Laboratory evolution of a sortase enzyme that modifies amyloid-ß protein.

Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase sortase A to recognize the LMVGG sequence in endogenous amyloid-ß (Aß) protein. Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aß in human cerebrospinal fluid, enabling the detection of Aß with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aß42, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of sortase-mediated labeling of Aß peptides.

PrP is a central player in toxicity mediated by soluble aggregates of neurodegeneration-causing proteins.

Neurodegenerative diseases are an enormous public health problem, affecting tens of millions of people worldwide. Nearly all of these diseases are characterized by oligomerization and fibrillization of neuronal proteins, and there is great interest in therapeutic targeting of these aggregates. Here, we show that soluble aggregates of α-synuclein and tau bind to plate-immobilized PrP in vitro and on mouse cortical neurons, and that this binding requires at least one of the same N-terminal sites at which soluble Aß aggregates bind. Moreover, soluble aggregates of tau, α-synuclein and Aß cause both functional (impairment of LTP) and structural (neuritic dystrophy) compromise and these deficits are absent when PrP is ablated, knocked-down, or when neurons are pre-treated with anti-PrP blocking antibodies. Using an all-human experimental paradigm involving: (1) isogenic iPSC-derived neurons expressing or lacking PRNP, and (2) aqueous extracts from brains of individuals who died with Alzheimer's disease, dementia with Lewy bodies, and Pick's disease, we demonstrate that Aß, α-synuclein and tau are toxic to neurons in a manner that requires PrPC. These results indicate that PrP is likely to play an important role in a variety of late-life neurodegenerative diseases and that therapeutic targeting of PrP, rather than individual disease proteins, may have more benefit for conditions which involve the aggregation of more than one protein.

Methods for the isolation and analysis of Aß from postmortem brain.

Amyloid ß-protein (Aß) plays an initiating role in Alzheimer's disease (AD), but only a small number of groups have studied Aß extracted from human brain. Most prior studies have utilized synthetic Aß peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. Here, we describe three distinct methods for studying Aß from cortical tissue. Each method allows the analysis of different ranges of species thus enabling the examination of different questions. The first method allows the study of readily diffusible Aß with a relatively high specific activity. The second enables the analysis of readily solubilized forms of Aß the majority of which are inactive. The third details the isolation of true Aß dimers which have disease-related activity. We also describe a bioassay to study the effects of Aß on the neuritic integrity of iPSC-derived human neurons. The combined use of this bioassay and the described extraction procedures provides a platform to investigate the activity of different forms and mixtures of Aß species, and offers a tractable system to identify strategies to mitigate Aß mediated neurotoxicity.

PrP-grafted antibodies bind certain amyloid ß-protein aggregates, but do not prevent toxicity.

BACKGROUND: The prion protein (PrP) is known to bind certain soluble aggregates of the amyloid ß-protein (Aß), and two regions of PrP, one centered around residues 19-33, and the other around 87-112, are thought to be particularly important for this interaction. When either of these sequences are grafted into a human IgG the resulting antibodies react with disease-associated PrP conformers, whereas the parental b12 IgG does not. METHODS: Human antibodies containing grafts of PrP 19-33 or 87-112 were prepared as before (Solforosi et al., 2007) and tested for their ability to recognize synthetic and Alzheimer's disease (AD) brain-derived Aß. Since aqueous extracts of AD brain contain a complex mixture of active and inactive Aß species, we also assessed whether PrP-grafted antibodies could protect against neuritotoxicity mediated by AD brain-derived Aß. For these experiments, human iPSC-derived neurons were grown in 96-well plates at 5000 cells per well and on post-induction day 21, AD brain extracts were added +/- test antibodies. Neurons were imaged for 3 days using an IncuCyte live-cell imaging system, and neurite number and density quantified. RESULTS: Grafted antibodies bound a significant portion of aggregated Aß in aqueous AD extracts, but when these antibodies were co-incubated with neurons treated with brain extracts they did not reduce toxicity. By contrast, the PrP fragment N1 did protect against Aß. CONCLUSIONS: These results further demonstrate that not all Aß oligomers are toxic and suggest that PrP derivatives may allow development of agents that differentially recognize toxic and innocuous Aß aggregates.