{"id":{"repo_id":"helsinki","oai_identifier":"oai:helda.helsinki.fi:10138/601463"},"canonical_url":"https://search.dev.ndltd.org/etd/helsinki/oai:helda.helsinki.fi:10138/601463","repository":{"repo_id":"helsinki","name":"University of Helsinki","base_url":"https://helda.helsinki.fi/server/oai/request"},"display":{"title":"The effects of the Parkinson’s disease-associated proteins LRRK2 and TMEM175 on the lysosomal profile of cultured mammalian cells under lysosomal stress conditions","abstract":"Lysosomal dysfunction is increasingly recognised as a central factor in the development of Parkinson’s disease (PD). At least 11 out of the 24 PD-associated genes have been identified by genome-wide association studies (GWAS) to be involved in autophagy-lysosome pathways. Leucine-rich repeat kinase 2 (LRRK2) has been found as the most common genetic risk factor for PD. LRRK2 is involved in various cellular processes, including cell signalling, vesicle trafficking, and the maintenance of lysosomal homeostasis. Another important PD-associated gene encodes the transmembrane protein 175 (TMEM175), a lysosomal cation channel essential for maintaining lysosomal pH and regulating other aspects of endolysosomal function. The mechanisms by which the mutations in LRRK2 or TMEM175 cause PD have not yet been established, which hinders the development of disease-modifying treatments for the cases caused by these mutations. The objective of this study was to investigate differences in lysosomal characteristics in mammalian A549 cells with distinct LRRK2 genotypes, and in human and mouse microglial lines under basal conditions and lysosomal stress. The pathogenic PD-associated LRRK2 mutations have been shown to increase LRRK2 kinase activity, causing increased phosphorylation of its Rab GTPase substrates. This study shows that A549 cells with PD-associated Y1699C LRRK2 mutation were more abundant in phosphorylated Rab10 compared to WT cells. Currently, LRRK2 is considered the major kinase confirmed to phosphorylate Rab10 at T73. However, this work reports increased levels of pRab10 also in LRRK2 KO cells, suggesting that the phosphorylation of Rab10 at the T73 site may be partly independent of LRRK2 activity. This study shows that LRRK2 has a role in the composition and glycosylation patterns of lysosome associated membrane proteins 1 (LAMP1) and 2 (LAMP2) in A549 cells, as well as in their response to lysosomal stress induced by the lysosome-disrupting agents, chloroquine and LLOME. It was found that A549 cells with different LRRK2 genotypes express distinct levels of glycosylated LAMP1 and LAMP2 proteins. Lysosomal stress induced by chloroquine and LLOME was observed to modulate the size and localisation of LAMP1 and LAMP2 depending on LRRK2 genotype. Additionally, preliminary data revealed 10-fold differences in the relative LAMP2 expression between mouse and human microglia cells. The results of this study support the importance of LRRK2 in regulating lysosomal function, showing that PD-associated mutations in LRRK2 may cause alterations in the lysosomal function of the cells. More effective methods are needed to study the role of TMEM175 in the lysosomal profile and the development of PD.","abstract_html":"Lysosomal dysfunction is increasingly recognised as a central factor in the development of Parkinson’s disease (PD). At least 11 out of the 24 PD-associated genes have been identified by genome-wide association studies (GWAS) to be involved in autophagy-lysosome pathways. Leucine-rich repeat kinase 2 (LRRK2) has been found as the most common genetic risk factor for PD. LRRK2 is involved in various cellular processes, including cell signalling, vesicle trafficking, and the maintenance of lysosomal homeostasis. Another important PD-associated gene encodes the transmembrane protein 175 (TMEM175), a lysosomal cation channel essential for maintaining lysosomal pH and regulating other aspects of endolysosomal function. The mechanisms by which the mutations in LRRK2 or TMEM175 cause PD have not yet been established, which hinders the development of disease-modifying treatments for the cases caused by these mutations. The objective of this study was to investigate differences in lysosomal characteristics in mammalian A549 cells with distinct LRRK2 genotypes, and in human and mouse microglial lines under basal conditions and lysosomal stress. The pathogenic PD-associated LRRK2 mutations have been shown to increase LRRK2 kinase activity, causing increased phosphorylation of its Rab GTPase substrates. This study shows that A549 cells with PD-associated Y1699C LRRK2 mutation were more abundant in phosphorylated Rab10 compared to WT cells. Currently, LRRK2 is considered the major kinase confirmed to phosphorylate Rab10 at T73. However, this work reports increased levels of pRab10 also in LRRK2 KO cells, suggesting that the phosphorylation of Rab10 at the T73 site may be partly independent of LRRK2 activity. This study shows that LRRK2 has a role in the composition and glycosylation patterns of lysosome associated membrane proteins 1 (LAMP1) and 2 (LAMP2) in A549 cells, as well as in their response to lysosomal stress induced by the lysosome-disrupting agents, chloroquine and LLOME. It was found that A549 cells with different LRRK2 genotypes express distinct levels of glycosylated LAMP1 and LAMP2 proteins. Lysosomal stress induced by chloroquine and LLOME was observed to modulate the size and localisation of LAMP1 and LAMP2 depending on LRRK2 genotype. Additionally, preliminary data revealed 10-fold differences in the relative LAMP2 expression between mouse and human microglia cells. The results of this study support the importance of LRRK2 in regulating lysosomal function, showing that PD-associated mutations in LRRK2 may cause alterations in the lysosomal function of the cells. More effective methods are needed to study the role of TMEM175 in the lysosomal profile and the development of PD.","abstract_has_math":false,"creators":["Himanka, Ella"],"institution":"Helsingin yliopisto","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-09-16","date_published":"2025-09-16","updated_at":"2026-07-27T19:56:04Z","subjects":["Parkinsonin tauti","lysosomaalinen stressi","LRRK2","TMEM175","klorokiini","LLOME","lysosomin kalvoproteiinit"],"languages":["eng"],"rights":["In Copyright 1.0"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10138/601463","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Himanka, Ella"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-09-16T15:16:36Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-06-24T21:00:14Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-09-16"]},{"key":"dc:publisher","label":"Institution","values":["Helsingin yliopisto","University of Helsinki","Helsingfors universitet"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Parkinsonin tauti","lysosomaalinen stressi","LRRK2","TMEM175","klorokiini","LLOME","lysosomin kalvoproteiinit"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright 1.0"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10138/601463"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Lysosomal dysfunction is increasingly recognised as a central factor in the development of Parkinson’s disease (PD). At least 11 out of the 24 PD-associated genes have been identified by genome-wide association studies (GWAS) to be involved in autophagy-lysosome pathways. Leucine-rich repeat kinase 2 (LRRK2) has been found as the most common genetic risk factor for PD. LRRK2 is involved in various cellular processes, including cell signalling, vesicle trafficking, and the maintenance of lysosomal homeostasis. Another important PD-associated gene encodes the transmembrane protein 175 (TMEM175), a lysosomal cation channel essential for maintaining lysosomal pH and regulating other aspects of endolysosomal function. The mechanisms by which the mutations in LRRK2 or TMEM175 cause PD have not yet been established, which hinders the development of disease-modifying treatments for the cases caused by these mutations. The objective of this study was to investigate differences in lysosomal characteristics in mammalian A549 cells with distinct LRRK2 genotypes, and in human and mouse microglial lines under basal conditions and lysosomal stress. The pathogenic PD-associated LRRK2 mutations have been shown to increase LRRK2 kinase activity, causing increased phosphorylation of its Rab GTPase substrates. This study shows that A549 cells with PD-associated Y1699C LRRK2 mutation were more abundant in phosphorylated Rab10 compared to WT cells. Currently, LRRK2 is considered the major kinase confirmed to phosphorylate Rab10 at T73. However, this work reports increased levels of pRab10 also in LRRK2 KO cells, suggesting that the phosphorylation of Rab10 at the T73 site may be partly independent of LRRK2 activity. This study shows that LRRK2 has a role in the composition and glycosylation patterns of lysosome associated membrane proteins 1 (LAMP1) and 2 (LAMP2) in A549 cells, as well as in their response to lysosomal stress induced by the lysosome-disrupting agents, chloroquine and LLOME. It was found that A549 cells with different LRRK2 genotypes express distinct levels of glycosylated LAMP1 and LAMP2 proteins. Lysosomal stress induced by chloroquine and LLOME was observed to modulate the size and localisation of LAMP1 and LAMP2 depending on LRRK2 genotype. Additionally, preliminary data revealed 10-fold differences in the relative LAMP2 expression between mouse and human microglia cells. The results of this study support the importance of LRRK2 in regulating lysosomal function, showing that PD-associated mutations in LRRK2 may cause alterations in the lysosomal function of the cells. More effective methods are needed to study the role of TMEM175 in the lysosomal profile and the development of PD.","Lysosomien toimintahäiriöiden on yhä useammin todettu olevan keskeinen tekijä Parkinsonin taudin kehittymisessä. Genomilaajuisissa assosiaatiotutkimuksissa (GWAS) on todettu ainakin yhdentoista Parkinsonin tautiin liittyvistä 24 geenistä liittyvän autofagia-lysosomi-reitteihin. LRRK2:n on todettu olevan yleisin geneettinen riskitekijä Parkinsonin taudissa. Se osallistuu useisiin soluprosesseihin, kuten solusignalointiin, vesikkelikuljetukseen ja lysosomien homeostaasin ylläpitämiseen. Toinen keskeinen geeni, TMEM175, koodaa lysosomaalista kationikanavaa, joka ylläpitää lysosomien pH-tasapainoa ja osallistuu erilaisten endolysosomaalisten toimintojen säätelyyn. Mekanismeja, joiden kautta LRRK2- ja TMEM175-geenimutaatiot vaikuttavat Parkinsonin taudin kehittymiseen, ei ole vielä täysin tunnistettu. Tämä rajoittaa taudin etenemisteen vaikuttavien kohdennettujen hoitomuotojen kehitystä. Tämän tutkimuksen tavoitteena oli selvittää lysosomaalisten ominaisuuksien eroavaisuuksia eri LRRK2-genotyyppien A549-nisäkässoluissa sekä ihmisen ja hiiren mikrogliasoluissa normaaleissa olosuhteissa ja lysosomaalisen stressin aikana. Parkinsonin tautiin liitettyjen LRRK2-mutaatioiden on osoitettu lisäävän LRRK2:n kinaasiaktiivisuutta, mikä johtaa sen Rab GTPaasi-substraattien lisääntyneeseen fosforylaatioon. Tässä tutkimuksessa havaittiin, että Parkinsonin tautiin liitetyn Y1699C LRRK2-mutaation A549-solulinjassa esiintyy enemmän fosforyloitunutta Rab10-proteiinia (pRab10) villityypin solulinjaan verrattuna. Nykyisen tiedon mukaan LRRK2 on ainoa kinaasi, joka fosforyloi Rab10:n T73-kohdasta. Tämän tutkimuksen tulokset kuitenkin osoittavat klorokiinin nostavan pRab10 (T73) -tasoja myös soluissa, joista LRRK2-geeni on poistettu. Tämä viittaa siihen, että Rab10-proteiinin fosforyloituminen kyseisestä kohdasta ei ole täysin LRRK2-riippuvaista. Tämä tutkimus osoitti myös, että LRRK2 vaikuttaa lysosomin kalvoproteiinien LAMP1:n ja LAMP2:n koostumukseen ja glykosylaatioon A549-soluissa sekä solujen vasteeseen klorokiinin ja LLOME:n aiheuttamaan lysosomaaliseen stressiin. Eri LRRK2-genotyyppien välillä havaittiin eroja glykosyloituneiden lysosomaalisten kalvoproteiinien määrässä. Lisäksi todettiin, että klorokiini ja LLOME säätelevät lysosomaalisten kalvoproteiinien kokoa ja sijaintia eri tavoin. Preliminäärinen data osoitti myös 10-kertaisen eron LAMP2:n ilmentymisessä hiiren ja ihmisen mikrogliasolujen välillä. Tutkimuksen tulokset tukevat käsitystä siitä, että LRRK2 on keskeinen lysosomien toiminnan säätelijä ja että Parkinsonin tautiin liittyvien LRRK2-mutaatioiden vaikutukset saattavat aiheuttaa muutoksia lysosomien toimintaan. Jatkotutkimuksia ja tehokkaampia menetelmiä tarvitaan erityisesti TMEM175:n vaikutusten selvittämiseksi lysosomaaliseen profiiliin ja sen rooliin Parkinsonin taudin patogeneesissä."]},{"key":"dc:title","label":"Title","values":["The effects of the Parkinson’s disease-associated proteins LRRK2 and TMEM175 on the lysosomal profile of cultured mammalian cells under lysosomal stress conditions"]}]}],"canonical_facts":{"dc:creator":["Himanka, Ella"],"dc:date.accessioned":["2025-09-16T15:16:36Z"],"dc:date.available":["2026-06-24T21:00:14Z"],"dc:date.issued":["2025-09-16"],"dc:description.abstract":["Lysosomal dysfunction is increasingly recognised as a central factor in the development of Parkinson’s disease (PD). At least 11 out of the 24 PD-associated genes have been identified by genome-wide association studies (GWAS) to be involved in autophagy-lysosome pathways. Leucine-rich repeat kinase 2 (LRRK2) has been found as the most common genetic risk factor for PD. LRRK2 is involved in various cellular processes, including cell signalling, vesicle trafficking, and the maintenance of lysosomal homeostasis. Another important PD-associated gene encodes the transmembrane protein 175 (TMEM175), a lysosomal cation channel essential for maintaining lysosomal pH and regulating other aspects of endolysosomal function. The mechanisms by which the mutations in LRRK2 or TMEM175 cause PD have not yet been established, which hinders the development of disease-modifying treatments for the cases caused by these mutations. The objective of this study was to investigate differences in lysosomal characteristics in mammalian A549 cells with distinct LRRK2 genotypes, and in human and mouse microglial lines under basal conditions and lysosomal stress. The pathogenic PD-associated LRRK2 mutations have been shown to increase LRRK2 kinase activity, causing increased phosphorylation of its Rab GTPase substrates. This study shows that A549 cells with PD-associated Y1699C LRRK2 mutation were more abundant in phosphorylated Rab10 compared to WT cells. Currently, LRRK2 is considered the major kinase confirmed to phosphorylate Rab10 at T73. However, this work reports increased levels of pRab10 also in LRRK2 KO cells, suggesting that the phosphorylation of Rab10 at the T73 site may be partly independent of LRRK2 activity. This study shows that LRRK2 has a role in the composition and glycosylation patterns of lysosome associated membrane proteins 1 (LAMP1) and 2 (LAMP2) in A549 cells, as well as in their response to lysosomal stress induced by the lysosome-disrupting agents, chloroquine and LLOME. It was found that A549 cells with different LRRK2 genotypes express distinct levels of glycosylated LAMP1 and LAMP2 proteins. Lysosomal stress induced by chloroquine and LLOME was observed to modulate the size and localisation of LAMP1 and LAMP2 depending on LRRK2 genotype. Additionally, preliminary data revealed 10-fold differences in the relative LAMP2 expression between mouse and human microglia cells. The results of this study support the importance of LRRK2 in regulating lysosomal function, showing that PD-associated mutations in LRRK2 may cause alterations in the lysosomal function of the cells. More effective methods are needed to study the role of TMEM175 in the lysosomal profile and the development of PD.","Lysosomien toimintahäiriöiden on yhä useammin todettu olevan keskeinen tekijä Parkinsonin taudin kehittymisessä. Genomilaajuisissa assosiaatiotutkimuksissa (GWAS) on todettu ainakin yhdentoista Parkinsonin tautiin liittyvistä 24 geenistä liittyvän autofagia-lysosomi-reitteihin. LRRK2:n on todettu olevan yleisin geneettinen riskitekijä Parkinsonin taudissa. Se osallistuu useisiin soluprosesseihin, kuten solusignalointiin, vesikkelikuljetukseen ja lysosomien homeostaasin ylläpitämiseen. Toinen keskeinen geeni, TMEM175, koodaa lysosomaalista kationikanavaa, joka ylläpitää lysosomien pH-tasapainoa ja osallistuu erilaisten endolysosomaalisten toimintojen säätelyyn. Mekanismeja, joiden kautta LRRK2- ja TMEM175-geenimutaatiot vaikuttavat Parkinsonin taudin kehittymiseen, ei ole vielä täysin tunnistettu. Tämä rajoittaa taudin etenemisteen vaikuttavien kohdennettujen hoitomuotojen kehitystä. Tämän tutkimuksen tavoitteena oli selvittää lysosomaalisten ominaisuuksien eroavaisuuksia eri LRRK2-genotyyppien A549-nisäkässoluissa sekä ihmisen ja hiiren mikrogliasoluissa normaaleissa olosuhteissa ja lysosomaalisen stressin aikana. Parkinsonin tautiin liitettyjen LRRK2-mutaatioiden on osoitettu lisäävän LRRK2:n kinaasiaktiivisuutta, mikä johtaa sen Rab GTPaasi-substraattien lisääntyneeseen fosforylaatioon. Tässä tutkimuksessa havaittiin, että Parkinsonin tautiin liitetyn Y1699C LRRK2-mutaation A549-solulinjassa esiintyy enemmän fosforyloitunutta Rab10-proteiinia (pRab10) villityypin solulinjaan verrattuna. Nykyisen tiedon mukaan LRRK2 on ainoa kinaasi, joka fosforyloi Rab10:n T73-kohdasta. Tämän tutkimuksen tulokset kuitenkin osoittavat klorokiinin nostavan pRab10 (T73) -tasoja myös soluissa, joista LRRK2-geeni on poistettu. Tämä viittaa siihen, että Rab10-proteiinin fosforyloituminen kyseisestä kohdasta ei ole täysin LRRK2-riippuvaista. Tämä tutkimus osoitti myös, että LRRK2 vaikuttaa lysosomin kalvoproteiinien LAMP1:n ja LAMP2:n koostumukseen ja glykosylaatioon A549-soluissa sekä solujen vasteeseen klorokiinin ja LLOME:n aiheuttamaan lysosomaaliseen stressiin. Eri LRRK2-genotyyppien välillä havaittiin eroja glykosyloituneiden lysosomaalisten kalvoproteiinien määrässä. Lisäksi todettiin, että klorokiini ja LLOME säätelevät lysosomaalisten kalvoproteiinien kokoa ja sijaintia eri tavoin. Preliminäärinen data osoitti myös 10-kertaisen eron LAMP2:n ilmentymisessä hiiren ja ihmisen mikrogliasolujen välillä. Tutkimuksen tulokset tukevat käsitystä siitä, että LRRK2 on keskeinen lysosomien toiminnan säätelijä ja että Parkinsonin tautiin liittyvien LRRK2-mutaatioiden vaikutukset saattavat aiheuttaa muutoksia lysosomien toimintaan. Jatkotutkimuksia ja tehokkaampia menetelmiä tarvitaan erityisesti TMEM175:n vaikutusten selvittämiseksi lysosomaaliseen profiiliin ja sen rooliin Parkinsonin taudin patogeneesissä."],"dc:identifier.uri":["http://hdl.handle.net/10138/601463"],"dc:language.iso":["eng"],"dc:publisher":["Helsingin yliopisto","University of Helsinki","Helsingfors universitet"],"dc:rights":["In Copyright 1.0"],"dc:subject":["Parkinsonin tauti","lysosomaalinen stressi","LRRK2","TMEM175","klorokiini","LLOME","lysosomin kalvoproteiinit"],"dc:title":["The effects of the Parkinson’s disease-associated proteins LRRK2 and TMEM175 on the lysosomal profile of cultured mammalian cells under lysosomal stress conditions"]},"updated_at":"2026-07-27T19:56:04Z"}