Enhancing neuronal inhibition by cell and gene therapy as a novel treatment for Epilepsy

Epilepsy is a family of heterogeneous and multifactorial neurological disorders, unified by the occurrence of spontaneous recurrent seizures. Overall, it affects 50 million people worldwide of all ages and genders. Available treatments are only symptomatic and have severe side effects, while they also fail to provide adequate seizure control in a third of the patients. Therefore, a cure is yet to be found. In this warrant for novel strategies to treat those refractory patients, this thesis evolved. The two approaches presented here based their goal on increasing inhibitory drive in the epileptic focus to reduce the pathological hyperexcitable neuronal network that characterizes epilepsy, and thus counteract seizures.First, based on evidence of loss and/or alteration of gamma-aminobutyric acid (GABA)-ergic interneurons in the epileptic neuronal network, cell-based therapy has been developed and tested in three different scenarios for restoration of the excitatory/inhibitory balance. GABAergic interneurons (hdINs) were generated in vitro from human embryonic stem cells and proved to survive and integrate into both human and rodent epileptic environments. Host neuronal activity could be modulated by light-activation of hdINs using optogenetics. Finally, grafted hdINs were able to reduce the seizure frequency and total time spent in seizures in a rat model of temporal lobe epilepsy (TLE), the most common form of refractory epilepsy in adults. However, grafted hdINs failed to improve the pathology in a genetic mouse model of cortical dysplasia-focal epilepsy syndrome associated with autism spectrum disorder, which highlights the diversity of epilepsies and the need for gaining a better understanding of the mechanisms underpinning the disease.Finally, direct inhibition of principal cells, similar to the one exerted by endogenous inhibitory interneurons, in the chronic epileptic hippocampus by using a chemogenetic approach delivered as gene therapy was also tested. Positive results were observed by decreasing ability to generate action potentials, although further investigation is required to evaluate the efficacy of this approach on seizures.Collectively, the work presented here has moved the field forward in testing two different therapeutic strategies in a TLE model, and also one of them in a genetic epilepsy model

High performance computational virtual screening tools: development and application to the discovery of kinase inhibitors

Ph.DDOCTOR OF PHILOSOPH

Two computational neural models : rodent perirhinal cortex and crab cardiac ganglion

Neural engineering research has been rapidly growing in prominence in the past two decades, with 'reverse engineer the brain' listed as one of the 14 grand challenges outlined by the National Academy of Engineering. The computational aspect of reverse engineering includes a study of how both single neurons and networks of neurons integrate diverse signals from both the environment and from within the animal and make complex decisions. Since there are many limitations on the experiments that can be performed in alive or isolated biological systems, there is a need of standalone computational models which can help perform 'in silico' experiments. This dissertation focuses on such 'in silico' neuronal models to predict underlying mechanisms of governing interactions and robustness. The first model investigated is that of a rodent perirhinal cortex area 36 (PRC), and its role in associative memory formation. A large-scale 520 cell biophysical model of the PRC was developed using biological data from the literature. We then used the model to shed light on the mechanisms that support associative memory in the perirhinal network. These analyses revealed that perirhinal associative plasticity is critically dependent on a specific subset of neurons, termed conjunctive cells. When the model network was trained with spatially distributed but coincident neocortical inputs, these conjunctive cells acquired excitatory responses to the paired neocortical inputs and conveyed them to widely distributed perirhinal sites via longitudinal projections. Ablation of conjunctive cells during recall abolished expression of the associative memory. The second model focuses on a model for crab cardiac system consisting of five Large Cells (LC) developed using firsthand biological data. The model is then used to study the features of its underlying oscillation in its membrane potential during a rhythm and to reverse engineer the experimentally discovered phenomenon related to network synchrony. The model predicted multiple mechanisms of compensation to restore network synchrony based on compensatory intrinsic conductances. Finally, a third model, related to the second one, was of an improved three-compartmental biophysical model of an LC that is morphologically realistic and includes provision for inputs from the SCs. To determine viable LC models, maximal conductances in three compartments of an LC are determined by random sampling from a biologically characterized 9D-parameter space, followed by a three-stage rejection protocol that checks for conformity with features in experimental single cell traces. Random LC models that pass the single cell rejection protocol are then incorporated into a network model followed by a final rejection protocol stage. Using disparate experimental data, the study provides hitherto unknown structure-function insights related to the crustacean cardiac ganglion large cell, including the differential roles of active conductances in the three compartments. The novel morphological architecture for the large cell was validated using biological data and used to make predictions about function. A testable prediction related to function was that active conductances, specifically, the persistent sodium current, is required in the neurite to transmit the spike waveforms from the spike initiation zone to the soma. Another pertains to the co-variation of maximal conductances of the persistent sodium current with that of the leak current

Pathway and biomarker discovery in a posttraumatic stress disorder mouse model

Posttraumatic stress disorder (PTSD), a prevalent psychiatric disorder, is caused by exposure to a traumatic event. Individuals diagnosed for PTSD not only experience significant functional impairments but also have higher rates of physical morbidity and mortality. Despite intense research efforts, the neurobiological pathways affecting fear circuit brain regions in PTSD remain obscure and most of the previous studies were limited to characterization of specific markers in periphery or defined brain regions. In my PhD study, I employed proteomics, metabolomics and transcriptomcis technologies interrogating a foot shock induced PTSD mouse model. In addition, I studied the effects of early intervention of chronic fluoxetine treatment. By in silico analyses, altered cellular pathways associated with PTSD were identified in stress-vulnerable brain regions, including prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala (BLA), central nucleus of amygdala(CeA), nucleus accumbens (NAc) and CA1 of the dorsal hippocampus. With RNA sequencing, I compared the brain transcriptome between shocked and control mice, with and without fluoxetine treatment. Differentially expressed genes were identified and clustered, and I observed increased inflammation in ACC and decreased neurotransmitter signaling in both ACC and CA1. I applied in vivo 15N metabolic labeling combined with mass spectrometry to study alterations at proteome level in the brain. By integrating proteomics and metabolomics profiling analyses, I found decreased Citric Acid Cycle pathway in both NAc and ACC, and dysregulated cytoskeleton assembly and myelination pathways in BLA, CeA and CA1. In addition, chronic fluoxetine treatment 12 hours after foot shock prevented altered inflammatory gene expression in ACC, and Citric Acid Cycle in NAc and ACC, and ameliorated conditioned fear response in shocked mice. These results shed light on the role of immune response and energy metabolism in PTSD pathogenesis. Furthermore, I performed microdialysis in medial prefrontal cortex and hippocampus to measure the changes in extracellular norepinephrine and free corticosterone (CORT) in the shocked mouse and related them to PTSD-like symptoms, including hyperaroual and contextual fear response. I found that increased free CORT was related to immediate stress response, whereas norepinephrine level, in a brain region specific manner, predicted arousal and contextual fear response one month after trauma. I also applied metabolomics analysis to investigate molecular changes in prefrontal microdialysates of shocked mice. Citric Acid Cycle, Glyoxylate and Dicarboxylate metabolism and Alanine, Aspartate and Glutamate metabolism pathways were found to be involved in foot shock induced hyperarousal. Taken together, my study provides novel insights into PTSD pathogenesis and suggests potential therapeutic applications targeting dysregulated pathways

Pathway and biomarker discovery in a posttraumatic stress disorder mouse model

Posttraumatic stress disorder (PTSD), a prevalent psychiatric disorder, is caused by exposure to a traumatic event. Individuals diagnosed for PTSD not only experience significant functional impairments but also have higher rates of physical morbidity and mortality. Despite intense research efforts, the neurobiological pathways affecting fear circuit brain regions in PTSD remain obscure and most of the previous studies were limited to characterization of specific markers in periphery or defined brain regions. In my PhD study, I employed proteomics, metabolomics and transcriptomcis technologies interrogating a foot shock induced PTSD mouse model. In addition, I studied the effects of early intervention of chronic fluoxetine treatment. By in silico analyses, altered cellular pathways associated with PTSD were identified in stress-vulnerable brain regions, including prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala (BLA), central nucleus of amygdala(CeA), nucleus accumbens (NAc) and CA1 of the dorsal hippocampus. With RNA sequencing, I compared the brain transcriptome between shocked and control mice, with and without fluoxetine treatment. Differentially expressed genes were identified and clustered, and I observed increased inflammation in ACC and decreased neurotransmitter signaling in both ACC and CA1. I applied in vivo 15N metabolic labeling combined with mass spectrometry to study alterations at proteome level in the brain. By integrating proteomics and metabolomics profiling analyses, I found decreased Citric Acid Cycle pathway in both NAc and ACC, and dysregulated cytoskeleton assembly and myelination pathways in BLA, CeA and CA1. In addition, chronic fluoxetine treatment 12 hours after foot shock prevented altered inflammatory gene expression in ACC, and Citric Acid Cycle in NAc and ACC, and ameliorated conditioned fear response in shocked mice. These results shed light on the role of immune response and energy metabolism in PTSD pathogenesis. Furthermore, I performed microdialysis in medial prefrontal cortex and hippocampus to measure the changes in extracellular norepinephrine and free corticosterone (CORT) in the shocked mouse and related them to PTSD-like symptoms, including hyperaroual and contextual fear response. I found that increased free CORT was related to diate stress response, whereas norepinephrine level, in a brain region specific manner, predicted arousal and contextual fear response one month after trauma. I also applied metabolomics analysis to investigate molecular changes in prefrontal microdialysates of shocked mice. Citric Acid Cycle, Glyoxylate and Dicarboxylate metabolism and Alanine, Aspartate and Glutamate metabolism pathways were found to be involved in foot shock induced hyperarousal. Taken together, my study provides novel insights into PTSD pathogenesis and suggests potential therapeutic applications targeting dysregulated pathways

Development of Virtual Screening and In Silico Biomarker Identification Model for Pharmaceutical Agents

Ph.DDOCTOR OF PHILOSOPH

Virtual Screening of Multi-Target Agents by Combinatorial Machine Learning Methods

Ph.DDOCTOR OF PHILOSOPH

Statistical learning approaches for predicting pharmacological properties of pharmaceutical agents

Ph.DDOCTOR OF PHILOSOPH

Novel Treatment Strategies for Glioblastoma

This book is a compilation of articles that brings together current knowledge from an international team of contributors who are dedicated investigators exploring novel strategies for the treatment of glioblastoma. These articles describe some of the latest concepts that will provide students, researchers and clinicians with an overview of the therapeutic approaches being developed in the field of neuro-oncology to combat this deadly disease

Transcriptomic cellular diversity of the early human developing brain

The complexity of the mammalian brain is partly reflected in its cell type diversity which influences the function of neurons that encode the behavior of animals. Brain cell type diversity emerges during embryonic stages, a critical period when neurons start to become functionally active and establish their connectivity across the brain. Since the pioneering of single-cell RNA-sequencing (scRNA-seq), we can question when and how cellular diversity arises in the brain in a large-scale manner. This thesis aims to study the human brain during the first trimester by using scRNA-seq to obtain a global view of the basic principles of the developing brain. First, I introduce human embryology from a historical perspective and summarize key concepts in central nervous system (CNS) development. I review few gaps in the field related to our findings, followed by current approaches and nomenclatures used in the field of single-cell genomics that applies to development. To put our work into perspective, I present an overview of the latest efforts to study human brain development at the single-cell level, both in the healthy and diseased brain. Then I present the following two papers and a manuscript: In Paper I we used scRNA-seq to construct a cell taxonomy of the adult mouse nervous system. We describe two major groups: neuronal- and non-neuronal cells that were subdivided into distinct cell types. Overall, the neurons were transcriptionally similar across brain regions, whereas non-neuronal cells such as astrocytes, formed subgroups and were regionally distinct. The whole dataset revealed an organization that reflects the developmental origin of all cell types. Paper II describes a method, RNA velocity, that infers temporal changes from static scRNAseq gene expression measurements. By realigning sequencing reads, this method detects and makes use of the unspliced and spliced mRNA, whose relative abundance is used to measure the change of rate in gene expression (the time derivative) in different tissues. This method is particularly suitable for developmental lineages, which was shown and validated both in vitro and in situ in this study. Paper III presents a single-cell atlas of the human developing CNS across all major brain regions during postconceptional weeks (p.c.w.) 5 to 14. We observe that major cell classes emerge during this period, most of them being regionally diverse and to a surprisingly high degree among glial cells. We display the high resolution of this data by resolving several lineages in the forebrain and validated the spatial location of transcriptional cell types at 5 p.c.w. by using single-molecule FISH. As a whole, this study represents a reference of human brain development during the first critical period in life. To tie these studies together, our findings on glial diversity were partially shared between the adult mouse and developing human CNS. We further showed that an RNA velocity-based method can be used to model the cell cycle dynamics in cortical tissue. To conclude, I discuss advantages and limitations of single-cell transcriptomics, its future challenges and how using this technology sheds light on the early human developing brain as is described in this thesis