The toxicity of the PrP106-126 prion peptide on cultured photoreceptors correlates with the prion protein distribution in the mammalian and human retina.

In patients affected by Creutzfeldt-Jakob disease and in animals affected by transmissible spongiform encephalopathies, retinal functions are altered, and major spongiform changes are observed in the outer plexiform layer where photoreceptors have their synaptic terminals. In the present study, the prion protein PrP(c) was found to form aggregates in rod photoreceptor terminals from both rat and human retina, whereas no labeling was observed in cone photoreceptors. Discrete staining was also detected in the inner plexiform layer where the prion protein was located at human amacrine cell synapses. In mixed porcine retinal cell cultures, the PrP106-126 prion peptide triggered a 61% rod photoreceptor cell loss by apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling, whereas cone photoreceptors were not affected. Amacrine cells were also reduced by 47% in contrast to ganglion cells. Although this cell loss was associated with a 5.5-fold increase in microglial cells, the strict correlation between the PrP(c) prion protein expression and the peptide toxicity suggested that this toxicity did not rely on the release of a toxic compound by glial cells. These results provide new insights into the retinal pathophysiology of prion diseases and illustrate advantages of adult retinal cell cultures to investigate prion pathogenic mechanisms.

Distribution of vesicular glutamate transporters in rat and human retina.

Central nervous system neurons have traditionally been thought to express exclusively membrane transporters and/or vesicular transporters for their transmitter. Three vesicular glutamate transporters have recently been cloned: BNPI/VGLUT1 (a brain-specific sodium-dependent inorganic phosphate (Pi) transporter), and its homologs DNPI/VGLUT2 (differentiation-associated sodium-dependent Pi transporter) and VGLUT3. We investigated the subcellular distributions of these three vesicular transporters in rat and human retina. VGLUT1 was present in the outer and inner plexiform layers (OPL and IPL), as shown by punctate staining in both human and rat retina. In the OPL, it was colocalized with synaptophysin, consistent with its expression in glutamatergic photoreceptor terminals, and it was present in PKC-alpha-labeled glutamatergic bipolar cell terminals in the IPL. By contrast, VGLUT2 was present in horizontal cells and ganglion cells in rat and human retina. In human retina, VGLUT2 was also found in some amacrine cells, including GAD-immunopositive amacrine cells. VGLUT3 was present in glycine-releasing amacrine cells in rat retina but was restricted to a few ganglion cells in human retina. The distribution of VGLUT1 in excitatory synaptic terminal was consistent with its involvement in glutamate release at excitatory synapses, whereas the cellular distributions of VGLUT2 and VGLUT3 suggested that these molecules may be involved in functions other than glutamate release, such as glutamate storage for GABA synthesis in non-glutamatergic neurons.

Excessive activation of cyclic nucleotide-gated channels contributes to neuronal degeneration of photoreceptors.

In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration.