Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel.

PURPOSE: Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology. METHODS: The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology. RESULTS: All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication. CONCLUSIONS: The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.

Transsynaptic virus tracing from host brain to subretinal transplants.

The aim of this study was to establish synapses between a transplant and a degenerated retina. To tackle this difficult task, a little-known but well-established CNS method was chosen: trans-synaptic pseudorabies virus (PRV) tracing. Sheets of E19 rat retina with or without retinal pigment epithelium (RPE) were transplanted to the subretinal space in 33 Royal College of Surgeons (RCS) and transgenic s334ter-5 rats with retinal degeneration. Several months later, PRV-BaBlu (expressing E. colibeta-galactosidase) or PRV-Bartha was injected into an area of the exposed superior colliculus (SC), topographically corresponding to the transplant placement in the retina. Twenty normal rats served as controls. After survival times of 1-5 days, retinas were examined for virus by X-gal histochemistry, immunohistochemistry and electron microscopy. In normal controls, virus was first seen in retinal ganglion cells and Müller glia after 1-1.5 days, and had spread to all retinal layers after 2-3 days. Virus-labeled cells were found in 16 of 19 transplants where the virus injection had retrogradely labeled the topographically correct transplant area of the host retina. Electron microscopically, enveloped and nonenveloped virus could clearly be detected in infected cells. Enveloped virus was found only in neurons. Infected glial cells contained only nonenveloped virus. Neurons in retinal transplants are labeled after PRV injection into the host brain, indicating synaptic connectivity between transplants and degenerated host retinas. This study provides evidence that PRV spreads in the retina as in other parts of the CNS and is useful to outline transplant-host circuitry.

Retinal transplantation-induced recovery of retinotectal visual function in a rodent model of retinitis pigmentosa.

PURPOSE: To map the spatiotemporal decline in retinally driven activity in the superior colliculus (SC) of transgenic S334ter-line-3 rats that express a mutated rhodopsin, which causes photoreceptor degeneration. To determine whether transplantation of fetal retinal sheets into the subretinal space of these rats can recover visual activity in the SC. METHODS: A visual stimulus was presented to the eye, and responses were recorded across the SC of untreated S334ter-line-3 rats aged 28 to 288 days. These data were used to draw a map of the developing scotoma. Intact retinal sheets from embryonic day 19 rats were transplanted into the subretinal space of S334ter-line-3 rats between 21 and 28 days of age. Responses to retinal stimulation were mapped in the SC of transplanted and sham control rats 78 to 163 days after surgery. The morphology of the retinas in all groups was examined. RESULTS: Photoreceptor cell loss in untreated rats matched the decline in visual activity in the SC. At 28 days, there was a scotoma in the area of the SC that represents the central retina and, by 63 days, it had enlarged to cover the entire retinal representation. Visual responses were evoked in 64% of rats with retinal transplants. These retinally driven responses were confined to a small, contiguous region of the SC that represents the sector of the retina where the transplant was placed. Visual responses were absent in the SC outside this area in transplant recipients and throughout the SC of untreated and sham control rats. CONCLUSIONS: Transplantation of fetal retinal sheets induced recovery of visual activity in the SC in this model of RP. The mechanisms underlying this functional recovery remain to be resolved, but these results suggest that transplantation should be further explored as a therapy for RP.