Loss of Melanopsin-Expressing Ganglion Cell Subtypes and Dendritic Degeneration in the Aging Human Retina

In mammals, melanopsin-expressing retinal ganglion cells (mRGCs) are, among other things, involved in several non-image-forming visual functions, including light entrainment of circadian rhythms. Considering the profound impact of aging on visual function and ophthalmic diseases, here we evaluate changes in mRGCs throughout the life span in humans. In 24 post-mortem retinas from anonymous human donors aged 10–81 years, we assessed the distribution, number and morphology of mRGCs by immunostaining vertical retinal sections and whole-mount retinas with antibodies against melanopsin. Human retinas showed melanopsin immunoreactivity in the cell body, axon and dendrites of a subset of ganglion cells at all ages tested. Nearly half of the mRGCs (51%) were located within the ganglion cell layer (GCL), and stratified in the outer (M1, 12%) or inner (M2, 16%) margin of the inner plexiform layer (IPL) or in both plexuses (M3, 23%). M1 and M2 cells conformed fairly irregular mosaics, while M3 cell distribution was slightly more regular. The rest of the mRGCs were more regularly arranged in the inner nuclear layer (INL) and stratified in the outer margin of the IPL (M1d, 49%). The quantity of each cell type decrease after age 70, when the total number of mRGCs was 31% lower than in donors aged 30–50 years. Moreover, in retinas with an age greater than 50 years, mRGCs evidenced a decrease in the dendritic area that was both progressive and age-dependent, as well as fewer branch points and terminal neurite tips per cell and a smaller Sholl area. After 70 years of age, the distribution profile of the mRGCs was closer to a random pattern than was observed in younger retinas. We conclude that advanced age is associated with a loss in density and dendritic arborization of the mRGCs in human retinas, possibly accounting for the more frequent occurrence of circadian rhythm disorders in elderly persons.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO-FEDER BFU2015-67139-R), Instituto de Salud Carlos III (RETICS-FEDER RD16/0008/0016) and Generalitat Valenciana (PROMETEO/2016/158)

Age and date for early arrival of the Acheulian in Europe (Barranc de la Boella, la Canonja, Spain)

The first arrivals of hominin populations into Eurasia during the Early Pleistocene are currently considered to have occurred as short and poorly dated biological dispersions. Questions as to the tempo and mode of these early prehistoric settlements have given rise to debates concerning the taxonomic significance of the lithic assemblages, as trace fossils, and the geographical distribution of the technological traditions found in the Lower Palaeolithic record. Here, we report on the Barranc de la Boella site which has yielded a lithic assemblage dating to ,1 million years ago that includes large cutting tools (LCT). We argue that distinct technological traditions coexisted in the Iberian archaeological repertoires of the late Early Pleistocene age in a similar way to the earliest sub-Saharan African artefact assemblages. These differences between stone tool assemblages may be attributed to the different chronologies of hominin dispersal events. The archaeological record of Barranc de la Boella completes the geographical distribution of LCT assemblages across southern Eurasia during the EMPT (Early-Middle Pleistocene Transition, circa 942 to 641 kyr). Up to now, chronology of the earliest European LCT assemblages is based on the abundant Palaeolithic record found in terrace river sequences which have been dated to the end of the EMPT and later. However, the findings at Barranc de la Boella suggest that early LCT lithic assemblages appeared in the SW of Europe during earlier hominin dispersal episodes before the definitive colonization of temperate Eurasia took place.The research at Barranc de la Boella has been carried out with the financial support of the Spanish Ministerio de Economı´a y Competitividad (CGL2012- 36682; CGL2012-38358, CGL2012-38434-C03-03 and CGL2010-15326; MICINN project HAR2009-7223/HIST), Generalitat de Catalunya, AGAUR agence (projects 2014SGR-901; 2014SGR-899; 2009SGR-324, 2009PBR-0033 and 2009SGR-188) and Junta de Castilla y Leo´n BU1004A09. Financial support for Barranc de la Boella field work and archaeological excavations is provided by the Ajuntament de la Canonja and Departament de Cultura (Servei d’Arqueologia i Paleontologia) de la Generalitat de Catalunya. A. Carrancho’s research was funded by the International Excellence Programme, Reinforcement subprogramme of the Spanish Ministry of Education. I. Lozano-Ferna´ndez acknowledges the pre-doctoral grant from the Fundacio´n Atapuerca. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

New Pyrenochaeta Species Causing Keratitis▿

We report a new fungus as an agent of fungal keratitis in a diabetic woman. The fungal etiology was established by classic microbiology and PCR following 3 months of antibacterial therapy. The morphological features of the isolate and sequence analysis of the internal transcribed spacer region indicate a new species of Pyrenochaeta (Coelomycetes)

Microglia activation and neuronal alterations in retinas from COVID-19 patients: correlation with clinical parameters

Background Different ocular alterations have been described in patients with coronavirus disease 2019 (COVID-19). Our aim was to determine whether COVID-19 affected retinal cells and establish correlations with clinical parameters. Methods Retinal sections and flat-mount retinas from human donors with COVID-19 (n = 16) and controls (n = 15) were immunostained. The location of angiotensin-converting enzyme 2 (ACE2) and the morphology of microglial cells, Müller cells, astrocytes, and photoreceptors were analyzed by confocal microscopy. Microglial quantification and the area occupied by them were measured. Correlations among retinal and clinical parameters were calculated. Results ACE2 was mainly located in the Müller cells, outer segment of cones and retinal pigment epithelium. Cell bodies of Müller cells in COVID-19 group showed greater staining of ACE2 and cellular retinaldehyde-binding protein (CRALBP). The 81.3% of COVID-19 patients presented disorganization of honeycomb-like pattern formed by Müller cells. Gliosis was detected in 56.3% of COVID-19 patients compared to controls (40%) as well as epiretinal membranes (ERMs) or astrocytes protruding (50%). Activated or ameboid-shape microglia was the main sign in the COVID-19 group (93.8%). Microglial migration towards the vessels was greater in the COVID-19 retinas (P < 0.05) and the area occupied by microglia was also reduced (P < 0.01) compared to control group. Cone degeneration was more severe in the COVID-19 group. Duration of the disease, age and respiratory failure were the most relevant clinical data in relation with retinal degeneration. Conclusions The retinas of patients with COVID-19 exhibit glial activation and neuronal alterations, mostly related to the inflammation, hypoxic conditions, and age.The study was supported from grants funded by the Spanish Ministry of Science and Innovation (FEDER-PID2019-106230RB-I00), Spanish Ministry of Universities (FPU16/04114 and FPU18/02964), National Institute of Health Carlos III (RETICS-FEDER RD16/0008/0016), Generalitat Valenciana (IDIFEDER/2017/064, PROMETEO/2021/024) and Valencia University General Hospital Foundation

Novel Candidate Genes and a Wide Spectrum of Structural and Point Mutations Responsible for Inherited Retinal Dystrophies Revealed by Exome Sequencing

<div><p>Background</p><p>NGS-based genetic diagnosis has completely revolutionized the human genetics field. In this study, we have aimed to identify new genes and mutations by Whole Exome Sequencing (WES) responsible for inherited retinal dystrophies (IRD).</p><p>Methods</p><p>A cohort of 33 pedigrees affected with a variety of retinal disorders was analysed by WES. Initial prioritization analysis included around 300 IRD-associated genes. In non-diagnosed families a search for pathogenic mutations in novel genes was undertaken.</p><p>Results</p><p>Genetic diagnosis was attained in 18 families. Moreover, a plausible candidate is proposed for 10 more cases. Two thirds of the mutations were novel, including 4 chromosomal rearrangements, which expand the IRD allelic heterogeneity and highlight the contribution of private mutations. Our results prompted clinical re-evaluation of some patients resulting in assignment to a syndromic instead of non-syndromic IRD. Notably, WES unveiled four new candidates for non-syndromic IRD: <i>SEMA6B</i>, <i>CEP78</i>, <i>CEP250</i>, <i>SCLT1</i>, the two latter previously associated to syndromic disorders. We provide functional data supporting that missense mutations in <i>CEP250</i> alter cilia formation.</p><p>Conclusion</p><p>The diagnostic efficiency of WES, and strictly following the ACMG/AMP criteria is 55% in reported causative genes or functionally supported new candidates, plus 30% families in which likely pathogenic or VGUS/VUS variants were identified in plausible candidates. Our results highlight the clinical utility of WES for molecular diagnosis of IRD, provide a wider spectrum of mutations and concomitant genetic variants, and challenge our view on syndromic vs non-syndromic, and causative vs modifier genes.</p></div

Identification of independent <i>PRPF31</i> deletion and duplication segregating in pedigree E4.

<p>Exome data indicated significant coverage differences of <i>PRPF31</i> exons in the E4 family, pointing to chromosomal rearrangements. Some patients (A) showed higher coverage in exons 2–5 compared to a control sample (C) whereas patients from another family branch showed a significant decrease of exons 1–13 (B). <i>CRX</i>, located a few Mb away from <i>PRPF31</i> gene, was used as a control gene (D-F). MLPA analysis confirmed a nearly full deletion of <i>PRPF31</i> (exons 1 to 13) in some patients of the family (G) and an internal duplication involving exons 2 to 5 in other affected members (H) (shown in grey in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168966#pone.0168966.g001" target="_blank">Fig 1</a>). I) Chromosomal region of <i>PRPF31</i> involved in the duplication, where the duplicated exons are coloured in orange. Green and red lines below indicate the extent of the duplication. Chromatogram of the rearranged allele is shown below. Alignment of the flanking sequences (boxed in orange and green) involved in the rearrangement shows no clear homology. Orange and green lines are the adjacent sequences to the breakpoint, which is indicated by a red square.</p

Identification of <i>EYS</i> and <i>CRX</i> deletions.

<p>A-F) Two different gross heterozygous deletions in genes <i>EYS</i> and <i>CRX</i> were respectively identified as the causative mutation in families 68ORG and 10NCE. The probands (B and D) showed a reduction in the coverage of some exons compared to the respective controls (A and C). The segregation of SNPs located in the expected deleted region showing that mother and child were homozygous for different alleles is indicated below. (E and F). G) Chromosomal deletion in family 10NCE is defined by genotyping common SNPs between <i>CRX</i> and <i>SULT2A1</i> genes in the affected probands. Heterozygous SNPs are indicated by △, whereas SNPs where mother and child were homozygous for different alleles are indicated by ∇. Adjacent breakpoint regions with high sequence similarity are boxed in orange and green and preserved sequences in the rearranged allele are indicated with orange and green lines. H) Sequence chromatogram of the rearranged allele is shown below. Alignment of the highly similar sequences of <i>CRX</i> intron 2 (CRX IVS2) and the intragenic region involved in the rearrangement is also indicated. Again, orange and green lines are the adjacent sequences to the breakpoint, which is indicated by a red square.</p

Segregation of mutations in selected pedigrees.

<p>Pedigrees bearing new IRD candidates and chromosomal rearrangements are shown. Pedigrees where mutations in several genes co-segregate with the disease are also depicted. Alleles and carrier status are indicated below each analysed individual. Grey symbols (in H) shown patients bearing a different chromosomal rearrangement. The rest of the pedigrees are available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168966#pone.0168966.s003" target="_blank">S3 Fig</a>.</p

Immunodetection of endogenous CEP250 in mouse retinal cryosections.

<p>Immunostaining of CEP250 with rod photoreceptor marker rhodopsin (A) and acetylated α-tubulin (B). CEP250 stained mainly the outer segment of photoreceptors (CEP250 is in red, Rhodopsin and acetylated-α-tubulin in green, nuclear counterstaining by DAPI in blue). <b>Cells expressing A609V CEP250-IT6 show longer cilia</b>. (C) Wild-type (Wt) and mutant (Mt) CEP250-IT6 (green) co-localize with acetylated α-tubulin (red) to primary cilia in serum-starved ARPE-19 cells. Immunolabelling of CEP250 and acetylated α-tubulin show longer cilia in cells transfected with the mutant A609V CEP250-IT6 compared to Wt-CEP250-IT6. (D) Cilia length quantification in Wt- and Mt- CEP250-IT6 transfected cells. Graph shows that cilia from cells expressing mutant CEP250 were one third longer than cilia from cells expressing Wt-CEP250 (n>30). Mean and error are shown. *** indicates high statistical significance by the t-Student test, p<0.001. (E) Distribution of cilium length represented as a cumulative frequency chart of the percentage of total cilia. OS—photoreceptor outer segments; CC—connecting cilium; IS—photoreceptor inner segments; ONL—outer nuclear layer; INL—inner nuclear layer; GCL—ganglion cell layer.</p