Transgenic Zebrafish as a Novel Animal Model to Study Tauopathies and Other Neurodegenerative Disorders in vivo

Our ageing society is confronted with a dramatic increase in patients suffering from tauopathies such as Alzheimer's disease, frontotemporal dementia and others. Typical neuropathological lesions including tangles composed of hyper-phosphorylated tau protein as well as severe neuronal cell death characterize these disorders. No mechanism-based cures are available at present. Genetically modified animals are invaluable models to understand the molecular disease mechanisms and to screen for modifying compounds. We recently introduced tau-transgenic zebrafish as a novel model for tauopathies. Our model allows recapitulating key pathological features of tauopathies within an extremely short time. Moreover, life imaging of tau-dependent neuronal cell death was performed for the very first time. This demonstrated tau-dependent neuronal cell loss independent of tangle formation. Finally, we exemplified that the zebrafish frontotemporal dementia model can be used to screen for drugs that prevent abnormal tau phosphorylation and neuronal cell death. Copyright (C) 2010 S. Karger AG, Base

Densities of almost-surely terminating probabilistic programs are differentiable almost everywhere

We study the differential properties of higher-order statistical probabilistic programs with recursion and conditioning. Our starting point is an open problem posed by Hongseok Yang: what class of statistical probabilistic programs have densities that are differentiable almost everywhere? To formalise the problem, we consider Statistical PCF (SPCF), an extension of call-by-value PCF with real numbers, and constructs for sampling and conditioning. We give SPCF a sampling-style operational semantics a la Borgstrom et al., and study the associated weight (commonly referred to as the density) function and value function on the set of possible execution traces. Our main result is that almost-surely terminating SPCF programs, generated from a set of primitive functions (e.g. the set of analytic functions) satisfying mild closure properties, have weight and value functions that are almost-everywhere differentiable. We use a stochastic form of symbolic execution to reason about almost-everywhere differentiability. A by-product of this work is that almost-surely terminating deterministic (S)PCF programs with real parameters denote functions that are almost-everywhere differentiable. Our result is of practical interest, as almost-everywhere differentiability of the density function is required to hold for the correctness of major gradient-based inference algorithms

Parkin Is Protective against Proteotoxic Stress in a Transgenic Zebrafish Model

Mutations in the gene encoding the E3 ubiquitin ligase parkin (PARK2) are responsible for the majority of autosomal recessive parkinsonism. Similarly to other knockout mouse models of PD-associated genes, parkin knockout mice do not show a substantial neuropathological or behavioral phenotype, while loss of parkin in Drosophila melanogaster leads to a severe phenotype, including reduced lifespan, apoptotic flight muscle degeneration and male sterility. In order to study the function of parkin in more detail and to address possible differences in its role in different species, we chose Danio rerio as a different vertebrate model system.We first cloned zebrafish parkin to compare its biochemical and functional aspects with that of human parkin. By using an antisense knockdown strategy we generated a zebrafish model of parkin deficiency (knockdown efficiency between 50% and 60%) and found that the transient knockdown of parkin does not cause morphological or behavioral alterations. Specifically, we did not observe a loss of dopaminergic neurons in parkin-deficient zebrafish. In addition, we established transgenic zebrafish lines stably expressing parkin by using a Gal4/UAS-based bidirectional expression system. While parkin-deficient zebrafish are more vulnerable to proteotoxicity, increased parkin expression protected transgenic zebrafish from cell death induced by proteotoxic stress.Similarly to human parkin, zebrafish parkin is a stress-responsive protein which protects cells from stress-induced cell death. Our transgenic zebrafish model is a novel tool to characterize the protective capacity of parkin in vivo

Membrane lipid remodeling modulates γ-secretase processivity

Imbalances in the amounts of amyloid-beta peptides (A beta) generated by the membrane proteases beta- and gamma-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of gamma-secretase have shown that increasing membrane thickness modulates A beta generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced gamma-secretase processivity, resulting in the increased production of the potentially beneficial A beta 37 and/or A beta 38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total A beta secretion by cells expressing WT gamma-secre-tase but lowered it for cells expressing an aggressive familial AD mutant gamma-secretase. We conclude that EA-mediated mod-ulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct gamma-secretase re-sponses, providing critical implications for lipid-based AD treatment strategies

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.This work was funded by the European Union’s Horizon 2020 research and innovation programme (BATCure, grant No. 666918 to DLM, JPB, SEM, TB and SS). JPB is funded by the Agencia Estatal de Investigación (PID2019-105699RB-I00/ AEI / 10.13039/501100011033 and RED2018-102576-T), Plan Nacional sobre Drogas (2020I028), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces. SS was funded by a grant from the Mila’s Miracle Foundation. TB was supported by German Research Council (DFG) grant FOR2625. SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards laboratory and office space. We acknowledge Marcella Cesana for providing the TFEB virus. Graphical abstract was created using BioRender.com

Effects of Combined Ketamine/Xylazine Anesthesia on Light Induced Retinal Degeneration in Rats

Objectives: To explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats. Methods: Rats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls. Results: Ketamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p,0.01) and significant reduction one week (p,0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p.0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p,0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wav

Reproducible and scalable differentiation of highly pure cortical neurons from human induced pluripotent stem cells

Summary: Human-induced-pluripotent-stem-cell (hiPSC)-derived neurons are valuable for investigating brain physiology and disease. Here, we present a protocol to differentiate hiPSCs into cortical neurons with high yield and purity. We describe neural induction via dual-SMAD inhibition, followed by spot-based differentiation to provide high quantities of neural precursors. We detail their enrichment, expansion, and purification to avoid unwanted cell fates and provide optimal conditions for neural rosette proliferation. These differentiated neurons are suitable for drug testing and co-culture studies.For complete details on the use and execution of this protocol, please refer to Paquet et al.1 and Weisheit et al..2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Spectral domain optical coherence tomography in mouse models of retinal degeneration

Purpose: Spectral domain optical coherence tomography (SD-OCT) allows cross-sectional visualization of retinal structures in vivo. Here, we report the efficacy of a commercially available SD-OCT device to study mouse models of retinal degeneration. Methods: C57BL/6 and BALB/c wild type mice and three different mouse models of hereditary retinal degeneration (Rho(-/-), rd1, RPE65(-/-)) were investigated using confocal scanning laser ophthalmoscopy (cSLO) for en face visualization and SD-OCT for cross-sectional imaging of retinal structures. Histology was performed to correlate structural findings in SD-OCT with light microscopic data. Results: In C57BL/6 and BALB/c mice, cSLO and SD-OCT imaging provided structural details of frequently used control animals (central retinal thickness, CRTC57BL/6 = 237+/-2microm and CRTBALB/c = 211+/-10microm). RPE65-/- mice at 11 months of age showed a significant reduction of retinal thickness (CRTRPE65 = 193+/-2microm) with thinning of the outer nuclear layer. Rho-/- mice at P28 demonstrated degenerative changes mainly in the outer retinal layers (CRTRho = 193+/-2microm). Examining rd1 animals before and after the onset of retinal degeneration allowed to monitor disease progression (CRTrd1 P11 = 246+/-4microm, CRTrd1 P28 = 143+/-4microm). Correlation of CRT assessed by histology and SD-OCT was high (r(2) = 0.897). Conclusion: We demonstrated cross sectional visualization of retinal structures in wild type mice and mouse models for retinal degeneration in vivo using a commercially available SD-OCT device. This method will help to reduce numbers of animals needed per study by allowing longitudinal study designs and facilitate characterization of disease dynamics and evaluation of putative therapeutic effects following experimental interventions