Research ArticleThe neurogenic basic helix-loop-helix transcription factor NeuroD6 enhances mitochondrial biogenesis and bioenergetics to confer tolerance of neuronal PC12-NeuroD6 cells to the mitochondrial stressor rotenone
Highlights
► NeuroD6 induces mitochondrial biogenesis in neuroprogenitor-like cells. ► NeuroD6 augments the bioenergetic reserve of the neuronal PC12-NeuroD6 cells. ► NeuroD6 increases the mitochondrial membrane potential and ATP levels. ► NeuroD6 confers tolerance to rotenone via an adaptive mitochondrial response.
Introduction
Mitochondrial dysfunction critically affects the developing and mature central nervous system given its high-energy demand [1], [2]. Autism spectrum disorder (ASD) is the latest identified neurodevelopmental disorder with mitochondrial pertubations [3]. Developing neurons require high levels of ATP to fuel plasmalemmal biogenesis and cytoskeletal assembly associated with axonal and dendritic growth, processes that are estimated to consume approximately 50% of cellular ATP [4]. Since mitochondria produce the large majority of intracellular ATP through the oxidative phosphorylation system (OXPHOS), developing neurons are vitally dependent on robust mitochondrial biogenesis and bioenergetics. OXPHOS involves a series of redox reactions during which electrons are transferred through several multimeric respiratory enzyme complexes I, III, and IV, establishing a mitochondrial membrane potential (ΔΨm) due to proton gradient between the mitochondrial matrix and the inner mitochondrial membrane, with ATP being synthesized by complex V, also called ATP synthase upon return of protons to the mitochondrial matrix [5]. The OXPHOS capacity of mitochondria is under a coordinated bi-genomic regulation due to the limited coding capacity of mitochondrial DNA (mtDNA), which only encodes 13 out of 80 protein subunits composing the OXPHOS system, with the remainder being nuclear-encoded and imported into mitochondria [6]. In contrast, the protein machinery responsible for mitochondrial biogenesis, more precisely mtDNA replication, is solely nuclear-encoded [7]. The mitochondrial-specific transcription factor Tfam is an essential regulator for in vivo mitochondrial biogenesis, as reflected by the early embryonic lethality of Tfam null mice and a limiting determinant of mtDNA copy number [8], [9]. Moreover, decreased Tfam expression levels in neurons of mutant mice result in mitochondrial respiratory chain defects [10], while mtDNA depletion in humans results in severe mitochondrial diseases, such as mtDNA depletion syndrome [11].
Although major progress has been made toward elucidating the transcriptional network regulating mitochondrial biogenesis and bioenergetics via the ubiquitous transcriptional factors NRF-1-NRF-2 and the co-activator PGC-1 [12], little is known about the identity of neuronal-specific transcriptional factors tailoring mitochondrial functions to the onset of neuronal differentiation. Our recent studies have addressed this gap in our knowledge by demonstrating a correlation between mitochondrial mass and expression of the neurogenic basic helix-loop-helix (bHLH) transcription factor NeuroD6 during the early stages of neuronal differentiation [13]. Furthermore, our gene set enrichment analysis of our genome-wide microarray studies has revealed a link between NeuroD6 and a cluster of mitochondrial bioenergetic-related genes [14]. Finally, NeuroD6 sustained the mitochondrial biomass and low levels of ROS during oxidative stress [15]. Thus, the goal of the present study was to determine whether NeuroD6 could coordinate mitochondrial biogenesis and bioenergetics with the onset of neuronal differentiation. This role would be in concordance with NeuroD6 embryonic expression being triggered at E11.5, a time when neuronal progenitor cells undergo cell cycle withdrawal and initiate neuronal differentiation [16], [17]. We found that NeuroD6 mediates mitochondrial biogenesis by concomitantly increasing mtDNA copy number and Tfam expression levels. Furthermore, NeuroD6 promotes mitochondrial bioenergetic functions by increasing the expression of key subunits of the respiratory complexes, the mitochondrial membrane potential and ATP levels, thereby generating an energetic reserve. Finally, NeuroD6 endows the PC12-NEUROD6 cells with tolerance to the mitochondrial stressor rotenone, an inhibitor of the respiratory complex I (NADH: nicotinamide adenine dinucleotide ubiquinone oxidoreductase) by utilizing this increased basal energetic capacity, thus preventing a deleterious mitochondrial bioenergetic deficit and subsequent cell death.
Section snippets
Cell culture
Control PC12 and PC12-NEUROD6 cells (previously called PC12-Nex1) were generated as described [18] and grown in the presence of F12K medium (Invitrogen) containing 15% horse serum (Invitrogen), 2.5% fetal bovine serum (Invitrogen). Since the three generated PC12-NEUROD6 clones (PC12-Nex1-M A, B and C) displayed similar response upon NGF exposure and withdrawal of trophic factors [18], [19], [20], we used the PC12-NEUROD6 clone A to remain consistent with our previous studies pertaining to
NeuroD6 expression Is associated with enhanced mitochondrial biogenesis
On the basis of the correlation between NeuroD6 expression and mitochondrial mass established in our previous studies [13], we investigated whether NeuroD6 could modulate the mitochondrial biomass by enhancing mitochondrial biogenesis. To address this question, we took advantage of our neuronal paradigm, the PC12-NeuroD6 cell line, which constitutively expresses NeuroD6 and spontaneously recapitulates the first three stages of neuronal differentiation with 44% of PC12-NeuroD6 cells being at
Discussion
This study documents for the first time a neurogenic modulation of mitochondrial biogenesis and bioenergetics by the bHLH differentiation factor NeuroD6, which induces neuronal differentiation and cell cycle exit of neuronal progenitor cells during brain development [16], [17]. We report three novel roles for NeuroD6: first, it promotes mitochondrial biogenesis during the early stages of neuronal differentiation. Second, NeuroD6 enhances the bioenergetic capacity of the neuronal-like
Conclusions
In summary, our findings demonstrate two novel roles for NeuroD6 in a neuronal-like context: (1) it stimulates mitochondrial biogenesis by concomitantly increasing mtDNA content and the key mitochondrial-specific regulator TFAM; and (2) it enhances mitochondrial bioenergetic functions by increasing expression levels of specific subunits of the respiratory complexes of the electron transfer chain, resulting in augmented mitochondrial membrane potential and levels of ATP produced by oxidative
Acknowledgments
We thank Dr. Anastas Popratiloff for his expertise in confocal microscopy and quantification analysis and Dr. Thomas Maynard for his expertise in qPCR. The work was supported in part by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (Grant Number R01-NS041391 to A.C.), the Medical Center Facilitating Funds to A.C., by the National Institute of Child Health and Development (P30HD40677), and by the National Center for Research Resources (S10RR025565).
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