An inherited night blindness in Wiltshire sheep

Twelve cases of adult-onset blindness were identified in a flock of 130 polled Wiltshire sheep in New Zealand over a 3-year period. Affected sheep developed night blindness between 2 and 3 years of age, which progressed to complete blindness by 4 to 5 years of age. Fundic examination findings included progressive tapetal hyperreflectivity and attenuation of retinal blood vessels. Histologically, the retinas had a selective loss of rod photoreceptors with initial preservation of cone photoreceptors. Retinal degeneration was not accompanied by any other ocular or central nervous system abnormalities, and pedigree analysis suggested an inherited basis for the disease. Mating an affected Wiltshire ram to 2 affected Wiltshire ewes resulted in 6 progeny that all developed retinal degeneration by 2 years of age, while mating of the same affected ram to 6 unaffected ewes resulted in 8 unaffected progeny, consistent with autosomal recessive inheritance. Homozygosity mapping of 5 affected Wiltshire sheep and 1 unaffected Wiltshire sheep using an OvineSNP50 Genotyping BeadChip revealed an identical-by-descent region on chromosome 5, but none of the genes within this region were considered plausible candidate genes. Whole-genome sequencing of 2 affected sheep did not reveal any significant mutations in any of the genes associated with retinitis pigmentosa in humans or progressive retinal atrophy in dogs. Inherited progressive retinal degeneration affecting rod photoreceptors has not been previously reported in sheep, but this disease has several similarities to inherited retinal dystrophies in other species.

Study of Protein Targeting and Mislocalization in Rod Photoreceptors

The photoreceptor outer segment (OS) is a highly specialized organelle for light absorption. Precise localization of OS resident proteins is important for photoreceptor function. Molecular mechanisms underlying OS targeting of proteins and their mislocalization, which frequently causes inherited retinal degeneration, have been intensely investigated. Rhodopsin, a major protein of the rod OS, is often mislocalized to the inner segment (IS) plasma membrane of rod photoreceptors in retinal degeneration patients. In the Xenopus laevis model of retinitis pigmentosa, we previously found that Na+/K+-ATPase (NKA), a major IS protein, was downregulated. The Imanishi lab recently created a novel retinitis pigmentosa mouse model carrying the Q344ter rhodopsin gene mutation, which causes rhodopsin mislocalization to the rod IS plasma membrane. In this summer program, we examined whether this mouse model also displays reduced NKA expression in the rod IS’s by immunohistochemistry at postnatal day 30. Although NKA was properly localized to the IS plasma membrane, expression of NKA was reduced in mutant photoreceptors compared to wildtype cells. In the rod OS, activation of rhodopsin eventually leads to the closure of the cyclic nucleotide gated (CNG) channel, which consists of a and b subunits. This channel localizes to the OS plasma membrane, and the N-terminal proline-rich region (R) of the b subunit (CNGb1) may be important for its interaction with peripherin (PRPH2), another OS resident protein. Currently, it is not well understood whether this interaction is necessary for the proper localization of CNGb1 to the OS plasma membrane. Using Xenopus as a model, we studied the role of the N-terminal proline-rich region in properly localizing CNGb1 to the OS plasma membrane by generating transgenic CNGb1(DR) tadpoles that expressed CNGb1(DR) in rods under the control of a rhodopsin promoter. We found that CNGb1(DR) properly localized to the OS plasma membrane.

Neuroinflammation as a Therapeutic Target in Retinitis Pigmentosa and Quercetin as Its Potential Modulator

The retina is a multilayer neuronal tissue located in the back of the eye that transduces the environmental light into a neural impulse. Many eye diseases caused by endogenous or exogenous harm lead to retina degeneration with neuroinflammation being a major hallmark of these pathologies. One of the most prevalent retinopathies is retinitis pigmentosa (RP), a clinically and genetically heterogeneous hereditary disorder that causes a decline in vision and eventually blindness. Most RP cases are related to mutations in the rod visual receptor, rhodopsin. The mutant protein triggers inflammatory reactions resulting in the activation of microglia to clear degenerating photoreceptor cells. However, sustained insult caused by the abnormal genetic background exacerbates the inflammatory response and increases oxidative stress in the retina, leading to a decline in rod photoreceptors followed by cone photoreceptors. Thus, inhibition of inflammation in RP has received attention and has been explored as a potential therapeutic strategy. However, pharmacological modulation of the retinal inflammatory response in combination with rhodopsin small molecule chaperones would likely be a more advantageous therapeutic approach to combat RP. Flavonoids, which exhibit antioxidant and anti-inflammatory properties, and modulate the stability and folding of rod opsin, could be a valid option in developing treatment strategies against RP.

A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation

ABSTRACT Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

Resolving the molecular architecture of the photoreceptor active zone by MINFLUX nanoscopy

Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology indicate that neurotransmission depends on such optimization strategies, yet the molecular topography of the presynaptic Active Zone (AZ) where transmitter is released upon synaptic vesicle (SV) fusion remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. To facilitate MINFLUX nanoscopy of the AZ, we developed and verified an immobilization technique, we name Heat Assisted Rapid Dehydration (HARD). Here fresh retinal slices are directly stamped onto glass coverslips yielding a single layer of rod AZs. These AZs exhibited excellent labeling efficiency and minimal signal redundancy in the Z-direction. Our data indicate that the SV release site is a molecular complex of bassoon-Rab3-binding molecule 2 (RIM2)-ubMunc13-2-CAST. The complexes are serially duplicated longitudinally, and reflected in register along the axis of symmetry of the synaptic ribbon.

Cone Rescue with Laser Photobiomodulation in Murine and Human Retinal Dystrophy

Retinitis pigmentosa (RP) encompasses a genetically diverse group of blinding inherited retinal diseases. In most subtypes the gene defect is expressed in the rod photoreceptors, yet in many affected individuals the cone photoreceptors undergo secondary degeneration, leading to loss of the remaining central vision. There is evidence that bioenergetic and oxidative stress are involved in this secondary cone loss. Photobiomodulation (PBM) uses low energy light in the far red or near-infrared spectrum to manipulate cellular activity. We have used a novel slit lamp-mounted retinal PBM laser to deliver precise energy levels to targeted retina. We showed that PBM laser attenuates oxidative and bioenergetic stress-induced photoreceptor loss in vitro and rescues cones in the rd1 murine model of RP. In a phase I trial (ACTRN12618000651280), foveal laser treatment was safe in humans with RP and temporarily recovered, on average, 5 letters of visual acuity.

Properties of multi-vesicular release from mouse rod photoreceptors support transmission of single photon responses

Vision under starlight requires rod photoreceptors transduce and transmit single photon responses to the visual system. Small single photon voltage changes must therefore cause detectable reductions in glutamate release. We found that rods achieve this by employing mechanisms that enhance release regularity and its sensitivity to small voltage changes. At the resting membrane potential in darkness, mouse rods exhibit coordinated and regularly timed multivesicular release events, each consisting of ~17 vesicles and occurring 2-3 times more regularly than predicted by Poisson statistics. Hyperpolarizing rods to mimic the voltage change produced by a single photon abruptly reduced the probability of multivesicular release nearly to zero with a rebound increase at stimulus offset. Simulations of these release dynamics indicate that this regularly timed, multivesicular release promotes transmission of single photon responses to post-synaptic rod bipolar cells. Furthermore, the mechanism is efficient, requiring lower overall release rates than uniquantal release governed by Poisson statistics.

Properties of multi-vesicular release from rod photoreceptors support transmission of single photon responses

Vision under starlight requires rod photoreceptors to transduce and transmit single photon responses to the visual system. This remarkable sensitivity depends on a small voltage change reliably reducing glutamate release such that post-synaptic rod bipolar cells can robustly detect the signal. To transmit this small signal, we have found that rod vesicle release deviates strongly from a Poisson process under conditions that mimic darkness. Specifically, at their resting membrane potential in darkness, rods exhibit coordinated and regularly timed multivesicular release events. Each release event consisted of ∼17 vesicles and occurred 2-3 times more regularly than expected from a Poisson process. Hyperpolarizing rods to mimic the voltage change produced by a single photon response abruptly reduced the probability of multivesicular release nearly to zero with a rebound increase in release probability at stimulus offset. Simulations of these release dynamics indicate that this regularly timed, multivesicular release promotes transmission of single photon responses to post-synaptic neurons. Furthermore, the mechanism is efficient, requiring fewer vesicles to be released per second than uniquantal release governed by Poisson statistics.