Extracellular vesicles in the study of Alzheimer's and Parkinson's diseases: Methodologies applied from cells to biofluids.

Extracellular vesicles (EVs) are gaining increased importance in fundamental research as key players in disease pathogenic mechanisms, but also in translational and clinical research due to their value in biomarker discovery, either for diagnostics and/or therapeutics. In the first research scenario, the study of EVs isolated from neuronal models mimicking neurodegenerative diseases can open new avenues to better understand the pathological mechanisms underlying these conditions or to identify novel molecular targets for diagnosis and/or therapeutics. In the second research scenario, the easy availability of EVs in body fluids and the specificity of their cargo, which can reflect the cell of origin or disease profiles, turn these into attractive diagnostic tools. EVs with exosome-like characteristics, circulating in the bloodstream and other peripheral biofluids, constitute a non-invasive and rapid alternative to study several conditions, including brain-related disorders. In both cases, several EVs isolation methods are already available, but each neuronal model or biofluid presents its own challenges. Herein, a literature overview on EVs isolation methodologies from distinct neuronal models (cellular culture and brain tissue) and body fluids (serum, plasma, cerebrospinal fluid, urine and saliva) was carried out. Focus was given to approaches employed in the context of Alzheimer's and Parkinson's diseases, and the main research findings discussed. The topics here revised will facilitate the choice of EVs isolation methodologies and potentially prompt new discoveries in EVs research and in the neurodegenerative diseases field.

Neural In Vitro Models for Studying Substances Acting on the Central Nervous System.

Animal models have been greatly contributing to our understanding of physiology, mechanisms of diseases, and toxicity. Yet, their limitations due to, e.g., interspecies variation are reflected in the high number of drug attrition rates, especially in central nervous system (CNS) diseases. Therefore, human-based neural in vitro models for studying safety and efficacy of substances acting on the CNS are needed. Human iPSC-derived cells offer such a platform with the unique advantage of reproducing the "human context" in vitro by preserving the genetic and molecular phenotype of their donors. Guiding the differentiation of hiPSC into cells of the nervous system and combining them in a 2D or 3D format allows to obtain complex models suitable for investigating neurotoxicity or brain-related diseases with patient-derived cells. This chapter will give an overview over stem cell-based human 2D neuronal and mixed neuronal/astrocyte models, in vitro cultures of microglia, as well as CNS disease models and considers new developments in the field, more specifically the use of brain organoids and 3D bioprinted in vitro models for safety and efficacy evaluation.

More dynamic, more quantitative, unexpectedly intricate: Advanced understanding on synaptic RNA localization in learning and memory.

Synaptic signaling exhibits great diversity, complexity, and plasticity which necessitates maintenance and rapid modification of a local proteome. One solution neurons actively exploit to meet such demands is the strategic deposition of mRNAs encoding proteins for both basal and experience-driven activities into ribonucleoprotein complexes at the synapse. Transcripts localized in this manner can be rapidly accessed for translation in response to a diverse range of stimuli in a temporal- and spatially-restricted manner. Here we review recent findings on localized RNAs and RNA binding proteins in the context of learning and memory, as revealed by cutting-edge in-vitro and in-vivo technologies capable of yielding quantitative and dynamic information. The new technologies include proteomic and transcriptomic analyses, high-resolution multiplexed RNA imaging, single-molecule RNA tracking in living neurons, animal models and human neuron cell models. Among many recent advances in the field, RNA chemical modification has emerged as one of the new regulatory layers of gene expression at synapse that is complex and yet largely unexplored. These exciting new discoveries have enhanced our understanding of the modulation mechanisms of synaptic gene expression and their roles in cognition.

mitoLUHMES: An Engineered Neuronal Cell Line for the Analysis of the Motility of Mitochondria.

Perturbations in the transport of mitochondria and their quality control in neuronal cells underlie many types of neurological pathologies, whereas systems enabling convenient analysis of mitochondria behavior in cellular models of neurodegenerative diseases are limited. In this study, we present a modified version of lund human mesencephalic cells, mitoLUHMES, expressing GFP and mitochondrially targeted DsRed2 fluorescent proteins, intended for in vitro analysis of mitochondria trafficking by real-time fluorescence microscopy. This cell line can be easily differentiated into neuronal phenotype and allows us to observe movements of single mitochondria in single cells grown in high-density cultures. We quantified the perturbations in mitochondria morphology and dynamics in cells treated with model neurotoxins: carbonyl cyanide m-chlorophenylhydrazone and 6-hydroxydopamine. For the first time we filmed the processes of fission, fusion, pausing, and reversal of mitochondria movement direction in LUHMES cells. We present a detailed analysis of mitochondria length, velocity, and frequency of movement for static, anterograde, and retrograde motile mitochondria. The observed neurotoxin treatment-mediated decreases in morphological and kinetic parameters of mitochondria provide foundation for the future studies exploiting mitoLUHMES as a new model for neurobiology.

Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells.

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge. Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture. Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of ß-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

An overview of brain-like computing: Architecture, applications, and future trends.

With the development of technology, Moore's law will come to an end, and scientists are trying to find a new way out in brain-like computing. But we still know very little about how the brain works. At the present stage of research, brain-like models are all structured to mimic the brain in order to achieve some of the brain's functions, and then continue to improve the theories and models. This article summarizes the important progress and status of brain-like computing, summarizes the generally accepted and feasible brain-like computing models, introduces, analyzes, and compares the more mature brain-like computing chips, outlines the attempts and challenges of brain-like computing applications at this stage, and looks forward to the future development of brain-like computing. It is hoped that the summarized results will help relevant researchers and practitioners to quickly grasp the research progress in the field of brain-like computing and acquire the application methods and related knowledge in this field.

Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy.

Accumulation of misfolded, aggregating proteins concurrent with disease onset and progression is a hallmark of neurodegenerative proteinopathies. An important class of these are tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD), associated with accumulation of aberrant forms of tau protein in the brain. Pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization and changes in solubility, cellular redistribution, and spreading. Development and testing of experimental therapeutics that target these pathological tau conformers requires use of cellular models that recapitulate neuronal endogenous, non-heterologous tau expression under genomic and physiological contexts relevant to disease. In this study, we employed FTD-patient induced pluripotent stem cells (iPSC)-derived neurons, expressing a tau variant or mutation, as primary models for driving a medicinal chemistry campaign around tau targeting degrader series. Our screening goal was to establish structure-activity relationships (SAR) for the different chemical series to identify the molecular composition that most efficiently led to tau degradation in human FTD ex vivo neurons. We describe the identification of the lead compound QC-01-175 and follow-up optimization strategies for this molecule. We present three final lead molecules with tau degradation activity in mutant neurons, which establishes potential disease relevance and will drive future studies on specificity and pharmacological properties.

A simple neuronal model with intrinsic saturation of the firing frequency.

We present a comparison of the intrinsic saturation of firing frequency in four simple neural models: leaky integrate-and-fire model, leaky integrate-and-fire model with reversal potentials, two-point leaky integrate-and-fire model, and a two-point leaky integrate-and-fire model with reversal potentials. "Two-point" means that the equivalent circuit has two nodes (dendritic and somatic) instead of one (somatic only). The results suggest that the reversal potential increases the slope of the "firing rate vs input" curve due to a smaller effective membrane time constant, but does not necessarily induce saturation of the firing rate. The two-point model without the reversal potential does not limit the voltage or the firing rate. In contrast to the previous models, the two-point model with the reversal potential limits the asymptotic voltage and the firing rate, which is the main result of this paper. The case of excitatory inputs is considered first and followed by the case of both excitatory and inhibitory inputs.

Activation of Interferon-Stimulated Genes following Varicella-Zoster Virus Infection in a Human iPSC-Derived Neuronal In Vitro Model Depends on Exogenous Interferon-α.

Varicella-zoster virus (VZV) infection of neuronal cells and the activation of cell-intrinsic antiviral responses upon infection are still poorly understood mainly due to the scarcity of suitable human in vitro models that are available to study VZV. We developed a compartmentalized human-induced pluripotent stem cell (hiPSC)-derived neuronal culture model that allows axonal VZV infection of the neurons, thereby mimicking the natural route of infection. Using this model, we showed that hiPSC-neurons do not mount an effective interferon-mediated antiviral response following VZV infection. Indeed, in contrast to infection with Sendai virus, VZV infection of the hiPSC-neurons does not result in the upregulation of interferon-stimulated genes (ISGs) that have direct antiviral functions. Furthermore, the hiPSC-neurons do not produce interferon-α (IFNα), a major cytokine that is involved in the innate antiviral response, even upon its stimulation with strong synthetic inducers. In contrast, we showed that exogenous IFNα effectively limits VZV spread in the neuronal cell body compartment and demonstrated that ISGs are efficiently upregulated in these VZV-infected neuronal cultures that are treated with IFNα. Thus, whereas the cultured hiPSC neurons seem to be poor IFNα producers, they are good IFNα responders. This could suggest an important role for other cells such as satellite glial cells or macrophages to produce IFNα for VZV infection control.

Creation of discontinuities in circle maps.

Circle maps frequently arise in mathematical models of physical or biological systems. Motivated by Cherry flows and 'threshold' systems such as integrate and fire neuronal models, models of cardiac arrhythmias, and models of sleep/wake regulation, we consider how structural transitions in circle maps occur. In particular, we describe how maps evolve near the creation of a discontinuity. We show that the natural way to create discontinuities in the maps associated with both threshold systems and Cherry flows results in a singularity in the derivative of the map as the discontinuity is approached from either one or both sides. For the threshold systems, the associated maps have square root singularities and we analyse the generic properties of such maps with gaps, showing how border collisions and saddle-node bifurcations are interspersed. This highlights how the Arnold tongue picture for tongues bordered by saddle-node bifurcations is amended once gaps are present. We also show that a loss of injectivity naturally results in the creation of multiple gaps giving rise to a novel codimension two bifurcation.

Novel test strategies for in vitro seizure liability assessment.

INTRODUCTION: The increasing incidence of mental illnesses and neurodegenerative diseases results in a high demand for drugs targeting the central nervous system (CNS). These drugs easily reach the CNS, have a high affinity for CNS targets, and are prone to cause seizures as an adverse drug reaction. Current seizure liability assessment heavily depends on in vivo or ex vivo animal models and is therefore ethically debated, labor intensive, expensive, and not always predictive for human risk. AREAS COVERED: The demand for CNS drugs urges the development of alternative safety assessment strategies. Yet, the complexity of the CNS hampers reliable detection of compound-induced seizures. This review provides an overview of the requirements of in vitro seizure liability assays and highlights recent advances, including micro-electrode array (MEA) recordings using rodent and human cell models. EXPERT OPINION: Successful and cost-effective replacement of in vivo and ex vivo models for seizure liability screening can reduce animal use for drug development, while increasing the predictive value of the assays, particularly if human cell models are used. However, these novel test strategies require further validation and standardization as well as additional refinements to better mimic the human in vivo situation and increase their predictive value.

The geometry of rest-spike bistability.

Morris-Lecar model is arguably the simplest dynamical model that retains both the slow-fast geometry of excitable phase portraits and the physiological interpretation of a conductance-based model. We augment this model with one slow inward current to capture the additional property of bistability between a resting state and a spiking limit cycle for a range of input current. The resulting dynamical system is a core structure for many dynamical phenomena such as slow spiking and bursting. We show how the proposed model combines physiological interpretation and mathematical tractability and we discuss the benefits of the proposed approach with respect to alternative models in the literature.

Towards animal-free neurotoxicity screening: Applicability of hiPSC-derived neuronal models for in vitro seizure liability assessment.

A sizeable proportion of drug attrition is due to drug-induced seizures. Current available animal models frequently fail to predict human seizure liability. Therefore, there is a need for in vitro alternatives, preferably based on human-derived neurons to circumvent interspecies translation. The increasing number of commercially available human induced pluripotent stem cell (hiPSC)-derived neuronal models holds great promise for replacing rodent primary cultures. We therefore tested three different hiPSC-derived neuronal models for their applicability for in vitro seizure liability assessment. Using immunofluorescent staining and multi-well micro-electrode arrays we show that all models develop functional neuronal networks that exhibit spontaneous activity and (network) bursting behavior. Developmental patterns differ between the models, probably due to differences in model composition and seeding density. Nevertheless, neuronal activity and (network) bursting can be reproducibly modulated with the seizurogenic compounds strychnine, picrotoxin (PTX) and 4-aminopyridine (4-AP). However, the sensitivity and degree of chemical-induced effects differs between the models, which can likely be explained by differences in seeding density, maturation and different ratios of inhibitory and excitatory cell types. Importantly, compared to rat primary cortical neurons, the hiPSC-derived neuronal models were equally, or even better in the case of 4-AP, suited to detect seizurogenicity. Overall, our data indicate that hiPSC-derived neuronal models may in the future be used as a first screening tool for in vitro seizure liability assessment. However, before hiPSC-derived neuronal models can fully replace animal experiments, more compounds should be tested and the available models must be further characterized to fully understand their applicability.

Applicability of hiPSC-Derived Neuronal Cocultures and Rodent Primary Cortical Cultures for In Vitro Seizure Liability Assessment.

Seizures are life-threatening adverse drug reactions which are investigated late in drug development using rodent models. Consequently, if seizures are detected, a lot of time, money and animals have been used. Thus, there is a need for in vitro screening models using human cells to circumvent interspecies translation. We assessed the suitability of cocultures of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared with rodent primary cortical cultures for in vitro seizure liability assessment using microelectrode arrays. hiPSC-derived and rodent primary cortical neuronal cocultures were exposed to 9 known (non)seizurogenic compounds (pentylenetetrazole, amoxapine, enoxacin, amoxicillin, linopirdine, pilocarpine, chlorpromazine, phenytoin, and acetaminophen) to assess effects on neuronal network activity using microelectrode array recordings. All compounds affect activity in hiPSC-derived cocultures. In rodent primary cultures all compounds, except amoxicillin changed activity. Changes in activity patterns for both cell models differ for different classes of compounds. Both models had a comparable sensitivity for exposure to amoxapine (lowest observed effect concentration [LOEC] 0.03 µM), linopirdine (LOEC 1 µM), and pilocarpine (LOEC 0.3 µM). However, hiPSC-derived cultures were about 3 times more sensitive for exposure to pentylenetetrazole (LOEC 30 µM) than rodent primary cortical cultures (LOEC 100 µM). Sensitivity of hiPSC-derived cultures for chlorpromazine, phenytoin, and enoxacin was 10-30 times higher (LOECs 0.1, 0.3, and 0.1 µM, respectively) than in rodent cultures (LOECs 10, 3, and 3 µM, respectively). Our data indicate that hiPSC-derived neuronal cocultures may outperform rodent primary cortical cultures with respect to detecting seizures, thereby paving the way towards animal-free seizure assessment.

A small molecule autophagy inducer exerts cytoprotection against α-synuclein toxicity.

α-synucleopathies are protein-misfolding disorders occur primarily due to aggregation and toxicity of α-synuclein. This study characterized the small molecule AGK2 as a modifier of α-synuclein mediated toxicity in an autophagy dependent manner in both yeast and mammalian cell line models. In yeast system, AGK2 enhances autophagy to clear toxic α-synuclein aggregates in an autophagy dependent manner. Autophagy flux analyses revealed that AGK2 induces autophagy especially autolysosomes. Importantly, AGK2 induces autophagy in an mTOR independent manner. These features enable AGK2 to exert cytoprotective potential against α-synuclein mediated toxicity in different model systems.

Inconvenient Truth to Principle of Neuroscience.

In 2004, Prinz et al. demonstrated that almost indistinguishable network activity can arise from widely different sets of underlying membrane and synaptic parameters, and, thus, likely arise from different cellular and network mechanisms. This now broadly accepted principle guides research into individual variation in neuronal and synaptic properties, and their homeostatic regulation.

Human iPSC-derived neuronal models for in vitro neurotoxicity assessment.

Neurotoxicity testing still relies on ethically debated, expensive and time consuming in vivo experiments, which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds, such as picrotoxin (PTX) and endosulfan, and the neurotoxicant methylmercury (MeHg). However, the chemical-induced effects on different parameters for neuronal activity, such as mean spike rate (MSR) and mean burst rate (MBR), may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening.

Input-to-output transformation in a model of the rat hippocampal CA1 network.

Here we use computational modeling to gain new insights into the transformation of inputs in hippocampal field CA1. We considered input-output transformation in CA1 principal cells of the rat hippocampus, with activity synchronized by population gamma oscillations. Prior experiments have shown that such synchronization is especially strong for cells within one millimeter of each other. We therefore simulated a one-millimeter it patch of CA1 with 23,500 principal cells. We used morphologically and biophysically detailed neuronal models, each with more than 1000 compartments and thousands of synaptic inputs. Inputs came from binary patterns of spiking neurons from field CA3 and entorhinal cortex (EC). On average, each presynaptic pattern initiated action potentials in the same number of CA1 principal cells in the patch. We considered pairs of similar and pairs of distinct patterns. In all the cases CA1 strongly separated input patterns. However, CA1 cells were considerably more sensitive to small alterations in EC patterns compared to CA3 patterns. Our results can be used for comparison of input-to-output transformations in normal and pathological hippocampal networks.