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Understanding the cytotoxicity of aberrantly folded proteins in neurodegeneration

Final Report Summary - FOLDTOX (Understanding the cytotoxicity of aberrantly folded proteins in neurodegeneration)

Protein aggregation diseases are associated with the intracellular or extracellular accumulation of aggregates of specific misfolded proteins (e.g. α-synuclein (α-Syn) in Parkinson’s disease (PD)). Such aberrant protein structures are implicated in cell dysfunction and pathology, but their specific cellular effects are still largely unknown. The proteins forming such deposits (aggregation-prone proteins, or APPs) strongly differ in terms of sequence, structure and properties, but the structural features of the mature aggregates are strongly conserved, irrespective of the protein they contain. Recent genome-wide genetic screens resulted in a set of ~100 genes that significantly reduce or enhance toxicity of each APPs, (genetic modulators, or GMs). These modulators might be extremely promising for therapeutic purposes, but their mechanisms of action are still unknown.
In this project we first applied integrated system-level analyses to a set of human and yeast models of protein aggregation to identify which responses cells activate upon the formation of intracellular protein aggregates, and uncover how cells counteract the proteotoxic insult. Strikingly, only some of these responses were conserved through the set of APPs considered (e.g. typical cellular stress responses and an increased activation of quality control systems), while the majority appeared to be strictly dependent on the sequence of the aggregating protein. This suggests that the toxicity mechanism of APPs are not generic, but depend on specific features of each APP.
Among the responses activated by cells in response to aggregate accumulation, was a set of proteins involved in neuronal protein degradation. Using molecular biology follow-up experiments, we demonstrated that this system selectively targets the toxic form of αSyn for ubiquitin-mediated degradation, and thereby protects cells from cytotoxicity. The protein complex also colocalizes with αSyn in PD patient brains. The discovery of a novel cellular mechanism of clearance of pathological protein aggregates of αSyn will contribute to improve our current knowledge on PD and aid the development of novel therapeutic strategies designed to inhibit PD progression across neurons.
Next, in order to evaluate the rescue mechanisms of known GMs, we developed a novel screening method, based on the concept of sentinel proteins, i.e. protein markers that report on the activation state of a given cellular pathway or module (e.g MAPK cascades, autophagy, apoptosis, etc). We constructed a targeted proteomics assay that measures ~200 such sentinels in about 1 hour, and thus reports on the activity of about 300 functional modules at high-throughput. We are now applying this assay to unravel which pathways each of the ~100 GMs activates or represses, to identify strategies to maximize the rescue and thus suggest suitable entry points for therapy intervention on protein aggregation diseases.
Last, to analyse the structural features of the pathological states of each APP, we developed a novel structural approach for the quantification and characterization of different conformational states of a protein directly in its cellular environment. The approach resulted in the identification of a set of conformotypic peptide markers, for the detection of the pathological and “healthy” forms of αSyn in a variety of biological samples, e.g. including patient specimens.
When I started the FoldTox project, I was about to setup my independent research group. Today the Picotti group includes 4 PhD students, 3 postdocs and a scientific lab manager and secured prestigious research funding, like the ERC Starting grant (1.5 Million Euro, starting after completion of FoldTox) or the Swiss National Science Foundation Professorship grant (1.5 Million CHF). Since its foundation and since the start of the FoldTox project we published 24 scientific articles, of which 13 without my post-doctoral supervisor. Four of the papers where I am corresponding author, and that are related to the FoldTox project, were published in journals with an impact factor above 23 (e.g. Feng et al., Nat Biotechnology, 2014, about the structural method described above; or Soste et al., Nat Methods 2014, about the sentinel screening).