The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Ig light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African American subjects, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 y causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the development of AG10, a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I-and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10, combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. drug design | crystal structure

The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration.

Advances in lipidomics technology have facilitated the precise detection, identification and profiling of lipid species within tissues. Mass spectrometry allows for identification of lipids as a function of the total number of carbons and double bonds in their acyl chains. Such detailed descriptions of lipid composition can provide a basis for further investigation of cell signaling and metabolic pathways, both physiological and pathological. Here, we applied phospholipid profiling to mouse models relevant to Parkinson’s disease, using mice that were transgenic for human α‐synuclein (αSyn) or deleted of endogenous αSyn. Proposed functions of αSyn include phospholipid binding, regulation of membrane composition, and regulation of vesicular pools. We investigated whether αSyn gene dosage interacts with differences in phospholipid composition across brain regions or with age‐related changes in brain phospholipid composition. The most dramatic phospholipid changes were observed in αSyn wild‐type animals as a function of age and gender. αSyn genotype‐specific changes were also observed in aged, but not young, mice. Our results provide a detailed and systematic characterization of brain phospholipid composition in mice and identify age‐related changes relevant both to Parkinson’s disease and to normal aging.

Abstractα-Synuclein (αSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding αSyn cause early-onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of αSyn. Several studies have reported evidence that αSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of αSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of αSyn, we sought to extend existing reports on this interaction.Using purified proteins, we tested the ability of αSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether αSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between αSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by αSyn in any of these systems. We propose that the previously reported effects of αSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with αSyn overexpression in cells, but are not likely due to a specific and direct interaction between αSyn and PLD.Parkinson's disease (PD 1 ) is a debilitating neurodegenerative disorder that affects over one million people in the United States (for review, see (1)). Although more than 90% of PD cases are believed to occur sporadically, several genes are known to cause familial forms of PD. Among these, point mutations or increased dosage of the α-synuclein (αSyn) gene are known to cause rare cases of early-onset familial PD (2-7). The αSyn protein is also the major component of Lewy bodies and Lewy neurites, the insoluble aggregates that are neuropathological hallmarks of both familial and sporadic PD (8). Thus, dysregulation of αSyn at either the genetic or protein level can contribute to PD-type neurodegeneration.