Thomas Ladewig

Cone Dystrophy with Supernormal Rod Response Is Strictly Associated with Mutations in KCNV2

PURPOSE Cone dystrophy with supernormal rod response (CDSRR) is a retinal disorder characterized by reduced visual acuity, color vision defects, and specific alterations of ERG responses that feature elevated scotopic b-wave amplitudes at high luminance intensities. Mutations in PDE6H and in KCNV2 have been described in CDSRR. A combined clinical and genetic study was conducted in a cohort of patients with CDSRR, to substantiate these prior RESULTS METHODS Seventeen patients from 13 families underwent a detailed ophthalmic examination including color vision testing, Goldmann visual fields, fundus photography, Ganzfeld and multifocal ERGs, and optical coherence tomography. The coding sequences and flanking intron/UTR sequences of PDE6C and KCNV2 were screened for mutations by means of DHPLC and direct DNA sequencing of PCR-amplified genomic DNA. results. Whereas no mutations were detected in the PDE6H gene, mutations in KCNV2 were identified in all patients, in either the homozygous or compound heterozygous state. Ten of the 11 identified mutations were novel, including three missense and six truncating mutations and one gross deletion. The mutations concordantly segregate in all available families according a recessive mode of inheritance. The CDSRR phenotype was associated with reduced visual acuity of variable degree and color vision defects. Macular defects ranging from mild pigmentary changes to distinct foveal atrophy were present in nine patients. Progression of the disease was observed in only three of seven patients with follow-up data. CONCLUSIONS The phenotype of cone dystrophy with supernormal rod response is tightly linked with mutations in KCNV2.

Functional analysis of human CNGA3 mutations associated with colour blindness suggests impaired surface expression of channel mutants A3R427C and A3R563C

Mutations in the CNGA3 gene have been associated with complete and incomplete forms of total colour blindness (achromatopsia), a disorder characterized by reduced visual acuity, lack of colour discrimination, photophobia and nystagmus. CNGA3 encodes the A‐subunit of the cone photoreceptor cyclic nucleotide‐gated (CNG) channel, an essential component of the phototransduction cascade. Here we report the identification of three new CNGA3 mutations in patients with achromatopsia. To assess the pathogenicity of these newly identified and four previously reported mutations, mutant CNGA3 channels were heterologously expressed in a human embryonic kidney cell line (HEK293 cells) and functionally analysed using calcium imaging. Channels with the mutations R427C and R563C showed a response in imaging experiments and were subsequently characterized in‐depth with the patch‐clamp technique. The mutant channels were analysed as homooligomers and also as heterooligomers with the wild‐type B‐subunit present in native channels. Overall, cyclic guanosine monophosphate (cGMP) maximum currents of mutant channels were profoundly reduced in homo‐ and heteromers. Treatment with the chemical chaperone glycerol effectively increased macroscopic currents, presumably by enhancing surface expression of mutant channels as confirmed by immunocytochemistry. These results suggest decreased channel density in the cell membrane due to impaired folding or trafficking of the channel protein as the main pathogenic effect of the mutations R427C and R563C. Moreover, A3R427C homomers showed distinctly increased cGMP and cyclic adenosine monophosphate (cAMP) sensitivities as well as cAMP fractional currents that were raised to over 90% of cGMP maximum currents. Co‐expression of A3R427C with the B3 subunit compensated for most of these aberrant properties, apart from the reduced cGMP maximum currents.

Age‐dependent changes in the regulation mechanisms for intracellular calcium ions in ganglion cells of the mouse retina

The purpose of this study was to investigate the role of intracellular calcium buffering in retinal ganglion cells. We performed a quantitative analysis of calcium homeostasis in ganglion cells of early postnatal and adult mice by simultaneous patch‐clamp recordings in sliced tissue and microfluorometric calcium measurements with Fura‐2. Endogenous calcium homeostasis was quantified by using the ‘added buffer’ approach which uses amplitudes and decay time constants of calcium transients to give a standard for intracellular calcium buffering. The recovery phase of depolarization‐induced calcium transients was well approximated by a mono‐exponential function with a decay time constant that showed a linear dependence on dye concentration. Endogenous calcium binding ratios were found to be 575 (n = 18 cells) in early postnatal and 121 (n = 18 cells) in adult retinal ganglion cells. With respect to ganglion cell degeneration at early postnatal stages, our measurements suggest that neuroprotection of a majority of developing ganglion cells partially results from a specialized calcium homeostasis based on high buffering capacities. Furthermore, the dramatic decrease of the intracellular calcium buffering capacity during ganglion cell development may enhance their vulnerability to neurodegeneration.

BDNF regulates NMDA receptor activity in developing retinal ganglion cells.

Brain-derived neurotrophic factor (BDNF) modulates glutamate receptors of the NMDA type in many areas of the brain. We assessed whether BDNF exerts an effect on NMDA receptor properties in retinal ganglion cells during early postnatal development. Electrophysiological responses to the glutamate agonist NMDA (500 μM–2 mM) in retinal slices of wildtype and BDNF deficient mice (bdnf−/−) were recorded using the whole-cell patch-clamp technique. Retinal ganglion cells of bdnf−/− mice displayed significantly smaller NMDA currents than those of age-matched wildtype mice. Remarkably, NMDA receptor activity was restored by incubating retinal slices of bdnf−/− mice in BDNF (50 ng/ml) for 1–3 h. We suggest that BDNF plays a role in the activation of functional NMDA receptors in early ganglion cell development.

Identification And Analysis Of CNGA3 And CNGB3 From Zebrafish

Cyclic nucleotide-gated (CNG) channels are a crucial component of the phototransduction cascade in vertebrate photoreceptors. The opening and closure of these channels and consequently the influx of sodium and calcium ions into the photoreceptor outer segment is directed by the intracellular light-dependent cGMP level. Cone CNG channels are heterooligomers consisting of two A3- and two B3-subunits, which are encoded by the CNGA3 and the CNGB3 gene. In both genes mutations have been identified, which can lead to a dysfunction of the CNG channels in cone photoreceptors. In humans this results in the autosomal-recessively inherited disease achromatopsia (color blindness).In order to characterize CNG channels in zebrafish, which possess four morphologically and physiologically distinct classes of cones, we have identified two homologous candidate genes for CNGA3 and two for CNGB3 by in silico database analyses. All four genes as well as a splice variant of CNGA3-1 have been cloned and were heterologously expressed in HEK293 cells. Subsequently, the zebrafish CNG channels were functionally characterized by calcium imaging and patch-clamp measurements.The retinal expression of all four genes has been confirmed by RT-PCR. In silico analyses revealed, that the two CNGA3 candidates are located at two different locations in the zebrafish genome and are presumably a result of the whole genome duplication as it is known for several genes in zebrafish. In contrast to that, the two CNGB3 candidates are located in a tandem as a result of an additional gene duplication event. ZfCNGA3-1 and zfCNGA3-2 have 62 % identity with the human CNGA3 protein. ZfCNGB3-1 has 43 % and zfCNGB3-2 has 49 % identity with human CNGB3.