Rubella virus (RUBV) is a significant human pathogen. RUBV infection takes an enormous toll due to congenital rubella syndrome (CRS), a constellation of birth defects including blindness, hearing defects and mental retardation. Little is known about RUBV-induced teratogenesis due to the absence of useful models. This research is now enabled by the availability of human embryonic stem cells (hESCs) and hESC-derived precursor cell lines. Human neural progenitor cells (hNPCs) serve as a particularly relevant model due to the symptoms and complications of CRS related to neural system development. The overarching question addressed in this dissertation is: what is the mechanism underlying the development of neurological abnormalities seen in CRS? In this context, we investigated the cellular responses of hNPCs to RUBV infection comprehensively by: 1) assessing susceptibility of the cells to RUBV infection; 2) analyzing the effect of infection on cell proliferation; and 3) examining the impact of RUBV infection on differentiation of hNPCs into neuronal and astroglial lineages . We found that hNPCs are susceptible to RUBV infection and that the percentage of infected cells closely mimics CRS in which few cells harbor virus. The virus was able to persist in culture for up to one month without significant alteration of cell morphology and stemness marker expression. In addition, RUBV infection moderately attenuated the proliferation of undifferentiated hNPCs by triggering cell cycle arrest, but not apoptosis or other cell death events commonly seen upon virus infection. This lack of apoptosis appeared to be due in part to virus-induced anti-apoptotic suppression. Interestingly, the virus only had a marginal effect on the induction of cell differentiation into both neuronal and astroglial phenotypes. In fact, RUBV infection promoted terminal differentiation of the culture due to depletion of precursor cells. With differentiation, viral replication was suppressed. We thus propose a model for RUBV-induced neurological defects in which the virus acts by depleting precursor cell pools. The results of this study provide clues for elucidating the mechanisms of RUBV teratogenicity at the cellular level and serves as a potential reference study for elucidating mechanisms of teratogenesis induced by other infectious agents.

Cache Valley virus (CVV) induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the CVV infection sequence of the cells targeted by the virus or the development of the antiviral response of the early, infected fetus that results in viral clearance before development of immunocompetency. To address these questions, ovine fetuses at 35 dg were inoculated in utero with CVV and euthanized at 7, 10, 14, 21 and 28 dpi. On postmortem examination arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphologic studies showed necrosis in the central nervous system (CNS) and skeletal muscle of earlier infected fetuses and hydrocephalus, micromyelia and muscular loss in later infected fetuses. Using immunohistochemistry and in situ hybridization, intense CVV viral antigenic signal was detected in the brain, spinal cord, skeletal muscles and fetal membranes of infected fetuses. Viral signal decreased in targeted and infected tissues with the progression of the infection. To determine specific cell types targeted by CVV in the CNS, indirect immunofluorescence was applied to sections of the CNS using a double labeling technique with antibodies against CVV together with antibodies against neurons, astrocytes and microglia. CVV viral antigen was shown within the cytoplasm of neurons in the brain and spinal cord. No viral signal was observed in microglial cells; however, infected animals had marked microgliosis. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time, quantitative PCR. Upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-?, TLR-7 and TLR-8. The amount of Mx protein, an interferon stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. This demonstrated that the infected ovine fetus is able to stimulate an innate and adaptive immune response before immunocompetency that presumably contributes to viral clearance in infected animals.

No description available.

To determine the characteristics, treatments and outcomes of patients with glioblastoma multiforme (GBM) or gliosarcoma (GS) and metastases outside of the central nervous system (CNS).

Background and Purpose: In 2007, the WHO classification of brain tumors was extended by three new entities of glioneuronal tumors: papillary glioneuronal tumor (PGNT), rosette-forming glioneuronal tumor of the fourth ventricle (RGNT) and glioneuronal tumor with neuropil-like islands (GNTNI). Focusing on clinical characteristics and outcome, the authors performed a comprehensive individual patient data (IPD) meta-analysis of the cases reported in literature until December 2012. Methods: PubMed, Embase and Web of Science were searched for peer-reviewed articles reporting on PGNT, RGNT, and GNTNI using predefined keywords. Results: 95 publications reported on 182 patients (PGNT, 71; GNTNI, 26; RGNT, 85). Median age at diagnosis was 23 years (range 4–75) for PGNT, 27 years (range 6–79) for RGNT, and 40 years (range 2–65) for GNTNI. Ninety-seven percent of PGNT and 69% of GNTNI were located in the supratentorial region, 23% of GNTNI were in the spinal cord, and 80% of RGNT were localized in the posterior fossa. Complete resection was reported in 52 PGNT (73%), 36 RGNT (42%), and 7 GNTNI (27%) patients. Eight PGNT, 3 RGNT, and 12 GNTNI patients were treated with chemo- and/or radiotherapy as the primary postoperative treatment. Follow-up data were available for 132 cases. After a median follow-up time of 1.5 years (range 0.2–25) across all patients, 1.5-year progression-free survival rates were 52±12% for GNTNI, 86±5% for PGNT, and 100% for RGNT. The 1.5-year overall-survival were 95±5%, 98±2%, and 100%, respectively. Conclusions: The clinical understanding of the three new entities of glioneuronal tumors, PGNT, RGNT and GNTNI, is currently emerging. The present meta-analysis will hopefully contribute to a delineation of their diagnostic, therapeutic, and prognostic profiles. However, the available data do not provide a solid basis to define the optimum treatment approach. Hence, a central register should be established.

Hypothalamic lactate metabolism regulates hepatic glucose and lipid homeostasis, however it remains unclear whether hypothalamic lactate also controls energy homeostasis. Furthermore, the precise downstream molecular and signaling pathway(s) involved in hypothalamic lactate-sensing is yet to be fully elucidated. To specifically address these two questions, we tested the hypothesis that hypothalamic lactate metabolism regulates energy homeostasis (Study 1) and assessed whether the activation of N-methyl-D-aspartate (NMDA) receptors in the nucleus of the solitary tract (NTS) of the brainstem is required for hypothalamic lactate, and sufficient per se, to regulate glucose homeostasis (Study 2). In an in vivo rat model, we reported in Study 1 that central lactate lowers food intake and body weight through its metabolism into pyruvate. In Study 2, we identified that hypothalamic lactate metabolism requires the activation of NMDA receptors in the NTS to lower hepatic glucose production. Moreover, we showed that the activation of NTS NMDA receptors per se lowers hepatic glucose production. In summary, these findings advance the understanding of central nutrient-sensing in the regulation of energy and glucose homeostasis, which is critical in bridging the therapeutic gap of obesity and type 2 diabetes.

An acute demyelinating syndrome (ADS) in a child may be a monophasic illness or may represent the incident attack of multiple sclerosis (MS) – an inflammatory demyelinating neurodegenerative disorder affecting the brain, spinal cord and optic nerves. The central objective of this dissertation was to identify MRI parameters present at ADS that predict MS diagnosis. A scoring tool was first created containing 14 parameters identified from the literature and demonstrating substantial inter-rater agreement (Cohen’s kappa values ≥0.6). Children aged <16 years were enrolled at incident ADS and are currently followed for five years at 23 Canadian centers. Standardized MRI scans were acquired at onset and serially. MS was defined based on the occurrence of a second demyelinating attack or MRI evidence of new lesions in accordance with McDonald criteria for dissemination in time. Multivariable Cox proportional hazards regression models were used to identify MRI parameters that predicted MS diagnosis. Over 1100 MRI scans in 284 children with ADS were evaluated. To date, 57(20%) children have been diagnosed with MS. For those that developed MS, the median (IQR) time from incident attack to diagnosis was 6.2 (4.7-11.1) months. The presence of ≥1 T1-hypointense lesion (HR 20.6, 95% CI 5.5-78.0) and ≥1 T2 periventricular lesion (3.3, 1.3-8.8) were associated with an increased likelihood for MS diagnosis (sensitivity 84%, specificity 93%, PPV 76%, NPV 96%). The predictive parameters were validated in an independent Dutch cohort of 45 children with ADS (n=15, 33% MS): sensitivity 93%, specificity 87%, PPV 78%, NPV 96%. Finally, it was determined that the 2010 McDonald criteria are applicable for diagnosis of pediatric-onset MS diagnosis in older children with non-ADEM presentations. The work embodied herein emphasizes the value of MRI in predicting MS diagnosis in children with incident ADS. Early identification of children with MS is important for planning clinical care and will be valuable in future pediatric MS treatment trials.

Hypothalamic lactate metabolism regulates hepatic glucose and lipid homeostasis, however it remains unclear whether hypothalamic lactate also controls energy homeostasis. Furthermore, the precise downstream molecular and signaling pathway(s) involved in hypothalamic lactate-sensing is yet to be fully elucidated. To specifically address these two questions, we tested the hypothesis that hypothalamic lactate metabolism regulates energy homeostasis (Study 1) and assessed whether the activation of N-methyl-D-aspartate (NMDA) receptors in the nucleus of the solitary tract (NTS) of the brainstem is required for hypothalamic lactate, and sufficient per se, to regulate glucose homeostasis (Study 2). In an in vivo rat model, we reported in Study 1 that central lactate lowers food intake and body weight through its metabolism into pyruvate. In Study 2, we identified that hypothalamic lactate metabolism requires the activation of NMDA receptors in the NTS to lower hepatic glucose production. Moreover, we showed that the activation of NTS NMDA receptors per se lowers hepatic glucose production. In summary, these findings advance the understanding of central nutrient-sensing in the regulation of energy and glucose homeostasis, which is critical in bridging the therapeutic gap of obesity and type 2 diabetes.

Injury to the central nervous system (CNS) results in the formation of a highly inhibitory glial scar consisting mainly of chondroitin sulfate proteoglycans (CSPGs). CSPGs are comprised of a protein core with covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. CSPGs and CS-GAGs have been implicated in the regenerative failure of the CNS, though the mechanism underlying inhibition is unclear. Sulfation affects both the physical and chemical characteristics of CS-GAGs and, therefore, it has been hypothesized that certain sulfation patterns are more inhibitory than others. To investigate this hypothesis, specific chondroitin sulfate sulfotransferases (CSSTs), the enzymes responsible for CS-GAG sulfation, were knocked down in vitro using siRNA. C4ST-1, C4ST-2, and C46ST were chosen as targets for gene knockdown in this study based on their expression in neural tissue and the extent of inhibition caused by their respective CS-GAG. It was hypothesized that transfection of primary rat astrocytes with siRNAs designed to prevent the expression of C4ST-1, C4ST-2, and C46ST would decrease specific sulfation patterns of CSPGs, resulting in improved neurite extension in a neurite guidance assay. Through optimization of siRNA dose, astrocyte viability was maintained while successfully knocking down mRNA levels of C4ST-1, C4ST-2, and C46ST and significantly reducing total levels of secreted CS-GAGs. However, no increase in the incidence of neurite extension was observed using conditioned media collected from siRNA transfected astrocytes compared to non-transfected controls. These data suggest that sulfation does not contribute to CSPG-mediated neurite inhibition, though further investigation is necessary to confirm these findings. Significantly, this work has established a paradigm for investigating the role of CSPG sulfation patterns in CNS regeneration.

The human brain (lat. cerebrum) is the most complex and heterogeneous organ in the human body. It is involved in a great number of body functions like movement, touch sensing, vision, hearing, smelling, hormone regulation and many more. In no other organ, the molecular communication mechanisms between different cells are so poorly understood. Due to the extensive diversity of processes that are controlled by the brain, diseases and injuries of the nervous system affect the human body significantly. Because of the immense complexity of the brain, the molecular mechanisms underlying the pathology of the diseases remain largely unknown. Hence, there is an urgent need for the development of new analytical strategies in order to investigate these conditions on a molecular level. Here, a central focus lies in the study of protein and peptide expression profiles, which can provide an insight in ongoing molecular mechanisms underlying the pathophysiology of the diseases. A powerful approach for studying proteins and peptide dynamics is mass spectrometry based proteomics, which is defined as the comprehensive study of all proteins expressed in a biological matrix at a certain point of time. The central objective of this thesis was to develop and employ different mass spectrometric techniques to study protein and peptide dynamics in the central nervous system in different neurological diseases. The individual studies comprise different aspects of proteome research. The first two studies included clinical proteomic applications for investigating protein dynamics in traumatic brain injury and amyotrophic lateral sclerosis. A further study was focused on method development for MS analysis of intact neural cells. The final three projects described in this thesis comprised MS based protein and peptide imaging in brain and spinal cord tissue samples. Here, the aim was to elucidate topological changes in protein expression in ALS as well as neuropeptide alterations in distinct brain structures in L-DOPA induced dyskinesia (LID) in Parkinson’s disease. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 713