Presnova in signaliziranje lipidov v podganjih astrocitih v kulturi : doktorska disertacija

Smolič, Tina (author), Vardjan, Nina (mentor)

Astrociti so celice nevroglije v centralnem živčnem sistemu (CŽS) s številnimi funkcijami, med drugim skrbijo za presnovno homeostazo v CŽS in dostavljajo hranila (laktat, glukoza) nevronom. V patoloških stanjih CŽS astrociti kopičijo lipidne kapljice (LK), ki so skladišča prostih maščobnih kislin (MK) in sterolov. Mehanizmi nastanka ter vloga LK v astrocitih so še slabo raziskani, prav tako signalizacija lipidov preko z G-proteini sklopljenih receptorjev za proste MK (npr. GPR40) na površini astrocitov in vpliv le-te na presnovo astrocitov. Z uporabo imunocitokemije in konfokalne mikroskopije smo ovrednotili subcelično lokalizacijo, mobilnost LK in vpliv presnovnega stresa in hipoksije (značilnih dejavnikov patoloških stanj CŽS) ter noradrenergične aktivacije, na tvorbo LK v podganjih astrocitih v kulturi in v organotipski možganski kulturi. Z uporabo fluorescenčne mikroskopije v realnem času smo raziskali vpliv agonistov GPR40, endogenih neselektivnih agonistov dokozaheksaenojske kisline (DHA) in oleinske kisline (OA) in sintetičnega selektivnega agonista TAK875, na znotrajcelično koncentracijo Ca2+, cAMP in laktata v astrocitih z uporabo fluorescenčnega Ca2+-indikatorja (Fluo-4, AM) ali genetsko kodirajočih FRET-nanosenzorjev za cAMP (Epac1-camps), PKA nanosenzor (AKAR2) in laktat (Laconic). Ugotovili smo, da je mobilnost LK v astrocitih glede na ostale organele majhna in prostorsko omejena, v bližini mitohondrijev in endoplazemskega retikuluma. Dolgotrajni presnovni stres in hipoksija ter noradrenergična aktivacija sprožijo kopičenje LK v astrocitih. LK lahko služijo astrocitom v stresnih situacijah kot alternativni vir energije (-oksidacija prostih MK), hkrati pa zaščitijo celice CŽS pred lipotoksičnostjo. Ugotovili smo, da astrociti izražajo GPR40 in da stimulacija astrocitov z agonisti GPR40 (DHA, OA, TAK75) povzroči prehoden porast v znotrajcelični koncentraciji Ca2+ ter dolgotrajen porast v znotrajcelični koncentraciji cAMP in laktata. To nakazuje, da v astrocitih zunajcelične MK kot signalne molekule uravnavajo aerobno glikolizo in tvorbo laktata. Slednji predstavlja pomemben vir energije nevronom.

• Language: Slovenian
• Keywords: Astrociti, presnova in signaliziranje lipidov, lipidne kapljice, GPCR, cAMP, Ca2+, FRET
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2021
• PID: 20.500.12556/RUL-125996
• COBISS.SI-ID: 61599747
• Publication date in RUL: 11.04.2021

Secondary language

• Language: English
• Title: Lipid metabolism and signalling in cultured rat astrocytes : doctoral dissertation

Abstract

Astrocytes are neuroglia cells in the central nervous system (CNS) with many functions, including metabolic homeostasis in the CNS and delivering nutrients (lactate, glucose) to neurons. During CNS pathologies, astrocytes accumulate lipid droplets (LDs), which serve as stores of free fatty acids (FA) and sterols. The mechanisms of LDs formation and their role in astrocytes are still poorly understood, as well as lipid signalling via G-protein coupled receptors for free FA (e.g., GPR40) on the surface of astrocytes and its effect on astrocyte metabolism. Using immunocytochemistry and confocal microscopy, we characterized subcellular localization, LD mobility and the effect of metabolic stress and hypoxia (typical stressors in CNS pathologies) and stress-related noradrenergic activation on LD formation in isolated and organotypic brain tissue astrocytes. Using real-time fluorescence microscopy we evaluated the effect of GPR40 agonists, endogenous non-selective agonist docosahexaenoic acid (DHA) and oleic acid (OA), and synthetic selective agonist TAK875, on intracellular Ca2+, cAMP and lactate concentration in astrocytes labelled with Ca2+-indicator (Fluo-4, AM) and genetically encoded FRET-based nanosensors for cAMP (Epac1-camps), PKA nanosensor (AKAR2) or lactate (Laconic). We found that LD mobility in astrocytes is, compared to other organelles, low and spatially limited, in close proximity to mitochondria and endoplasmic reticulum. Metabolic stress, hypoxia and noradrenergic activation triggers LD accumulation in astrocytes. Astroglial LD can serve in astrocytes as an alternative energy source (β-oxidation of free FA), but at the same time they protect CNS cells from lipototoxicity. We found that astrocytes express GPR40 and that stimulation with agonists of GPR40 (DHA, OA or TAK875) causes transient Ca2+ and persistent cAMP and lactate increases in astrocytes, suggesting that extracellular free FA can function as a signalling molecules regulating aerobic glycolysis and lactate production in astrocytes, which is an important source of energy to neurons.

• Keywords: Astrocytes, lipid metabolism and signaling, lipid droplets, GPCR, cAMP, Ca2+, FRET