A nanofluidic biosensor using surface-enhanced Raman scattering (SERS) was developed to detect the β-amyloid (Aβ) protein, one of the biomarkers of Alzheimer’s disease (AD). Recent studies have indicated that investigating changes in relative concentrations of structure specific Aβ oligomers in cerebral spinal fluid (CSF) during the progression of AD could be important indicators for diagnosing AD pre-mortem. However, there is no definitive pre-mortem diagnosis of AD thus far because of the lack of technology available for sensitive Aβ detection. Hence, the development of a system for detecting the structure specific Aβ oligomers, along with the concentrations of these oligomers in CSF, would be useful in the investigation of the molecular mechanisms of Aβ cytotoxicity associated with AD. In this thesis, a nanofluidic trapping device trapping system for detecting biomolecules at sub-picomolar concentrations was developed for using SERS. The device, with a microchannel leading to a nanochannel, carries out dual functions: encouraging sizedependent trapping of gold nanoparticles (60nm) at the entrance of the nanochannel as well as restricting the target molecules between the gaps created by the aggregated nanoparticles. Initially, the trapping capability of the nanofluidic device was tested using fluorescent polystyrene and gold nanoparticles. UV-vis absorption spectroscopy was used to characterize the gold nanoparticle clusters at the entrance to the nanochannel. The device established controlled, reproducible, SERS active sites within the interstices of gold nanoparticle clusters and shifted the plasmon resonance to the near infrared, in resonance with incident laser light. Two strongly Raman active molecules, adenine and Congo red, were used to test the feasibility of the SERS nanofluidic device as a platform for the detection of multiple analytes. The results showed that strong SERS signals were obtained from the nanoparticle clusters at the nanochannel entrance. Once the feasibility of the approach was determined with strong Raman molecules, Aβ was detected using this nanofluidic SERS platform. Distinct surface-enhanced Raman spectra of Aβ was observed in different conformational states as a function of concentration and structure (monomer versus oligomer form) due to Aβ refolding from α-helical to a predominantly β-pleated sheet form. The sensor was also shown to potentially distinguish Aβ from insulin and albumin, confounder proteins in cerebral spinal fluid. Thus, a novel platform was developed to detect picomoler levels of Aβ with the ultimate goal of facilitating the diagnosis and understanding of Alzheimer’s disease by means of detecting structure specific oligomers of Aβ.

We apply a molecular dynamics (MD) simulation technique to complex molecules at interfaces. Partitioning of dendritic surfactants into clay gallery and Ab protein behavior near hydrated lipids are chosen for the purpose. Using a full atomistic model of dendritic surfactants, the confinement force profiles featuring oscillatory fashion at moderate layer separation of 10 to 25 Å were observed. Integration of the confinement forces led to free energy profiles, which, in turn, were used to determine the final morphology of the nanocomposite. From the free energy profiles, smaller and linear surfactants (G1 and G2L) are expected to intercalate into the clay comfortably, while larger surfactants (G2 and G3) are expected to form frustrated intercalated structures due to the location and depth of the free energy minima. This would agree with the previous observations. As primary steps to understand the Ab protein behavior under biological conditions, simulations of bulk water and hydrated lipids were performed and the results were compared with the literature. Hydrated lipids were simulated using a full atomistic model of lipids (dipalmitoylphosphatidylcholine) and water with a cvff force-field and it was found that structural properties such as the molecular head group area and membrane thickness were accurately produced with MD simulation. Systems of the protein Ab(1-42) in bulk water were simulated and some secondary structural change, with loss of part of the a-helical structure, occurred during the 1 ns of simulation time at 323K. The fragment Ab(31-42) with b-sheet conformation was also simulated in bulk water, and the extended b-sheet structure became a bent structure. Simulations of Ab(1- 42) or Ab(31-42) near lipid bilayers have been performed to investigate the structural property changes under biological conditions. The different nature of structural change was observed from the simulations of the protein or fragment in water and near lipid bilayers due to the different solvent environment. The protein has close contacts with the membrane surface. It was impossible to observe the conformational change to b-sheet and protein entrance into the lipid bilayer within 1 ns simulations.

Recent advances in nanotechnology and the biotechnology revolution have created an immense opportunity for the use of noble metal nanoparticles as Surface Enhanced Raman Spectroscopy (SERS) substrates for biological sensing and diagnostics. This is because SERS enhances the intensity of the Raman scattered signal from an analyte by orders of 106 or more. This dissertation deals with the different aspects involved in the application of SERS for biosensing. It discusses initial studies performed using traditional chemically reduced silver colloidal nanoparticles for the SERS detection of a myriad of proteins and nucleic acids. It examines ways to circumvent the inherent aggregation problems associated with colloidal nanoparticles that frequently lead to poor data reproducibility. The different methods examined to create robust SERS substrates include the creation of thermally evaporated silver island films on microscope glass slides, using the technique of Nanosphere Lithography (NSL) to create hexagonally close packed periodic particle arrays of silver nanoparticles on glass substrates as well as the use of optically tunable gold nanoshell films on glass substrates. The three different types of SERS surfaces are characterized using UV-Vis absorption spectroscopy, Electron Microscopy (EM), Atomic Force Microscopy (AFM) as well as SERS using the model Raman active molecule trans-1,2-bis(4-pyridyl)ethylene (BPE). Also discussed is ongoing work in the initial stages of the development of a SERS based biosensor using gold nanoshell films for the direct detection of b-amyloid, the causative agent for Alzheimer's disease. Lastly, the use of gold nanoshells as SERS substrates for the intracellular detection of various biomolecules within mouse fibroblast cells in cell culture is discussed. The dissertation puts into perspective how this study can represent the first steps in the development of a robust gold nanoshell based SERS biosensor that can improve the ability to monitor biological processes in real time, thus providing new avenues for designing systems for the early diagnosis of diseases.

Retrospective studies suggest that lifestyle activities may provide protection against Alzheimer s Disease (AD). However, such studies can be inaccurate and prospective longitudinal studies investigating lifestyle protection against AD are both impractical and impossible to control for. Transgenic (Tg+) AD mice offer a model in a well controlled environment for testing the potential for environmental factors to impact AD development. In an initial study, Tg+ and non-transgenic (Tg-) mice were housed in either environmentally enriched (EE) or standard housing (SH) from 2-6 months of age, with a behavioral battery given during the last 5 weeks of housing. In the Morris maze, platform recognition, and radial arm water maze tasks, Tg+/EE mice were completely protected from cognitive impairment present in Tg+/SH mice and comparable to control Tg-/SH mice in cognitive performance. The current study utilized the same cognitive-based behavioral battery and multimetric statis statistical analysis to investigate the protective effects of "complete" environment enrichment (EE) versus several of its components (physical activity, social interactions) in AD transgenic mice. The AD transgenic mice utilized develop beta-amyloid (AB) deposition and cognitive impairment by 6-7 months of age. Similar to our initial study, results show that "complete" EE (physical, social, and cognitive activities) from 2 to 8 months of age completely protected AD transgenic mice from cognitive impairment in tasks representing different cognitive domains - working memory, reference learning, and search/recognition. In strong contrast, Tg+ mice reared in environments that included physical activity and social interaction, or only social interaction, were not protected from cognitive impairment in adulthood -- enhanced cognitive activity was required over and above that present in these other environments. Through use of discriminant function analysis, EE and/or NT mice were consistently discriminated from the poorer performing other housing groups. The cognitive benefits observed in EE-housed Tg+ mice occurred without significant changes in cortical AB levels, plasma cytokine levels, or plasma corticosterone levels, suggesting involvement of mechanisms independent of these endpoints. However, EE-housed Tg+ mice did have decreased dendritic length of neurons in the parietal cortex (but not hippocampus). Noteworthy is that plasma cytokine levels and hippocampal dendritic length consistently correlated with cognitive measures, suggesting their involvement in underlying mechanisms of cognitive performance. The present work provides the first evidence that "complete" EE (including enhanced cognitive activity) is needed to provide cognitive protection against AD in a Tg+ model of the disease, while the physical and social activity components of EE do not alone lead to protection. These results suggest that humans desiring to gain maximal environmental protection against AD should live a lifestyle high in cognitive, social, and physical activities together.

The α7 nicotinic acetylcholine receptor (nAChR) is involved in learning and memory, synaptic plasticity, neuroprotection, inflammation, and presynaptic regulation of neurotransmitter release. Alzheimer’s disease (AD), a neurodegenerative disease characterized by diminished cognitive abilities, memory loss, and neuropsychiatric disturbances, is associated with a loss of nAChRs. Similarly, traumatic brain injury (TBI) may result in long term neurobehavioral changes exemplified by cognitive dysfunction. Deficits in α7 nAChR expression have previously been shown in experimental TBI and may be related to cognitive impairment experienced in patients following TBI. The purpose of this dissertation was to investigate changes in α7 nAChR expression in models of neurodegeneration and determine if allosteric modulation of the nAChR facilitates functional recovery following experimental TBI through changes in nAChRs. Experimental models employed include a transgenic mouse model of AD that overexpresses the amyloid precursor protein (APPswe mice) and the controlled cortical impact injury model of TBI in rats. Quantitative receptor autoradiography using α-[125I]-bungarotoxin and [125I]-epibatidine and in situ hybridization were used to investigate changes in nAChR density and mRNA expression, respectively. In the first study, the effects of aging and β-amyloid on α7 nAChR expression were evaluated in APPswe mice. Hippocampal α7 nAChR density was significantly upregulated in APPswe mice compared to wild-type mice. It is postulated that elevated Aβ levels bind to the α7 nAChR resulting in upregulation. In a second study, galantamine, a medication used in the treatment of AD, was administered subchronically following experimental TBI to determine if treatment could facilitate cognitive recovery and affect nAChR expression. Interestingly, the results indicate TBI interferes with agonist mediated upregulation of nAChRs, and galantamine did not improve function in a behavioral task of learning a memory. In a third study, the regulation of TBI related deficits in α7 nAChRs was examined 48 hours following injury. α7 nAChR deficits occurred with a reduction in α7 mRNA in several hippocampal regions and non-α7 nAChR deficits occurred with a reduction in α4 mRNA in the metathalamus. The results of these studies suggest AD and TBI may involve complex but parallel processes contributing to the regulation of α7 nAChRs.

Tikslas. Ištirti įvairaus oligomerizacijos laipsnio Aβ1-40 ir Aβ1-42 peptidų agregatų poveikį smegenėlių ląstelėms ir išaiškinti toksinio veikimo mechanizmus. Uždaviniai: 1.Įvertinti Aβ1-40 ir Aβ1-42 monomerų, oligomerų ir fibrilių poveikį neuronų-glijos ląstelių kultūros gyvybingumui. 2.Nustatyti ryšį tarp Aβ1-42 oligomerų toksiškumo ir dydžio. 3.Ištirti Aβ1-42 oligomerų poveikį neuronų ir mikroglijos ląstelių plazminės membranos įtampos pokyčiams ir įvertinti, kokią reikšmę Aβ1-42 oligomerų sukeltoje neuronų žūtyje turi išorinio kalcio koncentracija, NMDA receptorių ir endocitozės aktyvumas. 4.Nustatyti Aβ1-42 oligomerų poveikį smegenėlių kultūrai, papildytai J774 makrofagais 5.Nustatyti Aβ1-42 oligomerų poveikį mitochondrijų oksidacinio fosforilinimo sistemai. Mokslinis naujumas Šiame darbe nustatytas ryšys tarp Aβ1-42 oligomerų dydžio ir toksiškumo neuronams. Pirmą kartą parodyta, kad maži (toksiški) Aβ1-42 oligomerai (1–2 nm), koncentracijose, kurios gali susidaryti smegenyse patologinėmis sąlygomis, yra toksiški ląstelių kultūroje esantiems neuronams, tačiau nekeičia kitų, ląstelių kultūroje esančių, ląstelių gyvybingumo (mikroglijos ir astrocitų). Pirmieji išsiaiškinome, kad maži ir dideli Aβ1-42 oligomerai gali sukelti neuronų žūtį skirtingais mechanizmais: maži tiesiogiai veikdami neuronus, o dideli veikdami per glijos ląsteles. Mūsų tyrimai atskleidė iki šiol neaprašytą reiškinį, kad maži Aβ1-42 oligomerai gali sąlygoti ne tik neuroninių ląstelių žūtį, bet ir jų... [toliau žr. visą tekstą] / Objective and tasks. The aim of this study is to investigate the effects of Aβ1-40 and Aβ1-42 peptide aggregates at various degrees of oligomerization on cultivated neurons of brain cells and their toxic mechanisms. 1.To investigate the effect of various Aβ1–40 and Aβ1-42 aggregates on the viability of neuronal-glial cell cultures. 2.To determine the relationship between the toxicity of Aβ1-42 oli-gomers and their size. 3.To analyze the effect of Aβ1-42 oligomers on membrane potential of neuronal and glial cells. To assess the effect of extracellular Ca2+ concentration, NMDA receptors and activity of endocytosis on neu¬ronal death caused by Aβ1-42 oligomers. 4.To determine the effect of Aβ1-42 oligomers (4–6 nm in size) on neurons in CGC cultures with added macrophages J774 cells. 5.To determine the effect of Aβ1-42oligomers on respiration of isolated brain mitochondria. Scientific novelty This study determined the relationship between size of amyloid and its toxicity to neurons. For the first time it was shown that small Aβ1-42 oligomers of the size 1–3 nm were toxic to neurons in cell cultures at the concentrations which occur in brain under pathological conditions. However, they did not affect the viability of other cells (microglia and astrocytes) present in the cell cultures. For the first time it has been shown that small and large Aβ1-42 oligo-mers can cause neuronal death by different mechanisms: small oligomers by affecting neurons directly, and large oligomers... [to full text]

Aβ oligomer-induced neurotoxicity has become an important area of therapeutic development in treating Alzheimer’s disease. Platelet-derived growth factor (PDGF) has been shown to be able to protect neurons against several neuronal insults such as ischemia and HIV1 toxin induced cytotoxicity. These neuroprotective effects correlate well with our previous results that demonstrate the neuroprotective effects of PDGF-BB, one of the PDGF receptor ligand subtypes, against NR2B containing NMDA receptor induced excitotoxicity, a possible underlying cause of Aβ oligomer induced synaptic dysfunction and neuronal death. This project examines the neuroprotective effect of PDGF-BB against Aβ1-42 oligomer induced cytotoxicity in both SH-SY5Y cells and primary hippocampal neurons. Cell viability was monitored by MTT assay and the affected signaling pathways were examined using pharmacological methods and Western blotting. The results demonstrated that Aβ1-42 oligomer elicited a dose-dependent toxicity with a sign of saturation at higher dosages, PDGF-BB failed to protect neurons against Aβ1-42 oligomer induced cytotoxicity. In contrast, Aβ1-42 oligomers strongly inhibit PDGF-BB induced mitogenesis in both SH-SY5Y cells and primary neurons. Further investigation using Western blotting to measure PDGF receptor expression and phosphorylation in SH-SY5Y cells showed that Aβ1-42 oligomer can inhibit PDGF-BB induced phosphorylation of PDGF β-receptor on Tyr1021, a site that is crucial for PLCγ mediated mitogenesis. These findings not only explained the poor neuroprotective effect elicited by PDGF-BB against Aβ1-42 oligomers, but also led to a novel hypothesis that Aβ1-42 oligomer may interfere with neurotrophic factor induced neuronal survival, either selectively or perhaps globally. Further exploration on this hypothesis will be able to shed light on this potentially novel mechanism of pathogenesis in Alzheimer’s disease.

Orientador: Wanda Pereira Almeida / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T15:17:44Z (GMT). No. of bitstreams: 1 Huber_PaulaCristina_M.pdf: 4520670 bytes, checksum: fd94b7f1261ebb6165efd24fe301909c (MD5) Previous issue date: 2012 / Resumo: A Doença de Alzheimer é um processo neurodegenerativo associado ao envelhecimento. Seu arsenal terapêutico é muito pobre. Dentre as características fisiopatológicas desta doença, destaca-se a formação de placas senis constituídas de fibrilas do peptídeo b-amilóide, principalmente do Ab42. Neste trabalho, avaliou-se o efeito de agentes antimicrobianos na inibição da agregação do peptídeo Ab42. Considerando a importância crescente do reposicionamento de fármacos, foram selecionados alguns antimicrobianos, que estão no mercado farmacêutico e tem o seu perfil farmacológico bastante conhecido, a fim de suprimir algumas etapas clínicas no caso destas substâncias apresentarem atividade anti-fibrilogênica. Levofloxacino e ciprofloxacino foram as fluorquinolonas selecionadas. Levofloxacino inibiu in vitro a agregação do peptideo Ab42 quando submetido a condições de fibrilogênese. As metodologias empregadas foram o ensaio de fluorescência da tioflavina-T, microscopia de força atômica e eletroforese em gel de poliacrilamida. Os resultados promissores obtidos nos motivaram a buscar o grupo farmacofórico responsável pela atividade observada. Para tanto, sintetizamos a 3-carboetoxi-4-quinolona, correspondente a porção quinolônica mais simples e planar do levofloxacino. Os ensaios indicaram que esta substância e ainda mais eficaz do que o levofloxacino. Sabendo que in vivo o processo de agregação e metal dependente (zinco e cobre divalentes), fizemos um estudo comparativo das propriedades geométricas destas substâncias com as de conhecidos agentes complexantes de metais. De acordo com a literatura, fluorquinolonas apresentam interações medicamentosas com antiácidos que apresentam em sua formulação cátions divalentes. Então, realizamos um estudo de formação de complexos de cobre e zinco tendo como ligantes as substâncias estudadas. Estes complexos foram obtidos e caracterizados, e, um estudo de propriedades importantes foi realizado, incluindo a solubilidade em água e coeficiente de partição. A atividade antimicrobiana e citotóxica dos complexos também foi determinada, o que e de extrema importância para o caso destes serem formados in vivo / Abstract: Alzheimer's disease is a neurodegenerative pathology associated with aging. The therapeutic arsenal to treat it is very poor. Among the main pathophysiological characteristics of this disease, there is the formation of senile plaques composed of fibrils of b-amyloid peptide, mainly of Ab42. This study assessed the effects of antimicrobial quinolones on peptide Ab42 aggregation inhibition. Considering the growing importance of drug repositioning, some antimicrobial agents available in the pharmaceutical market with a well known pharmacological profile were selected, in order to eliminate some Clinical Study steps in case they present anti-fibrilogenic activity. Levofloxacin and ciprofloxacin were the selected fluoroquinolones. Levofloxacin inhibited in vitro aggregation of the peptide Ab42 when subjected to fibrillogenesis conditions. The methods used were thioflavin T fluorescence, atomic force microscopy and polyacrylamide gel electrophoresis. The promising results obtained motivated us to seek the pharmacophore group responsible for the observed activity. For this purpose, we synthesized the 3-carboethoxy-4-quinolone, quinolone portion corresponding to the planar portion of levofloxacin. The tests indicated that this substance is more effective than levofloxacin. Knowing that in vivo aggregation process is metal dependent (divalent zinc and copper), we made a comparative study of the geometric properties of these substances with known metal complexing agents. According to the literature, fluoroquinolones have drug interactions with antacids that contain divalent cations in its formulation. So, we conducted a study of complex formation with copper and zinc using the studied substances as ligands. These complexes were obtained and characterized, and a study of important properties was conducted, including water solubility and partition coefficient. The antimicrobial and cytotoxic activities of the complexes were also determined, which is extremely important in case these are formed in vivo / Mestrado / Quimica Organica / Mestra em Química

The prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) is rising despite combination antiretroviral therapy (cART). Efavirenz (EFV) is among the most commonly used antiretroviral drugs globally, but causes neurological symptoms that may interfere with adherence and reduce tolerability, and may have central nervous system (CNS) effects that contribute in part to HAND in patients on cART. Thus we evaluated a commonly used EFV containing regimen: EFV/zidovudine (AZT)/lamivudine (3TC) in murine N2a cells transfected with the human “Swedish” mutant form of amyloid precursor protein (SweAPP N2a cells) to assess for promotion of amyloid-beta (Aβ) production (Chapter 3). Treatment with EFV or the EFV containing regimen generated significantly increased soluble Aβ, and promoted increased β-secretase-1 (BACE-1) expression while 3TC, AZT, or, vehicle control did not significantly alter these endpoints. Further, EFV or the EFV containing regimen promoted significantly more mitochondrial stress in SweAPP N2a cells as compared to 3TC, AZT, or vehicle control. We next tested the EFV containing regimen in Aβ - producing Tg2576 mice combined or singly using clinically relevant doses. EFV or the EFV containing regimen promoted significantly more BACE-1 expression and soluble Aβ generation while 3TC, AZT, or vehicle control did not. Finally, microglial Aβ phagocytosis was significantly reduced by EFV or the EFV containing regimen but not by AZT, 3TC, or vehicle control alone. These data suggest the majority of Aβ promoting effects of this cART regimen are dependent upon EFV as it promotes both increased production, and decreased clearance of Aβ peptide. Further (Chapter 4), there is evidence that neural stem cells (NSCs) can migrate to sites of brain injury such as those caused by inflammation and oxidative stress, which are pathological features of HAND. Thus, reductions in NSCs may contribute to HAND pathogenesis. Since the HIV non-nucleoside reverse transcriptase inhibitor EFV has previously been associated with cognitive deficits and promotion of oxidative stress pathways, we examined its effect on NSCs in vitro as well as in C57BL/6J mice. Here we report that EFV induced a decrease in NSC proliferation in vitro as indicated by MTT assay, as well as BrdU and nestin immunocytochemistry. In addition, EFV decreased intracellular NSC adenosine triphosphate (ATP) stores and NSC mitochondrial membrane potential (MMP). Further, we found that EFV promoted increased lactate dehydrogenase (LDH) release, activation of p38 mitogen-activated protein kinase (MAPK), and increased Bax expression in cultured NSCs. Moreover, EFV reduced the quantity of proliferating NSCs in the subventricular zone (SVZ) of C57BL/6 mice as suggested by BrdU, and increased apoptosis as measured by active caspase-3 immunohistochemistry. If these in vitro and in vivo models translate to the clinical syndrome, then a pharmacological or cell-based therapy aimed at opposing EFV-mediated reductions in NSC proliferation may be beneficial to prevent or treat HAND in patients receiving EFV. 1 Portions of this abstract have been previously published (Brown LAM, et al., 2014; Jin, J, et al, 2016) and are utilized with permission of the publisher.1

Die Alzheimersche Erkrankung (AD) ist die häufigste Demenzerkrankung bei älteren Menschen in den westlichen Industriestaaten mit ständig wachsender Zahl der Erkrankten. Trotz angestrengter wissenschaftlicher und medizinischer Forschung ist u. a. noch keine Möglichkeit der klinischen Frühdiagnose dieser Erkrankung etabliert. In der vorliegenden Arbeit wurden am transgenen Mausmodell Tg2576 mit beta-Amyloidplaque-Pathologie cholinerge und adrenerge Parameter sowie Einfluss-größen des Energiestoffwechsels untersucht, um transgen-induzierte neuro-chemische bzw. neuromorphologische Veränderungen im Hirngewebe zu erkennen. Außerdem sollte die Möglichkeit einer ex vivo Markierung solcher Moleküle getestet werden, von denen eine besondere Bindungsaffinität an beta-Amyloidablagerungen bereits bekannt ist. Das Ziel der vorliegenden Arbeit war, diese Mauslinie weiterhin hinsichtlich ihres Modellcharakters für die Alzheimersche Erkrankung zu beschreiben und potenzielle Plaque-assoziierte Markermoleküle aufzufinden, die einen in vivo Nachweis der b-Amyloidablagerungen erlauben. Die so gewonnenen Erkenntnisse könnten zur Entwicklung neuer Strategien zur Frühdiagnostik der Alzheimerschen Erkrankung beitragen. Genutzt wurden dazu v. a. die biochemischen Methoden der Rezeptorautoradiografie und der Immunhistochemie sowie der in situ Hybridisierung als molekularbiologische Methode. Weiterhin wurde eine radiochemische Methode zur ex vivo Darstellung der Azetylcholinesterase getestet. Bei der Untersuchung des cholinergen Systems konnte eine signifikante Verringerung in der [3H]Hemicholinium-3-Bindung (als Marker des hoch affinen Cholintransporters) bei den fünf Monate alten transgenen Mäusen im Vergleich zu deren nicht transgenen Wurfgeschwistern festgestellt werden. Es wird hier von einem modulatorischen Effekt des löslichen beta-Amyloids ausgegangen, da die jüngeren Tiere noch keine Plaqueablagerungen aufweisen, die Ursache solcher Veränderungen sein könnten. Beim vesikulären Azetylcholintransposter (VAChT) konnte eine signifikante Erhöhung in der [3H]Vesamicol-Bindung bei 17 Monate alten transgenen Mäusen im Vergleich zu nicht transgenen Geschwistertieren beobachtet werden, obwohl der gegenteilige Fall erwartet wurde. Das Ergebnis wird als Ausdruck einer erhöhten Vesikeldichte interpretiert. Die immunhistochemische Untersuchung der a4- und a7-Untereinheiten der nikoti-nischen Azetylcholinrezeptoren ergab, dass die beta-Amyloidplaqueablagerung keinen Einfluss auf morphologische Veränderungen der Neuronen hatte, die diese Rezep-toren tragen. Es gab keine Hinweise, dass solche Neuronen degenerieren. Hinsichtlich der untersuchten Parameter des Glukosestoffwechsels konnten keine Veränderungen zwischen transgenen Tieren und nicht transgenen Wurfgeschwistern festgestellt werden. Bei der Verteilung der Hirnkapillaren konnte eine verringerte Dichte in unmittelbarer Umgebung der b-Amyloidplaques, verglichen mit weiter entfernt liegenden Gebieten, ermittelt werden. Dieser Befund bedarf weiterer Unter-suchungen, da er Relevanz für die in vivo Diagnostik und Therapie besitzen könnte. Im Vergleich mit den zwar oft nicht einheitlichen Befunden bei Alzheimerpatienten wird deutlich, dass das Mausmodell Tg2576 als Modell der Alzheimerschen Erkrankung nicht alle Aspekte der Pathogenese simuliert. Übereinstimmungen ergeben sich bei dieser transgenen Maus hinsichtlich der beta-Amyloidproduktion und -ablagerung im Hirn und den auch beim Menschen vorkommenden entzündlichen Reaktion um die Plaques. Damit kann sie als geeignetes Modell zum Studium der Amyloidogenese und damit verbundener inflammatorischer Prozesse angesehen werden. / Alzheimer´s Disease (AD) is the most common form of dementia among the elderly in the Western world, with growing prevalence. In spite of intensive scientific and medical research, no possibility of early clinical diagnosis for this disease has yet been established. In this thesis cholinergic and adrenergic parameters, as well as energy metabolism, were studied in the transgenic mouse model Tg2576, to reveal transgene-induced neurochemical and neuromorphological changes in the brain tissue of these animals. Also, the ex vivo labelling of marker molecules with a known high affinity for beta-amyloid was to be tested. The objective was to further characterize the Tg2576 mouse strain as a model of AD, and to find plaque-associated marker molecules which could be used for the in vivo detection of b-amyloid plaques. Such findings could contribute to the development of new stategies for the early diagnosis of AD. Quantitative receptor autoradiography, immunohistochemistry and in situ hybridization were the main biochemical and molecular biological methods employed. Furthermore, a radiochemical method was used for ex vivo labelling of acetylcholinesterase. The 5 month-old transgenic mice, with no significant plaque load, demonstrated reduced [3H]hemicholinium-3 binding to choline uptake sites in anterior brain regions, as compared to non-transgenic littermates. This provides evidence of the modulatory effect of soluble beta-amyloid on high affinity choline uptake sites. However, a significant increase of [3H]vesamicol receptor binding to the vesicular acetylcholine transporter was detected in 17 month-old transgenic animals, as compared to non-transgenic mice. Even though the opposite was expected, the result could be interpreted as an elevated vesicle density. The immunhistochemical studies of a4 and a7 subunits of the nicotinic acetylcholine receptor revealed that neurons expressing these receptors do not undergo morphological changes in close proximity to beta-amyloid plaques. There was no sign of degeneration in these neurons. Concerning the examined parameters of glucose metabolism, no changes between transgenic animals and non-transgenic littermates were detected. This observation is in accordance with the data available in the literature, but in contrast to findings in AD patients. The density of brain capillaries in close vicinity of beta-amyloid plaques, compared to a more distant surrounding, is reduced. This finding needs further examination because it could be relevant for in vivo diagnosis and therapy. Comparing the transgenic Tg2576 mouse model to AD patients, it becomes apparent that the mouse model does not simulate all aspects of the pathogenesis of this disease. Consistent with the human disease this model is characterized by b-amyloid plaque production and deposition in the brain, as well as inflammatory processes around the plaques, which are also known in humans. Therefore, it represents a suitable model to study amyloidogenesis and inflammation.