Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 8 uppsatser.

La maladie d'Alzheimer (AD) est une pathologie neurodégénérative caractérisée par une atrophie cérébrale progressive associée à une mort neuronale. Plus récemment, il a été suggéré que la perte des fonctions cognitives survenant pendant la maladie s'explique principalement par une atteinte au niveau synaptique préalable à la mort neuronale. Ainsi il a été observé que le peptide β-amyloïde ou Aβ constituant des plaques séniles, l'un des deux marqueurs histologiques de la maladie, existe sous une forme soluble/oligomérique (Aβo), et cette conformation lui confère des propriétés synaptotoxiques. L'Aβo agit préférentiellement sur le compartiment post-synaptique des synapses excitatrices également appelées épines dendritiques, structures sub-cellulaires dont la forme est régie par un cytosquelette d'actine riche et dynamique. Parmi les nombreuses hypothèses émises pour expliquer la synaptotoxicité de l'Aβo, il a été suggéré que la disparition des épines était due à une dépolymérisation anormale des filaments d'actine par une enzyme : la cofiline. Pourtant des données récentes ont montré à l'inverse une phosphorylation/inactivation de la cofiline dans le cortex frontal de patients AD, mais aussi dans le cerveau de la lignée de souris APP/PS-1, modèle de AD. De plus, des analyses morphologiques des synapses de la région CA1 chez la souris APP/PS-1 ont montré une réduction de la densité d'épines, associée à une augmentation du volume des épines survivantes. Les variations de volume de la tête de l'épine sont des phénomènes très fréquents lors d'une induction de potentialisation à long terme, le corrélat électrophysiologique de la mémoire.. Au cours de ma thèse, nous avons cherché dans un premier temps à caractériser les altérations morphologiques des épines dendritiques chez la souris APP/PS-1 par microscopie électronique. Nous avons pu confirmer que dès 3 mois, les synapses excitatrices sont moins nombreuses, que les épines restantes sont plus larges, mais surtout, que l'épaisseur de la densité post-synaptique n'est plus proportionnelle à la surface de l'épine, ce qui suggère un découplage entre modifications morphologiques et fonctionnelles. Nous avons également mis en évidence la présence de spinules anormales sur les épines.En utilisant des cultures primaires de neurones corticaux, nous avons pu montrer qu'un traitement aigu avec de l'Aβo induit la formation de protrusions riches en actine filamenteuse ressemblant aux spinules observés chez les animaux transgéniques. En purifiant la fraction post-synaptique, nous avons montré que cette formation de protrusions est concomitante à une phosphorylation anormale de la cofiline induite par l'Aβo. Ainsi l'inactivation de la cofiline qui en résulte pourrait être à l'origine d'une stabilisation et donc d'un allongement des filaments d'actine synaptique conduisant à la formation des protrusions. Cette inactivation de la cofiline a également été retrouvée chez la souris APP/PS-1 et chez l'humain. En conclusion, l'ensemble des résultats de cette thèse montre que l'Aβo induit des déformations morphologiques des épines, qui se caractérisent par la formation de protrusions membranaires ressemblant à des spinules. Ces protrusions ne sont pas activité-dépendantes, mais proviennent plutôt d'une dérégulation de l'activité enzymatique de la cofiline par l'Aβo. / Alzheimer's disease (AD) is a neurodegenerative pathology associated with progressive cerebral atrophy linked to neuronal death. It has been recently suggested that loss of cognitive functions occurring during the disease was a consequence of synapse dysfunction and prior to neuronal death. Thus, it has been observed that Amyloïd-β peptide (Aβ), the main component of senile plaques, one histological marker of the disease, also exists as soluble/oligomeric Aβ (Aβo). This Aβ conformation is known to be synaptotoxic. Aβo acts preferentially on the post-synaptic compartment of excitatory synapses, also named dendritic spines, sub-cellular micro-domains containing dynamic and filamentous actin as their main cytoskeleton component. Among numerous theories explaining Aβo synaptotoxicity, it has been suggested that spine collapsing was due to an abnormal actin depolymerisation through Cofilin enzyme. Yet, recent evidences inversely showed Cofilin phosphorylation/inactivation in frontal cortex of AD patients and in the APP/PS-1 transgenic mice brain, an AD animal model. Moreover, synapse morphological analysis in the CA1 region of APP/PS-1 mice showed a reduction in spine density and an increase in spine head volume of remaining ones. Spine head volume variations are commonly occurring during induction of Long Term Potentiation, the electrophysiological correlate of memory.During my thesis, we firstly characterized APP/PS-1 mice dendritic spine morphological alterations using electron microscopy. We confirmed that even at 3 month-old, excitatory synapses are fewer, but also that remaining ones display larger surfaces. In addition, PSD thickness is not proportional to spine surface anymore, which suggests an uncoupling between functional and morphological modifications. We also demonstrated the presence of abnormal shaped spinules onto spines.Using primary cortical neuron cultures, we demonstrated that acute Aβo treatment induces the formation of filamentous actin enriched protrusions, resembling spinules observed in transgenic mice. By purifying post-synaptic protein fraction, we showed that protrusions formation is correlated to an abnormal Cofilin phosphorylation/inactivation by Aβo. Thus, resulting Cofilin inactivation could trigger actin filament stabilization, leading to protrusion formation. We also found Cofilin phosphorylation in APP/PS-1 mice and in AD brains. Taken together, these results show that Aβo triggers dendritic spine abnormal alterations, characterized by the formation of membrane protrusions ressembling spinules. These protrusions are not activity-dependant, but may instead originate from a disregulation of Cofilin enzymatic activity by Aβo.

Alzheimer’s Disease (AD) is the leading cause of dementia in the world and the World Health Organization has recognized AD as a global public health priority. One of the pathological hallmarks of AD is amyloid plaques formed from amyloid β (Aβ) fibrils. Aβ is formed when amyloid precursor protein is cleaved by secretase enzymes. Cleavage by different secretases causes Aβ to occur in different forms, mainly as 40 and 42 residue long proteins, called Aβ1-40 and Aβ1-42, where Aβ1-42 is more likely to form amyloid fibrils and is therefore considered more harmful. Fluorescent probes are currently used to stain Aβ fibrils for their detection and characterization.  We performed a literature study analysing which fluorescent probes are used for imaging of amyloid fibrils and present both the most commonly used probes but also newer probes that have been recently synthesized. Fluorescence spectra of a selection of probes were analysed in order to suggest some new combinations of probes for double-staining with the aim to be able to distinguish between Aβ1-40 and Aβ1-42. Microscopy images of the probe combinations were obtained in order to analyse the double staining results and the fluorescence intensities of the probes were plotted in different ways. All selected combinations were able to distinguish between Aβ1-40 and Aβ1-42, because of differently stained fibrils, and also displayed differences in fluorescence intensity at peak emission wavelength. The obtained results show that double-staining of amyloid fibrils with fluorescent probes can give additional information compared to staining fibrils with only one probe.

Alzheimer’s disease (AD) is symptomized by amyloid-beta plaques in the brain and accounts for more than 65 percent of dementia cases. Vitamin B12 (cobalamin) deficiency can result in similar cognitive impairment and roughly 15% of the elderly are vitamin B12 deficient. Vitamin B12 deficiency results in the accumulation of toxic methylmalonic acid and homocysteine. Hyperhomocysteinemia is a strong risk factor for AD. To test if vitamin B12 deficiency stimulates amyloid-beta toxicity, Caenorhabditis elegans expressing amyloid-beta in muscle were fed either vitamin B12-deficient OP50-1 or vitamin B12-rich HT115(DE3) E. coli bacteria. Increased amyloid-beta toxicity was found in worms fed the 0P50-1 diet. Supplementation of the OP50-1 diet with vitamin B12 did not rescue the increased C. elegans toxicity. Knockdown of either of the only two C. elegans vitamin B12-dependent enzymes metr-1 or mmmc-1 protected against toxicity. Therefore, vitamin B12 deficiency does not stimulate Alzheimer’s amyloid-beta-mediated toxicity in C. elegans.

La maladie d’Alzheimer (MA) est une neurodégénérescence liée à l’âge, caractérisée par l’agrégation intracérébrale du peptide Aβ. Plusieurs études ont montré que la signalisation insulin-like growth factor (IGF), régulateur clé de la longévité, est impliquée dans la progression de la MA. Nous avons récemment démontré que la suppression de la signalisation IGF neuronale à l’âge adulte chez un modèle de souris MA freine la progression de la pathologie amyloïde par une clairance de l’Aβ facilitée. L’objectif de ma thèse est d’identifier les mécanismes cellulaires et moléculaires liant signalisation IGF et neuroprotection contre la protéotoxicité Aβ. J’ai démontré que l’ablation de l’IGF-1R neuronal au cours du vieillissement protège des déficits cognitifs et de la neuroinflammation liée aux oligomères Aβ, et réduit substantiellement la taille du soma neuronal adulte. Pour identifier les voies impliquées dans cette neuroprotection, j’ai caractérisé le profil transcriptomique de neurones KO IGF-1R microdisséqués dans la région CA1 de l’hippocampe. J’ai démontré que la MA et l’ablation de l’IGF-1R neuronal impactent les mêmes fonctions biologiques, comme la neurotransmission, la croissance, et la transduction du signal, et presque tous les changements d’expression géniques communs à la MA et au KO IGF-1R neuronal se produisent dans le même sens. En revanche, dans les cerveaux MA, une proportion significative des gènes dérégulés par la MA sont inversés par le KO IGF-1R neuronal. Mes résultats révèlent que l’inhibition de la voie IGF neuronale à l’âge adulte protège de la pathologie Aβ en régulant l’organisation du cytosquelette, la neurotransmission, et la réponse au stress. / Alzheimer's disease (AD) is an age-related neurodegenerative disease, characterized by intracerebral amyloid-β (Aβ) peptide aggregation. Several studies have shown that insulin-like growth factor (IGF) signaling, a key regulator of longevity, is involved in AD progression. We recently showed that suppression of neuronal IGF signaling during adulthood alleviates amyloid pathology and cognitive deficits in AD mice through A clearance. In this context, the aim of my thesis was to identify the cellular and molecular mechanisms linking IGF signaling to neuronal protection against Aβ proteotoxicity. I demonstrated that ablation of neuronal IGF-1R during aging reduces cognitive deficits and neuroinflammation linked to A oligomers (AO), and induces a conspicuous decrease in neuronal soma size. To identify which pathways are involved in previously observed neuroprotection, I characterized the transcriptome profiling of microdissected hippocampal CA1 neurons from mice where neuronal IGF-1R was conditionally ablated at 3 months of age. I found that AD and neuronal IGF-1R inactivation impact on similar neuronal functions, namely neurotransmission, growth and differentiation, and signal transduction, and almost all of the changes in gene expression common to AD and IGF-1R ablation occurred in the same direction. However, in AD brains, a significant proportion of genes deregulated by AD were reversed by IGF-1R knockout. My results also reveal that inhibition of IGF signaling in adult neurons protects from A pathology by regulating cytoskeleton organization, neurotransmission, and response to stress.

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.

La maladie d'Alzheimer est la démence la plus répandue dans le monde. Le nombre de cas augmente de manière exponentielle et il est donc important de comprendre les mécanismes moléculaires donnant lieu à cette terrible maladie. Une des hypothèses les plus supportées est celle suggérant que la production et dégradation déséquilibrées de l'amyloïde-beta (Aß), un peptide de 42 acides aminés trouvé dans tous les individus sains, est un événement clé dans le déroulement de la maladie d'Alzheimer. En effet, une production accrue ou une dégradation faible du peptide ont pour conséquence son agrégation et accumulation dans des plaques de fibres entre les neurones des régions spécifiques du cerveau. C'est pourquoi la modulation de l'agrégation du peptide Aß est une des approches envisageables pour modifier l'évolution de la maladie d'Alzheimer. Les protéines chaperons dont une des fonctions est d'assister d'autres protéines dans leur repliement, sont parmi les molécules les plus étudiées pour leur capacité modulatrice de l'agrégation des protéines (inclus le peptide Aß). Plusieurs chaperons ont montré la capacité d'inhiber la formation des fibres par l'Aß. Cependant, du fait que les chaperons sont des molécules conservées et peu spécifiques, leur surexpression ou administration directe peut avoir des conséquences négatives si les chaperons interagissent avec des protéines autres que la protéine cible. Dans ce travail, nous nous sommes intéressés à une protéine chaperon bactérienne possédant une forte activité " holdase " (i.e., elle empêche le repliement précoce des protéines) comme possible modulateur de l'agrégation du peptide Aß. Le chaperon sauvage a une très faible capacité d'inhibition de la formation de fibres par le peptide Aß. Cependant, nous avons démontré qu'en modifiant légèrement la surface de liaison du chaperon, la protéine devient un puissant inhibiteur de l'agrégation d'Aß. En parallèle, nous nous sommes intéressés à l'influence des ions métalliques sur l'agrégation du peptide Aß. [...] / Alzheimer's disease is the most frequent type of dementia. With an exponentially growing number of cases, understanding the underlying molecular events leading to this devastating condition is of crucial importance. Much evidence points to a disequilibrium in the production and degradation of amyloid beta (Aß), a normally physiological 42 amino acid peptide, as an early key event in Alzheimer's etiology. Whether Aß is overproduced or poorly degraded, the overall result is an abnormally large pool of peptide that gradually aggregates forming extracellular deposits of fibrils, called amyloid plaques, in specific brain regions. Hence, modulation of Aß aggregation process is one of the suggested approaches to control the evolution of Alzheimer's disease. Universally conserved molecular chaperones have been intensively studied for their capacity to prevent aggregation of disease-related proteins, and many of them have proven to efficiently modulate Alzheimer's Aß aggregation. In a scenario where chaperones are overexpressed or directly administered into the affected tissue, the universal conservation and the relatively poor client-specificity of generic chaperones can become a downside because of the risk of interaction with proteins other than the targeted one is not dismissible, and thus the consequences unpredictable. In the first part of this work, we looked upon a bacterial chaperone call SecB with an unusually robust holdase activity (i.e. it prevents early protein folding) as a promising modulator of Alzheimer's Aß peptide aggregation. [...]

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers.

Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia associated with amyloid-beta peptide deposition and loss of mitochondrial function and regulation. Currently, there is no cure for AD, thus, there is a need to continuously develop therapeutic strategies that could address the complex multifactorial causes of AD development. Due to this necessity, this study has investigated the role of vitamin B2 as a disease modifying drug for AD by employingamyloid-beta and mitochondrial based AD therapeutic strategies. Using a transgenic C. elegans AD worm model expressing amyloid-beta (Aβ1-42) in muscle cells at temperature upshift to 25°C, we screened for protective effect of dose-dependent concentrations of active forms of vitamin B2, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), against amyloid-beta mediated paralysis. Protective concentrations were then assayed for improvement of mitochondrial metabolic functions by performing ATP, oxygen consumption and reactive oxygen species (ROS) production assays. Consequently, we investigated for drug protective mechanisms of FMN and FAD using RNAi genetic screening technique. FMN and FAD significantly delayed amyloid-beta mediated paralysis and improved mitochondrial metabolic functions at final concentrations of 0.74mM and 0.74µM respectively. More so, both compounds induced activation of stress response FOXO transcription factor, daf-16. Specifically, FMN treatment induced mitochondrial unfolded protein response (UPRmt) pathway through ubiquitin-like protein (ubl-5) activation as well as other stress response pathway signature such as Activating Transcription Factor Associated with Stress (atfs-1). This study will be useful in understanding the importance of micronutrients such as vitamin B2 in normal cellular function as related to neurodegenerativediseases and aging. Therefore, vitamin B2 supplementation could be an important source of Alzheimer’s disease therapeutic strategy.

Alzheimer’s disease (AD) is associated with amyloid-beta peptide deposition and loss of mitochondrial function. Using a transgenic C. elegans AD worm model expressing amyloid-beta in body wall muscle, we determined that supplementation with either of the forms of vitamin B2, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) protected against amyloid-beta mediated paralysis. FMN and FAD were then assayed to determine effects on ATP, oxygen consumption, and reactive oxygen species (ROS) with these compounds not significantly improving any of these mitochondrial bioenergetic functions. Knockdown of the daf-16/FOXO transcriptional regulator or the FAD synthase enzyme completely abrogated the protective effects of FMN and FAD, while knockdown of the mitochondrial unfolded protein response factors ubl-5 or atfs-1 also blocked the protective effects. Therefore, vitamin B2 supplementation could lead to the activation of conserved signaling pathways in humans to delay the onset and progression of neurodegenerative diseases such as AD.