Edward N. Pugh

Safety and Efficacy of Gene Transfer for Leber’s Congenital Amaurosis

Lebers congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Superconductivity on the border of itinerant-electron ferromagnetism in UGe2

The absence of simple examples of superconductivity adjoining itinerant-electron ferromagnetism in the phase diagram has for many years cast doubt on the validity of conventional models of magnetically mediated superconductivity. On closer examination, however, very few systems have been studied in the extreme conditions of purity, proximity to the ferromagnetic state and very low temperatures required to test the theory definitively. Here we report the observation of superconductivity on the border of ferromagnetism in a pure system, UGe 2, which is known to be qualitatively similar to the classic d-electron ferromagnets. The superconductivity that we observe below 1 K, in a limited pressure range on the border of ferromagnetism, seems to arise from the same electrons that produce band magnetism. In this case, superconductivity is most naturally understood in terms of magnetic as opposed to lattice interactions, and by a spin-triplet rather than the spin-singlet pairing normally associated with nearly antiferromagnetic metals.

Amplification and kinetics of the activation steps in phototransduction

We can summarize our investigation of amplification in the activation steps of vertebrate phototransduction as follows. (1) A theoretical analysis of the activation steps of the cGMP cascade shows that after a brief flash of phi photoisomerizations the number of activated PDE molecules should rise as a delayed ramp with slope proportional to phi, and that, as a consequence, the cGMP-activated current should decay as a delayed Gaussian function of time (Eqn. 20). (i) Early in the response to a flash, the normalized response R(t) can be approximated as rising as 1/2 phi At2 (after a short delay), where A is the amplification constant characteristic of the individual photoreceptor. (ii) The delayed ramp behavior of PDE activation and the consequent decline of current in the form of the delayed Gaussian are confirmed by experiments in a variety of photoreceptors; the analysis thus yields estimates of the amplification constant from these diverse photoreceptors. (iii) Eqn. 20 further predicts that the response-intensity relation at any fixed time should saturate exponentially, as has been found experimentally. (2) The amplification constant A can be expressed as the product of amplification factors contributed by the individual activation steps of phototransduction, i.e., A = nu RG cGP beta sub n (Eqns. 9 and 21), where (i) nu RG is the rate of G* production per Rh*; (ii) cGP is the efficiency of the coupling between G* production and PDE* production; (iii) beta sub is the increment in hydrolytic rate constant produced by one PDE*, i.e., a single activated catalytic subunit of PDE; and (iv) n is the Hill coefficient of opening of the cGMP-activated channels. (3) The amplification factor beta sub includes the ratio kcat/Km, which characterizes the hydrolytic activity of the PDE in vivo where cG << Km. Two different analyses based upon photocurrents were developed which provide lower bounds for kcat/Km in vivo; these analyses establish that kcat/Km probably exceeds 10(7) M-1 s-1 (and is likely to be higher) in both amphibian and mammalian rods. Few biochemical studies (other than those using trypsin activation) have yielded such high values. A likely explanation of many of the relatively low biochemical estimates of kcat/Km is that Km may have been overestimated by a factor of about 4 in preparations in which stacks of disks are left intact, due to diffusion with hydrolysis in the stacks.(ABSTRACT TRUNCATED AT 400 WORDS)

A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors.

1. We have undertaken a theoretical analysis of the steps contributing to the phototransduction cascade in vertebrate photoreceptors. We have explicitly considered only the activation steps, i.e. we have not dealt with the inactivation reactions. 2. From the theoretical analysis we conclude that a single photoisomerization leads to activation of the phosphodiesterase (PDE) with a time course which approximates a delayed ramp; the delay is contributed by several short first‐order delay stages. 3. We derive a method for extracting the time course of PDE activation from the measured electrical response, and we apply this method to recordings of the photoresponse from salamander rods. The results confirm the prediction that the time course of PDE activation is a delayed ramp, with slope proportional to light intensity; the initial delay is about 10‐20 ms. 4. We derive approximate analytical solutions for the electrical response of the photoreceptor to light, both for bright flashes (isotropic conditions) and for single photons (involving longitudinal diffusion of cyclic GMP in the outer segment). The response to a brief flash is predicted to follow a delayed Gaussian function of time, i.e. after an initial short delay the response should begin rising in proportion to t2. Further, the response‐intensity relation is predicted to obey an exponential saturation. 5. These predictions are compared with experiment, and it is shown that the rising phase of the flash response is accurately described over a very wide range of intensities. We conclude that the model provides a comprehensive description of the activation steps of phototransduction at a molecular level.

Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation.

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.

Molecular mechanisms of vertebrate photoreceptor light adaptation

An important recent advance in the understanding of vertebrate photoreceptor light adaptation has come from the discovery that as many as eight distinct molecular mechanisms may be involved, and the realization that one of the principal mechanisms is not dependent on calcium. Quantitative analysis of these mechanisms is providing new insights into the nature of rod photoreceptor light adaptation.

Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup.

Charles Darwin appreciated the conceptual difficulty in accepting that an organ as wonderful as the vertebrate eye could have evolved through natural selection. He reasoned that if appropriate gradations could be found that were useful to the animal and were inherited, then the apparent difficulty would be overcome. Here, we review a wide range of findings that capture glimpses of the gradations that appear to have occurred during eye evolution, and provide a scenario for the unseen steps that have led to the emergence of the vertebrate eye.

The Proteome of the Mouse Photoreceptor Sensory Cilium Complex

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including ∼1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.

UV- and midwave-sensitive cone-driven retinal responses of the mouse: A possible phenotype for coexpression of cone photopigments

Molecular biological, histological and flicker electroretinographic results have established that mice have two cone photopigments, one peaking near 350 nm (UV-cone pigment) and a second near 510 nm [midwave (M)-cone pigment]. The goal of this investigation was to measure the action spectra and absolute sensitivities of the UV-cone- and M-cone-driven b-wave responses of C57BL/6 mice. To achieve this goal, we suppressed rod-driven signals with steady or flashed backgrounds and obtained intensity–response relations for cone-driven b-waves elicited by narrowband flashes between 340 and 600 nm. The derived cone action spectra can be described as retinal1pigments with peaks at 355 and 508 nm. The UV peak had an absolute sensitivity of ∼8 nV/(photon μm2) at the cornea, approximately fourfold higher than the M peak. In an attempt to isolate UV-cone-driven responses, it was discovered that an orange conditioning flash (λ > 530 nm) completely suppressed ERG signals driven by both M pigment- and UV pigment-containing cones. Analysis showed that the orange flash could not have produced a detectable response in the UV-cone pathway were their no linkage between M pigment- and UV pigment-generated signals. Because cones containing predominantly the UV and M pigments have been shown to be located largely in separate parts of the mouse retina (Szel et al., 1992), the most probable linkage is coexpression of M pigment in cones primarily expressing UV pigment. New histological evidence supports this interpretation (Gloesman and Ahnelt, 1998). Our data are consistent with an upper bound of ∼3% coexpression of M pigment in the cones that express mostly the UV pigment.

Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl −/− mice, using suction pipette recordings from their “inner segments” (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the α-subunit of the G-protein transducin (G tα−/−), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (λmax = 360 nm) and M- (λmax = 508 nm) cone opsins and to be maximally sensitive at 360 nm (“S-cones”); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm (“M-cones”). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was ∼70 ms, and ∼0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A ∼4 s−2) was found to be about twofold lower than in rods (A ∼8 s−2). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate “dark light.”