Genetski mehanizmi lizosomske disfunkcije u Parkinsonovoj bolesti [Genetic mechanisms of lysosomal dysfunction in Parkinson's disease]

Blažeković, Antonela (2019) Genetski mehanizmi lizosomske disfunkcije u Parkinsonovoj bolesti [Genetic mechanisms of lysosomal dysfunction in Parkinson's disease]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Accumulation of misfolded proteins in the brain is the main pathological hallmark of neurodegenerative diseases, including Parkinson’s disease (PD), suggesting that inadequate clearance of aggregation-prone proteins plays an important role in the disease pathogenesis. Studies on the rare inherited forms of PD have highlighted disturbed alpha-synuclein clearance through the autophagy-lysosomal pathway (ALP) as a key mechanism leading to PD. In order to characterize the putative underlying genetic mechanisms leading to ALP dysfunction in PD, we performed comprehensive analysis of genetic variants in ALP genes in PD patients using the custom LYSOGENE targeted next-generation sequencing panel, containing a set of 440 ALP related genes, as well as genes previously implicated in familial forms of PD. We identified a significant number of ALP gene variants among PD patients when compared to control subjects. These variants were involved in over 50 biological processes, with the greatest enrichment observed in categories of lysosome organization, organic substance transport, abnormal myelination and sphingolipid metabolism. In contrast, variants found exclusively in healthy subjects were not related to specific biological pathways. Based on the NGS data, we selected genes ARSD, GALC and LRBA with the most over-represented genetic variants in PD patients and investigated their effects on lysosomal impairment, alpha-synuclein accumulation and neurotoxicity using SH-SY5Y cell lines stably expressing human alpha-synuclein. The results were compared to the effects of the knock-down of a known lysosome-related gene, ATP13A2, which causes a rare autosomal recessive form of juvenile-onset atypical PD (PARK9). Our knock-down experiments described for the first time the association between ARSD and LRBA genes with the accumulation of aSyn and confirmed the association between GALC gene and PD. This study confirmed a link between lysosomal dysfunction and alpha-synuclein accumulation.

Abstract in Croatian

Glavni patološki znak Parkinsonove bolesti (PB) jest nakupljanje proteina alfa sinukleina (aSyn) što sugerira da neučinkovita razgradnja proteina podložnih agregaciji igra važnu ulogu u patogenezi bolesti. Studije o rijetkim naslijeĊenim oblicima PB ukazale su na smanjenu razgradnju aSyn putem autofagno-lizosomalnog puta (ALP) kao jednog od ključnih mehanizama u podlozi PB. Kako bismo karakterizirali pretpostavljene genetske mehanizme koji dovode do disfunkcije ALP-a u PB, proveli smo opsežnu analizu genetskih varijanti korištenjem ciljanog panela za sekvenciranje pod nazivom LYSOGENE. LYSOGENE panel sadrţži sveobuhvatni skup od 440 ALP srodnih gena, kao i gene prethodno opisane u familijarnim oblicima PB, sinukleinopatijama i drugim neurodegenerativnim bolestima. Identificirali smo značajan broj varijanti ALP gena među PB pacijentima u usporedbi s kontrolnim ispitanicima, s 396 varijanti prisutnih isključivo u PB bolesnika. Ove varijante uključene su u više od 50 bioloških procesa, ponajprije u putovima organizacije lizosoma, transporta organskih tvari, abnormalne mijelinizacije i metabolizma sfingolipida. Suprotno tome, varijante koje su pronađene isključivo u zdravih ispitanika nisu bile povezane sa specifičnim biološkim putovima. Na temelju podataka sekvenciranja, odabrali smo ARSD, GALC i LRBA gene s najviše zastupljenim genetskim varijantama kod pacijenata s PB i istražili njihove učinke na lizosomalno oštećenje, akumulaciju aSyn i neurotoksičnost primjenom SH-SY5Y staničnih linija koje stabilno eksprimiraju ljudski aSyn. Rezultati su uspoređeni s učincima utišavanja ATP13A2 gena za kojeg je opisano da dovodi do nakupljanja aSyn. Naši rezultati snažno upućuju na to da specifične varijante ALP gena mogu doprinijeti lizosomalnoj disfunkciji u PB. Ovim istraživanjem je po prvi puta opisana povezanost utišavanja ARSD i LRBA gena s nakupljanjem aSyn proteina te potvrđena povezanost utišavanja GALC gena s PB. Ovo istraživanje je potvrdilo vezu između lizosomalnog oštećenja i akumulacije aSyn.

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