Correction to: Retinal Degenerative Diseases.

The title of the chapter is "Melatonin as the Possible Link Between Age-Related Retinal Degeneration and the Disrupted Circadian Rhythm in Elderly" but degeneration was incorrectly published as regeneration. Now this has been corrected to degeneration.

A Non-Canonical Role for ß-Secretase in the Retina.

It has long been established that ß-Secretase (BACE) plays a critical role in the formation of amyloid plaques in Alzheimer's Disease patients, but it is only recently that the importance of ß-secretases in retinal pathophysiology has been recognized. BACE expression is elevated in response to stress, and downregulation results in lysosomal abnormalities and mitochondrial changes. Inhibition of BACE can lead to reduced retinal function, retinal thinning, lipofuscin accumulation and vascular dysfunction in mice. Furthermore, BACE inhibition accelerates choroidal neovascularization (CNV) in mice. We propose that BACE plays an important role in retinal homeostasis and that BACE upregulation in response to stress is a protective measure.

Truncated Complement Factor H Y402 Gene Therapy Cures C3 Glomerulonephritis.

Patients with both age-related macular degeneration (AMD) and C3 glomerulonephritis (C3G) are challenged by the absence of effective therapies to reverse and eliminate their disease burden. Capitalizing on complement dysregulation as both a significant risk factor for AMD and the known pathophysiology of C3G, we investigated the potential for adeno-associated virus (AAV) delivery of complement factor H (CFH) to rescue C3G in a Cfh-/- mouse model of C3G. While past efforts to treat C3G using exogenous human CFH resulted in limited success before immune rejection led to a foreign protein response, our findings demonstrate the capacity for long-term AAV-mediated delivery of truncated CFH (tCFH) to restore inhibition of the alternative pathway of complement and ultimately reverse C3G without immune rejection. Comparing results from the administration of several tCFH vectors also revealed significant differences in their relative efficiency and efficacy. These discoveries pave the way for subsequent development of AAV-mediated tCFH replacement therapy for patients with C3G, while simultaneously demonstrating proof of concept for a parallel AAV-mediated tCFH gene augmentation therapy for patients with AMD.

Src family kinases engage differential pathways for encapsulation into extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions.

Degenerative diseases of the outer retina, including age-related macular degeneration (AMD), are characterized by atrophy of photoreceptors and retinal pigment epithelium (RPE). In these blinding diseases, macrophages are known to accumulate ectopically at sites of atrophy, but their ontogeny and functional specialization within this atrophic niche remain poorly understood, especially in the human context. Here, we uncovered a transcriptionally unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and in human AMD. Using disease models, we found that conditional deletion of galectin-3 in microglia led to defects in phagocytosis and consequent augmented photoreceptor death, RPE damage and vision loss, suggestive of a protective role. Mechanistically, Trem2 signaling orchestrated the migration of microglial cells to sites of atrophy, and there, induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection, but only in a galectin-3-dependent manner, further signifying the functional interdependence of these two molecules. Likewise in elderly human subjects, we identified a highly conserved population of microglia at the transcriptomic, protein and spatial levels, and this population was enriched in the macular region of postmortem AMD subjects. Collectively, our findings reveal an atrophy-associated specialization of microglia that restricts the progression of retinal degeneration in mice and further suggest that these protective microglia are conserved in AMD.

Anionic Nanoplastic Contaminants Promote Parkinson's Disease-Associated α-Synuclein Aggregation.

Recent studies have identified increasing levels of nanoplastic pollution in the environment. Here we find that anionic nanoplastic contaminants potently precipitate the formation and propagation of α-synuclein protein fibrils through a high-affinity interaction with the amphipathic and non-amyloid component (NAC) domains in α-synuclein. Nanoplastics can internalize in neurons through clathrin-dependent endocytosis, causing a mild lysosomal impairment that slows the degradation of aggregated α-synuclein. In mice, nanoplastics combine with α-synuclein fibrils to exacerbate the spread of α-synuclein pathology across interconnected vulnerable brain regions, including the strong induction of α-synuclein inclusions in dopaminergic neurons in the substantia nigra. These results highlight a potential link for further exploration between nanoplastic pollution and α-synuclein aggregation associated with Parkinson's disease and related dementias.

Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of exosomes that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.

ApoER2 function in the establishment and maintenance of retinal synaptic connectivity.

The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells. ApoER2 knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.

Unraveling a multifactorial late-onset disease: from genetic susceptibility to disease mechanisms for age-related macular degeneration.

Aging-associated neurodegenerative diseases significantly influence the quality of life of affected individuals. Genetic approaches, combined with genomic technology, have provided powerful insights into common late-onset diseases, such as age-related macular degeneration (AMD). Here, we discuss current findings on the genetics of AMD to highlight areas of rapid progress and new challenges. We also attempt to integrate available genetic and biochemical data with cellular pathways involved in aging to formulate an integrated model of AMD pathogenesis.

Heparan sulfate, including that in Bruch's membrane, inhibits the complement alternative pathway: implications for age-related macular degeneration.

An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase-based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch's membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch's membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch's membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch's membrane/choroid.

Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.

Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

PURPOSE: To identify the genes expressed in normal human trabecular meshwork tissue, a tissue critical to the pathogenesis of glaucoma. METHODS: Total RNA was extracted from human trabecular meshwork (HTM) harvested from 3 different donors. Extracted RNA was used to synthesize individual SAGE (serial analysis of gene expression) libraries using the I-SAGE Long kit from Invitrogen. Libraries were analyzed using SAGE 2000 software to extract the 17 base pair sequence tags. The extracted sequence tags were mapped to the genome using SAGE Genie map. RESULTS: A total of 298,834 SAGE tags were identified from all HTM libraries (96,842, 88,126, and 113,866 tags, respectively). Collectively, there were 107,325 unique tags. There were 10,329 unique tags with a minimum of 2 counts from a single library. These tags were mapped to known unique Unigene clusters. Approximately 29% of the tags (orphan tags) did not map to a known Unigene cluster. Thirteen percent of the tags mapped to at least 2 Unigene clusters. Sequence tags from many glaucoma-related genes, including myocilin, optineurin, and WD repeat domain 36, were identified. CONCLUSIONS: This is the first time SAGE analysis has been used to characterize the gene expression profile in normal HTM. SAGE analysis provides an unbiased sampling of gene expression of the target tissue. These data will provide new and valuable information to improve understanding of the biology of human aqueous outflow.

Developing SDOCT to assess donor human eyes prior to tissue sectioning for research.

BACKGROUND: To compare spectral domain optical coherence tomography (SDOCT) cross-sectional images of human central retina obtained from donor eyes with and without age-related macular degeneration (AMD) to corresponding histopathology from light micrographs. To establish the utility of SDOCT for localizing pathology in the posterior eyecup, for identifying ocular disease in donor eyes, or for directing subsequent sectioning of retinal lesions for research. METHODS: Seven consecutive human donor eyes were selected based on age. The eyes, with the anterior segment removed, were imaged by SDOCT with a focusing aspheric lens. Four eyes were from donors with a clinical history of AMD, and three were from age-matched donors with no history of AMD. Histopathological correlation of morphological changes detected in three eyes by SDOCT was obtained for comparison to step serial-sectioned light microscopy images of the formalin-fixed, paraffin-embedded retina. A simplified imaging setup was tested on an enucleated porcine eye for comparison. RESULTS: AMD pathology was detected and localized in four eyes by SDOCT. The SDOCT images correlated with the histopathology observed by light microscopy in each sectioned eye. Pathologies included a subfoveal neovascular lesion with subretinal fluid, peripapillary neovascularization, epiretinal membrane, foveal cyst, choroidal folds, and drusen. Similar imaging was possible with the simplified setup. CONCLUSIONS: SDOCT imaging identified retinal disease of the posterior eyecup in human donor eyes. Pathology detected with SDOCT was verified by light microscopy in three eyes, supporting the utility of SDOCT as a screening tool for research.

Recurrent choroidal neovascularization after macular translocation surgery with 360-degree peripheral retinectomy.

PURPOSE: To evaluate the pattern of age-related macular degeneration in the new foveal location after macular translocation surgery with 360 degree peripheral retinectomy for neovascular age-related macular degeneration. METHODS: Clinical data, fundus photos, and fluorescein angiograms of patients in the Duke Macular Translocation Study were reviewed with 2-year follow-up data. RESULTS: With 56 patients completing follow-up, no patient developed de novo choroidal neovascularization (CNV), geographic atrophy, or drusen in the new subfoveal retinal pigment epithelium bed. By 2 years, 14 patients (25%) developed recurrent CNV and 13 of these 14 recurrences clearly arose from the old CNV bed. Of the 13 recurrences clearly arising from the old bed, 12 of them had recurrent CNV that involved the margin of the bed closest to the repositioned fovea. Smokers were 5.3 times (95% confidence interval: 1.2-24) more likely to develop recurrent CNV over 2 years. Despite treatment, median visual acuity for the 14 eyes with recurrent CNV was 20/200 compared with 20/80 in eyes without recurrence. CONCLUSIONS: Findings in this study support the hypotheses that the development of CNV occurs via a signaling mechanism from the fovea.

Why do mutations in the ubiquitously expressed housekeeping gene IMPDH1 cause retina-specific photoreceptor degeneration?

PURPOSE: The purpose of this study was to investigate retinal inosine monophosphate dehydrogenase 1 (IMPDH1) transcripts and proteins to gain an understanding of how mutations in IMPDH1 lead to retinal disease. Mutations in IMPDH1 cause the RP10 form of autosomal dominant retinitis pigmentosa (adRP) and are a rare cause of dominant Leber congenital amaurosis (LCA). IMPDH1 is a highly conserved, widely expressed housekeeping gene, the product of which catalyzes the rate-limiting step of de novo guanine synthesis. Despite its conservation and ubiquity, the clinical consequences of missense mutations in IMPDH1 are limited to the retina, and the disease mechanism is currently unknown. METHODS: A variety of methods were used to address the unique features of IMPDH1 in the retina, including Northern blot analysis, serial analysis of gene expression (SAGE), immunohistochemistry, transcript sequencing, and Western blot analysis. RESULTS: Results of the experiments showed that IMPDH1 levels are higher in the retina than in any other tissue tested. Specifically, IMPDH1 is found predominately in the inner segment and synaptic terminals of retinal photoreceptors. The predominant transcripts of IMPDH1 in human retina are the result of alternate splicing and alternate start sites of translation. They are significantly different from those in other tissues, and these variant transcripts encode distinct proteins. Further, the proportions of IMPDH1 transcripts and proteins in human retina are different from those in mouse retina. CONCLUSIONS: Identification of unique retinal isoforms supports the existence of a novel IMPDH1 function in the retina, one that is probably altered by disease-causing mutations. This alone, or coupled with the high levels of IMPDH1 in the retina, may explain the retina-specific phenotype associated with IMPDH1 mutations. Elucidating the functional properties of these unique, human retinal isoforms is crucial to understanding the pathophysiology of IMPDH1 mutations.

The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.

During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch's membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals.

Rapid and sensitive method for detection of Y402, H402, I62, and V62 variants of complement factor H in human plasma samples using mass spectrometry.

PURPOSE: Variations in the complement factor H (CFH) gene are tightly associated with age-related macular degeneration (AMD) across diverse populations. Of the many nonsynonymous coding variants in CFH, two are most strongly associated with increased risk of AMD: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in a patient's blood is important for a risk assessment of AMD and disease prognosis. However, traditional methods of genetic analysis cannot be used for measuring CFH allotypes in some sources of human plasma and other biological fluids not containing DNA. The purpose was to develop a protein-based method of detecting CFH allotypes. METHODS: A combination of a single-step affinity enrichment of CFH, gel separation, and mass spectrometry identification of the CFH peptides spanning amino acids at positions 62 and 402 was used to identify individual CFH allotypes. RESULTS: The CFH isoforms V62, I62, H402, and Y402 were reliably detected based on identification of tryptic peptides with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and 2057.88 Da, respectively, using MALDI-TOF-TOF. The presence or absence pattern of these peptides in mass spectra of different CFH samples robustly correlated with all nine genotypes of CFH, as a result of variations at positions 62 and 402. CONCLUSIONS: A rapid and sensitive method has been developed for detection of V62, I62, H402, and Y402 variants of CFH in human plasma samples using mass spectrometry. This method can be used in clinical laboratories equipped with a basic inexpensive mass spectrometer capable of performing peptide fingerprinting.

Evaluation of the X-linked high-grade myopia locus (MYP1) with cone dysfunction and color vision deficiencies.

PURPOSE: X-linked high myopia with mild cone dysfunction and color vision defects has been mapped to chromosome Xq28 (MYP1 locus). CXorf2/TEX28 is a nested, intercalated gene within the red-green opsin cone pigment gene tandem array on Xq28. The authors investigated whether TEX28 gene alterations were associated with the Xq28-linked myopia phenotype. Genomic DNA from five pedigrees (with high myopia and either protanopia or deuteranopia) that mapped to Xq28 were screened for TEX28 copy number variations (CNVs) and sequence variants. METHODS: To examine for CNVs, ultra-high resolution array-comparative genomic hybridization (array-CGH) assays were performed comparing the subject genomic DNA with control samples (two pairs from two pedigrees). Opsin or TEX28 gene-targeted quantitative real-time gene expression assays (comparative CT method) were performed to validate the array-CGH findings. All exons of TEX28, including intron/exon boundaries, were amplified and sequenced using standard techniques. RESULTS: Array-CGH findings revealed predicted duplications in affected patient samples. Although only three copies of TEX28 were previously reported within the opsin array, quantitative real-time analysis of the TEX28 targeted assay of affected male or carrier female individuals in these pedigrees revealed either fewer (one) or more (four or five) copies than did related and control unaffected individuals. Sequence analysis of TEX28 did not reveal any variants associated with the disease status. CONCLUSIONS: CNVs have been proposed to play a role in disease inheritance and susceptibility as they affect gene dosage. TEX28 gene CNVs appear to be associated with the MYP1 X-linked myopia phenotypes.