Neurogenin 2 (iNGN2) overexpression induces human induced pluripotent stem cells (hiPSCs) into neurons but results in a mixed population of peripheral and central nervous system neurons. Pre-differentiation using BMP, MEK, and WNT inhibition (“BMWi”) prior to iNGN2 promotes telencephalic neuron differentiation. We outline neural induction, neural progenitor replating/freezing/thawing, and telencephalic neuron maturation and describe protocols for patterning motor neurons, ventral midbrain, and sensory neurons (sensBMi).For complete details on the use and execution of this protocol, please refer to Habich et al.1

Although progressive neurodegenerative diseases like Alzheimer's disease (AD) have been extensively studied for decades, some underlying mechanisms of pathogenesis remain elusive. In addition, modeling neurodegenerative diseases in vitro has proven to be a challenging task. However, advances in the technique of using human induced pluripotent stem cells (hiPSCs) have enabled the scientific community to study hiPSC-derived neurons, astrocytes, or microglia. Despite this important progress, monocultures of individual cell types may not accurately reflect the complexities of modeling specific pathological mechanisms. Therefore, we present a robust protocol for co-cultivating hiPSC-derived cortical neurons, astrocytes, and microglia in a tri-culture system for the purpose of hypothesis testing and drug screening. This co-cultivation system may allow modeling the effect of astrocyte and/or microglia modulation on neuronal health or intra-neuronal Tau aggregation, a key feature of AD progression.

Human induced pluripotent stem cells (hiPSCs) are a promising tool for studying neurological diseases and developing therapies for neurodegenerative diseases. Differentiation of hiPSCs into neurons can be achieved by dual SMAD inhibition (dSMADi) or by induced neurogenin 2 (NGN2) overexpression ("iNGN2"). Starting directly from hiPSCs, iNGN2 shortens the time to a neuronal stage but leads to neurons partially resembling peripheral or posterior fates while dSMADi more faithfully recapitulates telencephalic development. To modify the iNGN2 approach, we applied an accelerated induction paradigm that is dependent on the inhibition of BMP, MEK, and WNT pathways ("BMWi"), to commit hiPSCs into a telencephalic fate before iNGN2. The resulting neurons showed strong expression of telencephalic markers, with decreased levels of peripheral and posterior marker genes compared to iNGN2 alone. The resulting telencephalic neurons are suitable for a tau aggregation assay. Furthermore, we could demonstrate that during BMWi treatment, the cells are amenable to additional regional patterning cues. This allowed the generation of neurons from different regions of the CNS and peripheral nervous system (PNS), which will significantly facilitate in vitro modeling of a range of neurodevelopmental and neurodegenerative disorders.

Neurodegenerative diseases such as Alzheimer's or Parkinson's are marked by progressive loss of affected neurons. Even with novel disease-modifying therapies, this loss cannot be reversed. In situ astrocyte-to-neuron (AtN) transdifferentiation may provide an opportunity to convert resident astrocytes into new neurons to revert the loss of neurons incurred. Currently, most studies investigating AtN transdifferentiation in vitro rely on the use of primary mouse astrocyte cultures which require sacrificing animals and come with uncertainty regarding species differences. Conversely, human induced pluripotent stem cell (hiPSC)-derived astrocytes offer the advantage of working in a human cell culture system which improves translatability and provides the opportunity to generate large, cryopreservable batches of cells to identify and study conversion factors. This protocol details a workflow for assessing the suitability of potential conversion factors for transdifferentiating hiPSC-derived astrocytes into neurons.

Alzheimer’s disease (AD) is characterized by the accumulation and spread of Tau intraneuronal inclusions throughout most of the telencephalon, leaving hindbrain regions like the cerebellum and spinal cord largely spared. These neuropathological observations, along with the identification of specific vulnerable sub-populations from AD brain-derived single nuclei transcriptomics, suggest that a subset of brain regions and neuronal subtypes possess a selective vulnerability to Tau pathology. Given the inability to culture neurons from patient brains, a disease-relevant in vitro model which recapitulates these features would serve as a critical tool to validate modulators of vulnerability and resilience. Using our recently established platform for inducing endogenous Tau aggregation in human induced pluripotent stem cell (hiPSC)-derived cortical excitatory neurons via application of AD brain-derived exogenous Tau aggregates, we explored whether Tau aggregates preferentially induce aggregation in specific neuronal subtypes. We compared Tau seeding in hiPSC-derived neuron subtypes representing regional identities across the forebrain, midbrain, and hindbrain. Higher susceptibility (i.e. more Tau aggregation) was consistently observed among cortical neuron subtypes, with CTIP2-positive, somatostatin (SST)-positive cortical inhibitory neurons showing the greatest aggregation levels across hiPSC lines from multiple donors. hiPSC-neurons also delineated between the disease-specific vulnerabilities of different protein aggregates, as α-synuclein preformed fibrils showed an increased propensity to induce aggregates in midbrain dopaminergic (mDA)-like neurons, mimicking Parkinson’s disease (PD)-specific susceptibility. Aggregate uptake and degradation rates were insufficient to explain differential susceptibility. The absence of a consistent transcriptional response following aggregate seeding further indicated that intrinsic neuronal subtype-specific properties could drive susceptibility. The present data provides evidence that hiPSC-neurons exhibit features of selective neuronal vulnerability which manifest in a cell autonomous manner, suggesting that mining intrinsic (or basal) transcriptomic signatures of more vulnerable compared to more resilient hiPSC-neurons could uncover the molecular underpinnings of differential susceptibility to protein aggregation found in a variety of neurodegenerative diseases.

Aberrant protein aggregation is a pathological cellular hallmark of many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), where the tau protein is aggregating, forming neurofibrillary tangles (NFTs), and propagating from neuron to neuron. These processes have been linked to disease progression and a decline in cognitive function. Various therapeutic approaches aim at the prevention or reduction of tau aggregates in neurons. Human induced pluripotent stem cells (hiPSCs) are a very valuable tool in neuroscience discovery, as they offer access to potentially unlimited amounts of cell types that are affected in disease, including cortical neurons, for in vitro studies. We have generated an in vitro model for tau aggregation that uses hiPSC - derived neurons expressing an aggregation prone, fluorescently tagged version of the human tau protein after lentiviral transduction. Upon addition of tau seeds in the form of recombinant sonicated paired helical filaments (sPHFs), the neurons show robust, disease-like aggregation of the tau protein. The model was developed as a plate-based high content screening assay coupled with an image analysis algorithm to evaluate the impact of small molecules or genetic perturbations on tau. We show that the assay can be used to evaluate small molecules or screen targeted compound libraries. Using siRNA-based gene knockdown, genes of interest can be evaluated, and we could show that a targeted gene library can be screened, by screening nearly 100 deubiquitinating enzymes (DUBs) in that assay. The assay uses an imaging-based readout, a relatively short timeline, quantifies the extent of tau aggregation, and also allows the assessment of cell viability. Furthermore, it can be easily adapted to different hiPSC lines or neuronal subtypes. Taken together, this complex and highly relevant approach can be routinely applied on a weekly basis in the screening funnels of several projects and generates data with a turnaround time of approximately five weeks.

In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding-competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.

AbstractBackgroundOne of the most prominent genetic risk factors for Alzheimer’s Disease (AD) is the Apolipoprotein E (APOE) ε4 allele. Unlike the “neutral” ε3 variant, or the protective ε2variant, the ε4 variant is strongly associated with late‐onset AD, albeit by mechanisms that are not well‐understood.MethodsTo shed light on the biology of APOE in the development of AD, as part of the IMI (Innovative Medicines Initiative) consortium “ADAPTED”, we acquired two human induced pluripotent stem cell (hiPSC) lines, that were isogenically modified to carry the alleles ε2/ε2, ε3/ε3, ε3/ε4, ε4/ε4, as well as a knockout line. Neurons and the phagocytic cells of the CNS, the microglia were differentiated from all ten lines and subjected to thorough phenotyping, including RNA‐seq and proteomics analysis.ResultsCortical neurons do not secrete apoE under normal conditions and treatment of these cells with microglia‐enriched factors and apoE increased their overall Abeta secretion, without significant differences between the various genotypes. Microglia, on the other hand, secrete large amounts of apoE in varying quantities depending on the APOE genotype. Additionally, microglia carrying the ε4/ε4 genotype have a significantly reduced phagocytic capacity compared to their ε3/ε3 counterparts. These observations were supported by whole transcriptome sequencing, which revealed major differences in inflammatory and phagocytic activity‐related gene expression between the different APOE genotypes.ConclusionsThis unique set of ten isogenic hiPSC lines enabled us to assess the cellular phenotypes in functional human neuronal cells. Microglia omics show changes in an array of AD‐relevant pathways, and being the main producers of apoE, these changes need to be understood in depth. Follow‐up experiments will validate candidate genes that might help design new therapies for APOE ε4 AD patients. Disclosure: This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115975. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This study was sponsored by AbbVie Deutschland GmbH & Co. KG. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the abstract. Some authors are currently employed by AbbVie and may own AbbVie stock.

AbstractBackgroundOne of the hallmarks of Alzheimer's Disease (AD), as with some other neurodegenerative diseases, is the misfolding and aggregation of proteins, such as amyloid‐beta and tau. Tau pathology is also believed to propagate trans‐synaptically from neuron to neuron. Either prevention of propagation, or the removal of aggregated tau, is a potential approach for AD modification.MethodDeubiquitinating enzymes (DUBs) maintain ubiquitin homeostasis by removing ubiquitin modifications from target proteins, thereby altering protein function, stability, and signaling. We hypothesize that the modulation of the ubiquitin‐proteasome system with DUB inhibitors will lead to decreased tau aggregation either directly (by inhibiting the removal of ubiquitin from tau thereby increasing its degradation) or indirectly (via modulation of other relevant pathways, like autophagy). To select relevant DUBs, we developed a phenotypic assay to assess the effect of knocking down ∼100 DUBs on the clearance of tau aggregates in hiPSC – derived neurons, assayed using high content image analysis.ResultWe show that addition of recombinant tau seeds to hiPSC – derived cortical neurons expressing a fluorescent tau reporter construct, leads to formation of tau aggregates, which we can robustly and reproducibly reduce by knocking down specific DUBs.ConclusionThe DUB enzymes revealed in our phenotypic screen have been validated in confirmatory studies and have the potential to become novel targets for the treatment of AD. In collaboration with Mission Therapeutics we will develop selective, potent DUB inhibitors for preclinical target validation.

To better understand and model neurological, in particular neurodegenerative diseases, human induced pluripotent stem cells (hiPSCs) offer a great source for generation of neural cells. We provide a protocol for the differentiation of hiPSc-derived astrocytes in vitro. This protocol not only is chemically defined, that is, it does not use serum, but also allows for the expansion of astrocyte progenitor cells and mature astrocytes. Large batches of hiPSc-derived astrocytes can be stored and used for defined in vitro disease models.

The apolipoprotein ε4 allele (apoE4) is a well-established genetic risk factor for late onset Alzheimer's disease (AD), while the apoE2 allele provides protection against AD development. However, the exact molecular mechanisms by which apoE plays its role in AD pathogenesis remain largely unknown. Aiming to investigate the role of apoE in AD, isogenic lines for its different isoforms were generated from four human induced pluripotent stem cell (iPSC) donors within the framework of the IMI2 project ADAPTED. The lines from the first donor were used to derive expandable cortical progenitor cells that can be differentiated into cortical neurons and astrocytes. After characterization of these cells, they were further utilized to study phenotypic differences induced by the various apoE isoforms. It had been previously reported that human apoE might play a role in the inflammatory response of cells. Since inflammation and neurodegeneration are closely linked, we aimed at analyzing the role of apoE in modulating the inflammatory response of isogenic astrocytes. Indeed, stimulating these astrocytes with a cytokine cocktail triggered a differential inflammatory response depending on the apoE genotype. ApoE4 carrying astrocytes displayed stronger pro-inflammatory responses compared to apoE3 cells indicating a potential role for neuro-inflammation in AD. In this study the analysis of these differences was extended to additional apoE genotypes, like apoE2/2 and also included the heterozygote form apoE3/4. ApoE has also been reported to influence the complexity of neuronal networks denoted by the neuron's ability to grow and extend neurites. However, this has not been tested in human-derived cells. Therefore, we investigated the impact of the various apoE isoforms on the differentiation and maturation of cortical neurons under basal conditions and in the presence of various stressors. Since apoE4 is a significant factor in the development of late onset AD, we believe that our mechanistic studies help understand its role in the disease course, and ultimately bring us closer to developing impactful treatments.

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

Alzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay.

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48 h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300 nM) with a maximal effect at 30 min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, β and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1 μM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.

High-content screening has brought new dimensions to cellular assays by generating rich data sets that characterize cell populations in great detail and detect subtle phenotypes. To derive relevant, reliable conclusions from these complex data, it is crucial to have informatics tools supporting quality control, data reduction, and data mining. These tools must reconcile the complexity of advanced analysis methods with the user-friendliness demanded by the user community. After review of existing applications, we realized the possibility of adding innovative new analysis options. Phaedra was developed to support workflows for drug screening and target discovery, interact with several laboratory information management systems, and process data generated by a range of techniques including high-content imaging, multicolor flow cytometry, and traditional high-throughput screening assays. The application is modular and flexible, with an interface that can be tuned to specific user roles. It offers user-friendly data visualization and reduction tools for HCS but also integrates Matlab for custom image analysis and the Konstanz Information Miner (KNIME) framework for data mining. Phaedra features efficient JPEG2000 compression and full drill-down functionality from dose-response curves down to individual cells, with exclusion and annotation options, cell classification, statistical quality controls, and reporting.

Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3) is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM), 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1). Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3(NH2) and R3/I5(NH2) with pEC50 values of 6.54 (6.46 to 6.64) and 6.07 (5.94 to 6.20), respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27) in the presence of a probe (10 nM) concentration of relaxin-3(NH2). 135PAM1 does not compete for binding with the orthosteric radioligand, [(125)I] R3I5 (amide), in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native) form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe molecules that can affect allosteric modulation of RXFP3.

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the IKs channel, which along with the transient outward current (Ito) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent IKs blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies.