Challenges in sample preparation and structure determination of amyloids by cryo-EM.

Amyloids share a common architecture but play disparate biological roles in processes ranging from bacterial defense mechanisms to protein misfolding diseases. Their structures are highly polymorphic, which makes them difficult to study by X-ray diffraction or NMR spectroscopy. Our understanding of amyloid structures is due in large part to recent advances in the field of cryo-EM, which allows for determining the polymorphs separately. In this review, we highlight the main stepping stones leading to the substantial number of high-resolution amyloid fibril structures known today as well as recent developments regarding automation and software in cryo-EM. We discuss that sample preparation should move closer to physiological conditions to understand how amyloid aggregation and disease are linked. We further highlight new approaches to address heterogeneity and polymorphism of amyloid fibrils in EM image processing and give an outlook to the upcoming challenges in researching the structural biology of amyloids.

Cryo-EM of Aß fibrils from mouse models find tg-APPArcSwe fibrils resemble those found in patients with sporadic Alzheimer's disease.

The use of transgenic mice displaying amyloid-ß (Aß) brain pathology has been essential for the preclinical assessment of new treatment strategies for Alzheimer's disease. However, the properties of Aß in such mice have not been systematically compared to Aß in the brains of patients with Alzheimer's disease. Here, we determined the structures of nine ex vivo Aß fibrils from six different mouse models by cryogenic-electron microscopy. We found novel Aß fibril structures in the APP/PS1, ARTE10 and tg-SwDI models, whereas the human type II filament fold was found in the ARTE10, tg-APPSwe and APP23 models. The tg-APPArcSwe mice showed an Aß fibril whose structure resembles the human type I filament found in patients with sporadic Alzheimer's disease. A detailed assessment of the Aß fibril structure is key to the selection of adequate mouse models for the preclinical development of novel plaque-targeting therapeutics and positron emission tomography imaging tracers in Alzheimer's disease.

Conformational heterogeneity coupled with ß-fibril formation of a scaffold protein involved in chronic mental illnesses.

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as ß-fibrils. We have been able to narrow down the ß-core region participating in fibrillization to residues 716-761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease.

Endo-lysosomal Aß concentration and pH trigger formation of Aß oligomers that potently induce Tau missorting.

Amyloid-ß peptide (Aß) forms metastable oligomers >50 kDa, termed AßOs, that are more effective than Aß amyloid fibrils at triggering Alzheimer's disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aß accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AßO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AßO formation and the high endo-lysosomal Aß concentration together enable extensive AßO formation of Aß42 under physiological conditions. Exploiting the enhanced AßO formation of the dimeric Aß variant dimAß we furthermore demonstrate targeting of AßOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AßOs.

The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid ß fibril formation.

Point mutations in the amyloid precursor protein gene (APP) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid ß (Aß). Here, we describe the Uppsala APP mutation (Δ690-695), the first reported deletion causing autosomal dominant AD. Affected individuals have an age at symptom onset in their early forties and suffer from a rapidly progressing disease course. Symptoms and biomarkers are typical of AD, with the exception of normal cerebrospinal fluid (CSF) Aß42 and only slightly pathological amyloid-positron emission tomography signals. Mass spectrometry and Western blot analyses of patient CSF and media from experimental cell cultures indicate that the Uppsala APP mutation alters APP processing by increasing ß-secretase cleavage and affecting α-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated Aß, AßUpp1-42Δ19-24, accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain.