Summary Human Presequence Protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing a ∼13,300Å3 catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and amyloid-β (Aβ), the latter of which contributes to Alzheimer's disease pathogenesis. Here we report crystal structures for hPreP alone and in complex with Aβ, which show that hPreP uses size-exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aβ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP.

Amyloid-β (Aβ) fibrils in neuritic plaques are a hallmark of Alzheimer’s disease (AD). Since the 42-residue Aβ (Aβ42) fibril is the most pathogenic among different Aβ species, its structural characterization is crucial to our understanding of AD. While several polymorphs have been reported for Aβ40, previous studies of Aβ42 fibrils prepared at neutral pH detected essentially only one structure, with an S-shaped β-sheet arrangement (e.g., Nat. Struct. Mol. Biol.201522499). Herein, we demonstrate the feasibility of characterizing the structure of trace amounts of brain-derived and synthetic amyloid fibrils by sensitivity-enhanced 1H-detected solid-state NMR (SSNMR) under ultrafast magic angle spinning. By taking advantage of the high sensitivity of this technique, we first demonstrate its applicability for the high-throughput screening of trace amounts of selectively 13C- and 15N-labeled Aβ42 fibril prepared with ∼0.01% patient-derived amyloid (ca. 4 pmol) as a seed. The comparison of 2D 13C/1H SSNMR data revealed marked structural differences between AD-derived Aβ42 (∼40 nmol or ∼200 μg) and synthetic fibrils in less than 10 min, confirming the feasibility of assessing the fibril structure from ∼1 pmol of brain amyloid seed in ∼2.5 h. We also present the first structural characterization of synthetic fully protonated Aβ42 fibril by 1H-detected 3D and 4D SSNMR. With procedures assisted by automated assignments, main-chain resonance assignments were completed for trace amounts (∼42 nmol) of a fully protonated amyloid fibril in the 1H-detection approach. The results suggest that this Aβ42 fibril exhibits a novel fold or polymorph structure.

Seeding of amyloid fibrils into fresh solutions of the same peptide or protein in disaggregated form leads to the formation of replicate fibrils, with close structural similarity or identity to the original fibrillar seeds. Here we describe procedures for isolating fibrils composed mainly of β-amyloid (Aβ) from human brain and from leptomeninges, a source of cerebral blood vessels, for investigating Alzheimer's disease and cerebral amyloid angiopathy. We also describe methods for seeding isotopically labeled, disaggregated Aβ peptide solutions for study using solid-state NMR and other techniques. These methods should be applicable to other types of amyloid fibrils, to Aβ fibrils from mice or other species, tissues other than brain, and to some non-fibrillar aggregates. These procedures allow for the examination of authentic amyloid fibrils and other protein aggregates from biological tissues without the need for labeling the tissue.

Objective Autosomal‐dominant familial Alzheimer disease ( AD ) is caused by by variants in presenilin 1 ( PSEN 1 ), presenilin 2 ( PSEN 2 ), and amyloid precursor protein ( APP ). Previously, we reported a rare PSEN 2 frameshift variant in an early‐onset AD case ( PSEN 2 p.K115Efs*11). In this study, we characterize a second family with the same variant and analyze cellular transcripts from both patient fibroblasts and brain lysates. Methods We combined genomic, neuropathological, clinical, and molecular techniques to characterize the PSEN 2 K115Efs*11 variant in two families. Results Neuropathological and clinical evaluation confirmed the AD diagnosis in two individuals carrying the PSEN 2 K115Efs*11 variant. A truncated transcript from the variant allele is detectable in patient fibroblasts while levels of wild‐type PSEN 2 transcript and protein are reduced compared to controls. Functional studies to assess biological consequences of the variant demonstrated that PSEN 2 K115Efs*11 fibroblasts secrete less A β 1–40 compared to controls, indicating abnormal γ ‐secretase activity. Analysis of PSEN 2 transcript levels in brain tissue revealed alternatively spliced PSEN 2 products in patient brain as well as in sporadic AD and age‐matched control brain. Interpretation These data suggest that PSEN 2 K115Efs*11 is a likely pathogenic variant associated with AD . We uncovered novel PSEN 2 alternative transcripts in addition to previously reported PSEN 2 splice isoforms associated with sporadic AD . In the context of a frameshift, these alternative transcripts return to the canonical reading frame with potential to generate deleterious protein products. Our findings suggest novel potential mechanisms by which PSEN variants may influence AD pathogenesis, highlighting the complexity underlying genetic contribution to disease risk.

Alzheimer’s disease is characterized by neuritic plaques, the main protein components of which are β-amyloid (Aβ) peptides deposited as β-sheet-rich amyloid fibrils. Cerebral Amyloid Angiopathy (CAA) consists of cerebrovascular deposits of Aβ peptides; it usually accompanies Alzheimer’s disease, though it sometimes occurs in the absence of neuritic plaques, as AD also occurs without accompanying CAA. Although neuritic plaques and vascular deposits have similar protein compositions, one of the characteristic features of amyloids is polymorphism, i.e., the ability of a single pure peptide to adopt multiple conformations in fibrils, depending on fibrillization conditions. For this reason, we asked whether the Aβ fibrils in neuritic plaques differed structurally from those in cerebral blood vessels. To address this question, we used seeding techniques, starting with amyloid-enriched material from either brain parenchyma or cerebral blood vessels (using meninges as the source). These amyloid-enriched preparations were then added to fresh, disaggregated solutions of Aβ to make replicate fibrils, as described elsewhere. Such fibrils were then studied by solid-state NMR, fiber X-ray diffraction, and other biophysical techniques. We observed chemical shift differences between parenchymal vs. vascular-seeded replicate fibrils in select sites (in particular, Ala2, Phe4, Val12, and Gln15 side chains) in two-dimensional 13C-13C correlation solid-state NMR spectra, strongly indicating structural differences at these sites. X-ray diffraction studies also indicated that vascular-seeded fibrils displayed greater order than parenchyma-seeded fibrils in the “side-chain dimension” (~ 10 Å reflection), though the “hydrogen-bond dimensions” (~ 5 Å reflection) were alike. These results indicate that the different nucleation conditions at two sites in the brain, parenchyma and blood vessels, affect the fibril products that get formed at each site, possibly leading to distinct pathophysiological outcomes.

A concise, biomimetic synthesis of the antifungal and antispasmodic natural product (+)-davanone is described. The key stereoselective reactions are a Sharpless asymmetric epoxidation, a thiazolium-catalyzed esterification, and a palladium-mediated cyclization. All carbons are derived from isoprene units and no protecting groups are used, permitting an atom- and redox-economical synthesis.

Context: The coronavirus disease 19 (COVID-19) pandemic is placing unparalleled burdens on regional and institutional resources in medical facilities across the globe. This disruption is causing unprecedented downstream effects to traditionally established channels of patient care delivery, including those of essential anatomic pathology services. With Washington state being the initial North American COVID-19 epicenter, the University of Washington in Seattle has been at the forefront of conceptualizing and implementing innovative solutions in order to provide uninterrupted quality patient care amidst this growing crisis. Objective: To conduct a rapid validation study assessing our ability to reliably provide diagnostic Neuropathology services via a whole slide imaging (WSI) platform as part of our departmental COVID-19 planning response. Design: This retrospective study assessed diagnostic concordance of neuropathological diagnoses rendered via WSI as compared to those originally established via traditional histopathology in a cohort of 30 cases encompassing a broad range of neurosurgical and neuromuscular entities. This study included the digitalization of 93 slide preparations, which were independently examined by groups of board-certified neuropathologists and neuropathology fellows. Results: There were no major or minor diagnostic discrepancies identified in either the attending neuropathologist or neuropathology trainee groups for either the neurosurgical or neuromuscular case cohorts. Conclusions: Our study demonstrates that accuracy of neuropathological diagnoses and interpretation of ancillary preparations via WSI are not inferior to those generated via traditional microscopy. This study provides a framework for rapid subspecialty validation and deployment of WSI for diagnostic purposes during a pandemic event.

The presence of viable SCC in cervical lymph nodes has prognostic import when taken in context with the patient's history. Viable SCC in lymph nodes was significantly associated with worse outcome among patients with non-oropharyngeal SCC who received preCRT. Nucleate keratin debris should not be considered viable SCC in lymph nodes.