1) Researchers injected a viral vector expressing either wild-type or mutant C1QTNF5 specifically into the retinal pigment epithelium (RPE) of mice to study the effects.
2) Expression of the mutant protein led to intracellular aggregate formation in RPE cells and accumulation of extracellular deposits, causing RPE thinning, cell loss, and retinal degeneration similar to the human condition.
3) In contrast, the wild-type protein was secreted apically from the RPE as normal, with no pathological effects observed. This mouse model recapitulates key features of the human late-onset retinal degeneration caused by the C1QTNF5 mutation.
- The crystal structure of the N-terminal half of the UL37 inner tegument protein from pseudorabies virus was determined, revealing an elongated molecule composed of multiple helical bundles.
- Structure-based mutagenesis identified a novel functional region in UL37 important for cell-to-cell virus spread.
- UL37 shares structural similarity with cellular multisubunit tethering complexes, which control vesicular trafficking, suggesting UL37 mimics these complexes and provides a structural basis for its role in virus trafficking.
Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
NAD+ and NADH play roles in many important biological processes such as energy metabolism, mitochondrial function, calcium homeostasis, and gene expression. They have emerged as one of the most influential couples in life. Poly (ADP-ribose) polymerase 1 (PARP1) activation leads to NAD+ depletion, which mediates cell death. Intranasal administration of NAD+ can decrease ischemic brain injury by reducing PARP1-induced cell death and is a potential treatment for diseases involving PARP1.
1. The document provides information about the educational and professional background of an individual, including degrees obtained from various institutions between 1973-2013 related to prenatal diagnosis, genetics, and rare syndromes.
2. It discusses various levels of genetics from DNA to populations and highlights key concepts like meiosis, mitosis, chromosomes, and mitochondrial DNA.
3. Genetics and genomics provides information at the cellular level about structure, expression, regulation and pathways/networks, but understanding the whole organism is still incomplete and requires integration of concepts from other fields like physics, chemistry and mathematics.
This document summarizes a large-scale genetic screening effort to identify genes implicated in vascular biology in mammals. The screening is done by systematically targeting and trapping genes in embryonic stem cells, then analyzing resulting mouse knockouts. Genes causing lethality are further studied using RNAi knockdown and cell cultures to analyze effects on blood vessel formation in vitro and in developing embryos in vivo. Over 4,000 mouse knockouts have been analyzed so far, with about 20% showing reduced viability or embryonic lethality. Several promising gene targets for neovascular diseases have been identified through this screening process.
This study examines the expression of nicotinic acetylcholine receptors (nAChRs) in neuroepithelial bodies (NEBs) of neonatal hamster lung. The key findings are:
1) NEB cells express mRNA for the β2 subunit of nAChRs and contain α4, α7, and β2 nAChR subunits, as shown by in situ hybridization and immunofluorescence.
2) Patch clamp recordings of NEB cells show that nicotine activates inward currents in a concentration-dependent manner, mediated by nAChRs.
3) The nicotine-induced currents have properties consistent with α3β2, α4β2, and α7
This study investigated the epitope recognized by the broadly neutralizing antibody PG16 against HIV-1. PG16 was found to bind soluble gp140 proteins from clades B and C, but binding was lost for the clade C protein after incubation with a CD4-mimicking protein. Further experiments revealed a spatial component to PG16's neutralization mechanism, as binding of a V2-specific antibody reduced PG16 binding, suggesting they recognize overlapping regions. Additionally, western blot analysis showed PG16 had higher affinity for the clade B protein compared to the clade C protein. Overall, the results suggest PG16 may not recognize a quaternary epitope as previously thought.
This document discusses oligodendrocytes, the cells that form myelin in the central nervous system. It describes their role in development, diseases like multiple sclerosis where remyelination fails, and potential therapies to promote remyelination. A clinical trial was conducted using neural progenitor cell transplantation to treat Pelizaeus-Merzbacher disease, a fatal leukodystrophy caused by myelin deficiency. Research suggests the Wnt signaling pathway inhibits oligodendrocyte differentiation and myelination, and a small molecule Wnt inhibitor was found to accelerate remyelination in mice.
- The crystal structure of the N-terminal half of the UL37 inner tegument protein from pseudorabies virus was determined, revealing an elongated molecule composed of multiple helical bundles.
- Structure-based mutagenesis identified a novel functional region in UL37 important for cell-to-cell virus spread.
- UL37 shares structural similarity with cellular multisubunit tethering complexes, which control vesicular trafficking, suggesting UL37 mimics these complexes and provides a structural basis for its role in virus trafficking.
Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
NAD+ and NADH play roles in many important biological processes such as energy metabolism, mitochondrial function, calcium homeostasis, and gene expression. They have emerged as one of the most influential couples in life. Poly (ADP-ribose) polymerase 1 (PARP1) activation leads to NAD+ depletion, which mediates cell death. Intranasal administration of NAD+ can decrease ischemic brain injury by reducing PARP1-induced cell death and is a potential treatment for diseases involving PARP1.
1. The document provides information about the educational and professional background of an individual, including degrees obtained from various institutions between 1973-2013 related to prenatal diagnosis, genetics, and rare syndromes.
2. It discusses various levels of genetics from DNA to populations and highlights key concepts like meiosis, mitosis, chromosomes, and mitochondrial DNA.
3. Genetics and genomics provides information at the cellular level about structure, expression, regulation and pathways/networks, but understanding the whole organism is still incomplete and requires integration of concepts from other fields like physics, chemistry and mathematics.
This document summarizes a large-scale genetic screening effort to identify genes implicated in vascular biology in mammals. The screening is done by systematically targeting and trapping genes in embryonic stem cells, then analyzing resulting mouse knockouts. Genes causing lethality are further studied using RNAi knockdown and cell cultures to analyze effects on blood vessel formation in vitro and in developing embryos in vivo. Over 4,000 mouse knockouts have been analyzed so far, with about 20% showing reduced viability or embryonic lethality. Several promising gene targets for neovascular diseases have been identified through this screening process.
This study examines the expression of nicotinic acetylcholine receptors (nAChRs) in neuroepithelial bodies (NEBs) of neonatal hamster lung. The key findings are:
1) NEB cells express mRNA for the β2 subunit of nAChRs and contain α4, α7, and β2 nAChR subunits, as shown by in situ hybridization and immunofluorescence.
2) Patch clamp recordings of NEB cells show that nicotine activates inward currents in a concentration-dependent manner, mediated by nAChRs.
3) The nicotine-induced currents have properties consistent with α3β2, α4β2, and α7
This study investigated the epitope recognized by the broadly neutralizing antibody PG16 against HIV-1. PG16 was found to bind soluble gp140 proteins from clades B and C, but binding was lost for the clade C protein after incubation with a CD4-mimicking protein. Further experiments revealed a spatial component to PG16's neutralization mechanism, as binding of a V2-specific antibody reduced PG16 binding, suggesting they recognize overlapping regions. Additionally, western blot analysis showed PG16 had higher affinity for the clade B protein compared to the clade C protein. Overall, the results suggest PG16 may not recognize a quaternary epitope as previously thought.
This document discusses oligodendrocytes, the cells that form myelin in the central nervous system. It describes their role in development, diseases like multiple sclerosis where remyelination fails, and potential therapies to promote remyelination. A clinical trial was conducted using neural progenitor cell transplantation to treat Pelizaeus-Merzbacher disease, a fatal leukodystrophy caused by myelin deficiency. Research suggests the Wnt signaling pathway inhibits oligodendrocyte differentiation and myelination, and a small molecule Wnt inhibitor was found to accelerate remyelination in mice.
This document reports on changes in expression of trk family neurotrophin receptors (trkA, trkB, trkC) during development and progression of medullary thyroid carcinoma (MTC). In normal thyroid C cells, a subset express trkB but not trkA or trkC. In C cell hyperplasia, cells consistently express trkB with variable expression of trkA and trkC. Later stage MTC tumors show substantially reduced trkB expression but increased trkC expression. Exogenous trkB expression in MTC cell cultures resulted in impaired tumorigenicity and lower levels of angiogenesis factor VEGF, suggesting trkB limits MTC growth. Changes in trk receptor expression are involved in M
Viral apoptotic mimicry is when viruses expose phosphatidylserine (PS) on their surface to mimic apoptotic cells and hijack the host cell's apoptotic clearance machinery. PS exposure allows viruses to bind to PS receptors on host cells and be endocytosed. Many viruses use classic apoptotic mimicry by directly exposing PS. Others use non-classic mimicry by mimicking PS receptors like Gas6. Targeting PS is a potential antiviral strategy, as antibodies against PS can opsonize viruses and induce immune clearance. However, more research is needed to fully understand the mechanisms and in vivo relevance of viral apoptotic mimicry.
CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1a...Enrique Moreno Gonzalez
Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.
This study examined the roles of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the development of pulmonary neuroendocrine cells (PNECs) in fetal mouse lung. The study found that:
1) Mash-1 is essential for initiating PNEC phenotype development, while Prox-1 expression downstream of Mash-1 correlates with PNEC maturation.
2) Hypoxia reduced Mash-1 expression and PNEC numbers in early fetal lung explants, but had no effect in mid-fetal lung explants, indicating developmental programming is locked in.
3) Disturbances in intrauterine oxygen levels could alter PNEC functional maturation and
Verifying the role of AID in Chronic Lymphocytic LeukemiaCharlotte Broadbent
This study aimed to verify the role of the enzyme activation-induced deaminase (AID) in chronic lymphocytic leukemia (CLL) using statistical bootstrapping methods. DNA sequences from CLL patients were analyzed before and after AID stimulation. Three tests found statistically significant evidence that AID is active in CLL: 1) more mutations in the variable region than constant region, 2) more mutations at AID hotspots than expected, and 3) fewer mutations at AID coldspots than expected. The results confirm AID's role in somatic hypermutation in CLL, furthering understanding of this disease.
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
This document describes a protocol for differentiating mouse induced pluripotent stem cells (miPSCs) into oligodendrocyte progenitor cells (OPCs). The protocol utilizes a defined, stepwise differentiation process over 8 days to generate high yields of OPCs from three different miPSC lines. The OPCs produced from this protocol are capable of further differentiation into mature, myelinating oligodendrocytes both in vitro and in vivo, making them a potentially useful cell source for modeling demyelinating disorders and developing cell-based therapies.
Whole exome sequencing was performed on a cohort of patients with DOORS syndrome to identify the genetic basis. Homozygous or compound heterozygous mutations in the TBC1D24 gene were identified in several patients. Further analysis showed that the mutations cause loss of function of the TBC1D24 protein, implicating defective vesicular trafficking as the potential cause of DOORS syndrome. However, further studies are still needed to fully understand the role of TBC1D24 and the genetic heterogeneity of this rare disorder.
The document summarizes the identification of six hemocyte cell types in Culex quinquefasciatus by light and transmission electron microscopy:
1) Prohemocytes, the smallest hemocytes, with a large central nucleus and few organelles. They represent 9.3% of hemocytes.
2) Spherulocytes, small hemocytes with numerous spherules containing a lamellar pattern and dense core. They are 1.6% of hemocytes.
3) Adipohemocytes, rare cells with a large nucleolated nucleus, cytoplasm containing organelles and lipid inclusions. They are 0.8% of hemocytes.
4) Oenocytoids
Next generation sequencing (NGS) can simultaneously detect aneuploidy and gene defects in single cells. It involves fragmenting DNA, sequencing short fragments, and using bioinformatics to map the fragments to the human genome. NGS can detect chromosome abnormalities and mutations with the same resolution as array comparative genomic hybridization. It also allows detection of mitochondrial DNA abnormalities and simultaneous analysis of aneuploidy and gene defects. In the future, whole gene sequencing may be possible. NGS is being used successfully for preimplantation genetic diagnosis and was used to produce the first baby born from NGS analysis.
This document summarizes a presentation on using CRISPR-Cas9 for crop improvement. It begins with an introduction to CRISPR-Cas9 and how it is used to edit genomes by removing, adding, or altering DNA sequences. It then discusses the mechanism of the CRISPR-Cas9 complex and how it creates breaks in DNA that are repaired. The document reviews several case studies where CRISPR was used to modify crops, including creating low-gluten wheat and improving rice. It finds that CRISPR can efficiently edit multiple genes simultaneously with few off-target effects. The conclusion states that CRISPR is revolutionizing agriculture by enabling the creation of higher yielding, more resistant crop varieties without transgenes.
Deep Sequencing Identifies Novel Circulating and Hepatic ncRNA Profiles in NA...James Nelson
Next-generation RNA sequencing has expedited the identification of new non-coding RNA species (ncRNAs), thus ushering in the emerging field of ncRNA biology. The goals of this study were to catalogue the spectrum of different ncRNAs in serum and liver of patients with NAFLD and to compare expression of serum exRNAs between NAFLD patients and healthy control subjects.
1) The study analyzed copy number variants (CNVs) in 60 cardiomyopathy-associated genes from over 2,000 individuals who underwent genetic testing.
2) They identified 35 unique CNVs in these individuals, 10 of which were classified as pathogenic and 24 as uncertain significance.
3) Pathogenic CNVs were found in 1.9% of individuals and included deletions in genes like DSP and PKP2 associated with arrhythmogenic right ventricular cardiomyopathy. The identification of pathogenic CNVs provides important clinical implications for affected families.
This document summarizes a project studying the CXCR4 co-receptor in CD4+ T cell normal and depleted sooty mangabeys. The project aims to sequence the CXCR4 gene in sooty mangabeys and compare it to human CXCR4 sequence. The hypothesis is that an amino acid difference in the V3 extracellular loop of sooty mangabey CXCR4 may make it more difficult for HIV to use as a co-receptor compared to humans. Blood samples were collected from normal and depleted CD4+ sooty mangabeys and CXCR4 was amplified by PCR and sequenced. Preliminary results found differences between sooty mangabey and human CXCR4 sequences that could impact HIV
This study aimed to determine if mutations in the human cystatin M/E gene (CST6) contribute to harlequin ichthyosis (HI) by sequencing the entire coding region and intron-exon boundaries of CST6 in 11 patients with HI. No mutations were found in CST6, indicating it is not a major gene for types 1 and 2 HI. However, cystatin M/E protein expression was normal in patient tissues by immunohistochemistry, suggesting regulatory or noncoding mutations were unlikely. While CST6 does not appear to cause HI, further study of its role in skin differentiation and other skin disorders may provide insights into cornification pathways.
Cells undergoing morphological changes during apoptosisAnimatedWorld
1) Researchers developed a cell-free system to induce apoptosis in isolated chicken liver cell nuclei similar to how it occurs in intact cells. The nuclei underwent morphological changes characteristic of apoptosis like chromatin condensation and DNA fragmentation within 60 minutes.
2) Electron microscopy was used to observe the ultrastructural changes during apoptosis and necrosis in different cell types, like nuclear fragmentation and chromatin condensation during apoptosis.
3) A study compared analyzing cell death using annexin V/propidium iodide staining flow cytometry to holographic microscopy coupled with fluorescence detection, finding the latter better identified oncosis due to ability to observe morphological changes.
1) Researchers studied the internal ribosomal entry site (IRES) in the 5' untranslated region of the canine dicistrovirus (CDV-A) genome.
2) Using computational prediction and SHAPE analysis, they determined the secondary structure of the CDV-A 5'UTR IRES, which resembles the poliovirus IRES structure.
3) In vitro translation assays in rabbit reticulocyte lysate showed that the CDV-A 5'UTR IRES can direct cap-independent translation, and requires the initiation factor eIF4A.
The document summarizes a study that found:
1) Interleukin-12 (IL-12) in dendritic cells traffics through late endocytic vesicles marked by the SNARE protein VAMP7.
2) Dendritic cells from VAMP7 knockout mice show partially impaired secretion of bioactive IL-12/p70 dimer.
3) At the immune synapse between dendritic cells and T cells, IL-12 containing vesicles rapidly redistribute and their release is entirely dependent on VAMP7, leading to reduced T cell acquisition of effector functions when stimulated with VAMP7 knockout dendritic cells.
1) Researchers studied the origin of the retrovirus XMRV, which has been detected in human prostate tumors and chronic fatigue syndrome patients.
2) They found two related murine endogenous retroviruses, PreXMRV-1 and PreXMRV-2, in the mouse strains used to passage the human CWR22 prostate tumor xenograft.
3) The researchers conclude that XMRV likely arose through recombination of PreXMRV-1 and PreXMRV-2 during passage of the CWR22 xenograft in mice, as the CWR22 tumor itself did not contain XMRV but later passages and cell lines derived from it did.
This study used an AAV vector to express clarin-1 (CLRN1) in the mouse retina to determine its subcellular localization. CLRN1 was expressed in all major retinal cell types when driven by a ubiquitous promoter. In photoreceptors, CLRN1 localized mainly to the inner segment and outer plexiform layer, similar to other usher proteins. High-titer subretinal delivery led to loss of retinal function, suggesting a critical limit for CLRN1 expression. The results imply CLRN1 expression may be supported by multiple retinal cells and that the dose, promoter, and delivery method need optimization for USH3 gene therapy.
This document summarizes a study investigating the mechanisms by which the nerve growth factor precursor (proNGF) and its receptor p75NTR induce endothelial cell death and the development of acellular capillaries in the retina. The study found that overexpressing a cleavage-resistant form of proNGF in rat retina increased expression of p75NTR and its intracellular domain, decreased levels of mature nerve growth factor (NGF), and induced retinal vascular cell death. Silencing p75NTR using short hairpin RNA prevented these effects of proNGF overexpression and restored NGF levels. In vitro, proNGF overexpression in human retinal endothelial cells reduced NGF levels and induced apoptosis, while silencing p75NTR prevented
This document reports on changes in expression of trk family neurotrophin receptors (trkA, trkB, trkC) during development and progression of medullary thyroid carcinoma (MTC). In normal thyroid C cells, a subset express trkB but not trkA or trkC. In C cell hyperplasia, cells consistently express trkB with variable expression of trkA and trkC. Later stage MTC tumors show substantially reduced trkB expression but increased trkC expression. Exogenous trkB expression in MTC cell cultures resulted in impaired tumorigenicity and lower levels of angiogenesis factor VEGF, suggesting trkB limits MTC growth. Changes in trk receptor expression are involved in M
Viral apoptotic mimicry is when viruses expose phosphatidylserine (PS) on their surface to mimic apoptotic cells and hijack the host cell's apoptotic clearance machinery. PS exposure allows viruses to bind to PS receptors on host cells and be endocytosed. Many viruses use classic apoptotic mimicry by directly exposing PS. Others use non-classic mimicry by mimicking PS receptors like Gas6. Targeting PS is a potential antiviral strategy, as antibodies against PS can opsonize viruses and induce immune clearance. However, more research is needed to fully understand the mechanisms and in vivo relevance of viral apoptotic mimicry.
CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1a...Enrique Moreno Gonzalez
Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.
This study examined the roles of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the development of pulmonary neuroendocrine cells (PNECs) in fetal mouse lung. The study found that:
1) Mash-1 is essential for initiating PNEC phenotype development, while Prox-1 expression downstream of Mash-1 correlates with PNEC maturation.
2) Hypoxia reduced Mash-1 expression and PNEC numbers in early fetal lung explants, but had no effect in mid-fetal lung explants, indicating developmental programming is locked in.
3) Disturbances in intrauterine oxygen levels could alter PNEC functional maturation and
Verifying the role of AID in Chronic Lymphocytic LeukemiaCharlotte Broadbent
This study aimed to verify the role of the enzyme activation-induced deaminase (AID) in chronic lymphocytic leukemia (CLL) using statistical bootstrapping methods. DNA sequences from CLL patients were analyzed before and after AID stimulation. Three tests found statistically significant evidence that AID is active in CLL: 1) more mutations in the variable region than constant region, 2) more mutations at AID hotspots than expected, and 3) fewer mutations at AID coldspots than expected. The results confirm AID's role in somatic hypermutation in CLL, furthering understanding of this disease.
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
This document describes a protocol for differentiating mouse induced pluripotent stem cells (miPSCs) into oligodendrocyte progenitor cells (OPCs). The protocol utilizes a defined, stepwise differentiation process over 8 days to generate high yields of OPCs from three different miPSC lines. The OPCs produced from this protocol are capable of further differentiation into mature, myelinating oligodendrocytes both in vitro and in vivo, making them a potentially useful cell source for modeling demyelinating disorders and developing cell-based therapies.
Whole exome sequencing was performed on a cohort of patients with DOORS syndrome to identify the genetic basis. Homozygous or compound heterozygous mutations in the TBC1D24 gene were identified in several patients. Further analysis showed that the mutations cause loss of function of the TBC1D24 protein, implicating defective vesicular trafficking as the potential cause of DOORS syndrome. However, further studies are still needed to fully understand the role of TBC1D24 and the genetic heterogeneity of this rare disorder.
The document summarizes the identification of six hemocyte cell types in Culex quinquefasciatus by light and transmission electron microscopy:
1) Prohemocytes, the smallest hemocytes, with a large central nucleus and few organelles. They represent 9.3% of hemocytes.
2) Spherulocytes, small hemocytes with numerous spherules containing a lamellar pattern and dense core. They are 1.6% of hemocytes.
3) Adipohemocytes, rare cells with a large nucleolated nucleus, cytoplasm containing organelles and lipid inclusions. They are 0.8% of hemocytes.
4) Oenocytoids
Next generation sequencing (NGS) can simultaneously detect aneuploidy and gene defects in single cells. It involves fragmenting DNA, sequencing short fragments, and using bioinformatics to map the fragments to the human genome. NGS can detect chromosome abnormalities and mutations with the same resolution as array comparative genomic hybridization. It also allows detection of mitochondrial DNA abnormalities and simultaneous analysis of aneuploidy and gene defects. In the future, whole gene sequencing may be possible. NGS is being used successfully for preimplantation genetic diagnosis and was used to produce the first baby born from NGS analysis.
This document summarizes a presentation on using CRISPR-Cas9 for crop improvement. It begins with an introduction to CRISPR-Cas9 and how it is used to edit genomes by removing, adding, or altering DNA sequences. It then discusses the mechanism of the CRISPR-Cas9 complex and how it creates breaks in DNA that are repaired. The document reviews several case studies where CRISPR was used to modify crops, including creating low-gluten wheat and improving rice. It finds that CRISPR can efficiently edit multiple genes simultaneously with few off-target effects. The conclusion states that CRISPR is revolutionizing agriculture by enabling the creation of higher yielding, more resistant crop varieties without transgenes.
Deep Sequencing Identifies Novel Circulating and Hepatic ncRNA Profiles in NA...James Nelson
Next-generation RNA sequencing has expedited the identification of new non-coding RNA species (ncRNAs), thus ushering in the emerging field of ncRNA biology. The goals of this study were to catalogue the spectrum of different ncRNAs in serum and liver of patients with NAFLD and to compare expression of serum exRNAs between NAFLD patients and healthy control subjects.
1) The study analyzed copy number variants (CNVs) in 60 cardiomyopathy-associated genes from over 2,000 individuals who underwent genetic testing.
2) They identified 35 unique CNVs in these individuals, 10 of which were classified as pathogenic and 24 as uncertain significance.
3) Pathogenic CNVs were found in 1.9% of individuals and included deletions in genes like DSP and PKP2 associated with arrhythmogenic right ventricular cardiomyopathy. The identification of pathogenic CNVs provides important clinical implications for affected families.
This document summarizes a project studying the CXCR4 co-receptor in CD4+ T cell normal and depleted sooty mangabeys. The project aims to sequence the CXCR4 gene in sooty mangabeys and compare it to human CXCR4 sequence. The hypothesis is that an amino acid difference in the V3 extracellular loop of sooty mangabey CXCR4 may make it more difficult for HIV to use as a co-receptor compared to humans. Blood samples were collected from normal and depleted CD4+ sooty mangabeys and CXCR4 was amplified by PCR and sequenced. Preliminary results found differences between sooty mangabey and human CXCR4 sequences that could impact HIV
This study aimed to determine if mutations in the human cystatin M/E gene (CST6) contribute to harlequin ichthyosis (HI) by sequencing the entire coding region and intron-exon boundaries of CST6 in 11 patients with HI. No mutations were found in CST6, indicating it is not a major gene for types 1 and 2 HI. However, cystatin M/E protein expression was normal in patient tissues by immunohistochemistry, suggesting regulatory or noncoding mutations were unlikely. While CST6 does not appear to cause HI, further study of its role in skin differentiation and other skin disorders may provide insights into cornification pathways.
Cells undergoing morphological changes during apoptosisAnimatedWorld
1) Researchers developed a cell-free system to induce apoptosis in isolated chicken liver cell nuclei similar to how it occurs in intact cells. The nuclei underwent morphological changes characteristic of apoptosis like chromatin condensation and DNA fragmentation within 60 minutes.
2) Electron microscopy was used to observe the ultrastructural changes during apoptosis and necrosis in different cell types, like nuclear fragmentation and chromatin condensation during apoptosis.
3) A study compared analyzing cell death using annexin V/propidium iodide staining flow cytometry to holographic microscopy coupled with fluorescence detection, finding the latter better identified oncosis due to ability to observe morphological changes.
1) Researchers studied the internal ribosomal entry site (IRES) in the 5' untranslated region of the canine dicistrovirus (CDV-A) genome.
2) Using computational prediction and SHAPE analysis, they determined the secondary structure of the CDV-A 5'UTR IRES, which resembles the poliovirus IRES structure.
3) In vitro translation assays in rabbit reticulocyte lysate showed that the CDV-A 5'UTR IRES can direct cap-independent translation, and requires the initiation factor eIF4A.
The document summarizes a study that found:
1) Interleukin-12 (IL-12) in dendritic cells traffics through late endocytic vesicles marked by the SNARE protein VAMP7.
2) Dendritic cells from VAMP7 knockout mice show partially impaired secretion of bioactive IL-12/p70 dimer.
3) At the immune synapse between dendritic cells and T cells, IL-12 containing vesicles rapidly redistribute and their release is entirely dependent on VAMP7, leading to reduced T cell acquisition of effector functions when stimulated with VAMP7 knockout dendritic cells.
1) Researchers studied the origin of the retrovirus XMRV, which has been detected in human prostate tumors and chronic fatigue syndrome patients.
2) They found two related murine endogenous retroviruses, PreXMRV-1 and PreXMRV-2, in the mouse strains used to passage the human CWR22 prostate tumor xenograft.
3) The researchers conclude that XMRV likely arose through recombination of PreXMRV-1 and PreXMRV-2 during passage of the CWR22 xenograft in mice, as the CWR22 tumor itself did not contain XMRV but later passages and cell lines derived from it did.
This study used an AAV vector to express clarin-1 (CLRN1) in the mouse retina to determine its subcellular localization. CLRN1 was expressed in all major retinal cell types when driven by a ubiquitous promoter. In photoreceptors, CLRN1 localized mainly to the inner segment and outer plexiform layer, similar to other usher proteins. High-titer subretinal delivery led to loss of retinal function, suggesting a critical limit for CLRN1 expression. The results imply CLRN1 expression may be supported by multiple retinal cells and that the dose, promoter, and delivery method need optimization for USH3 gene therapy.
This document summarizes a study investigating the mechanisms by which the nerve growth factor precursor (proNGF) and its receptor p75NTR induce endothelial cell death and the development of acellular capillaries in the retina. The study found that overexpressing a cleavage-resistant form of proNGF in rat retina increased expression of p75NTR and its intracellular domain, decreased levels of mature nerve growth factor (NGF), and induced retinal vascular cell death. Silencing p75NTR using short hairpin RNA prevented these effects of proNGF overexpression and restored NGF levels. In vitro, proNGF overexpression in human retinal endothelial cells reduced NGF levels and induced apoptosis, while silencing p75NTR prevented
This document discusses the role of the cell polarity regulator PARD3 in lung squamous cell carcinomas (LSCC). The key points are:
1. PARD3 was found to be somatically and biallelically inactivated through mutations, deletions, and promoter hypermethylation in 8% of examined LSCC tumors and cell lines. Most alterations resulted in truncated or non-functional PARD3 proteins.
2. Reconstitution of normal PAR3 activity in vivo reduced the invasive and metastatic properties of tumors, suggesting PARD3 acts as a tumor suppressor in LSCC.
3. PARD3 alterations prevented the formation of tight junctions between cells and the downstream signaling of STAT
Molecular Vision Publication by Anuradha SinghAnuradha Singh
This study identified a novel heterozygous mutation, c.525G>C (Q175H), in the VSX1 gene in one individual with keratoconus out of 66 patients studied. Bioinformatics tools predicted this mutation would be potentially damaging. This adds to evidence that VSX1 plays a role in keratoconus, though its exact pathogenic mechanism requires further investigation given the low frequency of this mutation. This is the first report of a VSX1 mutation identified in keratoconus patients from India.
This document summarizes a study that evaluated the potential of an adeno-associated virus (AAV) vector delivering a peptide derived from the Nrf2 protein to target the Nrf2 signaling pathway in the retina. The Nrf2 peptide was fused to a cell-penetrating peptide sequence (Tat-peptide) and expressed from an AAV vector. In vitro, the TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1β secretion, and protected cells from oxidative injury. In mouse models, TatNrf2mer expression partially protected photoreceptor function and decreased inflammatory cytokines and cells in models of retinal oxidative injury and uveitis. The results suggest this AAV-delivered TatNrf
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
LncRNA WARS2-IT1 Functions as an Oncogene and is Associated with Poor Outcome...semualkaira
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors in adults and has high mortality and relapse rates. Over the past few years, great advances have been made in the diagnosis and treatment of GBM, but unfortunately, the five-year overall survival rate of GBM patients is approximately 5.1%. Our study aimed to investigate the new mechanism of Long noncoding RNAs (lncRNAs) WARS2-IT1 regulate the malignant progression of Glioblastoma.
Ultraviolet B (UVB) radiation induces DNA damage and cell death in skin cells. The study investigated which programmed cell death pathways - apoptosis, necroptosis, ferroptosis, or parthanatos - are activated in human dermal fibroblasts following lethal UVB exposure. The results showed that apoptosis is the only cell death pathway induced by UVB in these cells. Additionally, UVB caused a drastic loss of cellular metabolic activity through PARP-dependent overuse of NAD+.
Mitochondrial DNA encodes 13 proteins that are essential components of the oxidative phosphorylation system for producing cellular energy. Over 100 mutations in mitochondrial DNA have been linked to human disease. Mitochondrial DNA is inherited maternally and diseases often involve the muscles, brain, or both. Presentation can occur at any age and involves a variety of neurological or systemic symptoms depending on the mutation. Diagnosis involves clinical evaluation, blood or tissue analysis, and imaging or biopsy. Treatment focuses on supporting affected systems; more targeted therapies are under development.
Heinrich_et_al-2003-International_Journal_of_CancerBianca Heinrich
This study investigated the molecular basis of sporadic and familial multiple meningiomas in 7 unrelated patients without clinical evidence of neurofibromatosis type 2 (NF2). Mutational analysis of the NF2 and DAL-1 genes was performed on tumor and blood samples. Truncating NF2 mutations were found in 3 tumors but not blood, and 2 tumors showed loss of the NF2 locus. In contrast, 5 non-truncating changes were found in DAL-1 in both tumor and blood samples, with no loss of the DAL-1 locus. The findings provide evidence that the molecular basis of sporadic and familial multiple meningiomas differs, with NF2 inactivation involved in sporadic
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
This document reports on a study investigating the effect of the ABCA4 gene variant c.5461-10T/C, a frequent cause of Stargardt disease (STGD1), on mRNA splicing. The study found that in patient-derived photoreceptor progenitor cells, this variant resulted in skipping of exon 39 or exons 39 and 40 in the ABCA4 mRNA. In vitro minigene assays confirmed the variant caused these splicing defects. Patients who were homozygous for the variant displayed a severe phenotype, with early onset of STGD1, poor vision, and legal blindness by age 25. The variant was found to be located on a founder haplotype lacking other disease-causing variants,
This research article describes how plant pathogens have evolved to counteract central nodes of plant immune receptor networks. The researchers screened 165 pathogen effectors and identified 5 that suppressed cell death triggered by NLR immune receptors called NRCs. Further analysis showed that a cyst nematode effector and an oomycete effector specifically inhibited the function of two NRC proteins, NRC2 and NRC3, but not NRC4. The nematode effector bound directly to NRC2 and NRC3, while the oomycete effector acted through a host membrane trafficking protein to suppress NRC responses. This suggests that different pathogens have independently evolved effectors that target central nodes of the plant NLR network to promote infection. Coevolution with such
This document summarizes a study investigating how the N- and C-terminal regions of rotavirus nonstructural protein 4 (NSP4) influence its biochemical and biological properties. NSP4 is a multifunctional protein that plays a key role in rotavirus infection and virulence. The study found that cooperation between a putative amphipathic alpha-helix near the N-terminus and the C-terminal diarrhea-inducing region generates a unique conformational state in NSP4. This state promotes multimerization and enhances the protein's resistance to digestion and biological functions. Mutations disrupting the interaction between these terminal regions compromised NSP4's properties, providing insight into how its structure modulates vir
This document summarizes a research study that investigated the role of long non-coding RNA H19, microRNA miR-29b, and the gene FOXO4 in the apoptosis of retinal Müller cells in diabetic retinopathy. The study found that H19 and FOXO4 expression were increased, while miR-29b expression was decreased, in retinal tissue from diabetic rats and in retinal Müller cells treated with high glucose. Overexpression of H19 promoted retinal Müller cell apoptosis. Knockdown of H19 reversed the effects of high glucose by decreasing cell apoptosis and FOXO4 upregulation. Further experiments indicated that H19 is a target of miR-29b and inhibition of miR-29b
This document provides personal and professional information about Dr. Teh-Ying Chou. It summarizes her educational background, current positions, work experience, research interests and topics. Her research focuses on the pathobiology of lung diseases, including studies on EGFR mutations in lung cancer patients treated with gefitinib, genetic profiling of in situ and invasive lung adenocarcinomas, and the role of Clusterin in epithelial-mesenchymal transitions and metastasis of lung cancer cells. She has published papers on biomarkers for acute respiratory distress syndrome outcomes and the predictive value of EGFR mutations for gefitinib treatment responses in lung cancer.
1) The C-terminal carboxylate group of integrin β1 is necessary for its association with the kindlin-2 adapter protein. Affinity measurements indicate this interaction is coordinated by a putative carboxylate-binding motif in the FERM subdomain F3 of kindlin-2.
2) Injection of mRNA encoding mutant integrin β1 cytoplasmic tails that lack the C-terminal carboxylate group perturbed laterality organ development in zebrafish, indicating the carboxylate group is required for physiological functions.
3) The unusual interaction between integrin β1 and kindlin-2 identified here represents a novel protein-protein interaction mode governed mainly by the integrin
This document discusses microsatellite instability (MSI), which refers to changes in repeated sequences of DNA called microsatellites during DNA replication due to defects in the DNA mismatch repair system. The key mechanisms, detection methods, clinical significance and applications of MSI in various cancers are described. MSI is an important factor in tumor development and progression. Detection of MSI using techniques such as PCR and immunohistochemistry can aid in cancer diagnosis and predict response to immunotherapy. Cancers with high MSI (MSI-H) often respond well to immune checkpoint inhibitors.
Similar to Dinculescu IOVS i1552-5783-56-11-6971 (20)
1. Retina
Pathological Effects of Mutant C1QTNF5 (S163R)
Expression in Murine Retinal Pigment Epithelium
Astra Dinculescu, Seok-Hong Min, Frank M. Dyka, Wen-Tao Deng, Rachel M. Stupay,
Vince Chiodo, W. Clay Smith, and William W. Hauswirth
Department of Ophthalmology, University of Florida, Gainesville, Florida, United States
Correspondence: Astra Dinculescu,
Department of Ophthalmology, Uni-
versity of Florida, 1600 SW Archer
Road, Gainesville, FL 32610-0284,
USA;
astra@ufl.edu.
Submitted: April 24, 2015
Accepted: September 30, 2015
Citation: Dinculescu A, Min S-H, Dyka
FM, et al. Pathological effects of
mutant C1QTNF5 (S163R) expression
in murine retinal pigment epithelium.
Invest Ophthalmol Vis Sci.
2015;56:6971–6980. DOI:10.1167/
iovs.15-17166
PURPOSE. The mutation S163R in complement C1q tumor necrosis factor–related protein-5
(C1QTNF5) causes an autosomal dominant disorder known as late-onset retinal degeneration
(L-ORD). In this study, our goal is to evaluate the consequences of mutant S163R C1QTNF5
expression in mouse RPE following its delivery using an adeno-associated viral (AAV) vector.
METHODS. We generated AAV vectors containing either human wild-type C1QTNF5 or mutant
S163R C1QTNF5 driven by an RPE-specific BEST1 promoter, and delivered them subretinally
into one eye of adult C57BL/6 mice. Transgene expression was detected by immunohisto-
chemistry. Retinal function was assessed by full-field ERG. Pathological changes were further
examined by digital fundus imaging and spectral-domain optical coherence tomography (SD-
OCT).
RESULTS. We show that the AAV-expressed mutant S163R leads to pathological effects similar
to some of those found in patients with advanced L-ORD, including RPE thinning, RPE cell
loss, and retinal degeneration. In addition, we provide in vivo evidence that mutant S163R
C1QTNF5 can form large, transparent, spherical intracellular aggregates throughout the RPE,
which are detectable by light microscopy. In contrast to AAV-expressed wild-type C1QTNF5,
which is secreted apically from the RPE toward the photoreceptor cells and the outer limiting
membrane, the S163R mutant is primarily routed toward the basal side of RPE, where it forms
thick, extracellular deposits over time.
CONCLUSIONS. Adeno-associated viral–targeted expression of mutant S163R in the RPE
represents a useful approach for quickly generating animal models that mimic pathological
features of L-ORD and offers the potential to understand disease mechanisms and develop
therapeutic strategies.
Keywords: retina, adeno-associated virus, age-related macular degeneration, retinal pigment
epithelium, late-onset retinal degeneration
The retinal pigment epithelium is a polarized monolayer of
postmitotic epithelial cells located between the neural
retina and the vascular choroid. On its apical side, it contains
numerous microvilli extending into the interphotoreceptor
matrix, and surrounding the outer segments of rods and cones.
On the basal side is Bruch’s membrane, a thin, multilayered,
extracellular matrix1 in which pathological changes are integral
to AMD.2,3
The retinal pigment epithelium performs functions
essential for both the structural integrity and survival of
photoreceptor cells, including transport of nutrients, retinoids
and waste products, phagocytosis of outer segments, synthesis
of the visual chromophore11-cis retinal, and production of
growth factors.4,5
The mutation S163R in the globular gC1q domain of
complement C1q tumor necrosis factor–related protein-5
(C1QTNF5/CTRP5) causes a rare autosomal dominant disorder
known as late-onset retinal degeneration (L-ORD).6
The earliest
symptoms appear in midlife, manifested as difficulties in dark
adaptation and loss of night vision.7–9
Over time, the disorder
progresses to severe loss of both central and peripheral vision,
with some patients also developing choroidal neovasculariza-
tion in late stages of the disease10
that mimic neovascular AMD.
In addition, patients also present with abnormally long anterior
zonular insertions on the lens capsule which serve as an early
disease phenotype.11,12 The hallmark of L-ORD is the presence
of abnormal extracellular deposits between the RPE and
Bruch’s membrane.7,13
These thick deposits, which are
histopathologically similar to those found in AMD patients,
have a widespread distribution, extending outside the macular
region into the peripheral retina.14 Although there is evidence
that supplementation with vitamin A may be beneficial early in
the disease course when patients exhibit dark-adaptation
abnormalities,11
there is no current treatment that prevents
the progressive retinal degeneration leading to vision loss.
C1QTNF5 belongs to the C1q and tumor necrosis factor-
related superfamily (C1q/TNF), characterized by an N-terminal
signal peptide, a short variable region, a collagen repeat
domain, and a C-terminal globular domain (gC1q) that is
homologous to the immune complement component 1q. All
members of the C1q/TNF family are secreted multimeric
proteins, with specific tissue expression profiles, and many
are found circulating in plasma, with levels influenced by
genetic background, sex, and metabolic status. Members of the
C1q/TNF family are of broad clinical interest since they play
numerous roles in immunity and inflammation, glucose and
lipid metabolism, and vascular maintenance.15,16
C1QTNF5 is a
Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.
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2. membrane-associated and secreted protein of unknown
function, whose expression is the highest in the RPE and
ciliary epithelium.11,17,18
It colocalizes with ZO1, an RPE tight-
junction protein, at the hexagonal RPE membrane, and is also
secreted into the interphotoreceptor matrix.17,19
C1QTNF5
can also be found in other ocular tissues, including the lens.17
It is secreted by the adipose tissue, and circulates abundantly in
human sera, potentially playing an important role in energy
homeostasis and obesity-related inflammation.16,20
The crystal
structure of the trimeric globular domain of C1QTNF5 was
determined at 1.34 ˚A resolution by Tu and Palczewski,21
who
also showed that wild-type C1QTNF5 can multimerize into a
bouquet-like octadecameric assembly containing six trimers.22
Previous in vitro studies have demonstrated that the S163R
mutant is not secreted, has the tendency to aggregate, and is
retained primarily within the endoplasmic reticulum.17,23
Interestingly, C1QTNF5 is expressed as a dicistronic
transcript with MFRP, encoding the membrane-type frizzled
related protein.6,24
The complete open reading frame of
C1QTNF5 is located within the 30
untranslated region of the
final exon of MFRP. The two proteins were shown to directly
interact; however, the functional significance of this interac-
tion is not currently understood.18,22,23
Mutations in the two
genes are associated with distinct disease phenotypes in
humans, with the dominant S163R mutation in C1QTNF5
leading to L-ORD, whereas mutations in MFRP cause autosomal
recessive retinitis pigmentosa (RP), nanophthalmos, foveoschi-
sis, and optic disc drusen.25–27
We have previously shown that subretinal delivery of a fast-
acting AAV8-733YF vector expressing wild-type MFRP led to
robust transgene expression at its normal RPE location on the
apical membrane and microvilli, prevented retinal degenera-
tion, and rescued photoreceptor function in the rd6 mouse
model of RP.28
In this study, we used the recombinant AAV
vector technology to examine the pattern of mutant S163R
transgene expression and its pathological effects following
specific RPE targeting. We show that AAV-expressed wild-type
C1QTNF5 is secreted apically toward photoreceptor cells and
outer limiting membrane (OLM). In contrast, AAV-expressed
S163R mutant forms large spherical intracellular aggregates
within the RPE cells of normal mice, and gradually accumulates
as thick, widespread extracellular basal deposits, leading to
local regions of RPE atrophy and retinal degeneration. These
findings have important mechanistic and therapeutic implica-
tions for L-ORD and perhaps for AMD as well.
METHODS
AAV Vector Production and Subretinal Injections
Self-complementary AAV2 quadruple capsid tyrosine mutant
(Y272,444,500,730F) vectors (scAAV2quadYF) were used for
packaging and expressing either human wild-type C1QTNF5
(hC1QTNF5WT-HA) or the S163R mutant (hC1QTNF5(S163R)-
HA), driven by an RPE-selective human BEST1 promoter.29
Both wild-type and mutant S163R C1QTNF5 were tagged with
hemagglutinin (HA) at their C-terminal end for subsequent
detection by immunostaining. The quadruple capsid tyrosine
mutant vector AAV2 containing point mutations of surface-
exposed tyrosine residues was chosen for transgene delivery in
all these experiments based on its higher transduction
efficiency following a single injection, compared to wild-type
AAV2.30 The viral vectors were packaged, purified, and titered
according to previously reported methods.31 The vectors were
then subretinally injected in adult C57BL/6 mice (Jackson
Laboratory, Bar Harbor, ME, USA) as previously described.32
Each eye received 1 lL vector at a titer of 1 3 1012 genome
copies/mL, leaving the left eye as an untreated control. Animals
were maintained on a 12-hour light and 12-hour dark cycle in
the University of Florida Health Science Center Animal Care
Services Facility (Gainesville, FL, USA). All experiments were
approved by University of Florida Institutional Animal Care and
Use Committees and conducted in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision
Research.
Immunostaining and Histologic Analysis
Eyes were harvested, processed for paraffin embedding, and
sectioned at a thickness of 4 lm, at the same retinal locations
for treated and untreated eyes. For morphologic analysis, the
sections were stained with hematoxylin and eosin, and
visualized by light microscopy. For immunostaining, deparaffi-
nized tissue sections were dewaxed in xylene and rehydrated
in a graded series of ethanol, then incubated with 0.3% Triton
X-100 for 15 minutes, followed by blocking with a solution of
2% albumin for 30 minutes. Hemagglutinin-tagged C1QTNF5
proteins in the AAV-treated eyes were detected by immuno-
staining with a high-affinity anti-HA–fluorescein antibody
(3F10; Roche Diagnostics, Indianapolis, IN, USA). Some
sections were also incubated with mouse MFRP affinity-
purified polyclonal antibody (AF3445, R&D Systems, Minneap-
olis, MN, USA), followed by the Alexa Fluor 594 secondary
antibody (Molecular Probes/Invitrogen, Eugene, OR, USA).
Negative controls to check for any potential autofluorescence
in AAV-expressed S163R-treated retinas were also performed by
omitting the anti-HA antibody, and purposefully overexposing
those images to detect any background autofluorescence. The
mouse monoclonal antibody B6-30 against rod opsin33
was
used to label the outer segments. Fluorescence micrographs at
higher magnification were also acquired using a 3100 oil
immersion objective. All sections were examined by fluores-
cence microscopy using a microscope (Axiophot; Carl Zeiss
Microscopy, LLC, Thornwood, NY, USA). Cryosections for Oil
red O staining to detect neutral lipids in the AAV-expressed
S163R-treated eyes were also used. Frozen liver sections were
used as positive control for the lipid staining. The liver was
from a mouse that received intraperitoneal injections with
carbon tetrachloride (CCl4) to cause hepatocyte damage and
liver fibrosis.34
Cryosections were air dried at room tempera-
ture for 30 minutes and rinsed with distilled water. Sections
were then placed in polyethylene glycol (PEG), stained with
Oil red O (Abcam, Cambridge, UK), rinsed with 85% PEG, then
with distilled water, before adding an aqueous mounting
medium (Dako, Carpinteria, CA, USA). Images were acquired
using brightfield microscopy.
Transmission Electron Microscopy
For ultrastructural analysis, the injected eyes containing S163R
aggregates and basal deposits that were identified at the light
level were further examined by transmission electron micros-
copy (TEM). Paraffin blocks were submitted to the Electron
Microscopy Core Laboratory, Interdisciplinary Center for
Biotechnology Research, University of Florida. Areas of interest
containing the RPE–choroid interface were excised from the
paraffin blocks and placed in 100% xylene overnight. The
dewaxed tissues were rehydrated through a decreasing ethanol
series, water washed, buffer washed, and fixed with Trumps
(Electron Microscopy Sciences, Hatfield, PA, USA). Fixed
tissues were processed with the aid of a laboratory microwave
(Pelco BioWave Pro; Ted Pella, Redding, CA, USA). The samples
were washed in 0.1 M cacodylate buffer pH 7.24, postfixed
with 2% OsO4, water washed and dehydrated in a graded
ethanol series 25%, 50%, 75%, 95%, 100%. Dehydrated tissues
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3. were infiltrated with 1:1 ethanol/LR White hard acrylic resin
(Electron Microscopy Sciences, Hatfield, PA, USA) then
embedded in 100% LR White resin and polymerized at 608C
for 48 hours. The cured resin blocks were hand-trimmed and
prepared for ultramicrotomy. Semi-thick (500 nm) and ultra-
thin sections (100–120 nm) were cut using a diamond knife.
Semi-thick sections were dried onto a glass slide and stained
with toluidine blue. Ultra-thin sections were collected on
carbon-coated Formvar copper slotted grids, poststained with
2% aq. uranyl acetate, and Reynolds lead citrate. Sections were
examined with a FEI Tecnai Spirit LaB6 at 120 kV (FEI,
Hillsboro, OR, USA) and images acquired with a charge-
coupled device camera (AMT XR41, 2k X 2k; Advanced
Microscopy Techniques, Woburn, WA).
Western Blot Analysis
Whole eyecups from AAV-S163R–treated eyes (1 month post
injection) were homogenized by sonication in RIPA buffer
(Sigma-Aldrich Corp., St. Louis, MO, USA) containing a
complete protease inhibitor cocktail (Roche Diagnostics,
Mannheim, Germany). After centrifugation, the supernatant
was mixed with 2X Laemmli’s sample buffer containing
dithiothreitol (DTT) and subjected to SDS-PAGE. Protein
extracts from AAV–wild-type C1QTNF5 or AAV-S163R–injected
eyecups were also analyzed under nondenaturing conditions
by sonicating the tissues in a sample buffer lacking the SDS and
DTT, and loading the supernatants onto 4% to 15% gradient
Mini Protean TGX precast polyacrylamide gels (Bio-Rad
Laboratories, Inc., Hercules, CA, USA). After protein transfer
onto polyvinylidene difluoride membranes (Millipore, Bedford,
MA, USA) and blocking by incubation for 1 hour in Odyssey
blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), the
membranes were probed with a mouse monoclonal primary
antibody against the HA tag (Covance, Gaithersburg, MD, USA).
Signals were detected with the Odyssey infrared fluorescence
imaging system (LI-COR Biosciences), using an anti-mouse
antibody conjugated with CW800 dye (LI-COR Biosciences).
Electroretinographic Analysis and Fundoscopy
Treated C57BL/6 mice were dark-adapted (>12 hours) and
anesthetized by intraperitoneal injection. Full-field ERGs were
recorded as previously described,32 using a UTAS Visual
Diagnostic System (LKC Technologies, Gaithersburg, MD,
USA). Dark-adapted ERGs were elicited with 0.02, 0.2 and 2
scot cdÁsÁmÀ2
stimuli. Cone-isolated ERGs were elicited with a
25 phot-cdÁsÁmÀ2 (10 dB) stimulus on a rod-suppressing
background light after a preadaptation period. Differences in
b-wave maximum amplitudes between AAV hC1QTNF5(S163R)-
HA–injected and uninjected contralateral left eyes were
analyzed by Student’s t-test for paired samples (GraphPad Prism
6.0; GraphPad Software, San Diego, CA, USA), and considered
statistically significant if P < 0.05. Electroretinographic data are
presented as mean 6 SEM. The Micron III fundus imaging
system (Phoenix Research Laboratories, Pleasanton, CA, USA)
was used to obtain digital images of the central retina.
SD-OCT
Spectral-domain optical coherence tomography (SD-OCT) was
performed at 9 months post injection using a high-resolution
SD-OCT instrument (Bioptigen, Inc., Research Triangle Park,
Durham, NC, USA) for image capture and to measure the
thickness of the retina, as previously described.35
Neural retina
thickness was determined by measuring the distance from the
vitreal face of the ganglion cell layer to the retinal pigment
epithelium apical side. Four measurements at the same
distance from the optical nerve head were recorded from
each eye and averaged. Statistical analysis was performed by
paired t-test, with P < 0.05 considered statistically significant.
RESULTS
Distribution of AAV-Expressed Wild-Type C1QTNF5
and S163R Mutant
AAV2quadYF vectors containing either hC1QTNF5WT-HA or
hC1QTNF5(S163R)-HA transgenes were delivered to wild-type
mouse eyes in order to compare the distribution patterns of
the wild-type and S163R mutant following specific RPE
targeting, and to document potential pathological effects. In
order to avoid detecting endogenous C1QTNF5, a C-terminal
HA tag was added for the reliable detection of transgene
expression by immunostaining in each case. We determined
that wild-type C1QTNF5 was secreted apically from the RPE,
reaching the outer limiting membrane and interdigitating with
photoreceptor cells inner segments (Fig. 1A). Adeno-associated
viral-expressed C1QTNF5 appears to diffuse further, beyond
the outer limiting membrane, being detectable between
photoreceptor nuclei (Fig. 1B).
In stark contrast to vector expressed wild-type protein,
AAV-expressed S163R is predominantly retained within RPE
cells, where it forms large spherical or oval-shaped intracel-
lular aggregates (Figs. 1C–E). In some regions containing the
aggregates, RPE microvilli were missing, as revealed by
double immunostaining with an anti-MFRP antibody, which
detects the endogenous MFRP found abundantly across the
entire length of RPE apical processes (Fig. 1D). Aggregates of
S163R are transparent when examined under brightfield
microscopy (Fig. 1F). Interestingly, S163R mutant protein was
also secreted toward the basolateral side of the RPE, where it
formed a continuous layer (Fig. 2). Sections from AAV-S163R–
treated eyes were examined at higher magnification (3100)
using differential interference contrast (DIC) microscopy (Fig.
2A) and merged with the fluorescent image (Fig. 2B) to
provide a clear view of the S163R extracellular basal deposit
and globular aggregates. Transmission electron microscopy
was also used to examine the ultrastucture of the RPE-Bruch’s
membrane interface in S163R-injected eyes and confirmed
that the S163R layer accumulates between the basement
membrane of the RPE and its cell membrane, with large
S163R aggregates being present inside the RPE cytoplasm
(Figs. 2C, 2D).
Staining the AAV-S163R–treated sections with hematoxylin
and eosin (H & E stain) showed that the S163R aggregates
appear eosinophilic (pink; Fig. 3). Aggregates of S163R were
found throughout the RPE after a single subretinal delivery of
hC1QTNF5(S163R)-HA vector at 4 months post injection. The
retinal pigment epithelium appears vacuolated in many
regions, although some of these may be where the aggregates
were lost during tissue processing (Fig. 3, center and right
panels).
Detection of S163R Deposits in Older Mice
By 9 months post injection, AAV-S163R–treated eyes display
regions of RPE thinning and atrophy, as well as photoreceptor
cell loss, particularly in those areas where thick, dome-shaped
extracellular basal RPE deposits are present (Fig. 4B). In
contrast, uninjected control eyes show normal RPE and retinal
morphology (Fig. 4A). The basal RPE deposits consistently
formed a continuous layer in every AAV-S163R–treated eye
(Figs. 4C, 4D), and were determined to consist of S163R
mutant protein by immunofluorescence following staining
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4. with the anti-HA antibody (Fig. 5A, Supplementary Fig. S1). We
also tested for the presence of lipids following staining of
cryosections with oil red O, and did not detect lipid
accumulation in the AAV-S163R–injected eyes
(Supplementary Fig. S1).
The thick S163R deposits located between the Bruch’s
membrane and the deteriorating RPE layer appear transparent
under brightfield microscopy (Fig. 5B) and pink following H &
E staining (Fig. 5C). Interestingly, the spherical S163R
C1QTNF5 aggregates are mostly absent at this stage, suggesting
FIGURE 1. Detection of AAV-expressed wild-type C1QTNF5 and mutant S163R by immunofluorescence microscopy. (A) Vector-expressed wild-type
C1QTNF5 (green) is present throughout the RPE, and apically secreted toward the OLM, where it interdigitates with photoreceptor inner segments.
(B) Same image without DAPI nuclear stain, showing AAV-expressed wild-type C1QTNF5 diffusing beyond the OLM, between photoreceptor nuclei
(2 months post injection). (C–E) Detection of AAV-expressed mutant S163R C1QTNF5 (green) by immunofluorescence microscopy. Round or oval-
shaped S163R aggregates (arrows) are present throughout the RPE (4 months post injection). Note the continuous layer of S163R mutant protein
deposit on the basal side of the RPE membrane. (D) Double-labeling with an anti-MFRP antibody (red) and anti-HA (green) following AAV-
hC1QTNF5(S163R)-HA treatment. Note the reduced MFRP immunostaining in some areas containing large intracellular S163R aggregates (green),
indicating loss of RPE microvilli (arrow). (F) Transparent spherical S163R aggregates as examined by brightfield light-microscopy (arrow). Scale
bars: 30 lm. IS, inner segments; ONL, outer nuclear layer; OS, photoreceptor outer segments.
FIGURE 2. Examination of the S163R C1QTNF5 deposits at high magnification. (A) Image acquired using DIC microscopy at 3100 magnification.
(B) Differential interference contrast image merged with the fluorescent image obtained following immunostaining with an anti-HA antibody. Note
the intracellular globular S163R aggregate (red arrows) and the extracellular (green) basal RPE deposit. Scale bars: 25 lm. (C) Ultrastructural
examination of the RPE/Bruch’s membrane interface after AAV-S163R treatment using transmission electron microscopy. (D) Inset from (C) at
higher magnification showing the area containing the intracellular and basal laminar deposits.
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5. that the RPE may have developed a mechanism for their
transport and deposition on their basal surface. The remaining
photoreceptors retained their outer segments, as demonstrated
by immunostaining with a rod opsin antibody (Fig. 5D). Low
magnification images show that the S163R deposit above the
RPE is continuous and widespread (Figs. 6A–C). The endoge-
nous MFRP protein is expressed throughout the RPE apical
membrane (Fig. 6C).
Detection of AAV-Expressed S163R C1QTNF5 by
Western Blot Analysis
We also evaluated the properties of AAV-expressed S163R
mutant protein by Western blot analysis of whole eyecup
protein extracts (Fig. 7). The adeno-associated viral-delivered
mutant protein was detected primarily as a single immunore-
active band corresponding to the expected size of C1QTNF5
FIGURE 3. Histologic examination of AAV-hC1QTNF5(S163R)-HA–treated eyes by light microscopy at 4 months post injection. Staining with H & E
of paraffin sections reveals the presence of eosinophilic (pink) aggregates (arrows) throughout the RPE cytoplasm of AAV-S163R–treated eyes. The
choroid is detached from RPE (all panels) and photoreceptor outer segments have become disorganized (higher magnification images, middle and
right). Scale bars: 20 lm.
FIGURE 4. Histologic examination of AAV-hC1QTNF5(S163R)-HA–treated eyes by light microscopy at 9 months post injection. (A) Section stained
with H & E of an untreated wild-type mouse eye showing normal RPE and retinal structure. (B) Section stained with H & E of an AAV-
hC1QTNF5(S163R)-HA–injected eye. Note the thick deposit on the basal side of the RPE, the photoreceptor cell loss, and the RPE thinning and
atrophy in contrast to the normal eye. (C, D) Higher magnification images showing the RPE and the eosinophilic (pink) basal deposits in two
different eyes. Scale bars: 15 lm.
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6. (slightly larger than 25 kDa) under denaturing and reducing
conditions (Fig. 7A). We found S163R protein was present as
high molecular weight aggregates when eyecups were
sonicated in a buffer without SDS, a property consistent with
results from previous in-vitro studies6,23
(Fig. 7B, first lane). In
contrast, the AAV-expressed wild-type C1QTNF5 was detected
as primarily a monomer, dimer, and trimer, as well as less
abundant higher order oligomers in samples processed without
SDS (Fig. 7B, second lane). In the presence of SDS, AAV-
expressed wild-type C1QTNF5 was detected primarily as a
strong monomeric immunoreactive band, and as less abundant
signals corresponding to oligomeric and posttranslationally
modified forms in a smeared appearance of immunoreactive
bands (Fig. 7B, last lane).
Noninvasive Assessment of Pathological Effects
Noninvasive approaches were also used to analyze the
structural and functional consequences of the mutant
S163R expression in the RPE (Fig. 8). Full field ERG analysis
was performed under scotopic dark-adapted, and photopic
light adapted cone-mediated conditions to evaluate the
effects of S163R protein on retinal function (Fig. 8A). There
was a small but significant decrease in b-wave amplitudes in
treated versus untreated mouse eyes by 2 months post
injection under scotopic conditions at all light-intensities
(Fig. 8A, left panel). For example, AAV-injected eyes had a
rod-isolated b-wave amplitude (at 0.02 scot cdÁsÁmÀ2
) of
238.5 6 19.46 lV, which was 20% lower than the untreated
controls, with a maximum b-wave amplitude average of 296.5
6 26.12 lV (Average 6 SEM, n ¼ 13, P ¼ 0.001). At 5 months
FIGURE 5. Examination of S163R expression at 9 months following AAV-hC1QTNF5(S163R)–HA delivery. (A) Detection of the AAV-expressed S163R
by immunofluorescence. Note the thick, continuous S163R basal deposit, which appears transparent in the brightfield above the pigmented RPE
(B). (C) Staining with H & E showing the thick basal deposit above a highly vacuolated, deteriorated RPE. (D) Double-labeling with an antibody
against rod opsin showing the outer segments (red), and anti-HA (green) detecting the thick S163R deposit basal to the RPE. Scale bars: 20 lm.
FIGURE 6. Widespread distribution of basal S163R deposits at 9 months following AAV-hC1QTNF5(S163R)–HA delivery. (A–C) Low magnification
images of retinal sections showing continuous, widespread S163R expression (green) above the RPE by immunofluorescence (A, C). (B) Brightfield
image showing the transparent basal S163R deposit between the choroid and the pigmented RPE. (C) The RPE apical membrane is shown in red as
labeled by the anti-MFRP antibody.
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7. post injection (Fig. 8A, right panel), the maximum ERG b-
wave amplitudes reflecting the rod-mediated function (at
0.02 scot cdÁsÁmÀ2
flashes) further declined, being approx-
imately 30% lower relative to control eyes (average of 134.3
6 14.59 lV in AAV-treated versus 186 6 9.02 lV in
contralateral eyes, n ¼ 5, P ¼ 0.036). Maximum cone b-wave
amplitudes recorded under photopic conditions were similar
in treated and untreated eyes. Similar to other cases of RPE-
based retinal degeneration, such as the rd6 model caused by
lack of functional MFRP, cone-driven ERG amplitudes are
initially less affected compared with that of rods, perhaps
because cones can also rely on an alternate visual cycle using
M¨uller cells.36,37
Pathological changes in vector-treated eyes were also seen
by fundoscopy and OCT (Figs. 8B, 8C). Eyes treated with
AAV-S163R display an abnormal fundus with disperse
pigmentary changes (Fig. 8B, right panel). By imaging with
OCT, retinal thinning was seen in treated eyes by 9 months
post injection, consistent with a loss of photoreceptor cells
relative to untreated partner retinas (Fig. 8C). Comparison of
retinal thicknesses showed that AAV-S163R treated eyes were
0.137 6 0.021 mm thick, while untreated contralateral
controls were 0.206 6 0.014 mm (average 6 SEM, n ¼ 4, P ¼
0.01).
DISCUSSION
Although L-ORD is a rare inherited disorder, some of its clinical
features resemble AMD, a very common retinal degenerative
disease that causes progressive loss of central vision that
represents a major cause of blindness in the elderly.2,38,39
Specifically, both L-ORD and AMD exhibit age-related formation
of drusen between the RPE and Bruch’s membrane, a key
predictor of eventual AMD.6
In spite of the identification of
numerous mouse models that exhibit some aspects of AMD,
none mimic the disease accurately due to the lack of an
anatomical macula in rodents and the complex nature of the
disease, which results from an incompletely understood
interplay among multiple genetic and environmental fac-
tors.39–42 One feature that makes L-ORD a potentially
informative model for AMD is its well-established monogenic
etiology that leads to retinal pathology due to sub-RPE deposits
FIGURE 8. Evaluation of pathological effects following AAV-hC1QTNF5(S163R)–HA delivery using noninvasive approaches. (A) Comparison of
retinal function in AAV-S163R-C1QTNF5–treated and untreated eyes following ERG analysis. Bar graphs show the maximum ERG b-wave
amplitudes in AAV-S163R injected eyes (black) compared with untreated control eyes (gray) at 2 months (left panel) and 5 months (right panel)
post injection. Maximum b-wave amplitudes in AAV-S163R injected eyes were significantly lower than untreated controls at all light intensities under
scotopic conditions (*P < 0.05), and were similar under light-adapted conditions. (B) Fundus photographs show pigmentary abnormalities in AAV-
S163R–injected eyes (right) in contrast to the normal untreated age-matched control (left). (C) Evaluation of AAV-S163R–treated eyes by OCT
imaging. The OCT scan intersecting the optic nerve head shows the normal retinal laminar architecture in the untreated eye (left). The ONL
thickness was markedly reduced in the AAV-S163R–treated eye (right) at 9 months post injection, with numerous irregular deposits visible.
FIGURE 7. Western blot analysis of AAV-expressed S163R C1QTNF5.
(A) Detection of AAV-expressed S163R mutant protein from whole
eyecup extracts following SDS-PAGE separation and immunoblotting
with an anti-HA antibody. (B) Detection of AAV-expressed S163R and
wild-type proteins following resolution on nondenaturing gels. Note
the distinct immunoreactive pattern of the S163R mutant compared to
that of wild-type C1QTNF5. Prestained protein standards in kilodaltons
are shown on the left.
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8. that extend into the peripheral retina, causing widespread
photoreceptor cell loss and RPE atrophy.6,7
Consequently,
mouse models of L-ORD represent a novel platform for
developing treatment strategies that may apply to key aspects
of AMD as well.
Of the two knock-in mouse models of L-ORD generated to
date, only one was found to display an ocular phenotype
reflecting many of the early-stage characteristics of the
disease, with the other model showing no abnormalities
throughout its lifespan.43,44 Recombinant AAV vectors have
several advantages over transgenic approaches, including the
potential for quickly generating animal models, often with a
faster time course of disease progression, controlling mutant
protein expression using the appropriate vector serotype,
promoter and titer, and producing graded levels of pathology,
from early to late-stage disease phenotypes.45
Adeno-associ-
ated viral technology, combined with the use of an RPE-
specific VMD2 (bestrophin) promoter, enabled us to examine
the in vivo consequences of S163R C1QTNF5 mutant
expression in wild-type mice. Our results show that S163R
accumulates as intracellular aggregates within RPE cells, and
gradually forms thick and widespread deposits on the basal
side of the RPE. Interestingly, the S163R basal deposits are
strikingly similar to those described in Efemp1 knock-in mice
harboring the R345W mutation in the EFEMP1 protein, also
known as fibulin-3.46,47
Efemp1 knock-in mice are a model
for Malattia leventinese, also known as Doyne honeycomb
retinal dystrophy, an autosomal dominant inherited macular
degenerative disorder that shares many similarities with
AMD.48,49 Less prominent basal laminar deposits were also
observed by electron microscopic evaluation in a previously
characterized knock-in Ctrp5þ/À
mouse model of L-ORD
carrying the S163R mutation.43 The consequences of S163R
C1QTNF5 overexpression were also examined in a previous-
ly described experiment using a lentiviral system.44
In that
study, both photoreceptors and RPE appeared morphologi-
cally normal at 5 weeks following subretinal delivery in
C57Bl6 mice. In addition to using a different delivery vector
and promoter for transgene expression, most significantly,
the postinjection analysis time was much longer in our
current study, with the phenotypic effects becoming readily
apparent by 4 months post injection. Similarly to the
lentivirus study, we did not see apparent changes at earlier
time points by morphologic examination of the eyes using
light microscopy, supporting the idea that the phenotype is
moderately slow to develop.
Expressed AAV wild-type C1QTNF5 displays a directional
secretion from RPE, moving apically toward photoreceptor
inner segments, and reaching the outer limiting membrane, a
pattern similar to that of the endogenous protein.17,19 Its
interaction with MFRP and its polarized secretion toward the
photoreceptor layer suggests that wild-type C1QTNF5 plays a
significant role on the apical side of the RPE. Consistent with
this view, S163R aggregates clearly have a detrimental impact
on the apical RPE membrane, as seen by RPE microvilli being
absent or sparse in some regions of AAV-hC1QTNF5(S163R)–
HA treated eyes. The accumulation of mutant protein
intracellular globules with time and the abnormal distribution
of S163R toward the basal side of the RPE, suggest its presence
could interfere with not only the transport of nutrients—
including oxygen and vitamin A from the choroid to the
photoreceptor layer—but also secretion of waste materials
from the RPE, thus causing degeneration of both RPE and
photoreceptor cells.7
Previous studies demonstrated that sub-
RPE deposits from L-ORD patients are rich in oil red O-positive
lipids, protein, and calcium.7,13
However, we did not detect the
presence of lipids following staining of cryosections from AAV-
S163R–injected mouse eyes, suggesting that the pathogenesis
of L-ORD is incompletely recapitulated in this AAV-based
mouse model.
Many factors that alter the stratified extracellular matrix at
the RPE/choroid interface could lead to drusen forma-
tion.50–52
Critically, C1QTNF5 interacts with complement
factor H, a secreted glycoprotein that functions as a negative
regulator of complement activation.53
Single-nucleotide poly-
morphisms in genes encoding CFH and related complement
proteins have been associated with an increased risk of
developing AMD.54–57
In addition, other systemic factors may
also contribute to the disease pathology in patients, as
C1QTNF5 was found to circulate in the human sera with
broad interindividual variation.20
C1QTNF5 has a collagen
domain (residues 30–98) containing 23 uninterrupted Gly-X-Y
repeats,21
and this is perhaps the reason why both S163R
aggregates and basal deposits appear pink when stained with
hematoxylin and eosin, similar to other collagen-containing
structures. It is interesting to note that in L-ORD patients
collagen deposits are detected within the inner part of the
thick drusen accumulating between the basolateral side of the
RPE and the Bruch’s membrane,7 suggesting that S163R
deposits could be present at those sites in patients and act as
a stimulus for drusen formation. By extension therefore, AAV-
expressed S163R mutant, being misdirected toward the basal
side of the RPE, may alter its normal binding to CFH,
triggering complement activation over time, and thus
accelerating the formation of the drusen seen in patients.58
Protein aggregation is considered a pathological trigger in
many neurodegenerative disorders, including Alzheimer,
Parkinson, and Huntington diseases.59,60
Future experiments
aimed at enhancing the autophagic pathway will determine if
the disease progression can be slowed by increasing the
ability of the RPE to degrade mutant S163R efficiently.61
Another approach is the use of small interfering RNAs to
inhibit mutant protein expression.62
In conclusion, we show that RPE-targeted expression of the
S163R mutant of C1QTNF5 can generate a mouse model that
displays pathological features similar to some of those found in
patients with advanced L-ORD, including RPE thinning, RPE
cell loss, and retinal degeneration. S163R mutant protein forms
large, transparent, spherical aggregates throughout the RPE,
and thick basal deposits, in contrast to the wild-type protein
which is secreted in the opposite direction, toward the apical
microvilli and photoreceptor cells. The mechanism leading to
the formation of S163R basal deposits remains to be elucidated,
but promises to yield valuable clues as to how to treat L-ORD
and perhaps some aspects of AMD.
Acknowledgments
Supported by NIH Grants EY021721 and EY018331, and by Grants
from Foundation Fighting Blindness, Research to Prevent Blind-
ness, Inc., and Macula Vision Research Foundation.
Disclosure: A. Dinculescu, None; S.-H. Min, None; F.M. Dyka,
None; W.-T. Deng, None; R.M. Stupay, None; V. Chiodo, None;
W.C. Smith, None; W.W. Hauswirth, AGTC, Inc. (I, C)
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