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

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

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..

A microglial activity state biomarker panel differentiates FTD-granulin and Alzheimer's disease patients from controls

BackgroundWith the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states.MethodsUsing mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study.ResultsWe identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer's (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals.ConclusionsThe identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer's Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders

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

Tackling neurodegeneration in vitro with omics: a path towards new targets and drugs

Drug discovery is a generally inefficient and capital-intensive process. For neurodegenerative diseases (NDDs), the development of novel therapeutics is particularly urgent considering the long list of late-stage drug candidate failures. Although our knowledge on the pathogenic mechanisms driving neurodegeneration is growing, additional efforts are required to achieve a better and ultimately complete understanding of the pathophysiological underpinnings of NDDs. Beyond the etiology of NDDs being heterogeneous and multifactorial, this process is further complicated by the fact that current experimental models only partially recapitulate the major phenotypes observed in humans. In such a scenario, multi-omic approaches have the potential to accelerate the identification of new or repurposed drugs against a multitude of the underlying mechanisms driving NDDs. One major advantage for the implementation of multi-omic approaches in the drug discovery process is that these overarching tools are able to disentangle disease states and model perturbations through the comprehensive characterization of distinct molecular layers (i.e., genome, transcriptome, proteome) up to a single-cell resolution. Because of recent advances increasing their affordability and scalability, the use of omics technologies to drive drug discovery is nascent, but rapidly expanding in the neuroscience field. Combined with increasingly advanced in vitro models, which particularly benefited from the introduction of human iPSCs, multi-omics are shaping a new paradigm in drug discovery for NDDs, from disease characterization to therapeutics prediction and experimental screening. In this review, we discuss examples, main advantages and open challenges in the use of multi-omic approaches for the in vitro discovery of targets and therapies against NDDs

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