Microglia activation in a model of retinal degeneration and TUDCA neuroprotective effects

Background: Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound. Methods: For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified. Results: Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space. Conclusions: These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness-FEDER (BFU2012-36845), Instituto de Salud Carlos III (RETICS RD12/0034/0010), Organización Nacional de Ciegos Españoles (ONCE), FUNDALUCE, Asociación Retina Asturias and Fundación Jesús de Gangoiti

ZMYND10 Is Mutated in Primary Ciliary Dyskinesia and Interacts with LRRC6

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function

Spata7 is a retinal ciliopathy gene critical for correct RPGRIP1 localization and protein trafficking in the retina

Item does not contain fulltextLeber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients

Mutations In Emp2 Cause Childhood-Onset Nephrotic Syndrome

Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.WoSScopu

Exome resequencing reveals ADCK4 mutations as novel causes of steroid-resistant nephrotic syndrome

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of its pathogenesis. However, the genetic causes of a significant proportion of childhood and adult-onset SRNS are still molecularly unidentified. Here, we combined homozygosity mapping (HM) with whole human exome resequencing (WER) and identified 9 different mutations for both alleles in 14 individuals from 7 non-related families in the ADCK4 (aarF domain containing kinase 4) gene. All mutations segregated with the affected status and were absent from >60 ethnically matched healthy controls. ADCK4 has a high sequence similarity to ADCK3, the ortholog of yeast Abc1/Coq8, which participates in the biosynthesis of coenzyme Q10 (CoQ10). We recapitulated the nephrotic syndrome phenotype in zebrafish upon knockdown of adck4. Interestingly, an affected individual with a homozygous frameshift mutation showed partial remission when treated with the innocuous supplement CoQ10. ADCK4 is strongly expressed in glomerular podocytes, and partially localizes to mitochondria and foot processes of podocytes. By coimmunoprecipitation, we demonstrated that ADCK4 interacts with other members of the CoQ10 biosynthesis pathway, including COQ7 and most importantly, COQ6, a known SRNS protein in human culture podocytes. Knockdown of ADCK4 in cultured podocytes resulted in decreased migratory phenotype, which was reversed by addition of CoQ10 or overexpression of mouse Adck4. We, thus, identified recessive mutations of ADCK4 as causing SRNS in humans. Identification of this SRNScausing gene emphasizes the importance of recognizing individuals with mutations of genes that participate in CoQ10 biosynthesis, because they may represent a treatable form of SRNS

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

PURPOSE: The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. METHODS: Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. RESULTS: Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. CONCLUSIONS: This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE

<em>ADCK4</em> mutations promote steroid-resistant nephrotic syndrome through CoQ₁₀ biosynthesis disruption.

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q(10) (CoQ(10)) biosynthesis. Mutations in ADCK4 resulted in reduced COQ(10). levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ(10) biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ(10) addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ(10) treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ(10) biosynthesis may be treatable with CoQ(10)