optimized-minimally-invasive-transscleral-subretinal-injection

Optimized Minimally Invasive Transscleral Subretinal Injection Technique in Mouse

The conventional method of material delivery to the subretinal space in the mouse involves dual perforation of the neural retina, which causes extensive ...

Duke University School of Medicine

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin-proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness.

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration.

The heterotrimeric G-protein transducin mediates visual signaling in vertebrate photoreceptor cells. Many aspects of the function of transducin were learned from knock-out mice lacking its individual subunits. Of particular interest is the knockout of its rod-specific γ-subunit (Gγ). Two studies using independently generated mice documented that this knockout results in a considerable >60-fold reduction in the light sensitivity of affected rods, but provided different interpretations of how the remaining α-subunit (Gα) mediates phototransduction without its cognate Gβγ-subunit partner. One study found that the light sensitivity reduction matched a corresponding reduction in Gα content in the light-sensing rod outer segments and proposed that Gα activation is supported by remaining Gβ associating with other Gγ subunits naturally expressed in photoreceptors. In contrast, the second study reported the same light sensitivity loss but a much lower, only approximately sixfold, reduction of Gα and proposed that the light responses of these rods do not require Gβγ at all. To resolve this controversy and elucidate the mechanism driving visual signaling in Gγ knock-out rods, we analyzed both mouse lines side by side. We first determined that the outer segments of both mice have identical Gα content, which is reduced ∼65-fold from the wild-type (WT) level. We further demonstrated that the remaining Gβ is present in a complex with endogenous Gγ and Gγ subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea that Gα alone supports light responses of Gγ knock-out rods and suggest that Gβγ is not unique in its ability to mediate vertebrate phototransduction.

Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors.

Inherited retinal degenerations originate from mutations in >300 genes, many of which cause the production of misfolded mutant photoreceptor proteins that are ultimately degraded by the ubiquitin-proteasome system (UPS). It was previously shown that rod photoreceptors in multiple mouse models of retinal degeneration suffer from proteostatic stress consisting of an insufficient cellular capacity for degrading UPS substrates. In this study, we focused on a specific UPS component required for the degradation of a subset of proteasome targets: the substrate-processing complex formed by the AAA+ ATPase P97/VCP and associated cofactors. To assess whether P97 capacity may be insufficient in degenerating rods, we employed two complementary  proteasomal activity reporters whose degradation is either P97-dependent or P97-independent. Retinal accumulation of each reporter was measured in two models of retinal degeneration: the transducin -subunit knock-out ( ) and P23H rhodopsin knock-in (P23H) mice. Strikingly, the patterns of reporter accumulation differed between these models, indicating that the proteostatic stress observed in  and P23H rods likely originates from different pathobiological mechanisms, in which UPS substrate degradation may or may not be limited by P97-dependent substrate processing. Further, we assessed whether P97 overexpression could ameliorate pathology in  mice, in which proteostatic stress appears to result from P97 insufficiency. However, despite P97 overexpression being aphenotypic in other tissues, the ∼2.4-fold increase in retinal P97 content was toxic to rods, which complicated the interpretation of the observed phenotype. Our results highlight the complexity of pathophysiological mechanisms related to degrading misfolded proteins in mutant photoreceptors.

Probing Proteostatic Stress in Degenerating Photoreceptors Using Two Complementary  Reporters of Proteasomal Activity.

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes (), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific  and  to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single  KO showed no phenotype, and single  KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double  KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses.

The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3.

Rod photoreceptors are light-sensitive neurons of the retina that support vision in dim light. A rod cell consists of an outer segment for phototransduction, an inner segment and soma for energy production and protein synthesis, and a synaptic terminal for vesicle release onto second-order neurons-bipolar and horizontal cells. Mouse rods contain a single ribbon synaptic release site, where vesicles filled with glutamate are released at a rate of up to ~20 vesicles/synapse/second. This high release rate requires a fine balance between synaptic vesicle exocytosis and endocytosis. Here, we review the properties of synaptic transmission and highlight proteins essential for synaptic vesicle recycling at the rod ribbon synapse.

Article	Year
Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration. Nature communications\| PubMed ID: 29712894	2018
Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors. eNeuro\| PubMed ID: 29911170	2018
Probing Proteostatic Stress in Degenerating Photoreceptors Using Two Complementary Reporters of Proteasomal Activity. eNeuro\| PubMed ID: 31826915	2020
The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 38641407	2024
Molecular Components of Vesicle Cycling at the Rod Photoreceptor Ribbon Synapse. Advances in experimental medicine and biology\| PubMed ID: 39930217	2025

Stella Finkelstein

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