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Amyloidogenic diseases, such as Alzheimer's disease (AD) and Type II Diabetes (T2D), are characterized by the accumulation of amyloid aggregates. Despite having very different amino-acid sequences, the underlying amyloidogenic proteins form similar supramolecular fibril structures that are highly stable and resistant to physical and chemical denaturation. AD is characterized by two toxic lesions: extracellular amyloid β-peptide (Aβ) plaques and intracellular neurofibrillary tangles composed of microtubule-associated protein tau. Similarly, a feature of T2D is the deposition of islet amyloid polypeptide (IAPP) aggregates in and around the pancreas. The mechanisms by which Aβ, tau, and IAPP aggregate, and cause cell death is unknown; thus, gaining greater insight into the stabilizing forces and initial unfolding events is crucial to our understanding of these amyloidogenic diseases. This work uses molecular dynamics (MD) simulations to study the secondary, tertiary, and quaternary structure of Aβ, tau, and IAPP. Specifically, this work used the Drude polarizable force field (FF), which explicitly represents electronic polarization allowing charge distributions to change in response to perturbations in local electric fields. This model allows us to describe the role charge plays on protein folding and stability and how perturbations to the charge state drive pathology. Studies were conducted to address the following questions: 1) What are the stabilizing forces of fibril and oligomeric structures? 2) How do charge-altering mutations modulate the conformational ensemble and thermodynamic properties of Aβ? 3) How do charge-altering post-translational modifications of Aβ and tau modulate changes in the conformational ensembles? These studies establish that shifts in local microenvironments play a role in fibril and oligomer stability. Furthermore, these studies found that changes in protein sequence and charge are sufficient to disrupt and change the secondary and tertiary structure of these amyloidogenic proteins. Overall, this dissertation describes how charge modulates protein unfolding and characterizes the mechanism of those changes. In the long term, this work will help in the development of therapeutics that can target these changes to prevent protein aggregation that leads to cell death. Doctor of Philosophy Protein aggregation is the hallmark of many chronic diseases, such as Alzheimer's disease (AD) and Type II Diabetes (T2D). The formation of two toxic aggregates: amyloid β-peptide (Aβ) plaques and neurofibrillary tangles composed of microtubule-associated protein tau are some of the key characteristics of AD. In addition, the formation of islet amyloid polypeptide (IAPP) aggregates in the pancreas is thought to play a role in the development of T2D. The pathways by which the proteins Aβ, tau, and IAPP aggregate are unknown; thus, gaining a greater insight into the properties that may cause these diseases is necessary to develop treatments. By studying these proteins at the atomistic level, we can understand how small changes to these proteins alter how they misfold in a way that promotes toxicity. Herein, we used a computational technique called molecular dynamics (MD) simulations to gain new insights into how protein structure changes. We explored the dynamics of these proteins and investigated the role that charge plays in protein folding and described how charge modulates protein folding and characterized the mechanism of those changes. This work serves as a characterization of protein folding and sets the ground for future structural studies and drug development.

Amyloid β-peptide (Aβ) misfolding aggregates with β-sheet structures and surplus reactive oxygen species (ROS) are both considered to be the culprit of neuronal toxicity in Alzheimer’s disease (AD). Therefore, modulating the misfolding mode of Aβ and inhibiting ROS simultaneous has become an important method for anti-AD. Herein, a nanoscale manganese-substituted polyphosphomolybdate (H2en)3[Mn(H2O)4][Mn(H2O)3]2[P2Mo5O23]2·14.5H2O (abbreviated as MnPM) (en = ethanediamine) was designed and synthesized by single crystal to single crystal transformation method. MnPM can modulate the β-sheet rich conformation of Aβ aggregates, and thus reduce the formation of toxic species. Moreover, MnPM also possesses the ability to eliminate the free radicals produced by Cu2+-Aβ aggregates. It can inhibit the cytotoxicity of β-sheet-rich species and protect synapses of PC12 cells. MnPM combines the conformation modulating ability of Aβ and anti-oxidation ability, which makes a promising multi-funcational molecular with a composite mechanism for the new conceptual designing in treatment of such protein-misfolding diseases.

The effect of Aβ25–35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C14] galactose and results showed that Aβ25–35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25–35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 μM was able to prevent the toxicity triggered by the fibrillar Aβ25–35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24 h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer’s disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer’s disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer’s treatment.

Genetic AD (Alzheimer's disease) accounts for only few AD cases and is almost exclusively associated with increased amyloid production in the brain. Instead, most patients are affected with the sporadic form of AD and typically have altered clearance mechanisms. The identification of factors that influence the onset and progression of sporadic AD is a key step towards understanding its mechanism(s) and developing successful therapies. An increasing number of epidemiological studies describe a strong association between AD and cardiovascular risk factors, particularly hypertension, that exerts detrimental effects on the cerebral circulation, favouring chronic brain hypoperfusion. However, a clear demonstration of a pathophysiological link between cardiovascular risk factors and AD aetiology is still missing. To increase our knowledge of the mechanisms involved in the brain's response to hypertension and their possible role in promoting amyloid deposition in the brain, we have performed and investigated in depth different murine models of hypertension, induced either pharmacologically or mechanically, leading in the long term to plaque formation in the brain parenchyma and around blood vessels. In the present paper, we review the major findings in this particular experimental setting that allow us to study the pathogenetic mechanisms of sporadic AD triggered by vascular risk factors. Abbreviations: Aβ, amyloid β-peptide; AD, Alzheimer's disease; APP, amyloid precursor protein; BBB, blood–brain barrier; CAA, cerebral amyloid angiopathy; CBF, cerebral blood flow; LPS, lipopolysaccharide; LRP-1, low-density lipoprotein receptor-related protein-1; NSAID, non-steroidal anti-inflammatory drug; TAC, transverse aortic coarctation

The Amyloid β-peptide (Aβ) is accumulated in several diseases including GNE myopathy and Alzheimer’s disease (AD). GNE myopathy is a skeletal muscle disorder caused by biallelic mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which codifies for a key enzyme in sialic acid biosynthesis. The mutated GNE gene encodes a hypofunctional enzyme causing a decrease in cellular sialic acid. We have found that hyposialylation favors Aβ endocytosis in skeletal muscle cells, which is dependent on clathrin and heparan sulfate proteoglycan. Furthermore, we have observed that intracellular Aβ induces apoptosis in skeletal muscle cells through the impairment of Akt signaling pathway. Accordingly, Akt phosphorylation is reduced and apoptosis is enhanced in GNE myopathy myoblasts. Finally, through a genome-wide screen in Saccharomyces cerevisiae we have identified novel modulators of Aβ toxicity including components of the mitochondrial respiratory chain and members of the Ca2+ signaling pathways. In conclusion, this study provides a better understanding of the Aβ relevance in the pathophysiology of GNE myopathy and AD., El pèptid β-amiloide (Aβ) s’acumula en diverses malalties com la miopatia de GNE i la Malaltia d’Alzheimer (MA). La miopatia de GNE és una malaltia del múscul esquelètic causada per mutacions al gen UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), que codifica un enzim clau en la biosíntesis de l’àcid siàlic. El GNE mutat produeixen un enzim hipofuncional i una conseqüent disminució de l’àcid siàlic cel·lular. Hem vist que la hiposialització afavoreix l’endocitosi de l’Aβ en cèl·lules musculars, la qual és dependent de clatrina i de l’heparà sulfat proteoglicà. A més, hem observat que l’Aβ intracel·lular indueix l’apoptosi en cèl·lules musculars mitjançant la inhibició de l’Akt. Així, la fosforilació de l’Akt es troba reduïda i l’apoptosi induïda en mioblasts d’un pacient amb la miopatia de GNE. Finalment, a través d’un cribratge del genoma complert de Saccharomyces cerevisiae, hem identificat nous moduladors de la toxicitat per Aβ que inclouen components de la cadena respiratòria mitocondrial i membres de la via de senyalització del Ca2+. En resum, aquest estudi ofereix una millor comprensió del rol de l’Aβ en la miopatia de GNE i la MA.

The contribution of astrocytes to the pathophysiology of AD (Alzheimer's disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer's transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer's brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.

It has become clear that the products of several of the earliest identified myelin protein genes perform functions that extend beyond the myelin sheath. Interestingly, these myelin proteins, which comprise proteolipid protein, 2′, 3′-cyclic nucleotide 3′-phosphodiesterase and the classic and golli MBPs (myelin basic proteins), play important roles during different stages of oligodendroglial development. These non-myelin-related functions are varied and include roles in the regulation of process outgrowth, migration, RNA transport, oligodendrocyte survival and ion channel modulation. However, despite the wide variety of cellular functions performed by the different myelin genes, the route by which they achieve these many functions seems to converge upon a common mechanism involving Ca2+ regulation, cytoskeletal rearrangements and signal transduction. In the present review, the newly emerging functions of these myelin proteins will be described, and these will then be discussed in the context of their contribution to oligodendroglial development.

AD (Alzheimer's disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between GM1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells), but the precise effects of GM1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of GM1 and Aβ-(1–40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either GM1 or Aβ-(1–40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of GM1 and Aβ-(1–40). The exogenously added GM1 and Aβ-(1–40) were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with GM1 and Aβ-(1–40), but caspase 3 was activated. NECs treated with GM1 and Aβ-(1–40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that GM1 and Aβ-(1–40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40) and GM1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.