Fully Human Bifunctional Intrabodies Achieve Graded Reduction of Intracellular Tau and Rescue Survival of MAPT Mutation iPSC-derived Neurons.

Tau protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP), spurring development of tau-lowering therapeutic strategies. Here, we report fully human bifunctional anti-tau-PEST intrabodies that bind the mid-domain of tau to block aggregation and degrade tau via the proteasome using the ornithine decarboxylase (ODC) PEST degron. They effectively reduced tau protein in human iPSC-derived cortical neurons in 2D cultures and 3D organoids, including those with the disease-associated tau mutations R5L, N279K, R406W, and V337M. Anti-tau-hPEST intrabodies facilitated efficient ubiquitin-independent proteolysis, in contrast to tau-lowering approaches that rely on the cell's ubiquitination system. Importantly, they counteracted the proteasome impairment observed in V337M patient-derived cortical neurons and significantly improved neuronal survival. By serial mutagenesis, we created variants of the PEST degron that achieved graded levels of tau reduction. Moderate reduction was as effective as high reduction against tau V337M-induced neural cell death.

Identifying biomarkers of heterogeneity and transplantation efficacy in retinal pigment epithelial cells.

Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here, we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single-cell RNA sequencing (scRNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.

Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations.

Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.

Early identification of a COVID-19 outbreak detected by wastewater surveillance at a large homeless shelter in Toronto, Ontario.

SETTING: Toronto (Ontario, Canada) is a large urban centre with a significant population of underhoused residents and several dozen shelters for this population with known medical and social vulnerabilities. A sizeable men's homeless shelter piloted a facility-level SARS-CoV-2 wastewater surveillance program. INTERVENTION: Wastewater surveillance was initiated at the shelter in January 2021. One-hour composite wastewater samples were collected twice weekly from a terminal sanitary clean-out pipe. The genetic material of the SARS-CoV-2 virus was extracted from the solid phase of each sample and analyzed using real-time qPCR to estimate the viral level. Wastewater results were reported to facility managers and Toronto Public Health within 4 days. OUTCOMES: There were 169 clients on-site at the time of the investigation. Wastewater surveillance alerted to the presence of COVID-19 activity at the site, prior to clinical detection. This notification acted as an early warning signal, which allowed for timely symptom screening and case finding for shelter managers and the local health unit, in preparation for the declaration of an outbreak. IMPLICATIONS: Wastewater surveillance acted as an advanced notification leading to the timely deployment of enhanced testing prior to clinical presentation in a population with known vulnerabilities. Wastewater surveillance at the facility level is beneficial, particularly in high-risk congregate living settings such as shelters that house transient populations where clinical testing and vaccination can be challenging. Open communication, established individual facility response plans, and a balanced threshold for action are essential to an effective wastewater surveillance program.

RéSUMé: LIEU: Toronto (Ontario, Canada) est un grand centre urbain qui compte une importante population de résidents mal logés et plusieurs douzaines de refuges pour cette population aux vulnérabilités médicales et sociales connues. Un assez gros refuge pour hommes sans-abri a mis à l'essai dans ses installations un programme de surveillance des eaux usées pour le SRAS-CoV-2. INTERVENTION: La surveillance des eaux usées du refuge a commencé en janvier 2021. Des échantillons composites d'une heure ont été prélevés deux fois par semaine à partir d'un regard de nettoyage à l'extrémité du drain sanitaire. Le matériel génétique du virus du SRAS-CoV-2 a été extrait du support solide de chaque échantillon et analysé par PCR quantitative en temps réel pour estimer le niveau du virus. Les résultats des eaux usées ont été déclarés aux gestionnaires du refuge et à Santé publique Toronto dans un délai de quatre jours. RéSULTATS: Il y avait 169 usagers sur place au moment de l'enquête. La surveillance des eaux usées a averti de la présence d'une activité de la COVID-19 sur les lieux avant sa détection clinique. Cet avertissement a servi de signal d'alerte précoce, ce qui a permis aux gestionnaires du refuge et au bureau de santé local de procéder au dépistage rapide des symptômes et à la recherche des cas en préparation pour la déclaration d'une éclosion. CONSéQUENCES: La surveillance des eaux usées a servi de notification préalable et entraîné le déploiement opportun d'un dépistage complémentaire avant la manifestation clinique dans une population qui présente des vulnérabilités connues. La surveillance des eaux usées d'une installation est avantageuse, surtout dans des milieux d'hébergement collectif à risque élevé comme les refuges qui hébergent des populations de passage, où le dépistage clinique et la vaccination peuvent être difficiles. Une communication ouverte, des plans d'intervention établis pour chaque installation et un seuil d'intervention équilibré sont essentiels à l'efficacité d'un programme de surveillance des eaux usées.

Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol.

Mutations in the MAPT gene that encodes tau lead to frontotemporal dementia (FTD) with pathology evident in both cerebral neurons and glia. Human cerebral organoids (hCOs) from individuals harboring pathogenic tau mutations can reveal the earliest downstream effects on molecular pathways within a developmental context, generating interacting neurons and glia. We found that in hCOs carrying the V337M and R406W tau mutations, the cholesterol biosynthesis pathway in astrocytes was the top upregulated gene set compared with isogenic controls by single-cell RNA sequencing (scRNA-seq). The 15 upregulated genes included HMGCR, ACAT2, STARD4, LDLR, and SREBF2. This result was confirmed in a homozygous R406W mutant cell line by immunostaining and sterol measurements. Cholesterol abundance in the brain is tightly regulated by efflux and cholesterol biosynthetic enzyme levels in astrocytes, and dysregulation can cause aberrant phosphorylation of tau. Our findings suggest that cholesterol dyshomeostasis is an early event in the etiology of neurodegeneration caused by tau mutations.

The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue.

Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

Human RPE Stem Cell-Derived RPE Preserves Photoreceptors in the Royal College of Surgeons Rat: Method for Quantifying the Area of Photoreceptor Sparing.

PURPOSE: Numerous preclinical studies have shown that transplantation of stem cell-derived retinal pigment epithelial cell (RPE) preserves photoreceptor cell anatomy in the dystrophic Royal College of Surgeons (RCS) rat. How rescue is spatially distributed over the eye, relative to the transplantation site, is less clear. To understand spatial variations in transplant efficacy, we have developed a method to measure the spatial distribution of rescued photoreceptor cells. METHODS: Human RPE Stem Cell-derived RPE (RPESC-RPE) cells were subretinally injected into RCS rat eyes. After tissue recovery and orientating the globe, a series of retinal sections were cut through the injected area. Sections were stained with DAPI (4',6-diamidino-2-phenylindole) and a number of photoreceptor nuclei were counted across the nasal-temporal and superior-inferior axes. These data were used to construct 2D maps of the area of photoreceptor cell saving. RESULTS: Photoreceptor cell preservation was detected in the injected temporal hemisphere and occupied areas greater than 4 mm(2) centered near the injection sites. Rescue was directed toward the central retina and superior and inferior poles, with maximal number of rescued photoreceptor cells proximal to the injection sites. CONCLUSIONS: RPESC-RPE transplantation preserves RCS photoreceptor cells. The photoreceptor cell contour maps readily convey the extent of rescue across the eye. The consistent alignment and quantification of results using this method allow the application of other downstream statistical analyses and comparisons to better understand transplantation therapy in the eye.