Factors controlling the onset and progression of extracellular amyloid diseases remain largely unknown. Central to disease etiology is the efficiency of the endoplasmic reticulum (ER) machinery that targets destabilized mutant proteins for degradation and the enhanced tendency of these variants to aggregate if secreted. We demonstrate that mammalian cells secrete numerous transthyretin (TTR) disease-associated variants with wild-type efficiency in spite of compromised folding energetics. Only the most highly destabilized TTR variants are subjected to ER-associated degradation (ERAD) and then only in certain tissues, providing insight into tissue selective amyloidosis. Rather than a "quality control" standard based on wild-type stability, we find that ER-assisted folding (ERAF), based on global protein energetics, determines the extent of export. We propose that ERAF (influenced by the energetics of the protein fold, chaperone enzyme distributions, and metabolite chaperones) in competition with ERAD defines the unique secretory aptitude of each tissue.

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 --> methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, showed conformation-dependent optical properties and could be utilized for ex vivo spectral assignment of distinct prion deposits from two mouse-adapted prion strains. p-FTAA also revealed staining of transient soluble pre-fibrillar non-thioflavinophilic Aβ-assemblies during in vitro fibrillation of Aβ peptides. In brain tissue samples, Aβ deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localization with conventional antibodies (6E10 and AT8), indicating that p-FTAA detects all the immuno-positive aggregated proteinaceous species in Alzheimer disease, but with significantly shorter imaging time (100 fold) compared to immunofluorescence. In addition, a patchy islet-like staining of individual Aβ plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA, suggesting that pre-fibrillar species are likely an intrinsic component of Aβ plaques in human brain. The major hallmarks of Alzheimer's disease, namely Aβ aggregates versus NFTs could also be distinguished due to distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, Aβ−tau interactions and pathogenesis both ex vivo and in vivo. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe formation of highly ordered aggregates of intra-or extracellular proteins underlies a wide range of neurodegenerative conditions including prion, Parkinson's, Huntington's and Alzheimer's (AD) diseases. Hence, molecular probes that specifically target protein aggregates and allow in vitro or in vivo imaging of these pathological hallmarks, are of great importance. Small hydrophobic probes that cross the blood-brain barrier (BBB) can be monitored in vivo with positron emission tomography (PET), single-photon emission computerized tomography (SPECT) or multiphoton microscopy (1-7). The latter is especially applicable in transgenic mouse models where mechanistic insights regarding the pathological events involved in the formation of protein deposits can be obtained. Additionally, molecular imaging probes may also help in early diagnosis of neurodegenerative diseases and in monitoring the effect of therapeutic interventions. However, a major drawback of these conventional probes is that only a subset of aggregates that roughly corresponds to histologically identifiable amyloid ...

Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease; however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance-presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M͞T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.A myloid diseases are a large group of an even larger collection of misfolding disorders, the former including greater than 80 familial transthyretin (TTR)-based pathologies (1-11). The TTR missense mutations associated with familial amyloid disease display a wide range of diversity in age of disease onset, penetrance, etc. In diseases resembling and including the TTR amyloidoses, normally folded secreted proteins must first undergo partial denaturation to assemble into amyloid (7-10, 12). Although amyloidosis is modulated by numerous environmental and genetic factors (13), it is known that mutations that destabilize the native state predispose an individual to disease (7,8,14). However, thermodynamic stability alone does not reliably predict disease severity (15).Tetramer dissociation is required for TTR amyloid fibril formation (12,16,17). However, the resulting normally folded monomer cannot form amyloid without undergoing partial denaturation (12, 16, 17), yielding the so-called monomeric amyloidogenic intermediate composed of a three-stranded antiparallel ␤-sheet structure (18). Herein we use chaotropic denaturation studies in an attempt to understand energetic differences between single site variants of TTR, all of which are tetrameric u...

Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far-and near-UV circular dichroism spectroscopy, onedimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.