Periodic Reporting for period 4 - IMPRiND (Inhibiting Misfolded protein PRopagation in Neurodegenerative Diseases - Sofia ref.: 116060)
Reporting period: 2020-03-01 to 2021-02-28
Over the fourth year, we have made significant progress in delivering on our objectives. This has been facilitated by regular teleconferences and two virtual Consortium meetings.
The main research efforts of IMPRIND are focused on the following areas: (i) Generation of brain-derived or amplified assemblies and their characterisation using biochemical techniques, (ii) Cryo-EM structure of tau and α-syn assemblies, (iii) Conduction of CRISPR screens in cell lines followed by cross-validation in primary neuronal screens, (iv) bioinformatic integration and analysis of screens based on druggability and gene expression data, (v) development of advanced cell-based models or animals for validation of selected targets and (vi) Data dissemination.
(i) LMB, Lilly and Janssen have optimised and cross-validated protocols to isolate and quality control brain-derived tau assemblies from Alzheimer’s disease brain. CNRS has established methodologies for amplification of proteopathic assemblies from brain homogenates of -synucleinopathies.
(ii) LMB has published the Cryo-EM structures of tau filaments isolated from Alzheimer’s disease, Pick’s disease and chronic traumatic encephalopathy (Nature, 2017, 2018, 2019) as well as Cryo-EM structures of MSA filaments (Nature 2020). CNRS has published the Cryo-EM structure of de novo generated -syn fibrils (eLife 2019).
(iii) Development of assays suitable for high through-put screening and completion of primary screens were completed by UOXF, Novartis and HLU. These include focused CRISPR/Cas9 proteostasis screens for syn and tau, genome-wide CRISPR screens for tau, primary neuronal screens using siRNA for 300 targets for tau and the DUB screen for -syn.
(iv) Hits from these screens were further analysed in an integrated fashion by Lilly to prioritise targets for further validation in primary neurons.
(v) Validation of prioritised hits was performed by Novartis and HLU in primary neurons using siRNA or shRNA for Tau and siRNA for ⍺-syn. Further assessment of selected hits and additional targets for Tau was performed at Janssen and has been planned for -synuclein at Cellectricon.
(vi) A number of advanced models have been fully characterised and their suitability for further use has been established. These include the iPSC-based dopaminergic neuronal model (UOXF), organotypic cultures (DZNE) and in vivo mouse (BRFAA and HLU models for -syn. In vivo mouse tau seeding models have been established by Lilly and Janssen. The transplantation of iPSC-derived neurons for in vivo assessment of Tau propagation and the tau drosophila model (both VIB) have also been completed. iPSC-based organoids (UCAM), hNP-based tau neuronal model (DZNE) and the primate model (UBx) are on-going. For the in vivo models different viral mediated silencing approaches were tested and characterized.
(vii) We generated an on-line registry for critical reagents and initiated the deposition of data in public repositories to enable sharing of datasets or dissemination following publications. Website updates and news, social media messages, and a number of conference presentations and posters have completed the dissemination activities. We have also organised two international symposia during the biennial ADPD meetin