In this study we endeavored to gain new insight into the complexity and tissue specificity of mitochondrial-based diseases by studying the common human mtDNA mutation A1555G that has one primary outcome, deafness, and modeling its pathogenic mechanism in mice. Based on our results, we conclude that a major pathogenic driving force is cell death due to activation of the pro-apoptotic nuclear transcription factor E2F1 by a cell-type-specific mitochondrial stress pathway. Below we will discuss the results that led us to this conclusion and the potential broader implications of this study with regard to the documented environmental exacerbation of this form of deafness (Warchol, 2010
), age-related hearing loss (presbycusis), and other diseases where mitochondria are implicated in pathogenesis.
We propose that mitochondrial stress due the hyper-methylation of the mtDNA-encoded 12S rRNA is a critical component of the inner ear pathology associated with deafness caused by the human mtDNA A1555G mutation. This is perhaps best demonstrated by our results in the Tg-mtTFB1 transgenic mice, which exhibit 12S rRNA hyper-methylation in multiple tissues and activation of the pro-apoptotic transcription factor E2F1 that causes progressive hearing loss similar to that observed in human A1555G patients. Deafness is the primary effect of the A1555G mutation even though most patients are homoplasmic (i.e. have 100% mutant mtDNA) in all cells and tissues (Fischel-Ghodsian, 1999
; Prezant et al., 1993
). This implies a cell-type specific response to mitochondrial dysfunction is at play. Our results showing that up-regulation of E2F1 and increased cell death occurs in the stria vascularis and in the spiral ganglion neurons, but not in the organ of Corti, is consistent with activation of E2F1 by mitochondrial stress occurring only in certain critical cells in the inner ear and their subsequent apoptosis via this pathway.
E2F1-dependent cell death in the stria vascularis and spiral ganglion neurons is consistent with the pattern of hearing loss across all frequencies that we observe in the Tg-mtTFB1 mice. With regard to the irreversible nature of the deafness, neurodegeneration in spiral ganglion itself could itself be responsible, as has been proposed for age-related hearing loss (Someya et al., 2009
). Alternatively, or in addition, dysfunction of the stria vascularis and spiral ganglion neurons could ultimately result in a higher susceptibility of irreplaceable inner hair cells to undergo cell death (Fetoni et al., 2011
). Furthermore, since activation of the cell cycle in post-mitotic neuronal cells is an established trigger for apoptosis (Chen et al., 2003
; Hou et al., 2000
; Shadel, 2004a
) the mitochondrial stress pathway to E2F1 identified herein may also may make some cells of the inner ear inherently more susceptible to cell death due to being forced into the cell cycle, as opposed to activation the E2F1 apoptosis pathway per se. Based on these considerations, we propose that A1555G patients would likewise be more prone to eventual loss of critical inner ear cells via this mitochondrial stress pathway to E2F1, explaining their irreversible hearing loss either spontaneously, as a function of age, or in response to environmental stimuli such as noise or aminoglycosides. Even though our results strongly implicate 12S hyper-methylation per se in the deafness phenotype in mice, it is likely that defects in mitochondrial translation caused by the A1555G mutation, such as infidelity (Hobbie et al., 2008
), conspire with hyper-methylation to produce the precise deafness pathology in humans and/or mediate its exacerbation by aminoglycoside antibiotics. Therefore, it remains important to determine precisely which mechanisms and cell types within the inner ear instigate deafness pathology in this new mouse model and in human A1555G patients. A full understanding of these events may be therapeutic in this regard and may also help in advancing efforts to regenerate hearing function.
Our results in A1555G cybrids also provide key insight into the nature of the pathogenic mitochondrial stress-signaling pathway. Specifically, hyper-methylation of mitochondrial ribosomes, which are needed for translational and assembly of the respiratory chain (Bonawitz et al., 2006
), disrupts mitochondrial respiration in a manner that increases ROS production (). We propose that increased mitochondrial superoxide is sensed by AMPK (Emerling et al., 2009
; Quintero et al., 2006
), activation of which relays the stress signal to E2F1. Since E2F1 activity is associated with pro-apoptotic signaling and is necessary for the enhanced cell death on A1555G cybrids () and hearing loss in Tg-mtTFB1 mice (), we speculate ROS- and AMPK-dependent activation of E2F1 is the major mitochondrial stress-signaling pathway involved under these circumstances. Going forward it will be important to determine precisely how E2F1 is regulated by ROS and AMPK.
The marked tissue-specificity of E2F1 induction and, to a lesser degree, of AMPK activation (Figure S2A
), provides insight into the complex mechanisms underlying tissue-specificity of mitochondrial diseases. The requirement for multiple sequential steps to fully activate the mitochondrial ROS-AMPK-E2F1 apoptotic pathway provides a variety of opportunities for different tissues to suppress a pathogenic mechanism. For example, tissues with lower OXPHOS activity or better redox buffering could prevent initial activation of the pathogenic mechanism. Alternatively, in other tissues, AMPK may not respond to ROS, E2F1 may not be activated by AMPK, or E2F1 may not be a potent trigger of apoptosis, any of which would presumably prevent a pathogenic outcome. These observations may be generalizable, in that other pathogenic mitochondrial retrograde signaling pathways likely exist with their differential activation or readout in tissues contributing to the complex tissue specificity observed in mitochondrial diseases.
Finally, the induction of E2F1 in response to a mitochondrial malfunction may represent a paradigm in mitochondrial pathogenesis where the cause of the disease is not the immediate OXPHOS dysfunction, but instead the misinterpretation of resulting retrograde signals generated by mitochondria. The pivotal role of AMPK as a sensor of energy charge and now as a ROS-dependent regulator of E2F1 activity highlights the complexity of mitochondrial stress responses and the need for additional research to uncover other such pathways. Finally, it is tempting to speculate that aberrant mitochondrial stress signaling may also be of general significance in diseases and circumstances where mitochondrial dysfunction and apoptosis are implicated, such as heart disease, cancer, neurodegenerative diseases and aging.