Aß and tau prions feature in the neuropathogenesis of Down syndrome.

Down syndrome (DS) is caused by the triplication of chromosome 21 and is the most common chromosomal disorder in humans. Those individuals with DS who live beyond age 40 y develop a progressive dementia that is similar to Alzheimer's disease (AD). Both DS and AD brains exhibit numerous extracellular amyloid plaques composed of Aß and intracellular neurofibrillary tangles composed of tau. Since AD is a double-prion disorder, we asked if both Aß and tau prions feature in DS. Frozen brains from people with DS, familial AD (fAD), sporadic AD (sAD), and age-matched controls were procured from brain biorepositories. We selectively precipitated Aß and tau prions from DS brain homogenates and measured the number of prions using cellular bioassays. In brain extracts from 28 deceased donors with DS, ranging in age from 19 to 65 y, we found nearly all DS brains had readily measurable levels of Aß and tau prions. In a cross-sectional analysis of DS donor age at death, we found that the levels of Aß and tau prions increased with age. In contrast to DS brains, the levels of Aß and tau prions in the brains of 37 fAD and sAD donors decreased as a function of age at death. Whether DS is an ideal model for assessing the efficacy of putative AD therapeutics remains to be determined.

Emergence of distinct and heterogeneous strains of amyloid beta with advanced Alzheimer's disease pathology in Down syndrome.

Amyloid beta (Aß) is thought to play a critical role in the pathogenesis of Alzheimer's disease (AD). Prion-like Aß polymorphs, or "strains", can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of Aß that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and Aß strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various Aß and tau species and used conformation-sensitive fluorescent probes to detect differences in Aß strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of Aß emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sporadic AD.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Structural heterogeneity and intersubject variability of Aß in familial and sporadic Alzheimer's disease.

Point mutations in the amyloid-ß (Aß) coding region produce a combination of mutant and WT Aß isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aß determines WT Aß conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aß deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aß, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aß40 fibrils into transgenic mice expressing only WT Aß induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aß40 prions induce a conformation of WT Aß similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aß prion conformations, which kinetically dominate the spread of prions in the brain.

De novo design of a hyperstable non-natural protein-ligand complex with sub-Å accuracy.

Protein catalysis requires the atomic-level orchestration of side chains, substrates and cofactors, and yet the ability to design a small-molecule-binding protein entirely from first principles with a precisely predetermined structure has not been demonstrated. Here we report the design of a novel protein, PS1, that binds a highly electron-deficient non-natural porphyrin at temperatures up to 100 °C. The high-resolution structure of holo-PS1 is in sub-Å agreement with the design. The structure of apo-PS1 retains the remote core packing of the holoprotein, with a flexible binding region that is predisposed to ligand binding with the desired geometry. Our results illustrate the unification of core packing and binding-site definition as a central principle of ligand-binding protein design.