Granule Cell Dispersion in Human Temporal Lobe Epilepsy: Proteomics investigation of neurodevelopmental migratory pathways

Granule cell dispersion (GCD) is a common pathological feature observed in the hippocampus of patients with Mesial Temporal Lobe Epilepsy (MTLE). Pathomechanisms underlying GCD remain to be elucidated, but one hypothesis proposes aberrant reactivation of neurodevelopmental migratory pathways, possibly triggered by febrile seizures. This study aims to compare the proteomes of basal and dispersed granule cells in the hippocampus of eight MTLE patients with GCD to identify proteins that may mediate GCD in MTLE. Quantitative proteomics identified 1882 proteins, of which 29% were found in basal granule cells only, 17% in dispersed only and 54% in both samples. Bioinformatics analyses revealed upregulated proteins in dispersed samples were involved in developmental cellular migratory processes, including cytoskeletal remodelling, axon guidance and signalling by Ras homologous (Rho) family of GTPases (P<0.01). The expression of two Rho GTPases, RhoA and Rac1, was subsequently explored in immunohistochemical and in situ hybridisation studies involving eighteen MTLE cases with or without GCD, and three normal post mortem cases. In cases with GCD, most dispersed granule cells in the outer-granular and molecular layers have an elongated soma and bipolar processes, with intense RhoA immunolabelling at opposite poles of the cell soma, while most granule cells in the basal granule cell layer were devoid of RhoA. A higher density and percentage of cells expressing RhoA was observed in cases with GCD than without GCD (P<0.004). In GCD cases, the density and percentage of cells expressing RhoA was significantly higher in the inner molecular layer than granule cell layer (P<0.026), supporting proteomic findings. In situ hybridisation studies using probes against RHOA and RAC1 mRNAs revealed fine peri- and nuclear puncta in granule cells of all cases. The density of cells expressing RHOA mRNAs were significantly higher in the inner molecular layer of cases with GCD than without GCD(P=0.05). In summary, our study has found limited evidence for ongoing adult neurogenesis in the hippocampus of patients with MTLE, but evidence of differential dysmaturation between dispersed and basal granule cells has been demonstrated, and elevated expression of Rho GTPases in dispersed granule cells may contribute to the pathomechanisms underpinning GCD in MTLE

Developmental hypomyelination in Wolfram syndrome: New insights from neuroimaging and gene expression analyses

Wolfram syndrome is a rare multisystem disorder caused by mutations in WFS1 or CISD2 genes leading to brain structural abnormalities and neurological symptoms. These abnormalities appear in early stages of the disease. The pathogenesis of Wolfram syndrome involves abnormalities in the endoplasmic reticulum (ER) and mitochondrial dynamics, which are common features in several other neurodegenerative disorders. Mutations in WFS1 are responsible for the majority of Wolfram syndrome cases. WFS1 encodes for an endoplasmic reticulum (ER) protein, wolframin. It is proposed that wolframin deficiency triggers the unfolded protein response (UPR) pathway resulting in an increased ER stress-mediated neuronal loss. Recent neuroimaging studies showed marked alteration in early brain development, primarily characterized by abnormal white matter myelination. Interestingly, ER stress and the UPR pathway are implicated in the pathogenesis of some inherited myelin disorders like Pelizaeus-Merzbacher disease, and Vanishing White Matter disease. In addition, exploratory gene-expression network-based analyses suggest that WFS1 expression occurs preferentially in oligodendrocytes during early brain development. Therefore, we propose that Wolfram syndrome could belong to a category of neurodevelopmental disorders characterized by ER stress-mediated myelination impairment. Further studies of myelination and oligodendrocyte function in Wolfram syndrome could provide new insights into the underlying mechanisms of the Wolfram syndrome-associated brain changes and identify potential connections between neurodevelopmental disorders and neurodegeneration

Methodologies of Legacy Clinical Decision Support System -A Review

Information technology playing a prominent role in the field of medical by incorporating the Clinical Decision Support System(CDSS) in their routine practices. CDSS is a computer based interactive program to assist the physician to make the right decision at the right time. Now a day's Clinical decision support system is a dynamic research area in the field of computer, but the lack of the knowledge of the understanding as well as the functioning of the system ,make the adoption slow by the physician and patient. The literature review of this paper will focus on the overview of legacy CDSS, the kind of methodologies and classifier employed to prepare such decision support system using a non-technical approach to the physician and the strategy- makers . This study will provide the scope of understanding the clinical decision support along with the gateway to physician ,policy-makers to develop and deploy the decision support system as a healthcare service to make the quick, agile and right decision. Future direction to handle the uncertainties along with the challenges of clinical decision support system are also enlightened in this study

Behind the screens: Clinical decision support methodologies - A review

Clinical decision support systems (CDSSs) are interactive software systems designed to assist clinicians with decision making tasks, such as determining diagnosis of patient data. CDSSs are a widely researched topic in the Computer Science community but their workings are less well understood by clinicians. The purpose of this review is to introduce clinicians and policy makers to the most commonly computer-based methodologies employed to construct decision models to compute clinical decisions in a non-technical manner. We hope that a better understanding of CDSSs will open up discussion about the future of CDSSs as a part of healthcare delivery as well as engage clinicians and policy makers in the development and deployment of CDSSs that can meaningfully help with decision making tasks

International consensus recommendations on the diagnostic work-up for malformations of cortical development

Malformations of cortical development (MCDs) are neurodevelopmental disorders that result from abnormal development of the cerebral cortex in utero. MCDs place a substantial burden on affected individuals, their families and societies worldwide, as these individuals can experience lifelong drug-resistant epilepsy, cerebral palsy, feeding difficulties, intellectual disability and other neurological and behavioural anomalies. The diagnostic pathway for MCDs is complex owing to wide variations in presentation and aetiology, thereby hampering timely and adequate management. In this article, the international MCD network Neuro-MIG provides consensus recommendations to aid both expert and non-expert clinicians in the diagnostic work-up of MCDs with the aim of improving patient management worldwide. We reviewed the literature on clinical presentation, aetiology and diagnostic approaches for the main MCD subtypes and collected data on current practices and recommendations from clinicians and diagnostic laboratories within Neuro-MIG. We reached consensus by 42 professionals from 20 countries, using expert discussions and a Delphi consensus process. We present a diagnostic workflow that can be applied to any individual with MCD and a comprehensive list of MCD-related genes with their associated phenotypes. The workflow is designed to maximize the diagnostic yield and increase the number of patients receiving personalized care and counselling on prognosis and recurrence risk

Potential of activated microglia as a source of dysregulated extracellular microRNAs contributing to neurodegeneration in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron degeneration in adults, and several mechanisms underlying the disease pathology have been proposed. It has been shown that glia communicate with other cells by releasing extracellular vesicles containing proteins and nucleic acids, including microRNAs (miRNAs), which play a role in the post-transcriptional regulation of gene expression. Dysregulation of miRNAs is commonly observed in ALS patients, together with inflammation and an altered microglial phenotype. However, the role of miRNA-containing vesicles in microglia-to-neuron communication in the context of ALS has not been explored in depth. This review summarises the evidence for the presence of inflammation, pro-inflammatory microglia and dysregulated miRNAs in ALS, then explores how microglia may potentially be responsible for this miRNA dysregulation. The possibility of pro-inflammatory ALS microglia releasing miRNAs which may then enter neuronal cells to contribute to degeneration is also explored. Based on the literature reviewed here, microglia are a likely source of dysregulated miRNAs and potential mediators of neurodegenerative processes. Therefore, dysregulated miRNAs may be promising candidates for the development of therapeutic strategies

Peripheral blood biomarkers in multiple sclerosis.

Multiple sclerosis is the most common autoimmune disorder affecting the central nervous system. The heteroge-neity of pathophysiological processes in MS contributes to the highly variable course of the disease and unpre-dictable response to therapies. The major focus of the research on MS is the identification of biomarkers inbiologicalfluids, such as cerebrospinalfluid or blood, to guide patient management reliably. Because of the diffi-culties in obtaining spinalfluid samples and the necessity for lumbar puncture to make a diagnosis has reduced,the research of blood-based biomarkers may provide increasingly important tools for clinical practice. However,currently there are no clearly established MS blood-based biomarkers. The availability of reliable biomarkerscould radically alter the management of MS at critical phases of the disease spectrum, allowing for interventionstrategies that may prevent evolution to long-term neurological disability. This article provides an overview ofthis researchfield and focuses on recent advances in blood-based biomarker researc

A new diffusely infiltrating glioma mouse model reveals neuronal alterations in the brain tumor microenvironment

Gliomas are brain tumors that present with neurological symptoms including seizures and cognitive deficits. Starting at early stages of tumor development glioma cells diffusely infiltrate brain tissue where they interact with non-neoplastic cells including neurons and can perturb normal brain function. While the clinical consequences of glioma induced cortical dysfunction are well established, the neuronal alterations that underlie cortical dysfunction in glioma are unknown. We hypothesize that glioma cells infiltrate surrounding brain tissue and induce alterations in neurons that may contribute to the neurological symptoms associated with gliomas. Due to intermingling of glioma cells and neurons it has been challenging to isolate and characterize neurons from glioma brain tissue while preserving complex neuronal morphology. To address this issue we developed a new mouse glioma model that allowed us to obtain a neuron specific gene expression profile, otherwise obscured by the predominantly large population of glioma cells within the tumor. In this thesis I use this model to test the hypothesis that infiltrating glioma cells induce phenotypic alterations in neurons that contribute to the neurological symptoms associated with glioma. The Camk2a-Ribotag mouse glioma model enabled us to isolate neuron specific transcripts from glioma brain tissue. The Ribotag mouse has a conditional HA-tagged ribosomal protein (Rpl22) that can be expressed upon Cre-recombination. Camk2a is specifically expressed in excitatory neurons, the Camk2a-Cre mouse induces Cre-recombination in the Ribotag mouse so that Camk2a+ neurons selectively express HA-tagged Rpl22. We used the Camk2a-Ribotag glioma model to isolate neuron specific ribosome bound transcripts to characterize neuronal alterations in glioma. In chapter 2 of this thesis I describe how we developed and characterized the Camk2a-Ribotag mouse glioma model. We first obtained mouse glioma cells that have p53 deletion and overexpress PDGFRa, then we injected these cells in the Camk2a-Ribotag mouse and use this as our glioma model to extract neuron specific ribosome bound transcripts. This method is referred to as translating ribosome affinity purification (TRAP) which is used to obtain cell type specific translational profiles. Using this approach we identified alterations in neuronal gene expression, specifically we show that there is an upregulation of actin binding genes associated with dendritic spine morphology and a downregulation of synaptic genes associated synaptic regulation. We demonstrate that drebrin, an actin binding protein in dendritic spines, is upregulated in tumor brain synaptosomes, we also show a downregulation of dendritic spine density in HA-tagged neurons which suggests that these neuronal alterations contribute to synaptic dysfunction in our glioma model. Dendritic spines are dynamic structures that regulate synaptic function in response to diverse stimuli. mTOR signaling can regulate brain specific functions such as synaptic plasticity. Alterations in mTOR signaling can result in cognitive deficits, epilepsy and brain abnormalities that are associated with neurological disease. We hypothesized that mTOR regulates the neuronal alterations we identified in our glioma model. In chapter 3 of this thesis I describe how we tested this hypothesis by acutely inhibiting mTOR signaling with the ATP competitive inhibitor AZD8055 in the Camk2a-Ribotag mouse glioma model. Using TRAP we show that acute mTOR inhibition reverses many neuron specific alterations that occurs in the glioma infiltrated cortex, actin binding genes that were upregulated in tumor brains were downregulated after mTOR inhibition and synaptic genes that were downregulated in tumor brains were upregulated after mTOR inhibition. These results suggest that key neuron specific alterations are regulated by mTOR signaling in our glioma model. In chapter 4 of this thesis I describe how we used ribosome profiling to identify translational alterations in our Camk2a-Ribotag mouse glioma model. Ribosome profiling in an RNA sequencing based method that is used to measure translation efficiency by calculating the number of ribosomes per transcript. Using this approach we identified an upregulation in the translation of DNA methylation and demethylation gene ontologies. These results suggest that alterations in specific DNA methylation and demethylation gene ontologies are regulated at the level of translation and warrant further analysis of cell type specific translational alterations using ribosome profiling. The work described in this thesis demonstrates 1) use of the Camk2a-Ribotag mouse glioma model for the identification of neuron specific alterations, 2) neuron specific alterations include the upregulation of dendritic spine genes, downregulation of synaptic genes and downregulation of dendritic spine density, 3) acute mTOR inhibition reverses many of these neuronal alterations, 4) ribosome profiling revealed the translational upregulation of epigenetic genes in our mouse glioma model. The findings described in this thesis provide the first characterization of neuron specific transcriptional and translational alterations in glioma infiltrated cortex that and provide new insights into the mechanisms that underlie the devastating neurological symptoms in glioma patients

Multivariate Analysis of MR Images in Temporal Lobe Epilepsy

Epilepsy stands aside from other neurological diseases because clinical patterns of progression are unknown: The etiology of each epilepsy case is unique and so it is the individual prognosis. Temporal lobe epilepsy (TLE) is the most frequent type of focal epilepsy and the surgical excision of the hippocampus and the surrounding tissue is an accepted treatment in refractory cases, specially when seizures become frequent increasingly affecting the performance of daily tasks and significantly decreasing the quality of life of the patient. The sensitivity of clinical imaging is poor for patients with no hippocampal involvement and invasive procedures such as the Wada test and intracranial EEG are required to detect and lateralize epileptogenic tissue. This thesis develops imaging processing techniques using quantitative relaxometry and diffusion tensor imaging with the aiming to provide a less invasive alternative when detectability is low. Chapter 2 develops the concept of individual feature maps on regions of interest. A laterality score on these maps correctly distinguished left TLE from right TLE in 12 out of 15 patients. Chapter 3 explores machine learning models to detect TLE, obtaining perfect classification for left patients, and 88.9% accuracy for right TLE patients. Chapter 4 focuses on temporal lobe asymmetry developing a voxel-based method for assessing asymmetry and verifying its applicability to individual predictions (92% accuracy) and group-wise statistical analyses. Informative ROI and voxel-based informative features are described for each experiment, demonstrating the relative importance of mean diffusivity over other MR imaging alternatives in identification and lateralization of TLE patients. Finally, the conclusion chapter discuss contributions, main limitations and outlining options for future research