Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates

Abstract

Many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by abnormal accumulations of aggregated proteins. Brains in these diseases also show accumulation of autophagic vesicles in the neuronal cytoplasm, suggesting impairment of the autophagic process. As autophagy involves de novo membrane production and vesicle fusion, extensive changes in lipid molecules are necessary. However, the involvement of signaling lipid-modifying enzymes in autophagy and their roles in neurodegenerative diseases are not clear. Using specific inhibitor, we show that loss of phospholipase D1 (PLD1) activity resulted in an accumulation of microtubule-associated protein light chain 3 (LC3), p62, and polyubiquitinated proteins, signs representing malfunction in autophagic flux. Fluorescence and electron microscopic analyses demonstrated impaired fusion of autophagosomes with lysosomes, resulting in accumulation of autophagosomes. Within the cells with impaired autophagic flux, a-synuclein aggregates accumulated in autophagosomes. Knockdown of PLD1 expression using small interfering RNA also resulted in impaired autophagic flux and accumulation of a-synuclein aggregates in autophagosomes. Neuronal toxicity caused by a-synuclein accumulation was rescued by overexpression of PLD1; however, expression of activity-deficient mutant, PLD1-KRM, showed reduced rescue effects. Finally, we demonstrated that both PLD activity and expression levels were reduced in brain tissues of dementia with Lewy bodies (DLB) patients, whereas the amounts of a-synuclein and p62 were increased in the same tissue samples. Collectively, these results suggest that insufficient PLD activity, and therefore, the changes in phospholipid compositions within membranes, might be an important contributor to impaired autophagic process and protein accumulation in Lewy body diseases.

A lipid switch unlocks Parkinson’s disease-associated ATP13A2

“…PA also stimulates the activity of the mTOR complex in autophagy (31). Interestingly, PLD1 inhibition disturbs α-synuclein clearance via autophagy, whereas OE of PLD1 reduces α-synuclein toxicity (32). As such, PA might regulate ATP13A2 activity during autophagy, as ATP13A2 confers protection against α-synuclein toxicity (7) and also mediates mitochondrial clearance (5).…”

Distinguishing aggregate formation and aggregate clearance using cell-based assays

“…First, we introduce a novel method by which we can distinguish how a given pathway or modifier affects protein accumulation. Different degradation pathways have been implicated in affecting the accumulation of aggregation-prone proteins, from proteasome-mediated degradation (Mishra et al, 2008;Iwata et al, 2009;Zhang et al, 2010;Urushitani et al, 2010;Ortega and Lucas, 2014;Schipper-Krom et al, 2014) to the lysosome-mediated pathway or macroautophagy as being the predominant pathway for aggregate clearance (Ravikumar et al, 2002;Iwata et al, 2005b;Yamamoto et al, 2006;Pankiv et al, 2007;Lamark et al, 2009;Fan et al, 2010;Lamark and Johansen, 2012;Odagiri et al, 2012;Castillo et al, 2014;Bae et al, 2014). Our ability to interrogate how each pathway might contribute to aggregate turnover has been limited by our inability to temporally characterize aggregate formation and clearance in cells.…”

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…PLD-derived DAG was recently shown to be required for autophagy after infection (Shahnazari et al, 2010). Since PLD was only recently discovered to modulate autophagy (Liu et al, 2009;Dall'Armi et al, 2010;Moreau et al, 2012;Bae et al, 2014;Bruntz et al, 2014), future studies are needed clarify the detailed mechanism of autophagy regulation by PLD. It will also be important to ascertain whether other PtdOHgenerating pathways (see Fig.…”