Synaptosomal toxicity and nucleophilic targets of 4-hydroxy-2-nonenal

Abstract

4-Hydroxy-2-nonenal (HNE) is an aldehyde by-product of lipid peroxidation that is presumed to play a primary role in certain neuropathogenic states (e.g., Alzheimer disease, spinal cord trauma). Although the molecular mechanism of neurotoxicity is unknown, proteomic analyses (e.g., tandem mass spectrometry) have demonstrated that this soft electrophile preferentially forms Michael-type adducts with cysteine sulfhydryl groups. In this study, we characterized HNE synaptosomal toxicity and evaluated the role of putative nucleophilic amino acid targets. Results show that HNE exposure of striatal synaptosomes inhibited (3)H-dopamine membrane transport and vesicular storage. These concentration-dependent effects corresponded to parallel decreases in synaptosomal sulfhydryl content. Calculations of quantum mechanical parameters (softness, electrophilicity) that describe the interactions of an electrophile with its nucleophilic target indicated that the relative softness of HNE was directly related to both the second-order rate constant (k(2)) for sulfhydryl adduct formation and corresponding neurotoxic potency (IC(50)). Computation of additional quantum mechanical parameters that reflect the relative propensity of a nucleophile to interact with a given electrophile (chemical potential, nucleophilicity) indicated that the sulfhydryl thiolate state was the HNE target. In support of this, we showed that the rate of adduct formation was related to pH and that N-acetyl-L-cysteine, but not N-acetyl-L-lysine or beta-alanyl-L-histidine, reduced in vitro HNE neurotoxicity. These data suggest that, like other type 2 alkenes, HNE produces nerve terminal toxicity by forming adducts with sulfhydryl thiolates on proteins involved in neurotransmission.

FIG. 1.

This figure presents the line structures for HNE and several structurally related α,β-unsaturated carbonyl derivatives of the type 2 alkene class. Also shown is the line structure for the nonconjugated structural analog, allyl alcohol.

FIG. 2.

The concentration-dependent effects of HNE on ³H-DA uptake (A) and free sulfhydryl content (B) in synaptosomes isolated from rat striatum are presented in this figure. For comparative purposes, comparable data for acrolein, MVK, and ACR are shown (LoPachin et al., 2007a). Data are expressed as mean percentage of control ± SEM based on separate experiments (n =3–5). Calculated IC₅₀’s are provided in the parentheses.

FIG. 3.

The concentration-dependent effects of HNE on ³H-DA transport in striatal synaptic vesicles are presented in this figure. For comparative purposes, comparable data for acrolein, MVK, and ACR are shown (LoPachin et al., 2007a). Data are expressed as mean percentage of control ± SEM based on separate experiments (n =3–5). Calculated IC₅₀'s are provided in the parentheses.

FIG. 4.

This figure shows a plot of log[SH/SH₀] versus time (s) for the reaction of HNE with L-cysteine at pH 7.4 or pH 8.8, where SH₀ = initial sulfhydryl concentration at time zero. The respective second-order rate constants (k₂) for these reactions are provided in the figure.

FIG. 5.

The effects of NAC, NAL, and carnosine on the inhibition of ³H-DA transport (A) and loss of free sulfhydryl groups (B) in HNE-exposed striatal synaptosomes (n = 4–6 experiments) are presented in this figure. Control data are as follows: synaptosomal transport = 17 ± 2 nmol/mg protein/min; synaptosomal free sulfhydryl content = 132 ± 4 pmol/mg protein. Data are expressed as mean percentage control ± SEM. Calculated IC₅₀'s are provided in the parentheses.

FIG. 6.

The effects of carnosine on the inhibition of ³H-DA transport (A) and loss of free sulfhydryl groups (B) in acrolein- or MVK-exposed striatal synaptosomes (n = 4–6 experiments) are presented in this figure. Control data are provided in the legend of Figure 5. Data are expressed as mean percentage control ± SEM. Calculated IC₅₀'s are provided in the parentheses.