Role of Niemann Pick C2 protein and membrane contact sites in cholesterol export from endo-lysosomes

Alice Dupont Juhl

doi:10.21996/w1fh-rg26

Role of Niemann Pick C2 protein and membrane contact sites in cholesterol export from endo-lysosomes

Bidragets oversatte titel: Funktion af Niemann Pick type C2 protein og membran kontakt steder i kolesterol eksport fra endo-lysosomer

Alice Dupont Juhl

Institut for Biokemi og Molekylær Biologi

Publikation: Afhandling › Ph.d.-afhandling

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Abstrakt

Kolesterol regulerer flere biofysiske egenskaber af pattedyr cellemembraner, herunder barrierefunktionen. Kolesterolindholdet varierer meget fra organel til organel, og korrekt kolesterol transport er essentiel for homøostases og et godt helbred. Celler kan optage kolesterol fra blodbanen, via receptor medieret optag af lav-densitet lipoproteiner (LDL). Både LDL og dets receptor bliver internaliseret, men vil separeres i det lave pH miljø i endosomer. LDL vil med tiden ende i sene endosomer/lysosomer (SE/LYS), hvor det vil blive nedbrudt, og kolesterol estere vil blive hydrolyseret af lipaser, til fri kolesterol. Ved hjælp af de to lysosomale proteiner Niemann Pick type C1 og C2 (NPC1, NPC2) kan fri kolesterol forlade SE/LYS og komme til andre cellulære organeller. Hvordan og via hvilke intracellulære ruter kolesterol når til andre cellulære organeller, er dog ikke fuldt forstået endnu. Manglende funktionel NPC1 eller NPC2 giver ophav til den neurodegenerative Niemann Pick type C sygdom, hvor kolesterol og andre typer af lipider akkumulerer i SE/LYS. Uafhængigt af hvilket NPC protein der er dysfunktionelt, lader den kliniske fænotype til at være den samme. Dog varierer det mitokondrielle kolesterolindhold, alt efter hvilket protein der er defekt. På trods af det lave kolesterolindhold i mitokondrier, er kolesterol meget vigtigt for membran vedligeholdelsen, samt for produktionen af oxysteroler og steroid hormoner. Mitokondrier kan modtage kolesterol fra flere forskellige organeller, inklusiv SE/LYS via en proces som afhænger af NPC2. Sådan en overførelse, kunne finde sted over membran kontakt steder (MKS).

Denne afhandling præsenterer ny indsigt i NPC2s rolle i transport af kolesterol, samt flere ny udviklede og anvendte værktøjer. I publikation I har vi brugt en kombination af avanceret mikroskopi og kvantitativ analyse til at undersøge NPC2’s rolle i sterol effluks fra SE/LYS. Vi har vist at NPC2 accelerer sterol udstrømningen fra SE/LYS, i en proces som var akkompagneret med omfordelingen af SE/LYS til cellens periferi. Fra perifere SE/LYS kunne sterol nå plasmamembranen, hvorfra det kunne blive frigivet via extracellulære vesikler. Ultrastrukturen af sådanne extracellulære vesikler var undersøgt med blød røntgen tomografi under kryo-temperatur, en teknik vi også brugte i publikation II. Her afslørede den at mitokondrier er i tæt kontakt med endosome-lignende organeller. For videre undersøgelse af sådanne interaktioner, udviklede vi en kvantitativt billede-baseret strategi, som viste at ca. 30 % af NPC2 indeholdende SE/LYS danner kontakt med mitokondrier i primære fibroblaster. Ved at analysere bevægelsen af disse interaktioner, fandt vi at sådanne MKS kunne være enten transiente eller længerevarende, hvoraf den sidstnævnte kunne udgøre steder for sterol overføresel mellem organellerne. Endelig, i kapitel 3 viser vi upublicerede resultater af fluorescerende sterolprober inkorporeret i LDL, der kan optages af celler. Disse har stort potentiale for at blive værdifulde værktøjer i mikroskopi af intracellulær kolesterol transport og frigivelse.

Bidragets oversatte titel	Funktion af Niemann Pick type C2 protein og membran kontakt steder i kolesterol eksport fra endo-lysosomer
Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Wüstner, Daniel, Hovedvejleder
Dato for forsvar	31. aug. 2021
Udgiver	Syddansk Universitet. Det Naturvidenskabelige Fakultet
DOI	https://doi.org/10.21996/w1fh-rg26
Status	Udgivet - 11. apr. 2022

Note vedr. afhandling

Den fulde afhandling kan læses på SDUs bibliotek.

Dokumenter og links

10.21996/w1fh-rg26

PhD_Thesis_Alice_Dupont_JuhlForlagets udgivne version, 6,53 MB

2 Tidsskriftartikel

Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles
Juhl, A. D., Lund, F. W., Jensen, M. L. V., Szomek, M., Heegaard, C. W., Guttmann, P., Werner, S., McNally, J., Schneider, G., Kapishnikov, S. & Wüstner, D., 2021, I: Chemistry and Physics of Lipids. 235, 19 s., 105047.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells
Juhl, A. D., Heegaard, C. W., Werner, S., Schneider, G., Krishnan, K., Covey, D. F. & Wüstner, D., 2021, I: Scientific Reports. 11, 22 s., 8927.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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abstract = "In membranes of mammalian cells, cholesterol regulates several biophysical properties, including the barrier function. The cholesterol content of membranes varies greatly from organelle to organelle, and correct cholesterol transportation is essential for homeostasis and health. Cells can obtain cholesterol by receptor-mediated uptake of low-density lipoproteins (LDL) from the bloodstream. Both the LDL and its receptor are internalized into the cell but will separate in the low pH environment of endosomes. The LDL will eventually end up in the late endosome/lysosomes (LE/LYSs), where it is degraded, and cholesteryl esters are hydrolyzed by acid lipases. By the work of the two lysosomal proteins Niemann Pick type C1 and C2 (NPC1, NPC2) unesterified cholesterol can leave the LE/LYSs and reach other cellular organelles. How, and by which intracellular routes, cholesterol reaches other subcellular compartments, is not yet fully understood. The absence of functional NPC1 or NPC2 gives rise to the neurodegenerative Niemann Pick type C disease, in which cholesterol together with other lipid species accumulates within LE/LYSs. Independent of which NPC protein is dysfunctional, the clinical phenotype appears to be identical. However, the mitochondrial cholesterol content differs, depending on which protein is deficient. Despite this low content, the cholesterol is very important for the maintenance of the mitochondrial membranes and the production of oxysterols and steroid hormones. Mitochondria can receive cholesterol from several subcellular sources, including LE/LYSs in a process depending on NPC2. Such a transfer process could likely take place across membrane contact sites (MCSs). This thesis presents new insights into NPC2{\textquoteright}s role in cellular cholesterol traffic, along with several new tools that have been developed and applied. In publication I, we have used a combination of advanced microscopy and quantitative analyses to examine NPC2{\textquoteright}s role in sterol efflux from LE/LYSs. We showed that NPC2 protein accelerates sterol efflux from LE/LYSs, in a process that was accompanied by a reallocation of LE/LYSs to the cell periphery. From peripheral LE/LYSs sterol could reach the plasma membrane and from there be released by shedding of extracellular vesicles. The ultrastructure of extracellular vesicles was examined with cryo soft X-ray tomography, a technique we also applied in publication II. Here it revealed mitochondria in close contact with endosome-like organelles. To explore such interactions further, we developed a quantitative imaging strategy, which showed that approximately 30 % of NPC2-containing LE/LYSs form contact sites with mitochondria in primary human fibroblasts. By analyzing their movement, we found that these could either be transient or longer lasting, of which the latter could pave the way for sterol transfer between the organelles. Lastly, in chapter 3 we show unpublished results of fluorescence sterol probes that are incorporated in LDL and taken up by cells. These have a high potential to become valuable tools in imaging intracellular cholesterol trafficking and release. ",

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N2 - In membranes of mammalian cells, cholesterol regulates several biophysical properties, including the barrier function. The cholesterol content of membranes varies greatly from organelle to organelle, and correct cholesterol transportation is essential for homeostasis and health. Cells can obtain cholesterol by receptor-mediated uptake of low-density lipoproteins (LDL) from the bloodstream. Both the LDL and its receptor are internalized into the cell but will separate in the low pH environment of endosomes. The LDL will eventually end up in the late endosome/lysosomes (LE/LYSs), where it is degraded, and cholesteryl esters are hydrolyzed by acid lipases. By the work of the two lysosomal proteins Niemann Pick type C1 and C2 (NPC1, NPC2) unesterified cholesterol can leave the LE/LYSs and reach other cellular organelles. How, and by which intracellular routes, cholesterol reaches other subcellular compartments, is not yet fully understood. The absence of functional NPC1 or NPC2 gives rise to the neurodegenerative Niemann Pick type C disease, in which cholesterol together with other lipid species accumulates within LE/LYSs. Independent of which NPC protein is dysfunctional, the clinical phenotype appears to be identical. However, the mitochondrial cholesterol content differs, depending on which protein is deficient. Despite this low content, the cholesterol is very important for the maintenance of the mitochondrial membranes and the production of oxysterols and steroid hormones. Mitochondria can receive cholesterol from several subcellular sources, including LE/LYSs in a process depending on NPC2. Such a transfer process could likely take place across membrane contact sites (MCSs). This thesis presents new insights into NPC2’s role in cellular cholesterol traffic, along with several new tools that have been developed and applied. In publication I, we have used a combination of advanced microscopy and quantitative analyses to examine NPC2’s role in sterol efflux from LE/LYSs. We showed that NPC2 protein accelerates sterol efflux from LE/LYSs, in a process that was accompanied by a reallocation of LE/LYSs to the cell periphery. From peripheral LE/LYSs sterol could reach the plasma membrane and from there be released by shedding of extracellular vesicles. The ultrastructure of extracellular vesicles was examined with cryo soft X-ray tomography, a technique we also applied in publication II. Here it revealed mitochondria in close contact with endosome-like organelles. To explore such interactions further, we developed a quantitative imaging strategy, which showed that approximately 30 % of NPC2-containing LE/LYSs form contact sites with mitochondria in primary human fibroblasts. By analyzing their movement, we found that these could either be transient or longer lasting, of which the latter could pave the way for sterol transfer between the organelles. Lastly, in chapter 3 we show unpublished results of fluorescence sterol probes that are incorporated in LDL and taken up by cells. These have a high potential to become valuable tools in imaging intracellular cholesterol trafficking and release.

AB - In membranes of mammalian cells, cholesterol regulates several biophysical properties, including the barrier function. The cholesterol content of membranes varies greatly from organelle to organelle, and correct cholesterol transportation is essential for homeostasis and health. Cells can obtain cholesterol by receptor-mediated uptake of low-density lipoproteins (LDL) from the bloodstream. Both the LDL and its receptor are internalized into the cell but will separate in the low pH environment of endosomes. The LDL will eventually end up in the late endosome/lysosomes (LE/LYSs), where it is degraded, and cholesteryl esters are hydrolyzed by acid lipases. By the work of the two lysosomal proteins Niemann Pick type C1 and C2 (NPC1, NPC2) unesterified cholesterol can leave the LE/LYSs and reach other cellular organelles. How, and by which intracellular routes, cholesterol reaches other subcellular compartments, is not yet fully understood. The absence of functional NPC1 or NPC2 gives rise to the neurodegenerative Niemann Pick type C disease, in which cholesterol together with other lipid species accumulates within LE/LYSs. Independent of which NPC protein is dysfunctional, the clinical phenotype appears to be identical. However, the mitochondrial cholesterol content differs, depending on which protein is deficient. Despite this low content, the cholesterol is very important for the maintenance of the mitochondrial membranes and the production of oxysterols and steroid hormones. Mitochondria can receive cholesterol from several subcellular sources, including LE/LYSs in a process depending on NPC2. Such a transfer process could likely take place across membrane contact sites (MCSs). This thesis presents new insights into NPC2’s role in cellular cholesterol traffic, along with several new tools that have been developed and applied. In publication I, we have used a combination of advanced microscopy and quantitative analyses to examine NPC2’s role in sterol efflux from LE/LYSs. We showed that NPC2 protein accelerates sterol efflux from LE/LYSs, in a process that was accompanied by a reallocation of LE/LYSs to the cell periphery. From peripheral LE/LYSs sterol could reach the plasma membrane and from there be released by shedding of extracellular vesicles. The ultrastructure of extracellular vesicles was examined with cryo soft X-ray tomography, a technique we also applied in publication II. Here it revealed mitochondria in close contact with endosome-like organelles. To explore such interactions further, we developed a quantitative imaging strategy, which showed that approximately 30 % of NPC2-containing LE/LYSs form contact sites with mitochondria in primary human fibroblasts. By analyzing their movement, we found that these could either be transient or longer lasting, of which the latter could pave the way for sterol transfer between the organelles. Lastly, in chapter 3 we show unpublished results of fluorescence sterol probes that are incorporated in LDL and taken up by cells. These have a high potential to become valuable tools in imaging intracellular cholesterol trafficking and release.

U2 - 10.21996/w1fh-rg26

DO - 10.21996/w1fh-rg26

M3 - Ph.D. thesis

PB - Syddansk Universitet. Det Naturvidenskabelige Fakultet

ER -

Role of Niemann Pick C2 protein and membrane contact sites in cholesterol export from endo-lysosomes

Abstrakt

Note vedr. afhandling

Dokumenter og links

Fingeraftryk

Relaterede publikationer

Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles

Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells

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