Nuclear transcription factor MEF2s are involved in a growing number of critical cellular functions involving both neuronal and non-neuronal systems. The basic assumption has always been that MEF2s exert their control on cells solely through modulating the expression of nuclear target genes (20
). Indeed, MEF2A has previously been shown to affect mitochondrial function by regulating the expression of mitochondrial proteins encoded by the nuclear genes (21
). In this study, we provide the first evidence to our knowledge showing that MEF2D is present in neuronal mitochondria, where it binds to a discrete and well-conserved MEF2 consensus site within the coding region of mitochondrial gene ND6
to regulate its transcription, thereby directly modulating complex I activity and affecting a number of key mitochondrial functions and physiology. Thus, we believe MEF2D to qualify as a bona fide, novel mitochondrial transcription factor. Our findings broaden the cellular roles played by MEF2D and raise a series of interesting questions, such as how nuclear and mitochondrial MEF2 activities are coordinated; what signal pathways are involved specifically in modulating mitochondrial MEF2D; whether unique posttranslational modifications are required for regulation of MEF2D in mitochondria; and what interactions may exist between mitochondrial MEF2D and other components of mitochondrial transcriptional machinery. Our recent study showed that the autophagic pathway regulates the activity of nuclear MEF2D (29
); thus, it would be interesting to examine whether autophagy plays a similar role in controlling mitochondrial MEF2D activity. Although MEF2D levels in the whole cell are elevated in the brains of alpha-synuclein transgenic mice and PD patients (29
), its levels in the mitochondria are decreased in PD patients. Collectively, these data indicate that the decrease in MEF2D level in mitochondria is accompanied by a buildup of MEF2D in the cytoplasm. It is possible that reduced mitochondrial MEF2D may contribute to the overall increase of cytoplasmic MEF2D. But this effect should be small, since only a small fraction of MEF2D will normally go to mitochondria. Whether and how alpha-synuclein may affect mitochondrial MEF2D requires further investigation.
Our data suggest that MEF2D exclusively regulates expression of the ND6
gene without significant effects on several other protein-encoding mtDNA genes tested. The reason for this high degree of specification is not clear but quite intriguing. Since ND6
is the only protein-encoding gene present in the L strand of mtDNA, whereas the rest of the 13 protein-encoding mitochondrial genes all reside in the H strand (7
), this apparent specificity may be due, at least in part, to the unique organization of mtDNA. Indeed, cAMP response element–binding protein (CREB) has previously been shown to bind the D-loop, whereas p53 can also localize to mitochondria under stressful conditions. But none of them has been reported to affect L-strand transcription (30
). Insufficiency of ND6 protein is known to lead to severe disruption of complex I structure (6
). Therefore, maintaining adequate levels of ND6 is critical for the proper assembly of complex I (33
). Our data clearly showed that a reduction of MEF2D activity specifically in mitochondria resulted in substantial disorganization of complex I and subsequent loss of complex I activity without affecting other complexes. These findings highlight the critical and distinctive role of MEF2D in maintaining the function of complex I. Together with the reported mitochondrial localization by CREB and p53 (30
), these studies emphasize a previously underappreciated mechanism of interaction between nucleus and mitochondria. The selective degeneration of DA midbrain neurons in the substantia nigra (SN) is a hallmark of PD. DA neurons in the neighboring ventral tegmental area (VTA) are markedly less affected. The mechanisms for this differential vulnerability of DA neurons are unknown. Recent studies have identified several differences between them, including different transcriptional response to MPTP (34
), divergent electrophysiological features (35
), and selective activation of ATP-sensitive potassium channels (36
). It is therefore possible that SN and VTA DA neurons may have different sensitivity to mitochondrial malfunction induced by MEF2D defects.
Although the full biological significance of this added layer of regulation of ND6
gene expression by MEF2D requires further exploration, our studies offer multiple lines of evidence, including a cellular model, animal studies, and examination of human tissues, to implicate this regulatory mechanism in the pathogenic process of PD. Our results indicated that well-established toxins known to target complex I and induce parkinsonism in model systems also reduced MEF2D levels in mitochondria and disrupted MEF2D binding to the ND6
MEF2 site, offering an alternative mode of action by which these important toxins inhibit mitochondrial function. Because low-dose toxins preferentially reduce mitochondrial MEF2D without affecting its level in the nucleus, our data suggest that inhibition of the MEF2D-ND6 pathway could represent one of the earlier steps involved in pathologic changes at the subcellular level (37
). More importantly, the levels of both MEF2D and ND6 proteins were greatly reduced in brain mitochondria of both chronic MPTP-treated mice and human PD patients. These findings are consistent with the notion that reduced complex I activity secondary to toxic agent–induced inhibition of the MEF2D-ND6 pathway may contribute to the mitochondrial dysfunction and oxidative stress often observed in PD and possibly in other neurodegenerative diseases (3
). The molecular mechanisms by which complex I inhibitors disrupt MEF2D mitochondrial function are presently unclear. Since both MPP+ and rotenone directly bind complex I (38
), it is possible that these toxins may exert their effects on MEF2D via a mechanism involving complex I inhibition or disruption of mitochondrial redox balance.
A recent study found that mice with the Mef2d
gene conditionally deleted are viable and show no obvious phenotypic abnormalities (39
). However, under stress, these mice showed defects in cardiac remodeling. This is consistent with our data demonstrating that reduced MEF2D activity in mitochondria sensitized the cells to toxic stress. While our data have focused on the regulation of mitochondria by MEF2D in mediating PD-like injury, it is likely that mitochondrial MEF2D plays a role in other organ systems and disease processes as well.