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Parkinson's disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase-dependent apoptosis in dopaminergic neurons. Using the midbrain-derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over-expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase-9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase-independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large-scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large-scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase-independent cell death in MPP+-treated dopaminergic neurons.
Parkinson's disease is a debilitating neurodegenerative movement disorder characterized by loss of monoaminergic neurons, particularly dopaminergic neurons of the ventral midbrain. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a parkinsonian pattern of neuron loss in humans and other mammals. The active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+), is internalized by the dopamine transporter. Although MPP+ acts as a complex I inhibitor, other mechanisms may also contribute to toxicity (Dauer and Przedborski 2003). The observations that transgenic mice overexpressing anti-apoptotic Bcl-2 (Offen et al. 1998; Yang et al. 1998) or inhibitor of apoptosis protein (Eberhardt et al. 2000), and that knockout mice lacking pro-apoptotic bax (Vila et al. 2001), are all protected from MPTP toxicity suggests a role for regulated pathways of active cell death in MPTP/MPP+ toxicity.
Apoptosis inducing factor (AIF) is a more recently characterized apoptogenic factor that mediates caspase-independent cell death in other systems (Susin et al. 1999; Daugas et al. 2000; Cregan et al. 2002). AIF normally resides in the intermembrane space of mitochondria, and is ubiquitously expressed in brain tissues (Cao et al. 2003). AIF release from mitochondria precedes large-scale DNA fragmentation (50 kbp) (Daugas et al. 2000). Nuclear translocation of AIF is elicited by transient cerebral ischemia (Cao et al. 2003) or traumatic brain injury in rats (Zhang et al. 2002). AIF also mediates poly(ADP-ribose) polymerase-1-dependent forms of cell death (Yu et al. 2003). Caspase inhibitors do not affect mitochondrial release of AIF (Daugas et al. 2000; Cao et al. 2003). Thus, we hypothesized that AIF release may contribute to MPP+-elicited cell death.
We studied the potential role of AIF in MPP+ toxicity using a dopamine-producing midbrain-derived neuronal cell line and primary midbrain neuronal cultures. Our data indicate that MPP+ elicited nuclear translocation of AIF. Whereas caspase inhibitors were unable to reduce MPP+ toxicity, inhibiting the cellular expression of AIF using RNA interference [small interfering RNA (siRNA)] conferred significant protection against MPP+ toxicity. These data indicate an important role for AIF in dopaminergic neuronal cell death, suggesting that multiple pathways must be considered when developing neuroprotective therapies.
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- Materials and methods
The data presented here indicate that AIF redistribution plays an important role in caspase-independent cell death in dopaminergic neuronal cells. Although MPP+ activates pathways associated with both caspase-dependent and caspase-independent mechanisms, neither overexpression of AIP, a dominant negative apoptosome regulatory protein, nor use of broad-spectrum caspase inhibitors conferred protection. In contrast, siRNA-mediated knockdown of AIF expression effectively reduced large-scale DNA fragmentation, chromatin condensation and cell death in the neuronal differentiated MN9D cells.
The role of caspases in MPP+-elicited cell death has been inconsistent in the literature. Caspase inhibitors have been reported to protect against MPTP toxicity (Yang et al. 2004) and MPP+-elicited cell death in primary cultures, but there is no protection from neuritic dysfunction (Bilsland et al. 2002). However, other studies found little evidence to support either phosphatidylserine externalization or apoptotic DNA laddering (Lotharius et al. 1999; Han et al. 2003). These groups also found no protection using broad-spectrum caspase inhibitors, and others have reported that caspase inhibition potentiates necrotic cell death (Hartmann et al. 2001). Although studies using higher doses of MPP+ have suggested necrosis as an explanation for caspase-independent cell death (Choi et al. 1999), our data indicate that MPP+ elicits an active form of cell death in differentiated MN9D cells that requires transcription, translation and AIF. Expression profiling studies of MPP+-treated neuronal cell lines indicate induction of transcription factors such as CHOP/Gadd153 (Ryu et al. 2002; Holtz and O'Malley 2003). Interestingly, overexpression of CHOP promotes stress-related death, resulting in decreased cellular glutathione, increased production of reactive oxygen species and decreased levels of Bcl-2 (McCullough et al. 2001). Given the common role of mitochondrial oxidative stress in major models of parkinsonian injury (Betarbet et al. 2002; Dawson and Dawson 2003; Callio et al. 2005), and the ability of Bcl-2 overexpression to regulate AIF release (Cao et al. 2003), it is possible that similar mechanisms are linked to AIF-mediated death.
A multiplicity of cell death pathways is not surprising in pathological cell death associated with disease. The eventual decline in viability of MPP+-treated AIF-deficient cells at 72 h implies the existence of additional caspase-independent pathways. In addition to apoptosis and necrosis, caspase-independent forms of cell death involving increased autophagosomes have been reported in neurons (Zaidi et al. 2001; Tolkovsky et al. 2002; Gomez-Santos et al. 2003; Florez-McClure et al. 2004). We found that autophagy is induced during MPP+ toxicity (J.-h. Zhu and C. T. Chu, unpublished data), and that autophagocytosed mitochondria are present in human Parkinson's/Lewy body disease neurons (Zhu et al. 2003), suggesting the possibility of additional pathways not prevented by either AIF siRNA or caspase inhibitors. Although cytochrome c release was observed in both MN9D and primary TH neurons, caspase-3 activation was not robust. This may be explained by expression of endogenous caspase inhibitors in neuronal cells (Eberhardt et al. 2000; Potts et al. 2003), and/or reduced ATP levels in MPP+-treated cells (Han et al. 2003). Cellular context and pre-existing ATP levels determine available pathways of cell death (Eguchi et al. 1997; Nicotera et al. 2000; Han et al. 2003). It is interesting to note that AIF nuclear translocation can be triggered by ATP depletion itself (Daugas et al. 2000), suggesting that AIF-mediated cell death may occur under conditions where caspase activation is impaired.
Given that multiple pathways can be initiated in a given cell injury paradigm, the siRNA results are especially important in demonstrating a direct role for AIF in mediating MPP+ toxicity. A role for AIF as a caspase-independent mediator was derived from observations that overexpression of AIF induces neuronal cell death in a caspase-independent manner (Cregan et al. 2002). Other studies supporting a role for AIF as a death mediator include observations that AIF nuclear translocation corresponds with early commitment to apoptosis (Bidere et al. 2003), precedes cytochrome c release (Wang et al. 2004) and correlates with large-scale DNA fragmentation (Daugas et al. 2000; Cao et al. 2003). However, a temporal correlation does not necessarily predict causality. Although cytochrome c release precedes AIF release in our system, caspase activation is not necessary for MPP+-mediated cell death. Intracellular delivery of neutralizing antibodies (Cregan et al. 2002; Wang et al. 2004), genetic inactivation in embryonic stem cells (Joza et al. 2001) and our current siRNA studies directly support an executionary role for AIF.
The siRNA studies also support a mechanism involving AIF-mediated large-scale DNA fragmentation, as suggested by studies using purified nuclei (Daugas et al. 2000). An alternative possibility for detrimental effects of AIF release may include loss of some normal protective mitochondrial function. This possibility is not substantiated by the siRNA studies as the MN9D siRNA lines with reduced or absent AIF expression did not exhibit problems with viability. Moreover, there were no significant effects of AIF knockdown on mitochondrial complex I activity. The significance of the abnormal clumped pattern of AIF staining observed in primary neurons is unknown, but may reflect injury-induced alterations in mitochondrial distribution.
The in vivo evidence for a role of apoptosis in MPTP toxicity include studies using transgenic mice overexpressing Bcl-2 (Offen et al. 1998; Yang et al. 1998) and knockout mice lacking pro-apoptotic bax (Vila et al. 2001). It is interesting to note that overexpression of Bcl-2 reduces AIF nuclear translocation in other neuronal cell systems (Cao et al. 2003), and that Bax is capable of promoting AIF release (Bidere et al. 2003). Moreover, in contrast to the expression pattern of a number of apoptosis regulatory gene products during brain development, the expression of AIF increases with brain maturation and peaks in adulthood (Cao et al. 2003). Thus, the involvement of AIF in caspase-independent MPP+ toxicity to MN9D and primary dopaminergic neurons in culture could reconcile conflicting interpretations derived from transgenic mouse studies that support a role for active, regulated cell death and primary midbrain culture systems interpreted as ‘necrotic’ owing to inability of caspase inhibitors to confer protection.
To summarize, MPP+ elicits an active form of cell death in dopaminergic neuronal cells that involves AIF nuclear translocation, but is caspase independent. Given the multiplicity in potential cell death pathways in dopaminergic neurons, combination therapies targeted at multiple death pathways or those regulating signaling mechanisms that act before commitment to death may be beneficial (Chu et al. 2004; Horbinski and Chu 2005).