Mit über 35 Millionen Betroffenen weltweit ist die Alzheimersche Erkrankung (AD) die häufigste Demenzerkrankung in westlichen Industrieländern. Durch stetig steigende Prävalenz, gekoppelt mit der zunehmenden Lebenserwartung, erfährt die AD eine immer schwerwiegendere ökonomische und soziale Bedeutung, zumal es bis heute keine wirksame Therapie gibt. Entsprechend ist für mögliche Therapieansätze dieser Erkrankung ein genaues Verständnis der neuropathologischen Mechanismen unerlässlich. So werden neben Therapien, die die cholinerge Hypofunktion und damit auch die zwei weiteren Hauptmerkmale (Amyloid-Plaques und neurofibrilläre Tangles) der AD positiv beeinflussen, auch viele weitere Therapieansätze geprüft. So weiß man, dass einige Substanzen die Expression verschiedener Proteine im Gehirn beeinflussen können, die eine mögliche Rolle bei der Pathogenese der AD spielen. Vor allem untersucht die vorliegende Arbeit, ob eine chronische Behandlung mit dem Ginkgo biloba-Extrakt EGb761 eine veränderte Expression der Proteine Transthyretin (TTR), Prolaktin, Gesamt-Tau und Wachstumshormon (GH) in Hirnen von Mäusen bewirkt. Diese Marker zeigten sich in Mikroarray-Vorversuchen in Hirnmaterial von mit Ginkgo biloba behandelten Mäusen auf Ebene der Genexpression verändert. Veränderungen werden in verschiedenen Fütterungsgruppen dargestellt und diskutiert. Hierzu wird das Methodenspektrum der Immunhistochemie sowie der computergestützten Bildanalyse mittels Densitometrie genutzt. EGb761 sowie im Besonderen dessen Flavonoid-Fraktion bewirkten – im Vergleich zur Kontrollgruppe – eine signifikant erhöhte Immunreaktivität des Proteins Transthyretin in der hippokampalen Formation, während die Terpenoid-Fraktion keine Wirkung auslöste. Die übrigen Marker erschienen unbeeinflusst. Die Ergebnisse werden sowohl im Zusammenhang mit der aktuellen Literatur zu AD als auch mit verschiedenen, vermeintlich neuroprotektiven Proteinen diskutiert.

Wild-type transthyretin amyloidosis (ATTRwt) is a rare, sporadic protein misfolding disorder with no validated biomarkers or specific treatments. The disease is characterized by deposition of amyloid fibrils composed of wild-type transthyretin (TTR) in cardiac tissue, which leads to cardiomyopathy, heart failure, and death within 5 years. The hypothesis for the studies detailed in this dissertation was non-coding variants in the TTR gene regulatory regions impact expression and serum levels of the protein, thereby contributing to ATTRwt pathogenesis. Investigations included examination of 2 enhancer regions and the proximal promoter of the TTR gene for risk factors which could contribute to pathogenesis of ATTRwt amyloidosis. In total, 11 common and 20 rare variants were identified. The analyses demonstrated significant associations of 3 variants with increased disease risk and 4 variants with age at disease onset and/or survival. Functional studies using GFP and luciferase reporter assays in HepG2 cells were performed to examine the impact of nucleotide alterations in the TTR proximal promoter on reporter expression. Three ATTRwt-risk factors (rs3764479, rs72922940, rs3794885), caused significantly decreased reporter expression in both GFP and luciferase assays (p < 0.02). Moreover, serum TTR levels, measured by immunoturbidity and analyzed along with ATTRwt clinical data, demonstrated that lower serum TTR concentrations were associated with worse survival (hazard ratio = 0.89, p = 0.003). Follow-up analysis of an ATTRwt subset treated with diflunisal, a TTR stabilizer, showed increased serum TTR (p = 0.002) and organ improvement as assessed by cardiac biomarkers (p = 0.043). Unexpectedly, our genetic sequencing data suggested that the TTR G6S variant was disease-protective. Analysis of the TTR G6S protein using circular dichroism and aggregation assay corroborated these findings by demonstrating a higher structural stability and a lower aggregation propensity compared to L55P and V122I, two unstable amyloidogenic TTR variants. In summary, the major findings of this work were: 1) identification of genetic variants that confer risk for ATTRwt amyloidosis through changes in expression, 2) evidence in support of serum TTR as a candidate for monitoring disease progression and response to treatment, and 3) evidence suggesting that TTR G6S may confer protection from ATTRwt by slowing the amyloid cascade. / 2017-09-08T00:00:00Z

BACKGROUND: Studies in various chronic diseases have correlated health-related quality of life (HRQOL) with disease state and treatment outcomes. Limited data exists on the association between HRQOL, survival, and clinical biomarkers of disease in wild-type and familial TTR amyloidosis (ATTRwt & ATTRm) patient populations. OBJECTIVES: To assess the association between HRQOL and survival, as well as HRQOL and clinical biomarkers of disease in transthyretin-mediated amyloidosis (ATTR) patient populations. METHODS: Using a retrospective cohort study design, HRQOL was assessed via SF-36 health surveys collected from patients with ATTRwt and ATTRm presenting for their initial evaluation at the BU Amyloidosis Center between 1985 and 2015. Kaplan-Meier curves and hazard ratios (HRs) calculated using Cox proportional hazards regression analysis were used to examine the association between physical (PCS) and mental (MCS) component scores derived from the SF-36 health surveys and survival follow-up. All analyses were adjusted for potential confounders such as age at presentation, gender, and co-morbidities including diabetes mellitus, hypertension and hyperlipidemia. Spearman’s rank correlations were calculated to assess the association between PCS, MCS and clinical biomarkers of disease (mBMI, troponin I and BNP) also collected at time of initial evaluation visit. Statistical significance was set at a two-sided alpha=0.05. RESULTS: In the ATTRwt cohort, 133 white males, aged 74.6 +/- 6.0 years (mean + SD) presented with mean MCS (45.7 +/- 12.3) and PCS (36.7 +/- 10.8) that were respectively, 0.5 and 1.5 SD below 50. Patients with PCS or MCS scores < 35 had a significantly higher risk of death during follow-up than those with scores >= 35 for PCS (HR=2.45; p=0.002) and for MCS (HR=3.38, p<0.0001). BNP and troponin I associated with MCS (r= - 0.24; p=0.01 and p=0.02, respectively) and PCS (r= - 0.29; p=0.002 and r= - 0.25; p=0.012, respectively). In the ATTRm cohort, 331 white (82%) males (67%), aged 57.5 +/- 13.9 years presented with mean MCS (45.2 +/- 11.7) and PCS (37.2 +/- 13.3). Patients with PCS scores < 35 had a significantly higher risk of death during follow-up than those with scores >= 35 (HR=2.76; p= <0.0001). In contrast, MCS scores < 35 did not correlate with increased risk of death during follow-up (HR=1.38, p=0.13). BNP and troponin I most strongly associated with PCS (r= - 0.50; p<0.0001 and r= - 0.41; p<0.0001, respectively) and less with MCS (r= - 0.16; p=0.03 and r= - 0.24; p=0.007, respectively). mBMI did not associate with MCS or PCS in the ATTRwt and ATTRm cohorts. CONCLUSIONS: ATTR disease significantly decreased an individual’s physical and mental HRQOL. PCS and MCS were shown to be independent predictors of mortality but their ability to predict survival varied by cohort. Assessment of HRQOL may provide valuable prognostic information that could be of use in the management of ATTR disease.

Amyloidosis is a group of clinical disorders caused by the aggregation of specific proteins into abnormal extracellular deposits. Today, 31 different proteins have been linked to amyloid diseases including transthyretin-related amyloidosis (ATTR). ATTR occurs through the aggregation of either wild-type plasma protein transthyretin (TTR) or a mutated form. TTR is a homotetramer that under normal circumstances functions as a carrier of thyroxine and retinol binding protein. The aggregation cascade requires dissociation of the tetramer into monomers, and preventing this dissociation represents a potential mode of intervention. Interestingly, small molecules, referred as kinetic stabilizers, can bind to TTR’s thyroxine-binding site (TBS) and such molecules are currently being used as a therapeutic approach to impair tetramer dissociation. The efficacy of TTR stabilization is directly correlated to the binding affinity of the ligand to TBS. However, the binding of the ligand to TTR in vivo can be affected by other plasma components resulting in poor efficacy. Thus, the selectivity of ligands is an important parameter. We have designed an assay where the ability to stabilize TTR can be directly evaluated in plasma and we have investigated the stabilizing effect of nine potential TTR binders (Paper I). The results, surprisingly, revealed that the binding affinity of molecules has a poor correlation to its selectivity. However, the nature of protein-ligand complex formation can also be described by enthalpic (∆H) and entropic (∆S) energy contributions. ∆H represents the change in chemical bonds and frequently requires a higher order of orientation compared to the ∆S component, which mainly represents the hydrophobic effect via the exclusion of water. We hypothesized that ligands possessing high ΔH in binding to their co-partner would also be more specific in a complex environment such as plasma. By applying a thermodynamic analysis using isothermal titration calorimetry, we found that the selectivity in plasma correlates well with the ∆H contribution and might, therefore, be a better predictor for selectivity. Luteolin was found to be a highly selective stabilizer of TTR and was investigated further (Paper II). The ligand displayed a significant rescuing effect in both cell culture and animal models. However, luteolin undergoes rapid enzymatic degradation in the liver and this impairs its use as a potential therapeutic drug. To attempt to circumvent this issue, we modified the most exposed hydroxyl group thus rendering the molecule inert towards glucuronidation (Paper III). The substitutions resulted in higher stability in the face of hepatic degradation molecules, but they also affected the selectivity in a negative manner. The screening for new TTR stabilizers resulted in the discovery of tetrabromobisphenol A, which displayed a very high selectivity (Paper IV). This study also included a comparison with the drug Vyndaqel™ which currently is in clinically use, and showed how the dosage could be altered to acquire a better level of saturation and possibly also a better clinical effect. Taken together we present new molecules with the ability to stabilize TTR, and these can serve as scaffolds for the design of new drugs. We present a method to measure the efficacy of a TTR-stabilizing drugs in a complex matrix and as well as a way to adjust the dosage of existing drugs. We also show that the selectivity of a drug is affected by the relative proportion of ∆H and ∆S, and this is of interest for drug design in general.

Amyloid formation occurs when normally soluble proteins and peptides misfold and aggregate into intractable threadlike structures called fibrils. There are currently more than 30 proteins associated with this aberrant structure, including the Aβ peptide in Alzheimer’s disease (AD) and transthyretin (TTR) in TTR amyloidosis. TTR is a homotetrameric transporter protein present in both cerebrospinal fluid and plasma. Dissociation of its tetrameric structure is required for the formation of amyloid fibrils. Small molecule ligands able to bind and stabilize the tetrameric structure of TTR thus represent a potential therapeutic intervention. Interestingly, apart from TTR’s role as a toxic agent in TTR amyloidosis, it also has a role as an inhibitor of the Aβ toxicity associated with AD. The work presented in this thesis focused on small molecules that have the potential ability to prevent TTR amyloidosis. We also sought to gain a greater understanding of the interaction between TTR and the Aβ peptide with respect to Aβ fibril formation. The ability of a drug to stabilize TTR is directly correlated to its binding affinity. However, since TTR is a plasma protein, it is of great importance that the drug binds selectively to TTR. In paper I, we used a newly developed urea denaturation assay, in combination with isothermal titration calorimetry, to show that, in a complex environment such as plasma, the enthalpy of binding correlates better with a drug’s ability to stabilize TTR than the binding affinity. In paper II, we modified the highly selective but rapidly degraded TTR ligand luteolin in order to increase its resistance against biotransformation. Using a liver-based microsome assay, in combination with HPLC, we show how the luteolin analogues have gained increased stability. However, using the urea assay, we also show that the analogues have lost much of luteolin’s selectivity. In paper III, we show that tetrabromobisphenol A is a highly selective binder of TTR in plasma and is able to rescue cells from TTR-induced toxicity. In paper IV, we studied the interaction of TTR with Aβ and its effect on Aβ fibril formation. We used a ThT fluorescence-based assay and dot blotting to show that TTR inhibits Aβ amyloid formation and promotes the formation of high molecular weight assemblies with an open N-terminus. Using surface plasmon resonance, we further show how TTR is unable to inhibit fibril elongation and instead targets the nucleation processes, both primary and fibril-catalyzed secondary nucleation. To conclude, we present new molecules with the ability to selectively stabilize TTR that can serve as scaffolds in drug design. We also elucidate TTR’s inhibiting effects on toxic Aβ amyloid formation.

Amyloidoses comprise a group of diseases where normal or mutated protein precipitates into amyloid fibrils. The deposition of fibrils causes dysfunction of organs and toxicity to nervous tissue. Up to date, 24 different proteins and peptides are known to be able to form amyloid fibrils. The most well known are Amyloid beta peptide and Prione protein causing Alzheimer’s disease and Creutzfeld Jacob’s disease respectively. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the mechanisms involved. The model protein mostly used in the studies was the plasma protein transthyretin (TTR). Familial Amyloidotic Polyneuropathy (FAP) is a hereditary, autosomal-dominant neurodegenerative disease caused by point mutations in the TTR gene. One of the most common variants of FAP is a mutation in position 30 where alanine is exchanged for methonine. This gives rise to “Skellefteåsjukan” in Sweden. TTR is secreted into the plasma as a tetramer. Point mutations destabilize the tetramer leading to disassembled monomers, which undergo partial denaturation as an initiation step to aggregation and amyloid fibril formation. In vivo amyloidogenesis takes a long time and does not occur until late in adult life. Most of the clinical TTR mutations do not form amyloid in vitro under physiological conditions. We have created amyloidogenic TTR mutants that are prone to aggregate and form fibrils under physiological conditions. This provides us with a model system on the cellular level for studies of the mechanisms of amyloid associated cytotoxicity as we can control the aggregation process and capture defined stages in the TTR amyloidogenic pathway. We used Atomic Force Microscopy (AFM) to follow the morphology of aggregates during fibril formation. Initially, amorphous aggregates were formed that subsequently matured into fibrillar structures, denoted protofilaments. This observation was interpreted as an optimisation of ß-strand registers. In addition we identified a correlation between the presence of early-formed aggregates of TTR and cytotoxicity. The toxic response was mediated via an apoptotic mechanism. We were not able to more carefully determine the structure and size of the toxic TTR species. To address this problem we turned to another amyloidogenic protein, equine lysozyme (EL). Intermediate samples corresponding to the aggregation and growth phase of amyloid fibrils of EL were collected. These samples were subjected to cytotoxicity assays as well as monomeric starting material and mature amyloid fibrillar species. The results clearly showed that the soluble oligomers were cytotoxic in contrast to the monomers and fibrils. Our data indicate that the toxic properties of the oligomers are size dependent. In this thesis we asked the question whether all mutated forms of TTR can be expressed and secreted or if there is a selection against the most aggressive mutations in vivo? We transfected hematopoetic K562 cells with wild type or mutant TTR, with or without the N-terminal signal peptide, responsible for secretion, to generate both extra- and intracellular TTR. We show that the post-translational quality control of the cells does not allow intracellular mutant TTR outside the secretory pathway, possibly due to the cytotoxic effects, while translocated to the secretory pathway made it escape the quality control permitting secretion and amyloid formation outside the cells. We have further analyzed the cytotoxic mechanisms induced by TTR oligomers with a focus on intracellular apoptotic signalling pathways. We show that TTR oligomers bind to the surface of the target cells but are not taken up, that is in contrast to mature fibrils that do not bind them at all. The apoptotic response occurred in a caspase-independent and a free radical dependent way.

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.

The transthyretin-associated amyloidoses are a group of protein-folding disorders caused by deposition of the liver-secreted plasma protein transthyretin (TTR) in various tissues of the body. The sporadic form of the disease is caused by deposition of wild-type TTR whereas the inherited form is caused by deposition of mutated TTR; there are over 100 known amyloidogenic mutations of the TTR gene. The transcriptional regulation of the mouse transthyretin gene has been well studied. Organ-specific modulation of TTR mRNA is achieved by coordinated binding of hepatocyte-specific and ubiquitously expressed transcription factors to regulatory regions in the proximal promoter and upstream enhancer region. The hypothesis of this dissertation is that non-coding genetic sequence variations in the promoter of the transthyretin gene situated upstream of the regulatory regions, alter its transcriptional regulation, contributing to the onset and expression of transthyretin-associated amyloidosis. Previously, we identified a significant association of a non-coding polymorphism of the TTR promoter, rs3764479, with age of onset and survival in patients with ATTRwt amyloid disease. In this dissertation, electrophoretic mobility shift assays (EMSA) were used to investigate transcription factor binding of HepG2 nuclear proteins to short DNA probes with and without rs3764479. These mobility shift studies revealed that HepG2 nuclear extract proteins showed higher affinity to the wild-type TTR sequence than to one containing the rs3764479 SNP. Competition EMSAs suggested SNP-related changes in the binding of transcription factors hepatocyte nuclear factor-1 (HNF1) and hepatocyte nuclear factor-3b may alter TTR gene transcription. To investigate transthyretin gene regulation in V122I ATTR amyloid, the most prevalent TTR gene variant in the United States, the proximal promoter region from patients with V122I ATTR amyloidosis was sequenced and analyzed. In total, 8 SNPs were identified; one (rs955705399) was significantly associated with disease between the two V122I genotype-positive cohorts studied with and without cardiomyopathy. It is postulated that the presence of SNPs could influence gene expression and ultimately disease pathogenesis. In summary, these studies suggest that presence of disease-associated non-coding genetic variations modify transthyretin gene expression by disrupting transcription factor binding and may, in part, explain the clinical heterogeneity seen in patients with transthyretin-associated amyloidoses.

Transthyretin (TTR) is one of several proteins involved in amyloid disease in humans. Unknown conformational changes of the native state of TTR result in aggregation of TTR molecules into amyloid fibrils, which accumulate in extracellular tissues. This may result in different clinical symptoms, e.g. polyneuropathy or cardiomyopathy, depending on their site of accumulation. Our long-term goal is to identify structural changes associated with amyloid formation. For this work, structural characterization of TTR from other species than human may provide valuable information. The three-dimensional X-ray crystallographic structure of TTR from sea bream (Sparus aurata) was determined at 1.75 Å resolution. Human and sea bream TTR were found to be structurally very similar. However, interesting differences were present in the area at and around -strand D, which in fish forms an extended loop region. Interestingly, this area is believed to dissociate from the structure prior to amyloid formation, to allow -strands A and B to participate in polymerization. During evolution, TTR from different species have developed differences in preference to their natural ligands, the thyroid hormones 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4). While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution. A significantly wider entrance and narrower thyroid hormone binding channel provide a structural explanation to the differences in thyroid hormone preference between human and piscine TTR. In a separate work, we identified a novel protein family with structural similarity to TTR, which we named the transthyretin-related protein (TRP) family. To attain information about this protein family, we cloned, expressed, purified and characterized TRP from Escherichia coli (EcTRP). Furthermore, we solved the structure of EcTRP to 1.65 Å resolution. As predicted, EcTRP and human TTR are structurally very similar. Interesting structural differences are found in the area corresponding to the thyroid hormone binding site in TTR, which due to its amino acid conservation within the TRP family we identified as a putative ligand-binding site in TRPs. The function of the TRP is not known, however, recent studies suggest that it might be involved in purine catabolism. It has been shown that partial acid denaturation of human TTR results in amyloid-fibril formation. Interestingly, we have shown that sea bream TTR also forms amyloid-like fibrils in vitro, even though it shares only 52% sequence identity to human TTR. Corresponding studies on EcTRP did not generate amyloid-like fibrils. EcTRP has 30% sequence identity to human TTR. The fact that two of the proteins form amyloid fibrils and one does not means that they can serve as a model system for the study of amyloid formation. Further studies on these three proteins are currently performed to attain more information about the mechanism of amyloid formation.

Mit über 35 Millionen Betroffenen weltweit ist die Alzheimersche Erkrankung (AD) die häufigste Demenzerkrankung in westlichen Industrieländern. Durch stetig steigende Prävalenz, gekoppelt mit der zunehmenden Lebenserwartung, erfährt die AD eine immer schwerwiegendere ökonomische und soziale Bedeutung, zumal es bis heute keine wirksame Therapie gibt. Entsprechend ist für mögliche Therapieansätze dieser Erkrankung ein genaues Verständnis der neuropathologischen Mechanismen unerlässlich. So werden neben Therapien, die die cholinerge Hypofunktion und damit auch die zwei weiteren Hauptmerkmale (Amyloid-Plaques und neurofibrilläre Tangles) der AD positiv beeinflussen, auch viele weitere Therapieansätze geprüft. So weiß man, dass einige Substanzen die Expression verschiedener Proteine im Gehirn beeinflussen können, die eine mögliche Rolle bei der Pathogenese der AD spielen. Vor allem untersucht die vorliegende Arbeit, ob eine chronische Behandlung mit dem Ginkgo biloba-Extrakt EGb761 eine veränderte Expression der Proteine Transthyretin (TTR), Prolaktin, Gesamt-Tau und Wachstumshormon (GH) in Hirnen von Mäusen bewirkt. Diese Marker zeigten sich in Mikroarray-Vorversuchen in Hirnmaterial von mit Ginkgo biloba behandelten Mäusen auf Ebene der Genexpression verändert. Veränderungen werden in verschiedenen Fütterungsgruppen dargestellt und diskutiert. Hierzu wird das Methodenspektrum der Immunhistochemie sowie der computergestützten Bildanalyse mittels Densitometrie genutzt. EGb761 sowie im Besonderen dessen Flavonoid-Fraktion bewirkten – im Vergleich zur Kontrollgruppe – eine signifikant erhöhte Immunreaktivität des Proteins Transthyretin in der hippokampalen Formation, während die Terpenoid-Fraktion keine Wirkung auslöste. Die übrigen Marker erschienen unbeeinflusst. Die Ergebnisse werden sowohl im Zusammenhang mit der aktuellen Literatur zu AD als auch mit verschiedenen, vermeintlich neuroprotektiven Proteinen diskutiert.