Projeto Portugal 2030

Sumário

In this project we tackle two pressing challenges in PD research, viz. the lack of surrogate endpoints of disease progression and the lack of disease-modifying drugs against neurodegeneration Specifically, our main objectives are (Fig. 1B): - OBJECTIVE 1: To develop a quantitative, NASYNOs-based biomarker for PD. - OBJECTIVE 2: To validate an inhibitor of NASYNOs formation in preclinical models of PD. As a contingency plan in the case that NASYNOs are not detected or not inhibited in vivo, the following complementary objective is delineated: - OBJECTIVE 3: To quantify NASYNOs in human plasma and CSF. To accomplish OBJECTIVE 1, NASYNOs will be selectively digested using TTR free from posttranslational modifications (PTMs) that are known to markedly decrease the rates of aSyn proteolysis (Figs. 3G to 3K). The characterization of TTR’s PTMs will be assured by advanced mass spectrometry techniques available in the Unit of Proteomics of i3S. NASYNOs quantification will be further sought through ELISA and aSyn seed amplification (ASA) assays. We have access to a comprehensive panel of antibodies selective to different aSyn proteoforms. Because TTR selectively proteolyzes NASYNOs and the monomeric precursors of NASYNOs (Figs. 3 and 4), our methodology does not rely on the occurrence of a reduced number of large aggregates as in the case of seed amplification assays. For the same reason, it is expected that this quantitative assay will be robust with CSF but also with blood plasma samples in which the total amount aSyn is lower. To accomplish OBJECTIVE 2, we rely on solid experimental evidence showing, e.g., that NASYNOs are detrimental to undifferentiated SH-SY5Y cell cultures and that inhibiting NASYNOs formation rescues cell viability. Before the proof-of-concept tests in vivo, we will study cpds 16 and 19 using cell-based assays such as transwell models of Blood-Brain Barrier (BBB) permeability and additional cell models of PD, including differentiated SH-SY5Y cells. Finally, a mouse model recapitulating major PD features and showing aSyn-dependent toxicity will be used to test one of our lead compounds. The clinical validation of NASYNOs at a pilot scale (OBJECTIVE 3) is possible in collaboration with the Dutch cohort ProPark, of which project consultant WvB is the leader. Finding a novel biomarker and/or a candidate drug for PD is a «high-risk» enterprise only counterbalanced by the «high gain» represented by a possible curative treatment to this devastating disease. To answer the challenges posed by our ambitious objectives, we delineated a methodology based on (i) realistic cell models of neurodegeneration, (ii) biosamples obtained from PD patients and healthy controls, and (iii) well-established animal models of PD. Even considering the technological soundness of the proposed approach, there are evident risks that (1) NASYNOs are not identified in biological samples or that (2) NASYNOs inhibitors are considered toxic or ineffective after the preclinical tests are concluded. To mitigate these risks, naturally occurring aSyn oligomers generated in the presence of dopamine (DA) will be investigated in addition to NASYNOs. Not only DA-aSyn oligomers have been reported to be neurotoxic (Mor et al., 2019), as their formation is also inhibited by our compounds. Moreover, included in the list of hit compounds is a brain-permeable, non-toxic drug-repurposing compound (cpd R) that can be tested in alternative to the more potent cpds 16 and 19.