The reactions of reducing sugars with primary amino groups are the most common nonenzymatic modifications of proteins. Subsequent rearrangements, oxidations, and dehydrations yield a heterogeneous group of mostly colored and fluorescent compounds, termed "Maillard products" or advanced glycation end products (AGEs). AGE formation has been observed on long-lived proteins such as collagen, eye lens crystalline, and in pathological protein deposits in Alzheimer's (AD) and Parkinson's disease (PD) and dialysis-related amyloidosis. AGE-modified proteins are also involved in the complications of diabetes. AGEs accumulate in the the ß-amyloid plaques and neurofibrillary tangles (NFT) associated with AD and in the Lewy bodies characteristic of PD. Increasing evidence supports a role for oxidative stress in neurodegenerative disorders such as AD and PD. AGEs have been shown to contribute towards oxidative damage and chronic inflammation, whereby activated microglia secrete cytokines and free radicals, including nitric oxide (NO). Roles proposed for NO in the pathophysiology of the central nervous system are increasingly diverse and range from intercellular signaling, through necrosis of cells and invading pathogens, to the involvement of NO in apoptosis. Using in vitro experiments, it was shown that AGE-modified bovine serum albumin (BSA-AGE) and AGE-modified ß-amyloid, but not their unmodified proteins, induce NO production in N-11 murine microglia cells. This was mediated by the receptor for AGEs (RAGE) and upregulation of the inducible nitric oxide synthase (iNOS). AGE-induced enzyme activation and NO production could be blocked by intracellular-acting antioxidants: Ginkgo biloba special extract EGb 761, the estrogen derivative, 17ß-estradiol, R-(+)-thioctic acid, and a nitrone-based free radical trap, N-tert.-butyl-*-phenylnitrone (PBN). Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), common precursors in the Maillard reaction, were also tested for their ability to induce the production of NO in N-11 microglia. However, no significant changes in nitrite levels were detected in the cell culture medium. The significance of these findings was supported by in vivo immunostaining of AD brains. Single and double immunostaining of cryostat sections of normal aged and AD brains was performed with polyclonal antibodies to AGEs and iNOS and monoclonal antibodies to Aß and PHF-1 (marker for NFT) and reactive microglia. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localisation of AGE and iNOS in the upper neuronal layers of the temporal (Area 22) and entorhinal (Area 28, 34) cortices compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localised with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localised with AGE and iNOS as well as AGE and ß-amyloid were found surrounding mature but not diffuse ß-amyloid plaques in the AD brain. Parts of NFT were AGE-immunoreactive. Immunohistochemical staining of cryostat sections of normal aged and PD brains was performed with polyclonal antibodies to AGEs. The sections were counterstained with monoclonal antibodies to neurofilament components and a-synuclein. AGEs and a-synuclein were colocalized in very early Lewy bodies in the substantia nigra of cases with incidental Lewy body disease. These results support an AGE-induced oxidative damage due to the action of free radicals, such as NO, occurring in the AD and PD brains. Furthermore, the involvement of astrocytes and microglia in this pathological process was confirmed immunohistochemically in the AD brain. It is suggested that oxidative stress and AGEs participate in the very early steps of Lewy body formation and resulting cell death in PD. Since the iNOS gene can be regulated by redox-sensitive transcription factors, the use of membrane permeable antioxidants could be a promising strategy for the treatment and prevention of chronic inflammation in neurodegenerative disorders. / Die Glykierung oder Maillard-Reaktion ist neben der oxidativen Modifikation die bekannteste nicht-enzymatische posttranslationale Modifikation von Proteinen. Glykierung startet mit der Reaktion von reduzierenden Zuckern mit primären Aminogruppen. Nachfolgenden Umlagerungen, Oxidationen und Dehydra- tionen führen zu einer heterogenen Gruppe von farbigen und fluoreszierenden Verbindungen, den sogenannten "Maillard products" oder "advanced glycation end products" (AGEs). Diese Vorgänge werden besonders an langlebigen Proteinen wie Kollagen und Kristallin der Augenlinsen sowie in pathologischen Proteinab- lagerungen bei der Alzheimer-Demenz (AD), der Parkinson-Krankheit (PD) und bei Hämodialyse beobachtet. AGE-modifizierte Proteine sind auch aktiv beteiligt an den Spätkomplikationen des Diabetes mellitus. AGEs reichern sich im Alzheimer Gehirn in den ß-Amyloid-Plaques und den neurofibrillären Bündeln (tangles, NFT), sowie in den für PD charakteristisch- en Lewi-Körpern an. Immer mehr Befunde belegen die Rolle von oxidativem Stress in neurodegenerativen Erkrankungen wie AD und PD. Es konnte gezeigt werden, dass AGEs an oxidativer Schädigung und chronischer Entzündung Anteil haben, wobei aktivierte Mikroglia Cytokine und freie Radikale, inklusive Stickstoffmonoxid (NO) sezernieren. Vermutungen über die Rolle von NO in der Pathophysiologie des Zentralnervensystems gehen weit auseinander und reichen von interzellulärer Signalübertragung über nekrotischen Zelltod und eindringende pathogene Substanzen bis zur Beteiligung von NO an der Apoptose. In einem Zellkultur Modell konnte in vitro gezeigt werden, dass AGE- modifiziertes Albumin (BSA-AGE) und AGE-modifiziertes ß-Amyloid, aber nicht die unmodifizierten Proteine, die Synthese von NO in N-11 Maus-Mikrogliazellen induzieren. Diese wird möglicherweise vom Rezeptor für AGE (RAGE) und durch eine Steigerung der Expression der induzierbaren Stickstoffmonoxid-Synthase (iNOS) vermittelt. AGE-induzierte Enzymexpression und NO-Produktion konnten durch folgende intrazellulär wirkende Antioxidantien blockiert werden: Ginkgo biloba Spezialextrakt EGb 761, das Östrogenderivat 17ß-Estradiol, alpha- Liponsäure, und ein Radikalfänger, N-tert.-butyl-*-phenylnitrone (PBN). Neben AGEs wurden auch reaktive Dicarbonyle wie Methylglyoxal (MG) und 3- Deoxyglucosone (3-DG), Vorläufer der Maillard-Reaktion, auf ihre Fähigkeit untersucht, die Synthese von NO in N-11 Mikroglia zu induzieren. Es konnten jedoch keine Induktion der NO-Produktion festgestellt werden. Die Bedeutung dieser in vitro-Ergebnisse wurden durch in vivo Immunohisto- chemischen Untersuchungen an AD Gehirnen bestätigt. Einfache und doppelte Immunfärbungen wurden an Gefrierschnitten von normal gealterten Gehirnen und AD-Gehirnen mit polyklonalen Antikörpern gegen AGEs und iNOS und monoklonalen Antikörpern gegen Aß, PHF-1 (zur spezifischen Markierung der NFT) und reaktive Mikroglia angefertigt. Bei normal gealterten Personen sowie bei AD Erkrankten im Frühstadium (d.h. keine pathologischen Veränderungen in iso- kortikalen Gebieten) wiesen wenige Astrozyten eine Kolokalisation von AGE und iNOS in den oberen Neuronenschichten des temporalen (Area 22) und entorhinalen (Area 28, 34) Kortex auf. Im Vergleich dazu wurden keine Astrozyten in jungen Kontrollgehirnen gefunden. In fortgeschrittenen Alzheimergehirnen wurde eine viel dichtere Anreicherung von Astrozyten, kolokalisiert mit AGE und iNOS in den tieferen, und insbesondere in den oberen Neuronenschichten gefunden. Weiterhin konnten zahlreiche Neurone mit diffuser AGE-Reaktivität, aber ohne iNOS-Reaktivität, sowie einige AGE- und iNOS-positive Mikroglia gezeigt werden, im Vergleich zu nur wenigen AGE-reaktiven Neuronen und keinen Mikroglia in den Kontrollen. Schliesslich wurden in Astrozyten, die reife, aber nicht diffuse ß-Amyloid-Plaques im AD-Gehirn umlagern, AGE mit iNOS sowie mit ß-Amyloid kolokalisiert gefunden. Teile der NFT waren AGE-immunoreaktiv. Immunhistochemische Färbung an Gefrierschnitten von normal gealterten und PD- Gehirnen wurden mit polyklonalen Antikörpern gegen AGEs durchgeführt. Die Schnitte wurden mit monoklonalen Antikörpern gegen Neurofilamentkomponenten und a-Synuclein gegengefärbt. AGEs und a-Synuclein waren kolokalisiert in sehr frühen Lewi-Körpern der Substantia nigra in Gehirnen mit vorhandener Lewi-Körper-Demenz. Diese Ergebnisse unterstützen die These AGE-induzierter oxidativer Schädigung mittels freier Radikale wie z.B. NO in AD- und PD- Gehirnen. Ausserdem wurde die Beteiligung von Astrozyten und Mikroglia an diesem pathologischen Prozess immunhistochemisch im Alzheimergehirn bestätigt. Es liegt nahe, dass oxidativer Stress und AGEs an den sehr frühen Stufen der Lewi-Körper-Bildung und dem daraus resultierenden Zelltod in PD beteiligt sind. Da das iNOS-Gen durch redoxsensitive Transkriptionsfaktoren reguliert werden kann, könnte die Verwendung membranpermeabler Antioxidantien eine erfolgversprechende Strategie für die Behandlung und Prävention chronischer Entzündungen bei neurodegenera- tiven Erkrankungen darstellen.

Darmstadt, Techn. University, Diss., 2007. / Dateien im PDF-Format. - Enth. 4 Sonderabdr. aus verschiedenen Zeitschr. und Publ.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

Bakgrund: När en individ drabbas av Alzheimers, vilket orsakar minnessvårigheter påverkas även familjen då vardagen drastiskt förändras. För att öka de närståendes hälsa och bemöta dem på bästa sätt behövs en ökad förståelse om deras upplevelser.Syfte: Syftet med denna studie är att genom självbiografier belysa närståendes upplevelser av att leva med en person som har Alzheimers sjukdom i det dagliga livet.Metod: En litteraturstudie av självbiografier skrivna utav närstående till personer med Alzheimers.Resultat: I resultatet kunde vissa upplevelser urskiljas. Upplevelserna delades upp i olika kategorier. Huvudkategorier var Att börja förstå, Att förlora en livskamrat, Att leva med en person med Alzheimers, Att leva psykologiskt ensam.Diskussion: Många negativa upplevelser och likheter framkom i datamaterialet. Det som framkom i studien kan styrkas med tidigare studier. Huvudkategorin Att förlora en livskamrat visade sig vara framträdande hos de närstående.Implikationer för Omvårdnad: Sjuksköterskan kan genom en ökad förståelse för de närståendes negativa upplevelser ge stöd som kan bidra till mera positiva upplevelser. / Background: When an individual is diagnosed with Alzheimer's, which causes memory difficulties, this will also affect the family/relatives as everyday life drastically changes. To assure optimal approach of these relatives and to improve their health a greater understanding of their experiences is required.Aim: The aim of this review is to shed light upon relatives' experiences of everyday life living with an individual with Alzheimer's disease.Method: A literature review. Result: Analysis of autobiographies written by relatives of individuals with Alzheimer's revealed certain specific experiences. The experiences were categorized. Main categories were: Beginning to understand, To loose a life-partner, To live with an individual with Alzheimer's, To live alone psyhologically. Discussion: A lot of negative experiences and similarities appeared in the material. What emerged from the study is substantiated by previous studies. Main Categories To loose a life-partner was found to be prominent in the related. Implications for Nursing: Nurse can by a better understanding of the related negative experiences support that can contribute to more positive experiences.

The enzyme monoamine oxidase (MAO) is linked to mental disorders such as depression and neurodegenerative diseases. Our laboratory has recently demonstrated that increases in calcium (Ca2+) can enhance MAO activity and that this might contribute to Alzheimer disease (AD). AD has been linked to gain-of-function mutations in the presenilin-1 (PS-1) protein that not only promote the generation of the toxic amyloid-â peptide, but that also alter intracellular Ca2+ availability. <p>Radioenzymatic MAO assays were used to demonstrate that over-expression of different AD-related PS-1 mutant proteins, i.e. Y115H, ÄEx9 and M146V, in hippocampal-derived HT-22 cells alter either basal and/or Ca2+-sensitive MAO-A activity. The effects of PS-1 mutant proteins on the availability of intracellular Ca2+ are not consistent suggesting that this may not be the primary means of regulating MAO activity. The sensitivity of MAO to Ca2+ was also demonstrated in cortical (both MAO-A and MAO-B responded to Ca2+) and cerebellar (only MAO-A responded to Ca2+) samples from transgenic mice overexpressing the PS-1 (M146V) mutation. These changes in MAO coincided with changes in the availability of the neurotransmitters dopamine, noradrenaline and serotonin in the cerebellum, but not in the cortex, and reflect the known regional differences in neurotransmitter regulation. Immunoprecipitation studies and the observed increase in MAO-A activity following in vitro chemical inhibition of the ã-secretase complex (comprising several proteins including PS-1) support the notion that PS-1 constitutively associates with MAO-A. These effects on Ca2+-sensitive MAO function could contribute to AD-related pathology and could also contribute to the depression often associated with AD.

Alzheimer disease (AD) and related dementias affect nearly one in thirteen Canadians over the age of 65. Difficulties in communicating are frequently cited as the greatest source of stress for individuals who have a diagnosis of dementia and their families. Despite the wide recognition that the family is affected by a relative’s diagnosis of dementia, there has been little research aimed at understanding the experience of the family as a unit. The purpose of this study is to gain insight into the family experience of communicating with a relative who has dementia. This study used a qualitative single case study design, drawing on the theory of Symbolic Interactionism. Three members of one family participated in two individual interviews and a family interview. The family member who was experiencing symptoms associated with AD but whose diagnosis was referred to as “cognitive impairment” (CI), participated in one individual interview. Interviews were transcribed and the data was analyzed using constant comparative analysis. The findings that emerged from the data indicate that the participating family approached communication with the intention of achieving three particular goals in their interactions. These goals were to include, protect, and bring happiness to their afflicted family member. Three strategies were identified as the primary strategies used to achieve these goals: interpreting, scripting, and translating. Further, the family was organized in such a way that members were positioned either as part of the ‘core’ of the family or on the ‘outskirts’ of the family. Family members that pursued and achieved the three goals in their interactions with the relative who has CI were considered to be part of the core while others who were not willing or capable of interacting in this way were positioned on the outskirts of the family. Understanding the communication experience of the family as a unit offers a vital link to meet the needs of families dealing with the effects of CI. This knowledge will aid in formulating important new questions and insights for researchers and clinicians to provide the care and support necessary to promote the well-being of families affected by CI.

El objetivo de esta tesis fue estudiar el efecto de las manipulaciones farmacológicas y genéticas sobre el complejo gamma-secretasa. El estudio de estos cambios en el complejo gamma-secretasa es un buen modelo de las alteraciones proteicas que ocurren durante el avance de la enfermedad de Alzheimer. Se ha sugerido que los niveles de colesterol celular pueden modular el metabolismo de la proteína precursora de amiloide (APP). Estudios epidemiológicos han mostrado una menor incidencia y prevalencia de Enfermedad de Alzheimer (EA) en individuos tratados con estatinas. En el presente trabajo, investigamos en detalle la relación existente en la reducción de los niveles de colesterol, el procesamiento de APP y la función de &#947;-secretasa en cultivos celulares. Nuestros resultados indican que la disminución de los niveles de colesterol reduce la asociación de APP a los rafts lipídicos e interrumpe la interacción APP-PS1. En conjunto, nuestros resultados muestran que una reducción leve del colesterol de membrana altera el procesamiento de APP en los pasos previos al procesamiento por &#947;-secretasa. Esta alteración puede ser debida a cambios en el tráfico de APP y su distribución en los rafts lipídicos. Mutaciones en el gen de la APP son una de las principales causas de EA familiar hereditaria. Estas mutaciones alteran el procesamiento de APP y la generación de &#946;-amiloide. Recientemente nuestro grupo describió el hallazgo de una mutación en APP I716F en una familia con dos portadores. El probando presentaba un EA de inicio precoz (31 años) y murió a los 36 años de edad. El análisis neuropatológco muestra abundantes placas difusas compuestas de &#946;-amiloide (A&#946;) 42 y ovillos neurofibrilares extensos. Se observaron cuerpos de Lewy localizados en la amígdala. El estudio de esta alteración se llevó a cabo en una línea celular a la cual transfectamos la mutación de interés. Estos datos sugieren que la mutación provoca una menor proteólisis de la proteína APP por &#947;-secretasa. En conjunto, nuestros resultados indican que la mutación APP I716F está asociada con la edad de inicio más temprana de la enfermedad y muestra la correlación inversa existente entre la ratio A&#946;42/ A&#946;40 y la edad de inicio de la EA. Esta pérdida de función puede representar un mecanismo adicional mediante el cual, algunas mutaciones localizadas alrededor del lugar de procesamiento de la &#947;-secretasa provocan EA familiar.

La formació de fibres amiloides és un tret característic dels sistemes nerviosos afectats per malalties com la d'Alzheimer o les priòniques. En els teixits, les fibres es troben associades a les membranes cel·lulars i a elements de la matriu extracel·lular com els glicosaminoglicans. En les diferents malalties amiloidogèniques, pèptids i proteïnes amb una homologia seqüencial molt baixa, donen lloc a la formació d'agregats amiloides, de característiques físico-químiques molt semblants. Aquest fet implica la possibilitat que existeixi un mecanisme comú de formació d'agregats amiloides i, per tant, una possible via d'intervenció comuna a les diferents malalties. En aquest sentit, ha estat descrit recentment un hipotètic motiu estructural d'unió a esfingolípid que seria comú al fragment 1-28 del pèptid &#946; amiloide, relacionat amb la malaltia d'Alzheimer, i el fragment 185-208 de la proteïna priònica humana. A més, les dues seqüències contenen residus d'His, involucrats en possibles motius d'unió a heparina. El fragment 1-28 del pèptid &#946; amiloide ha estat molt estudiat pel que fa a la seva capacitat per formar fibres amiloides, a la influència de l'heparina en el procés d'agregació i, fins i tot, en la seva capacitat d'interacció amb membranes biològiques. Les propietats amiloidogèniques del fragment 185-208 de la proteïna priònica, en canvi, eren fins ara desconegudes. Combinant l'espectroscòpia d'IR i de fluorescència amb la microscòpia electrònica, hem procedit a la determinació de les característiques amiloidogèniques del fragment priònic. Els resultats mostren que aquest fragment és capaç de formar fibres amiloides, seguint el característic procés de polimerització nucleada, només en presència d'heparina i de membranes biològiques amb càrrega superficial negativa. Les fibres formades mostren la típica morfologia amiloide. A diferència del fragment 1-28 del pèptid de l'Alzheimer, el fragment priònic és citotòxic, reduint en un 25% la viabilitat cel·lular en una línia de neuroblastoma. Un cop caracteritzat el fragment priònic, s'han estudiat les capacitats anti-amiloidogèniques dels dendrímers, uns polímers ramificats amb càrrega superficial positiva capaços d'interferir en el procés d'agregació amiloide, modulant la formació de fibres. / La formación de fibras amiloides es una característica fundamental de los sistemas nerviosos afectados por la Enfermedad de Alzheimer o las enfermedades priónicas. En estos tejidos, las fibras están asociadas a las membranas celulares y a glicosaminoglicanos. En las diferentes enfermedades amiloidogénicas, péptidos y proteínas con homología secuencial muy baja dan lugar a la formación de agregados amiloides, los cuales tienen características fisicoquímicas muy similares. Este hecho implica la posibilidad de que exista un mecanismo común de formación de este tipo de agregados y, por tanto, una posible via de intervención común a las diferentes patologías. En este sentido, se ha descrito recientemente un hipotético motivo estructural de unión a esfingolípidos que seria común al fragmento 1-28 del péptido &#946; amiloide, relacionado con la enfermedad de Alzheimer, y el fragmento 185-208 de la proteína priónica humana. Además, las dos secuencias contienen residuos de His, involucrados en posibles motivos de unión a heparina. El fragmento 1-28 del péptido &#946; amiloide ha sido ampliamente estudiado en lo que se refiere a su capacidad de formar fibras amiloides, la influencia de la heparina en su proceso de agregación e, incluso, en su capacidad de interacción con las membranas biológicas. Las propiedades amiloidogénicas del fragmento 185-208 de la proteína priónica humana, en cambio, actualmente eran desconocidas. La combinación de la espectroscopia de IR y de fluorescencia con la microscopia electrónica, nos ha permitido determinar las características amiloidogénicas del fragmento priónico. Los resultados obtenidos ponen de manifiesto que este fragmento es capaz de formar fibras amiloides, siguiendo el característico proceso de polimerización nucleada, sólo en presencia de heparina y de membranas biológicas con carga superficial negativa. Las fibras formadas muestran la típica morfología amiloide. A diferencia del fragmento 1-28 del péptido del Alzheimer, el fragmento priónico resulta citotóxico, reduciendo en un 25% la viabilidad celular en una línea celular de neuroblastoma. Una vez caracterizado el fragmento priónico, se han estudiado las propiedades anti-amiloidogénicas de los dendrímeros, unos polímeros ramificados y con carga superficial positiva capaces de interferir en el proceso de agregación amiloide, modulando la formación de fibras. / Amyloid fibril formation is a hallmark of nervous systems affected by Alzheimer's and prion diseases. In both pathologies, fibrils are found associated to cellular membranes and glycosaminoglycans. In this kind of pathologies, amyloid peptides and proteins with very poor sequence homology originate very similar aggregates. This fact implies the possible existence of a common amyloid formation mechanism, and therefore, common pathogenic mechanisms. In this sense, a homologous structural sphingolipid-binding motif has been described for the Alzheimer's peptide A&#946;(1-28) and the human prion protein fragment PrP(185-208). Both sequences contain His residues in a cluster of basic residues proposed as a heparin binding motif.The A&#946;(1-28) fragment has been widely described as an amyloid peptide and the influence of heparin in its aggregation process and its interaction with cellular membranes have been studied. The amyloidogenic properties of prion fragment were still unknown. In the present work, we have used a combination of spectroscopic techniques (Fourier-Transform Infrared Spectroscoy and Fluorescence Spectroscopy) complemented with electron microscopy in order to characterize PrP(185-208) as an amyloid peptide.The results show that PrP(185-208) is able to form amyloid aggregates following a nucleation-dependent polymerization only in the presence of heparin or negatively charged model membranes. The formed fibers show the typical amyloid morphology. Whereas A&#946;(1-28) fragment is not toxic, as already known, 25% of the viability cell population in a neuronal cell line is reduced in the presence of PrP(185-208). Once the prion peptide was characterized as an amyloid, we checked the possible anti-amyloid properties of dendrimers, branched and globular polymers with a densely positive surface. Dendrimers ability to interfere with the aggregation process, modulating fibril formation is shown.

El pèptid amiloide Aβ(1-40) és un dels principals components de les plaques dels cervells afectats per la malaltia d’Alzheimer, on normalment es troba associat als lípids de la membrana cel·lular. Les membranes poden tenir un rol important en la toxicitat del pèptid, tant com a possible diana com influenciant la formació d’agregats peptídics. Un punt particularment interessant de la influència de les membranes en el procés d’agregació, del que existeixen evidències experimentals, és la peroxidació lipídica com a factor important en la neurodegeneració. L’objectiu general d’aquest treball ha estat la caracterització molecular de la interacció del pèptid amiloide amb les membranes biològiques, parant especial esment en el rol de la peroxidació lipídica. Els resultats han mostrat que en presència de membranes de lípid d’extracte de cervell s’observa una clara acceleració de formació de fibres pel pèptid Aβ(1-40) i una major formació d’aquestes. Per altra banda, l’agregació del pèptid en presència de membranes no oxidades mostra un alentiment i una menor formació de fibres. S’ha observat que aquesta diferència en l’agregació del pèptid segons el grau d’oxidació de les membranes, és deguda a l’aparició de càrregues negatives a la superfície de les membranes durant el procés d’agregació. L’aparició de càrregues negatives a la superfície de la membrana implica un augment de la concentració de protons i per tant una davallada del pH a la superfície de la membrana. Aquesta baixada de pH afecta al plegament del pèptid Aβ(1-40). A pH neutre, aquest pèptid tendeix a formar fibres, passant per una sèrie d’agregats oligomèrics. A pH inferior a 6, quan les histidines del pèptid es troben protonades, aquest pèptid forma agregats amorfs d’alt pes molecular. En aquesta tesi, s’ha vist com aquests agregats tenen una capacitat més gran de desestabilitzar la membrana que no els agregats que es formen a pH neutre. La comparació amb els sistemes vius s’ha dut a terme utilitzant dues línies cel·lulars amb característiques diferents. S’ha vist com el pèptid és més tòxic en cèl·lules PC12 que en cèl·lules SH-5YSY. Les cèl·lules PC12 són crònicament apoptòtiques, la qual cosa implica un augment de fosfatidilserina a la superfície, lípid amb cap polar negatiu, que aporta càrregues negatives a la superfície, i que, per tant, implica una davallada del pH a la superfície cel·lular d’aquestes cèl·lules. El treball conclou que aquests agregats amorfs que es formen a pH baix podrien ser els responsables de la toxicitat del pèptid. Els resultats són rellevants ja que donen un possible mecanisme molecular a la relació que els processos oxidatius i les malalties vasculars en la malaltia d’Alzheimer. Tant els processos oxidatius, que implicarien una acidificació a la superfície cel·lular degut a la peroxidació lipídica, com les malalties vasculars, que poden implicar una acidificació local del teixit, podrien afavorir la formació d’aquests agregats amorfs d’alt pes molecular augmentant la toxicitat del pèptid. / Aβ peptides are the main components of the characteristic fibrilar amyloid plaques found in central nervous systems affected by Alzheimer’s disease. Amyloid plaques are usually found associated to lipids from the plasma cell membrane. Membranes may play a role in amyloid toxicity, either as a peptide’s target or by influencing the peptide aggregation state. A particularly interesting aspect of the possible influence of lipid bilayers is the existence of an important amount of experimental evidence pointing to lipid peroxidation as an important factor in neurodegeneration. The general objective of this thesis was the molecular characterization of the peptide interaction with biological membranes. Granular non-fibrillar aggregates (GNAs) are identified as possible toxic species in Alzheimer’s disease. GNAs form on the surface of negatively charged biological membranes and as a consequence of an acidic environment, off the polymerization pathway at neutral pH. Aβ(1-40) GNAs disturb the bilayer structure of model membranes and seem to be more toxic to cells with negatively charged membranes (consequence of chronic pre-apoptosis). GNAs may be relevant in physiological situations associated to Alzheimer’s disease: a local acidic pH at the cell surface (consequence of lipid oxidation or other cell insults) and acidification as a consequence of vascular events causing hypoxia. Together with previous descriptions of granular aggregates with poly-glutamine peptides related to Huntington’s disease and the SH3 domain of PI3, GNAs related to Alzheimer’s disease are a further example of a possible common aggregation and toxicity mechanism in conformational diseases. GNAs may represent a new pharmacological target in Alzheimer’s disease.

El compuesto PF9601N es un derivado de propargilamina y un potente inhibidor irreversible de la enzima monoamino oxidasa B (IMAO-B) el cual fue identificado por nuestro grupo tras una extensiva búsqueda de potenciales IMAOs. Además de su potente capacidad inhibidora, el PF9601N posee varias propiedades neuroprotectoras demostradas en varios modelos animales y celulares de la enfermedad de Parkinson (EP). Estos efectos, los cuales han sido relacionados con la presencia de la propargilamina en su estructura, están mediados por acciones en vías involucradas en la neurodegeneración observada en otras enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA). Así, para estudiar más en detalle las propiedades beneficiosas del PF9601N investigamos sus efectos en un modelo in vivo de excitotoxicidad, un mecanismo implicado en el daño neuronal observado en las enfermedades neurodegenerativas. El hallazgo de que el PF9601N era capaz de evitar el daño excitotóxico mediante la disminución de la liberación inducida de glutamato y aspartato, y el aumento de la liberación de taurina así como mediante la prevención de la activación glial y la apoptosis proporcionó un valor añadido a este compuesto para ser considerado en la terapia de estas enfermedades. El tratamiento actual para la EA se basa principalmente en el uso de inhibidores de las enzimas colinesterasas (IChEs). Sin embargo, estos fármacos no son capaces de disminuir la progresión de la enfermedad y sólo producen una mejora temporal de los síntomas. Actualmente está ampliamente aceptado que la EA es una enfermedad multifactorial. En este contexto, la aproximación farmacológica más novedosa, conocida como aproximación de los MTDL (de las siglas en inglés “ligandos dirigidos hacia múltiples dianas”), propone el uso de compuestos multifuncionales capaces de abrazar varias propiedades biológicas. Esta tesis se centra en el estudio de la relación estructura-actividad (REA) así como la evaluación biológica de varios compuestos híbridos especialmente diseñados y sintetizados para actuar sobre múltiples factores involucrados en la EA. Los compuestos híbridos combinan la porción de bencilpiperidina de Donepecilo, un anticolinesterásico ampliamente utilizado en el tratamiento de la enfermedad, con el grupo propargilamina o indolil propargilamina presente en PF9601N, con el objetivo de obtener un compuesto capaz de retener la capacidad inhibidora de MAO así como las propiedades neuroprotectoras y antiapoptóticas de PF9601N. El trabajo presentado en esta tesis demuestra que algunos de los compuestos híbridos son potentes IMAOs (rango nM) y moderadamente potentes IChEs (rango subM). De entre todos los compuestos evaluados, ASS234 resultó ser un potente inhibidor de la agregación del péptido β−amiloide (A) y fue capaz de ejercer una acción protectora frente a la toxicidad inducida por Aβ y H2O2 en células neuronales. En resumen, los datos presentados en esta tesis doctoral sugieren que el compuesto ASS234 es un compuesto multidiana muy prometedor que podría tener un papel modificador en la EA dada su demostrada capacidad de interactuar con varias dianas involucradas en la patogénesis de esta enfermedad. / PF9601N is a propargylamine-containing irreversible monoamine oxidase B inhibitor (MAOBI) previously identified by our group in an extensive screen of potential MAOIs. Besides its potent inhibitory capacity, it possesses several neuroprotective properties demonstrated in different animal and cellular models of Parkinson’s disease (PD). The beneficial effects of PF9601N, which have been related to the propargylamine group present in the molecule, are mediated through actions in pathways that are commonly involved in the neurodegeneration observed in other neurodegenerative disorders such as Alzheimer’s disease (AD), thus making this molecule a promising agent in the therapy of this disease as well. Thus, to study the beneficial properties of PF9601N in depth, we investigated its effects against an in vivo model of excitotoxicity, an important mechanism involved in the neuronal damage observed in neurodegenerative diseases. The finding that PF9601N was able to prevent the induced excitotoxic damage by decreasing the evoked release of excitatory neurotransmitters and decreasing the output of the inhibitory and neuroprotective taurine as well as preventing the induced glial activation and apoptosis gave more value to this compound to be considered in the therapy. The current treatment for AD is the use of cholinesterase inhibitors (ChEIs) although there is also a NMDA receptor antagonist. However, far from stopping the disease’s progression, these drugs only produce a temporary symptomatic benefit, thus highlighting an urgent need to provide real disease-modifying drugs. At present, the most accepted notion is that AD is a multifactorial disease caused by many different factors and thus drug therapy with multifunctional compounds, the so-called multi-target-directed ligand (MTDL) approach, embracing diverse biological properties will have noticeable advantages over individual-target drugs or cocktails of drugs. In this context, this thesis focuses on the structure-activity relationship (SAR) study and the biological evaluation of different hybrid compounds specifically designed and synthesised to target multiple factors involved in AD. The hybrid molecules combine the benzyl piperidine moiety of Donepezil, a commonly used anticholinesterasic for the treatment of AD, with the propargylamine or the indolyl propargylamine substructure of PF9601N, with the aim of retaining the MAO inhibitory capacity as well as the neuroprotective and antiapoptotic properties observed for this compound. The work presented in this thesis demonstrates that some hybrid compounds are potent MAOIs (nM range) and moderately potent ChEIs (submicroM range). Among them, ASS234 has also been shown to reduce Αβ fibrillogenesis, and to protect neuronal cells from A and H2O2 toxicity. Thus, this compound has proved to be able to block the Aβ-induced cell death in two ways: by preventing caspase cleavage and activation and blocking LDH release. Overall, the present data suggest ASS234 as a promising MTDL that may have a potential disease-modifying role in the treatment of AD since it is able to interact with diverse targets involved in the pathogenesis underlying AD.