Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy (CAR) syndrome. In these studies we examined the effect of anti-recoverin antibodies from the sera of patients with CAR and rat monoclonal antibody on the retinas of Lewis rats. Anti-recoverin autoanti-bodies penetrated into the photoreceptor and bipolar cell layers following intravitreal injection. Their presence in the retina could be detected by immunofluorescence 24 h after injection. At the same time, individual cells undergoing apoptosis were identified throughout photoreceptor and bipolar cell layers using terminal transferase-mediated dUTP nick-end labeling (TUNEL) and electron microscopy. Normal antibodies used in control experiments did not produce TUNEL labeling. At 24 h, DNA fragmentation was confirmed by DNA ladder electrophoresis. At the electron microscopic level, there was clear evidence of cells undergoing apoptotic cell death in the retinas treated with anti-recoverin antibodies. At 24 and 96 h, nuclear chromatin condensation and increased vacuolization of photoreceptor outer segments were observed. An examination of retinas from animals receiving anti-retinal antibodies revealed a loss of 1-2 rows of nuclei in the outer and inner nuclear layers whereas all controls (sham, normal IgG, phosphate buffered saline) showed an unchanged number of nuclei rows. In addition, there was an increase in spacing between the rows of nuclei of the outer nuclear layer in retinas treated with anti- recoverin antibodies, indicating additional cell loss. These studies provide clear evidence that anti-recoverin antibodies are capable of penetrating photoreceptor and bipolar cells, the normal site of recoverin expression in the retina, and that anti-recoverin antibodies produce apoptotic cell death. A similar mechanism may occur in patients with CAR, which may lead to visual loss and blindness.

Spatial and temporal expression of AP-1 responsive rod photoreceptor genes and bZIP transcription factors during development of the rat retina.

PURPOSE: The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes contains highly conserved cis-acting elements, which include an AP-1 and/or Nrl response element (NRE: An extended AP-1 like sequence). Transactivation of AP-1 or NRE appears necessary to drive expression of these rod-specific genes during adulthood, however, their role during development is relatively unknown. Therefore, we determined the spatial and temporal relationships between rod morphological and functional development, rod-specific gene expression, and expression of the bZIP transcription factors c-fos, junD and Nrl. METHODS: Retinas from 0-45 day old (PN0-45) dark- and light-adapted Long-Evans rats were used. Morphological development was monitored by light and electron microscopy. Whole retinal trypsin-activated cGMP-PDE activity and rhodopsin content were measured biochemically. The expression of opsin, beta-PDE, c-fos, junD and Nrl mRNAs were determined by Northern blot analysis. The cellular localization of Nrl was examined with in situ hybridization. RESULTS: The mRNAs for opsin, beta-PDE and c-fos were observed at PN0-2, while cGMP-PDE activity and rhodopsin were detected first at PN5: coincident with rod outer segment development. The developmental pattern of cGMP-PDE activity and rhodopsin accumulation paralleled the expression of beta-PDE and opsin mRNA and all reached their maximal levels by PN45. Nrl expression, for all three transcripts found in the rat retina, was low on PN2 and reached its maximal level at PN14. The c-fos and Nrl expression preceded beta-PDE and opsin mRNA expression by 1-2 days. Nrl expression was detected first in the distal post-mitotic retina at PN5 and then in all nuclear layers during retinal development. Maximal expression shifted from the ganglion cells to the outer nuclear layer as the neural retina matured. In contrast, junD expression was highest at PN0 and declined to a stable level by PN10. CONCLUSIONS: Colocalization of Nrl and c-Fos suggests that expression of rod-specific genes, which utilize AP-1 or NRE sites in their promoter, could be regulated through the formation of Nrl-Fos dimers. We hypothesize that Nrl and c-Fos play a fundamental role in the initiation and regulation of the rod-specific gene expression in developing and adult rod photoreceptors.

Lead-induced alterations in retinal cGMP phosphodiesterase trigger calcium overload, mitochondrial dysfunction and rod photoreceptor apoptosis.

Lead exposure results in the selective apoptotic loss of rods and bipolar cells. During and following developmental lead exposure rod/retinal cGMP phosphodiesterase expression and activity are delayed in onset and decreased, [Ca2+] is elevated, and mitochondrial ATP synthesis is decreased. In vitro studies, using retinas incubated in Ca2+ and/or Pb2+, demonstrate that rods selectively die by apoptosis, retinal mitochondrial ATP synthesis is decreased, mitochondrial cytochrome c is released and caspase activity is increased. These results suggest that lead-induced rod and bipolar cell apoptosis is triggered by Ca2+ and Pb2+ overload due to altered cGMP phosphodiesterase activity and that mitochondrial alterations play a central role in this process.

Functional alterations and apoptotic cell death in the retina following developmental or adult lead exposure.

Long-term visual system deficits occur in man and animals following developmental and occupational lead exposure. Recent experimental data suggests that the adult brain is not altered following lead exposure. Therefore, the aim of these studies was to use the retina as a CNS model to examine and compare the morphological, biochemical and electroretinographic (ERG) changes occurring in rats exposed to low or moderate levels of lead during development (0-21 days of age) with those occurring in adult rats with equivalent blood and retinal levels of lead for three or six weeks. Five main results were obtained. First, developmental and adult lead exposure for six weeks produced age- and dose-dependent retinal degeneration such that rods and bipolar cells were selectively lost. At the ultrastructural level, all dying cells exhibited the classical morphological features of apoptotic cell death. Second, in the lead-exposed groups, the decrease in the number of rods was correlated with the loss of rhodopsin content per eye confirming that rods were directly affected by lead. Third, single-flash rod ERGs and cone ERGs obtained from developmentally and adult lead-exposed rats demonstrated that there were age- and dose-dependent decreases in the rod a-wave and b-wave sensitivity and maximum amplitudes without any effect on cones. In adult rats exposed to lead for three weeks, qualitatively similar ERG changes occurred in the absence of cell loss or decrease in rhodopsin content. Fourth, developmental and adult lead exposure for three and six weeks produced age- and dose-dependent decreases in retinal cGMP phosphodiesterase (PDE) activity resulting in increased cGMP levels. Fifth, picomolar to micromolar concentration of free lead directly inhibited rat retinal and purified bovine rod cGMP PDE. In summary, there are three main conclusions. First, the retinas of developing and adult rats exposed to lead exhibit qualitatively similar rod-mediated ERG alterations as well as rod and bipolar apoptotic cell death. Thus, developing and mature retinas are both sensitive to the adverse effects of lead: albeit to significantly different extents. Second, a similar biochemical mechanism such as the inhibition of rod and bipolar cell cGMP PDE, varying only in degree and duration, underlies both the lead-induced ERG rod-mediated deficits and the rod and bipolar apoptotic cell death. Third, the composite results from our experiments and those of others suggest that the developing retina might be more sensitive to preweaning lead exposure than the hippocampus and thus may serve as a good model for studying the cellular and molecular mechanisms underlying developmental and adult lead neurotoxicity.