Dissertationen zum Thema „Neurodegenerative disease modeling“

IPSC are potent tools in the creation of disease models for both basic studies on the disease and testing of potential therapeutical drugs. In this context, it has been developed the derivation of iPSC from patient fibroblasts suffering from Gaucher Disease (GD) or Tay Sachs disease (TS). GD is a recessive autosomic disease which is characterized by the deficiency of the enzyme called glucocerebrosidase (GBA), which leads to the accumulation of its substrate glucosylceramide in macrophages and neurons. This disease has 3 forms of clinical presentation: type I, which is systemic; type II, the most severe with a neuronopathic acute presentation; type III, which is a combination of the former two, but without the severity of the type II. Tay Sachs is an autosomic recessive disease which is characterized by the Hexosaminidase A (HexA) deficiency, which leads to GM2 accumulation on the lysosomes of neurons. Patients present neurodegeneration and severe impairment of the brain which ultimately leads to their death. In this project iPSC derived from GD and TS patient fibroblasts. Pluripotent state of the derived iPSC has been characterized. Later, iPSC have been differentiated to neurons in order to confirm the disease phenotype on the in vitro differentiated tissue. In GD the phenotype was corroborated by enzymatic assays and GBA detection by Western blot. A lower GBA activity on GD neurons compared to WT was found, consistent with the minor GBA levels in GD neurons detected in the western blot. In TS, derived neurons were analyzed by immunofluorescence for Lamp2 (lysosome marker), observing an increase in size and number on the TS neurons in contrast to WT. Also, TS neurons were analyzed by transmission electron microscopy, presenting membranous lamellar bodies in the cytosol of TS. Both iPSC diseases have been used as a platform for testing therapeutical drugs efficiency on the iPSC derived neurons.
Las iPS (células pluripotentes inducidas) se han revelado como potentes herramientas en la creación de modelos de enfermedades humanas para su estudio y el testeo de potenciales drogas. En este marco hemos desarrollado un proyecto para derivar iPS de fibroblastos de pacientes de Gaucher y Tay Sachs, ambas enfermedades monogénicas recesivas. La enfermedad de Gaucher se caracteriza por la deficiencia de la glucocerebrosidasa (GBA), lo que conlleva la acumulación de su substrato, la glucosilceramida, en macrófagos y neuronas. Esta enfermedad tiene tres presentaciones I, que es sistémica; II, que es una forma neuronopática aguda, tiene efectos fatales ya que los pacientes rara vez sobreviven a los dos años de edad; y III, que es una mezcla de las dos anteriores, siendo neuronopática crónica, sin llegar a la severidad del tipo II. Tay Sachs es una enfermedad que se caracteriza por la deficiencia de la Hexosaminidasa A (HexA) lo que conlleva el almacenamiento en el lisosoma del gangliósido GM2. Los pacientes de esta enfermedad presentan daños neurológicos, provocando la muerte en la mayoría de los casos. En este proyecto se han desarrollado las iPS derivadas de fibroblastos de un paciente de Gaucher tipo II, y de otro de Tay Sachs. Las iPS resultantes de ambas enfermedades han sido caracterizadas para constatar su estado pluripotente y diferenciadas a neuronas para comprobar que presentan el fenotipo característico de las enfermedades. En el caso de Gaucher, mediante ensayos enzimáticos y detección de la GBA1 por western blot, detectando una menor actividad en las neuronas gaucher que en las WT, lo que es consecuente con la menor cantidad de GBA1 detectada. En el caso de Tay Sachs, las neuronas se han analizado mediante inmunohistoquímica, marcando Lamp2, típico de lisosomas y se ha observado un aumento de tamaño y cantidad respecto de las células WT diferenciadas en paralelo. También han sido analizadas por microscopía electrónica, presentando una acumulación de cuerpos laminares en los lisosomas y aumento de número y tamaño de éstos. Ambas enfermedades han sido utilizadas como modelos para probar compuestos en las neuronas derivadas de las iPS derivadas de fibroblastos del paciente y comprobar su eficacia.

Mitochondria plays a crucial role in cells by maintaining energy metabolism and directing cell death mechanisms by buffering calcium (Ca2+ )from cytosol. Therefore, the Ca2+ overload of mitochondria due to the upregulated cytosolic Ca2+ , observed in many neurological disorders is hypothesized to be a key pathway leading to mitochondrial dysfunction and cell death. In particular, Ca2+ homeostasis disruptions due to Alzheimer’ s disease (AD)-causing presenilins (PS1/PS2) and oligomeric forms of β-amyloid peptides Aβ commonly found in AD patients are presumed to cause detrimental effects on the mitochondria and its ability to function properly. We begin by showing that Familial Alzheimer’s disease (FAD)-causing PS mutants affect intracellular Ca2+ ([Ca2+]i) homeostasis by enhancing the gating of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channels on the endoplasmic reticulum (ER), leading to exaggerated Ca2+ release into the cytoplasm. Using experimental IP3R-mediated Ca2+ release data in conjunction with a computational model of mitochondrial bioenergetics, we explore how the differences in mitochondrial Ca2+ uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca2+ ([Ca2+ ]m). We find that as a result of exaggerated [Ca2+]i in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca2+ dependent enzymes that stimulate NADH production. This leads to decreased rates of proton pumping due to diminished membrane potential (Ψm) along with less ATP and enhanced ROS production. These results show that through Ca2+ signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially cell death. Next, the model for the mitochondria is expanded to include the mitochondrial uniporter (MCU) that senses Ca2+ in the microdomain formed by the close proximity of mitochondria and ER. Ca2+ concentration in the microdomain ([Ca2+] mic) depends on the distance between the cluster of IP3R channels (r) on ER and mitochondria, the number of IP3R in the cluster (nIP3R), and open-probability (Po) of IP3R. Using the same experimental results for Ca2+ release though IP3R due to FAD-causing PS mutants, in conjunction with a computational model of mitochondrial bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore the difference in mitochondrial Ca2+ uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Ca2+]m due to the gain-of-function enhancement of IP3R channels in the cell expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to nIP3R. Furthermore, we observe diminished inner mitochondrial Ψm, [NADH], [Ca2+]m, and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na+/ Ca2+ -exchanger affect the latency of PTP to open in PTPh. Intracellular accumulation of oligomeric forms of Aβ are now believed to play a key role in the early phase of AD as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular Aβ oligomers. To study the effect of intracellular Aβ on Ca2+ signaling and the resulting mitochondrial dysfunction, we employed data-driven modeling in conjunction with total internal reflection fluorescence (TIRF) microscopy (TIRFM). High resolution fluorescence TIRFM together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the IP3R. Achieving this requires a close agreement between Ca2+ signals from computational models and TIRFM experiments. However, we found that elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel opening and closing during x and between puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10 ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature. Once a close agreement between experiment and model is achieved, we use multiscale modeling in conjunction with patch-clamp electrophysiology of IP3R and fluorescence imaging of whole-cell Ca2+ response, induced by intracellular Aβ42 oligomers to show that Aβ42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the ER by intracellular Aβ42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aβ42 content together with the whole-cell model allows us to show that Aβ42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and [H2O2]. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of Aβ42 cytotoxicity by EGTA in our live cells experiments. Finally, our modeling study was extended to other pathological phenomena such as epileptic seizures and spreading depolarizations (SD) and their effects on mitochondria by incorporating conservation of particles and charge, and accounting for the energy required to restore ionic gradients to the neuron. By examining the dynamics as a function of potassium and oxygen we can account for a wide range of neuronal hyperactivity from seizures, normoxic SD, and hypoxic SD (HSD) in the model. Together with a detailed model of mitochondria xi and Ca2+ -release through the ER, we determine mitochondrial dysfunction and potential recovery mechanisms from HSD. Our results demonstrate that HSD causes detrimental mitochondrial dysfunction that can only be recovered by restoration of oxygen. Once oxygen is replenished to the neuron, organic phosphate and pH gradients along the mitochondria determine how rapid the neuron recovers from HSD.

Neurodegenerative disease is an umbrella term for pathologies that primarily damage neurons. As their incidence increases with age it is becoming of a greater concern for the west, due to its aging population. Due to their chronic nature and the difficulty to create reliable and reproducible animal models of these diseases their pathophysiologies are still poorly understood. For all these reasons, a mathematical modelling approach is suggested. The methodology of the work here consisted of identifying the state of the art models that describe the healthy behaviour of cells (e.g. metabolism and ionic regulation) and adapting them for pathological environments. With these models hypotheses provided by clinicians and pathologists were tested. The work focuses on developing models of mechanisms common to neurodegenerative diseases, which include: glutamate excitotoxicity, aquaporin water kinetics, inflammatory complement lysis and acute inflammation. Glutamate excitotoxicity was modelled by creating a compartmental model of glutamate exchange between neurons and astrocytes. This model was the first model of glutamate kinetics validated in an ischaemic stroke context. The aquaporin water kinetics and complement lysis models were developed in the context of the autoimmune disease Neuromyelitis Optica. Through this project a hypothesised trigger for the pathology was confirmed. Additionally, the first model of astrocytic cytotoxic oedema due to complement lysis was developed. Finally, a preventative drug for complement lysis was simulated. Acute inflammation was explored in the context of understanding the potential of chemerin as a pro-resolving cytokine. To that effect, a model of acute inflammation was developed where pro-resolving mechanisms were included. This model was the first to attempt model the effects of an intervention in inflammation. The results indicated that there is a maximum inhibitory effect of chemerin on inflammation. Additionally, two preventive avenues for chronic inflammation were found. With this work, the first attempts of capturing relevant mechanisms of neurodegenerative diseases were presented. These models can now be further developed and adapted to other pathological environments.

The neurodegenerative disorders Alzheimer??s Disease (AD) and Parkinson??s Disease (PD) are characterised by the accumulation of misfolded amyloid beta 1-42 peptide (Aβ1-42) or α-synuclein, respectively. In both cases, there is extensive evidence to support a central role for these aggregation-prone molecules in the progression of disease pathology. However, the precise mechanisms through which Aβ1-42 and α-synuclein contribute to neurodegeneration remain unclear. Organismal, cellular and in vitro models are under development to allow elucidation of these mechanisms. A cellular system for the study of intracellular Aβ1-42 misfolding and localisation was developed, based on expression of an Aβ1-42-GFP fusion protein in the model eukaryote Saccharomyces cerevisiae. This system relies on the known inverse relationship between GFP fluorescence, and the propensity to misfold of an N-terminal fusion domain. To discover cellular processes that may affect the misfolding and localisation of intracellular Aβ1-42, the Aβ1-42-GFP reporter was transformed into the S. cerevisiae genome deletion mutant collection and screened for fluorescence. 94 deletion mutants exhibited increased Aβ1-42-GFP fluorescence, indicative of altered Aβ1-42 misfolding. These mutants were involved in a number of cellular processes with suspected relationships to AD, including the tricarboxylic acid cycle, chromatin remodelling and phospholipid metabolism. Detailed examination of mutants involved in phosphatidylcholine synthesis revealed the potential for phospholipid composition to influence the intracellular aggregation and localisation of Aβ1-42. In addition, an existing S. cerevisiae model of α-synuclein pathobiology was extended to study the effects of compounds that have been hypothesized to be environmental risk factors leading to increased risk of developing PD. Exposure of cells to aluminium, dieldrin and compounds generating reactive oxygen species enhanced the toxicity of α- synuclein expression, supporting suggested roles for these agents in the onset and development of PD. Expression of α-synuclein-GFP in phosphatidylcholine synthesis mutants identified in the Aβ1-42-GFP fluorescence screen resulted in dramatic alteration of α-synuclein localisation, indicating a common involvement of phospholipid metabolism and composition in modulating the behaviours of these two aggregation-prone proteins.

The aim of my thesis was to develop and characterise PrP transgenic Drosophila melanogaster of various genotypes to study the process of prion-induced neurodegeneration in this model. Prion diseases are caused by the occurrence of an abnormally-folded form of PrP (PrPSc) protein that arises either from the environment as an acquired disease, from mutation in the PrP-coding gene as a genetic disease or sporadically from causes unknown. The PrPSc then recruits PrPC, the normal form of PrP, that is ubiquitously present in the mammalian CNS and triggers neurotoxicity and neurodegeneration that is transmissible between individuals of the same or even different species. All prion diseases are currently incurable, fatal and the mechanism of prion-induced neurodegeneration remains to be discovered. In this thesis, Drosophila transgenic for ovine (chromosome 3 and dual PrP transgenic flies), hamster, humanised murine, human and cervid PrP were characterised for expression and biochemical properties. The ultimate goal of my thesis was investigation of cell-to-cell spread of misfolded PrP in Drosophila CNS. To achieve this, a mutant form of PrP that is thought to misfold was co-expressed with the normal form PrPC that served as a substrate in the same dual PrP-transgenic fly. The process was modelled using hamster, humanised murine or ovine PrP transgenes that carry human mutations associated with the spontaneous onset of transmissible neurodegeneration in the natural host. Various approaches towards independent spatial expression of PrP in Drosophila were exploited here in both single and dual PrP expressing flies. Moreover, the ability to initiate misfolding and the impact of this on the fly phenotype was investigated. Both apparent misfolding and phenotypic changes were seen in different fly models suggesting the models were successful. To this extent, PrP transgenic Drosophila were developed to allow for relatively rapid modelling of mammalian prion disease in this invertebrate organism.

Huntington's disease (HD) is a progressive neurodegeneration which has symptoms such as movement dysfunction, cognitive abnormalities, and psychiatric disturbances. Neurodegeneration in HD is characterised by pathology that spreads throughout the cortico-striatal network. There is growing recognition that the transfer of mutant huntingtin (mHTT) protein is cellular and shaped by structural organisation of the brain, provides a general framework underlying progressive spread of degeneration in HD. However, relatively little is known regarding how such progression occurs over time. This knowledge is critical to inform future drug discovery efforts where neuroimaging methodology can be used to develop the potential therapeutic compounds in targeting vulnerable neural circuits in HD. This research aimed to develop and apply a novel graph theoretical network diffusion model to predict how and where in the brain neurodegenerative process is seeded in HD. We used longitudinal MRI scans (N=106 Premanifest HD (pre-HD) and N=89 Healthy Controls), collected 12 and 24 months from the Track-On HD study. We implemented Network diffusion model (NDM), which was previously applied to symptomatic individuals with HD, in the Premanifest HD population. To evaluate the spatial patterns in change in brain volume of HD brain regions compared to controls, we segmented the T1-weighted structural MRI scans into 82 brain regions using FreeSurfer based tools. A linear mixed-effects model is statistically used to evaluate the brain volume differences (across 82 brain regions) in different time visits. The outcome of this statistical approach revealed that degeneration over time in pre-HD compared to controls shows extensively higher (p<0.05). The putamen and caudate showed the highest atrophy sub-cortically. Cortically, the highest atrophy is observed in the visual cortex (lateral occipital) and temporal cortex (superior and middle temporal). Next, we implemented NDM to simulate the longitudinal pattern of degeneration across the 82 brain regions. The NDM assumes that the linear diffusion of pathological proteins facilitates pathology spread in the HD brain via the brain's structural connectome. Therefore, we used a canonical connectome from the healthy brain (82 x 82 IIT connectome) to simulate the brain's spread process. We found that initiating the diffusion process from pallidum, thalamus, and putamen predicts a pattern of degenerations (predicted atrophy) which is significantly correlated (p<0.05, corrected) with longitudinal degeneration measured using MRI scans. However, the predictive ability of NDM was moderate at best (maximum Pearson correlation between predicted and measured atrophy = 0.48, p <0.0001). These findings suggest that NDM can only moderately predict the pattern of degeneration in the HD brain. Overall, this thesis explores how NDM can better understand the progression of degeneration in HD. Our comparative study between healthy and patient’s groups statistically showed a progressive pattern of atrophy in the cerebrum. Furthermore, the results from NDM suggests that NDM could be helpful to model the progression of atrophy in subcortical and cortical regions in HD. Thus, NDM may build bio-physically informed machine learning algorithms for predicting future degeneration from baseline MRI scans.

Le langage est une caractéristique déterminante de l'être humain et depuis des siècles les chercheurs s'intéressent à l'organisation fonctionnelle du langage et aux substrats neuronaux sous-tendent son fonctionnement normal. Le dysfonctionnement des mécanismes permettant un bon fonctionnement langagier est présent dans différentes maladies neurodégénératives, devenues ainsi un modèle majeur pour explorer les capacités langagières. En l'absence de traitement efficace pour les troubles du langage dans différentes maladies neurodégénératives, les techniques non invasives de stimulation cérébrale gagnent du terrain. Parmi ces techniques, la stimulation transcrânienne à courant continu (STCC) module l'activité neuronale via l'induction de faibles courants électriques dans le cerveau, et des effets bénéfiques ont été démontrés chez des patients aphasiques victimes d'accidents vasculaires cérébraux et des patients neurodégénératifs. Les études incluses dans cette thèse ont utilisé des modèles de lésions neurodégénératives pour étudier les mécanismes comportementaux de l'accès et du traitement des mots, pour étudier leur impact sur les capacités langagières et pour explorer la possibilité de moduler le langage à travers la STCC afin de définir sa valeur en tant qu'outil thérapeutique. Le manuscrit est divisé en 4 chapitres s'articulant autour de trois axes principaux : (1) recherche fondamentale sur le langage, (2) recherche clinique sur le dysfonctionnement du langage et approches thérapeutiques et (3) impact de facteurs individuels sur la variabilité de la réponse à ces thérapies, ainsi qu’un chapitre d’Introduction et un chapitre de Discussion Générale
Language is one of the most defining features of human beings and for centuries researchers have been interested on the functional organization of language and which neural substrates subtend its normal functioning. A breakdown of mechanisms subtending normal language abilities characterizes different neurodegenerative conditions, which have become models to study the neural basis and mechanisms of language processing. In the absence of effective treatments for language deficits in different neurodegenerative diseases, non-invasive brain stimulation approaches have been gaining momentum. Transcranial Direct Current Stimulation (tDCS) modulates neural activity via the induction of weak electrical intracranial currents, showing benefits in post-stroke and neurodegenerative aphasic patients. In this context, the studies included in this thesis analyzed neurodegenerative lesion models to characterize the behavioral mechanisms of word access and processing, address their impact on language abilities and explore the modulation of language impairment by means of tDCS to define its therapeutic value. The manuscript is divided in 4 chapters organized along three main axes: (1) fundamental research on language (2) clinical research on language breakdown and therapies and (3) impact of individual factors on the variability of the response to such therapies, an Introduction chapter and a General Discussion chapter

Alzheimer's disease (AD), as one of the most common neurodegenerative diseases, is characterized by the loss of neuronal dysfunction and death resulting in progressive cognitive impairment. The main histopathological hallmark of AD is the accumulation and deposition of misfolded Aβ peptide as amyloid plaques, however the precise role of Aβ toxicity in the disease pathogenesis is still unclear. Moreover, at early stages of the disease the important clinical features of the disorder, in addition to memory loss, are the disruptions of circadian rhythms and spatial disorientation. In the present work I first studied the role of Aβ toxicity by comparing the findings of genome-wide association studies in sporadic AD with the results of an RNAi screen in a transgenic C. elegans model of Aβ toxicity. The initial finding was that none of the human orthologues of these worm genes are associated with risk for sporadic Alzheimer’s disease, indicating that Aβ toxicity in the worm model may not be equivalent to sporadic AD. Nevertheless, comparing the first degree physical interactors (+1 interactome) of the GWAS and worm screen-derived gene products have uncovered 4 worm genes that have a +1 interactome overlap with the GWAS genes that is larger than one would expect by chance. Three of these genes form a chaperonin complex and the fourth gene codes for actin, a major substrate of the same chaperonin. Next I have evaluated the circadian disruptions in AD by developing a new system to simultaneously monitor the oscillations of the peripheral molecular clock and behavioural rhythms in single Drosophila. Experiments were undertaken on wild- type and Aβ-expressing flies. The results indicate the robustness of the peripheral clock is not correlated with the robustness of the circadian sleep and locomotor behaviours, indicating that the molecular clock does not directly drive behaviour. This is despite period length correlations that indicate that the underlying molecular mechanisms that generate both molecular and behavioural rhythms are the same. Rhythmicity in Aβ-expressing flies is worse than in controls. I further investigated the mechanism of spatial orientation in Drosophila. It was established that in the absence of visual stimuli the flies use self-motion cues to orientate themselves within the tubes and that in a Drosophila model of Aβ toxicity this control function is disrupted.

Alzheimer's disease is the leading cause of senile dementia in the elderly, and as life expectancy increases across the globe, incidence of the disease is continually increasing. Current estimates place the number of cases at 25-30 million worldwide, with more than 5.4 million of these occurring in the United States. While the exact cause of the disease remains a mystery, it has become clear that the amyloid β-peptide (Aβ) is central to disease pathogenesis. The aggregation and deposition of this peptide in the brain is known to give rise to the hallmark lesions associated with Alzheimer's disease, but its exact mechanism of toxicity remains largely uncharacterized. Molecular dynamics (MD) simulations have achieved great success in exploring molecular events with atomic resolution, predicting and explaining phenomena that are otherwise obscured from even the most sensitive experimental techniques. Due to the difficulty of obtaining high-quality structural data of Aβ and its toxic assemblies, MD simulations can be an especially useful tool in understanding the progression of Alzheimer's disease on a molecular level. The work contained herein describes the interactions of Aβ monomers and oligomers with lipid bilayers to understand the mechanism by which Aβ exerts its toxicity. Also explored is the mechanism by which flavonoid antioxidants may prevent Aβ self-association and destabilize toxic aggregates, providing insight into the chemical features that give rise to this therapeutic effect.
Ph. D.

Parkinson’s disease (PD) is an incurable, chronically progressive neurodegenerative disease leading to premature invalidity and death. The locomotor disability of PD patients is mainly rooted in the gradual and insidious degeneration of dopaminergic neurons (DA) projecting from the midbrain substantia nigra (SN) to the basal ganglia striatum, a pathological process highlighted microscopically by the formation of insoluble cytosolic protein aggregates, known as Lewy bodies and Lewy neurites. The pathogenic mechanisms leading to PD remain poorly understood, arguably owing to the lack of suitable animal and cellular experimental models of the disease. Therefore, there is an urgent need for developing reliable experimental models that recapitulate the key features of PD. The recent development of induced pluripotent stem cell (iPSC) technology has enabled the generation of patient-specific iPSC and their use to model human diseases, although it is currently unclear whether this approach could be useful to successfully model age-related conditions. Importantly, disease modeling using iPSC largely relies on the existence of efficient protocols for the differentiation of disease-relevant cell types. Here, we first developed an efficient protocol for the differentiation of iPSC to authentic midbrain-specific DA neurons with SN properties by forced expression of LMX1A using a lentivirus-mediated gene delivery system. Next, we generated an iPSC-based cellular model of PD that recapitulates key phenotypic features of PD, such as DA neuron loss and α-synuclein accumulation in DA neurons from PD patients. Overall, our results demonstrate that we have developed a valuable tool for elucidating the pathogenic mechanisms leading to PD, as well as an experimental platform for screening new drugs that may prevent or rescue neurodegeneration in PD.
La malaltia de Parkinson (MP) és una malaltia neurodegenerativa incurable que causa invalidesa i mort prematura. Els pacients de la malaltia de Parkinson presenten alteracions motores degudes a una degeneració gradual de les neurones dopaminèrgiques que projecten des de la substància nigra fins a l’estriat. A nivell microscòpic s’observa la presència d’agregats proteics insolubles en el citosol de les neurones coneguts com cossos o neurites de Lewy. Els mecanismes patològics responsables de la MP no es coneixen bé, possiblement a causa de la manca de models animals i cel•lulars adequats. Per tant, existeix una gran necessitat de desenvolupar models experimentals fiables que recapitulin les característiques bàsiques de la MP. El recent desenvolupament de les cèl•lules mare pluripotents induïdes (iPSC) ha permès la generació de iPSC específiques de pacient i el seu ús per modelar malalties humanes, ara bé, no és clar si aquesta estratègia es pot utilitzar per modelar exitosament malalties d’origen tardà, com ara la MP. És important destacar que el modelatge de malalties utilitzant iPSC, es basa, en gran mesura en l'existència de protocols eficients per a la diferenciació de les iPSC cap al tipus cel•lular rellevant per a la malaltia. Durant aquest període, per primera vegada, s’ha desenvolupat un protocol per a l’eficient diferenciació de les iPSC cap a neurones dopaminèrgiques amb les propietats característiques de neurones dopaminèrgiques nigrostriatals, mitjançant l’expressió forçada de LMX1A utilitzant vectors lentivirals. A continuació, s’ha generat un model cel•lular usant iPSC derivades de pacients de MP que recapitula les principals característiques fenotípiques de la malaltia, com ara la pèrdua de neurones dopaminèrgiques i l'acumulació de α-sinucleïna en les neurones dopaminèrgiques. En general, els nostres resultats demostren que hem desenvolupat una eina valuosa per a l’estudi dels mecanismes patològics que condueixen a la MP, així com una nova plataforma pel descobriment de nous fàrmacs encaminats a prevenir o evitar la neurodegeneració.

Dans cette thèse, nous avons développé des outils pour simuler des imageslongitudinales réalistes de cerveau présentant de l’atrophie ou de lacroissance. Cette méthode a été spécifiquement élaborée pour simuler leseffets de la maladie d’Alzheimer sur le cerveau. Elle se fonde sur un modèle dedéformation du cerveau qui décrit les effets biomécaniques d’une perte detissue due à une carte d’atrophie prescrite. Nous avons élaboré une méthodepour interpoler et extrapoler les images longitudinales d’un patient en simulantdes images avec une carte d’atrophie spécifique au sujet. Cette méthode a étéutilisée pour interpoler des acquisitions temporelles d’Images par RésonnanceMagnétique (IRM) de 46 patients souffrant de la maladie d’Alzheimer. Pour cefaire, des cartes d’atrophie sont estimées pour chaque patient, d’après deuxacquisitions IRM temporelles distinctes. Les IRM cliniques présentent du bruitet des artefacts. De plus, les acquisitions longitudinales présentent desvariations d’intensité d’une image à l’autre. Nous avons donc élaboré uneméthode qui combine le modèle de déformation du cerveau, ainsi que lesdifférentes images cliniques disponibles d’un patient donné, afin de simuler lesvariations d’intensité des acquisitions longitudinale. Pour finir, les outils desimulation d’images réalistes développés au cours de cette thèse sont mis àdisposition en open-source
This thesis develops a framework to simulate realistic longitudinal brainimages with atrophy (and potentially growth), particularly in the case ofAlzheimer's Disease (AD). The core component of the framework is a braindeformation model: a carefully designed biomechanics-based tissue loss modelto simulate the deformations having the prescribed atrophy. The thesispresents a method to interpolate or extrapolate longitudinal images of asubject by simulating images with subject-specific atrophy patterns. Themethod was used to simulate interpolated time-point Magnetic ResonanceImages (MRIs) of 46 AD patients by prescribing atrophy estimated for eachpatient from the available two time-point MRIs. Real MRIs have noise andimage acquisition artefacts, and real longitudinal images have variation ofintensity characteristics among the individual images. In this thesis, wepresent a method that uses our brain deformation model and different availableimages of a subject to add realistic variations of intensities in the syntheticlongitudinal images. Finally, the software developed during the thesis tosimulate realistic longitudinal brain images with our brain deformation modelis released open-source

Neurodegeneration with Brain Iron Accumulation (NBIA) disorders, such as Phospholipase A2G6-Associated Neurodegeneration (PLAN) and Pantothenate Kinase-Associated Neurodegeneration (PKAN), are a group of rare early-onset, genetic disorders characterized by neurodegeneration and iron accumulation inside of the basal ganglia (BG), which is accompanied by progressive motor symptoms. In order to address the limitations in available models of NBIA, a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN was characterized. This model demonstrated key hallmarks of the disease presentation in NBIA, including a severe and early-onset motor deficit, neurodegeneration inside of the substantia nigra (SN) including a loss of dopaminergic function and the formation of abnormal spheroid inclusions as well as iron accumulation. The capture of these hallmarks of NBIA makes this an ideal animal research model for NBIA. Additionally, exploration of candidate systemic biomarkers of NBIA was performed in a case study of a patient with PLAN and in a cohort of 30 patients with PKAN. These investigations demonstrated reductions in transfer and slight, but not significant elevations in soluble transferrin receptor. No significant difference was seen in serum iron parameters. A systemic disease burden including chronic oxidative stress; elevated malondialdehyde, and inflammation; elevated C-reactive protein (CRP), IL-6 and TNFα was noted in both investigations. A number of candidate protein biomarkers including: fibrinogen, transthyretin, zinc alpha-2 glycoprotein and retinol binding protein were also identified. These markers correlated with measures of the severity of iron loading in the globus pallidus (GP); based on R2* magnetic resonance imaging (MRI) and the severity of motor symptoms (Barry-Albright Dystonia Rating Scale) making them potential candidates markers of dysfunction in NBIA. In the patient with PLAN, 37 weeks of therapy with the iron chelator deferiprone (DFP) as well as 20 months of therapy with the antioxidants alpha lipoic acid (ALA) and n-acetylcysteine (NAC) were efficacious in reducing the systemic oxidative and inflammatory disease burden, but it did not significantly alter the progression of the disease. In the antioxidant therapy, this efficacy was primarily due to ALA. When the cohort of patients with PKAN were treated with DFP for 18 months it was highly efficacious in lowering brain iron accumulation in the GP. No significant reduction in the speed of disease progression was seen in DFP treated patients compared to placebo based on initial analysis. Similar to the PLAN patient, DFP also mitigated the systemic disease burden in PKAN patients. In both cases DFP was well tolerated and had minimal impact on serum iron levels, TIBC and transferrin saturation. Collectively these investigations provide valuable insights into disease progression in NBIA. They also provide tools to aid further investigations in NBIA. These are provided in the form of a well-characterized B6.C3-Pla2g6m1J/CxRwb model of PLAN, which robustly captures the disease presentation seen in patients, as well as a panel of systemic blood-based markers of disease burden in NBIA and candidate markers of dysfunction in NBIA. These markers were used to assess two novel therapies in NBIA chelation with DFP and antioxidant therapy with ALA and NAC.
Graduate
2019-04-19

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
Nos dias que correm, as doenças neurodegenerativas afetam milhões de pessoas em todo o mundo, havendo mais de 600 doenças diferentes que incidem sobre o sistema nervoso, sendo as doenças de Alzheimer e Parkinson as mais comuns. Estudos indicam que 60-70% das pessoas que sofrem de distúrbios cerebrais, são casos de Alzheimer. Se olharmos para os Estados Unidos como exemplo, mais de 5 milhões de pessoas sofrem de Alzheimer e pelo menos 500 mil pessoas sofrem de Parkinson. As doenças neurodegenerativas são doenças caracterizadas pela deterioração progressiva do sistema nervoso, que ocorre quando as suas células, os neurónios, começam a degenerar e a morrer. Isso é preocupante pois os neurónios são a base do funcionamento do sistema nervoso e não se reproduzem, não podendo ser substituídos, o que faz com que as doenças neurodegenerativas sejam, normalmente, incuráveis e debilitantes. A doença de Alzheimer, descrita pela primeira vez por Alois Alzheimer, é um tipo de demência que afecta o cérebro, provocando problemas ao nível da memória, pensamento e comportamento. Os seus sintomas desenvolvem-se lentamente, em geral, com pioras progressivas que acabam por interferir com as tarefas do dia-a-dia. Esta doença afecta cerca de 44 milhões de pessoas a nível mundial, sendo que este número é esperado aumentar até três vezes mais nos próximos 50 anos. É uma doença geralmente associada à idade, embora haja casos de incidência antecipada, com pacientes nos seus 40s ou 50s anos de idade. Embora as causas para a doença de Alzheimer não estejam ainda completamente compreendidas, os cientistas acreditam que, para a maioria das pessoas, esta doença resulta duma combinação de factores genéticos, ambientais e de estilo de vida, que vão afectando o cérebro ao longo do tempo. Por ser a doença neurodegenerativa mais comum no Mundo, a doença de Alzheimer acaba também por ser a doença com maior impacto financeiro nos países desenvolvidos, representando também um enorme peso para os pacientes e para a sua família, tanto a nível económico, como a nível mental e psicológico, visto que esta devastadora doença, apesar de ter medicação disponível para eventualmente reduzir o efeito dos sintomas, não tem ainda cura. Com estes dados em mente, podemos verificar a importância de se começar a direcionar a investigação no sentido de melhorar o diagnóstico e terapia clínica destas doenças neurodegenerativas. Uma das maneiras mais eficazes de alcançar este objetivo é tentar centralizar todo o conhecimento e informação sobre essas doenças e reformar sistemas de classificação de doenças previamente estabelecidos, tais como a CID (Classificação Internacional de Doenças). A doença de Alzheimer é actualmente classificada pelos seus sintomas e severidade, embora seja claro hoje em dia que este sistema não representa as diferentes causas possíveis para a doença, pois os mesmo sintomas podem ser gerados por diferentes causas genéticas ou moleculares. Existe então um reconhecimento geral de que os sistema de classificação tem de mudar, por exemplo para um que utilize uma taxonomia baseada em mecanismos (mechanism-based taxonomy), a qual se baseia no conhecimento sobre as vias biológicas envolvidas na etiologia de uma doença, de modo a guiar a classificação das classes e subclasses da doença. É portanto neste contexto que surge o projeto AETIONOMY, cujo principal objetivo incide no desenvolvimento de uma taxonomia baseada em mecanismos para as doenças de Alzheimer e Parkinson, sendo que o seu maior desafio reside no facto de que, para a maioria das doenças neurodegenerativas, as vias biológicas disfuncionais subjacentes à doença não são conhecidas. O AETIONOMY pretende identificar e recolher todos os dados disponíveis mundialmente, incluíndo dados clínicos, de imagem ou genéticos, sobre a doença de Alzheimer, com o fim de desenvolver uma estrutura comum que combine todos os dados obtidos, de maneira a ser possível identificar padrões que tornem possível a divisão da doença em sub-grupos de pacientes com causas semelhantes da sua doença, na expectativa de identificar mudanças patofisiológicas durante a doença a nível molecular, que possam conduzir a uma nova taxonomia da doença. O presente estudo incidiu sobre a doença de Alzheimer, mais especificamente sobre os dados clínicos da ADNI (Alzheimer’s Disease Neuroimaging Initiative), e como processar e tratar estes dados para que seja possível integrá-los numa plataforma de biomedicina translacional chamada tranSMART, cujo intuito é a criação de novas rotas para a identificação dos mecanismos subjacentes à doença, antes de os organizar e propor uma taxonomia racional da mesma. Este estudo teve como principal objetivo obter os dados da ADNI, analisá-los e estudar toda a envolvência desta iniciativa, de maneira a conseguir uma melhor perceção do panorama geral da mesma, fazendo de seguida o processamento, modelação e limpeza destes dados clínicos para integração no tranSMART. Ao mesmo tempo procurou-se aprofundar o conhecimento sobre a doença de Alzheimer, inclusive os seus sintomas, possíveis causas e diagnóstico. Recorreu-se neste trabalho a diferentes pacotes de software e linguagens de programação, tais como o OpenRefine e o Python. Este estudo propõe então uma revisão completa dos conceitos e dados estruturais da ADNI, ao mesmo tempo que obtém e armazena localmente todos os dados, valores e variáveis utilizados no âmbito desse mesmo projecto. Foi também criado um software para a transformação dos dados da ADNI, com o objectivo de os integrar numa plataforma de biomedicina translacional. No entanto, primeiro é indispensável compreender os princípios de tratamento de dados para o carregamento de dados no tranSMART para poder ser possível a integração dos dados da ADNI, fazendo com que estejam disponíveis para outros projectos a nível mundial, tal como o AETIONOMY e o eTRIKS, por exemplo. Com este estudo tornou-se possível adicionar os dados da ADNI à base de dados do tranSMART, de forma a torná-los disponíveis para terceiros e outros projetos, ajudando então a tão desejada centralização do conhecimento sobre esta doença, o que representa um passo importante na direção dos objectivos traçados por este projecto, ajudando na recolha de dados, e, sendo a ADNI uma das maiores iniciativas de estudo clínico sobre Alzheimer alguma vez feita, o seu carregamento no traSMART é crucial para uma melhor compreensão da doença e dos seus mecanismos. A dissertação encontra-se dividida em 5 capítulos. O primeiro consiste numa introdução ao tema da dissertação e aos seus objetivos, e no segundo são descritos os principais conceitos inerentes ao trabalho desenvolvido --- como por exemplo conceitos sobre doenças neurodegenerativas, mais especificamente a doença de Alzheimer --- analisando ao pormenor as suas causas, as diferentes fases, compreender a sua neuropatologia e estudar o seu diagnóstico. São abordados também temas e conceitos relacionados com a bioinformática por detrás deste estudo, especificando também algumas das plataformas e pacotes de software utilizados. O terceiro capítulo descreve os materiais e metodologias utilizadas, explicando pormenorizadamente todos os passos e processos involvidos neste projecto, em cada uma das quatro principais fases dos mesmo (aquisição, tratamento, modelação e integração dos dados da ADNI), sendo que no quarto capítulo se encontram os resultados obtidos após a conclusão da parte prática deste estudo, apresentando também uma discussão sobre os mesmos, à medida que vão sendo demonstrados. No quinto e último capítulo encontram-se a Conclusão e Trabalho Futuro, ondese apresentam as ideias finais deste estudo, juntamente com sugestões de trabalho futuro que poderá dar seguimento a este estudo. Existe ainda uma secção de Anexos, onde se encontram os dicionários criados para este projecto e o script de Python criado para a modelação dos dados.
Neurodegenerative diseases affect millions of people worldwide, and Alzheimer’s disease (AD) is the most common type. For example in America, more than five million people are living with AD, although some estimates are much higher. With that in mind, it is more and more important to start working towards the improvement of these neurodegenerative diseases’ diagnosis and clinical therapy. One way to achieve that goal is to centralize all the knowledge on those diseases and reform established disease classification systems, such as ICD [International Classification of Disease], using a “mechanism-based taxonomy”, based upon the knowledge about the biological pathways involved in the aetiology of a disease to guide the classification of disease classes and subclasses, and that is the main goal of the AETIONOMY project, with its greatest challenge lying on the fact that, for most neurodegenerative diseases, the dysfunctional biological pathways underlying the disease are not known. This project will focus on the Alzheimer’s Disease Neuroimaging Initiative (ADNI) Clinical Data, and how to properly curate this clinical data and process it for integration in a translational biomedicine platform (tranSMART), analyzing and studying its relevance, as well as having a better understanding of this initiative’s global overview, with the purpose of defining new routes towards the identification of the underlying disease mechanisms, before organizing the latter, as well as proposing a rational disease taxonomy. This study made it possible to add the ADNI clinical data to the tranSMART’s database, making it available to third-part projects and thus helping the centralization of the current knowledge about this disease.