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Pan, Tak Yin. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Content --- p.v / Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease --- p.1 / Chapter 1.1.1 --- Cholinergic hypothesis --- p.2 / Chapter 1.1.2 --- p-amyloid hypothesis --- p.2 / Chapter 1.1.3 --- Taupathy hypothesis --- p.3 / Chapter 1.1.4 --- Current therapies --- p.4 / Chapter 1.2 --- Proteins Involved in Alzhemer's Disease --- p.5 / Chapter 1.2.1 --- Acetylcholinesterase (AChE) --- p.5 / Chapter 1.2.2 --- p-amyloid --- p.6 / Chapter 1.2.3 --- Paired helical filaments (PHF) --- p.7 / Chapter 1.2.4 --- Protein kinases --- p.8 / Chapter 1.2.4.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.9 / Chapter 1.2.4.2 --- Cyclin-dependent kinase-5 (CDK-5) --- p.9 / Chapter 1.2.5 --- Protein phosphatase (PP) --- p.10 / Chapter 1.2.5.1 --- Protein phosphatase 1 (PP-1) --- p.11 / Chapter 1.2.5.2 --- Protein phosphatise 2A (PP-2A) --- p.12 / Chapter 1.2.5.3 --- Protein phosphatise 2B (PP-2B) --- p.13 / Chapter 1.2.6 --- Apoptotic and Anti-apoptotic proteins --- p.14 / Chapter 1.2.6.1 --- Caspase-3 --- p.15 / Chapter 1.2.6.2 --- Bcl-2 --- p.15 / Chapter 1.3 --- Flavonoids --- p.16 / Chapter 1.3.1 --- Biosynthesis of flavonoids --- p.17 / Chapter 1.3.2 --- Biological functions of flavonoids in plants --- p.18 / Chapter 1.3.3 --- Beneficial effects of flavonoids on human health --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- Differentiation of SHSY-5Y cells --- p.20 / Chapter 2.1.1 --- SHSY-5Y cell culture --- p.20 / Chapter 2.1.2 --- Counting cells --- p.20 / Chapter 2.1.3 --- Retinoic acid differentiation --- p.21 / Chapter 2.2 --- Western blot analysis --- p.21 / Chapter 2.2.1 --- Extraction of proteins from mammalian cells --- p.21 / Chapter 2.2.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.22 / Chapter 2.2.3 --- Semi-dry protein transfer to nitrocellulose membrane --- p.23 / Chapter 2.2.4. --- Membrane blocking and immunostaining --- p.24 / Chapter 2.3 --- MTT assay --- p.25 / Chapter 2.4 --- Hoechst 33342 Nuclei staining --- p.25 / Chapter 2.5 --- Cell cycle analysis --- p.25 / Chapter 2.5.1 --- Ethanol fixation --- p.25 / Chapter 2.5.2 --- Propidium iodide staining --- p.26 / Chapter 2.6 --- Annexin V-FITC & Propidium iodide staining --- p.26 / Chapter 2.7 --- DNA fragmentation analysis --- p.26 / Chapter 2.7.1 --- Phenol/Chloroform extraction of DNA --- p.26 / Chapter 2.7.2 --- Ethanol precipitation of DNA --- p.27 / Chapter 2.7.3 --- Agarose gel electrophoresis of DNA --- p.27 / Chapter 2.8 --- Proteomic analysis --- p.28 / Chapter 2.8.1 --- First dimension: isoelectric focusing --- p.28 / Chapter 2.8.2 --- Second dimension: SDS PAGE --- p.29 / Chapter 2.8.3 --- Gel staining --- p.30 / Chapter 2.8.3.1 --- Silver staining --- p.30 / Chapter 2.8.3.2 --- SYBRO Ruby staining --- p.31 / Chapter 2.8.4 --- Gel scanning and image analysis --- p.31 / Chapter 2.8.5 --- ln-gel digestion --- p.32 / Chapter 2.8.6 --- Zip Tip for desalting the digested sample --- p.33 / Chapter 2.8.7 --- Protein identification with mass spectrometry and database search --- p.33 / Chapter Chapter 3: --- Characterization of Okadaic acid-induced tail hyperphosphorylation in SHSY-5Y cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Objectives --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Differentiation of SH-SY5Y cell --- p.38 / Chapter 3.3.2 --- Changes of protein expression after okadaic acid treatment --- p.40 / Chapter 3.3.3 --- Neurite Retraction Induced by okadaic acid --- p.42 / Chapter 3.3.4 --- Okadaic acid-induced Cell Death measured by MTT assay --- p.44 / Chapter 3.3.5 --- Hoechst 33342 Nuclei Staining --- p.44 / Chapter 3.3.6 --- Cell cycle analysis by propidium iodide staining --- p.47 / Chapter 3.3.7 --- Early Apoptotic cells detection by Annexin V/PI staini --- p.49 / Chapter 3.3.8 --- DNA fragmentation --- p.51 / Chapter 3.4 --- Discussion --- p.53 / Chapter Chapter 4: --- Flavonoids screening for protecting neuronal death by preventing tau hyperphosphorylation --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Objectives --- p.58 / Chapter 4.3 --- Tested flavonoids --- p.59 / Chapter 4.4 --- Results --- p.60 / Chapter 4.4.1 --- Toxicity of flavonoids --- p.60 / Chapter 4.4.2 --- Effects of flavonoid pre-treatment on OA-induced neu retractions and cell death --- p.62 / Chapter 4.4.3 --- Western blot analysis --- p.65 / Chapter 4.4.4 --- The effect of different concentrations of hesperidin or OA treatment --- p.70 / Chapter 4.4.5 --- Proteomic analysis --- p.74 / Chapter 4.5 --- Discussion --- p.78 / Chapter Chapter 5: --- General Discussion --- p.82 / References

Tauopathies are a group of neurodegenerative diseases characterized by the abnormal deposition of the protein tau, a microtubule stabilizing protein. Under normal physiological conditions tau is a highly soluble protein that is not prone to aggregation. In disease states alterations to tau lead to enhanced fibril formation and aggregation, eventually forming neurofibrillary tangles (NFTs). The exact cause for NFT deposition is unknown, but increased post-translational modifications and mutations to the tau gene can increase tangle formation. Tauopathic brains are stuck in a detrimental cycle, with cellular dysfunction contributing to the development of tau pathology and the development of tau pathology contributing to cellular dysfunction. The exact mechanisms by which each part of the cycle contributes to the other are still being explored. To investigate the unique contributions of each part of this cycle we utilized two separate models of tauopathy: one chronic and one acute. Overall this project provides novel insight into the role of pathological tau as both a cause, and a consequence, of cellular dysfunction. To understand how development of tau pathology contributes to cellular dysfunction we studied chronic disease models. Using human brain tissue we found that under normal conditions tau associates with ribosomes but that this interaction is enhanced in Alzheimer’s disease brains. We then used in vitro and in vivo models of tauopathy to show that this association causes a decrease in protein synthesis. Finally, we show that wild type tau and mutant tau reduce protein translation to similar levels. To understand how general cellular dysfunction contributes to development of pathology we used an acute model of tauopathy through traumatic brain injury (TBI). We injured rTg4510 tau transgenic mice at different ages to investigate the effect of TBI on tau fibrillization (2 month old) and the effect of TBI on tau already in NFTs (4.5 month old). In 2 month old mice, we found that tau hyperphosphorylation was decreased at 24 hours and increased at 7 days post injury, and that tau oligomerization was decreased at 24 hours post injury. We also found that tau fibrillization was not increased after 24 hours or 7 days post injury. In 4.5 month old mice, we found that TBI did not increase or decrease tangle counts in the brain, but we did qualitatively observe decreased variability within groups. Overall these studies contribute novel understanding of tau’s role in different disease states. We identified a functional consequence of the interaction between tau and ribosomes, and demonstrated that a single head impact did not increase tau fibril formation within 7 days of injury. While human diseases associated with TBI show neurofibrillary tangle deposition, we have yet to recreate that aspect of the disease in research models of TBI. Our findings support the need for further investigation into the nuances of tau in disease, especially following TBI.

The aging brain is prone to the development of pathology and dementia. With a rapidly growing elderly population diagnoses of neurodegenerative diseases, such as Alzheimer’s disease (AD), frontotemporal dementia (FTD), and Parkinson’s disease are on the rise. Additionally, diabetes and obesity are linked to an increased risk of dementia. The convergence of this increasingly aged population with the obesity and diabetes epidemic give rise to new concerns regarding the future of prevention and treatment of neurodegenerative diseases. Our lab has previously shown that leptin, an adipokine involved in signaling satiety to the hypothalamus, can modulate the generation of the amyloid beta (Aβ) peptide (a toxic peptide associated with neurologic disease) and attenuate hyperphosphorylation of the tau protein (another peptide prone to forming large insoluble structures causing neurodegeneration). From these studies we have elucidated that leptin resistant mice (which develop severe obesity and type-2 diabetes mellitus) with knock-in mutations for the amyloid precursor protein (APP) and presenilin-1 (PS1) genes develop extensive vascular pathology and cognitive impairments. Interestingly, these mice do not display increased levels of amyloid deposition in the brain. Additionally, increased tau phosphorylation occurs in these mice with leptin resistance. As a follow up to this study db mice were transduced, via adeno-associated virus, with the tau P301L mutant to induce the development of tangle pathology. These mice displayed no cognitive deficits, yet they displayed increases in both tau phosphorylation and tangle count within the hippocampus. Collectively, these studies indicate leptin resistance, obesity, and type-2 diabetes mellitus promote the development of cerebrovascular and neurofibrillary tangle pathologies associated with neurodegeneration and dementia. These observations open many previously unexplored avenues for developing novel therapeutics to treat these devastating diseases.

Les mécanismes physiopathologiques liés aux maladies neurodégénératives restent encore largement méconnus. Deux processus semblent être particulièrement en cause dans les phénomènes de neurodégénérescence : la neurotoxicité par afflux massif de calcium due à une activation excessive des récepteurs NMDA (GluN) et la neurotoxicité par déstabilisation du cytosquelette du neurone par le biais de la phosphorylation anormale de la protéine tau. L'imagerie moléculaire par le biais de la tomographie par émission de positons (TEP) et de radiotraceurs, en étudiant les mécanismes moléculaires in vivo, permet de détecter et quantifier ces phénomènes. Ce travail a eu pour objet d'étudier un dérivé de la mémantine, un antagoniste des GluN se fixant sur un site intra-canal accessible uniquement lorsque ces récepteurs sont activés ce qui en fait donc un vecteur d'imagerie intéressant pour étudier leur activation. Nous avons mis au point la synthèse d'un nouveau radiotraceur dérivé de la mémantine : la [18F]-FNM (Fluoroéthylnormémantine). Il s'agit d'une synthèse par substitution nucléophile d'un groupement tosylate par du [18F], suivie d'une hydrolyse acide. Cette synthèse est reproductible avec un rendement de 10%, son activité spécifique est > 355 GBq/µmol. Chez le rat, le traceur passe la barrière hémato-encéphalique et sa distribution cérébrale est bien corrélée avec la localisation des GluN (r=0.622, p<0.0001). Sa cinétique de fixation (40 minutes) est compatible avec son utilisation en TEP. En ce qui concerne les tauopathies, la protéine tau stabilise l'organisation microtubulaire. Lors d'une phosphorylation anormale, l'interaction avec les microtubules diminue et les protéines tau vont s'accumuler en formant des Paires de Filaments en Hélice (PHF). Nous avons optimisé la radiosynthèse de l' [18F]-AV1451 ciblant les PHF. Notre rendement de synthèse est de 30% et l'activité spécifique du traceur > 10 GBq/µmol. Nous avons réalisé des autoradiographies sur des coupes de cerveaux atteints de tauopathie et nous avons constaté la capacité du traceur à différencier les coupes saines des coupes malades. La production de cet outil dans notre centre va nous permettre d'étudier la présence de PHF chez le marmouset, un primate particulièrement intéressant dans l'étude du vieillissement. Nous avons donc réalisé la synthèse de deux radiotraceurs innovants : la [18F]-FNM et le [18F]-AV1451, les synthèses sont reproductibles et les rendements compatibles avec des productions de doses en recherche pré-clinique et clinique. / The pathophysiological mechanisms associated with neurodegenerative diseases remain largely unknown. Two processes appear to be particularly involved in the phenomena of neurodegeneration: neurotoxicity induced by massive influx of calcium caused by excessive activation of NMDA receptors (GluN) and neurotoxicity by destabilization of neuron cytoskeleton through abnormal protein tau phosphorylation. Molecular imaging through positron emission tomography (PET) and radiotracers, by studying the molecular mechanisms in vivo, allows to detect and quantify these phenomena. This work was intended to study a memantine derivative, a GluN antagonist. We chose to develop a ligand that selectively binds to the ion channel in the open and active state which therefore makes it an interesting vector to study their overactivation. We have developed the synthesis of a new memantine analogue radiotracer: the [18F]-FNM (Fluoroéthylnormémantine). This is a synthesis by nucleophilic substitution of a tosylate with [18F], followed by acid hydrolysis. This synthesis is reproducible with a yield of 10%, its specific activity was> 355 GBq / µmol. In rats, the tracer cross the blood-brain barrier and brain distribution correlates well with the location of GluN (r = 0.622, p <0.0001). The binding kinetics (40 minutes) is compatible with its use in PET. Regarding tauopathies, the tau protein stabilizes microtubule organization. During abnormal phosphorylation, interaction with microtubules and tau proteins decreases and tau will accumulate to form Paired helical Filament (PHF). We optimized the radiosynthesis of [18F] AV1451 targeting 3 tau PHF. Our yield of synthesis is 30% and the specific activity of the tracer> 10 GBq / µmol. We made autoradiography on brains sections and have shown tracer ability to differentiate healthy and pathological slices. This tool will allow us to study the presence of PHF in marmosets, a particularly interesting primate in the study of aging. So we performed the synthesis of two innovative radiotracers: the [18F]-FNM and [18F]-AV1451, syntheses are reproducible and yields compatible with doses manufacturing in pre-clinical and clinical research.

Alzheimer’s disease is a debilitating, progressive neurodegenerative disorder in the elderly, characterized by severe loss of memory and higher cognitive functions. In the hundred years since its discovery, Alzheimer’s disease (AD) has traversed from the status of a ‘rare neurological oddity’ to one of the greatest challenges faced by healthcare and medicine in this millennium. A reported 44 million people currently suffer from AD but only 1 in 4 people have been diagnosed. Although AD has been an area of intense research for almost 50 years now, most studies have focused on the end stage disease. Years of study on the pathological cause underlying AD; have conclusively shown that the accumulation of the sticky peptide, Aβ, is one of the major triggers of AD pathogenesis. However, after the initial Aβ trigger, multiple processes contribute to disease progression, so that by the time a patient is diagnosed on the basis of overt behavioral phenotypes, it is difficult to understand and differentiate between the causative mechanisms and the consequential effects of the disease. It is, perhaps, because of this, that we are still struggling to find therapies for AD which will stop or at the very least slow the course of the disease. In the 2015 report on AD, issued by the Alzheimer’s association, much emphasis has been placed on the early diagnosis of AD and the revision of the diagnostic criteria for AD. According to the new guidelines proposed in 2011, AD has been divided into three stages where the first stage occurs before the appearance of overt behavioral symptoms such as memory loss, whereas by the 1984 guidelines, cognitive disabilities must have already occurred for diagnoses of AD. This proposed preclinical stage of AD has been defined, reflecting the current belief that AD pathogenesis begins almost 20 years before the occurrence of behavioral dysfunction. However, no diagnostic criteria are currently available to establish this stage. Hence, there is a need to understand the early pathogenic mechanisms of AD, which will yield early therapeutic targets as well as early diagnostic markers of AD. One of the earliest documented events in AD pathogenesis is synaptic dysfunction, which is later manifested as loss of dendritic spines. Deficits in long term potentiation (LTP) has been demonstrated in Aβ exposed hippocampal slices as well as in mouse models of AD, much before the appearance of pathological hallmarks such as plaques and tangles as well as overt behavioral phenotypes. While these and other studies indicate clearly that elevated levels of soluble Aβ peptide leads to impairment of synaptic function, the underlying molecular mechanisms are yet to be elucidated. One of the purported mediators of Aβ induced dysfunction is oxidative stress. The Aβ peptide, especially the Aβ42, is a self aggregating peptide with a propensity to form peptidyl radicals. Interaction of the peptidyl radicals with biomolecules leads to the generation of more free radical species via cascading chain reactions. Additionally, Aβ peptide has also been demonstrated to have synaptotoxic effects via its effect on NMDA receptors and calcium influx leading to deregulated reactive oxygen species (ROS) production as well as excitotoxicity. Hence, with a view to understanding Aβ mediated early synaptic dysfunction in AD, we studied early signaling changes in the synaptosomes derived from the cortex of APP/PS1 mice model of AD at various ages. The APP/PS1 model contains a mouse/human chimeric APP gene bearing the KM670/671NL Swedish mutation and the human PS1 gene with an exon 9 deletion. These mice exhibit behavioral deficits from 7 months of age while plaque deposition and gliosis become apparent by 9 months of age. We chose to study both pre-symptomatic ages (1 and 3 months old) as well as post symptomatic (9 months old) mice. Post nuclear supernatant (PNS) as well as synaptosomes were isolated from the cortex of APP/PS1 and age matched control mice. We assayed the levels of reactive oxygen species (ROS) in the PNS and the synaptosomes of post symptomatic 9 months old APP/PS1 mice and age matched controls. In contrast to reports of enhanced oxidative stress markers in the brains of AD patients, we did not find any increase in the levels of ROS in the PNS of post symptomatic APP/PS1 mice compared to age matched controls. However, synaptosomes from the cortex of these animals exhibited a significant increase in ROS levels in APP/PS1 mice compared to controls. We further found that there was significant increase in the ROS levels in synaptosomes, but not PNS, of very young asymptomatic 1 and 3 months old APP/PS1 mice. This is a first demonstration of synapse specific increase in oxidative stress in AD mice, as young as 1 month of age, indicating that disease specific mechanisms operate at the synapse much before the appearance of any overt cellular or behavioral symptoms. The increase in synaptic ROS levels correlated with a small but significant increase in the levels of Aβ42 in the brains of APP/PS1 mice compared to controls. We also found a concurrent change in the redox status of the cytoskeletal protein, actin, at the synapse. As early as 1 month of age, there was a significant decrease in the protein level of reduced actin indicating that there is an increase in the level of oxidized actin at the synapse. This loss of reduced actin was specific to the fibrillar pool of actin while no significant change was observed in the redox status of the monomeric globular pool of actin. Oxidation of actin has been demonstrated to lead to its depolymerization. Concurrently, we found a significant loss of fibrillar actin in the synaptosomes of APP/PS1 mice. Actin is the major cytoskeletal protein at the synapse. Changes in the globular to fibrillar actin ratio at the synapse at early pre-symptomatic ages in APP/PS1 mice will likely lead to structural and consequent functional changes at the synapse. This could potentially be one of the triggers of synaptic dysfunction in AD. Furthermore, changes in the Akt-mTOR signaling pathway was also observed in the synaptosomes of 1 month old APP/PS1 mice, which is sustained at 9 months. There was a significant loss of the mTOR-pS6K-4EBP1 axis in the synaptosomes, but not PNS, of APP/PS1 mice. We found that loss of Akt signaling, as evinced by loss of Akt phosphorylation, Akt kinase activity as well as loss of phosphorylation of downstream effector GSK3β, potentially underlies the loss of mTOR signaling. Further, the loss of Akt signaling is mediated by synapse specific redox modification of Akt and consequent interaction with the protein phosphatase PP2a. Loss of the Akt-mTOR signaling at the synapse is indicative of deficits in local protein translation. Loss of this essential synaptic function, which plays critical roles in synapse maintenance as well as synaptic plasticity during learning and memory, at an early age, will have long ranging impact on synaptic function such as long term potentiation (LTP) in APP/PS1 mice. Our study is the first demonstration of oxidative stress and consequent signaling changes which occur specifically at the synapse of very young 1 month old APP/PS1 mice. These changes occur much before the appearance of overt phenotype such as plaque deposition and behavioral dysfunction but sustain till the appearance of classical pathological hallmarks. Hence, the study demonstrates that disease progression starts much before previously thought and provides us a critical time window during which therapeutic strategies designed to delay or stop these changes might change the course of AD.

No description available.

O presente estudo comparou o efeito do tratamento crônico com lítio em doses subterapêuticas (0,02mM e 0,2mM) e dose terapêutica (2mM) em cultura primária de neurônios corticais e hipocampais. As amostras foram analisadas e comparadas com o grupo controle (sem tratamento) tanto para os neurônios corticais, como para os neurônios hipocampais. O objetivo do estudo foi: 1) avaliar, nessas culturas celulares, a atividade de diferentes quinases (PKA, CaMKII, AKT e GSK3beta), diferentes sítio de fosforilação da Tau (Ser, 199, 205, 214, 396, C-terminal e seis isoformas), a partir da inibição da PLA2 pelo lítio; 2) Investigar as vias de sinalização envolvidas na modulação do estado de fosforilação da proteína tau a partir da inibição da PLA2 em culturas primárias de neurônios; 3) avaliar, simultaneamente, a expressão de fatores neurotróficos (BDNF) e citocinas (GM-CSF, IL-1b, IL-2, IL-4, IL-5, IL6, IL-10, IL-12, IFN-y e TNF-alfa) mediante o tratamento de neurônios primários com lítio e 4) avaliar expressão gênica por microarray das culturas tratadas com diferentes doses, subterapêuticas e terapêutica, de cloreto de lítio. Nossos resultados sugerem uma dissociação de efeitos em neurônios corticais dos observados em neurônios hipocampais. O lítio aumentou a atividade enzimática da iPLA2 e da cPLA2, tanto em neurônios corticais como em neurônios hipocampais. A atividade da GSK3beta foi inibida pelo tratamento crônico com lítio em neurônios hipocampais e apresentou efeito contrário em neurônios corticais. Observamos comportamentos diferentes das diferentes proteínas analisadas em culturas de neurônios corticais e hipocampais, e não tivemos significância estatística para as alterações na proteína tau. O tratamento nas doses subterapêuticas aumentou a secreção de citocinas pró-inflamatórias tanto em neurônios corticais quanto em neurônios hipocampais / The present study compared the effect of lithium chronic treatment with subtherapeutic doses (0.02mM and 0.2mM) and therapeutic dose (2mM) in primary cortical and hippocampal neurons cell culture. This samples were analyzed and compared with the control group (no treatment). The study\'s goal was: 1) to evaluate in these Cell Culture a different activity kinases (PKA, CaMKII, AKT and GSK3beta), different phosphorylation site of the Tau (199, 205, 214, 396, C-terminal and Six isoforms) and PLA2 inhibition; 2) To investigate how signaling pathways involved in modulation of tau phosphorylation from the inhibition of PLA2 in primary cultures of neurons; 3) analyze an expression of neurotrophic factors (BDNF) and cytokines (GM-CSF, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFNy TNFalfa 4) to evaluate gene expression via microarray with different doses of lithium treatment. Our results suggest a dissociation effects on cortical and hippocampal neurons cell culture. Lithium increased the enzyme activity of iPLA2 and cPLA2, in both cortical and hippocampal neurons. The GSK3beta the activity was inhibited by chronic treatment with lithium in hippocampal neurons and presented contrary effect on cortical neurons. We observe not statistics significance on tau protein. The treatment at subtherapeutic and therapeutic doses increased the secretion of pro and anti-inflammatory cytokines both in cortical and hippocampal neurons

Perineuronale Netze (PN) sind eine spezialisierte Form der neuronalen extrazellulären Matrix. Es wird vermutet, dass sie neuroprotektive Eigenschaften besitzen und die von ihnen umschlossenen Neurone gegenüber Degeneration schützen. Mehrere Studien untersuchten bereits die PN in Zusammenhang mit der Pathologie der Alzheimer-Erkrankung. Für die Frontotemporalen Demenzen, die nach der Alzheimer-Erkrankung und der vaskulären Demenz zu den häufigsten dementiellen Erkrankungen gehören, ist die Beziehung von PN und Tau-Protein (TP)-Pathologie noch nicht untersucht worden. Die Dissertation beschäftigt sich mit der Korrelation von netztragenden Neuronen mit TP-Pathologie in einem Mausmodel der Frontotemporalen Demenz mit Parkinsonismus des Chromosoms 17 (FTDP-17). Sie geht der Frage nach, ob netztragende Neuronen vor Degeneration und intrazellulären Ablagerungen von verändertem TP, in Form von Hyperphosphorylierungen (HP) und Neurofibrillären Tangles (NFT), geschützt sind. Mit Hilfe immunhistochemischer Fluoreszenzmarkierung und Western Blot wurden die Gehirne transgener Mäuse mit der P301L-Mutation des TP im Alter von drei und achteinhalb Monaten untersucht. Dabei wurden sechs Hirnregionen ausgewählt: Primärer Somatosensorischer Kortex (PSC), Entorhinaler Kortex (EC), Hippokampus (Hipp), Pars magnocellularis des Nucleus ruber (NR-m), Pars reticulata der Substantia nigra (SN-r) und Motorischer Trigeminuskern (Mo5). Die Untersuchungen zeigen, dass die PN bei der FTDP-17 die Neurone nicht explizit vor TP-Pathologie schützen. Jedoch kommt es zu keiner signifikanten Reduktion der netztragenden Zellen im Altersgang, sodass eine neuroprotektive Funktion der PN weiterhin vermutet werden kann.

O presente estudo comparou o efeito do tratamento crônico com lítio em doses subterapêuticas (0,02mM e 0,2mM) e dose terapêutica (2mM) em cultura primária de neurônios corticais e hipocampais. As amostras foram analisadas e comparadas com o grupo controle (sem tratamento) tanto para os neurônios corticais, como para os neurônios hipocampais. O objetivo do estudo foi: 1) avaliar, nessas culturas celulares, a atividade de diferentes quinases (PKA, CaMKII, AKT e GSK3beta), diferentes sítio de fosforilação da Tau (Ser, 199, 205, 214, 396, C-terminal e seis isoformas), a partir da inibição da PLA2 pelo lítio; 2) Investigar as vias de sinalização envolvidas na modulação do estado de fosforilação da proteína tau a partir da inibição da PLA2 em culturas primárias de neurônios; 3) avaliar, simultaneamente, a expressão de fatores neurotróficos (BDNF) e citocinas (GM-CSF, IL-1b, IL-2, IL-4, IL-5, IL6, IL-10, IL-12, IFN-y e TNF-alfa) mediante o tratamento de neurônios primários com lítio e 4) avaliar expressão gênica por microarray das culturas tratadas com diferentes doses, subterapêuticas e terapêutica, de cloreto de lítio. Nossos resultados sugerem uma dissociação de efeitos em neurônios corticais dos observados em neurônios hipocampais. O lítio aumentou a atividade enzimática da iPLA2 e da cPLA2, tanto em neurônios corticais como em neurônios hipocampais. A atividade da GSK3beta foi inibida pelo tratamento crônico com lítio em neurônios hipocampais e apresentou efeito contrário em neurônios corticais. Observamos comportamentos diferentes das diferentes proteínas analisadas em culturas de neurônios corticais e hipocampais, e não tivemos significância estatística para as alterações na proteína tau. O tratamento nas doses subterapêuticas aumentou a secreção de citocinas pró-inflamatórias tanto em neurônios corticais quanto em neurônios hipocampais / The present study compared the effect of lithium chronic treatment with subtherapeutic doses (0.02mM and 0.2mM) and therapeutic dose (2mM) in primary cortical and hippocampal neurons cell culture. This samples were analyzed and compared with the control group (no treatment). The study\'s goal was: 1) to evaluate in these Cell Culture a different activity kinases (PKA, CaMKII, AKT and GSK3beta), different phosphorylation site of the Tau (199, 205, 214, 396, C-terminal and Six isoforms) and PLA2 inhibition; 2) To investigate how signaling pathways involved in modulation of tau phosphorylation from the inhibition of PLA2 in primary cultures of neurons; 3) analyze an expression of neurotrophic factors (BDNF) and cytokines (GM-CSF, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFNy TNFalfa 4) to evaluate gene expression via microarray with different doses of lithium treatment. Our results suggest a dissociation effects on cortical and hippocampal neurons cell culture. Lithium increased the enzyme activity of iPLA2 and cPLA2, in both cortical and hippocampal neurons. The GSK3beta the activity was inhibited by chronic treatment with lithium in hippocampal neurons and presented contrary effect on cortical neurons. We observe not statistics significance on tau protein. The treatment at subtherapeutic and therapeutic doses increased the secretion of pro and anti-inflammatory cytokines both in cortical and hippocampal neurons