Dissertations / Theses on the topic 'Tau Effect'

This thesis investigates the concept of psychological time and, in particular, the concept of psychological present. Starting by the studies of the main authors who dealt with these issues, I delved into the relationship that space and time have within the present. The tau effect, a perceptual phenomenon discovered by Vittorio Benussi in 1907, is of particular interest for this field. The tau effect indeed shows that the duration of temporal intervals influences the perception of spatial distances in the sense of “equation” (e.g. briefer temporal intervals corresponded to perceptual shorter spatial distances). Even though this effect has been extensively investigated in the last decades, a conclusive theory doesn’t exist. Five experiments were hence performed to better understand this effect. The main result is that the tau effect could be found only when the Standard Stimulus (i.e. the Stimulus that does not vary during the whole experiment, Ss) is presented as first. Furthermore, results proved that this effect emerged only within the temporal limits of the psychological present (i.e. 2‐3 sec. on average). Since, according to the authors examined in this work, one of the features of present is its flexibility depending on individual differences, the possible correlations between the tau effect and personality traits have been also investigated. Results proved that the way space and time are intertwined is linked to Extroversion and Emotional Stability, as defined by the Big Five model. These results are in line with the literature on time perception. To explain the results about the order of presentation of Ss, an alternative explanation based on the general theories developed by Benussi is discussed. According to this alternative explanation, in our experiments the tau effect occurs only when Ss is presented as first because its temporal interval used is perceived as “indifferent” or “indeterminate”, that is not “long‐” or “short‐lived”. This condition would allow participants to judge only the spatial distances without being influenced by the corresponding temporal intervals of the first stimulus presented.

Decreased amyloid-ß42 (Aß42), increased total tau (t-tau) and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) reflect histopathological core changes in the most common dementia disorder, Alzheimer’s disease (AD). They discriminate AD from healthy controls and predict conversion to AD with a relatively high accuracy. Memantine, an uncompetitive NMDA-receptor antagonist, is indicated for symptomatic treatment of AD. The first aim of this thesis was to investigate effects of memantine on CSF concentrations of Aβ42, tau and p-tau. Secondly, the aim was to explore the relation between these CSF biomarkers and retention of the amyloid biomarker Pittsburgh compound B using positron emission tomography (PIB PET), regional glucose metabolism measured with 18Fluoro-2-deoxy-d-glucose (FDG) PET and neuropsychological test performance. The third aim was to investigate their possible utility as predictors of future rate of AD dementia deterioration. All patients in the studies were recruited from the Memory Clinic, Uppsala University Hospital. In study I CSF p-tau concentrations in 11 AD patients were reduced after twelve months treatment with memantine, indicating that this compound may affect a key pathological process in AD. Results from study II showed that the concentrations of CSF Aß42 are lower in PIB+ patients than in PIB- patients, and that the PIB retention was stable during 12 months. In study III 10 patients with the diagnoses AD (6 PIB+/4 PIB-) and 8 subjects (1 PIB+/7 PIB-) with frontotemporal dementia were included. PIB+ patients had lower psychomotor speed measured by performance on the Trail Making Test A and impaired visual episodic memory compared to the PIB- patients. The initial clinical diagnoses were changed in 33% of the patients (6/18) during follow-up. Study IV is the first-ever report of an association between high CSF tau and dying in severe dementia. These 196 AD patients were followed up to nine years after baseline lumbar puncture. Moreover, CSF t-tau concentrations above median was associated with an increased risk of rapid cognitive decline (OR 3.31 (95% CI 1.53-7.16), independently of baseline functional stage. Thus, a clear association between high levels of CSF t-tau and p-tau and a more aggressive course of the disease was shown.

La maladie d’Alzheimer (MA) représente aujourd’hui la forme de démence la plus commune pour laquelle il n’existe toujours pas de traitement curatif. Dans le cerveau, elle se caractérise par la présence de deux agrégats protéiques majeurs : les plaques amyloïdes qui résultent de l’accumulation extracellulaire d’un peptide nommé peptide amyloïde, et les enchevêtrements neurofibrillaires correspondant à l’agrégation intra-neuronale d’une protéine nommée Tau qui se trouve dans un état anormalement hyperphosphorylé. La pathologie Tau est importante puisque son étendue corrèle avec le degré du déficit cognitif retrouvé dans la MA. Seule une petite proportion des cas de la MA est causée par des mutations génétiques. En revanche, l’étiologie de la majorité des cas (~99%), qui est d’origine sporadique et à apparition tardive, semble être multifactorielle, avec des facteurs externes pouvant interagir avec des susceptibilités biologiques ou génétiques afin d’accélérer la manifestation de la maladie. Au cours de la dernière décennie, des données cliniques et précliniques émergentes suggèrent que le diabète sucré et la dysfonction de l’insuline qui l’accompagne pourrait représenter l’un de ces facteurs. En plus de son rôle métabolique, l’insuline a été rapportée pour avoir un rôle neurotrophique et régulateur dans le cerveau humain. Plus particulièrement, des études in vitro ont montré que l’insuline est capable de moduler la phosphorylation de Tau dans les cellules neuronales. Cette hypothèse a été par la suite renforcée par les observations d’hyperphosphorylation de Tau dans les cerveaux de souris montrant des anomalies au niveau de la signalisation de l’insuline. Malgré toutes ces données, on connaît très peu concernant l’impact du diabète sur la pathologie Tau in vivo. Le but global de ce projet de doctorat a été donc de clarifier l’impact du diabète sur la pathogenèse de la protéine Tau, dans deux modèles génétiques de diabète de type 1 (DT1) et diabète de type 2 (DT2), qui sont les souris NOD (non-obese-diabetic) et les souris ob/ob, respectivement. Nos résultats montrent que le DT1 entraine une hyperphosphorylation progressive de Tau qui commence à être détectée même en absence de toute dérégulation dans le métabolisme du glucose. De plus, cette hyperphosphorylation est plus prononcée en présence des caractéristiques du DT1 (hyperglycémie et glycosurie) et encore plus amplifiée en présence de l’hypothermie. D’une manière intéressante, nos résultats suggèrent que l’hyperphosphorylation de Tau chez ces souris corrèle avec une dérégulation de PP2A (protein phosphatase 2A), l’une des phosphatases les plus importantes de Tau in vivo. Quant au DT2, nos résultats montrent une hyperphosphorylation de Tau chez les souris ob/ob à 4 et 26 semaines, au niveau de plusieurs sites spécifiques. De plus, ces souris développent une hypothermie modérée, mais le rétablissement de la normothermie ne restaure pas les niveaux de phosphorylation de Tau, ce qui suggère que cette hyperphosphorylation serait plutôt la conséquence des composantes du DT2, et non pas de l’hypothermie qui en résulte. D’une manière intéressante, nos résultats ne montrent pas de dérégulation au niveau des protéines impliquées dans la voie de signalisation de l’insuline, suggérant par conséquent que, d’autres facteurs, probablement associés à l’obésité, pourraient contribuer à l’hyperphosphorylation de Tau dans le DT2. La compréhension des mécanismes qui sous-tendent la corrélation entre la dysfonction de l’insuline et la pathologie Tau aidera par la suite à trouver de nouvelles cibles thérapeutiques visant à contrôler la progression de la maladie.
Alzheimer’s disease (AD) is the leading form of dementia. There is actually no cure for AD, but even a treatment that would slow down the progression of the disease by 5 or 10 years will have a tremendous socio-economic impact for Canada. The neuropathological hallmarks of Alzheimer's disease include senile plaques of -amyloid (A) peptides (a cleavage product of the amyloid precursor protein, or APP), and neurofibrillary tangles (NFT) of hyperphosphorylated Tau protein assembled in paired helical filaments (PHF). NFT pathology is important since it correlates with the degree of cognitive impairment in AD. Only a small proportion of AD is due to genetic variants, the large majority of cases (~99%) is late onset and sporadic in origin. The cause of sporadic AD is likely to be multifactorial, with external factors interacting with biological or genetic susceptibilities to accelerate the manifestation of the disease. Diabetes mellitus (DM) might be such factor, as there is extensive data from epidemiological studies suggesting that DM is associated with an increased relative risk for AD. Type 1 diabetes (T1DM) and type 2 diabetes (T2DM) are known to affect multiple cognitive functions in patients. However, the consequences of both type of diabetes on AD pathology are not well understood. The challenge is therefore to better understand the mechanisms of AD pathology and how they are affected by factors such as diabetes. The overall goal of this project is therefore to clarify the impacts that diabetes have on Tau protein pathogenesis, in two well-characterized mouse models of T1DM and T2DM: NOD (non-obese diabetic) and ob/ob mice, respectively. Our data suggest that spontaneous T1DM provokes a progressive Tau hyperphosphorylation that begin to be detectable in adult mice even during the non-diabetic stage, where there is no apparent deregulation of glucose metabolism. We further show that Tau phosphorylation is greatly exacerbated in the presence of principal T1DM features, notably hyperglycemia and glycosuria, and further amplified by hypothermia. Finally, we demonstrate that Tau hyperphosphorylation during T1DM is likely attributable to a deregulation in PP2A (protein phosphatase 2A), the major Tau phosphatase in vivo. Furthermore, we show that ob/ob male mice aged 4 and 26 weeks present Tau hyperphosphorylation at specific sites, but also have mild hypothermia. However, restoring normothermia did not rescue Tau hyperphosphorylation to control levels. These data indicate that Tau hyperphosphorylation accompanies major features of T2DM. Interestingly, we did not observe any deregulation in the proteins implicated in the insulin-signaling pathway, suggesting that other obesity-associated factors, contribute to Tau phosphorylation in ob/ob mice. In turn, this understanding will help the development of treatments or life-style strategies destined to check the advance of the disease.

Systemic inflammation is thought to be an important driver in chronic neurodegeneration. During systemic infection, the inflammatory status of the periphery is communicated to the brain and conserved sickness behaviours initiated. However, in the context of dementia the same inflammatory stimulus might trigger delirium. Delirium is a severe, transient neuropsychiatric condition characterised by altered levels of arousal, inattention, cognitive deficits and psychoses. Delirium and systemic inflammation exacerbate the trajectory of pre-existing dementia, and are associated with increased risk of future dementia. Accumulating experimental studies suggest microglia are “primed” by chronic neurodegeneration, such that a subsequent inflammatory insult – central or systemic – induces an increased inflammatory response which manifests as exaggerated sickness behaviours. To date there have been no studies of microglial priming in the context of pure tau pathology, without amyloid pathology, and none investigating acute sickness behaviour in such a model. The overarching aim of this thesis is to address this gap in the literature and further our understanding of the interactions between systemic inflammation, neuroinflammation and neurodegeneration in the context of tauopathy. The P301S mouse over-expresses human mutant tau protein under the Thy1.2 promoter. It develops hyperphosphorylated and insoluble tau accumulations and progressive neuronal loss. Consequently, P301S mice develop progressive hind limb paralysis. This study identified the horizontal bar task, a test of motor control and coordination, conducted at weekly intervals from 8-22 weeks of age, as a non-invasive measure of disease progression. In addition, a detailed temporal profile of pathological hallmarks at 8, 9, 10, 11, 12, 16 and 20 weeks of age was determined. Key results presented here demonstrate progressive, superficial neuronal loss in the cortex of P301S mice, with associated astrogliosis and surprisingly this occurs in the absence of apparent cortical microgliosis. In stark contrast, there is progressive microgliosis in the spinal cord of P301S mice. On this background, lipopolysaccharide (LPS), a chemical moiety found on the outer surface of gram-negative bacteria, was used to mimic a systemic bacterial infection. P301S mice and C57BL/6 control mice were injected, at 10 or 16 weeks of age, intraperitoneally with 500 μg/kg LPS or saline and were monitored in the following hours and weeks. Acutely, P301S mice showed signs of an exaggerated, longer lasting sickness response. Importantly, exaggerated acute symptoms extended beyond those typically associated with sickness behaviour; LPS induced an exaggerated acute impairment of horizontal bar performance in P301S mice and not C57BL/6 mice – a function which is known to be impaired in P301S mice later in disease. Impairments were age-dependent in terms of timing of injection. These data suggest an interaction between acute infection and existing CNS vulnerability leading to acute neurological dysfunction that is not a feature observed in sickness in a normal animal. LPS-injected P301S mice also showed, again age-dependent, increased rate of decline in motor performance compared with controls. There was no evidence of microglial priming in P301S mice. LPS caused an acute increase in AT8-positive phospho-tau however this did not persist until end stage. At 22 weeks of age there was significant disease-associated cortical neuronal loss in the vehicle-injected P301S mice, and additional superficial cortical neuronal loss in LPS-injected P301S mice and control mice. There was significant IBA1-positive microgliosis in the spinal cord of P301S mice at end stage which was further increased in LPS-injected P301S mice. Taken together these data indicate a clear and clinically relevant interaction between systemic inflammation and tau-associated neuropathology with acute and long-term functional consequences. In the absence of evidence of microglial priming, future work will explore potential mechanisms.

Tauopathy, which results from the oligomerization of misfolded tau protein in neurons, is a feature present in a number of neurodegenerative diseases and a hallmark of Alzheimer’s Disease (AD). Tau is an important phosphoprotein that regulates the assembly of microtubules, but tauopathy can occur when tau becomes hyperphosphorylated. Phosphorylation prevents tau from binding to tubulin, which results in cytosolic accumulation of tau and eventual oligomerization. This abnormal accumulation of tau leads to the spreading of hyperphosphorylated tau to downstream synaptically connected neurons through an unknown mechanism. In AD, the hippocampus is one of the first brain structures to be affected by tauopathy in humans. According to previous research, tauopathy occurs primarily between principal cells in the hippocampus. The involvement of local inhibitory interneurons in tauopathy and their potential role in AD is more controversial. Previous research suggests that tau pathogenesis primarily affects principal cells; however, given the importance, diversity, and function of interneurons in the hippocampus, it is important to gain a better understanding of the interneuron subtypes that may be impacted by the spread of trans-synaptic tau into the hippocampus. Understanding the involvement of interneurons in trans-synaptic tau transmission is important to understanding neurodegeneration in AD and other neurodegenerative disorders. To investigate this, both male and female genetically-modified mice underwent surgery to examine the trans-synaptic spread of pathogenic tau (EGFP-Tau P301L) from the entorhinal cortex to hippocampal neurons. Histology and imaging analysis of brain sections were performed to examine the hippocampal cells impacted by trans-synaptic spread of tau. Results show that pathogenic tau can trans-synaptically spread from presynaptic neurons in the entorhinal cortex into downstream hippocampal interneurons and also that hippocampal interneurons are capable of trans-synaptically spreading tau. Future studies examining the specific subtypes of hippocampal interneurons vulnerable to trans-synaptic spread of tau will be important for a better understanding of disease progression, which could lead to uncovering new therapeutic targets for neurodegenerative diseases, like AD, which are associated with tauopathy.

L’hyperphosphorylation de la protéine Tau, à l’origine de la formation de dégénérescences neurofibrillaires et de la mort cellulaire observée, chez les patients atteints de la maladie d’Alzheimer, est causée par un déséquilibre entre l’activité des kinases et des phosphatases. Cette modification post traductionnelle touche de nombreux sites dans la Région Riche en Proline (RRP) de Tau. Or, des études RMN, réalisées au sein du laboratoire, ont montré que les polyphénols sont capables d’interagir avec des fragments de la protéine issues de la RRP avec des affinités de l’ordre de grandeur de celles décrites pour les kinases. Dans ce contexte, il a été envisagé de synthétiser une bibliothèque de polyphénols et de mettre au point une réaction de phosphorylation sur des peptides modèles afin d’étudier, par spectrométrie de masse, la capacité de ces composés à protéger Tau contre l’attaque des kinases
Hyperphosphorylation of Tau protein, which leads to their abnormal aggregation into neurofibrillary tangles and to neuronal loss observed in patients with Alzheimer disease, is regulated by a disequilibrium between kinases and phosphatases activities. This post traductional modification affects several residues, in particular in the Proline Rich Region of Tau (PRR). But, NMR studies, realized in our laboratory, have shown that polyphenols are able to interact with peptide Tau models issued from the PRR and the affinity are in the same range that those described for the kinases. In this context, we have envisaged to synthetize several polyphenols and to develop a phosphorylation reaction of peptide models in order to study, by mass spectrometry, the ability of these compounds to protect Tau against kinase attack

The purpose of this dissertation is to chart how the development of visual effects has changed popular cinema¡¯s vision of the real, producing the powerful reality effect. My investigation of the history of visual effects studies not only the industrial and economic context of visual effects, but also the aesthetic characteristics of the reality effect. In terms of methodology, this study employs a theoretical discourse which compares the parallels between visual effects and the discourse of modernity/postmodernity, utilizing close textual analysis to understand the symptomatic meanings of key texts. The transition in the techniques and meanings of creating visual effects reflects the cultural transformation from modernism to postmodernism. Visual effects have developed by adapting to the structural transformation of production systems and with the advance of technology. The studio system strongly controlled the classical Hollywood cinema by means of the modern economic production system of Fordism. Breakdown of Hollywood classicism as a production system gave rise to the creation of digital effects with the rise of the concept of the blockbuster and with the development of computer technologies. I argue that the characteristic feature of time-space compression, occurring in the process of the transition from Fordism to flexible accumulation, clearly reflects that of compression of multi-layered time and space, generated in the development process from analog visual effects, such as trick, rear and front projection, to the digital effects, such as rotoscoping and CGI animation. While the aesthetics of analog visual effects, without computing, can be compared to a Fordist production system, digital effects, which hugely rely on CGI manipulation, are examples of flexible accumulation. As a case study of the local resistance or alternative of Hollywood today, I examine the effects-oriented Korean nationalist blockbuster. The Korean nationalist blockbuster films have sought large-scale filmmaking and presentation of spectacular scenes, including heavy dependence on the use of special effects, which is frequently considered a Hollywood style. This paradoxical combination of peculiar Korean subjects and Hollywood style can be viewed as a form of cultural jujitsu, taking advantage of the force of the dominant culture in order to resist and subvert it.

This research examines the presence and effectiveness of the ‘normalising effect’, traditionally offered as the main justification for tax effect accounting’s (TEA) adoption. TEA can be seen as a technical facet of accounting practice, ‘normalising’ the timing differences between the accounting and taxation systems. That is, income tax is recognised according to when transactions are recognised for accounting purposes in order to ‘normalise’ reported profits, thereby reflecting an income statement focus. It has been contended that this will improve the usefulness of financial reports by ‘correcting’ misleading and ‘unreal’ fluctuations in income tax. Australia’s adoption of AIFRS in 2005 entailed a major conceptual re-alignment of the methodology underpinning TEA, moving away from the income statement focus in favour of a balance sheet focus. This implied a different normalisation emphasis. It is within this contemporary setting, based on a study of 90 companies over the two regulatory periods between 2002 and 2011 (AGAAP and AIFRS), that a quantitative measure of the presence and effectiveness of the normalising effect was undertaken, additionally considering the subsequent balance sheet impact. Effective normalisation was revealed during the AGAAP period, whilst only effective after the removal of loss makers during the AIFRS period. These findings suggest that the relaxation of recognition criteria under AIFRS may have had a meaningful impact on the effectiveness of the new standard. However, when normalisation was given a more narrow definition in light of prima facie tax, deferred taxes had a more substantial impact, particularly during the AIFRS period. Such findings are consistent with the notion thatTEA enables reported tax to be ‘as if’ it were a function of accounting, without a substantial build up on the balance sheet as a consequence. These findings have implications for evaluating the efficacy of TEA and comprehending the nature of contemporary financial statements.
Doctor of Philosophy

More than 1.2 million people are estimated to be currently living with the human immunodeficiency virus (HIV) in the United States of America. The gastrointestinal (GI) tract is both a major target and an important component of HIV pathogenesis. The GI processes that are dysregulated during HIV infection are controlled by the enteric nervous system (ENS). Indeed, both clinical and experimental studies have implicated the ENS in HIV and simian immunodeficiency virus (SIV) pathogenesis. In addition to direct viral effects, the HIV virus also indirectly affects the GI tract via cellular and/or viral toxins released by infected cells. Trans-activator of transcription (Tat) is a viral toxin that plays an important role in replication of the HIV virus. While, the HIV virus does not directly infect neurons, Tat has been shown to modulate neuronal function. HIV infection in the gut is accompanied by: translocation of bacteria and bacterial products from the gut lumen to peripheral blood, immune activation and inflammation. Lipopolysaccharide (LPS) is a major bacterial product that is used to determine the rate of bacterial translocation and to drive inflammation. Despite reports of enteric ganglionitis in SIV infected monkeys and autonomic denervation in the jejunum of HIV patients, little is known of the mechanism underlying enteric neuropathogenesis in HIV and the role of the ENS in HIV pathogenesis. In the present study, we assessed the effects of Tat on enteric neuronal excitability and how Tat and LPS interact in the ENS to bring about inflammation and GI motility problems observed in HIV patients. We show that Tat significantly increased enteric neuronal excitability by modulating sodium channels expressed on enteric neurons. Tat sensitized ENS cells to LPS-mediated increase in pro-inflammatory cytokines via a TLR4-mediated pathway involving MyD88. Mice expressing the tat transgene (Tat+) had faster GI transit rates and significantly higher frequencies of diameter changes in the proximal ileum than controls (Tat-). Tat+ mice were also more sensitive to LPS-mediated decreases in colonic transit rate. This study highlights the role of viral and bacterial proteins in HIV pathogenesis in the gastrointestinal tract and also demonstrates a critical role of the ENS in HIV pathogenesis.

Acquired immunodeficiency syndrome (AIDS) is a major problem in our current society. There are over 35 million of the population that are currently living with human immunodeficiency virus (HIV), and the number of HIV-infected patients are still rising every year in spite of our efforts to control it. Furthermore, within the AIDS affected population, opportunistic infection is a major cause of complications and is the number one cause of death. The HIV trans-activator (Tat) protein plays a major role in the AIDS pathogenesis. HIV-1 Tat is known to cause dysregulation of cytokines such as TNF-α, IL-6, and IL-10 in AIDS patients. In this study we recognized a proto-oncogene, c-Myc, could regulate the cytokine dysregulation caused by HIV-1 Tat in primary blood monocyte derived macrophages (PBMac). By knocking down the expression of c-Myc with gene specific small-interfering RNA (siRNA), we demonstrated that c-Myc may be critical for the expression of the pro-inflammatory cytokines TNF-α and IL-6. HIV-1 Tat was subsequently found to regulate the expression of c-Myc via the activation of dsRNA-activated protein kinase (PKR), ERK1/2 and p38 mitogen-activated protein kinase (MAPK). Furthermore, c-Myc regulation of the pro-inflammatory cytokines was demonstrated to have a role in AIDS related opportunistic infections. HIV-1 Tat was shown to increase the intracellular growth of Mycobacteria avium complex (MAC) within PBMac. This increase in MAC growth was in turn found to be regulated by TNF-α expression controlled by c-Myc. HIV-1 Tat was also demonstrated to induce the expression of RIG-I, a common pattern recognition receptor of double stranded RNA viruses, in PBMac. RIG-I is known to activate the viral immune responses such as the type-I interferon (IFN) and pro-inflammatory cytokine pathways. This induction of RIG-I by HIV-1 Tat was found to be regulated by c-Myc, as well as through other signalling kinases such as p38 MAPK and PKR. Tat induction of RIG-I ultimately led to the induction of IFN-α2 and IFN-β through the expression and nuclear translocation of the interferon regulatory factor-7 (IRF-7). This alteration in type-I IFN expression regulated by HIV-1 Tat and RIG-I was also found to play a role against AIDS related opportunistic infections. HIV-1 Tat is known to increase the infectivity of Kaposi’s sarcoma-related herpesvirus (KSHV), a common opportunistic viral infection. We were able to demonstrate that this increase in KSHV infectivity was regulated by RIG-I and type-I IFN induced by HIV-1 Tat. Lastly, this study also demonstrated how HIV-1 Tat was able to manipulate the expression of IL-8 induced by KSHV in PBMac. HIV-1 Tat was able to mediate the production of IL-8 induced by KSHV by altering the phosphorylation of the p38 MAPK and the signal transducer and activator of transcription-1 (STAT-1). Taken together, the results of this study showed how c-Myc and RIG-I may be able to play critical roles in HIV-1 Tat induced cytokine dysregulation. Furthermore, the importance of these pathways is further demonstrated in their roles in regulating the immune responses against opportunistic infections in AIDS patients.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

This dissertation raises a number of policy concerns from a macroeconomic policy point of view and provides additional insights and implications in terms of the effects of fiscal policy and its macroeconomic effects that have kept the open economy macroeconomics literature busy since the early 2000s. The first essay develops a dynamic stochastic general equilibrium (DSGE) model for analyzing the impact of various capital income tax policies in a small open economy that is populated by households possessing endogenous time preferences. I contribute to the literature by studying the impacts of: i) anticipated tax shocks under stochastically growing output, ii) stochastic tax shocks under deterministic output, on a dynamic general equilibrium framework. With the model's specifications, this is the first attempt to integrate uncertainty in the study of taxation and welfare. The results suggest that under certain conditions welfare paradoxes may exist, in the sense that increases in tax instruments may improve welfare. The second essay characterizes the dynamic effects of net tax and government spending shocks on prices, interest rate, GDP and its private components in four OECD countries using structural vector autoregressive regressions (SVAR) approach. For the first time in this literature, I propose a structural decomposition of total net taxes into four components: corporate income taxes, income taxes, indirect taxes and social insurance taxes. The paper provides estimates of the responses of macroeconomic aggregates to innovations in these net tax components. Decompositions of total net tax innovations show that net tax components have different impacts on economic variables. Moreover, the size and persistence of these effects vary across countries depending upon the strength of wealth, substitution, and income effects reflecting the structure of the economies. The last essay estimates the wealth effects of housing and stock market wealth using time-series data for eight developed countries. In estimation I employ the SVAR, which articulate the dynamic interactions of shocks to housing prices, stock values, and disposable incomes. The results show that for these countries the initial consumption response to housing price shocks is greater than to stock market capitalization shocks, but the long-run consumption response to the latter is more persistent than to the former. My findings suggest balanced monetary policies for the developments of housing markets and equity markets.

TAT peptide is one of the best-characterised cell penetrating peptides (CPPs) derived from the transactivator of transcription protein from the human immunodeficiency virus 1 (HIV-1). TAT peptide is able to cross the cell membrane and deliver various biomolecules into cells with low immunogenicity and no toxicity. However, the exact mechanism of internalization still remains a subject of controversy. Lamellar neutron scattering was used to determine the location of TAT peptide in the negativelycharged phospholipids bilayers. The results reveal two locations, one in the peripheral aqueous phase between the adjacent bilayers and the second one below the glycerol backbone region of the lipid bilayer. A concentrationindependent membrane thinning above a peptide concentration threshold (1mol%) and a contiguous transbilayer water channel at the largest peptide concentration (10mol%) were also found. This evidence led to the suggestion that the toroidal pore model might be involved in the transmembrane mechanism at high peptide concentration. Another set of neutron diffraction experiments examined the interaction between the TAT peptide and neutral phospholipids showed that TAT peptide preferentially intercalated into the hydrophobic core and the glycerol backbone region of the neutral lipid bilayer at the lowest peptide concentration investigated (0.1mol%), indicating that the insertion did not require negatively-charged phospholipids. There was also clear evidence for the concentration-dependent reorientation of TAT peptide. A plasmid containing the human copper-zinc SOD gene linked with the coding sequence for a 11-aa HIV-TAT peptide (pGEX-TAT-SOD, 513bp) was constructed and used to express a recombinant fusion protein in Escherichia coli strain BL21 (DE3). High-level expression of TAT-SOD soluble protein with a GST tag (44-kDa) was achieved under optimal expression conditions and a small-scale glutathione affinity column or large-scale ion-exchange chromatography used for its purification. The potential protective effect of TAT-SOD against UV-induced cell damage was studied on UVC-irradiated MDCK epithelial cells. Before any further clinical study, the UV full-length absorption of TAT-SOD protein was measured. The results showed the potential UV protective effect of TAT-SOD was not due to the physical absorption of UV irradiation. In a preclinical study with five healthy volunteers, the penetration of TAT-SOD through human stratum corneum on the inner upper arm was identified by the tape stripping and specific SOD activity analysis. Significant increases on SOD activity were found on the outer layers of stratum corneum in TAT-SOD treated group, compared to placebo treated control, indicating that the TAT peptide assisted SOD to penetrate into the human stratum corneum . In a clinical study with ten healthy volunteers, eight showed a significant increase of minimal erythema dose (MED) with TAT-SOD pre-treatment. The median blood flow value of ten subjects at the UVB-irradiated site decreased with TAT-SOD pretreatment. Taken together, this evidence showed that TATvi SOD did have a marked protective effect against UVB induced skin damage. In a second clinical study, five healthy volunteers were challenged with a series of UVB doses. Skin punch biopsies were taken from four test sites on the lower back for H&E and immunohistochemical staining analysis. UVB-induced apoptotic sunburn cell (SBC) formation, p53 up-regulation and thymine dimer formation in epidermis were not attenuated by pretreatment with TAT-SOD. These data suggest that transdermal superoxide scavenger TAT-SOD reduced the UVB-induced inflammation, but did not abrogate the direct DNA damage of UVB irradiation on the skin. However, the hope of TAT-SOD could reduce UVA indirect DNA damage remains.

Questo lavoro propone dei temi sulla tax compliance in un contesto come quello italiano, caratterizzato da un basso livello della stessa. Il primo capitolo propone una ricerca concernente gli effetti dei controlli. Nella letteratura, l’esito degli stessi è ancora discusso, alla luce del fatto che sono possibili due effetti opposti: il target effect ed il bomb-crater effect. Grazie all’impiego di un database dell’Agenzia delle Entrate italiana, con una combinazione di tecniche di of matching e difference – in – difference, questo lavoro mostra come in un particolare contesto, come quello italiano, i controlli possono avere un effetto positivo sulla tax compliance. Il secondo capitolo mostra gli effetti dell’introduzione di una presumptive tax nella forma di una minimum tax. L’obiettivo principale è quello di studiare l’effetto di tale introduzione quando si hanno particolari condizioni. Nello specifico, questa analisi confronta le tasse italiane pagate da un particolare gruppo di contribuenti, con quelle che verrebbero pagate con l’introduzione di una presumptive tax nella forma di una minimum tax. Il presente lavoro include due differenti metodologie di stima di una presumptive tax, sviluppate dall’ISTAT e dall’Agenzia delle Entrate italiana, includendo anche le possibili reazioni dei contribuenti.
This work investigates issues of tax compliance in a context like the Italian one, characterized by a low level of it. The first chapter investigates the role of audits. In the present literature, the outcome of them is an open question since two opposite effects are possible: the target effect and the bomb-crater effect. Using a database provided by the Italian Revenue Agency, with a combination of matching with difference – in – difference techniques, this work shows how in a particular context, such as Italy, audits can have a positive effect on tax compliance. The second chapter explores the effects of implementing a presumptive tax in the form of a minimum tax. The main aim is to study the effect of a change in the policy from a particular starting condition. More specifically, this analysis compares the taxes collected in Italy from a particular group of taxpayers, to the ones that would be collected if Italy implements a presumptive tax in the form of a minimum tax. This work implements two different methodologies to estimate a presumptive tax, provided by ISTAT and the Italian Revenue Agency, and reactions of taxpayers are included as well.

Les mecanismes de regulation des syntheses de prostaglandines sont etudies dans les cellules de l'epithelium endometrial ovin en culture primaire. L'intervention de l'interferon embryonnaire (otp) dans ces mecanismes est precisee. Les conditions de culture des cellules epitheliales des glandes uterines sont etablies. La confluence est obtenue apres 5-6 jours ; la croissance des cellules differe selon l'etat endocrinien des animaux. La quantite de proteines cellulaires caracterise le mieux une colonie confluente de cellules epitheliales. La chromatographie liquide a haute performance montre que 95% de l'acide arachidonique est incorpore dans les phosphatidylethanolamines, les phosphatidylserines et les phosphatidylcholines. Parmi eux, les plasmalogenes d'ethanolamine constituent la forme majoritaire qui esterifie l'acide arachidonique. Le stockage de l'acide arachidonique est plus important dans les plasmalogenes des cellules de brebis gestantes par comparaison a celles de brebis non gestantes. Les cellules epitheliales forment presqu'exclusivement des prostaglandines f2 et e2. La secretion par les cellules de brebis non gravides est superieure a celles de brebis gravides. La synthese de prostaglandine-f2, determinee par dosage radio-immunologique, est stimulee par l'addition d'acide arachidonique dans le milieu de culture. L'utilisation de la mepacrine, inhibiteur de la phospholipase-a2, indique que l'acide arachidonique est esterifie dans les phospholipides avant sa conversion en prostaglandines. L'ocytocine stimule la secretion de prostaglandine-f2 des cellules provenant uniquement de brebis en cycle. L'emploi d'un analogue du dag ou des esters de phorbol demontre l'implication de la pkc dans la stimulation des secretions de prostaglandines. Ce processus necessite une neosynthese de proteines. A l'aide d'anticorps monoclonaux, nous mettons en evidence l'induction de la cyclooxygenase-2, forme inductible de la prostaglandine synthetase, sans modification des quantites de cyclooxygenase-1, forme constitutive de l'enzyme. L'otp reduit la secretion basale de prostaglandine-f2. Elle empeche la stimulation par l'ocytocine et les analogues du dag, suggerant qu'elle bloque l'effet stimulant de l'activation de la pkc. L'action de l'otp n'affecte pas l'incorporation d'acide arachidonique dans les phospholipides. Cependant, elle modifie la conversion du precurseur probablement en agissant sur la cyclooxygenase. La reduction de l'activite phospholipase-a2 qui se produit en debut de gestation est due a l'inhibition competitive de la phospholipase-a2 dans l'endometre des brebis gravides. Nous suggerons que cette inhibition peut etre induite par l'otp

Det har länge hävdats att bostadsbristen i Stockholm i stora delar beror på att Sveriges sedan 1968 rådande hyreslagstiftning har inneburit ett pristak på marknaden, vilket motverkat en tillfredsställande nybyggnation av hyreslägenheter. Syftet med denna uppsats är undersöka om så är fallet. Hyreslagens grundläggande intentioner och antaganden har utifrån grundläggande teori noggrant synats. Dessutom har den utveckling som skett på marknaden jämförts med de symptom som ett efterfrågeöverskott och ett pristak skulle innebära. Lagstiftningen har visat sig bestå av vad som i förlängningen kan argumenteras ha inneburit en icke-adekvat prisbildningsmekanism. Rimligtvis kan detta ha inneburit ett pristak vilket i sin tur det uppenbara efterfrågeöverskottet i teorin kan härledas till. De symptomatiska tillstånd som visat sig gällande i Stockholm kan därmed på många punkter förklaras av att lagen i praktiken har inneburit ett pristak.

Ett av målen för svensk familjepolitik är att utjämna den sneda fördelningen i uttaget av familjeförsäkringen. Inom familjeförsäkringen hittas segmentet för den tillfälliga föräldrapenningen och i denna, ersättningen för vård av sjukt barn. Två modeller, med olika antal variabler, används för att åskådliggöra sambandet mellan andel barn som bor i traditionell kärnfamilj och andel nettodagar män tar ut för vård av sjukt barn. Resultatet påvisar att uttaget av nettodagar könen emellan är jämnare fördelat inom gruppen traditionell kärnfamilj än för den totala gruppen föräldrar. I Sverige lever idag omkring 78 procent av barn i åldern ett till elva år inom den traditionella kärnfamiljen.

This dissertation analyzes the optimal mix of direct and indirect taxes in an economy with multiple tax collecting authorities when both the taxes are subject to evasion and to what extent the tax compliance behavior of individuals in the United States are persistent and spatially dependent. Essay I derives and provides an intuitive interpretation of: (i) impact of the changes in the government instruments on tax evasion by firms, the expected prices they charge, and the expected tax rates they face; (ii) a generalized version of Ramsey rule for optimal commodity taxation which accounts for income tax evasion from either or both the tax authorities; (iii) generalized formulae for the optimal income tax rate for each of the tax authorities; and (iv) the tradeoff between optimal tax rates and audit probabilities for each of the tax authorities. It also re-examines controversies surrounding the uniform income taxes and the differentiated commodity taxes, and investigates how income tax evasion affects the progressivity of the income tax rates. It concludes that whether or not tax evasion calls for reductions in the optimal income tax rates hinges on how tax evasion and the associated concealment costs vary across individual taxpayers. Essay II introduces the twin issues of spatiality and persistence in the individual income tax evasion. While the issue of persistence arises through accumulated learning over time, spatiality arises for several reasons. Some these include the exchange of information between taxpayers; the social norm of tax compliance: an individual would comply if everybody in the society complies and vice versa; individuals faced with dynamic stochastic decision problems that pose immense computational challenges may simply look to others to infer satisfactory policies and interpersonal dependence works through learning by imitating rather than learning by doing. State-level annual per return evasion of individual income tax and related data were used to examine the above hypotheses and found supports for both of them in the individual income tax evasion in the United States.

碩士
國立清華大學
生醫工程與環境科學系
102
Nanoparticles have been used in biomedical application extensively. For iron oxide nanoparticles, known as SPIO or USPIO, it has applied to be a negative contrast agent to investigate the reticuloendothelial system such as liver, spleen and lymph nodes in MRI. For gold nanoparticles, it had been used for the optical imaging or color sensing. The combined of gold and iron oxide nanoparticles, Au-Fe3O4 had been synthesized as dumbbell-like (DBNPs) and flower-like (FLNPs) composites. The heterostructures of Fe3O4 have not been analyzed the ability of contrast by the morphological effect. In this study, we had evaluated the morphological effect on T2 relaxation. In biomedical application, we had used DBNPs to reaction with tau protein, related with Alzheimer’s disease. Tau protein is the normal component in structure in the neuron cell. In unknown pathogenesis, tau protein release to cerebral spinal fluid (CSF) and the neuron cell would breakdown. The disease severity is highly correlated with concentration of tau protein in CSF. The tau protein sensing at the prophase of disease is very important for prevent medicine. Even though the tau protein sensing was limited by the sensor design in this study, the sensor could be revising at the future work.

博士
國立成功大學
基礎醫學研究所
102
Alzheimer’s disease (AD) is an age-related neurodegenerative disease. AD occurs gradually and results in memory loss, behavior and personality changes, and a decline in thinking abilities. Pathologically, AD is characterized by intracellular aggregation of neurofibrillary tangles and extracellular deposition of amyloid plaque. The amyloid plaques primarily consist of β-Amyloid (Aβ) peptides and the neurofibrillary tangles comprise of hyperphosphorylated tau proteins. Results from epidemiological and pathological research showed a strong link between cardiovascular diseases and AD. It has been suggested that chronic brain hypoperfusion is the common denominator among cardiovascular diseases. One major consequence of hypoperfusion is insufficient supply of glucose to brain, hence results in cerebral hypometabolism. However, whether energy deficiency contributes to the development of AD remains unclear. To investigate the causal relationship, we performed the following three experiments. 1) We cultured the differentiated N2a neuroblastoma cells in media containing no glucose or pyruvate (NGM). Shortly after the N2a cells cultured in the NGM, the mitochondria membrane potential was reduced and the AMP-activated-protein-kinase (AMPK), an energy sensor, was activated. Treatment of NGM not only increased the levels of tau phosphorylation at Ser262 and Ser396, but also increased the levels of active forms of GSK3α and GSK3β, two major tau kinases. 2) The effect of energy deficiency was further examined in vivo by intracerebroventricular (icv) injection of streptozotocin (STZ) to the Wistar rats. STZ selectively injuries glucose transporter type 2-bearing cells which are primarily astrocytes in the rat brain, hence, interrupts glucose transportation from blood vessel to neuron. STZ-icv injection induced energy crisis in the brain regions surrounding the ventricles, as indicated by elevated pAMPK levels in the hippocampus. STZ-icv treatment increased the levels of phosphorylated tau and activated GSK3β in the hippocampus. The hippocampus-dependent spatial learning and memory was impaired by the STZ-icv treatment. 3) Because the levels of Aβ in the N2a cells and Wistar rats were too low to be accurately quantified, we therefore used human amyloid precursor protein overexpressed N2a cells (APP cells) to investigate the effect of energy deficiency on Aβ production. The results showed that concentrations of glucose in culture media negatively associated with the levels of pAMPK in the APP cells, but positively correlated with the levels of Aβ in the condition media. When APP cells were cultured in glucose-containing media, drug-induced activation of AMPK decreased the levels of Aβ in the condition media. However, if APP cells were incubated in media containing no glucose, inhibition of AMPK activity increased the levels of Aβ, while the levels of full-length APP, APPα, APPβ, APP C-terminal fragment α and C-terminal fragment β were unchanged. Taken together, these studies suggest that energy deficiency increases the levels of tau phosphorylation. Furthermore, when energy is deficient and AMPK activation is inhibited, the levels of Aβ are increased, probably due to reduced clearance of Aβ. Thus, our studies support the premise that metabolic disorders contribute to AD pathogenesis.

碩士
高雄醫學大學
醫學檢驗生物技術學研究所
102
Alzheimer''s disease (AD) is a neurodegenerative disease with progressive loss of neuronal functions, which in turn results in memory deficits, cognitive deterioration, and impaired motor coordination. The growing aged population has led to an increased prevalence of AD. However, there is no effective treatment to halt the progression or prevent the onset of AD. Previous studies have indicated angiotensin-converting enzyme (ACE) () inhibitor improved the cognitive impairment, but the mechanisms are still unclear. Our previous finding indicated that Alu sequence in human ACE gene possesses a regulatory function on the ACE promoter activity in neurons. Moreover, a previous study indicated that ACE inhibitor enhanced ACE gene expression. We aimed to investigate the pharmacogenetic effect of ACE inhibitors on ACE I/D polymorphism. We used transient transfection and reporter assay to examine the intracellular ACE signaling pathway in SH-SY5Y cells. We find that lisinopril , a ACE inhibitor, enhances ACE promoter transcriptional activity and that the ACE I/D polymorphism differently responds to lisinopril in regulating ACE promoter activity in neurons. The hyper-phosphorylated tau protein has been one of hallmarks for AD. In this study, we also aimed to investigate whether ACE inhibitors regulate hyper-phosphorylated tau protein in neuron and to observe whether the regulators of phosphorylated tau are affected by ACE inhibitors, including GSK3β (glycogen synthase kinase 3 beta), AKT(protein kinase B), PP2A(protein phosphatase 2A). We find that lisinoplril could decrease the hyper-phosphorylation of tau protein induced by okadaic acid. Our results show ACE inhibitors have pharmacogentic effect on ACE gene and might protect neurodegeneration from hyperphosphorlated of tau protein.

Hung Shieh-Jung Fanny.
Thesis submitted in: August 2002.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 97-109).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
摘要 --- p.iv
Content --- p.vi
List of Abbreviations --- p.xiii
List of Figure --- p.xv
List of Tables --- p.xix
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Alzheimer's Disease (AD) --- p.1
Chapter 1.1.1 --- Clinical features --- p.2
Chapter 1.2 --- Histopathological studies of AD --- p.2
Chapter 1.2.1 --- Neuritic plaques --- p.2
Chapter 1.2.2 --- Neurofibrillary tangles (NFT) --- p.4
Chapter 1.2.3 --- Tau --- p.5
Chapter 1.3 --- Kinases and Alzheimer's Disease --- p.7
Chapter 1.4 --- Free radical damage --- p.8
Chapter 1.5 --- Available treatment for AD --- p.7
Chapter 1.6 --- A Chinese medicinal material 一 Danshen （（Salviae miltiorrhizcie) --- p.11
Chapter 1.6.1 --- Chemical constituents --- p.11
Chapter 1.6.1.1 --- Lipophilic Compounds of Danshen --- p.12
Chapter 1.6.1.2 --- Water-soluble Compounds of Danshen --- p.17
Chapter 1.6.2 --- Pharmacological usage --- p.20
Chapter 1.6.2.1 --- Action on Coronary system --- p.20
Chapter 1.6.2.2 --- Bacteriostatic action --- p.21
Chapter 1.6.2.3 --- Actions on the immune system --- p.21
Chapter 1.6.3 --- Biological activity on brain --- p.22
Chapter 1.7 --- Objectives and scope of the project --- p.23
Chapter Chapter 2 --- General Materials and Method --- p.24
Chapter 2.1 --- Recombinant DNA techniques --- p.24
Chapter 2.1.1 --- Preparation of E. coli strain DH-5a competent cells --- p.24
Chapter 2.1.2 --- Transformation of plasmid DNA into competent cells --- p.25
Chapter 2.1.3 --- Preparation of plasmid DNA using QIAGEN Plasmid Maxipreps kit --- p.25
Chapter 2.1.4 --- Phenol/ choroform extraction of DNA --- p.26
Chapter 2.1.5 --- Spectrophotometric quantitation of the amount and purity of DNA --- p.27
Chapter 2.2 --- Drugs preparation --- p.27
Chapter 2.2.1 --- Preparation of aqueous extracts of Traditional Chinese Medicine (TCM) --- p.27
Chapter 2.2.2 --- Preparation of ethanol extracts of Traditional Chinese Medicine (TCM) --- p.27
Chapter 2.3 --- "3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay " --- p.28
Chapter 2.4 --- Analysis of proteins from culture cells --- p.28
Chapter 2.4.1 --- Extraction of total proteins from culture cells --- p.28
Chapter 2.4.2 --- Quantitation of protein by Bradford method --- p.29
Chapter 2.4.3 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29
Chapter 2.4.4 --- Western blot analysis --- p.31
Chapter 2.5 --- Reagents and buffers --- p.32
Chapter 2.5.1 --- Reagents for competent cell preparation --- p.32
Chapter 2.5.2 --- Reagents provided by QIAGEN Plasmid Maxipreps kit --- p.33
Chapter 2.5.3 --- Reagents for SDS-PAGE --- p.34
Chapter 2.5.4 --- Reagents and buffers for Western Blotting --- p.35
Chapter 2.5.5 --- Cell lines --- p.36
Chapter 2.5.6 --- Antibodies --- p.37
Chapter 2.5.7 --- Plasmids --- p.37
Chapter 2.5.8 --- Other Chemicals --- p.38
Chapter Chapter 3 --- The effect of Danshen on GSK-3 induced hyperposphorylation of tau in Cos7 cells
Chapter 3.1 --- Introduction --- p.39
Chapter 3.1.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.39
Chapter 3.1.2 --- Structure of GSK-3 --- p.39
Chapter 3.1.3 --- The importance of GSK-3 in AD --- p.39
Chapter 3.2 --- Materials and Methods --- p.41
Chapter 3.2.1 --- Transfection of Gsk-3 and tau into Cos7 monkey kidney cells --- p.43
Chapter 3.2.2 --- Extraction of total proteins from culture cells --- p.44
Chapter 3.2.3 --- Quantitation of protein by Bradford method --- p.44
Chapter 3.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44
Chapter 3.2.5 --- Western blot analysis --- p.44
Chapter 3.3 --- Results --- p.45
Chapter 3.3.1 --- Toxicity test on Cos7 cells --- p.45
Chapter 3.3.2 --- The effect of ethanol extract of Danshen on GSK-3 β induced tau phosphorylation --- p.45
Chapter 3.3.3 --- The effect of aqueous extract of Danshen on GSK-3 β induced tau phosphorylation --- p.48
Chapter 3.3.4 --- The effect of Protocatechualdehyde on GSK-3β induced tau phosphorylation --- p.48
Chapter 3.3.5 --- The effect of Salvianolic acid B on GSK-3β induced tau phosphorylation --- p.49
Chapter 3.4 --- Discussion --- p.60
Chapter Chapter 4 --- Cdk5 induced hyperposphorylation of tau in CHO cells
Chapter 4.1 --- Introduction --- p.63
Chapter 4.1.1 --- Cyclin dependent kinase 5 (Cdk5) --- p.63
Chapter 4.1.2 --- Structure of Cdk5 --- p.63
Chapter 4.1.3 --- Neurological functions of Cdk5 --- p.64
Chapter 4.2 --- Materials and Methods --- p.66
Chapter 4.2.1 --- Transfection of p35 and tau into CHO cells --- p.66
Chapter 4.2.2 --- Extraction of total proteins from culture cells --- p.67
Chapter 4.2.3 --- Quantitation of protein by Bradford method --- p.67
Chapter 4.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.67
Chapter 4.2.5 --- Western blot analysis --- p.67
Chapter 4.3 --- Results --- p.68
Chapter 4.3.1 --- Toxicity test on CHO cells --- p.68
Chapter 4.3.2 --- Tau transfection in Cdk5/p35 and TauON3R transiently transfected in CHO cells --- p.68
Chapter 4.3.3 --- Effect of roscovitine treatment on the transiently tau and p35 transfection in CHO cells --- p.74
Chapter 4.3.4 --- "Effects of aqueous active components of Danshen, PCAH and SAB on the transiently tau and p35 transfection in CHO cells " --- p.74
Chapter 4.4 --- Discussion --- p.79
Chapter Chapter 5 --- Antioxidant effect of Danshen and its active components on lipid peroxidation
Chapter 5.1 --- Introduction --- p.81
Chapter 5.1.1 --- Red-blood-cell hemolysis model --- p.82
Chapter 5.2 --- Materials and methods --- p.84
Chapter 5.2.1 --- Red-blood-cell hemolysis model --- p.84
Chapter 5.2.2 --- Materials --- p.85
Chapter 5.2.2.1 --- Animals --- p.85
Chapter 5.2.2.2 --- Chemicals --- p.85
Chapter 5.3 --- Results --- p.86
Chapter 5.3.1 --- Aqueous and ethanol extracts of Danshen --- p.86
Chapter 5.3.2 --- Active components ´ؤ Protocatechualdehyde and Salvianolic acid B --- p.87
Chapter 5.4 --- Discussion --- p.91
Chapter Chapter 6 --- General discussion and Outlook
Chapter 6.1 --- General discussion --- p.93
Chapter 6.2 --- Proposed study in the future --- p.95
Chapter 6.2.1 --- In vitro kinase assay using gamma32 P ATP and substrate with or without TCM --- p.95
Chapter 6.2.2 --- Use of neuroblastoma cells (SHSY-5Y) to study the effect of Danshen and its active components on tau phosphorylation --- p.95
Chapter 6.2.3 --- Thiobarbituric acid-reacting substances (TBARS) assay --- p.96
Chapter 6.2.4 --- In vitro phosphatase kinase assay --- p.96

碩士
國立陽明大學
生物藥學研究所
97
Alzheimer’s disease (AD), the most common neurodegenerative dementia, is neuropathologically characterized by the extracellular Amyloid ���n�vA���w�npeptides accumlation forming a senile plaque and the intracellular accumulation of neurofibrillary tangles composed of hyperphosphorylated tau. Amyloid plaques were considered as the prime pathogenic driver of neurodegeneration in AD. However, many studies suggested that amyloid plaques alone may not be able to explain the neuron loss and cognitive ability decline in AD. Therefore, both Aβand tau were equally important on AD pathogenesis, and tau phosphorylation may be induced by Aβ, microglia, excitotoxicity and oxidative stress. The aim of this thesis is to investigate the effects of tau hyperphosphorylation mediated by Aβ, microglia, excitotoxicity and oxidative stress. Futhermore, the mechanism of Aβ- and microglia– mediated tau phosphorylation was also investigated. We found that Aβ25-35 fibril and Aβ1-42 oligomer significantly increased tau phosphorylation at Ser404, but not at Ser413, Ser214 and Thr212 in cortical neurons, Aβ1-42 fibril and oligomer significantly increased tau phosphorylation at Ser404 in the co-culture of cortical neuons with microglia-derived cell line (BV2). The results suggest that Aβ may induce neuronal tau phosphorylation directly or indirectly through microglia activation by Aβ. In respect of tau phosphorylation mediated by excitotoxicity and oxidative stress, cortical neurons were treated with N-methyl-D-asparate (NMDA) and H2O2, respectively. NMDA significantly increased tau phosphorylation of 72-kDa band at Ser404, but not at Ser413 and Ser214. Nevertheless, tau phosphorylation Thr212 decreased in a concentration and time dependent manner. On the other hand, H2O2 failed to induce tau phosphoryaltion at Ser404 in cortical neurons. Regarding the mechanism of tau phosphoryaltion, GSK- 3�� is one of the tau-protein kinases. We found that Aβ1-42 oligomer decreased the phosphorylation of GSK-3���nat Ser9 (the inactive from of GSK-3��). On the contrary, the GSK-3�� activation induced by Aβ1-42 oligomer and fibril were not observed in the co-culture of cortical neurons and BV2 cells.

Alzheimer’s disease (AD) is a devastating worldwide disease and the main cause of dementia. In its pathology, are include two main hallmarks: the extracellular deposition of Aβ peptides, formed in amyloidogenic processing of APP, that aggregate into senile plaques (SP) and the intracellular formation of neurofibrillary tangles (NFT), aggregates of hyperphosphorylated tau protein. Tau hyperphosphorylation occurs due to increased kinase activity (including GSK3β and CDK5) and impaired protein phosphatase activity (PP2A, PP1 and PP2B). Neuroinflammation is a crucial event in AD pathogenesis, during which microglia and astrocytes are constitutively activated, leading to massive production of cytokines. These molecules may alter APP amyloidogenic processing, Aβ aggregation or tau phosphorylation. Among cytokines, chemokines seem to have an important role in AD progression. In the present work, chemokines interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) impact on tau phosphorylation was addressed. Additionally, the effect of these chemokines on kinases (GSK3β and CDK5) and phosphatase (PP1 and PP2A), important in this process, was evaluated. For both chemokines, a significant increase in ptau S396 levels was observed at longer incubation periods and higher chemokines concentration. Further, results regarding GSK3β support that this is not the main involved kinase in this process. However, preliminary results suggest that CDK5, PP1 and PP2A can have a role in tau increased phosphorylation, promoted by chemokines. This work supports the involvement of chemokines on tau hyperphosphorylation and potentially in NFT formation, that consequently result in AD neurodegeneration. At a disease view, this thesis allowed a better understanding of molecular mechanisms that underling the disease pathology, highlighting the role of inflammation in this process.
A doença de Alzheimer (DA) é uma doença devastadora e a principal causa de demência a nível mundial. Na sua patologia estão incluídas duas principais características: a deposição extracelular de péptidos Aβ, formados durante o processamento amiloidogénico da APP, que agregam em placas senis (SP) e a formação de traças neurofibrilares (NFT), agregados de proteína tau hiperfosforilada. A hiperfosforilação da tau ocorre devido ao aumento da atividade de proteínas cinases (incluindo a GSK3β e a CDK5) e à diminuição da atividade de proteínas fosfatases (PP2A, PP1 e PP2B). A neuroinflamação é um evento crucial na patogénese da DA, durante o qual, a microglia e os astrócitos são constitutivamente ativados, o que leva a uma produção massiva de citocinas. Estas moléculas podem alterar o processamento amiloidogénico da APP, a agregação do Aβ ou a fosforilação da tau. Entre as citocinas, as quimiocinas parecem ter um papel importante na progressão da DA. No presente trabalho foi avaliado o impacto das quimiocinas, interleucina 8 (IL-8) e proteína quimiotática de monócitos 1 (MCP-1), na fosforilação da tau. Para além disso, o efeito destas quimiocinas na atividade de cinases (GSK3β e CDK5) e nas fosfatases (PP1 e PP2A) importantes neste processo também foi avaliado. Para as duas quimiocinas, foi observado um aumento significativo nos níveis de pTau S396 para os períodos de incubação mais longos e nas concentrações de quimiocinas mais elevadas. Além disso, os resultados em relação à GSK3β indicam que esta não é a cinase maioritariamente envolvida neste processo. No entanto, resultados preliminares sugerem que a CDK5, a PP1 e a PP2A podem desempenhar um papel no aumento da fosforilação da tau, promovido pelas quimiocinas. Este trabalho suporta o envolvimento das quimiocinas no estado de hiperfosforilação da tau e potencialmente na formação de NFT que, consequentemente resultam em neurodegeneração na DA. Do ponto de vista da doença, esta tese permite uma melhor compreensão dos mecanismos moleculares que estão na base da patologia, evidenciando o papel da inflamação neste processo.
Mestrado em Biomedicina Molecular

Alzheimer s disease is characterized by synaptic alterations and neurodegeneration. Histopathological hallmarks represent amyloidplaques composed of amyloid-beta (Abeta) and neurofibrillary tangles containing hyperphosphorylated tau. To determine whether synaptic changes and neurodegeneration share common pathways we established an ex vivo model using organotypic hippocampal slicecultures from amyloid precursor protein transgenic mice combined with virus-mediated expression of EGFP-tagged tau constructs. Confocal high-resolution imaging, algorithm-based evaluation of spines and live imaging was employed to determine spine changes and neurodegeneration. We report that Abeta but not tau induces spine loss and shifts spine shape from mushroom to stubby through a mechanism involving NMDA receptor (NMDAR), calcineurin and GSK-3beta activation. In contrast, Abeta alone does not cause neurodegeneration but induces toxicity by phosphorylation of wt tau in a NMDAR-dependent pathway. We show thatGSK-3beta levels are elevated in APP transgenic cultures and that inhibiting GSK-3beta activity or use of phosphorylation-blocking tau mutations prevent Abeta-induced toxicity of tau. FTDP-17 tau mutants are differentially affected by Abeta. While R406W tau shows increased toxicity in the presence of Abeta, no change is observed with P301L tau. While blocking NMDAR activity abolishes toxicity of both wt and R406W tau, the inhibition of GSK-3beta only protects against toxicity of wt tau but not of R406W tau induced by Abeta. Tau aggregation does not correlate with toxicity. We propose that Abeta-induced spine pathology and tau-dependent neurodegeneration are mediated by divergent pathways downstream of NMDA receptor activation and suggest that Abeta affects wt and R406W tau toxicity by different pathways downstream of NMDAR activity.

碩士
國立中正大學
化學工程研究所
106
The adjective of this study involves to produce double emulsion solid lipid nanoparticles with transferrin to load curcumin (CURC), quercetin (QU), rosmarinic acid (RA) and nerve growth factor (NGF) and study their synergic activity against beta-amyloid (Aβ) induced tau hyperphosphorylation in SK-N-MC cells. Double layered solid lipid nanoparticles (SLNs) containing beeswax (BW), cacao butter (CB), compritol®888 ATO (CT), tricaprin (TC) and cardiolipin (CL) as lipids and Tween®80 and lecithin (LEC) as surfactants were modified with transferrin (Tf) to carry CURC, QU, RA and NGF across the blood–brain barrier (BBB) for Alzheimer’s disease (AD) treatment. The particle size and stability of SLNs, and grafting efficiency of drugs in SLNs and releasing rate of drugs from SLNs were measured to find out the most appropriate composition of SLNs via mathematical statistics. From the results, drug-loaded SLNs were used to treat the Aβ induced neurotoxicity in SK-N-MC cells. The results showed that the addition of CL improved the therapeutic efficiency through persuasively targeting Aβ. In addition, the combination of CURC, QU, RA and NGF enhanced their role by interacting with each other with sufficient reduction of tau hyperphosphorylation induced neurotoxicity in SK-N-MC cells. Immunoflurorescence images and westerm blot analysis revealed the suppressed expressions of c-Jun N terminal kinases (JNK), caspase-3, extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinases (p38), extracellular signal-regulated kinases 5 (ERK5) and cAMP response element-binding protein (CREB) after treatment with CURC-QU-RA-NGF-SLNs. SLNs were not only act as a carrier for drugs, and also serve to reduce the drugs induced toxicity to the cells by slow releasing. Moreover, CURC-QU-RA-NGF-SLNs surface modified with Tf facilitated the permeability of drugs across the BBB with the interaction of LEC in SLNs and Tf. Hence, drug-loaded SLNs grafted with Tf could cross the BBB and might help AD brain to achieve the inhibition of Aβ deposition.

Dottorato di Ricerca in Molecular Bio-Pathology, XXI Cycle, 2008
Frontotemporal lobar degeneration (FTLD) is a heterogeneous syndrome encompassing different nosological entities characterized by behavioural and personality change, accompanied by deterioration of executive function, language and movement. Clinically FTLD results in at least three distinct syndromes: Frontotemporal dementia (FTD), Semantic dementia (SD) and Primary progressive aphasia (PPA), while the pathological classification is based on histopathological presence or absence of neuronal inclusions of tau and/or ubiquitin proteins accumulating in the neuronal/glial inclusions, being forms of FTLD differentiated in tau-positive, ubiquitin-positive and tau-negative. The most common clinical manifestation of FTLD is FTD, characterized by atrophy of the frontal and temporal lobes, with neuronal loss, gliosis and spongiosis of the superficial layers. FTD is mostly a presenile disorder showing changes in personality, impaired social conduct, emotional blunting, loss of insight, disinhibition, perseverative behaviour and hyperorality; cognitive deterioration, especially in language and in executive functions, appear later. Despite most cases of FTD are sporadic, approximately 10%-50% of FTD patients have a positive family history for dementia. Familial FTDL was associated to mutations in four genes: Microtubules associated protein tau (MAPT) and Progranulin (PGRN) genes that are responsible for the most genetic forms of FTD; instead, Valosin containing protein (VCP) gene is involved in rare forms of FTD with inclusion body myopathy and Paget’s disease of the bone and Charged multivescicolar body protein 2B (CHMP2B) is mutated in some families with a combination of FTD and Amyotrophic lateral sclerosis (ALS). Mutations in MAPT gene are responsible for 10%-20% of familial FTD. Alternative splicing of exons 2, 3 and 10 in MAPT pre m-RNA results in the expression of six isoforms. Exclusion or inclusion of Exon 10 gives rise to tau isoforms with three (tau3R, E10-) o four (tau4R, E10+) microtubule-binding repeats. In normal adult human brain the overall ratio of 3R to 4R tau is generally 1, whereas in fetal brain only the shortest tau isoform with 3R is expressed, indicating that tau expression is developmentally regulated.To date, 44 different potential pathogenic MAPT mutations have been reported, divided into two groups depending on the primary molecular mechanism involved: missense or deletion mutations that commonly modify tau interaction with microtubules and splicing mutations that affect the alternative splicing of exon 10, leading to changes of the ratio of 3R-tau/4R-tau. However, a third group of mutations exists that might have effects at protein and RNA levels. In the present study we report the molecular effect of two novel heterozygous MAPT gene mutations, a T to C transition at position -15 of intron 9 [T(-15)C] and an A to C transversion at position +4 of intron 10 (E10+4), identified in a patient with sporadic FTD, clinically and neuropathologically ascertained. Considering that both mutations are located in the splicing regulatory regions surrounding Exon 10, we analyzed their molecular effect on the alternative splicing of MAPT pre-mRNA in a minigene model system and in brain tissue. Semi-quantitative RT-PCR analyses, in minigene costructs and in brain tissue, have shown that the two novel mutations cause a novel Exon 10 splicing effect giving rise to a higher increase of mRNAs transcripts lacking Exon 10 (E10- or Tau3R) when compared with FTD-Ub+ control. Immunohistochemical and biochemical analyses on brain tissue evidenced neuronal and oligodendroglial tau deposits mostly made of Tau3R isoforms and an increased increased availability of shorter Tau3R isoform respectively. Data obtained with minigenes derived by the phenotipically healthy patient’s parents demonstrate that when the mutations are inherited in a non compound heterozygous condition the ratio of E10 including/E10 excluding transcripts is quite normal. Although the molecular mechanism underlying exon 10 splicing regulation remain to be completely elucidated, the exon 10 splice donor site is predicted to give rise to a RNA stem loop structure considered crucial for the quantitative regulation of exon 10 alternative splicing. Most of previously characterized mutations identified in the upper part of the stem loop strongly alter mRNA splicing by destabilizing the secondary structure, with a corresponding increase of E10 inclusion and 4RTau expression. Considering that the E10+4 mutation is located into the exon 10 splice donor site, we also investigated the effect of the E10+4 mutation on the thermodynamic stability of the RNA stem loop structure. Our data, based on bioinformatic prediction of the stem loop sequence thermostability and Ultraviolet Melting experiments demonstrated a strong increasing of stability in the stem-loop structure carrying the E10+4 mutation. This higher stability could be important for the skipping of exon 10, even though the E10+4 mutation alone is not able to give rise to a pathologic phenotype. We cannot exclude that the T to C transition, localized in a regulatory region upstream of exon 10, could also alter the binding of specific trans-splicing factors increasing the effect of the E10+4 mutation, giving rise when both mutation are present in the compound heterozygous condition (namely the FTD patient) to the E10 exclusion and the altered 4R/3R tau ratio observed. Thus, we can hypothesize a trans-acting regulatory effect of both mutations with known, or unknown splicing factors, which might have contributed to the very atypical clinical and pathological FTD phenotype of the patient.
Università della Calabria

Tese de doutoramento em Ciências Biomédicas (Neurociências Básicas), apresentada à Universidade de Lisboa através da Faculdade de Medicina, 2007
Alzheimer’s disease (AD) is a major disease of aging affecting about 10% of the elderly population, is the leading cause of dementia and a major health care concern. AD patients develop a gradual and progressive decline in cognitive and functional abilities as well as behavioral and psychiatric symptoms, eventually leading to a vegetative state and ultimately death. In 1907, Dr. Alois Alzheimer published the first report linking the clinical features of AD to plaque and tangle neuropathology. Neurofibrillary tangles in the brain consist primarily of hyperphosphorylated tau. Neuritic plaques consist primarily of aggregated amyloid β (Aβ) peptides and are surrounded by dystrophic neurites, microglia, and reactive astrocytes. Plaques are increasingly viewed as tombstones, and do not correlate well with cognitive impairment. The progressive accumulation of soluble β-amyloid (Aβ) in vulnerable brain areas with aging does parallel the progressive dementia found in AD patients. Plaques are not considered to be responsible for the initial impairments. Therefore, reduction of Aβ is currently the leading approach of experimental therapies for AD. Purification and sequencing of Aβ from AD brain led to the cloning of the amyloid precursor protein (APP) gene and triggered research on trying to understand how the age-dependent accumulation of Aβ in the brain leads to the downstream pathologies and progressive dementia. Aβ is generated by two sequential cleavages of APP by β- and γ-secretases, two major forms of Aβ are produced, a long one with 42 amino acids, Aβ42, and a shorter one with 40 amino acids, Aβ40. Aβ42 is more hydrophobic; it easily aggregates and associates to membranes, likely facilitating its retention intracellularly. Aβ40 is more soluble and is mostly secreted. In 1993, the first biochemical evidence for appreciable levels of endogenous intracellular Aβ was reported in vitro. In 2000, with the development of antibodies that differentiated Aβ42 peptide from its precursor, it was reported that Aβ42 accumulated intracellularly in neurons even before plaque formation. Since then, several studies have implicated the accumulation of Aβ within neurons as important for AD pathogenesis. Loss of synapses occurs early in AD and is considered the best pathological correlate of cognitive decline. Several lines of evidence suggest a relationship between Aβ accumulation and synaptic dysfunction. Depressed synaptic transmission, including impaired LTP (long-term potentiation), has been reported in AD mouse models of β- amyloidosis, and occurs concomitantly with the appearance of intraneuronal Aβ accumulation even prior to appearance of extracellular plaques. This observation suggests that the intracellular accumulation of Aβ42 may have an important role in AD pathogenesis, especially in the initial stages of the disease. Tg2576 mice harbor the human APP transgene with the familial AD Swedish mutation and develop AD-like cerebral amyloidosis. Aβ accumulates and oligomerizes within neurons of Tg2576 mice with aging in vivo, especially within neuronal processes and synaptic compartments. To study the cell biology of Aβ-dependent neuronal dysfunction, we turned to cultured neurons from Tg2576 mice. Tg2576 neurons were previously characterized to recapitulate the accumulation of Aβ with time in culture. We set out to better characterize the consequences of this aberrant Aβ42 accumulation on synapses. In this study, we found both structural and functional alterations in pre- and postsynaptic compartments in Tg2576 compared to wild-type neurons at 19 days in vitro (DIV). The presynaptic alterations included reductions in the number of active presynaptic compartments and in the levels of synaptophysin, a synaptic vesicle protein. The postsynaptic alterations included reductions in the levels of the glutamate receptor subunit, GluR1, and of the postsynaptic density protein, PSD-95. Since Aβ42 accumulation progresses with time in culture, at 19 DIV there is higher concentration of Aβ42 with more probability of aggregation and oligomerization than at 12 DIV. We hypothesized that the changes at 19 DIV could correspond to a more severe phenotype paralleling a later stage of disease. Therefore, we studied synapses at 12 DIV in Tg2576 compared to wild-type neurons. Presynaptically there was no difference in the number of active compartments or in the levels of synaptophysin however, postsynaptically spines were reduced. Specifically, the postsynaptic changes included reduction in the levels of PSD-95 and of glutamate receptors subunits GluR1 and NR1 at the postsynaptic density but not in total cellular lysates. These results suggest that early on the alterations at synapses are due to an impaired trafficking of glutamate receptors. The glutamate receptors are critical determinants for LTP and long-term depression (LTD), forms of synaptic plasticity thought to be involved in the establishment of new memories. Our results shed light on the consequences of Aβ acumulation on synapses, specifically on the earliest alterations that lead to synaptic dysfunction and cognitive decline in AD. However, although we found Aβ42 to be responsible for the synaptic alterations in Tg2576 neurons, the mechanism whereby Aβ42 affects synapses needs further clarification. Over the past few years, increasing evidence supports that intraneuronal Aβ accumulation is involved in early AD pathogenesis. Although the precise location(s) of Aβ production in neurons remains controversial, the subcellular site of Aβ42 accumulation in AD is better accepted. Aβ42 accumulation in the brains of Tg2576 mice and human AD occurs especially in multivesicular endosomes (MVBs). Alterations in the endocytic pathway are among the earliest pathological changes in AD. The molecular mechanisms by which abnormalities in the endocytic pathway arise and are involved in the pathogenesis of AD remain unknown. In the brain, Aβ42- accumulating MVBs often localize close to degenerating synapses. Since Aβ42 accumulation with aging occurs in the outer limiting membranes of MVBs, we hypothesized that this accumulation could alter the MVB sorting pathway in neurons. MVBs are vesicular organelles morphologically defined by a limiting membrane and characteristic inner vesicles. MVBs are considered to be late endosomes, formed by maturation of early endosomes by membrane invaginations that generate inner vesicles with an acidic pH. The inner vesicles have a different content of lipids and proteins: they are rich in cholesterol, LBPA (lysobisphosphatidic acid) and lysosomal glycoproteins. MVBs are involved in many cellular functions, including the regulated trafficking of several proteins and membrane receptors. The trafficking of the epidermal growth factor receptor (EGFR) to MVBs has been the most extensively studied. EGFR upon stimulation with EGF is internalized into early endosomes. The EGF-EGFR complex is stable at the pH of early endosomes, and therefore the activated receptor follows the MVB pathway. During endosomal maturation, inward invagination of the outer membrane leads to MVB formation and traps receptors in the MVB interior, terminates signaling and promotes their subsequent degradation in the more acidic environment of inner vesicles of MVBs and lysosomes. In neurons, the functional role of MVBs is less well understood. MVBs are thought to have an important role in retrograde transport, carrying receptors from neuronal terminals to the cell body for signaling and/or degradation in lysosomes. For example, neurotrophin receptors after ligand binding are internalized presynaptically and transported in MVBs to the cell body where signaling to the nucleus can occur, thereby preventing the loss of signaling during transport. The sorting of neurotrophin receptors to MVBs has been described for TrkB, which upon brain derived neurotrophic factor (BDNF) activation is quickly removed from the cell surface by endocytosis and delivered to the MVB sorting pathway. Since Aβ42 accumulates especially in the outer limiting membrane of MVBs with AD pathogenesis, we postulated that this could impair MVB sorting. We set out to study the MVB sorting pathway in cultured Tg2576 neurons following the trafficking of EGFR after EGF binding, which is well characterized. We demonstrated that Aβ accumulating Tg2576 neurons revealed normal internalization and recycling but altered MVB sorting of EGFR upon activation. The accumulation of EGF-EGFR in MVBs of Tg2576 compared to wild-type neurons suggested reduced degradation of EGFR. We verified altered MVB sorting and degradation of another ligand-receptor complex, BDNF-TrkB, which provided similar results. To obtain more information on the trafficking of EGFR within MVBs, we assessed the levels of phosphorylated EGFR (P-EGFR), since dephosphorylation is thought to occur at the low pH of the inner vesicles of MVBs, and found elevated P-EGFR in Tg2576 neurons. These data suggested impairment in the trafficking of EGFR within MVBs that accumulate Aβ42. Ubiquitination is required for trafficking of EGFR to MVBs, subsequent translocation within MVBs, and later degradation of EGFR via the MVB sorting pathway. It was therefore possible that reduced ubiquitination of EGFR might be blocking EGFR from translocating to inner MVB membranes. Instead, we found increased ubiquitin conjugated EGFR in Tg2576 neurons, suggesting that ubiquitination of EGFR was not impaired. Therefore, we hypothesized that deubiquitination activity might be compromised. Analysis of deubiquitinating enzyme activity did reveal reduced deubiquitination in Tg2576 neurons, although the modest change seemed insufficient for the more marked elevation in ubiquitinated EGFR. Considerable evidence over the past few years has indicated that the ubiquitinproteasome system (UPS) regulates the trafficking of EGFR through the MVB sorting pathway. Proteasome activity has been described to be necessary for deubiquitination to occur prior to EGFR translocation, also since proteasome inhibition leads to decreased EGFR degradation. Therefore, we next examined proteasome activity in Tg2576 compared to wild-type neurons, which revealed a marked reduction in proteasome activity in Tg2576 neurons. Since proteasome inhibition also inhibits deubiquitination, reduced proteasome activity therefore could be responsible for the cellular alterations that we observed in the Tg2576 neurons. We provide evidence that Aβ is responsible for the alterations in the MVB sorting pathway and the UPS in Tg2576 neurons, since the inhibition of Aβ accumulation blocked the cellular alterations in Tg2576 neurons, including impairment in proteasome activity. Moreover, in support of Aβ-induced proteasome inhibition occurring in Tg2576 neurons, we found that proteasome inhibition of wild-type neurons produced alterations in the MVB sorting pathway paralleling those seen in Tg2576 neurons. The findings that the disease-linked Aβ42 peptides localize and accumulate especially in MVBs and thereby alter the trafficking of membrane receptors by inhibiting the UPS, suggest a mechanism whereby Aβ42 accumulation contributes to AD pathogenesis. The UPS is a complex system of interconnecting biochemical pathways that are increasingly being found to have major cellular functions even beyond the degradation of proteins. The UPS is being studied in the regulated recycling and degradation of membrane receptors and has been linked to the regulation of synaptic plasticity. Indeed, we hypothesize that the inhibition of the UPS by Aβ42 impairs the endocytic trafficking of neuronal receptors, and thereby may be the cause of synaptic dysfunction in AD. Our data supports a novel mechanism whereby Aβ accumulation alters the cell biology of vulnerable neurons in AD.
A doença de Alzheimer é a principal causa de demência na população idosa, afectando cerca de 10% das pessoas com mais de 65 anos, constituindo um problema grave de saúde pública. Os doentes com doença de Alzheimer sofrem um gradual declínio cognitivo e decréscimo das suas capacidades funcionais, progredindo para alterações psiquiátricas e comportamentais, eventualmente para um estado vegetativo e finalmente à morte. Em 1907, o Dr. Alois Alzheimer publicou um artigo onde, pela primeira vez, estabelecia uma ligação entre as características clínicas da doença de Alzheimer e as alterações neuropatológicas, os novelos neurofibrilhares e as placas neuríticas. Os novelos neurofibrilares são constituídos principalmente por proteína tau hiperfosforilada. As placas neuríticas são principalmente formadas pelo péptido β-amilóide (Aβ) e encontram-se normalmente envolvidas por neurites distróficas, microglia, e astrócitos activados. As placas neuríticas são o marcador por excelência da doença de Alzheimer, mas não se correlacionam directamente com o declínio cognitivo. Considera-se, portanto, que as placas neuríticas não são responsáveis pelo início do declínio cognitivo na doença de Alzheimer. Por outro lado, a acumulação progressiva de Aβ na sua forma solúvel em áreas vulneráveis do cérebro correlaciona-se bastante bem com o declínio cognitivo dos pacientes com doença de Alzheimer. Por isso, a diminuição dos níveis de Aβ é neste momento o principal objectivo das terapêuticas experimentais na doença de Alzheimer. A purificação do Aβ a partir de cérebro de doentes com doença de Alzheimer e a sua sequenciação conduziu à clonagem do gene da proteína precursora de amilóide (APP) e desencadeou a investigação sobre os mecanismos pelos quais o Aβ está envolvido na patogénese da doença de Alzheimer. O Aβ é gerado por duas clivagens do APP pela β- e γ-secretase sequencialmente, dando origem a duas formas de Aβ, uma mais longa de 42 aminoácidos, o Aβ42, e uma mais curta de 40 aminoácidos, o Aβ40. O Aβ42 é a forma mais hidrofóbica, mais xx propensa a agregar-se e associar-se a membranas intracelulares, e assim mais provável de acumular intracelularmente. O Aβ40 é mais solúvel sendo principalmente secretado. Em 1993, foi identificada pela primeira vez a presença de níveis apreciáveis de Aβ42 intracelularmente in vitro. Alguns anos mais tarde, com o desenvolvimento de anticorpos capazes de distinguir o péptido Aβ42 da proteína percursora, foi possível detectar também in vivo a acumulação intraneuronal de Aβ42 mesmo antes do aparecimento de placas. Desde então, vários estudos têm sugerido ser a acumulação intraneuronal de Aβ42 importante para a patogénese da doença de Alzheimer. A perda de sinapses é um dos eventos iniciais na doença de Alzheimer e é considerada a característica patológica que melhor se correlaciona com o declínio cognitivo. Várias linhas de investigação sugerem uma relação entre a acumulação de Aβ42 e a disfunção sináptica. A depressão da transmissão sináptica, incluindo redução da potenciação de longa duração (LTP), tem sido descrita em ratinhos transgénicos que constituem modelos de doença de Alzheimer, e ocorre concomitantemente com o aparecimento de Aβ42 intraneuronal, muito antes da presença das primeiras placas neuríticas. Esta observação sugere que a acumulação intracelular de Aβ42 pode ter um papel importante no desenvolvimento da doença de Alzheimer, especialmente nas fases iniciais da doença. Os ratinhos transgénicos Tg2576 possuem o transgene humano para o APP com a mutação Sueca da doença de Alzheimer familiar e desenvolvem β-amiloidose semelhante à doença de Alzheimer. Com o envelhecimento, o Aβ42 acumula-se e oligomeriza intracelularmente em neurónios de ratinhos Tg2576 in vivo, especialmente nas neurites, e compartimentos sinápticos. Para estudarmos a biologia celular da disfunção neuronal dependente de Aβ42, utilizámos culturas primárias de neurónios de ratinhos Tg2576, que reproduzem a acumulação progressiva de Aβ42 em cultura. Para melhor compreendermos as consequências da acumulação aberrante de Aβ42 começámos por caracterizar as sinapses destes neurónios. Neste estudo, encontrámos alterações estruturais e funcionais tanto pré- como pós-sinápticas em neurónios mutantes para o APP (Tg2576) aos 19 dias in vitro (DIV) quando comparados com neurónios de ratinhos não transgénicos (wild-type). As alterações pré-sinápticas incluíram a redução no número de terminais pré-sinápticos propensa a agregar-se e associar-se a membranas intracelulares, e assim mais provável de acumular intracelularmente. O Aβ40 é mais solúvel sendo principalmente secretado. Em 1993, foi identificada pela primeira vez a presença de níveis apreciáveis de Aβ42 intracelularmente in vitro. Alguns anos mais tarde, com o desenvolvimento de anticorpos capazes de distinguir o péptido Aβ42 da proteína percursora, foi possível detectar também in vivo a acumulação intraneuronal de Aβ42 mesmo antes do aparecimento de placas. Desde então, vários estudos têm sugerido ser a acumulação intraneuronal de Aβ42 importante para a patogénese da doença de Alzheimer. A perda de sinapses é um dos eventos iniciais na doença de Alzheimer e é considerada a característica patológica que melhor se correlaciona com o declínio cognitivo. Várias linhas de investigação sugerem uma relação entre a acumulação de Aβ42 e a disfunção sináptica. A depressão da transmissão sináptica, incluindo redução da potenciação de longa duração (LTP), tem sido descrita em ratinhos transgénicos que constituem modelos de doença de Alzheimer, e ocorre concomitantemente com o aparecimento de Aβ42 intraneuronal, muito antes da presença das primeiras placas neuríticas. Esta observação sugere que a acumulação intracelular de Aβ42 pode ter um papel importante no desenvolvimento da doença de Alzheimer, especialmente nas fases iniciais da doença. Os ratinhos transgénicos Tg2576 possuem o transgene humano para o APP com a mutação Sueca da doença de Alzheimer familiar e desenvolvem β-amiloidose semelhante à doença de Alzheimer. Com o envelhecimento, o Aβ42 acumula-se e oligomeriza intracelularmente em neurónios de ratinhos Tg2576 in vivo, especialmente nas neurites, e compartimentos sinápticos. Para estudarmos a biologia celular da disfunção neuronal dependente de Aβ42, utilizámos culturas primárias de neurónios de ratinhos Tg2576, que reproduzem a acumulação progressiva de Aβ42 em cultura. Para melhor compreendermos as consequências da acumulação aberrante de Aβ42 começámos por caracterizar as sinapses destes neurónios. Neste estudo, encontrámos alterações estruturais e funcionais tanto pré- como pós-sinápticas em neurónios mutantes para o APP (Tg2576) aos 19 dias in vitro (DIV) quando comparados com neurónios de ratinhos não transgénicos (wild-type). As alterações pré-sinápticas incluíram a redução no número de terminais pré-sinápticos activos e nos níveis de sinaptofisina, uma proteína de membrana das vesículas sinápticas. As alterações pós-sinápticas incluíram reduções nos níveis da subunidade GluR1 do receptor do glutamato do tipo AMPA e da proteína da densidade pós-sináptica, PSD-95. Uma vez que a acumulação de Aβ42 aumenta com o tempo em cultura, aos 19 DIV existe uma concentração mais elevada de Aβ42 com uma maior probabilidade de agregar e oligomerizar do que aos 12 DIV. Portanto, as alterações observadas aos 19 DIV podem corresponder a um fenótipo mais severo equivalente a um estádio avançado da doença. Assim sendo, na pesquisa das alterações iniciais, estudámos as sinapses de neurónios Tg2576 comparados com os neurónios wild-type aos 12 DIV. Présinapticamente, não encontrámos diferenças nem no número de terminais activos nem nos níveis de sinaptofisina, no entanto, pós-sinapticamente encontrámos uma redução no número e tamanho das espinhas dendríticas. Especificamente, as alterações pós-sinápticas incluíram a redução de PSD-95 e das subunidades dos receptores de glutamato AMPA ou NMDA (GluR1 ou NR1), na densidade pós-sináptica apesar de não haver diferença nos níveis totais celulares destas proteínas. Estes resultados sugerem que as primeiras alterações sinápticas surgem devido a uma alteração no tráfico de receptores de glutamato para as sinapses. Os receptores de glutamato são determinantes para a LTP e para a depressão de longa duração (LTD), fenómenos de plasticidade sináptica que estão associados aos processos de criação de novas memórias. Os nossos resultados elucidam as consequências da acumulação de Aβ nas sinapses, identificando as alterações iniciais que conduzem à disfunção sináptica e ao declínio cognitivo na DA. No entanto, apesar de termos identificado o Aβ como responsável pelas alterações sinápticas dos neurónios Tg2576, o mecanismo pelo qual a acumulação de Aβ afecta as sinapses necessita ser clarificado. Nos últimos anos, as evidências de que a acumulação intraneuronal de Aβ42 está envolvida na patogénese inicial da doença de Alzheimer têm aumentado. Apesar da controvérsia acerca de qual o organelo celular onde se origina o Aβ42 em neurónios, a localização preferencial da acumulação intracelular de Aβ42 na doença de Alzheimer é mais consensual. A acumulação de Aβ42 ocorre especialmente em endosomas multivesiculares (MVBs) no cérebro de ratinhos transgénicos para o APP e de humanos com doença de Alzheimer. Os endosomas têm sido descritos como anómalos na DA e estas alterações encontra-se entre as detectáveis na doença de Alzheimer. No entanto, os mecanismos moleculares responsáveis pelo aparecimento e participação destas alterações na patogénese da doença de Alzheimer permanecem desconhecidos. No cérebro, a acumulação de Aβ42 com o envelhecimento ocorre predominantemente na membrana exterior dos MVBs e junto a sinapses em degenerescência. Colocámos, por isso, a hipótese de que a acumulação de Aβ42 poderá afectar a via de sorting dos MVBs em neurónios. Os MVBs são organelos vesiculares morfologicamente caracterizados por uma membrana exterior e vesículas internas características. MVBs são considerados endosomas tardios, que se formam por maturação de endosomas primários por invaginações membranares que geram as vesículas internas. As vesículas internas têm uma composição lípidica e proteica particularmente rica em colesterol, LBPA (ácido lisobisfosfatídico) e glicoproteínas lisosomais, e um pH mais ácido. Os MVBs estão envolvidos em muitas funções celulares, que incluem o tráfico de várias proteínas e receptores membranares. O tráfico do receptor do factor de crescimento epidérmico (EGFR) para os MVBs tem sido extensivamente estudado. O EGFR, após activação pelo EGF, é internalizado para os endosomas primários. O complexo EGF-EGFR é estável ao pH dos endosomas primários e por isso segue a via dos MVBs. Durante a maturação endosomal, a formação dos MVBs através da invaginação da membrana exterior dos endosomas, retém os receptores no interior dos MVBs, prevenindo a sinalização dos receptores, e promovendo a sua subsequente degradação no meio mais ácido das vesículas internas e nos lisosomas. Nos neurónios, o papel funcional dos MVBs é pouco conhecido. Pensa-se que os MVBs têm um papel importante no transporte retrógrado, conduzindo receptores dos terminais nervosos para o corpo celular onde a sinalização ou degradação lisosomal ocorre. Por exemplo, os receptores de neurotrofinas são activados por ligação de factores de crescimento, internalizados pré-sinapticamente e transportados em MVBs para o corpo celular onde a sinalização para o núcleo ocorre, prevenindo-se assim a perda de sinal durante o transporte. O sorting dos receptores de neurotrofinas para os MVBs é particularmente importante para o receptor TrkB, que após ligação do factor neurotrófico derivado do cérebro (BDNF) é rapidamente removido da superfície celular por endocitose e segue a via dos MVBs. Resolvemos estudar a via de sorting dos MVBs em neurónios primários Tg2576 para avaliar as consequências da acumulação de Aβ42 nos MVBs. Para estudarmos a via de sorting dos MVBs seguimos o tráfico do EGFR após activação por ligação de EGF, cujo tráfico endocítico está melhor caracterizado. Demonstrámos que o processo de internalização e reciclagem é normal nos neurónios Tg2576 mas o sorting do EGFR nos MVBs estava afectado. Observámos que havia acumulação de EGF-EGFR nos MVBs de neurónios Tg2576 quando comparados com neurónios wild-type, sugerindo que a degradação de EGFR está diminuída. Verificámos, ainda, que o sorting nos MVBs e a degradação de um outro complexo receptor-ligando, BDNF-TrkB, estavam também alterados de forma análoga. Para obtermos mais informação acerca do tráfico do EGFR nos MVBs, medimos os níveis de EGFR fosforilado (P-EGFR), uma vez que a desfosforilação do receptor ocorre após a translocação do EGFR para o interior dos MVBs, e encontrámos um aumento de P-EGFR nos neurónios Tg2576. Estes resultados sugerem que o tráfico do EGFR nos MVBs que acumulam Aβ42 se encontra alterado. A ubiquitinação do EGFR é necessária ao tráfico para os MVBs, para a translocação para o interior dos MVBs, e para a eventual degradação do EGFR. Uma diminuição da ubiquitinação do EGFR, com redução da translocação do EGFR para as vesículas interiores e subsequente degradação, seria uma justificação possível para a redução da degradação observada. No entanto, os níveis de ubiquitina conjugada ao EGFR encontravam-se aumentados nos neurónios Tg2576, sugerindo que a conjugação de ubiquitina não se encontra alterada. Assim sendo, uma outra hipótese para justificar a acumulação de EGFR ubiquitinado seria uma alteração na actividade de desubiquitinação nos neurónios Tg2576. Efectivamente, a análise da actividade de enzimas desubiquitinantes confirmou uma redução na desubiquitinação em neurónios Tg2576, no entanto a redução, sendo modesta, pareceu-nos insuficiente para a elevada ubiquitinação do EGFR. Nos últimos anos, o sistema da ubiquitina-proteasoma (UPS) tem sido implicado na regulação do tráfico do EGFR e da via dos MVBs. Por um lado, a actividade do proteasoma foi descrita como necessária à desubiquitinação do EGFR, sendo um passo necessário para a sua translocação para o interior dos MVBs, por outro lado, a inibição do proteasoma leva a uma redução da degradação do EGFR. Por isso, medimos a actividade do proteasoma em neurónios Tg2576 em comparação com neurónios wild-type. A actividade do proteasoma revelou-se bastante reduzida nos neurónios Tg2576, sendo esta inibição devida à acumulação de Aβ. Uma vez que a inibição do proteasoma também inibe a desubiquitinação, a inibição da actividade do proteasoma pode ser responsável pela alteração no tráfico de EGFR e também pelas restantes alterações celulares que descrevemos em neurónios Tg2576. Assim, a acumulação de Aβ42 é responsável pelas alterações da via dos MVBs e do UPS em neurónios Tg2576, uma vez que a inibição da acumulação de Aβ preveniu as alterações celulares nestes neurónios, incluindo a inibição do proteasoma. Para suportar a evidência de que o proteasoma está inibido pelo Aβ42 em neurónios mutantes para o APP, descrevemos ainda que a inibição do proteasoma em neurónios wild-type produz alterações na via dos MVBs que são equivalentes às que observámos em neurónios Tg2576. A descoberta de que o Aβ42, o péptido associado à doença de Alzheimer, que se localiza e acumula especialmente nos MVBs, altera o tráfico de receptores da membrana através da inibição do UPS, sugere um mecanismo pelo qual a acumulação de Aβ42 contribui para a patogénese da doença de Alzheimer. O UPS é um sistema complexo que participa em diferentes vias bioquímicas que se interceptam, e tem sido progressivamente considerado como tendo importantes funções celulares para além da degradação de proteínas. As funções celulares do UPS incluem a regulação da reciclagem e degradação de receptores membranares e a regulação da plasticidade sináptica. Concluímos que a inibição do tráfico endocítico de receptores neuronais pelo Aβ42 poderá ser a causa da disfunção sináptica na doença de Alzheimer. Os nossos resultados propõem um novo mecanismo pelo qual a acumulação de Aβ42 altera a biologia celular de neurónios vulneráveis na doença de Alzheimer.
Fundação para a Ciência e a Tecnologia (FCT, SFRH/6270/2001) e Fundo Social Europeu