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

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 (Dnm1-3), 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 Dnm1 and Dnm3 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 Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 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.

Disrupted Blood-Retina Lysophosphatidylcholine Transport Impairs Photoreceptor Health But Not Visual Signal Transduction.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

Proteasome overload is a common stress factor in multiple forms of inherited retinal degeneration.

Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying mutations cause protein misfolding or mistargeting in affected photoreceptors. This places an increased burden on the protein folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are affected in degenerating rods of the transducin γ-subunit (Gγ1) knockout mouse. These rods produce large amounts of transducin ß-subunit (Gß1), which cannot fold without Gγ1 and undergoes intracellular proteolysis instead of forming a transducin ßγ-subunit complex. Our data revealed that the most critical pathobiological factor leading to photoreceptor cell death in these animals is insufficient capacity of proteasomes to process abnormally large amounts of misfolded protein. A decrease in the Gß1 production in Gγ1 knockout rods resulted in a significant reduction in proteasomal overload and caused a striking reversal of photoreceptor degeneration. We further demonstrated that a similar proteasomal overload takes place in photoreceptors of other mutant mice where retinal degeneration has been ascribed to protein mistargeting or misfolding, but not in mice whose photoreceptor degenerate as a result of abnormal phototransduction. These results establish the prominence of proteasomal insufficiency across multiple degenerative diseases of the retina, thereby positioning proteasomes as a promising therapeutic target for treating these debilitating conditions.

Mechanistic basis for the failure of cone transducin to translocate: why cones are never blinded by light.

The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of transducin activation and inactivation were altered, we demonstrate that, like in rods, transducin translocation in cones can be triggered when transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of transducin translocation at any light intensity.

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

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 in vivo 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 γ-subunit knock-out (Gγ1-/- ) and P23H rhodopsin knock-in (P23H) mice. Strikingly, the patterns of reporter accumulation differed between these models, indicating that the proteostatic stress observed in Gγ1-/- 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 Gγ1-/- 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.

Phosphoinositide Profile of the Mouse Retina.

Phosphoinositides are known to play multiple roles in eukaryotic cells. Although dysregulation of phosphoinositide metabolism in the retina has been reported to cause visual dysfunction in animal models and human patients, our understanding of the phosphoinositide composition of the retina is limited. Here, we report a characterization of the phosphoinositide profile of the mouse retina and an analysis of the subcellular localization of major phosphorylated phosphoinositide forms in light-sensitive photoreceptor neurons. Using chromatography of deacylated phosphatidylinositol headgroups, we established PI(4,5)P2 and PI(4)P as two major phosphorylated phosphoinositides in the retina. Using high-resolution mass spectrometry, we revealed 18:0/20:4 and 16:0/20:4 as major fatty-acyl chains of retinal phosphoinositides. Finally, analysis of fluorescent phosphoinositide sensors in rod photoreceptors demonstrated distinct subcellular distribution patterns of major phosphoinositides. The PI(4,5)P2 reporter was enriched in the inner segments and synapses, but was barely detected in the light-sensitive outer segments. The PI(4)P reporter was mostly found in the outer and inner segments and the areas around nuclei, but to a lesser degree in the synaptic region. These findings provide support for future mechanistic studies defining the biological significance of major mono- (PI(4)P) and bisphosphate (PI(4,5)P2) phosphatidylinositols in photoreceptor biology and retinal health.

Multimodal Coherent Imaging of Retinal Biomarkers of Alzheimer's Disease in a Mouse Model.

We acquired depth-resolved light scattering measurements from the retinas of triple transgenic Alzheimer's Disease (3xTg-AD) mice and wild type (WT) age-matched controls using co-registered angle-resolved low-coherence interferometry (a/LCI) and optical coherence tomography (OCT). Angle-resolved light scattering measurements were acquired from the nerve fiber layer, outer plexiform layer, and retinal pigmented epithelium using image guidance and segmented thicknesses provided by co-registered OCT B-scans. Analysis of the OCT images showed a statistically significant thinning of the nerve fiber layer in AD mouse retinas compared to WT controls. The a/LCI scattering measurements provided complementary information that distinguishes AD mice by quantitatively characterizing tissue heterogeneity. The AD mouse retinas demonstrated higher mean and variance in nerve fiber layer light scattering intensity compared to WT controls. Further, the difference in tissue heterogeneity was observed through short-range spatial correlations that show greater slopes at all layers of interest for AD mouse retinas compared to WT controls. A greater slope indicates a faster loss of spatial correlation, suggesting a loss of tissue self-similarity characteristic of heterogeneity consistent with AD pathology. Use of this combined modality introduces unique tissue texture characterization to complement development of future AD biomarker analysis.

Transducin gamma-subunit sets expression levels of alpha- and beta-subunits and is crucial for rod viability.

Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the alpha- and betagamma-subunits. Here we demonstrate that the knock-out of transducin gamma-subunit leads to a major downregulation of both alpha- and beta-subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining alpha- and beta-subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the alpha-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the gamma-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin beta-subunit without its constitutive gamma-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.

Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.

Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin alpha subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.

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

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 (Gγ1). 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 (Gαt) mediates phototransduction without its cognate Gß1γ1-subunit partner. One study found that the light sensitivity reduction matched a corresponding reduction in Gαt content in the light-sensing rod outer segments and proposed that Gαt activation is supported by remaining Gß1 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 Gαt and proposed that the light responses of these rods do not require Gßγ at all. To resolve this controversy and elucidate the mechanism driving visual signaling in Gγ1 knock-out rods, we analyzed both mouse lines side by side. We first determined that the outer segments of both mice have identical Gαt content, which is reduced ∼65-fold from the wild-type (WT) level. We further demonstrated that the remaining Gß1 is present in a complex with endogenous Gγ2 and Gγ3 subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea that Gαt alone supports light responses of Gγ1 knock-out rods and suggest that Gß1γ1 is not unique in its ability to mediate vertebrate phototransduction.

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration.

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.

Dendritic supramolecular assemblies for drug delivery.

Dendritic supramolecular assemblies were formed in water with Reichardt's dye or the anticancer drug 10-hydroxycamptothecin and the dendritic macromolecule, ([G4]-PGLSA-OH)2-PEG3400.

HLA-A and -B allele expression and ability to develop anti-Gag cross-clade responses in subtype C HIV-1-infected Ethiopians.

A cohort of 35 human immunodeficiency virus type 1 (HIV-1) subtype C-infected Ethiopians was studied to define the HLA phenotype in all 35 subjects and highly conserved Gag protein regions involved in cross-clade cell-mediated immunity. Full-length Gag virus sequences were determined in 15 individuals. CD8 cell-mediated immune responses were detected by interferon-gamma ELISpot assay. HLA-A*03, -B*49, and -B*57 allelic frequencies were relatively higher than in other African populations. Anti-p17 (aa 1-60) CD8+ were detectable in the highest number of individuals. Anti-p17 (aa 1-60 and 51-110) cross-clade responses against subtype B and C were detected in 50% of the tested subjects. The p24 KF11 (aa 162-172) epitope was found to be immunodominant among the HLA-B*5703--positive individuals. These data represent the first report of correlating HLA phenotype and HIV-specific cell-mediated immune responses among infected Ethiopians and may be useful in designing cytotoxic T lymphocyte-inducing vaccines for this part of Africa.

CCR3 is required for tissue eosinophilia and larval cytotoxicity after infection with Trichinella spiralis.

The CCR3 binds at least seven different CC chemokines and is expressed on eosinophils, mast cells (MC), and a subset of Th cells (Th2) that generate cytokines implicated in mucosal immune responses. Using mice with a targeted disruption of CCR3 (CCR3(-/-)) and their +/+ littermates, we investigated the role of CCR3 in the amplification of tissue eosinophilia and MC hyperplasia in the mouse after infection with Trichinella spiralis. In CCR3(-/-) mice, eosinophils are not recruited to the jejunal mucosa after infection and are not present in the skeletal muscle adjacent to encysting larvae. In addition, the number of cysts in the skeletal muscle is increased and the frequency of encysted larvae exhibiting necrosis is reduced. The CCR3(-/-) mice exhibit the expected MC hyperplasia in the jejunum and caecum and reject the adult worms from the small intestine at a normal rate. This study is consistent with distinct functions for MC (adult worm expulsion) and eosinophils (toxicity to larvae) in immunity to a helminth, T. spiralis, and defines the essential requirement for CCR3 in eosinophil, but not MC recruitment to tissues.

Dendritic molecular capsules for hydrophobic compounds.

Reichardt's dye, a highly solvatochromic dye, was encapsulated within poly (glycerol succinic acid) ([Gn]-PGLSA-OH) dendrimers to investigate the interior environment of these dendritic macromolecules. The absorption maximum for the encapsulated Reichardt's dye in water was indicative of a relatively high dielectric constant present within the dye/dendrimer complex. (1)H NMR of the encapsulated complex showed the presence of aromatic protons from Reichardt's dye along with the aliphatic protons of the dendrimer. Additionally, there were substantial changes in T(1) and T(2) times of the encapsulated dye when compared with the free dye, and (1)H NOESY spectra for the complex showed a significant number of intermolecular NOE cross-peaks. These data reveal the close through-space proximity of the dye to the dendrimer and the restricted motion of the encapsulated dye. To demonstrate the potential use of these macromolecules as drug delivery vehicles, the poorly water-soluble anticancer drug 10-hydroxycamptothecin (10HCPT) was encapsulated within a carboxylated PGLSA dendrimer ([G4]-PGLSA-COONa). Cytotoxicity assays with human breast cancer cells showed a significant reduction of cell viability, demonstrating that 10HCPT retains activity upon encapsulation.