Sunday, May 11, 2025
5/11/2025
Probing the biochemical mechanism of amyloid disease - We seek to understand how the process of transthyretin (TTR) aggregation, and which aggregate structures lead to the dysfunction and ultimately the death of post-mitotic tissue in the TTR amyloid diseases. Understanding this structure-proteotoxicity relationship would enable the development of novel therapeutic strategies, and the establishment of early diagnostic biomarkers that quantify response to therapy. A mutation in one TTR allele results in destabilized heterotetramers comprising mutant and wild type (WT) TTR subunits being secreted from the liver, which exhibit faster rate-limiting tetramer dissociation and monomer misfolding, affording a spectrum of aggregate structures, including amyloid fibrils linked to autosomal dominant familial TTR amyloid cardiomyopathy. There is also sporadic WT TTR cardiomyopathy, affecting as much as 10% of the elderly male population. In a clinical trial assessing the potential of tafamidis (a drug that binds to and stabilizes the native TTR tetramer preventing misfolding and aggregation of newly synthesized TTR) for treating TTR cardiomyopathy, the amyloid load in the heart did not detectably change on the timescale of the tafamidis clinical response, rendering it unlikely that amyloid infiltration of the heart is the main driver of mortality. In Specific Aim 1, we will take a fresh look at the pathology of the human heart using the CLARITY method to probe the 3D relationships between the non-native TTR structures present, the host molecules to which the aggregates are colocalized, discern whether aggregates are inside or outside heart cells, characterize infiltration by immune cells such as macrophages, and probe whether there are any discernable autonomic nervous system deficiencies. Another key question is why does WT TTR aggregate? We will test the hypothesis that a minor population of a less kinetically stable, alternatively folded TTR tetramer forms during non-optimal cellular folding or is produced during failed lysosomal degradation, affording a TTR tetramer that is unstable and leads to aggregation. In Specific Aim 2, we will use antibodies and peptide probes to isolate non-native TTR from polyneuropathy patient plasma to characterize the circulating non- native structure(s) by atomic force microscopy and cryo-electron microscopy in collaboration with the Lander Lab, to test the hypothesis that distinct aggregate structures drive tissue tropism. We will compare the structure of aggregates from V30M plasma (a pure polyneuropathy) vs aggregates from a pure cardiomyopathy to assess their relative cytotoxicities to DRG neurons vs cardiomyocytes, to test the idea that the tissue tropism of these diseases is aggregate structure-based. With the Coelho group, we will continue to improve early polyneuropathy diagnosis by studying V30M carriers as they progress to polyneuropathy patients using unbiased plasma proteomics. Proteomics will also be used to attempt to understand why ≈ 30% of TTR polyneuropathy patients do not respond to any approved therapies, focusing on inflammatory and immune-modulatory molecules and cells that may become drivers of polyneuropathy.
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