Browsing by Subject "Mitophagy"

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Open Access
The effects of ER stress on glial cells: evidence from both in vivo and in vitro models
(2022-01) Mutlu, Duygu
Consuming high fat diet for long periods of time increases neuroinflammation, which may result in cognitive decline and loss in memory formation. Excessive free fatty acid influx stresses the ER and mitochondria, two important organelles taking part in protein folding and energy production. The ER responds to stress in part by activating Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) pathway. It has been shown that PERK pathway activation inhibits mitophagy (autophagy of mitochondria) in macrophages. Since microglia are the immune cells of the Central Nervous System (CNS), first I investigated the effects of high fat diet in the cortex of wild type (C57BL/6) and ApoE -/- mice by looking at microglial marker Iba1. Western Blot analysis showed no significant effects of diet and genotype on Iba1 level. I also found that there is no correlation between Iba1 and GFAP (astrocyte marker) levels for these mice. Brain contains many other types of cells and in order to effects of ER stress directly on the microglia, I moved on to the BV2 mouse microglial cell line. There were differential effects of ER stress induction with thapsigargin and palmitate treatments. An increase in ER stress markers such as CHOP and p-IRE1 has been observed with both treatments. While CHOP protein levels could not reach significance, there was an increasing numerical trend. p-IRE1 was marginally significant for both treatments (p=0.087 for thapsigargin and p=0.061 for 100 M palmitate). Mitophagy indicators (Pink1 and p62) were assessed with Western Blot analysis after successful mitochondria isolation from BV2 cells. The data indicated that p62 marginally increased with both palmitate (p=0.61) and thapsigargin (p=0.1) treatments. Following both treatments, very subtle effects of ER stress were observed. This suggests that further experiments examining optimal dosage and duration need to be performed. Overall, the induction of ER stress appears to induce mitophagy and alter microglia, which likely leads to altered cellular and synaptic function.
Open Access
Investigating hyperlipidemia-driven organelle stress and neuroinflammation on the mouse cerebral cortex: insights into the intervention of perk pathway
(2024-09) Kızıldağ, Fulya
Deficits in the metabolism of lipids called hyperlipidemia have been linked to a higher risk of developing neurodegenerative diseases. Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) signaling is crucial in cellular homeostasis. Abnormalities in the PERK have been associated with neurodegeneration. Mitophagy and the PERK pathway emphasize how cellular stress responses are regulated to preserve cellular homeostasis and mitochondrial quality control. The activity of main mitophagy regulators, such as Parkin and PINK1 (PTEN-induced kinase 1), is regulated by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) by PERK. If lipid metabolism is at a high level, abnormalities in the mitochondria and endoplasmic stress (ER) emerge. During the ER stress activation, the PERK pathway is induced, and mitophagy is blocked, causing an enhancement in the neuroinflammation. The underlying molecular mechanism by which hyperlipidemia impacts the PERK pathway and mitophagy in the cerebral cortex, as well as the relationship between mitophagy and neuroinflammation, is not fully understood. In this study, Apoe-/- and C57BL/6 mice were given a chow or western diet to stimulate hyperlipidemia. Moreover, western diet-fed Apoe-/- mice were injected with PERK inhibitors, GSK2606414 and Trans-ISRIB, intraperitoneally for six weeks to suppress the PERK pathway. This study explores the effects of hyperlipidemia on the PERK pathway, inflammatory and mitophagy markers in the cerebral cortex of chow and western diet-fed C57BL/6 and Apoe-/- mice and investigates whether the inhibition of the PERK pathway can change the levels of inflammatory and mitochondrial markers in the cerebral cortex of hyperlipidemic mice subjects. mRNA and protein expression levels of mitophagy and inflammatory markers were assessed using the RT-qPCR and western blot, respectively. PERK pathway activation under hyperlipidemia conditions was not determined. Nevertheless, significant alterations in mitophagy markers and inflammation were detected in Apoe-/- mice apart from the diet. Furthermore, significant alterations were not seen in the PERK pathway markers; however, mitophagy was stimulated, and some inflammation markers were significantly decreased mildly at the cortical tissue of WD-fed Apoe-/- mice administrated with PERK pathway inhibitors, GSK2606414 and Trans-ISRIB. Besides, no statistically significant changes were observed in the transcript levels of the inflammatory markers. Taken together, hyperlipidemia did not cause the PERK pathway to be activated in the cerebral cortex of mice; nevertheless, it mildly altered inflammation and caused mild effects of the dysregulation of the mitochondria by hyperlipidemia independent from the PERK pathway. Furthermore, although the PERK pathway was not inhibited by the administration of PERK pathway inhibitors, mitophagy was induced, and inflammation was decreased mildly. Targeting the PERK pathway with GSK2606414 and Trans-ISRIB inhibitors from the cerebral cortex would not be a therapeutic approach for neurodegenerative diseases.