Brett G. Jeffrey

TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1−/−) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

Subretinal Transplantation of Forebrain Progenitor Cells in Nonhuman Primates: Survival and Intact Retinal Function

PURPOSE Cell-based therapy rescues retinal structure and function in rodent models of retinal disease, but translation to clinical practice will require more information about the consequences of transplantation in an eye closely resembling the human eye. The authors explored donor cell behavior using human cortical neural progenitor cells (hNPC(ctx)) introduced into the subretinal space of normal rhesus macaques. METHODS hNPC(ctx) transduced with green fluorescent protein (hNPC(ctx)-GFP) were delivered bilaterally into the subretinal space of six normal adult rhesus macaques under conditions paralleling those of the human operating room. Outcome measures included clinical parameters of surgical success, multifocal electroretinogram (mfERG), and histopathologic analyses performed between 3 and 39 days after engraftment. To test the effects of GFP transduction on cell bioactivity, hNPC(ctx)-GFP from the same batch were also injected into Royal College of Surgeons (RCS) rats and compared with nonlabeled hNPC(ctx). RESULTS Studies using RCS rats indicated that GFP transduction did not alter the ability of the cells to rescue vision. After cells were introduced into the monkey subretinal space by a pars plana transvitreal approach, the resultant detachment was rapidly resolved, and retinal function showed little or no disturbance in mfERG recordings. Retinal structure was unaffected and no signs of inflammation or rejection were seen. Donor cells survived as a single layer in the subretinal space, and no cells migrated into the inner retina. CONCLUSIONS Human neural progenitor cells can be introduced into a primate eye without complication using an approach that would be suitable for extrapolation to human patients.

RGS7 and -11 complexes accelerate the ON-bipolar cell light response.

PURPOSE The retinal ON-bipolar cell (ON-BPC) light response is initiated upon deactivation of the metabotropic glutamate receptor mGluR6 and the G protein Go. G protein-based signaling cascades are typically accelerated by interaction of the G protein alpha subunit with a member of the regulator of G protein signaling (RGS) protein family. The goal of this study was to determine whether RGS7 and/or -11 serve this function in retinal ON-BPCs. METHODS Retinas from mice lacking RGS11 (RGS11(-/-)), or with a deletion mutation in RGS7 (RGS7(Delta/Delta)), or both, were compared to wild-type (WT) by immunofluorescence confocal microscopy. The retinal light response was measured with the electroretinogram (ERG). The kinetics of simulated light responses from individual rod bipolar cells were recorded by whole-cell patch-clamp electrophysiology. RESULTS Levels of the R7 RGS interaction partners, Gbeta5 and R9AP, were reduced in the outer plexiform layer of the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. ERG recordings demonstrated a delay in the rising phase of the ERG b-wave, larger photopic b-wave amplitudes, and increased scotopic threshold response sensitivity in the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. The ERG measured from the RGS7(Delta/Delta) retina was normal. Patch-clamp recordings of chemically simulated light responses of rod BPCs revealed a 25-ms delay in the onset of the ON-BPC response in the RGS7(Delta/Delta)/RGS11(-/-) mouse compared with the WT. CONCLUSIONS RGS11 plays a role in the deactivation of Galphao, which precedes activation of the depolarizing current in ON-BPCs. RGS7 must also serve a role as changes in RGS7(Delta/Delta)/RGS11(-/-) mice were greater than those in RGS11(-/-) mice.

The electroretinogram in infants and young children.

Electroretinography (ERG), especially when combined with visual evoked potential recording, can provide valuable information to the pediatric clinician assessing a young child with nystagmus who appears not to see. ERG changes are described in clinical conditions affecting rod and/or cone function, vitreoretinal disease, maculopathies, and disorders involving the retina and central nervous system. The role of ERG in monitoring treatment, and in excluding retinal disease prior to eye surgery, is also covered.

Ophthalmologic Assessment of Young Patients with Alport Syndrome

BACKGROUND Alport syndrome is an X-linked disease affecting basement membrane collagen. It is characterized by nephritis associated with high-tone sensorineural hearing impairment and ophthalmic signs. Although ocular changes have been described in adults, few data exist regarding the incidence of abnormal ocular features in adolescence and childhood. METHODS Fifteen male and five female patients with Alport syndrome underwent ophthalmologic, audiologic, and nephrologic assessments. All patients studied had hematuria and a positive family history of Alport syndrome. Thirteen patients had a renal biopsy that showed characteristic electron microscopic changes of the disease. Eleven patients had high-tone sensorineural impairment. Electrophysiologic investigations performed included electroretinography, visual-evoked potentials, and electro-oculography. RESULTS Two patients had early signs of anterior lenticonus, three had flecks in the retina, and two patients also had posterior subcapsular cataracts. None of the patients had significant electrophysiologic abnormalities. CONCLUSION These findings indicate that ocular changes are uncommon and subtle in young patients with Alport syndrome, and suggest that the signs increase in frequency and severity with age.

Visual development: Neural basis and new assessment methods

The strongest evidence for beneficial effects of long-chain polyunsaturated fatty acid (LCPUFA) supplementation comes from measures of visual development. In particular, two measures of visual function, the electroretinogram (ERG) and grating acuity, have provided the primary basis for the addition of LCPUFA to infant formulas. However, the neural mechanisms underlying dietary effects on these visual functions are not well understood. Furthermore, the influence of dietary LCPUFA on other aspects of visual development is largely unknown. Additional measures of visual function would help to clarify the extent and nature of the effects of LCPUFA on the developing visual system, and several new methods promise to provide more detailed information about the sites of these effects. A number of recent reviews have described methods for visual assessment and the effects of infant LCPUFA supplementation on visual development. Therefore, the following review will emphasize newer developments in infant visual assessment and in understanding the neural mechanisms that may underlie the effects of LCPUFA.

R9AP stabilizes RGS11-G beta5 and accelerates the early light response of ON-bipolar cells.

The rate-limiting step in the recovery of the photoreceptor light response is the hydrolysis of GTP by transducin, a reaction that is accelerated by the RGS9-Gbeta5 complex, and its membrane anchor, R9AP. Similar complexes, including RGS7, RGS11, and Gbeta5, are found in retinal ON-bipolar cell dendrites. Here, we present evidence that R9AP is also expressed in the dendritic tips of ON-bipolar cells. Immunofluorescent staining for R9AP revealed a punctate pattern of labeling in the outer plexiform layer, where it colocalized with mGluR6. In photoreceptors, R9AP is required for proteolytic stability of the entire regulator of G protein signaling complex, and we found that genetic deletion of R9AP also results in a marked reduction in the levels of RGS11 and Gbeta5 in the bipolar cell dendrites; the level of RGS7 was unaffected, suggesting the presence of another interaction partner to stabilize RGS7. To determine the effect of R9AP deletion on the response kinetics of ON-bipolar cells, we compared the electroretinogram (ERG) between wild-type and R9AP-deficient mice. The ERG b-wave, reflecting ON-bipolar cell activity, was delayed and larger in the R9AP-deficient mice. Our data indicate that R9AP is required for stable expression of RGS11-Gbeta5 in ON-bipolar cell dendrites. Furthermore, they suggest that the RGS11-Gbeta5-R9AP complex accelerates the initial ON-bipolar cell response to light.

CNGB3-achromatopsia clinical trial with CNTF: diminished rod pathway responses with no evidence of improvement in cone function.

PURPOSE Ciliary neurotrophic factor (CNTF) protects rod photoreceptors from retinal degenerative disease in multiple nonhuman models. Thus far, CNTF has failed to demonstrate rod protection in trials for human retinitis pigmentosa. Recently, CNTF was found to improve cone photoreceptor function in a canine CNGB3 achromatopsia model. This study explores whether this finding translates to humans with CNGB3 achromatopsia. METHODS A five-subject, open-label Phase I/II study was initiated by implanting intraocular microcapsules releasing CNTF (nominally 20 ng/d) into one eye each of CNGB3 achromat participants. Fellow eyes served as untreated controls. Subjects were followed for 1 year. RESULTS Pupil constriction in treated eyes gave evidence of intraocular CNTF release. Additionally, scotopic ERG responses were reduced, and dark-adapted psychophysical absolute thresholds were increased, attributable to diminished rod or rod pathway activity. Optical coherence tomography revealed that the cone-rich fovea underwent structural changes as the foveal hyporeflective zone (HRZ) became diminished in CNTF-treated eyes. No objectively measurable enhancement of cone function was found by assessments of visual acuity, mesopic increment sensitivity threshold, or the photopic ERG. Careful measurements of color hue discrimination showed no change. Nonetheless, subjects reported beneficial changes of visual function in the treated eyes, including reduced light sensitivity and aversion to bright light, which may trace to decreased effective ambient light from the pupillary constriction; further they noted slowed adaptation to darkness, consistent with CNTF action on rod photoreceptors. CONCLUSIONS Ciliary neurotrophic factor did not measurably enhance cone function, which reveals a species difference between human and canine CNGB3 cones in response to CNTF. (ClinicalTrials.gov number, NCT01648452.).

Age-Related Decline in Rod Phototransduction Sensitivity in Rhesus Monkeys Fed an n-3 Fatty Acid―Deficient Diet

PURPOSE Docosahexaenoic acid (DHA), an n-3 fatty acid, is the major polyunsaturate in rod outer segments. The effect of long-term n-3 fatty acid deficiency on rod and cone phototransduction was investigated in the rhesus monkey. METHODS From birth to approximately = 9 years rhesus monkeys were fed an n-3-deficient diet (n = 9) known to reduce retinal DHA by 80%. Monkeys in the control group (n = 12) received either 8% alpha-linolenic acid (ALA) or 0.6% DHA, both of which support normal retinal DHA levels. None of the diets contained carotenoids. Photoactivation kinetics were assessed from the rate of increase and a P3 model fit of the ERG a-wave. Maximal cone amplitude and sensitivity were measured from the cone a-wave at 4 ms. The rod photoresponse and rod recovery were derived by using a paired flash METHOD RESULTS Rod sensitivity was reduced by 40% in the n-3-deficient monkeys at 9 but not 4.5 years. The onset of the rising phase of the photoresponse was significantly delayed (P < 0.004) at 9 years. Rod recovery was delayed by 20% in n-3-deficient monkeys at both ages, but only for bright saturating flashes. Cone phototransduction was not altered by n-3 deficiency. CONCLUSIONS Long-term dietary n-3 deficiency in the rhesus monkey was associated with two changes in retinal function. First, there was a delay in rod recovery that has remained relatively constant throughout life. Second, there was an age-dependent loss in rod phototransduction sensitivity; the lack of dietary carotenoids may have contributed to this decline.

Serum TRPM1 Autoantibodies from Melanoma Associated Retinopathy Patients Enter Retinal ON-Bipolar Cells and Attenuate the Electroretinogram in Mice

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1−/− mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1−/− mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.