Dissertations / Theses on the topic 'Tuberous sclerosis 1 protein'

Retinoblastoma tumour suppressor Rb is a cell cycle regulator that is active during early G1 preventing the transition from G1 to S-phase. This is achieved by Rb inhibiting E2F transcription factors from activating expression of genes required for G1 to S-phase progression and DNA synthesis. In our initial genetic test searching for genes that interact with mutations of rbf1, the homologue of rb in Drosophila melanogaster, one of the genes identified was tsc1, which is also a tumour suppressor gene that regulates translation and cell growth. We found that in Drosophila eye imaginal disc cells, tsc1 and rbf1 mutations have a synergistic effect on increasing the level of cell death and promoting ectopic S-phase entry. In addition, I found that dE2F1 protein level increased in tsc1 mutant eye disc cells, which implies that Tsc1 is a negative regulator of dE2F1 expression. The goal of my thesis study was to characterize the synergistic relation between Rbf1 and Tsc1 as well as the regulation of dE2F1 expression by Tsc1. In cells triple-mutant for rbf1, tsc1, and de2f1, I found that the observed elevation in cell death in rbf1 and tsc1double-mutant cells was suppressed, which suggests that the cooperation between Rbf1 and Tsc1 is dE2F1-dependent. Moreover, by using a reporter construct for dE2F1 activity, PCNA-GFP, and performing in situ hybridization with anti-sense RNA probes of dE2F1 target genes, rnrS, Cyclin E, and PCNA, I showed that activities of de2f1 downstream target genes were activated by tsc1 mutations, suggesting that Tsc1 also regulates dE2F1 target gene expression. Through clonal analysis of loss-of-function mutant alleles of the canonical Tsc pathway genes, I found that Tsc1 regulates dE2F1 via the Tsc pathway, specifically tsc/rheb/Tor/s6k. Finally, my RTq-PCR result showed that the regulation of dE2F1 protein expression by Tsc1 is at post-transcriptional level. To address whether the regulation is at the level of translation, I cloned the 5' untran<br>Le suppresseur de tumeur du Rétinoblastome, Rb, est un régulateur du cycle cellulaire qui est actif dans la phase précoce G1, prévenant le passage en phase S. Pour ce faire Rb inhibe le facteur de transcription E2F, l'empêchant d'activer l'expression de gènes requis pour le passage de la phase G1 à la phase S et pour la synthèse d'ADN. Dans nos tests génétiques initiaux, cherchant des gènes interagissant avec la mutation rbf1, l'homologue de rb chez Drosophila Melanogaster, un des gènes identifié fut tsc1, qui est également un gène suppresseur de tumeur qui régule la traduction et la croissance cellulaire. Nous avons découvert que dans les cellules des disques imaginaux des yeux, les mutations tsc1 et rbf1 ont un effet synergique sur l'augmentation du taux de mort cellulaire et promeuvent l'entrée en phase ectopique S. Il fut également découvert que le taux de la protéine dE2f1 augmente dans les cellules mutantes du disque des yeux, ce qui implique que Tsc1 est un régulateur négatif de l'expression de dE2F1. Le but de ma thèse était de caractériser la régulation de l'expression de dE2F1 par Tsc1 et la relation de synergie entre Rbf1 et Tsc1. Dans les cellules triples mutantes pour rbf1, tsc1, et de2f1, j'ai trouvé que l'augmentation du taux de mort cellulaire observé disparaissait dans les cellules doubles mutantes rbf1 et tsc1, ce qui suggère que la coopération entre Rbf1 et Tsc1 est dE2F1-dependante. Egalement, en utilisant un gène rapporteur de l'activité de de2f1, PCNA-GFP, et en réalisant des hybridations in situ avec des sondes ARN anti sens, rnrS, Cyclin E, and PCNA, j'ai montré que l'activité de la région en aval des gènes cibles de de2f1 était activé par la mutation tsc1, suggérant que Tsc1 régule également l'expression des gènes cible de dE2F1. Par l'analyse de clones possédant des allèles mutants perte de fonction pour les gènes de la cascade canonique Tsc, j'ai trouvé que Tsc1 régule dE2F1 par le biais$

Amyotrophic lateral sclerosis (ALS) is a disease in which the motor neurons die in a progressive manner, leading to paralysis and muscle wasting. ALS is always fatal, usually through respiratory failure when the disease reaches muscles needed for breathing. Most cases are sporadic, but approximately 5–10% are familial. The first gene to be linked to familial ALS encodes the antioxidant enzyme superoxide dismutase-1 (SOD1). Today, more than 160 different mutations in SOD1 have been found in ALS patients.  The mutant SOD1 proteins cause ALS by gain of a toxic property that should be common to all. Aggregates of SOD1 in motor neurons are hallmarks of ALS patients and transgenic models carrying mutant SOD1s, suggesting that misfolding, oligomerization, and aggregation of the protein may be involved in the pathogenesis. SOD1 is normally a very stable enzyme, but the structure has several components that make SOD1 sensitive to misfolding. The aim of the work in this thesis was to study misfolded SOD1 in vivo. Small amounts of soluble misfolded SOD1 were identified as a common denominator in transgenic ALS models expressing widely different forms of mutant SOD1, as well as wild-type SOD1. The highest levels of misfolded SOD1 were found in the vulnerable spinal cord. The amounts of misfolded SOD1 were similar in all the different models and showed a broad correlation with the lifespan of the different mouse strains. The misfolded SOD1 lacked the C57-C146 intrasubunit disulfide bond and the stabilizing zinc and copper ions, and was prinsipally monomeric. Forms with higher apparent molecular weights were also found, some of which might be oligomers. Misfolding-prone monomeric SOD1 appeared to be the principal source of misfolded SOD1 in the CNS. Misfolded SOD1 in the spinal cord was found to interact mainly with chaperones, with Hsc70 being the most important. Only a minor proportion of the Hsc70 was sequestered by SOD1, however, suggesting that chaperone depletion is not involved in ALS.  SOD1 is normally found in the cytoplasm but can be secreted. Extracellular mutant SOD1 has been found to be toxic to motor neurons and glial cells. Misfolded SOD1 in the extracellular space could be involved in the spread of the disease between different areas of the CNS and activate glial cells known to be important in ALS. The best way to study the interstitium of the CNS is through the cerebrospinal fluid (CSF), 30% of which is derived from the interstitial fluid. Antibodies specific for misfolded SOD1 were used to probe CSF from ALS patients and controls for misfolded SOD1. We did find misfolded SOD1 in CSF, but at very low levels, and there was no difference between ALS patients and controls. This argues against there being a direct toxic effect of extracellular SOD1 in ALS pathogenesis. In conclusion, soluble misfolded SOD1 is a common denominator for transgenic ALS model mice expressing widely different mutant SOD1 proteins. The misfolded SOD1 is mainly monomeric, but also bound to chaperones, and possibly exists in oligomeric forms also. Misfolded SOD1 in the interstitium might promote spread of aggregation and activate glial cells, but it is too scarce to directly cause cytotoxicity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome of unknown etiology that most commonly affects people in middle and high age. The hallmark of ALS is a progressive and simultaneous loss of upper and lower motor neurons in the central nervous system that leads to a progressive muscle atrophy, paralysis and death usually by respiratory failure. ALS is not a pure motor neuronal syndrome; it extends beyond the motor system and affects extramotor areas of the brain as well. The majority of the patients suffer from a sporadic ALS disease (SALS) while in at least ten percent the disease appears in a familial form (FALS). Mutations in the gene encoding the antioxidant enzyme superoxide dismutase-1 (SOD1) are the most common cause of FALS. More than 165 SOD1 mutations have been described, and these confer the enzyme a cytotoxic gain of function. Evidence suggests that the toxicity results from structural instability which makes the mutated enzyme prone to misfold and form aggregates in the spinal cord and brain motor neurons. Recent studies indicate that the wild-type human SOD1 protein (wt-hSOD1) has the propensity to develop neurotoxic features. The aim of the present study was to investigate if wt-hSOD1 is involved in the pathogenesis of SALS and FALS patients lacking SOD1 mutations and to evaluate the neurotoxic effect of misfolded wt-hSOD1 protein in vivo by generating a transgenic wt-hSOD1 mice model. We produced specific SOD1-peptide-generated antibodies that could discriminate between the misfolded and native form of the enzyme and optimized a staining protocol for detection of misfolded wt-hSOD1 by immunohistochemistry and confocal microscopy of brain and spinal cord tissue. We discovered that aggregates of misfolded wt-hSOD1 were constitutively present in the cytoplasm of motor neurons in all investigated SALS patients and in FALS patients lacking SOD1 gene mutations. Interestingly, the misfolded wt-hSOD1 aggregates were also found in some motor neuron nuclei and in the nuclei of the surrounding glial cells, mainly astrocytes but also microglia and oligodendrocytes, indicating that misfolded wt-hSOD1 protein aggregates may exert intranuclear toxicity. We compared our findings to FALS with SOD1 mutations by investigating brain and spinal cord tissue from patients homozygous for the D90A SOD1 mutation, a common SOD1 mutation that encodes a stable SOD1 protein with a wild-type-like enzyme activity. We observed a similar morphology with a profound loss of motor neurons and aggregates of misfolded SOD1 in the remaining motor neuron. Interestingly, we found gliosis and microvacuolar degeneration in the superficial lamina of the frontal and temporal lobe, indicating a possible frontotemporal lobar dementia in addition to the ALS disorder. Our morphological and biochemical findings were tested in vivo by generating homozygous transgenic mice that over expressed wt-hSOD1. These mice developed a fatal ALS-like disease, mimicking the one seen in mice expressing mutated hSOD1. The wt-hSOD1 mice showed a slower weight gain compared to non-transgenic mice and developed a progressive ALS-like hind-leg paresis. Aggregates of misfolded wt-hSOD1 were found in the brain and spinal cord neurons similar to those in humans accompanied by a loss of 41 % of motor neurons compared to non-transgenic litter mates. In conclusion, we found misfolded wt-hSOD1 aggregates in the cytoplasm and nuclei of motor neurons and glial cells in all patients suffering from ALS syndrome. Notable is the fact that misfolded wt-hSOD1 aggregates were also detected in FALS patients lacking SOD1 mutations indicating a role for SOD1 even when other genetic mutations are present. The neurotoxicity of misfolded wt-hSOD1 protein was confirmed in vivo by wt-hSOD1 transgenic mice that developed a fatal ALS-like disease. Taken together, our results support the notion that misfolded wt-hSOD1 could be generally involved and play a decisive role in the pathogenesis of all forms of ALS.

Systemic sclerosis (scleroderma, SSc) is a complex autoimmune disease with a highly variable array of clinical features and often fatal outcome. The disease is characterized by microvascular dysfunction, immune abnormalities, chronic inflammation and tissue fibrosis in the skin and internal organs. Chi3L1 is part of the innate stress response of connective tissue cells and elevated serum levels have been observed in SSc. It has long been established that the degree of skin involvement is useful to classify SSc patients, as it often associates with distinct patterns of organ involvement, disease severity and survival. Dermal cells are one of the main effector cells involved in the development of fibrotic lesions in the skin, and their biological activity is regulated by a variety of cytokines and growth factors. In this study the capacity of dermal cells from SSc patients and from healthy individuals to synthesize Chi3L1 and the regulation of this process by growth factors and cytokines were investigated. The cell types that express Chi3L1 in the dermis of SSc patients were also characterized.This study involved 61 dermal cell preparations from skin biopsies taken from the forearm and abdomen of 41 SSc patients and 15 dermal cell preparations from 10 control individuals. The cells were maintained in monolayer cell culture, and stimulated with cytokines and growth factors: IL-1, IL-4, IL-10, IL-13, IL-6, IL-17, OSM, ET-1, TNF-α, TGF-β or PDGF in the absence of serum. Chi3L1 in culture media was analyzed by SDS-PAGE and immunoblotting. Results demonstrated that some SSc dermal cells endogenously express Chi3L1; Chi3L1 expression is variably upregulated in SSc in response to IL-1 and OSM, with OSM having a more prominent effect; Chi3L1 is never found in healthy control skin cell preparations; dermal cells from forearm skin are more likely to produce Chi3L1 than those derived from abdominal skin; and patients with early disease are more likely to produce Chi3L1 consistently in dermal cells derived from both arm and abdomen irrespective of other disease characteristics. Further characterization of Chi3L1-expressing SSc dermal cells showed that only about 5-10% of the total cell population produced Chi3L1 spontaneously. While OSM, a pro-inflammatory cytokine nearly doubled Chi3L1-expressing cells; TGF-β, a pro-fibrotic growth factor downregulated Chi3L1 expression by the cells. Cells that express Chi3L1 also did not stain positively for α-smooth muscle actin, which identifies mature myofibroblasts, suggesting that Chi3L1 is not directly associated with fibrosis. The cells that endogenously produced Chi3L1 also expressed nestin, CD73, STRO-1, TIE2, LSP-1 and NG2 (all of which are progenitor/stem cell markers). These cells were not detected in normal dermal cell preparations. Some SSc dermal cell preparations also contain a population of fibroblast-related cells capable of secreting Chi3L1 when stimulated by OSM. Thus endogenous and OSM induced expression of Chi3L1 identified cell-population(s) in the dermis of SSc patients which may be associated with the transient stage in the transition of progenitor/stem cells to fibroblast-related cells to mature fibroblasts. Therefore, Chi3L1 expression may be a marker of "active" cells in the early establishment of SSc to expand the pool of fibroblasts in the dermis of SSc patients.<br>La sclérodermie systémique (sclérodermie ou SSc) est une maladie auto-immune complexe qui présente un ensemble très variable de caractéristiques cliniques et est souvent fatale. La maladie est caractérisée par un disfonctionnement micro vasculaire, une inflammation chronique ainsi qu'une fibrose dans le tissu cutané et les organes internes.Chi3L2 fait partie de la réponse inhérente au stress des cellules des tissus conjonctifs et des niveaux sériques ont été observés dans des cas de SSc. Il a été établi, il y a longtemps, que le niveau d'atteinte cutanée aide à la classification des patients atteints de SSc étant donné qu'il est associé aux différents schémas d'atteinte des organes, sévérité de la maladie et le taux de survie. Les cellules dermiques sont impliquées dans le développement des lésions fibriques de la peau et leur activité biologique est régularisée par nombre de cytokines et de facteurs de croissance. La capacité des cellules dermiques de patients atteints de SSc et de sujets normaux à synthétiser du Chi3L1 et le régulation de ce processus par des facteurs de croissance et des cytokines a été au centre de cette étude. Les cellules exprimant le Chi3L1 dans le derme des personnes atteintes de SSc a également été caractérisé.Cette étude comporte 61 préparations de cellules dermiques prises de biopsie de peau de l'avant-bras et d'abdomen provenant de 41 patients atteints de SSc et de 15 préparations cde cellules provenant de 10 sujets témoins. Les cellules ont été maintenues en culture cellulaire monocouche et stimulées par des cytokines et facteurs de croissance suivants : IL-1, IL-4, IL-10, IL-13, IL-6, IL-17, OSM, ET-1, TNF-, TGF- ou PDGF en absence de sérum. La présence de Chi3L1 en milieu de culture a été analysée par SDS-PAGE et immunoblot. Les résultats démontrent que certaines cellules SSc dermiques expriment du Chi3L1 de façon endogène; le Chi3L1 est exprimé positivement de façon variable en réponse au IL1 et l'OSM, ce dernier ayant un effet plus prononcé. Chi3L1 n'est jamais trouvé dans les cellules de sujets témoins; les cellules dermiques d'avant-bras sont plus sujettes à produire du Chi3L1 que celles de l'abdomen et il est probable que les patients atteints plus tôt produisent du Chi3L1 d'une façon plus constante dans les cellules dermiques des avant-bras et de l'abdomen nonobstant les autres caractéristiques de la maladie. Une caractérisation plus poussée des cellules dermiques exprimant le Chi3L1 a démontré qu'à peine de 5 à 10% des cellules totales produisent du Chi3L1 de façon spontanée. Tandis que l'OSM, une cytokine pro-inflammatoire a doublé le nombre de cellules dermiques exprimant le Chi3L1, le TGF-b, un facteur de croissance profibrotique a régulé à la baisse l'expression de Chi3L1. Les cellules exprimant le Chi3L1 ne colorent pas pour l'actine de muscle lisse, qui caractérise les myo-fibroblastes matures, suggérant que le Chi3L1 n'est pas associé à la fibrose. Les cellules produisant le Chi3L1 de façon endogène ont aussi exprimé pour le nestine, CD73, STRO-1, TIE2, LSP-1 et NG-2 (qui sont tous des marqueurs de cellules souches/progénitrices). Ces cellules n'ont pas été détectées dans les préparations de cellules dermiques témoins. Quelques préparations cellulaires dermiques SSc contiennent aussi une population de cellules associées aux fibroblastes qui peuvent sécréter Chi3L1 quand elles sont stimulées par l'OSM. Donc, l'expression de Chi3L1 endogène ainsi que induite par l'OSM a permis d'identifier des populations de cellules dermiques dans les patients atteints de SSc qui pourraient être associés à une étape passagère dans la transition des cellules souches/progénitrices qui se développent en cellules de type fibroblastes en fibroblastes matures. En résumé, l'expression du Chi3L1 pourrait être un marqueur des cellules « actives » pour permettre un pool de fibroblastes dans le derme de patients atteints de SSc.

The mammary gland is a dynamic organ that undergoes the majority of its development in the postnatal period in four stages; mature virgin, pregnancy, lactation, and involution. Every stage relies on tightly regulated cellular proliferation, programmed cell death, and tissue remodeling mechanisms. Misregulation of autophagy, an intracellular catabolic process to maintain energy stores, has long been associated with mammary tumorigenesis and other pathologies. We hypothesize that appropriate regulation and execution of autophagy are necessary for proper development of the mammary ductal tree and maintenance of the secretory epithelia during late pregnancy and lactation. To test this hypothesis we examined the role of two genes during development of the mammary gland. Beclin1 (Becn1) is an essential autophagy gene. Since the Becn1 knockout model is embryonic lethal, we have generated a Becn1 conditional knockout (cKO). We used two discrete mammary gland-specific Cre transgenic lines to interrogate the role of BECN1 during development. We report that MMTV-CreD; Becn1fl/fl mice have a hyper-branching phenotype and WAP-Cre; Becn1fl/- mice are unable to sustain a lactation phase. Becn1 mutants exhibit abnormal glandular morphology during pregnancy and after parturition. Moreover, when autophagy is chemically inhibited in vitro, mammary epithelial cells have an increased mean number of lipid droplets per cell. MTOR inhibits autophagy upstream of BECN1; we looked higher in the regulatory pathway for regulatory candidates. It has been well characterized that Tuberous sclerosis complex 1 (TSC1), in a heterodimer with its primary binding partner TSC2, inhibits MTOR signaling via inhibition of RHEB. Using the Tsc1 floxed model we generated a mammary gland specific Tsc1 cKO and found that these mice phenocopy the Becn1 cKO mice, including a gross lactation failure. Tsc1 cKO glands have altered morphology, retained lipid droplets in secretory epithelia, and an overall increase in MTOR signaling. We show that TSC1 and BECN1 are interacting partners, and that the interaction is nutrient responsive. These results suggest that Becn1 and Tsc1 are necessary for proper mammary gland development and differentiation. Furthermore, we have demonstrated a novel murine protein-protein interaction and an important link between regulation of MTOR pathway and regulation of autophagy in a developmental context.

This thesis describes a new approach for molecular imaging of neuroinflammation in Multiple Sclerosis (MS). My aim was to use the 2nd generation TSPO radioligand 18F-PBR111 to explore the potential of Positron Emission Tomography (PET) targeting the 18-kDa Translocator Protein (TSPO), as an in vivo biomarker of activated microglia in MS patients. This thesis addresses three research objectives. First, I characterised 18F-PBR111 PET signal in healthy controls’ brains and tested how it is affected by the TSPO gene polymorphism at rs6971. Second, I measured 18F-PBR111 uptake across white matter volumes segmented using structural MRI measures related to MS neuropathology. Third, I explored how 18F-PBR111 uptake in the hippocampus correlated with depressive symptoms and to the brain functional connectivity of the hippocampus. Eleven patients with relapsing-remitting MS and 22 age-matched healthy controls underwent 18F-PBR111 PET and MRI scans. Structural and functional MRI sequences were used to define conventional MS neuropathological markers and for the assessment of functional connectivity, respectively. I discovered that white matter 18F-PBR111 PET signal in healthy volunteers varied with TSPO genotype and correlated positively with age. In patients with MS, signal intensity in MRI-defined lesions was higher than that in normal-appearing white matter and correlated with the historical rate of progression of their disability. Hippocampal 18F-PBR111 uptake was higher in the MS patient group than in healthy volunteers and correlated with both depressive symptoms and functional connectivity of the hippocampus with frontal, temporal and parietal cortex. I thus discovered that this 2nd generation TSPO PET radiotracer, used in humans for the first time in our study, is sensitive to MS neuropathology consistent with recognized patterns of microglial activation and that differences between subjects can be related to disability progression. I also have discovered a novel relationship between this measure of hippocampal microglial activation and affective symptoms of MS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, late-onset, progressive neurodegenerative disease. A familial form of ALS, autosomal dominant ALS8, is characterized by a mutation in an ER membrane protein, VAPB. To characterize the role of VAPB in motor neurons, two C. elegans models were generated: one expressing human VAPB-P56S and another with the knockdown of C. elegans VAPB ortholog, VPR-1. Overexpression of human VAPB in DA neurons caused backward locomotion defects, enhanced vulnerability to oxidative stress and premature neuronal death. Knockdown of vpr-1 in C. elegans recapitulated the loss of protein function believed to be associated with human cases of ALS8. It caused backward locomotion defects, such as uncoordination and slowed rates of movement, as well as age-dependent motor neuronal death. In both models, DA6 and DA7 were the most vulnerable motor neurons. Because of the unexpected developmental defects associated with the VAPB transgenic model, the knockdown of vpr-1 may be a better model to recapitulate the human disease. This model provides further support that ALS8 pathogenesis is due to a loss of VAPB protein function and can also be used to test drugs or treatments that may delay the onset of neuronal death.

Magnetic resonance imaging (MRI) of the brain is the mainstay of diagnosis and monitoring of multiple sclerosis (MS) patients. Conventional brain MRI imaging captures characteristic multifocal demyelinating white matter lesions (WML). However, these imaging measures do not effectively predict an individual patient’s future clinical condition, limiting the ability of clinicians to rationalize therapy. In part, this is because cMRI lacks sensitivity and specificity in detecting chronic diffuse and multi-focal inflammation mediated by activated microglia/macrophages. In this work, molecular imaging using second generation translocator protein (TSPO) positron emission tomography (PET) and advanced MRI sequences are used to investigate inflammation in the brains of MS patients. A reference tissue based approach is used to quantify uptake of the TSPO PET ligands, as a marker of activated microglia/macrophages. The TSPO PET brain signal in extra-lesional white matter (so-called “normal appearing white matter” or NAWM) and individual WML of MS patients are evaluated with two TSPO radioligands, [11C]PBR28 and [18F]PBR11, and compared with a group of healthy volunteers. Individual lesions are classified based on TSPO binding to test whether heterogeneity of microglial/macrophage activation in lesions could be detected in vivo in a way analogous to histopathological studies in MS brains. An exploratory analysis is performed of the relationships between [11C]PBR28 binding and the putative astrocyte marker [myo-inositol] using single voxel proton magnetic resonance spectroscopy (MRS). Finally, a longitudinal analysis is performed to explore how differences in baseline [11C]PBR28 binding relate to future changes in WML and brain volume changes after 1 year. TSPO binding was higher in the MRI NAWM for both second generation radioligands than in the white matter of healthy volunteers. Both radioligands demonstrated considerable heterogeneity in TSPO binding in NAWM and between lesions in the patients. When considering the lesions individually, lesions with diffusely high TSPO radioligand uptake were seen in all disease stages and treatment groups. This indicates that second generation TSPO PET imaging detects a range of active inflammation not detected by conventional MRI in MS patients and that the inflammation is ongoing at all disease stages and may not be completely suppressed even in patients on the most efficacious treatments with good clinical control of symptoms. In the exploratory analysis of MRS [myo-inositol] and PET [11C]PBR28 uptake, there was no correlation between the two measures for the whole cohort of MS patients. This is consistent with the notion that changes in the two measures are related to distinct processes or to elements of a common process with different time courses. In the longitudinal analyses, NAWM and WML [11C]PBR28 binding at baseline was correlated to enlarging T2 lesion volume change after 1 year, driven by the subgroup of relapsing remitting MS patients (RRMS). Baseline white matter TSPO binding was associated with the longitudinal brain atrophy changes in the secondary progressive MS (SPMS) subgroup of patients. These observations together suggest that during the earlier disease microglial activation may contribute to greater neuroaxonal loss locally in and around lesions, while in the later stages of disease, brain innate immune inflammation becomes more diffuse and contributes to global neurodegeneration. These findings were combined into a single multimodal model where baseline MRI and [11C]PBR28 binding independently contribute to the description of the subsequent enlarging T2 lesion volume changes after 1 year.

O complexo da esclerose tuberosa (TSC) é um transtorno clínico, com expressividade variável, caracterizado por hamartomas que podem ocorrer em diferentes órgãos. Tem herança autossômica dominante e é devido a mutações em um de dois genes supressores de tumor, TSC1 ou TSC2. Estes codificam para as proteínas hamartina e tuberina, respectivamente, que se associam formando um complexo macromolecular que regula funções como proliferação, diferenciação, crescimento e migração celular. As lesões cerebrais podem ser muito graves em pacientes com TSC e caracterizam-se por nódulos subependimários (SEN), astrocitomas subependimários de células gigantes (SEGA), tuberosidades corticais e heterotopias neuronais, podendo relacionar-se clinicamente à epilepsia refratária à terapia medicamentosa, deficiência intelectual, desordens do comportamento e hidrocefalia. O potencial de crescimento de SEGA até os 21 anos de idade dos pacientes exige acompanhamento periódico por exame de imagem e condutas clínicas ou cirúrgicas, conforme indicação médica. As lesões subependimárias têm sido explicadas por déficits de controle da proliferação, crescimento e diferenciação de precursores neuro-gliais na zona subventricular telencefálica. Embora a capacidade da tuberina em inibir a proliferação celular pela repressão do alvo da rapamicina em mamíferos (mTOR) esteja bem documentada, outros aspectos celulares do desenvolvimento de SEGA ainda não foram examinados. Assim, é importante estabelecer um sistema in vitro para o estudo de células da zona subventricular e testá-lo na análise das proteínas hamartina e tuberina. Neste sentido, o cultivo de neuroesferas em suspensão é muito apropriado. Neste estudo, buscamos relacionar a expressão e distribuição subcelular da hamartina e tuberina à capacidade proliferativa e de diferenciação das células de neuroesferas cultivadas in vitro a partir da dissociação da vesícula telencefálica de embriões de ratos normais. Analisamos a expressão e distribuição subcelular da hamartina e tuberina por imunofluorescência indireta em células entre a primeira e a quarta passagens das neuroesferas, sincronizadas nas fases G1 ou S do ciclo celular e após a reentrada no ciclo celular, através da incorporação de 5-bromo-2\'-desoxiuridina (BrdU) e imunofluorescência com anticorpo anti-BrdU. Em geral, células de neuroesferas apresentaram baixa colocalização entre hamartina e tuberina in vitro. A expressão da tuberina foi elevada em basicamente todas as células das esferas e fases do ciclo celular; ao contrário, a hamartina apresentou-se principalmente nas células da periferia das esferas. A colocalização entre hamartina e tuberina foi observada em células mais periféricas das esferas, sobretudo no citoplasma e, em G1, no núcleo celular. A proteína rheb, que conhecidamente interage diretamente com a tuberina, apresentou distribuição subcelular muito semelhante à desta. Ao carenciamento das células visando à parada do ciclo celular na transição G1/S, tuberina distribuiu-se ao núcleo celular em quase todas as células avaliadas e, de forma menos frequente, a hamartina também. À reentrada no ciclo celular pelo reacréscimo dos fatores de crescimento, avaliaram-se células com incorporação de BrdU ao seu núcleo celular, após 72 e 96 horas. Nestas, tuberina mostrou-se novamente no citoplasma de forma preponderante e hamartina manteve-se citoplasmática, em geral subjacente à membrana plasmática, em níveis mais baixos. Os grupos cujas células reciclaram por 72 ou 96 horas diferiram quanto ao aumento significativo da expressão da hamartina em células proliferativas no último. À diferenciação neuronal, aumentaram-se os níveis de expressão de hamartina observáveis à imunofluorescência indireta, tornando-se equivalentes àqueles da tuberina. Concluímos que as células de neuroesferas cultivadas em suspensão apresentam-se como um sistema apropriado ao estudo da distribuição das proteínas hamartina e tuberina e sua relação com o ciclo celular<br>The tuberous sclerosis complex (TSC) is a clinical disorder with variable expressivity, characterized by hamartomas that can occur in different organs. It has autosomal dominant inheritance and is due to mutations in one of two tumor suppressor genes, TSC1 or TSC2. These encode for the proteins hamartin and tuberin, respectively, which are associated in a macromolecular complex which functions as a regulator of cell proliferation, differentiation, growth and migration. TSC brain lesions may be severe and are characterized by subependymal nodules (SEN), subependymal giant cell astrocytomas (SEGA), neuronal heterotopias and cortical tubers, and may be clinically related to refractory epilepsy, intellectual disability, behavioral disorders and hydrocephaly. The growth potential of SEGA up to 21 years of age in TSC patients requires regular monitoring by imaging. Clinical and surgical interventions may be medically indicated. Subependymal lesions have been explained by deficient control of proliferation, growth and differentiation of neuro-glial progenitors from the telencephalic subventricular zone. While tuberin ability to inhibit cell proliferation by repressing the mammalian target of rapamycin (mTOR) has been well documented, other cell aspects of SEGA development have not been thoroughly examined. Therefore, it is important to establish conditions for an in vitro system to study the cells from the subventricular zone and to test its suitability for the study of the TSC proteins. In this regard, the neurosphere suspension culture is very appropriate. We evaluated the expression and subcellular distribution of hamartin and tuberin in relation to the proliferation and differentiation capability of neurosphere cells derived in vitro from the dissociation of the telencephalic vesicle of normal E14 rat embryos. These analyses were performed by indirect immunofluorescence in cells from first through fourth passages of neurospheres, synchronized in G1 or S phases of the cell cycle, and after reentry into the cell cycle by the addition of 5-brome-2\'-desoxyuridine (BrdU) and immunolabeling with anti-BrdU antibody. In general, neurosphere cells presented low colocalization between hamartin and tuberin in vitro. Tuberin expression was relatively high in basically all neurosphere cells and cell cycle phases, whereas hamartin distributed mainly to cells from the periphery of the spheres. In these cells, hamartin and tuberin colocalization was evident mostly in the cytoplasm and, in G1, also in the cell nucleus. Rheb, which is known to interact directly with tuberin, had subcellular distribution very similar to tuberin. Cell starvation indicating cell cycle arrest at G1/S redistributed tuberin to the cell nucleus in virtually all cells examined, what was accompanied by nuclear location of hamartin in a small subset of cells. When cells were allowed to reenter cell cycle by adding growth factors, we evaluated BrdU-labeled nuclei 72 and 96 hours later. In the two groups, tuberin was shown to move back to the cytoplasm as well as hamartin, which apparently maintained its lower expression levels distribution underneath the plasma membrane. Group of cells that recycled for 96 hours had significantly more expression of hamartin than those cells that cycled for only 72 hours. After neuronal differentiation, hamartin expression levels observed by immunofluorescence were similar to those of tuberin. We conclude that neurosphere cells cultured in suspension showed to be an appropriate cell system to study hamartin and tuberin distribution in respect to the cell cycle

Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.

Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.

La presenza di disturbi cognitivi nei pazienti con Sclerosi Multipla (MS) era già stata descritta nel 1877 da Charcot; comunque solo negli ultimi 30 anni si è assistito ad una attenzione degli studiosi in merito alla comprensione delle compromissioni cognitive quantitative e qualitative presenti in questi pazienti. Circa il 45-65% dei pazienti con MS mostra disfunzioni cognitive di una certa entità, tali disturbi vanno da disturbi selettivi di specifiche funzioni a una compromissione grave e diffusa. Le disfunzioni cognitive frequentemente osservate nei pazienti con MS, sono associate con alterazioni della materia grigia, atrofia cerebrale e alterazioni della connettività e plasticità sinaptica. Recenti studi mostrano come anchvi e l'infiammazione acuta può esacerbare i deficit cognitivi indipendentemente dal sistema funzionale primariamente coinvolto.In questo studio noi misuriamo i livelli di amiloide beta 1-42 e proteina tau in pazienti con MS e CIS (Clinical Isolated Syndrome) , da sempre le due proteine sono state associate con il deterioramento cognitivo presente nei pazienti con malattia di Alzheimer (AD). Nella AD, l'amiloide beta 1-42 si accumula nel tessuto nervoso in placche extracellulari insolubili; questo fenomeno rende possibile la spiegazione del fatto che la forma solubile della beta 1-42 amiloide sia ridotta nel liquido cefalo rachidiano di questi pazienti. Nel nostro campione di pazienti affetti da MS, i livelli di amiloide beta 1-42 erano significativamente più bassi in pazienti con deficit cognitivi e erano inversamente correlati con il numero di lesioni GD+ alle immagini della risonanza magnetica (MRI). Sono state inoltre evidenziate correlazioni positive tra i livelli di amiloide beta 1-42 e i deficit di attenzione e concentrazione. Inoltre, l'anormale neuroplasticità della corteccia cerebrale è stata esplorata con TBS (theta brust magnetic stimulation). Da questa analisi è emerso che in pazienti con deficit cognitivi c'è una correlazione positiva tra i livelli di amiloide beta 1-42 presenti nel CSF e l'ampiezza dei long-term-potentiation (LPT) indotti dalla TBS. Nessuna correlazione è stata invece individuata tra la concentrazione della proteina tau, MRI, parametri cognitivi e gli effetti della TBS in questi pazienti. Insieme questi risultati indicano che nella MS l'infiammazione del SNC è capace di alterare il metabolismo della beta amiloide, riducendo la sua concentrazione nel liquido cerebro spinale e portando ad un danneggiamento della plasticità sinaptica e delle funzioni cognitive.<br>Cognitive dysfunction is of frequent observation in Multiple Sclerosis (MS). It is associated with grey matter pathology, brain atrophy and altered connectivity, and recent evidence showed that acute inflammation can exacerbate mental deficits independently of the primary functional system involved. In the present study, we measured cerebrospinal fluid (CSF) levels of amyloid-1-42 and tau protein in MS and in Clinical Isolated Syndrome (CIS) patients, since both proteins have been associated with cognitive decline in Alzheimer's disease (AD). In AD, amyloid-1-42 accumulates in the brain as insoluble extracellular plaques, possible explaining why soluble amyloid-ß1–42 is reduced in the CSF of these patients. In our sample of MS patients, amyloid-1-42 levels were significantly lower in patients cognitively impaired and were inversely correlated with the number of Gadolinium-enhancing (Gd+) lesions at the magnetic resonance imaging (MRI). Positive correlations between amyloid-1-42 levels and measures of attention and concentration were also found. Furthermore, abnormal neuroplasticity of the cerebral cortex, explored with theta burst magnetic stimulation (TBS), was observed in cognitively impaired patients, and a positive correlation was found between amyloid-1-42 CSF contents and the magnitude of long-term potentiation (LTP)-like effects induced by TBS. No correlation was conversely found between tau protein concentrations and MRI findings, cognitive parameters, and TBS effects in these patients. Together, our results indicate that in MS central inflammation is able to alter amyloid- metabolism, by reducing its concentration in the CSF, and leading to impairment of synaptic plasticity and cognitive function.

La sclérose latérale amyotrophique est une maladie neurodégénérative associée à un dysfonctionnement métabolique. Des altérations du métabolisme des lipides, décrites chez les patients SLA et les animaux modèles, pourraient participer à la mise en place des premières étapes de la maladie. L’objectif de cette thèse était d’étudier le rôle de la stéaroyl-coenzyme A désaturase 1 (SCD1), une enzyme clé du métabolisme des lipides, dans la SLA. En étudiant le profil d’acides gras périphériques dans un modèle de souris SLA, les souris SOD1m, nous avons vu une diminution de l’activité de la SCD1 dès les stades précoces (subcliniques) de la maladie. Cette diminution pourrait expliquer, à elle seule, les altérations du métabolisme des lipides caractéristiques de la SLA. La répercussion de la perte de l’activité de la SCD1 sur l’axe moteur a été étudiée. Une délétion du gène ou une inhibition pharmacologique de la SCD1 améliore la récupération fonctionnelle après lésion du nerf sciatique chez la souris sauvage. Nous avons cherché à voir si la perte d’activité de la SCD1 trouvée chez les souris SOD1m est un mécanisme de protection mis en place pour lutter contre l’évolution de la SLA. Nous avons traité des souris SOD1m avec un inhibiteur de l’activité de la SCD1. Le traitement a conduit à une augmentation du métabolisme oxydatif, une préservation de l’intégrité neuromusculaire ainsi qu’une amélioration de la survie des motoneurones. Nousconcluons que l’inhibition de la SCD1 représente une cible thérapeutique prometteuse dans la SLA<br>Amyotrophic lateral sclerosis is a neurodegenerative disease, associated with metabolic dysfunction. Alteration of lipid metabolism has been documented in ALS patients and animal models, and could participate to the first pathological steps of the disease. The objective of this thesis was to study the role of stearoyl-CoA desaturase 1 (SCD1), a key enzyme of lipid metabolism, in ALS. By studying the profile of peripheral fatty acids in an animal model of ALS, the SOD1 mice, we found that SCD1 activity was strongly reduced at early (sub-clinical) disease stage, and that this reduction could explain in itself the alteration of lipid metabolism characteristic of ALS. The impact of loss of SCD1 activity for the motor axis was then studied. Genetic deletion or pharmacological inhibition of SCD1 enhanced functional recovery after sciatic nerve injury in mice. Wefurther explored if the loss of SCD1 activity found in SOD1 mice is a protective mechanism elicited in response to ALS. We treated SOD1 mice with an inhibitor of SCD1 activity. The treatment resulted in exacerbated muscular oxidative metabolism,preservation of neuromuscular integrity and enhanced motor neuron survival. We conclude that inhibition of SCD1 represents a promising therapeutic target for ALS

Indiana University-Purdue University Indianapolis (IUPUI)<br>Sequestration at the aggresome and degradation through autophagy are two approaches by which a cell can counteract the toxic effect of misfolded proteins. Tuberous sclerosis (TS) and cancer cells can become dependent on autophagy for survival due to the high demand for protein synthesis, thus making protein clearance a potential therapeutic target. Because of its histone deacetylase (HDAC) inhibitory activity, we hypothesized that 4-phenylbutyrate (4-PBA) inhibits HDAC6 and aggresome formation to induce TS cell death. We found that 4-PBA treatment increases cell death and reduces bortezomib-induced aggresome formation. To link these results with HDAC inhibition we used two other HDAC inhibitors, trichostatin A (TSA) and tubastatin, and found that they also reduce bortezomib-induced protein aggregation. Because tubulin is a target of HDAC6, we next measured the effect of the HDAC inhibitors and 4-PBA treatment on tubulin acetylation. As expected, tubastatin increased tubulin acetylation but surprisingly TSA and 4-PBA did not. Because 4-PBA did not significantly inhibit HDAC6, we next hypothesized that 4-PBA was alternatively inducing autophagy and increasing aggresome clearance. Surprisingly, autophagy inhibition did not prevent the 4-PBA-induced reduction in protein aggregation. In conclusion, we found 4-PBA to induce cell death and reduce aggresome levels in TS cells, but we found no link between these phenomena. We next hypothesized that loss of the Ral guanine nucleotide exchange factor Rgl2 induces cell death via autophagy inhibition in pancreatic adenocarcinoma (PDAC) cells. KRas is mutationally activated in over 90% of PDACs and directly activates Rgl2. Rgl2 activates RalB, a known regulator of autophagy, and Rgl2 has been shown to promote PDAC cell survival. We first confirmed that loss of Rgl2 does increase cell death in PDAC cells. Initial experiments using doubly tagged fluorescent p62 and LC3 (autophagy markers) suggested that loss of Rgl2 inhibited autophagosome accumulation, but after developing a more sophisticated quantitation method we found loss of Rgl2 to have no effect. We also measured endogenous LC3 levels, and these experiments confirmed loss of Rgl2 to have no effect on autophagy levels. Therefore, loss of Rgl2 increases cell death in PDAC cells, but does not have a significant effect on autophagy.

A number of proteins have been identified which are pathologically and/or genetically associated with both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). A hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene is the largest genetic factor associated with FTD and ALS. Additionally, pathological inclusions of the TDP-43 protein are found in brains of approximately 50% of FTD cases and 90% of ALS cases. However, the normal function of these two proteins and how this relates to the degeneration of neurons in disease are not yet well understood. Accumulating evidence suggests that both proteins may have involvement with the neuronal cytoskeleton. The cytoskeleton is of particular interest in FTD/ALS due to significant axon pathology and loss observed in post mortem cases. It is known that the cytoskeleton is often a key effector to changes in axon integrity in many neurodegenerative diseases. This thesis examined the normal functions of TDP-43 and C9ORF72, and their links with the cytoskeleton through use of mouse primary cell culture, histological analysis of intact mouse brain, and viral-mediated expression of proteins of interest in a retina model in the mouse. TDP-43 is a predominantly nuclear protein, with much known about its nuclear roles in DNA and RNA binding, and regulation of transcription and translation. Evidence suggests that TDP-43 is also important for neurite outgrowth, remodelling and can regulate many components of the neuronal cytoskeleton. The effects of overexpression of TDP-43 in primary cortical neurons were examined and demonstrated distinct alterations to actin-associated cellular processes including neurite branching and growth cone morphology, as well as down-regulation of actin-binding proteins in the proteome of these cells. To examine the effect of pathogenic alterations to TDP-43 in vivo AAV2 virus was used to mediate the expression of wildtype human TDP-43 and human TDP-43 with a mutation to the nuclear localisation signal (NLS) in mouse retinal ganglion cells, through intraocular injection of the virus. These changes induced axon pathology in the optic nerve, with indications of perturbed axonal transport due to presence of organelle accumulation. Several mechanisms for how the non-coding repeat expansion in C9ORF72 may lead to disease have been described, however little is known about the normal function and the expression pattern of the C9ORF72 protein. This thesis characterized the expression pattern and cellular localisation of the three reported mouse isoforms of C9ORF72 in cell culture and in vivo, and demonstrated that C9ORF72 was present in synaptosomes and within actin-rich structures of neurons. In summary, these results indicate that both C9ORF72 and TDP-43 may have links to the neuronal cytoskeleton, and in particular the actin cytoskeleton. Modulation of the neuronal cytoskeleton is a compelling target for providing therapeutic protection to vulnerable cellular components, such as the axon. Studies such as those described in this thesis may provide insight for whether TDP-43- and C9ORF72-related FTD/ALS are candidates for these types of interventions.

Oral cancer or oral squamous cell carcinoma (OSCC) is a subset of head and neck cancer (excluding non-melanoma skin cancer), with an estimated 354,864 new cases and 177,384 deaths reported annually. India accounts for 34% of the world's oral cancer burden with an incidence of 119,992 and mortality of 72,616 cases annually, second only to breast cancer. Despite advancements in the therapeutic approaches, the prognosis of OSCC fares poorly, with an overall 5-year survival rate ranging from 34% to 62.9%. The PI3K/AKT/mTOR pathway, which regulates fundamental cellular processes like proliferation, cell cycle, metabolism, development, and differentiation, is most commonly deregulated in cancers, including OSCC. TSC1 (Tuberous sclerosis complex 1), by forming a complex with TSC2 and TBC1D7, negatively regulates this pathway by inhibiting the activation of mTORC1. Downregulation of TSC1 is reported in oral as well as several other cancers and is associated with an unfavorable clinical outcome in patients. The expression and function of TSC1 are regulated by various mechanisms like promoter methylation, loss-of-heterozygosity (LOH), loss of function mutations, and phosphorylation. In recent years, increasing evidence suggests the involvement of microRNAs (miRNAs) in oral carcinogenesis by acting as tumor suppressors or oncogenes. In this study, we worked on the post-transcriptional regulation of TSC1 by miR-130a and its role in the pathogenesis of OSCC. The current study clearly illustrates that miR-130a represses the expression of TSC1 at both the transcript and protein levels. The dual-luciferase reporter assay confirms the direct interaction between miR-130a and TSC1 3'UTR. Overexpression of miR-130a activates the PI3K/AKT/mTOR pathway, while its inhibition via antagomiR-130a repressed the pathway. Expression analysis of miR-130a and TSC1 in matched normal and oral tumor tissues through RT-qPCR analysis shows an inverse correlation in a majority (19/36) of OSCC samples, suggesting the physiological significance of their interaction. Further, miR-130a increases the cell proliferation, invasion, and anchorage-independent growth of OSCC cells by targeting the 3'UTR of TSC1. The mice injected with antagomiR-130a treated OSCC cells showed reduced tumor weight and volume compared to those injected with mock treated cells, which confirms the oncogenic role of miR-130a. Taken together, these results suggest that miR-130a exerts its oncogenic function in OSCC, in part, by regulating TSC1, whereby the inhibition of miR-130a holds potential in OSCC therapeutics and perhaps in other cancers as well.

Human profiin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profiin-1 is monomeric and possesses a well-defied secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profiin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic florescence. Refolding from the urea unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic florescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS florescence, which indicates the conversion into a transiently populated collapsed state possessing solvent exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profiin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as five mutations, namely C71G, M114T, G118V, A20T and T109M have been found in patients with familial forms of the disease and others, E117G and Q139L, has been proposed to be a moderate risk factor for disease onset. In this second part of the thesis we have purified the four profilin-1 variants along with the wild type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina<br>Oligodendrócitos são as células da glia do sistema nervoso central (CNS) responsáveis pela produção das bainhas de mielina. Estas estruturas permitem a insolação dos neurónios, melhorando a transmissão dos impulsos elétricos. Danos nas bainhas de mielina, acompanhados por uma incorreta diferenciação dos oligodendrócitos, são a base de um grupo de patologias conhecidas como doenças desmielinizantes. Estas são caraterizadas por uma transmissão anormal do impulso nervoso, o que leva a sintomas como convulsões e paralisia. Este é o caso da esclerose múltipla (MS), onde falhas na diferenciação de oligodendrócitos e na remielinização culminam na morte de células neurais. A MS é a doença desmielinizante mais comum no planeta, afetando mais de 2.3 milhões de pessoas no mundo. Estudos recentres identificaram alterações nas propriedades biofísicas da matriz extracelular (ECM) do CNS de pacientes com MS, o que sugere que a rigidez da ECM possa ser importante na fisiopatologia desta doença. Em contraste, outras doenças desmielinizantes, como a distrofia muscular congénita tipo 1A (MDC1A), originam de uma falta de merosina (MN) na ECM, a qual é uma proteína que promove a diferenciação de oligodendrócitos. A rigidez da ECM tem sido extensivamente estudada como uma forma de modular mecanicamente a diferenciação e as funções celulares. Em oligodendrócitos de rato, a diferenciação é otimizada quando as células são diferenciadas em substratos compatíveis com a rigidez do cérebro de 6.5 kPa. Isto levou a um aumento da expressão da proteína básica da mielina (MBP) e este efeito foi acentuado pela presença de MN. Resultados não publicados do nosso laboratório relacionam o aumento da expressão de MBP com a translocação nuclear da proteína de especificidade 1 (Sp1), de uma forma dependente da rigidez do substrato. O Sp1 já está descrito como um promotor da síntese de MBP. No entanto, esta foi a primeira vez que este processo foi descrito em resposta a um estímulo mecânico. Recorrendo a géis de poliacrilamida (PAHs) com diferentes graus de rigidez como substratos para cultura celular, nós propusemo-nos a investigar o mecanismo por detrás da ativação do Sp1 em condições compatíveis com o cérebro. Para tal, avaliámos se a transdução de sinal dependia de integrinas, funcionalizando os géis com poli-d-lisina (PDL) ou uma combinação de PDL/MN. Ademais, a fosforilação do Sp1pela ERK foi estudada como uma possível forma de regulação da atividade do Sp1. Por fim, desafiámo-nos a revelar possíveis alvos do Sp1 para além da transcrição de MBP. O nosso interesse foi focado em determinar se o Sp1 conseguia promover a saída do ciclo celular durante a diferenciação de oligodendrócitos. Os nossos resultados revelaram que a mecanotransdução acionada pela rigidez extracelular que leva à translocação nuclear do Sp1 é independente da ativação de integrinas. Além disso, a fosforilação do Sp1 não está relacionada com a sua translocação nuclear. Contudo, a ERK foi capaz de regular a atividade do Sp1 indiretamente, evidenciando a existência de moléculas intermediárias que faltam identificar nesta via. A atividade do Sp1 não levou a um aumento de p21, todavia, substratos menos rígidos promoveram a síntese desta proteína, o que sugere que estas rigidezes favorecem a saída do ciclo celular em oligodendrócitos em diferenciação. Tomados em conjunto, estes resultados elucidam uma nova via que pode melhorar a diferenciação de oligodendrócitos. A aquisição de mais conhecimentos sobre a mecano-modulação da síntese de MBP mediada pelo Sp1 em resposta às propriedades biofísicas do ambiente extracelular pode levar à descoberta de um novo alvo terapêutica que promova a diferenciação de oligodendrócitos em doenças desmielinizantes.<br>Oligodendrocytes are the glial cells from the central nervous system (CNS) responsible for the production of myelin sheaths. These structures allow for neuron insolation, improving the transmission of electric impulses. Damages in myelin sheaths accompanied by defective oligodendrocyte differentiation are the foundation of a group of diseases known as demyelinating disorders. These disorders are characterized by abnormal nervous impulse transmission, which leads to symptoms such as convulsions and paralysis. This is the case of multiple sclerosis (MS) where failed oligodendrocyte differentiation and remyelination culminate in neuronal cell death. MS is the most common demyelinating disease in the world, affecting over 2.3 million people worldwide. Recent studies identified changes in the biophysical properties of the extracellular matrix (ECM) of the CNS in MS patients, which suggests that ECM stiffness may be important in the pathophysiology of this disease. In contrast, other demyelinating disorders, such as congenital muscle dystrophy type 1A (MDC1A) result from a lack of merosin (MN) in the ECM, which is a protein that promotes oligodendrocyte differentiation. ECM stiffness has been extensively studied as a way to mechanically-modulate cell fate and function. In rat oligodendrocytes, differentiation is optimized when cells are differentiated in brain-complaint substrates of 6.5 kPa. This resulted in an increased expression of the myelin basic protein (MBP). This effect was further accentuated by the presence of MN. Unpublished data from our lab correlated the increased MBP expression with the nuclear translocation of the specificity protein 1 (Sp1), in a substrate stiffness dependent manner. Sp1 has been known to promote MBP synthesis, however this is the first time this process was described as a response to mechanical stimuli. Using poly-acrylamide hydrogels (PAHs) of varying stiffnesses as substrates for cell culture, we set out to investigate the mechanism behind Sp1 activation in brain-complaint conditions. To accomplish this, we assessed if the signal transduction was integrin-dependent, by functionalizing the substrates with poly-d-lysine (PDL) or a combination of PDL/MN. Furthermore, Sp1 phosphorylation via ERK was studied as a possible regulator of Sp1 activity. Finally, we set out to uncover possible downstream targets of Sp1 besides MBP. We were mainly interested in determining if Sp1 could promote cell cycle exit during oligodendrocyte differentiation. Our results revealed that the mechanotransduction triggered by extracellular stiffness that leads to Sp1’s nuclear translocation is integrin-independent. Moreover, the phosphorylation of Sp1 did not correlate with its nuclear translocation. However, ERK was able to regulate Sp1 activity indirectly, evidencing the presence of intermediary unidentified molecules in this pathway. Sp1’s activity did not lead to an increase in p21, however, soft substrates promoted the synthesis of this protein, suggesting that these stiffnesses favor cell cycle arrest of differentiating oligodendrocytes. Taken together, these results shed light on a new uncharacterized pathway that can improve oligodendrocyte differentiation. Further understanding on how the biophysical properties of the extracellular environment can modulate Sp1-mediated MBP synthesis could uncover a new therapeutic target to promote oligodendrocyte differentiation in demyelinating disorders.<br>FCT

TDP-43 est une protéine multifonctionnelle possédant des rôles dans la transcription, l'épissage des pré-ARNm, la stabilité et le transport des ARNm. TDP-43 interagit avec d'autres hnRNP, incluant hnRNP A2, via son extrémité C-terminale. Plusieurs membres de la famille des hnRNP étant impliqués dans la réponse au stress cellulaire, alors nous avons émis l’hypothèse que TDP-43 pouvait y participer aussi. Nos résultats démontrent que TDP-43 et hnRNP A2 sont localisés au niveau des granules de stress, à la suite d’un stress oxydatif, d’un choc thermique, et lors de l’exposition à la thapsigargine. TDP-43 contribue à la fois à l'assemblage et au maintien des granules de stress en réponse au stress oxydatif. TDP-43 régule aussi de façon différentielle les composants clés des granules de stress, notamment TIA-1 et G3BP. L'agrégation contrôlée de TIA-1 est perturbée en l'absence de TDP-43. En outre, TDP-43 régule le niveau d`ARNm de G3BP, un facteur de granule de stress de nucléation. La mutation associée à la sclérose latérale amyotrophique, TDP-43R361S, compromet la formation de granules de stress. Ainsi, la fonction cellulaire de TDP-43 s'étend au-delà de l’épissage; TDP-43 est aussi un composant de la réponse cellulaire au stress central et un acteur actif dans le stockage des ARNs.<br>TDP-43 is a multifunctional protein with roles in transcription, pre-mRNA splicing, mRNA stability and transport. TDP-43 interacts with other hnRNPs, including hnRNP A2, via its C-terminus and several hnRNP family members are involved in the cellular stress response. This relationship led us to investigate the role of TDP-43 in cellular stress. Our results demonstrate that TDP-43 and hnRNP A2 are localized to stress granules, following oxidative stress, heat shock, and exposure to thapsigargin. TDP-43 contributes to both the assembly and maintenance of stress granules in response to oxidative stress and differentially regulates key stress granules components including TIA-1 and G3BP. The controlled aggregation of TIA-1 is disrupted in the absence of TDP-43. In addition, TDP-43 regulates G3BP mRNA levels, a stress granule nucleating factor. A mutation associated with amyotrophic lateral sclerosis, TDP-43R361S, compromises stress granule formation. Thus, the cellular function of TDP-43 extends beyond splicing and places TDP-43 as a participant of the central cellular response to stress and an active player in RNA storage.

La sclérose en plaques (SEP) est caractérisée par des infiltrations périvasculaires de cellules immunitaires et par de la démyélinisation au sein du système nerveux central (SNC). Ces deux paramètres de la maladie sont associés à la fragilisation de la barrière hémato-encéphalique (BHE). En ce sens, le recrutement des cellules présentatrices d’antigène (CPA) myéloïdes, telles que les monocytes, les macrophages et les cellules dendritiques, dans le SNC à travers la BHE, est une étape cruciale dans l’initiation et la persistance de l’inflammation cérébrale. Nerve injury-induced protein (Ninjurin)-1 est une nouvelle molécule d’adhérence qui médie une interaction de type homophilique et dont l’expression sur l’endothélium vasculaire de la BHE humaine fut identifiée grâce à une analyse protéomique des protéines associées à la BHE. Les résultats présentés dans ce mémoire montrent que l’expression de Ninjurin-1 augmente dans un contexte inflammatoire dans les cultures primaires de cellules endothéliales de la BHE (CE-BHE) et sur les CPA myéloïdes humaines ex vivo et générées in vitro. De plus, les CPA infiltrantes retrouvées dans les lésions cérébrales de patients atteints de SEP et dans le SNC des souris atteintes d’encéphalomyélite autoimmune expérimentale (EAE), le modèle murin de la SEP, expriment de hauts niveaux de Ninjurin-1. À l’aide du modèle in vitro de la BHE, la neutralisation de Ninjurin-1 restreint spécifiquement la migration des monocytes à travers les CE-BHE sans affecter le recrutement des lymphocytes, ni la perméabilité des CE-BHE. Enfin, les souris atteintes d’EAE et traitées avec un peptide bloquant dirigé contre Ninjurin-1 présentent une maladie moins sévère ainsi qu’une diminution des CPA infiltrant le SNC et ce comparé au groupe contrôle. Ces résultats suggèrent que Ninjurin-1 est une molécule d’adhérence de la BHE impliquée dans le recrutement de CPA myéloïdes au sein du SNC et qu’elle peut être considérée comme une cible thérapeutique potentielle en SEP.<br>Multiple Sclerosis (MS) is characterized by perivascular infiltrations of immune cells and by demyelination in the central nervous system (CNS). These two hallmarks of the disease are associated with the disruption of the blood-brain barrier (BBB). The recruitment of monocytes, macrophages and dendritic cells, the so-called myeloid antigen-presenting cells (APCs), in the CNS through the BBB is thought to play a crucial role in the initiation and the persistence of the disease. Therefore the identification of the molecular mechanisms involved in the migration of myeloid APCs into the CNS is considered a valid therapeutic option in MS. Nerve injury-induced protein (Ninjurin)-1, a novel adhesion molecule that mediates homophilic binding, was found to be expressed in the vascular endothelium of the BBB following a proteomic screen of human BBB-associated proteins. Ninjurin-1’s expression increases during an inflammatory context in primary cultures of endothelial cells of the BBB (BBB-ECs) and on ex vivo and in vitro generated myeloid APCs. In addition, infiltrating APCs in human MS lesions and in the CNS of the murine model of MS, the mice affected with experimental autoimmune encephalomyelitis (EAE), express high levels of Ninjurin-1. Using an experimental model of the BBB, the neutralization of Ninjurin-1 specifically restricts the migration of monocytes across the BBB-ECs without affecting the recruitment of lymphocytes or the permeability of the BBB-ECs. Finally, EAE mice treated with a Ninjurin-1 blocking peptide have reduced disease severity and a reduced infiltration of myeloid APCs in the CNS, as compared to the control group. Our results show that Ninjurin-1 is an adhesion molecule of the BBB involved in the recruitment of myeloid APCs to the CNS and is also a potential therapeutic target to dampen CNS inflammatory processes, as occurs in MS.

La sclérose en plaques (SEP) est une maladie inflammatoire du système nerveux central (SNC) caractérisée par une infiltration périvasculaire de cellules mononucléaires, telles que les lymphocytes T CD4+ et CD8+, les lymphocytes B ainsi que les cellules myéloïdes qui comprend les monocytes, les macrophages et les cellules dendritiques (DCs). Ce phénomène d’infiltration est dû à une fragilisation de la barrière hémato-encéphalique (BHE). L’entrée des cellules immunitaires au SNC va mener à la destruction de la gaine de myéline et donc à l’apparition de plaques de démyélinisation. Ainsi, nous avons émis l’hypothèse que la migration des divers sous-types de cellules immunitaires du sang périphérique à travers la BHE est contrôlée par des mécanismes moléculaires distincts et spécifiques à chaque type cellulaire. Afin de répondre à cette hypothèse, quatre différentes études ont été mises sur pieds. En premier lieu, nous démontrons un effet bénéfique des statines sur la BHE en SEP, en diminuant la migration des lymphocytes T et des monocytes, et en diminuant la diffusion de marqueurs moléculaire soluble. Ce phénomène s’opère via la suppression du processus d’isoprenylation, et en empêchant probablement la contraction des cellules endothéliales de la BHE. De plus, nous démontrons que les monocytes qui migrent au SNC en condition inflammé sont en mesures de se différencier en DCs et d’induire une réponse inflammatoire de la part des lymphocytes T CD4+. La migration des monocytes à travers la BHE est contrôlée par une nouvelle molécule d’adhérence nommée Ninjurin-1. Le blocage de Ninjurin-1 conduit à une inhibition spécifique de la migration des monocytes in vitro, ainsi qu’à une amélioration des signes cliniques du modèle animal de la SEP, soit l’encéphalomyélite auto-immune expérimentale (EAE). Finalement, nous démontrons que la migration des lymphocytes T CD8+ au SNC s’effectue via l’intégrine alpha-4. De plus, la majorité des lymphocytes T CD8+ que l’on retrouve dans le liquide céphalo-rachidien de patients SEP, dans le SNC de souris EAE ainsi que dans le SNC de souris infectée au virus de l’hépatite murine portent un phénotype effecteur mémoire. Ces données pourraient expliquer l’émergence de leucoencéphalopathie multifocale progressive observée chez certains patients SEP traités au natalizumab, un anticorps dirigé contre l’intégrine alpha-4. En conclusion, notre étude a permis de démontrer l’importance des monocytes provenant de la périphérie dans le processus inflammatoire prenant part au SNC en SEP. L’inhibition d’entrée de ces cellules pourrait s’avérer bénéfique en SEP tout en permettant l’immuno-surveillance du cerveau, ce que l’anti-alpha-4 intégrine ne permet pas. Les statines pourraient s’avérer une autre option intéressante puisqu’elles agissent sur les processus inflammatoires impliqués dans la SEP.<br>Multiple sclerosis (MS) is an immune-mediated disorder of the central nervous system (CNS) characterized by multifocal areas of leukocyte infiltration and demyelination associated with a breakdown of the blood-brain barrier (BBB). Typically, demyelination is centered around perivascular accumulation of CD4+ and CD8+ T lymphocytes, monocytes, macrophages and dendritic cells (DCs) that arise from migration of peripheral blood immune cells across the CNS microvascular endothelium. We have thus suggested that the migration across the BBB of immune cells subsets from the blood is controlled by molecular mechanism specific for each cell type. To answer this hypothesize, we have performed four different studies. We first show a beneficial effect of statins on the BBB, restricting the migration of lymphocytes and monocytes as well as the diffusion of soluble molecular tracers. This phenomenon is mediated through abrogation of isoprenylation processes that is probably inhibiting the ability of endothelial cells of the BBB to contract. We also show that CD14+ monocytes migrate across the inflamed human blood BBB and differentiate into DCs in response to BBB-secreted TGF-beta and GM-CSF. These DCs then promote the proliferation and expansion of inflammatory CD4+ T lymphocytes. We demonstrate that the migration of monocytes is controlled by a new adhesion molecule called Ninjurin-1. Ninjurin-1 neutralization specifically abrogated the adhesion and migration of human monocytes across endothelial cells of the BBB, without affecting lymphocyte recruitment. Moreover, Ninjurin-1 blockade reduced clinical disease activity and histopathological indices of experimental allergic encephalomyelitis (EAE). Finally we show that migration of CD8+ T lymphocytes across BBB is dependent on alpha-4 integrin. Also, the majority of CD8+ T lymphocytes found in the cerebrospinal fluid of MS patients, and in the CNS of EAE mice as well as the CNS of mouse infected with hepatitis virus are showing an effector memory phenotype. These data could explain the numerous cases of progressive multifocal leukoencephalopathy seen in natalizumab treated MS patients. In conclusion, our study unveils an important role of peripheral monocytes in MS. The inhibition of migration of these cells to the CNS could be a beneficial therapy since it would allow immune surveillance of the brain. The statins could also be a very interesting option since these molecules would reduce the inflammatory processes involved in MS.