Development of novel human cellular models for neurotoxicity studies

Dissertation to obtain master degree in Genética Molecular e BiomedicinaInformation currently available on neurotoxicity of chemicals is scarce and there are a growing number of new compounds to be tested. Therefore, new strategies are necessary to identify neurotoxic agents with speed, reliability and respect for animal welfare. The limited availability of primary human brain cells means that there is a need for human cell lines that reliably model human neurons and astrocytes. Despite the advances in stem cell research, numerous challenges must be overcome before this technology can be widespread used, such as low differentiation efficiency. Human pluripotent embryocarcinoma NTera2/cloneD1 (NT2) cell line is an alternative cell source from which neurons and astrocytes can be derived in vitro. The aim of this work was to develop scalable and reproducible novel human cellular models using NT2 cells as source of differentiated neural phenotypes. A 2D culture system for astrocytic differentiation was implemented. After 4 weeks of differentiation with retinoic acid followed by 5 weeks maturation with mitotic inhibitors, astrocytes obtained expressed vimentin, GFAP, S100- and GLT-1 as characterized by immunodetection and qRT-PCR. Then, a 3D culture approach was adopted, using stirred suspension culture systems, in which cell-cell and cell-extracellular matrix interactions occur, mimicking better the in vivo situation. NT2 cells, inoculated as single cells, spontaneously aggregated without compromising their pluripotency. Optimization of stirring rate allowed control of aggregate size along time. After 3 weeks of RA treatment and 2 weeks of maturation, neurons expressing βIII-tubulin, MAPs and synaptophysin and astrocytes expressing vimentin, GFAP, S100- and GLT-1 were detected, as characterized by immunodetection and qRT-PCR. Furthermore, astrocytes presented a 2.5-fold higher yield than that observed in 2D culture systems. Results showed that NT2 differentiated cells are promising models for neurotoxicity testing. Furthermore, the 3D culture systems developed herein can contribute to increase the relevance of these studies, recapitulating human neuron-astrocyte interactions in a 3D cellular context.Fundação para a Ciência e Tecnologia - PTDC/EEB-BIO/112786/200

Exploiting 3D differentiation of human stem cells

"Neurological disorders are a major public health problem and are expected to rise dramatically together with the higher life expectancy and the shift towards an ageing society. Current therapeutic options can only ameliorate some of the symptoms and there are no effective treatments to target pathological mechanisms and stop disease progression. The human brain complexity hampers the understanding of the brain functioning at the molecular, cellular, and pathophysiological levels for many neurological disorders. This highlights the need for new brain models, which can contribute to unveil molecular mechanisms of neurological disorders, identify therapeutic targets and evaluate preclinically new therapies in a more adequate and predictive basis, withstanding its successful translation to the clinics. Despite their undeniable value, traditional animal models diverge from humans at biochemical and genetic levels. Moreover, 2D in vitro cell-based models do not mimic important aspects of brain cellular heterogeneity, architecture and microenvironment (...)".N/

Refining iPSC-based 3D neural cell models and characterization tools to address brain microenvironment-related diseases

Brain microenvironment plays important roles in neurodevelopment and pathology and can affect therapy efficacy. Neural cell culture typically relies on the use of heterologous matrices that poorly resemble brain extracellular matrix (ECM) or reflect its pathological features. We have shown that perfusion bioreactor-based 3D differentiation of iPSC-derived human neural stem cells (hiPSC-NSC) sustains the concomitant differentiation of the three neural lineages (neurospheroids). If this neurospheroid culture strategy also allows deposition of native neural ECM it would be possible to (i) mimic cellular and microenvironment remodeling during neural differentiation, without the confounding effects of exogenous matrices and (ii) recapitulate pathological phenotypic features of diseases in which homotypic/ heterotypic cell-cell interactions and ECM are relevant. To characterize the neural extracellular space we employed quantitative transcriptomic (NGS) and proteome (SWATH-MS) analysis. Neurogenic developmental pathways were recapitulated in neurospheroids, with significant changes in cell membrane and ECM composition along differentiation; a significant enrichment in structural proteoglycans, typical of brain ECM, a downregulation of basement membrane proteins constituents and a higher expression of synaptic and ion transport machinery were observed. Neurospheroids were generated using hiPSC-NSC derived from Mucopolysaccharidosis type VII (MPS VII) patients. MPS VII is a rare neuronopathic lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity, leading to glycosaminoglycan (GAGs) accumulation in the brain. The main MPS VII molecular hallmarks were recapitulated, e.g. accumulation og GAGs. By combining the neurospheroid culture with a 3D neuronal connectivity assay based on calcium imaging analysis we refined a new analytical strategy to characterize neuronal connectivity defects in a more predictive setting. We showed that MPS VII neurospheroids presented reduced neuronal activity and disturbances in network functionality, with alterations in connectivity and synchronization. These data provide insights into the interplay between reduced β-gluc activity, GAGs accumulation, alterations in neuronal network and its impact on MPS VII-associated cognitive defects Applying the characterization tools refined in this work to cope with 3D neurospheroid cultures, namely the neuronal connectivity assay, we provide a new platform to unveil the cellular processes responsible for brain dysfunction in neurological disorders and to test and optimize new therapies. Acknowledgments: iNOVA4Health – UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement, is acknowledged. PD/BD/52473/2014, PD/BD/52481/2014, and PD/BD/128371/2017 PhD fellowships funded by FCT, Portugal. N.R. is supported by the European Research Council Starting Grant 337327. MS data were obtained by UniMS – Mass Spectrometry Unit, ITQB and iBET, Oeiras, Portugal

iPSC-derived neurospheroids recapitulate development and pathological signatures of human brain microenvironment

Brain microenvironment plays an important role in neurodevelopment and pathology, where extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on the use of heterologous matrices that poorly resemble the brain ECM or reflect its pathological features. We have previously demonstrated that perfusion stirred-tank bioreactor-based 3D differentiation of human neural stem cells (NSC) - pSTR-neurospheroids, sustains the concomitant differentiation of the three neural cell lineages (neurons, astrocytes and oligodendrocytes) and the establishment of physiologically relevant cell-cell interactions. Here, we hypothesized that if the pSTR-neurospheroid strategy would also allow the deposition of native neural ECM components and diffusion of secreted factors, it would be possible to: (i) mimic the cellular and microenvironment remodeling occurring during neural differentiation without the confounding effects of exogenous matrices; (ii) recapitulate the pathological phenotypes of diseases in which alteration of homotypic and heterotypic cell-cell interactions and ECM components are relevant. To demonstrate the first point, we analyzed pSTR-neurospheroid differentiation by quantitative transcriptome (NGS) and proteome (SWATH-MS). Data showed that neurogenic developmental pathways were recapitulated, with significant changes at cell membrane and ECM composition, diverging from the 2D differentiation profile. A significant enrichment in structural proteoglycans typical of brain ECM, along with downregulation of basement membrane constituents was observed. Moreover, higher expression of synaptic and ion transport machinery in pSTR-neurospheroids suggest higher neuronal maturation than in 2D. Having shown recapitulation of neural microenvironmental dynamics in pSTR-neurospheroids, we used Mucopolysaccharidosis VII (MPSVII) as a disease case study. MPS VII is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity, which leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. In pSTR-neurospheroids generated from hiPSC of a MPS VII patient, the main molecular disease hallmarks were recapitulated, namely accumulation of GAGs. Notably, MPS VII neurospheroids showed reduced neuronal activity and a disturbance in network functionality, with alterations both in connectivity and synchronization, not observed in 2D cultures. These data provide insight into the interplay between reduced β-gluc activity, GAG accumulation, alterations in the neural network, and its impact on MPS VII-associated cognitive defects. Overall we demonstrate that neural cellular and extracellular developmental and pathological features are recapitulated in healthy and diseased pSTR-neurospheroids, respectively. These can be valuable in vitro models to address molecular defects associated with neurological disorders that affect neural microenvironment homeostasis. Moreover, the 3D neuronal connectivity assay developed is a new tool with potential to assess other lysosomal storage diseases and neurodegenerative diseases that have variable phenotypes. Acknowledgements: SFRH/BD/78308/2011, SFRH/BD/52202/2013 and SFRH/BD/52473/2014 PhD fellowships from FCT, Portugal and iNOVA4Health-UID/Multi/04462/2013, supported by FCT/ MEC, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement

Neuroprotection in a 3D Cell Model of Parkinson's Disease

Funding Information: This work was supported by European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under grant agreement No. 804229; iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through national funds. FCT/MCTES, through the project PTDC/BTM‐ORG/29580/2017. Authors would like to acknowledge FCT for financial support of R.C. (PD/BD/135492/2018) and J.G.‐P. (SFRH/BD/145522/2019). M.L. was supported by the Land‐BW (NAM‐ACCEPT) and funding by the European Union's Horizon 2020 research and innovation program under grant agreements No. 964537 (RISK‐HUNT3R), No. 964518 (ToxFree), and No. 825759 (ENDpoiNTs). Publisher Copyright: © 2022 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbHScope: Diets rich in (poly)phenols have been associated with positive effects on neurodegenerative disorders, such as Parkinson's disease (PD). Several low-molecular weight (poly)phenol metabolites (LMWPM) are found in the plasma after consumption of (poly)phenol-rich food. It is expected that LMWPM, upon reaching the brain, may have beneficial effects against both oxidative stress and neuroinflammation, and possibly attenuate cell death mechanisms relate to the loss of dopaminergic neurons in PD. Methods and Results: This study investigates the neuroprotective potential of two blood-brain barrier permeant LMWPM, catechol-O-sulfate (cat-sulf), and pyrogallol-O-sulfate (pyr-sulf), in a human 3D cell model of PD. Neurospheroids were generated from LUHMES neuronal precursor cells and challenged by 1-methyl-4-phenylpyridinium (MPP+) to induce neuronal stress. LMWPM pretreatments were differently neuroprotective towards MPP+ insult, presenting distinct effects on the neuronal transcriptome. Particularly, cat-sulf pretreatment appeared to boost counter-regulatory defense mechanisms (preconditioning). When MPP+ is applied, both LMWPM positively modulated glutathione metabolism and heat-shock response, as also favorably shifting the balance of pro/anti-apoptotic proteins. Conclusions: Our findings point to the potential of LMWPM to trigger molecular mechanisms that help dopaminergic neurons to cope with a subsequent toxic insult. They are promising molecules to be further explored in the context of preventing and attenuating parkinsonian neurodegeneration.publishersversionepub_ahead_of_prin

Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neural cells

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects

Using High-Pressure Technology to Develop Antioxidant-Rich Extracts from Bravo de Esmolfe Apple Residues

Bravo de Esmolfe (BE) is a traditional Portuguese apple highly appreciated by consumers due to its peculiar flavor and aroma. This apple contains higher concentration of phenolic compounds than other cultivars and is thus considered a rich source of antioxidants. Its sensorial and functional properties have attracted farmers' associations to increase BE production. However, a large quantity of apples is wasted due to storage/transportation procedures that impact BE's quality attributes. In this work, we applied high-pressure extraction methodologies to generate antioxidant-rich fractions from BE residues aiming at adding high value to these agro-food by-products. We performed a first extraction step using supercritical CO2, followed by a second extraction step where different CO2 + ethanol mixtures (10-100% v/v) were tested. All experiments were carried out at 25 MPa and 50 °C. Extracts were characterized in terms of global yield, phenolic content and antioxidant activity using chemical (ORAC, HOSC, HORAC) and cell-based assays (CAA). We demonstrated that, although the pressurized 100% ethanol condition promoted the highest recovery of phenolic compounds (509 ± 8 mg GAE/100 g BE residues), the extract obtained with 40% ethanol presented the highest CAA (1.50 ± 0.24 µmol QE/g dw) and ORAC (285 ± 16 µmol TEAC/g dw), as well as HOSC and HORAC values, which correlated with its content of epicatechin and procyanidin B2. Noteworthy, this fraction inhibited free radical production in human neurospheroids derived from NT2 cells, a robust 3D cell model for neuroprotective testing

Functional metabolic interactions of human neuron-astrocyte 3D in vitro networks

The generation of human neural tissue-like 3D structures holds great promise for disease modeling, drug discovery and regenerative medicine strategies. Promoting the establishment of complex cell-cell interactions, 3D culture systems enable the development of human cell-based models with increased physiological relevance, over monolayer cultures. Here, we demonstrate the establishment of neuronal and astrocytic metabolic signatures and shuttles in a human 3D neural cell model, namely the glutamine-glutamate-GABA shuttle. This was indicated by labeling of neuronal GABA following incubation with the glia-specific substrate [2-13C]acetate, which decreased by methionine sulfoximine-induced inhibition of the glial enzyme glutamine synthetase. Cell metabolic specialization was further demonstrated by higher pyruvate carboxylase-derived labeling in glutamine than in glutamate, indicating its activity in astrocytes and not in neurons. Exposure to the neurotoxin acrylamide resulted in intracellular accumulation of glutamate and decreased GABA synthesis. These results suggest an acrylamide-induced impairment of neuronal synaptic vesicle trafficking and imbalanced glutamine-glutamate-GABA cycle, due to loss of cell-cell contacts at synaptic sites. This work demonstrates, for the first time to our knowledge, that neural differentiation of human cells in a 3D setting recapitulates neuronal-astrocytic metabolic interactions, highlighting the relevance of these models for toxicology and better understanding the crosstalk between human neural cells

Up-regulation of APP endocytosis by neuronal aging drives amyloid dependent-synapse loss

Neuronal aging increases the risk of late-onset Alzheimer's disease. During normal aging, synapses decline, and β-amyloid (Aβ) accumulates intraneuronally. However, little is known about the underlying cell biological mechanisms. We studied normal neuronal aging using normal aged brain and aged mouse primary neurons that accumulate lysosomal lipofuscin and show synapse loss. We identify the up-regulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing and Aβ production in vitro and in vivo. The increased APP endocytosis may contribute to the observed early endosomes enlargement in the aged brain. Mechanistically, we show that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increase with neuronal aging. Finally, Aβ production inhibition reverts synaptic decline in aged neurons while Aβ accumulation, promoted by endocytosis up-regulation in younger neurons, recapitulates aging-related synapse decline. Overall, we identify APP endocytosis up-regulation as a potential mechanism of neuronal aging and, thus, a novel target to prevent late-onset Alzheimer's disease

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators