Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity.

The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders.

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is selectively toxic to primary dopaminergic neurons in vitro.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.

Functional oligomerization of the Saccharomyces cerevisiae isoprenylcysteine carboxyl methyltransferase, Ste14p.

The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26-kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CAAX post-translational processing pathway. Sequence analysis reveals a putative GXXXG dimerization motif located in transmembrane 1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminal-tagged His(10)-myc(3)-Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species.