Poly-GA triggers TDP-43 pathology by inhibiting the proteasome and nucleocytoplasmic import in C9orf72 ALS/FTD

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two fatal neurodegenerative disorders that share overlapping clinical and pathological features (Kato, Hayashi et al. 1993, Lomen-Hoerth, Anderson et al. 2002). Although both diseases occur mostly sporadically, several disease-associated mutations have been identified in >25 different genes, many of which are encoding RNA binding proteins (RBP), components of the ubiquitin proteasome system (UPS), and the autophagy pathway (Kapeli, Martinez et al. 2017, Bartoletti, Bosco et al. 2019). Cytoplasmic inclusions of the multifunctional RNA-binding protein TDP-43 (TAR DNA-binding protein) that normally resides predominantly in the nucleus are found in ~90% of ALS and ~45% of FTD cases. TDP-43 dyshomeostasis including aberrations in its nucleocytoplasmic shuttling and aggregation has been shown to induce toxicity, explaining a crucial role in the process of neurodegeneration (Araki, Minegishi et al. 2014, Leibiger, Deisel et al. 2018, Prasad, Bharathi et al. 2019). The most common pathogenic mutation found in ~10% of ALS/FTLD patients is the massive expansion of a hexanucleotide repeat (G4C2)n in the first intron of C9orf72 gene (DeJesus-Hernandez, Mackenzie et al. 2011, Renton, Majounie et al. 2011). C9orf72 patients show, in addition to typical TDP-43 pathology, nuclear RNA foci containing the repeat RNA from both sense and antisense transcripts (DeJesus-Hernandez, Mackenzie et al. 2011) and unique inclusions of five different species of dipeptide repeat (DPR) proteins (poly-GA/-GP/-GR/-PA and -PR). DPR inclusions are derived from an unconventional non-AUG translation of the intronic repeat RNA in all reading frames (Ash, Bieniek et al. 2013, Gendron, Bieniek et al. 2013, Mori, Arzberger et al. 2013, Mori, Weng et al. 2013, Zu, Liu et al. 2013). Nearly all inclusions are poly-GA positive and often contain also poly-GP/-GR and far less frequently poly-PA/-PR. In vitro, poly-GA sequesters large amounts of stalled proteasomes suggesting a deleterious effect on cellular proteostasis (Guo, Lehmer et al. 2018). DPR pathology has been shown to precede TDP-43 pathology in patients, thereby considered as a major driver of neurodegeneration associated with C9ORF72 expansion (Baborie et al. 2015; Mann 2015; Mori, Arzberger, et al. 2013; Mori, Weng, et al. 2013; Proudfoot et al. 2014). How C9orf72‐specific pathology triggers TDP‐43 pathology, despite not being spatially correlated in human studies (Schludi et al. 2017), was the primary objective of my PhD studies. When I started my PhD project, several groups reported impaired global nucleocytoplasmic transport possibly through a direct effect on the nuclear pore complex (NPC) in different C9orf72 models and connected it to poly-GR/PR (Jovicic, Mertens et al. 2015), repeat RNA (Zhang, Donnelly et al. 2015), or both (Freibaum, Lu et al. 2015), however in these studies trafficking of TDP-43 itself was not analyzed. Furthermore, artificial aggregating β-sheet proteins were shown to inhibit nucleocytoplasmic transport related to sequestration of the THOC complex and RNA binding proteins (Woerner, Frottin et al. 2016). Since GA15 peptides, but not 15-mers of the other DPR species, form amyloid-like fibrils (Chang, Jeng et al. 2016), I asked whether poly-GA may also impair nucleocytoplasmic transport. I therefore compared the impact of individual expression of poly-GA, poly-GR and poly-PR on the nuclear import, specifically of TDP-43 to understand the link between the C9orf72 mutation and TDP-43 pathology. I quantitatively analyzed cytoplasmic mislocalization of endogenous TDP-43 and of a reporter containing the established bipartite classical nuclear localization signal (NLS) of TDP-43. Interestingly, poly-GA blocked nuclear import of TDP-43 more robustly than poly-GR/PR, while none of the DPR proteins affected the localization of a reporter containing a transportin-dependent PY-NLS in our in vitro models arguing DPR proteins mainly impair nuclear transport through the classical importin α/β pathway mediating TDP-43 import. In addition, I found that overexpression of two NPC components (NUP54 and NUP62) can fully rescue nuclear localization of the TDP-43 reporter. NUP54 and NUP62 were interestingly shown to be essential for nuclear import of TDP-43 (Nishimura, Zupunski et al. 2010) and NUP62 knockdown enhances (PR)25 toxicity in flies (Boeynaems, Bogaert et al. 2016). Thus, inhibition of nuclear import of TDP-43 by poly-GA may link the C9orf72 mutation to TDP-43 pathology in C9orf72 ALS/FTD cases. Since poly-GA precedes symptom onset by many years (Vatsavayai, Yoon et al. 2016) and rarely co-localize with TDP-43 inclusions, chronic toxicity and possibly non‐cell‐autonomous effects were proposed (Edbauer and Haass 2016). For example, cell‐to‐cell transmission of cytoplasmic Tau and α‐synuclein aggregates results in stereotypic spreading during the progression of Alzheimer's and Parkinson's disease, respectively (Jucker and Walker 2018). Thus, I analyzed non‐cell‐autonomous effects of DPRs as a potential trigger of TDP‐43 pathology using co‐culture assays and antibody treatment experiments to inhibit poly-GA cell‐ to‐cell transmission. Importantly, I discovered that poly‐GA transmission inhibits the proteasome even in neighboring cells. Activating the proteasome pharmacologically (with the PDE4 inhibitor rolipram) or genetically (using PSMD11 overexpression) rescues nuclear import of the TDP‐43 NLS reporter. Therefore, I hypothesized that poly‐GA inclusions may block the import of TDP‐43 via ubiquitination directly within its NLS, which accumulate through impaired proteostasis. I could show that mutagenizing lysine 95 in the NLS largely prevents ubiquitination of TDP-43 and did not impair basal nuclear import, but completely prevented poly-GA mediated inhibition of TDP-43 import. In contrast, mutation of lysine 84 severely blocks interactions with nuclear import receptors such as importin‐α5/KPNA1, independent of poly-GA expression, which is in consistence with previous reports (Hans, Eckert et al. 2018). Importantly, poly-GA antibodies reduce poly-GA transmission and cytoplasmic TDP-43 mislocalization in HeLa cells and primary neurons, suggesting poly-GA antibodies could be potentially used to treat C9orf72 ALS/FTD. Taken together, my work shows that poly-GA promotes cytoplasmic mislocalization of TDP-43 cell- and non-cell-autonomously due to impaired proteasomal clearance of TDP-43 ubiquitinated within its NLS at lysine 95. My work also indicates that pharmacologically boosting proteasome activity (e.g. by rolipram) or inhibiting poly-GA transmission using antibodies are promising therapeutic approaches for C9orf72 ALS/FTD. Indeed, our group (Zhou, Mareljic et al. 2020) and others (Nguyen, Montrasio et al. 2020) have reported promising results using anti-GA antibodies or vaccination in mouse models confirming my data in vivo

Comparative Evaluation of Ultraviolet and Visible Light Transmittance through Prescriptive Ophthalmic Minus Lenses

Background: Wearing spectacles is the most common approach in correcting the refractive errors worldwide. Due to harmful effects of overexposure to solar ultraviolet radiations, the usage of multi-layer coatings in ophthalmic lenses has recently been increased. These lenses can reduce the reflections and hence increase the transmission of visible light; they can also decrease the transmission of ultraviolet rays. This study aims to compare the transmission of ultraviolet (A and B) and visible rays through coated and uncoated prescriptive ophthalmic plastic lenses.Materials and Methods: In this study, 39 minus non-photochromic multi-coated white plastic single-vision lenses; 9 similar lenses but without any coatings were assessed by spectral transmittancemeter for evaluation of the transmission of visible and ultraviolet rays.Results: The transmission of visible light was 97.9%±1.07% for coated lenses and 93.5%±0.54% for lenses without coating. Ultraviolet-A transmission was 12.15%±8.02% for coated lenses compared to 66.27%±23.92% in lenses without coating. The transmission of ultraviolet-B rays was 1.21%±0.4% and 23.0%±15.97% for lenses with and without coatings, respectively.Conclusion: The transmission of visible light was significantly higher in multi-coated lenses compared to uncoated samples; whereas the transmissions of ultraviolet rays in multi-coated lenses were significantly lower than uncoated ones. Therefore, it is recommended that, except for particular cases, prescribed lenses be equipped with this multi-layer coating

The Effect of Contact Lens Induced Myopia and Hyperopia on Retinal Thickness and Volume Measured by Optical Coherence Tomography

Purpose:To determine the effect of induced myopia and hyperopia in emmetropic eyes using soft contact lenses on retinal parameters, measured by optical coherence tomography.Patients and Methods: In this quasi-experimental self-controlled study 57 emmetropic participants, 18 - 42 years of age, were studied. Each subject underwent a complete ophthalmic examinations including, measurement of best corrected visual acuity, intraocular pressure, dry and cycloplegic refractions, and axial length. Optical coherence tomography scans to measure foveal thickness, parafoveal thickness and perifoveal thickness were performed while different refraction powers were induced in each eye by wearing soft contact lenses of five different diopter (- 10.00, - 5.00, plano, + 5.00, + 10.00).Results: Fifty seven normal emmetropic participants with a mean age of 25.78 ± 6.50 years participated in the present study. Average foveal thickness was 246.02 ± 22.03 μm, 245.47 ± 22.78 μm, 246.47 ± 24.38 μm, 246.42 ± 22.96 μm, and 246.18 ± 22.46 μm in high-induced- myopic (CL: + 10.00 D), mild-induced-myopic (CL: + 5.00 D), emmetropic (CL: Plano), mild-induced-hyperopic (CL: - 5.00 D), and high-induced-hyperopic (CL: - 10.00 D) groups, respectively. Average parafoveal thickness was 329.21 ± 16.31 μm, 329.24 ± 16.36 μm, 328.86 ± 16.46 μm, 328.92 ± 16.57 μm, and 328.80 ± 16.76 μm in high-induced-myopic, mild-induced-myopic, emmetropic, mild-induced-hyperopic, and high-induced-hyperopic groups, respectively. Corresponding numbers for perifoveal thickness was 312.25 ± 14.39 μm, 311.84 ± 14.91 μm, 312.46 ± 16.55 μm, 311.57 ± 14.88 μm, and 311.77 ± 14.96 μm. Conclusion: Contact lens induced myopia and hyperopia had no significant effect on foveal thickness,parafovealthickness and perifoveal thickness readings in Fourier domain optical coherence tomography.Keywords: Optical coherence tomography; myopia; hyperopia; retinal parameters; contact lens.

Study of Post Operative Regression after Photorefractive Keratectomy for Treatment of Hyperopia and Hyperopic Astigmatism

Purpose: To study post operative regression rates after photorefractive keratectomy among patients with hyperopia and hyperopic astigmatism with follow-up of at least six months.Patients and Methods: In this historical cohort study, 171 eyes from 91 patients with moderate hyperopia and hyperopic astigmatism were treated using Bausch and Lomb Technolas 217 Z Excimer Laser. Pre-operation evaluation included; best spectacle corrected visual acuity, manifest and cycloplegic refraction, diameter of optical zone, central corneal thickness and simulated keratometry. Postoperative evaluation, performed at least six months after the procedure, included measurement of corneal curvature, manifest and cycloplegic refraction, best corrected visual acuity, uncorrected visual acuity, refraction manifest, haze, and any pathologic finding.Results: The mean regression was 0.35 ± 1.04. Post surgical manifest refraction equivalent in ± 0.5 diopter range of surgeon’s desired refraction was observed in 57.1 % of eyes. Manifest refraction equivalent in ± 1.00 diopter range was observed in 85.7 % of eyes, and manifest refraction equivalent in ± 2.00 diopter range in 96.6% of eyes. Uncorrected visual acuity of 20/20 or better was reported in 37.1 % and 20/40 or better in 92 % of patients. Loss of the best spectacle corrected visual acuity of one line was observed in 13.4 % and 2 lines or more in 5.7 % of patients.Conclusion: In patients with moderate hyperopia and hyperopic astigmatism undergoing PRK the rate of regression was in ± 1 diopter range of surgeon’s intended correction in 85.7 % of patients at least six months postoperatively, which is in line with other studies findings.Keywords: Photorefractive keratectomy; Hyperopia astigmatism; Regression.

Changes in Corneal Asphericity after MyoRing Implantation in Moderate and Severe Keratoconus

Purpose: To evaluate the effect of MyoRing implantation on corneal asphericity in moderate and severe keratoconus (KCN). Methods: This cross-sectional observational study comprised 32 eyes of 28 patients with KCN, who had femtosecond-assisted MyoRing corneal implantation. The primary outcome measures were preoperative and six-month postoperative corneal asphericity in 6-, 7-, 8-, 9-, and 10-mm optical zones in the superior, inferior, nasal, temporal, and central areas. The secondary outcome measures included uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction, thinnest location value, and keratometry readings. Results: A significant improvement in the UDVA and CDVA was observed six months after the surgery (P < 0.001) with a significant reduction in the spherical (4.67 diopters (D)) and cylindrical (2.19 D) refractive errors. A significant reduction in the corneal asphericity in all the optical zones and in the superior, inferior, nasal, temporal, and central areas was noted (P < 0.001). The mean thickness at the thinnest location of the cornea decreased from 437.15 ± 30.69 to 422.81 ± 36.91 μm. A significant corneal flattening was seen. The K1, K2, and Km changes were 5.32 D, 7 D, and 6.17 D, respectively (P < 0.001). Conclusion: MyoRing implantation is effective for improving corneal asphericity in patients with KCN. It allows successful corneal remodeling and provides a significant improvement in UDVA, CDVA, and refractive errors

Characteristics of Astigmatism after MyoRing Implantation

Considering the rising number of MyoRing implantation procedures in keratoconic corneas and the refractive outcomes associated with this treatment modality, this study aimed to evaluate and compare the magnitude and axis orientation of total and corneal astigmatism between before and after MyoRing implantation in 34 eyes of 28 patients with keratoconus (KCN) (mean age: 29.41 ± 7.0 years). The inclusion criterion was a reliable diagnosis of clinical KCN based on corneal biomicroscopic and tomographic findings. The mean total astigmatism of ocular refraction decreased significantly from -4.27 ± 3.15 D (before MyoRing implantation) to -2.18 ± 1.63 D (after MyoRing implantation) (P < 0.001). The mean astigmatism in the anterior and posterior surface of the cornea decreased significantly by 1.16 D (P = 0.001) and 0.24 D (P = 0.009), respectively, after MyoRing implantation. Before MyoRing implantation, the axis orientation of total ocular astigmatism for with-the-rule, oblique, and against-the-rule astigmatism was 21%, 42%, and 37%, respectively; at 6 months after MyoRing implantation, it was 18%, 24%, and 58%, respectively. Before MyoRing implantation, the axis orientation for with-the-rule, against-the-rule, and oblique astigmatism of the anterior surface of the cornea was 59%, 24%, and 17%, respectively; at 6 months after MyoRing implantation, it was 52%, 24%, and 24%, respectively. Before MyoRing implantation, the axis orientation of with-the-rule, oblique, and against-the-rule astigmatism of the posterior surface of the cornea was 68%, 29%, and 3%, respectively; at 6 months after MyoRing implantation, it was 67%, 12%, and 12%, respectively. MyoRing implantation significantly decreased the amount of total, anterior, and posterior corneal astigmatism

The Effect of Soft Contact Lenses on Optic Nerve Head Measurements Using Optical Coherence Tomography

Purpose: To evaluate the effect of soft contact lens induced myopia and hyperopia on optic nerve head measurements of normal eyes using spectral domain optical coherence tomography (SD-OCT). Methods:  This cross sectional study was performed on 114 emmetropic eyes of 57 participants. Each participant underwent a complete ophthalmic examination including determination of best-corrected visual acuity, intraocular pressure, dry and cycloplegic refraction as well as axial length measurement. SD-OCT measurement was performed in all ayes while different levels of refraction strength were induced by wearing soft contact lenses of five different diopters (- 10.00, - 5.00, Plano, + 5.00, + 10.00). Results:  The mean measured thicknesses of retinal nerve fiber layer were 123.29 ± 10.56 micrometer, 123.17 ± 11.61 micrometer, 122.77 ± 11.61 micrometer, 123.37 ± 11.15 micrometer and 123.42 ± 11.45 micrometer in contact lens induced high myopia, moderate myopia, emmetropic, moderate hyperopia, and high hyperopia groups, respectively (P = 0.721). Also, corresponding evaluations for mean rim area (P = 0.781), mean optic disc area (P = 0.601), mean cup area (P = 0.53), and mean cup to disc area ratio (P = 0.414) showed no statistically significant difference. Conclusion: Our findings indicate that refractive error variation at the corneal plane caused by contact lens wear has no significant effect on thickness of retinal nerve fiber layer, disc area, cup area, rim area and mean cup to disc area ratio measured by SD-OCT.Keywords: Contact Lenses; Hyperopia; Myopia; Tomography, Optical Coherence; Optic Nerv

Cytoplasmic poly-GA aggregates impair nuclear import of TDP-43 in C9orf72 ALS/FTLD

A repeat expansion in the non-coding region of C9orf72 gene is the most common mutation causing frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Sense and antisense transcripts are translated into aggregating dipeptide repeat (DPR) proteins in all reading frames (poly-GA,-GP,-GR,-PA and -PR) through an unconventional mechanism. How these changes contribute to cytoplasmic mislocalization and aggregation of TDP-43 and thereby ultimately leading to neuron loss remains unclear. The repeat RNA itself and poly-GR/PR have been linked to impaired nucleocytoplasmic transport. Here, we show that compact cytoplasmic poly-GA aggregates impair nuclear import of a reporter containing the TDP-43 nuclear localization (NLS) signal. However, a reporter containing a non-classical PY-NLS was not affected. Moreover, poly-GA expression prevents TNF alpha induced nuclear translocation of p65 suggesting that poly-GA predominantly impairs theimportin-alpha/beta-dependent pathway. In neurons, prolonged poly-GA expression induces partial mislocalization of TDP-43 into cytoplasmic granules. Rerouting poly-GA to the nucleus prevented TDP-43 mislocalization, suggesting a cytoplasmic mechanism. In rescue experiments, expression of importin-alpha (KPNA3, KPNA4) or nucleoporins (NUP54, NUP62) restores the nuclear localization of the TDP reporter. Taken together, inhibition of nuclear import of TDP-43 by cytoplasmic poly-GA inclusions causally links the two main aggregating proteins in C9orf72 ALS/FTLD pathogenesis

Mapping geographical inequalities in access to drinking water and sanitation facilities in low-income and middle-income countries, 2000-17

Background: Universal access to safe drinking water and sanitation facilities is an essential human right, recognised in the Sustainable Development Goals as crucial for preventing disease and improving human wellbeing. Comprehensive, high-resolution estimates are important to inform progress towards achieving this goal. We aimed to produce high-resolution geospatial estimates of access to drinking water and sanitation facilities. Methods: We used a Bayesian geostatistical model and data from 600 sources across more than 88 low-income and middle-income countries (LMICs) to estimate access to drinking water and sanitation facilities on continuous continent-wide surfaces from 2000 to 2017, and aggregated results to policy-relevant administrative units. We estimated mutually exclusive and collectively exhaustive subcategories of facilities for drinking water (piped water on or off premises, other improved facilities, unimproved, and surface water) and sanitation facilities (septic or sewer sanitation, other improved, unimproved, and open defecation) with use of ordinal regression. We also estimated the number of diarrhoeal deaths in children younger than 5 years attributed to unsafe facilities and estimated deaths that were averted by increased access to safe facilities in 2017, and analysed geographical inequality in access within LMICs. Findings: Across LMICs, access to both piped water and improved water overall increased between 2000 and 2017, with progress varying spatially. For piped water, the safest water facility type, access increased from 40·0% (95% uncertainty interval [UI] 39·4–40·7) to 50·3% (50·0–50·5), but was lowest in sub-Saharan Africa, where access to piped water was mostly concentrated in urban centres. Access to both sewer or septic sanitation and improved sanitation overall also increased across all LMICs during the study period. For sewer or septic sanitation, access was 46·3% (95% UI 46·1–46·5) in 2017, compared with 28·7% (28·5–29·0) in 2000. Although some units improved access to the safest drinking water or sanitation facilities since 2000, a large absolute number of people continued to not have access in several units with high access to such facilities (>80%) in 2017. More than 253 000 people did not have access to sewer or septic sanitation facilities in the city of Harare, Zimbabwe, despite 88·6% (95% UI 87·2–89·7) access overall. Many units were able to transition from the least safe facilities in 2000 to safe facilities by 2017; for units in which populations primarily practised open defecation in 2000, 686 (95% UI 664–711) of the 1830 (1797–1863) units transitioned to the use of improved sanitation. Geographical disparities in access to improved water across units decreased in 76·1% (95% UI 71·6–80·7) of countries from 2000 to 2017, and in 53·9% (50·6–59·6) of countries for access to improved sanitation, but remained evident subnationally in most countries in 2017. Interpretation: Our estimates, combined with geospatial trends in diarrhoeal burden, identify where efforts to increase access to safe drinking water and sanitation facilities are most needed. By highlighting areas with successful approaches or in need of targeted interventions, our estimates can enable precision public health to effectively progress towards universal access to safe water and sanitation

Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.

The Global Burden of Diseases, Injuries and Risk Factors 2017 includes a comprehensive assessment of incidence, prevalence, and years lived with disability (YLDs) for 354 causes in 195 countries and territories from 1990 to 2017. Previous GBD studies have shown how the decline of mortality rates from 1990 to 2016 has led to an increase in life expectancy, an ageing global population, and an expansion of the non-fatal burden of disease and injury. These studies have also shown how a substantial portion of the world's population experiences non-fatal health loss with considerable heterogeneity among different causes, locations, ages, and sexes. Ongoing objectives of the GBD study include increasing the level of estimation detail, improving analytical strategies, and increasing the amount of high-quality data. METHODS: We estimated incidence and prevalence for 354 diseases and injuries and 3484 sequelae. We used an updated and extensive body of literature studies, survey data, surveillance data, inpatient admission records, outpatient visit records, and health insurance claims, and additionally used results from cause of death models to inform estimates using a total of 68 781 data sources. Newly available clinical data from India, Iran, Japan, Jordan, Nepal, China, Brazil, Norway, and Italy were incorporated, as well as updated claims data from the USA and new claims data from Taiwan (province of China) and Singapore. We used DisMod-MR 2.1, a Bayesian meta-regression tool, as the main method of estimation, ensuring consistency between rates of incidence, prevalence, remission, and cause of death for each condition. YLDs were estimated as the product of a prevalence estimate and a disability weight for health states of each mutually exclusive sequela, adjusted for comorbidity. We updated the Socio-demographic Index (SDI), a summary development indicator of income per capita, years of schooling, and total fertility rate. Additionally, we calculated differences between male and female YLDs to identify divergent trends across sexes. GBD 2017 complies with the Guidelines for Accurate and Transparent Health Estimates Reporting