Dequalinium-Induced Cell Death of Yeast Expressing α-Synuclein-GFP Fusion Protein

Abstract

Intracellular toxic effects of the dequalinium-induced protofibrils of alpha-synuclein have been investigated with the yeast system expressing alpha-synuclein-GFP fusion protein in single copy, which appears in the green halo around the plasma membrane. Intracellular responses of the green fluorescent protein were analyzed as the cells were treated with dequalinium (DQ) and lactacystin. Yeast cells expressing alpha-synuclein-GFP were susceptible to both compounds in alpha-synuclein-dependent manner. Upon DQ treatment, the green halo became smeared throughout the cytoplasm while lactacystin induced a few discrete green dots, reflecting intracellular formation of the protofibrils and the protein inclusions, respectively. The DQ-treated yeast cells were intensely stained with the nucleic acid stains of cell-permeable Hoechst 33342 and cell-impermeable propidium imidione, indicating that nucleus has been disrupted in addition to plasma membrane destabilization. Those DQ-treated yeast cells, however, still contained active mitochondria identified with MitoTracker Red. Therefore, the DQ-induced protofibrillar state of alpha-synuclein-GFP has been suggested to cause the nuclear damage either independently or in combination with the membrane destabilization without affecting mitochondria.

Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins

“…Treatment of yeast cells expressing α-synuclein with the proteasome inhibitor lactacystin resulted in increased α-synuclein aggregation (Zabrocki et al, 2005; Lee et al, 2008). Consistently, expression of α-synuclein in the yeast strain sen3-1 , which harbors a mutation in the gene encoding the regulatory proteasome subunit Rpn2, led to increased steady-state levels of α-synuclein (using untagged α-synuclein) and to increased formation of aggregates (using GFP-tagged α-synuclein) (Sharma et al, 2006).…”

“…Yeast cells overexpressing wild-type and disease-associated α-synuclein demonstrated growth deficits and age-dependent loss of clonogenic cell survival paralleled by the emergence of morphological markers of apoptosis and necrosis (Willingham et al, 2003; Flower et al, 2005; Witt and Flower, 2006; Büttner et al, 2008, 2013a,b; Lee et al, 2008; Su et al, 2010). The cellular accumulation of ROS and mitochondrial dysfunction are pivotal for the execution of α-synuclein-triggered cell death.…”

“…Moderate α-synuclein expression, which is non-toxic for wild-type yeast cells, resulted in severe growth deficits in yeast cells bearing mutations in the 20S proteasomal barrel ( pre1-1001, pre2-1001, doa3-1 ) (Dixon et al, 2005; Sharma et al, 2006) and in the 19S regulatory particle of the 26S proteasome ( sen3-1 ) (Sharma et al, 2006), or treated with the proteasome inhibitor lactacystin (Lee et al, 2008). Consistently, expression of Rpt5, a component of the 19S regulatory particle, and expression of the ERAD ubiquitin ligase Hrd1 suppressed α-synuclein-triggered cytotoxicity (Liang et al, 2008; Gitler et al, 2009).…”

Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration

“…Yeast cells overexpressing wild-type and disease-associated α-synuclein demonstrated growth deficits and age-dependent loss of clonogenic cell survival (Willingham et al, 2003; Flower et al, 2005; Witt and Flower, 2006; Büttner et al, 2008; Lee et al, 2008) (Table 1). α-Synuclein-triggered cytotoxicity in yeast was characterized by increased levels of ROS, and by the emergence of morphological markers of both apoptosis and necrosis (Flower et al, 2005; Büttner et al, 2008; Su et al, 2010).…”

“…Mitochondria are critically damaged upon α-synuclein expression: (1) The mitochondrial network was fragmented (Sampaio-Marques et al, 2012), (2) mitochondria were found to be abnormally swollen (Su et al, 2010), (3) Cyt. c was released from mitochondria into the cytosol (Flower et al, 2005), (4) α-synuclein-expressing cells treated with the proteasome inhibitor lactacystin demonstrated loss of mitochondrial membrane potential (Lee et al, 2008), and (5) mRNA profiling revealed that 60% of the downregulated genes encode proteins localized to mitochondria (Yeger-Lotem et al, 2009). Mitochondrial damage pivotally contributes to α-synuclein-triggered cytotoxicity, because α-synuclein expression in ρ 0 cells, which lack mtDNA and are devoid of respiratory competent mitochondria, significantly relieved the loss of cell survival, reduced the number of apoptotic and necrotic cells and markedly decreased ROS levels (Büttner et al, 2008).…”