Structure-based inhibitors of amyloid beta core suggest a common interface with tau.

Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline

The Freshman, vol. 6, no. 8

The Freshman was a weekly, student newsletter issued on Mondays throughout the academic year. The newsletter included calendar notices, coverage of campus social events, lectures, and athletic teams. The intent of the publication was to create unity, a sense of community, and class spirit among first year students

The Freshman, vol. 6, no. 1

The Freshman was a weekly, student newsletter issued on Mondays throughout the academic year. The newsletter included calendar notices, coverage of campus social events, lectures, and athletic teams. The intent of the publication was to create unity, a sense of community, and class spirit among first year students

Structural characterization of the functional amyloid protein Orb2A, and evaluation of structure-based inhibitors of amyloid assembly

Amyloids are stable protein assemblies characterized by their β-sheet rich secondary structure and unbranched, fibrillar morphology. Whereas formation of amyloid is traditionally associated with neurodegenerative diseases such as Alzheimer’s disease (AD), the amyloid fold has also been adapted for beneficial biological functions. These so-called functional amyloids exhibit similar structural characteristics to pathogenic amyloids, but are not toxic to their hosts. The study of functional amyloid structure and mechanisms may provide insight into amyloid disease pathogenesis, and aid in the development of therapeutics. In chapter one, I present the atomic-resolution structure of the amyloid-driving N-terminal segment of the functional amyloid protein Orb2A. Using micro-electron diffraction (micro-ED), I determined the structure of this nine-residue segment, which I term M9I, and found that it forms a classical amyloid steric zipper structure, with phenylalanine side chains playing a critical role in the formation of the self-complementary dry interface. Using electron microscopy, x-ray diffraction, and Thioflavin-T binding assays I show that the M9I segment is sufficient to form amyloid-like fibrils, and replacement of phenylalanine residues with tyrosine reduces fibril formation of the Orb2A prion-like domain (PLD). I also propose a structural model for full-length Orb2A that incorporates both this M9I steric zipper structure, and a previously published cryo-EM structure of the downstream glutamine/histidine (Q/H)-rich region from the related Orb2B isoform. In chapters two and three, I evaluate amyloid aggregation inhibitors that were rationally designed using steric zipper structures of segments derived from full-length proteins. Chapter two describes the amyloidogenic and phase-separating behavior of the nucleocapsid (NCAP) protein of SARS-CoV-2, and the structure-based design of peptide inhibitors against steric zipper-forming segments from its central low-complexity domain (LCD). My contribution to this work included high-throughput screening of this inhibitor panel in a cell culture model of SARS-CoV-2 infection, and subsequent evaluation of inhibitor hits. I identified an inhibitor termed G12 that robustly reduced SARS-CoV-2 infection in a dose-dependent manner, and disrupted phase-separation of NCAP in vitro; other weaker inhibitors of SARS-CoV-2 infection had no effect on NCAP phase separation, thereby correlating disruption of NCAP condensation with reduced infection in cultured cells. This work demonstrates that amyloid fibrils formed by NCAP can be targeted for antiviral drug design, and the G12 peptide inhibitor is a prototype molecule that may be further optimized for the treatment of severe COVID-19 disease. Chapter three describes the structure of the AD-associated protein amyloid-β (residues 16-26 D23N), and the design of peptide inhibitors that reduce cytotoxicity and aggregation of full-length Aβ(1-42) and cross-seeding of tau by Aβ. My contribution to this work was in evaluation of inhibitor panels for reduction of Aβ-induced cytotoxicity in N2a cells. I identified one inhibitor termed D1 that reduced cytotoxicity when co-incubated with Aβ(1-42) prior to exposure to cells; a second generation of inhibitors was designed based on D1, and I identified two (D1b and D1d) that reduced cytotoxicity both when co-incubated with Aβ, and when added to pre-formed Aβ fibers directly before exposure to N2a cells. These inhibitors may be considered as lead molecules that can be further optimized for treatment of AD

Micro-electron diffraction structure of the aggregation-driving N terminus of Drosophila neuronal protein Orb2A reveals amyloid-like β-sheets

Amyloid protein aggregation is commonly associated with progressive neurodegenerative diseases, however not all amyloid fibrils are pathogenic. The neuronal cytoplasmic polyadenylation element binding protein is a regulator of synaptic mRNA translation and has been shown to form functional amyloid aggregates that stabilize long-term memory. In adult Drosophila neurons, the cytoplasmic polyadenylation element binding homolog Orb2 is expressed as 2 isoforms, of which the Orb2B isoform is far more abundant, but the rarer Orb2A isoform is required to initiate Orb2 aggregation. The N terminus is a distinctive feature of the Orb2A isoform and is critical for its aggregation. Intriguingly, replacement of phenylalanine in the fifth position of Orb2A with tyrosine (F5Y) in Drosophila impairs stabilization of long-term memory. The structure of endogenous Orb2B fibers was recently determined by cryo-EM, but the structure adopted by fibrillar Orb2A is less certain. Here we use micro-electron diffraction to determine the structure of the first 9 N-terminal residues of Orb2A, at a resolution of 1.05 Å. We find that this segment (which we term M9I) forms an amyloid-like array of parallel in-register β-sheets, which interact through side chain interdigitation of aromatic and hydrophobic residues. Our structure provides an explanation for the decreased aggregation observed for the F5Y mutant and offers a hypothesis for how the addition of a single atom (the tyrosyl oxygen) affects long-term memory. We also propose a structural model of Orb2A that integrates our structure of the M9I segment with the published Orb2B cryo-EM structure

De novo designed protein inhibitors of amyloid aggregation and seeding.

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-β (Aβ) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aβ, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD

Structure-based inhibitors of amyloid beta core suggest a common interface with tau.

Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies