in 1. 5-mm-long microcapsules made of the biocompatible polymer AN69 for at least 90 days under in vitro and in vivo conditions because of their stable transfection with the gene for the 18-kDa form of the human basic fibroblast growth factor (hFGF-2). Furthermore, when transferred surgically into the vitreous cavity of 21-day-old Royal College of Surgeons rats, the microencapsulated hFGF-2-secreting fibroblasts provoked a local delay of photoreceptor cell degeneration, as seen at 45 days and 90 days after transplantation. This effect was limited to 2.08 mm2 (45 days) and 0.95 mm2 (90 days) of the retinal surface. In both untreated eyes and control globes with encapsulated hFGF-2-deficient fibroblasts, the rescued area (of at most 0.08 mm2) was significantly smaller at both time points. Although, in a few ocular globes, surgical trauma induced a reorganization of the retinal cytoarchitecture, neither microcapsule rejection nor hFGF-2-mediated tumor formation were detected in any treated eyes. These findings indicate that encapsulated fibroblasts secreting hFGF-2 or perhaps other agents can be applied as potential therapeutic tools to treat retinal dystrophies.

A human tyrosine hydroxylase isoform associated with progressive supranuclear palsy shows altered enzymatic activity.

A novel human tyrosine hydroxylase (HTH) messenger RNA subgroup generated by alternative splicing and characterized by the absence of the third exon was recently identified. The corresponding putative protein lacks 74 amino acids including Ser31 and Ser40, two major phosphorylation sites implicated in the regulation of HTH activity. These mRNA species are detected in adrenal medulla and are overexpressed in patients suffering from progressive supranuclear palsy, a neurodegenerative disease mostly affecting catecholaminergic neurons of the basal ganglia. In the present work, an HTH protein isoform lacking exon 3 was identified in human adrenal medulla. For this purpose, an antibody was raised against the HTH exon 3. The effect of the removal of exon 3 on the enzymatic activity of HTH was studied in vitro by comparing a purified recombinant fusion protein without exon 3 (glutathione S-transferase (GST)-HTHDelta3) to the equivalent protein containing exon 3 (GST-HTH3). In initial velocity conditions, GST-HTHDelta3 has 30% of the maximal velocity of GST-HTH3. Moreover, the skipping of exon 3 results in the absence of activation of GST-HTH by heparin and increases by 10-fold the retroinhibition constant for dopamine, demonstrating the involvement of exon 3 in the regulation of HTH enzymatic activity. The identification of a variably expressed HTH isoform that lacks an exon implicated in activity regulation supports the view that HTH alternative splicing contributes to the functional diversity within the catecholaminergic system and may be implicated in some neurological diseases.

Identification of novel PAX6 mutations in two families with bilateral aniridia. Mutations in brief no. 167. Online.

We report two novel PAX6 mutations in aniridia patients of two Swiss pedigrees (We, Sc) which give rise to different phenotypes. An SSCP analysis of the PAX6 14 exons reveals electrophoretic mobility shifts exclusively in exons 5 and 12 of aniridia patients. As determined by bidirectional sequencing and restriction digest analysis, these shifts are caused by mono-allelic base transitions in exon 5 (c.547C-->T; R44X; We) and intron 12 (IVS12+5G-->A; Sc). Each mutation co-segregates with the trait in the affected family with complete penetrance. The Sc mutation in the splicing donor site of intron 12 may result in either intron inclusion or exon skipping, both giving rise to a truncated PAX6 protein which may retain a residual transactivating activity. In contrast, the We genetic alteration is a loss-of-function mutation leading to a more severe phenotype than that observed in the Sc pedigree.

Characterization and sleep deprivation-induced expression modulation of dendrin, a novel dendritic protein in rat brain neurons.

We report on the characterization of the novel rat brain protein dendrin which is encoded by the brain-specific transcript 464. On immunoblots, two protein variants (81 kD, 89 kD) were identified in cytosolic and membraneous protein fractions. The variants are most abundant in the hippocampus, notably in apical dendrites of CA1 pyramidal cells. Dendritic and perikaryal immunolabelling is apparent in neurons of the cerebral cortex, dentate gyrus, subiculum, amygdala, and preoptic areas. In cortical and hippocampal dendrites, electron-dense immunoreaction is associated with the endoplasmic reticulum, the plasma membrane, and spine heads. An association of dendrin with polyribosomes and the presence of its mRNA in dendrites both provide evidence for dendritic mRNA translation. In the rat forebrain, dendrin expression is altered after an extended period of wakefulness. Twenty-four-hour sleep deprivation decreases the mRNA and protein concentrations of both variants in subcortical forebrain plus midbrain areas by 24 +/- 11% (P < 0.05) and 40 +/- 14% (P < 0.1), respectively, as measured relative to beta-actin mRNA and neural actin. In the cerebral cortex and hippocampus, the relative mRNA level remains unchanged whereas the cortical protein concentration is reduced by 42 +/- 10% (P < 0.05). Thus, dendrin belongs to a new class of dendritic proteins whose expression is differentially modulated by prolonged behavioral activity.

Neurogranin is locally concentrated in rat cortical and hippocampal neurons.

The rat protein kinase C substrate neurogranin has a granular distribution in cortical and hippocampal neurons. We demonstrate that in these cells, granular labelling corresponds to a local concentration of neurogranin-immunoreactivity at both the membranes of mitochondria and trans-Golgi vesicles and soma-proximal dendritic shaft and spinal head structures. Our findings suggest that the function of neurogranin could be affected by protein assembly at these cellular sites.

Sleep deprivation differentially alters the mRNA and protein levels of neurogranin in rat brain.

The mRNA level of the 17-kDa protein neurogranin (NG), a postsynaptic substrate of the protein kinase C, has previously been found to be decreased in rat forebrain after 24-h sleep deprivation (SD). To investigate the functional significance of this finding in various forebrain regions, the effect of 24-h SD on the mRNA level and the protein level of NG was determined in the cerebral cortex, hippocampus, and the total of the remaining subcortical forebrain plus midbrain areas (SFMA) of rats. In these areas, high levels of both NG mRNA and NG protein were detected by in situ hybridization and immunohistochemistry, respectively. NG protein was recognized in brain tissue by newly developed polyclonal antibodies. As determined by RNase protection assays, the level of NG mRNA was decreased in SFMA by 34 +/- 7% (P < 0.05) after 24-h SD, and was not significantly affected in the cerebral cortex and hippocampus. In contrast, on Western blots, the protein concentration of NG was reduced in the cerebral cortex by 37 +/- 7% (P < 0.05) whereas no significant changes were present in other brain areas tested. The results indicate that the mRNA and protein levels of NG are differentially modulated in rat brain by the prolongation of the waking period.

24-h variation of vigilance in the cockroach Blaberus giganteus.

Our objective was to investigate whether sleep-like states occur in the cockroach, Blaberus giganteus by applying the methods formerly used for another cockroach species, Leucophaea maderae and the scorpions, Heterometrus and Pandinus. The behaviour of isolated animals (n = 10) kept under LD 12:12 h was recorded by time-lapse video for three consecutive 24-h periods. Nine behavioural states were scored for 1-min real-time epochs. Rest was subdivided into 4 sub-states on the basis of body posture and the position of the antennae. The cockroaches showed a nocturnal behaviour exhibiting a bout of locomotion at dark onset which lasted several hours, and a preference for rest in the light period. Immobility with both the body and the antennae touching the substrate (state 1) was the predominating state in the light period. In order to establish whether the sub-states of rest represented different levels of vigilance the arousal threshold was measured by determining the latency of a behavioural response to a vibration stimulus. The levels of arousal differed significantly in four behavioural states in the light period but not in the dark period. In state 1 the animals exhibited the lowest arousal whereas in the activity states arousal was the highest. The state with the highest arousal threshold occurred in the beginning of the light period. Thereafter, arousal progressively increased and remained relatively high during the dark period. The effect of 6-h deprivation of rest by the gentle shaking of the cages whenever the animals were immobile, resulted in a reduced latency to state 1, a small increase of state 1 and a more prominent initial increase of activity during recovery. In conclusion, this study provides evidence for the existence of a 24-h variation of vigilance in the cockroach. It further indicates that a 'rest deficit' gives rise to a compensatory response. The data support the notion that sleep-like states are present in these insects.