Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology
Parkinson’s disease (PD) is the second most common neurodegenerative disorder for which there is no disease-modifying therapy. Pathologically, the neuronal loss correlates with abnormal accumulation, aggregation and toxic gain of function of the small pre-synaptic protein alpha-Synuclein (aSyn). Whe...
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| Format: | Thesis |
| Language: | English |
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2020
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| _version_ | 1826302509998669824 |
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| author | Lewin, R |
| author2 | Tofaris, G |
| author_facet | Tofaris, G Lewin, R |
| author_sort | Lewin, R |
| collection | OXFORD |
| description | Parkinson’s disease (PD) is the second most common neurodegenerative disorder for which there is no disease-modifying therapy. Pathologically, the neuronal loss correlates with abnormal accumulation, aggregation and toxic gain of function of the small pre-synaptic protein alpha-Synuclein (aSyn). Whether neurons degrade aggregated aSyn or, only its soluble monomers, remains elusive. This thesis combines hypothesis-driven and unbiased genetic screens to identify pathways that specifically regulate the pathological fraction of aSyn. Initial data in cellular PD models show that intracellular aSyn inclusions that form over a period of days are conjugated to K48-linked ubiquitin (Ub) chains and that the pharmacological activation of the proteasome, but not of macroautophagy, reduces the relative levels of aggregated aSyn. Subsequent studies involving large-scale CRISPR/Cas9 knockout (KO) screens targeting the human ubiquitination machinery (1,247 genes: almost all E2s, E3s and DUBs) aim to map novel Ub effectors whose KO modify either pathological levels of aSyn or seeded aggregation. A major hit from these screens is ARIH1, a RBR type E3 ligase. ARIH1 KO increases the levels and aggregation of aSyn. ARIH1 interacts with aSyn in a neddylation-dependent manner suggesting that an ARIH1-cullin-RING ligase (ARIH1-CRL) regulates the ubiquitination of aggregated aSyn. Such complexes typically form K48-linked Ub chains. Accordingly, upon inhibition of NEDD8 transfer, the co-localisation of K48-linked Ub chains and the recruitment of p97 to aSyn inclusions is reduced. Furthermore, the knockdown of either p97 or ARIH1 prevents the clearance of aggregated aSyn by the proteasome. Thus, in this cell-based model, a Ub-dependent and p97-mediated proteasomal pathway appears to be an early attempt to clear misfolded proteins, that upon further validation may constitute a target for the development of novel therapeutics against aSyn aggregation and toxicity. |
| first_indexed | 2024-03-07T05:48:38Z |
| format | Thesis |
| id | oxford-uuid:e810b4c4-4a4e-4843-b6f1-6813dcabbe20 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:48:38Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | oxford-uuid:e810b4c4-4a4e-4843-b6f1-6813dcabbe202022-03-27T10:44:00ZMapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathologyThesishttp://purl.org/coar/resource_type/c_db06uuid:e810b4c4-4a4e-4843-b6f1-6813dcabbe20Medical scienceEnglishHyrax Deposit2020Lewin, RTofaris, GD'Angiolella, VParkinson’s disease (PD) is the second most common neurodegenerative disorder for which there is no disease-modifying therapy. Pathologically, the neuronal loss correlates with abnormal accumulation, aggregation and toxic gain of function of the small pre-synaptic protein alpha-Synuclein (aSyn). Whether neurons degrade aggregated aSyn or, only its soluble monomers, remains elusive. This thesis combines hypothesis-driven and unbiased genetic screens to identify pathways that specifically regulate the pathological fraction of aSyn. Initial data in cellular PD models show that intracellular aSyn inclusions that form over a period of days are conjugated to K48-linked ubiquitin (Ub) chains and that the pharmacological activation of the proteasome, but not of macroautophagy, reduces the relative levels of aggregated aSyn. Subsequent studies involving large-scale CRISPR/Cas9 knockout (KO) screens targeting the human ubiquitination machinery (1,247 genes: almost all E2s, E3s and DUBs) aim to map novel Ub effectors whose KO modify either pathological levels of aSyn or seeded aggregation. A major hit from these screens is ARIH1, a RBR type E3 ligase. ARIH1 KO increases the levels and aggregation of aSyn. ARIH1 interacts with aSyn in a neddylation-dependent manner suggesting that an ARIH1-cullin-RING ligase (ARIH1-CRL) regulates the ubiquitination of aggregated aSyn. Such complexes typically form K48-linked Ub chains. Accordingly, upon inhibition of NEDD8 transfer, the co-localisation of K48-linked Ub chains and the recruitment of p97 to aSyn inclusions is reduced. Furthermore, the knockdown of either p97 or ARIH1 prevents the clearance of aggregated aSyn by the proteasome. Thus, in this cell-based model, a Ub-dependent and p97-mediated proteasomal pathway appears to be an early attempt to clear misfolded proteins, that upon further validation may constitute a target for the development of novel therapeutics against aSyn aggregation and toxicity. |
| spellingShingle | Medical science Lewin, R Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology |
| title | Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology |
| title_full | Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology |
| title_fullStr | Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology |
| title_full_unstemmed | Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology |
| title_short | Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology |
| title_sort | mapping networks of alpha synuclein proteostasis in cellular models of parkinson s disease pathology |
| topic | Medical science |
| work_keys_str_mv | AT lewinr mappingnetworksofalphasynucleinproteostasisincellularmodelsofparkinsonsdiseasepathology |