[Autophagy 2:3, 228-230, July/August/September 2006]; ©2006 Landes Bioscience
Autophagy, Bafilomycin and Cell Death Addenda
The “A-B-Cs” of Plecomacrolide-Induced Neuroprotection ABSTRACT
Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2703
KEY WORDS ABBREVIATIONS
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THE DICHOTOMY OF BAFILOMYCIN A1 IN CELL DEATH
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bafilomycin A1 bafilomycin B1 cerebellar granule neurons vacuolar ATPase
Bafilomycins (including A1, B, C and D) were isolated originally from the mycelium of Streptomyces gresius and were identified as macrolide antibiotics that prevented the growth of yeast, gram-negative bacteria and fungi.1 Bafilomycins were defined functionally by their inhibition of vacuolar or V-type ATPase (V-ATPase), an enzyme responsible for maintaining vesicular acidification.2 Subsequently, bafilomycin A1 (BafA1) was shown to prevent the fusion between lysosomes and autophagosomes, an effect attributed to its inhibition of V-ATPase.3 This inhibition of fusion between autophagosomes and lysosomes has been suggested to promote an “inhibition” of autophagy, as demonstrated by the ability of high concentrations of BafA1 (≥10 nM) to induce a robust accumulation of autophagosomes concomitant with apoptotic death.4,5 Chloroquine is a lysosomotropic agent that as a weak base attracts to lysosomes, which compromises their normal degradation and recycling capacity, thus also resulting in an inhibition of autophagy and subsequent Bax-dependent apoptotic death.4-6 Our laboratory has shown that BafA1 at concentrations ≤1 nM does not appear to inhibit vesicular acidification, nor does it induce accumulation of autophagosomes or apoptotic death.5 However, 1 nM BafA1 significantly attenuates chloroquine-induced apoptosis of cultured cerebellar granule neurons (CGNs) without preventing the chloroquine-induced inhibition of macroautophagy, evidenced biochemically by measuring the degradation of long-lived proteins and morphologically by the persistent accumulation of autophagosomes in neurons lacking apoptotic morphology.5 Because 1 nM BafA1 does not attenuate the chloroquine-induced inhibition of macroautophagy, we propose that the protective action(s) of BafA1 lie downstream of autophagosome formation and either upstream of or at the level of Bax-dependent activation of the intrinsic apoptotic pathway. In addition, we have shown that BafA1 does not prevent the death of CGNs induced by staurosporine, a broad-spectrum kinase inhibitor and widely-used inducer of apoptosis.5 These results suggest that the protective effects of BafA1 may be specific to stimuli that induce autophagic stress/inhibition (such as chloroquine) and lead to downstream induction of apoptosis. A wide range of death-inducing agents must be tested in future studies to further characterize this stimulus-specific pattern of BafA1-mediated protection.
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ACKNOWLEDGEMENTS
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autophagy, apoptosis, bafilomycin
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Received 03/15/06; Accepted 03/16/06
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*Correspondence to: John J. Shacka; Department of Pathology; Neuropathology Division; University of Alabama at Birmingham; SC 961 Birmingham, Alabama 35294-0017 USA; Tel.: 205.996.7252; Fax: 205.934.6700; Email:
[email protected]
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Department of Pathology; Neuropathology Division; University of Alabama at Birmingham; Birmingham, Alabama USA
BafA1 BafB1 CGNs V-ATPase
Bafilomycin A1 (BafA1), which is a member of the plecomacrolide sub-class of macrolide antibiotics, has differential, concentration-dependent effects on neuronal cell viability. When used at high concentrations, BafA1 inhibits vacuolar ATPase (V-ATPase), promotes the accumulation of autophagic vacuoles and triggers Bax-dependent apoptosis. These effects are similar to those induced by the lysosomotropic agent chloroquine. Conversely, at concentrations below its reported ability to completely inhibit V-ATPase, BafA1 dramatically attenuates chloroquine-induced apoptosis. The protective effects of BafA1 appear to be independent of the chloroquine-induced accumulation of autophagosomes. Rather, BafA1 appears to inhibit events downstream of chloroquine-induced autophagosome accumulation, such as the loss of mitochondrial or lysosomal integrity. Our finding that BafA1 inhibits the death of neurons induced by autophagic stress/inhibition suggests a potentially novel mechanism of action apart from its ability to inhibit V-ATPase. Here we provide further evidence of neuroprotection against chloroquine-induced death by plecomacrolide antibiotics that are structurally similar to BafA1, including bafilomycin B1 and concanamycin A, and discuss potential mechanism(s) of neuroprotection against autophagic stress.
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John J. Shacka* Barbara J. Klocke Kevin A. Roth
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Addendum to:
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We thank Angela Schmeckebier for assistance in preparation of this manuscript. The research in this and the target paper was supported by NINDS 35107 (K.A.R.) and an award from the Batten Disease Support and Research Association (J.J.S.).
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Bafilomycin A1 Inhibits Chloroquine-Induced Death of Cerebellar Granule Neurons
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John J. Shacka, Barbara J. Klocke, Masahiro Shibata, Yasuo Uchiyama, Geeta Datta, Robert E. Schmidt and Kevin A. Roth
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AUTOPHAGY, BAFILOMYCIN AND CELL DEATH
Figure 1. Bid deficiency does not prevent chloroquine-induced death of CGNs. Cultured CGNs were prepared from Bid-deficient vs. wild-type brains and cultured as previously described.16 After four days in vitro, CGNs were treated with chloroquine (20 µM) for 24 h then measured for viability using the fluorescent calcein conversion assay as previously described.5,16
POTENTIAL MECHANISMS OF BafA1-INDUCED PROTECTION There are many possible mechanisms mediating BafA1-induced neuroprotection against the inhibition of autophagy that are currently being tested in our laboratory. It has been shown previously that the preservation of mitochondrial membrane potential significantly attenuates apoptotic death associated with the pharmacological inhibition of autophagy.4 Currently it is not known whether BafA1 interacts directly with mitochondria or preserves mitochondrial function. Chloroquine has also been shown to disrupt the colocalization of mitochondria and lysosomes.4 Although the functional significance of this colocalization is not entirely clear, its disruption may be detrimental to cell viability and thus the interaction of mitochondria and lysosomes should be explored as a potential target of BafA1-mediated protection. Bax has been shown to colocalize to lysosomal membranes7 and Bcl-2 overexpression has been shown to prevent stress-induced lysosomal de-stabilization.8 Although the functional significance of these interactions remains elusive, it is tempting to speculate that BafA1 may affect interactions of Bcl-2 family proteins with lysosomes and/or mitochondria that help preserve mitochondrial and lysosomal membrane integrity in the face of autophagic stress, with a resultant decrease in Bax-dependent apoptosis. The release of lysosomal cathepsins has been shown previously to induce cleavage of Bid and Bax-dependent apoptosis.9 However, in our hands Bid deficiency did not prevent chloroquineinduced death of CGNs (Fig. 1), thus ruling out this pathway as a potential target of BafA1-mediated protection. In cultured neurons, BafA1 has also been shown to inhibit both the release of vesicular glutamate10 and the uptake of several neurotransmitters including GABA, glutamate and serotonin.11,12 However, it is unlikely that alterations in neurotransmitter uptake/release play a role in BafA1-mediated protection against the inhibition of autophagy, since this effect of BafA1 is observed at micromolar concentrations and 1 nM bafilomycin A1, its most effective neuroprotective concentration, does not inhibit glutamate uptake.12
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Figure 2. Plecomacrolide-specific inhibition of chloroquine-induced death. Cultured CGNs were prepared and treated after four days in vitro for 24 h with chloroquine (20 µm) in the presence or absence of 1 nM BafA1 (Sigma), BafB1 (AG Scientific), concanamycin A (Calbiochem), erythromycin (Sigma) or oligomycin A (Sigma), and measured for viability using the fluorescent calcein conversion assay. BafA1, BafB1 and concanamycin induced a twofold, significant increase in viability compared to treatment with chloroquine alone (*p < 0.05 via ANOVA and Bonferroni’s post hoc test).
PLECOMACROLIDE-INDUCED NEUROPROTECTION The structure of bafilomycins consists of an identical 16-membered macrolactone ring and a hemiacetal side chain separated by a three-carbon linker, and differ with respect to side chain substitutions at carbon 21.2 Concanamycins are compounds isolated originally from Streptomyces diastatochromogenes13 that are structurally similar to bafilomycins and also potently and selectively inhibit V-ATPase,14 but possess 18-membered macrolactone rings.15 Together, the 16and 18-membered macrolactones, bafilomycins and concanamycins respectively, form the plecomacrolide subclass of macrolide antibiotics. Treatment with 1 nM BafA1, BafB1 or concanamycin A all significantly inhibited the chloroquine-induced death of CGNs, whereas treatment with macrolide antibiotics that are not members of the plecomacrolide subclass, including erythromycin (the first clinically available macrolide antibiotic) and oligomycin A (a potent inhibitor of the mitochondrial F1F0 ATPase) did not alter the death-inducing effects of chloroquine (Fig. 2). While more rigorous dose-response analyses are required, these results certainly suggest that the neuroprotective actions of macrolide antibiotics against autophagic stress-induced death may be limited to the plecomacrolide subclass. In addition, since our studies also suggest that ≤1 nM BafA1 does not decrease vesicular acidification,5 the putative mechanism of plecomacrolide-induced neuroprotection is potentially novel and independent of their ability to inhibit V-ATPase. Comprehensive structureactivity relationship studies are needed to definitively preclude the contribution of V-ATPase inhibition in plecomacrolide-induced neuroprotection.
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