Elsevier

Neurobiology of Aging

Volume 25, Issue 8, September 2004, Pages 1057-1066
Neurobiology of Aging

Divergence of the apoptotic pathways induced by 4-hydroxynonenal and amyloid β-protein

https://doi.org/10.1016/j.neurobiolaging.2003.10.003Get rights and content

Abstract

In this paper, we examine the hypothesis that 4-hydroxynonenal (HNE), a product of lipid peroxidation, is a key mediator of cell death resulting from β-amyloid exposure. We revisit the effects of HNE on different neuronal cell types to determine which caspase or caspases are required for HNE-induced death, and to compare these results with the known caspase requirements in other death paradigms. We have previously shown that in a given neuronal cell type different death stimuli can evoke stimulus-specific apoptotic pathways. We now show that HNE treatment of neuronal cells induced dose-dependent death and caspase activity which were blocked by inhibition of caspases. Antisense down-regulation of caspases-3, -7 or -9 provided complete protection from HNE-induced death, as did down-regulation of the caspase regulators APAF-1 and DIABLO. Conversely, this work and our previous studies of three other death paradigms show that caspase-3 is not required for death induced by β-amyloid, SOD1 down-regulation, or trophic factor deprivation. We also show that HNE accumulated in settings where death does not ensue. We conclude that HNE toxicity is mediated via a caspase-9-dependent pathway but that HNE accumulation need not induce cell death nor is it an obligate mediator of Aβ-induced cell death.

Introduction

While programmed cell death is a feature of a variety of neurodegenerative disorders, the stimuli which trigger the neurons to die in specific diseases remain the subject of active investigation. Oxidative stress has been proposed to play a role in a variety of diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) [1], [8]. It has been proposed by others that 4-hydroxynonenal (4-HNE), an aldehyde generated during lipid peroxidation, is responsible for all or part of the pathophysiological consequences of oxidative stress [21], [44]. This proposal is supported by reports of an increase in HNE in postmortem samples of the brains of patients succumbing to Alzheimer’s disease [18], [21], and in the spinal cord of patients with amyotrophic lateral sclerosis [26]. HNE has been suggested as a down-stream mediator of β-amyloid toxicity and free radical-induced neuronal death [18], [22], [23], [30].

A number of studies have pointed out a requirement for caspase activation in HNE-induced death [4], [14], [19], [49]. These studies are based on the use of pseudosubstrate caspase inhibitors. These inhibitors are only partially specific for individual caspases and cannot definitively identify the caspase or caspases involved in a specific death cascade.

In the studies presented here we use vector-facilitated antisense oligonucleotides to specifically knock-down the levels of the individual caspases in cell exposed to HNE to see which caspases execute HNE-mediated apoptosis. We will compare these findings to those obtained with other death initiators, including peroxynitrite-mediated oxidative stress [35], [41], β-amyloid toxicity (proposed to have a causal role in AD) [25], [42], [47] and trophic factor deprivation [12], [39], [43].

Section snippets

Cell culture

Sympathetic neuron cultures were prepared from 2-day-old rat pups, as previously described [39]. Cultures were grown in 24-well collagen-coated dishes in RPMI 1640 medium plus 10% horse serum with mouse NGF (100 ng/ml). One day following plating, uridine and 5-fluorodeoxyuridine (10 μM each) were added to the cultures and left for 3 days to eliminate non-neuronal cells (less than 1% non-neuronal cells present after 3 days). On the sixth day following plating HNE was added. Each culture was

HNE induces dose-dependent death in multiple neuronal cell types

It has been previously shown that HNE induces apoptosis in PC12 cells [18], in primary cultures of hippocampal neurons [18] and sympathetic neurons [3]. We have extended these data to include neuronal cell lines derived from a central source (CAD cells). Sympathetic neurons and PC12 cells are neural crest derivatives. CAD cells are a catecholaminergic cell line derived from the CNS which contains markers of CNS neurons [24], and differentiates upon removal of serum. The dose-dependence after 1

Discussion

Apoptotic death has been suggested to play a role in neurodegenerative diseases [6], [7], [27], [48], though its importance is still hotly debated. Certainly neuronal loss is an outstanding feature of neurodegenerative diseases and if this loss is a result of apoptosis then molecules which are crucial in multiple cell death paradigms are enticing therapeutic targets. One such molecule is HNE. HNE has been proposed as a downstream mediator of cell death initiated by different stimuli, such as

Acknowledgements

This work was supported in part by grants from NIH-NINDS (C.M.T., W.J.F., M.L.S.), NSF (W.J.F.) and the Muscular Dystrophy Association (C.M.T.).

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