Donato, Stella (2018) Embryonic stem cells and 3D Minibrains as model systems to study the role of oxidative stress in neurological diseases. [Tesi di dottorato]

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Oxidative stress is a common issue in neurodegenerative diseases such as Parkinson´s disease (PD), Alzheimer´s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington´s disease (HD), and fronto-temporal dementia (FTD). Oxidative stress occurs when the cellular redox balance is impaired leading to excessive production of reactive oxygen and nitrogen species (ROS/RNS). ROS/RNS and oxidative modified molecules accumulate in the cells over the time and contribute to cellular senescence and to the aging process. Since most neurodegenerative diseases are late-onset it is likely that oxidative stress-mediated damage accumulation occurring over the time contribute in the onset of the pathology. However, numerous evidences of oxidative stress-mediated damage have been found in developmental brain diseases, such as Rett Syndrome (RTT), in which there is not aging contribution. Rett Syndrome is an X-linked pathology mainly caused by the loss of function (LoF) mutations in the gene encoding Methyl CpG binding Protein 2 (MeCP2). Despite the studies done so far, the molecular mechanisms that link MeCP2 loss of function to the pathology outcome are still not fully understood. Oxidative stress is looked at as a possible actor involved in the disease. High level of oxidatively modified proteins and lipids and altered expression of genes involved in both oxidative stress defense and mitochondrial activity have been observed in RTT patients. Moreover, MeCP2 reactivation in mouse models not only attenuates neural dysfunction but also restores the redox homeostasis. However, the molecular pathways that link the absence of MeCP2 protein to oxidative stress state in the pathophysiology of this brain disease are still unknown. I investigated the role of oxidative stress in Rett syndrome, taking advantage from a mouse embryonic stem (mES) cell line carrying a deletion of MeCP2 gene (MeCP2-/Y mES). In particular, I analyzed the effect of oxidative stress, induced via treatment with the enzyme Glucose oxidase (GOX) that produces H2O2 in the culture medium, on WT and MeCP2-/Y mES derived neurons. I studied the mitogen-activated protein kinase (MAPK) cellular response pathways and the apoptotic activation in mES derived neurons upon oxidative stress stimulation. I observed that MeCP2-/Y mES derived neurons resided in an active cellular response state and that the response rate increased upon treatment with GOX. Moreover, GOX treated MeCP2-/Y neurons did not die via apoptosis as shown by the lack of activation of caspase 3 protein and the high level of the antiapoptotic Bcl2 protein. Interestingly, MeCP2 protein level increased in GOX treated WT mES derived neurons compared to untreated condition thus giving strength to the possible role of MeCP2 protein in the regulation of cellular response against oxidative stress. So far, studies on Rett Syndrome, as well as other brain diseases, have been carried out using mainly rodent animal models. However, it is important to take into account the differences that exist between humans and rodents. During the very last years, new strategies to model and study human brain diseases have been emerging. Relying on the use of human embryonic or induced pluripotent stem cells, it is possible to generate complex three-dimensional (3D) structures, the so-called cerebral organoids (Minibrain/MB). The use of human specific stem cells allows following the species-specific differentiation patterning and the presence of multiple human brain-like structures allows studying the interactions between several areas of the brain in an in vitro model system that resembles human brain development and architecture. I was fascinated by the possibility to use MBs to analyze the role of oxidative stress in neurological disease. To this aim, I joined the lab directed by Prof. Dr. Peter Heutink at the German Center for Neurodegenerative Diseases (DZNE) in Tübingen where I experienced the methodology to generate cerebral organoids in order to evaluate their possible use to model brain developmental and neurodegenerative diseases. In particular, I sought to establish a 3D model for Parkinson’s disease (PD). PD is the most common neurodegenerative disease with motor symptoms. This complex multifaceted disease has long been believed to be only environmental. However, the recent genome wide association studies (GWAS) on PD patients have identified hundreds of genetic risk factors and many of them are involved in mitochondrial function and/or oxidative stress related. Alpha-synuclein (SNCA) gene is the most common genetic risk factor associated with both familiar and sporadic forms of PD. The alpha-synuclein (-syn) protein function is still unclear, but it is known that mutations of -syn proteins lead to aggregates formation in dopaminergic neurons (Lewy bodies and Lewy neurites) that cause toxicity in the cells. The molecular mechanisms that lead to -syn aggregation and toxicity are not fully defined. I aimed to establish 3D Minibrains using lentiviral-transduced induced pluripotent stem (iPS) cells overexpressing SNCA in order to study the molecular mechanisms leading to -syn aggregation and toxicity. I reproduced a previously established 3D MB differentiation protocol and characterized MBs growth and differentiation up to three months in culture. 3D MBs acquired a good degree of neural maturation starting from two months in culture and retained the human cell identity specification. Indeed, differentiated neurons showed a spatial patterning similar to that observed in the in vivo developing human brain. However, the MB generation was highly variable between and within the experiments and this highlighted the necessity to make MB generation more reproducible. I tested the effect of SMAD inhibitors, to modulate the TGF-beta and BMP signaling pathways responsible for cell differentiation. SMAD inhibitors are known to repress cell differentiation towards mesoderm and endoderm lineages and they have already been successfully used in two-dimensional (2D) iPS neural differentiation protocols. The SMAD inhibitors treatment reduced significantly the expression of early mesodermal (BRACHYURY) and endodermal (GATA4) genes and increased the expression of neural precursor genes (OTX1 and PAX6) in one-month old MBs. Nevertheless, this treatment did not significantly improve the mature neuron genes expression at any of the time points analyzed. Interestingly, mature neuron genes were less expressed in MBs overexpressing SNCA compared to WT, suggesting an impairment of differentiation in SNCA overexpressing MBs that might occur in the late stages of differentiation. Moreover, I observed that three-months old SNCA overexpressing MBs exhibited higher level of heme oxygenase 1 (HMOX1) mRNA compared to age-matched WT MBs. In line with previous studies, these results suggested the enhanced activation of nuclear factor E2-related factor 2 (Nrf2) signaling pathway in response to SNCA overexpression. Therefore, these results showed that MBs are an interesting tool to study some aspects of neurodegenerative diseases, such as the link between -syn protein aggregation and oxidative stress response in late onset Parkinson´s disease. The generation of 3D organoid cultures opened the way to a new human-specific model system for brain research that compensates the limitations derived from both animal models and 2D in vitro systems. In this perspective, MBs might be incredibly useful to model Rett Syndrome and to test possible therapeutic strategies in a complex human-specific model system.

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