Excess membrane binding of monomeric alpha-, beta- and gamma-synuclein is invariably associated with inclusion formation and toxicity

Abstract

α-synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains—Lewy bodies and Lewy neurites—are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs β-synuclein (βS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases, and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-β component of plaques (NAC) domain, constituting amino acids 61 to 95, has been identified to be critical for aggregation in vitro. This domain is partially absent in βS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS-membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that βS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased βS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic, and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Synucleins: New Data on Misfolding, Aggregation and Role in Diseases

“…The C-terminal domain differs in amino acid sequence among synucleins and regulates their solubility depending on its length and charge. A stretch of amino acids located between positions 61–95 in α-synuclein is called NAC ( Figure 1 ), and a stretch of amino acids 71–82 within the α-synuclein NAC region is critical for β-sheet-rich aggregation in vitro [ 8 , 9 ]. This amino acid sequence is VTGVTAVAQKTV in α-synuclein, FSGAGNIAAATG in β-synuclein, and VSSVNTVATKTV in γ-synuclein.…”

“…This amino acid sequence is VTGVTAVAQKTV in α-synuclein, FSGAGNIAAATG in β-synuclein, and VSSVNTVATKTV in γ-synuclein. Amino acids belonging to the 71–82 stretch are necessary and sufficient for α-synuclein fibrillization [ 9 ].…”

“…Furthermore, the substitutions of amino acids (E35K + E46K + E61K = ‘3K’) are render all synucleins considerably more toxic than their wild-type counterparts [ 9 ]. These substitutions increase α-synuclein-membrane interactions pointing to a mechanism of synuclein toxicity associated with membrane binding.…”

“…Remarkably, increasing β-synuclein and γ-synuclein affinity to the membrane by point mutations converts these members of the synuclein family into monomers associated with the membrane, increases their cytotoxicity, and predisposes them to form round cytoplasmic inclusions [ 9 ].…”

“…The absence of this region in β-synuclein decreases its tendency to aggregate. Amino acids 71VTGVTAVAQKTV82 are the most essential for α-synuclein aggregation [ 8 , 9 ]; it is. This domain is partly absent in β-synuclein and to some extent conserved in γ-synucleins, which might clarify why both homologs of α-synuclein are not aggregation prone as α-synuclein.…”