Vloga akvaporina 4 v uravnavanju volumna astrocitov

Lisjak, Marjeta (author), Jorgačevski, Jernej (mentor)

Vzdrževanje primernega ionskega in osmotskega ravnotežja je za normalno aktivnost možganov ključnega pomena. Astrociti se na spremembe v zunajcelični osmolarnosti odzovejo s hitrimi spremembami celičnega volumna. Mehanizem uravnavanega zmanjšanja volumna (angl. regulatory volume decrease; RVD) je pomemben pri preprečevanju poškodb celic, ki nabrekajo, saj vodi v normaliziranje celičnega volumna. Pri nabrekanju in sprožitvi RVD so v astrocitih udeleženi akvaporini 4 (AQP4), in sicer je bil fenomen RVD potrjen pri izooblikah AQP4a (M1) in AQP4c (M23). V doktorski disertaciji smo raziskali celično lokalizacijo in vpliv izražanja še neraziskanih izooblik AQP4, in sicer AQP4b, AQP4d in AQP4e, na uravnavanje celičnega volumna osamljenih podganjih astrocitov v hipoosmotskih razmerah. Spremembe celičnega volumna smo merili z barvilom sulforodamin 101. Rezultati so pokazali, da ima AQP4e pomembno vlogo pri nabrekanju in uravnavanju celičnega volumna astrocitov. Vpliv AQP4d na nabrekanje celic je bil znatno manjši, kot v primeru AQP4e, poleg tega AQP4d ni vplival na RVD. AQP4b ni imel neposredne vloge v uravnavanju celičnega volumna astrocitov pri do 10-minutni izpostavljenosti hipoosmotskim razmeram. AQP4 se v celični membrani združujejo v ortogonalne skupke (angl. orthogonal arrays of particles; OAP). Izražanje izooblik AQP4b in AQP4d v astrocitih je povzročilo zmanjšanje gostote OAP-jev, hkrati pa je izražanje AQP4d povzročilo povečanje OAP-jev. Pokazali smo, da sta AQP4b in AQP4d v astrocitih v znotrajcelični obliki, torej je njun vpliv na uravnavanje celičnega volumna astrocitov in reorganizacije OAP-jev posreden. V isoosmotskih razmerah je bila gostota OAP-jev nižja v razmerah povečanega izražanja AQP4b in AQP4d, medtem ko je bila gostota OAP-jev višja v razmerah povečanega izražanja AQP4e. V hipoosmotskih razmerah se je značilno spremenila gostota OAP-jev le v razmerah povečanega izražanja AQP4e, ne pa AQP4b in AQP4d. Hipoosmotske razmere so imele vpliv na razporeditev AQP4b in AQP4d le v zgodnjih endosomih, v katerih se je povečal delež obeh izooblik. Pri do 10-minutni izpostavitvi hipoosmotskim razmeram AQP4b in AQP4d nista vplivala na volumen Golgijevega aparata in lizosomov. Pokazal pa se je vpliv AQP4b na povečanje zgodnjih endosomov po daljši izpostavitvi hipoosmotskim razmeram. Hipoosmotske razmere tudi niso vplivale na hitrost potovanja mešičkov z AQP4b in AQP4d, in s tem povezanim znotrajceličnim prerazporejanjem AQP4b in AQP4d. Zadnje raziskave so pokazale vlogo lipidnih splavov pri uravnavanju aktivnosti AQP4. Z uporabo označevalca mCherry-D4, ki se veže na holesterolne domene, smo potrdili prisotnost AQP4 v lipidnih splavih. Preverili smo, ali bi ketamin lahko vplival na prerazporejanje holesterolnih domen v celični membrani. V celični membrani astrocitov je povzročil povečanje gostote lipidnih splavov, v fibroblastih pa povečanje površin lipidnih splavov. Dokazali smo vpliv ketamina na uravnavanje domen z visoko vsebnostjo holesterola v celični membrani astrocitov, kar bi lahko odsevalo robustni antidepresivni učinek tega zdravila.

• Language: Slovenian
• Keywords: astrociti, vodni kanali, akvaporin 4, AQP4, AQP4b, AQP4d, AQP4e, holesterol, ketamin
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Lisjak]
• Year: 2019
• PID: 20.500.12556/RUL-111852
• UDC: 616.8(043.3)
• COBISS.SI-ID: 936823
• Publication date in RUL: 16.10.2019

Secondary language

• Language: English
• Title: The role of aquaporin 4 in the regulation of astrocyte volume

Abstract

Proper functioning of the brain is dependent on the regulation of ionic and osmotic gradients. Astrocytes respond to changes in extracellular osmolarity with rapid changes in volume. Regulatory volume decrease (RVD) is important in preventing damage in swelling cells, as it leads to the normalization of their volume. Aquaporin 4 (AQP4) has essential role in cell swelling as well as in RVD in astrocytes, as it was described for AQP4a (M1) and AQP4c (M23) isoforms. We investigated the localization of yet unexplored AQP4 isoforms AQP4b, AQP4d in AQP4e and assesed their role in the regulation of cell volume of rat astrocytes in hypoosmotic conditions. Changes in cell volume were detected by sulforhodamine 101 dye. The results showed that AQP4e plays an important role in the swelling and cell volume regulation of astrocytes. AQP4d had a significantly smaller effect on cell swelling than AQP4e and did not affect RVD. AQP4b had no direct role in cell volume regulation of astrocytes in hypoosmotic conditions in the timeframe of 10 minutes. In the plasma membrane AQP4 aggregates into orthogonal arryas of particles (OAPs). The overexpression of AQP4b and AQP4d resulted in decreased density of OAPs, and at the same time, overexpression of AQP4d resulted in an increase of the OAPs size. We showed that isoforms AQP4b and AQP4d are intracellular, therefore they exert an indirect effect on astrocyte volume regulation and OAPs reorganization. Overexpression of AQP4b and AQP4d in isoosmotic conditions decreased density of OAPs, while overexpression of AQP4e increased density of OAPs. In hypoosmotic conditions only overexpression of AQP4e affected the density of OAPs in the plasma membrane, but not overexpression of AQP4b and AQP4d. Hypoosmotic conditions affected the distribution of AQP4b and AQP4d only in early endosomes, where the percentage of both isoforms increased. AQP4b and AQP4d did not affect the volume of the Golgi apparatus and lysosomes within a 10-minute exposure to the hypoosmotic conditions. However, the increase in the size of early endosomes, observed in AQP4b transfected cells, has been demonstrated after prolonged exposure to hypoosmotic conditions. Hypoosmotic conditions also did not affect the speed of the vesicle mobility of AQP4b and AQP4d-laden vesicles and the related intracellular re-allocation of AQP4b and AQP4d. Recent studies have shown the role of lipid rafts in the regulation of AQP4 activity. Using the mCherry-D4 probe, which binds to cholesterol domains, we confirmed the presence of AQP4 in lipid rafts. We tested whether ketamine could influence the redistribution of cholesterol domains in the plasma membrane. Ketamine caused an increase in the density of lipid rafts in the astrocytes' plasma membrane, and an increase in size of lipid rafts in fibroblasts. We have confirmed that ketamine affects the high cholesterol-containing domains in plasma membrane in astrocytes, which could reflect the robust antidepressant effect of this medication.

• Keywords: astrocytes, water channels, aquaporin 4, AQP4, AQP4b, AQP4d, AQP4e, cholesterol, ketamine