Rozprawy doktorskie na temat „Amyloid Proteins (Amyloidosis)”

Amyloidosis is a group of diseases caused by proteins that have lost their correct three-dimensional conformation and instead assemble into insoluble fibrils in various tissues and organs. Today, more than 30 different proteins that can give rise to amyloid fibrils have been identified. Each protein that assembles into fibrils causes a specific disease. For clinical diagnosis of amyloid, antibodies are one of the most important tools. In this study, antibodies generated towards various amyloid-specific peptides were characterized and validated. This was assessed by immunohistochemistry, slot blot and SDS-PAGE and western blot. Congo red, an amyloid specific dye, was used for detection of amyloid. Immunohistochemical staining and slot blot analysis indicated that each antiserum used in this study was amyloid-specific. Antigen retrieval can facilitate staining by the techniques ability to break cross-linkages caused by fixation in formaldehyde. The results from the characterization of antisera in this study should be a great helpin clinical work on amyloid, and ensure correct diagnosis.

Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect.

Serum amyloid A protein (SAA) is an acute phase protein that has recently gained increasing interest as a potential marker for disease and treatment monitoring. We investigated SAA and CRP levels in (a) patients with various common infectious diseases (n=98), (b) patients with pyelonephritis (n=37) versus patients with cystitis (n=32), (c) healthy individuals of varying ages (n=231), (d) very immature newborn infants with or without nosocomial infections (NIs) (n=72) and (e) patients with bacterial infections treated with cefuroxime (n=81).

SAA significantly correlated with CRP in viral as well as in bacterial infections (for the total group: r²=0.757, p<0.0001) and showed a systemic inflammatory response in 90% of the patients with cystitis as compared with 23% for CRP. Equally high efficiencies (0.96 and 0.94 for SAA and CRP, respectively) were observed in discriminating between pyelonephritis and cystitis. SAA and high sensitive (hs) CRP were lower in umbilical cords (p<0.0001) and higher in elderly adults (p<0.0001-0.03) than in the other age groups; higher in immature newborn infants than in term infants; and higher in the NI group than in the non-NI group. Interindividual variabilities of the time course of the biomarkers SAA and CRP were considerable. Because of the smoothed distribution of SAA and CRP (i.e. elevations were both essentially unchanged during the first 3 days of cefuroxime treatment), these markers were not useful when deciding parenteral-oral switch of therapy, which occurred within this time period in most cases.

SAA is a sensitive systemic marker in cystitis. SAA and hsCRP in umbilical cord blood are close to the detection limit and increase with age. They increase in relation to NI in very immature newborn infants and might therefore be used in diagnosis and monitoring. Finally, SAA and CRP in adults with bacterial infections could not predict an early parenteral-oral switch of antimicrobial therapy.

The aggregation of misfolded proteins into amyloid fibrils is implicated in the pathogenesis of several human degenerative diseases, including Alzheimer’s, Parkinson’s and Type II diabetes. Links between the deposition of amyloid fibrils and the progression of these diseases are poorly understood, with much of the current research focused on monomer misfolding and subsequent assembly of oligomers and mature fibrils. This project examines the formation of human apolipoprotein (apo) C-II amyloid fibrils, with a focus on the stability and reversibility of amyloid fibril assembly.
The initial stages of the project were to develop a model for apoC-II amyloid fibril formation. This was achieved by analysis of the concentration dependent kinetics of apoC-II amyloid fibril formation, and correlation of these data with the final size distribution of the fibrils, determined by sedimentation velocity experiments. On the basis of these studies, a new reversible model for apoC-II amyloid fibril formation is proposed that includes fibril breaking and re-joining as integral parts of the assembly mechanism. The model was tested by rigorous experimentation, with antibody-labelling transmission electron microscopy providing direct evidence for spontaneous fibril breaking and re-joining.
The development of this model for apoC-II fibril assembly provided the foundation for experiments to investigate factors that promote, inhibit or reverse amyloid fibril formation. Factors that were considered include a molecular chaperone protein, αB-crystallin, and a chemical modification, methionine oxidation. Investigations on the effect of αB-crystallin revealed that the inhibition of apoC-II fibril formation occurs by two distinct mechanisms: transient interaction with monomer preventing oligomerisation, and binding to mature fibrils, which inhibits fibril elongation. Studies on the effect of methionine oxidation on apoC-II fibril formation showed that both the assembly and stability of the fibrils was affected by this modification. ApoC-II contains two methionine residues (Met-9 and Met-60), and upon oxidation of these residues fibril formation was inhibited. In addition, the treatment of pre-formed fibrils with hydrogen peroxide caused dissociation of the fibrils via the oxidation of Met-60, located with the fibril core structural region. The final chapter details the development of antibodies that specifically recognise the conformation of apoC-II amyloid fibrils, which provide the foundation for future studies to examine the role that apoC-II amyloid fibrils play in disease.
Overall, this thesis reveals the dynamic and reversible nature of amyloid fibril formation. New insight is also obtained of the general stability of amyloid fibrils and the processes that may regulate their formation, persistence and disease pathogenesis in vivo.

Herein, immunoglobulin light chains (LCs) native state was studied in the context of the pathology known as light chain amyloidosis (AL). This pathology is characterized by LCs overexpression, which leads to toxicity and aggregation into amyloid fibrils in target organs, with heart being the most affected one. Due to genetic rearrangement and somatic hypermutation, virtually, each AL patient presents a different amyloidogenic LC (Merlini, 2017). Because of such complexity, the fine molecular determinants of LC aggregation propensity and proteotoxicity are, to date, unclear; significantly, their decoding requires investigating large sets of cases. This project is aimed to unravel the molecular determinants linked with LCs toxicity. First, we screened several independent biophysical and structural properties of the LCs native state. In particular, we considered hydrophobicity, fold stability, flexibility and 3D structure. Our experimental approach considered two LCs sets called ‘H’ and ‘M’. The H set is composed of eight LCs from AL patients while the M set by LCs from multiple myeloma (MM) patients. M LCs were chosen as control since they are overexpressed as the toxic H LCs but they do not lead to toxicity or aggregation. To date, the molecular bases leading to LC proteotoxicity remain to be elucidated. Our data show that low fold stability and high protein flexibility correlate with amyloidogenic LCs, while hydrophobicity, structural rearrangements and nature of the LC dimeric association interface (as observed in seven crystal structures here presented) do not appear to play a significant role in protein aggregation. Additionally, it has been demonstrated that the LCs toxicity in vivo is linked to copper (Cu2+) (Diomede et al., 2017a) by increasing the radical oxygen species (ROS) production. We aimed our studied to clarify Cu2+ LCs interaction. Moreover, we wanted to assess whether Cu2+ is able to alter the biophysical properties of the native state to more aggregation prone states. Our findings reveal that H LCs interacts with Cu2+ with a higher affinity than M LCs and that His residues may be involved in Cu2+ binding. Indeed the affinity decreases in presence of protonated His residues. Moreover, data suggest that the interaction with Cu2+ increases the molecular flexibility and decreases the fold stability. These observations suggests that protein aggregation cannot be evaluated through one single parameters but by the co-action of several biophysical traits. Moreover, our results suggest that the presence of Cu2+ can alter the native LCs properties leading to a higher toxicity in vivo.

Brain inflammation is a hallmark of Alzheimer's Disease (AD) neuropathology. This involves microglial activation which has been well characterized around fibrillar plaques in humans and in transgenic mouse models. Plaques are initiated by the accumulation of Abeta1-42 (beta-amyloid) which is a highly aggregating, neurotoxic protein derived from alternative processing of APP (amyloid precursor protein). Genetic factors involved in influencing the intensity of an inflammatory response have been shown clearly in other pathologies but not in AD. Therefore, an in vitro model of microglial activation by Abeta1-42, was developed using mouse strains having a low (A/J) or high (C57BL/6) inflammatory response. Interferon-gamma-primed C57BL/6 microglia underwent morphological changes from resting (ramified) morphology to activated (ameboid) morphology, increases in nitric oxide (NO) production, and very high levels of tumor necrosis factor alpha (TNF-alpha) when stimulated with Abeta1-42. On the other hand, A/J microglia underwent few morphological changes, had increased levels of NO and had minimal increases in TNF-alpha levels when submitted to the same treatment. Thus, microglial activation to Abeta1-42 was influenced by the magnitude in inflammatory response which may be determined by genetic factors. The timing of an inflammatory response in the process of Abeta deposition is still under debate. Therefore, to define the kinetics of the inflammatory response in brain amyloidogenesis, TgCRND8 mice which overexpress hAPP and aggressively develop amyloidosis, were used to characterize the evolution of diffuse and fibrillar plaque formation and gliosis. Histopathological analysis revealed diffuse plaques as early as 12 weeks of age. Fibrillar (senile) plaques and microgliosis were seen at 13 weeks of age whereas astrocytic clustering began at 14--15 weeks of age. Microglial activation was found to be correlated strongly to Abeta deposition and fibril

A cross-sectional and a longitudinal design were employed to address whether PDAPP mice exhibit any plaque-related learning deficit. Firstly, the cross-sectional study indicated that PDAPP mice simultaneously displayed an early (plaque-independent) and age-related learning impairment. Further analysis showed that the age-related learning deficit was highly correlated with plaque burden in the hippocampus of aged PDAPP mice, suggesting that amyloid plaques play a very important role in memory loss of AD. Second, the longitudinal study showed that the same PDAPP mice exhibited significant age-related learning deficits in trials to criterion and learning capacity tasks when they aged. Interestingly, cued navigation and object recognition in both cross-sectional and longitudinal studies were unaffected, indicating normal sensorimotor function and recognition memory of PDAPP mice. The longitudinal study further showed that the age-related learning impairment was significantly correlated with significantly reduced size of field potentials, suggesting important roles of amyloid plaques in disturbing synaptic transmission and cognitive function. Preclinical data indicated that Aβ immunotherapy is very effective to improve both AD-like neuropathology and cognitive impairment in APP transgenic mice. Using active Aβ immunisation, long-term (9 months: prevention study) and short-term (5 months: treatment study) effects of Aβ vaccines (AN-1792) on synaptic transmission and spatial learning of PDAPP mice were investigated. Although neither of the two studies showed that Aβ vaccines had significant improvement on either synaptic transmission or spatial learning of PDAPP mice, both long-term and short-term Aβ vaccinations effectively reduced the levels of total Aβ. Thus, these two studies indicated that active Aβ immunotherapy might be very effective to improve AD-like pathology, but ineffective to rescue the learning impairment in APP mice.

Moduler l’agrégation de protéines amyloïdes est thérapeutiquement pertinent (p. ex. la polyneuropathie amyloïde familiale traitée avec le Tafamidis). Cependant, pour de nombreuses amyloïdoses, il n’existe pas encore de modulateur d'agrégation efficace pour thérapie. Il a été récemment montré que combiner des composés qui modulent l’agrégation d’amyloïdes peut résulter en des effets synergiques. Une stratégie de criblage combinatoire, pour identifier des cocktails synergiques de composés, pourrait donc conduire à une percée thérapeutique pour de nombreuses amyloïdoses. Cependant, les technologies à haut débit existantes ne sont pas adaptées pour le criblage combinatoire.J’ai développé SynAggreg – une technologie in vitro à haut débit très sensible, précise, reproductible, peu coûteuse et flexible - qui permet d'identifier à la fois des inhibiteurs et des accélérateurs d'agrégation, de caractériser leur mécanisme d'action sur la cinétique d'agrégation et de les classer par leur efficacité. SynAggreg est également la première technologie adaptée au criblage combinatoire et pour l’étude d’effets synergiques de manière fiable. Enfin, cette nouvelle technologie peut être facilement adaptée à plusieurs amyloïdoses en remplaçant la partie amyloïde de la protéine de fusion par des techniques de biologie moléculaire. Ainsi, SynAggreg apparaît comme une boîte à outils pour la recherche fondamentale et appliquée et possède un fort potentiel de valorisation
Modulating amyloid proteins aggregation is therapeutically relevant (e.g. the familial amyloid polyneuropathy treated with Tafamidis). However, for many amyloidoses, there is yet no efficient aggregation modulator for therapy. It was recently shown that combining compounds that modulate the aggregation of amyloids can result in synergistic effects. A combinatorial screening strategy to identify synergistic cocktails of compounds could thus lead to a therapeutic break through for many amyloidoses. However, existing high-throughput technologies are not adapted for combinatorial screening.I developed SynAggreg - a very sensitive, accurate, reproducible, cost effective, flexible and high-throughput in vitro technology - which allows identifying both aggregation inhibitors and accelerators, characterizing their mechanism of action on aggregation kinetics and ranking them by their efficiency. SynAggreg is also the first technology suitable for combinatorial screening and for studying reliably synergistic effects of combinations of compounds. Finally, this new technology can be easily adapted to several amyloidoses by replacing the amyloid part of the fusion protein with molecular biology techniques. Thus, SynAggreg appears as a toolbox for fundamental and applied research, and has a high potential for valorization

Transthyretin-related amyloidosis (ATTR) is a disease involving the formation of a misfolded transthyretin (TTR) protein and resulting insoluble aggregates that deposit in extracellular regions of various tissues and organs. There are hereditary forms of the disease, referred to as ATTRm, and more than 100 TTR amyloid-forming mutants have been reported. The major goal of this work was to analyze the biochemical and biophysical properties of a unique and recently identified TTR mutant protein, TTR Glu51_Ser52dup, found in a patient with ATTRm. Unlike other single nucleotide replacements that have been described as amyloidogenic, the gene abnormality in the present case is the first identification of a TTR duplication mutation. The patient with TTR Glu51_Ser52dup exhibited an extremely aggressive form of ATTRm; clinical symptoms included peripheral neuropathy at baseline evaluation and rapid disease progression to early death from pneumonia and congestive heart failure. We hypothesized that the TTR Glu51_Ser52dup variant would be less stable than the wild-type protein and similar in stability to another highly amyloidogenic mutant, TTR L55P; moreover, the highly unstable nature of this TTR variant would provide a basis for understanding the extremely aggressive clinical phenotype observed in this case. Using Escherichia coli (E. coli) as an expression system and an appropriately modified expression vector, we produced histidine-tagged recombinant human TTR Glu51_Ser52dup protein in high yield and purified to homogeneity. Structural and stability studies were performed by circular dichroism (CD) spectroscopy and SDS-PAGE analysis. We demonstrated that TTR Glu51_Ser52dup was less stable than the wild-type or L55P proteins when measured under different types of denaturing conditions, including thermal and chemical stress. The presence of diflunisal, a drug that stabilizes tetrameric TTR and is currently approved for treatment of ATTRm, was also investigated; our results indicated that diflunisal stabilized the TTR Glu51_Ser52dup protein. Collectively, the data obtained from these studies suggest that Glu51_Ser52dup is one of the least stable and most amyloidogenic TTR variant described to date. Future investigations are necessary to determine which specific structural elements of the protein destabilize the TTR tetramer, and precisely characterize the binding of small molecules, including diflunisal, to the protein.

Protein β2-microglobulin (β2-m) is the causative agent of dialysis-related amyloidosis (DRA), a prevalent pathology affecting individuals undergoing long-term hemodialysis. The goal of this PhD project is to explore the early stage of the aggregation mechanism of β2-m with molecular simulations, using two model systems: the ΔN6 variant, a cleaved form lacking the six N-terminal residues, which is a major component of ex vivo amyloid plaques from DRA patients, and the single point D76N mutant, recently identified as the cause of an hereditary systemic amyloidosis affecting visceral organs. Methodologically, the main goal of this research project is the development of a Monte Carlo Ensemble Docking method with a cost function that considers shape, hydrophobic and electrostatic complementarity, the major drivers of protein-protein association. The D76N mutant populates two folding intermediates called I1 and I2, which display an unstructured C-terminus and two unstructured termini, respectively. The ΔN6 variant populates one folding intermediate, with an unstructured N-terminus. Protein-protein docking simulations predict an essential role for the termini and for the DE-loop (both variants), EF-loop (D76N mutant) and BC-loop (ΔN6 variant) in the dimerization mechanism of β2-m. The terminal regions are more relevant under acidic conditions while the BC-, DE- and EF-loops gain importance at physiological pH. Our results recapitulate experimental evidence according to which Phe30 and His31 (BC-loop), Arg45 (CD-loop), and Trp60 and Phe62 (DE-loop) are dimerization hotspots (i.e. residues triggering dimerization). Additionally, we predicted the involvement of new residues such as Tyr10 (A-strand), Lys75 (EF-loop), and Trp95 and Arg97 (C-terminus), thus providing new testable predictions to guide the research on β2-m amyloidogenesis. Finally we predicted that β2-m tetramerization is mainly driven by the self-association of dimers via the N- and C-terminal regions and the DE-loop, and identify Arg3 (N-terminus), Tyr10, Arg45, Phe56 (D-strand), Trp60 and Arg97 as essential residues in the process.

No
Beta-2-microglobulin (2m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (~1 m), straight morphology are produced by incubation of the protein at pH=<3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of 2m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

Proteins are the molecular machines that control and regulate most of the vital cellular functions in living organisms, and account for about half of the total dry mass of the cell. For a protein to function properly, it must attain its correct conformation and location within the crowded milieu of the cell. A complex set of molecular chaperone proteins assist polypeptides to acquire their native functional fold within the relevant biological timescale. However, the intricacy and diverse nature of the protein folding process and various environmental factors present numerous opportunities for error, which may lead to its misfolding and aggregation. Misfolded, aggregated, non-functional proteins and peptides within the cell are implicated with more than 55 pathological conditions including various neurodegenerative diseases such as Alzheimer’s,Parkinson’s and haemodialysis-related amyloidosis, which together affect more than 24 million people worldwide. Collectively, these disorders are classified as protein conformational or protein aggregation diseases. Over the past few years, significant progress has been made to understand the various types and processes of protein aggregation, identify the toxic species, structurally characterise various species generated during their assembly and comprehend the underlying mechanisms which regulate protein conformational diseases. This thesis has attempted to address some of the issues discussed above, to assist in advancing the understanding of the complexities of protein misfolding, aggregation and their mitigation. In this thesis, I have found that various fragments of mouse Acyl co-enzyme thioesterase 7 (mAcot7) and human full-length ACOT7 form amyloid fibrils under physiological conditions. Acot7 is a brain cytosolic protein, involved in fatty acid metabolism, and has a putative role during inflammation. Using various mouse Acot7 constructs, I demonstrated that mAcot7 undergoes nucleation-independent, multi-stranded polymerisation, leading to the formation of globular oligomeric species and subsequently amyloid fibrils. Arachidonoyl-CoA, the substrate of mAcot7 did not prevent the fibrillation of mAoct7, and the protein remained enzymatically active throughout the assembly process. Understanding the biological significance of fibrillar and other forms of Acot7 is one of the avenues to be explored in the future. After putting forth a unique molecular model of mouse Acot7 fibrillation, the next objective was to identify and characterise the heterogeneous population of protein aggregates formed during polymerisation. Bis(Triphenylphosphonium) tetraphenylethene (TPE-TPP), a novel aggregation-induced emission luminogen aided in detecting early-stage protein aggregates; the species that are considered to be the most toxic entities during the development and progression of protein conformational diseases. Compared to traditional amyloid binding fluorescent dyes such as Thioflavin T (ThT), TPE-TPP showed a broader applicability in monitoring the process of fibrillation in various conditions such as acidic pH, elevated temperature, presence of potential amyloid inhibitors, and its ability to detect variations in amyloid fibril morphologies. Once I could differentiate between the different species generated during protein aggregation, I attempted to explore ways to modify these entities. Utilising the intra- and extra-cellular molecular chaperones αB-crystallin (αB-C), clusterin, α2-macroglobulin and haptoglobin, I observed that αB-C and clusterin stabilise various forms of D76N β2-microglobulin (a potent amyloidogenic variant of β2-microglobulin) generated during the fibrillation process. It is likely that these chaperones prevent primary and secondary nucleation of D76N β2-microglobulin (D76N β2m) fibrillation in vivo and decelerate the proliferation of amyloid fibril plaques. Experiments performed in vitro are usually less confined compared to the crowded cellular environment, and such crowding conditions further confound the process of protein aggregation. Moreover, oligomers of various amyloid proteins such as α-synuclein (αS) are believed to be the primary cause of cellular damage, for example in Parkinson’s disease. Utilising the highly identical presynaptic α- and β-synuclein and crowding agents such as Ficoll 400, I analysed the impact of crowding on the association of the synuclein proteins. Under physiologically relevant conditions, I found that β-synuclein (βS) exists as positively charged oligomers, which gradually polymerise to form self-assembled, non-amyloid fibril-like structures. Furthermore, βS destabilises αS and induces its aggregation, which is significantly moderated under the crowded cell-like environment. The results documented in this dissertation have assisted in advancing our knowledge and understanding of the molecular, biophysical and biochemical mechanisms of amyloid fibril formation. In addition, this thesis determined the broader applicability of TPE-TPP compared to the standard amyloid dye (i.e. ThT) as a fluorescent probe to monitor amyloid fibril formation, detect early-stage aggregates, variations in fibril morphologies, and can be deemed useful in screening amyloid inhibitors. Part of the aim to identify and characterise various protein aggregate species was to develop a method for regulating potentially toxic entities. I showed that intra- and extra-cellular molecular chaperones were capable of acting on different stages of D76N β2m aggregation, thereby reducing the effects of undesirable, misfolded toxic protein forms. Furthermore, I demonstrated that there could be other potentially hazardous forms of unwanted protein aggregates (i.e. βS self-assembled structures) that are not necessarily amyloid fibrils. Greater comprehension of protein aggregation will not only shed light on protein conformational disorders and aid in the development of therapeutics against their toxic effects, but may also offer insights into opportunities for exploiting stable, non-toxic protein conformations to our advantage.

Description of a possible approach to interfere with amyloid aggregation of alpha synuclein by molecules of natural origin and study of cellular autophagy in the presence of Beta2 microglobulin D76N aggregates to identify modulation strategies for this process.

Oligodendrocytes produce and wrap an insulating, fatty substance called myelin around axons to increase the conduction velocity of action potentials along these axons and to provide them with critical metabolic support. Highly myelinated white matter regions are amongst the first to be damaged in Alzheimer’s disease (AD), and early oligodendrocyte damage has been detected in transgenic mice carrying human genetic variants associated with the development of AD. In diseases such as multiple sclerosis, immature brain cells called oligodendrocyte progenitor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes in an attempt to replace oligodendrocytes lost to the disease, restore action potential conduction speed and protect neurons from further damage. However, OPC fate in the early stages of ADlike pathology is unknown. In this thesis, I have shown that the cells of the oligodendrocyte lineage respond differently to hyperphosphorylated microtubule-associated protein tau (tau) (Chapter 3) and amyloidosis (Chapter 4), two major pathological features of AD, respectively recapitulated in transgenic mice by the overexpression in neurons of a human genetic variant of microtubule-associated protein tau (MAPT) or the amyloid precursor protein (APP). Overexpression of MAPT in neurons indirectly increased new oligodendrocyte addition throughout the brain; however, this was not associated with a change in total oligodendrocyte number, proportion of myelinated axons and myelin thickness in MAPT mice compared to WT (Chapter 3). The OPC response to glutamate and GABA was unchanged in pre-symptomatic MAPT mice; however, OPC responded more robustly to GABA in early amyloid pathology. By overexpressing APP in neurons and oligodendrocytes throughout life, developmental myelin thickness was increased, but amyloid plaque formation was also coincident with an increase in oligodendrogenesis between 5 and 6 months of age in APP mice (Chapter 4). Together, this thesis suggests that oligodendrocyte turnover is an early feature of both tau and amyloid pathology, while myelin remodelling only occurs in amyloid pathology, which may result from neurotransmitter or excitatory-inhibitory signalling imbalance within the neuronal network. My data suggest that OPCs and oligodendrocytes are affected by both tauopathy and amyloidosis, which are critical aspects of pathology in people diagnosed with AD. More specifically, my data suggest that oligodendrocytes are particularly susceptible to undergo cell death in response to these pathological insults. The lost oligodendrocytes are replaced by OPCs early on, suggesting that early interventions that promote oligodendrocyte survival and oligodendrogenesis could be very beneficial for preserving neural circuit function in people with AD, and slowing neurodegeneration in other tauopathies.

High-density lipoproteins and their major protein, apolipoprotein A-I (apoA-I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in arteries contributing to atherosclerosis. In hereditary amyloidosis (AApoAI), a potentially fatal disease, N-terminal fragments of variant apoA-I deposit in vital organs and damage them. There is no cure for apoA-I amyloidosis and its structural basis is unknown. Previously, AApoAI mutations were mapped on the crystal structure of the human C-terminally truncated Δ(185-243)apoA-I. The results suggested that the mutation-induced destabilization of the lipid-free protein initiates β-aggregation. Our biophysical studies showed that amyloidogenic mutations G26R, W50R, F71Y and L170P did not necessarily destabilize the native structure, prompting us to search for additional triggers of apoA-I misfolding. We mapped residue segments predicted to promote β-aggregation (termed amyloid hot spots) on the atomic structure of ∆(185-243)apoA-I. The results suggested that perturbed packing of these hot spots, particularly residues 14-22, triggers amyloidosis. This enabled us to propose the first molecular mechanism of apoA-I misfolding. To explore a potential mechanism, we combined structural, stability, dynamics and functional studies of several amyloidogenic mutants and a non-amyloidogenic control, L159R. All mutants reduced structural protection of the segment 14-22, supporting our hypothesis that increased dynamics of this segment triggers AApoAI. The non-amyloidogenic mutant showed helical unfolding near the mutation site indicating susceptibility to proteolysis. We propose that the major factors that make apoA-I amyloidogenic are reduced protection of the major amyloidogenic segments combined with the structural integrity of the four-helix bundle to facilitate protein aggregation. Together, our results suggest that the fate of apoA-I in vivo depends on the balance between its misfolding, proteolysis, and protective protein-lipid interactions. Our structural and bioinformatics analysis of other members of the apolipoprotein family (A-II, A-IV, A-V, B, C-I, C-II, C-III, E, SAA) showed that apolipoproteins’ propensity to form amyloid is rooted in the proteins’ hydrophobicity, which is key to the lipid binding ability. The overlap of functional and pathologic interfaces suggests competition between normal protein function and misfolding. Therefore, increasing apolipoprotein retention on the lipid surface provides a potential therapeutic strategy against amyloidosis.