Abstract
Neuronal differentiation involves extensive modification of biochemical and morphological properties to meet novel functional requirements. Reorganization of the mitochondrial network to match the higher energy demand plays a pivotal role in this process. Mechanisms of neuronal differentiation in response to nerve growth factor (NGF) have been largely characterized in terms of signaling, however, little is known about its impact on mitochondrial remodeling and metabolic function. In this work, we show that NGF-induced differentiation requires the activation of autophagy mediated by Atg9b and Ambra1, as it is disrupted by their genetic knockdown and by autophagy blockers. NGF differentiation involves the induction of P-AMPK and P-CaMK, and is prevented by their pharmacological inhibition. These molecular events correlate with modifications of energy and redox homeostasis, as determined by ATP and NADPH changes, higher oxygen consumption (OCR) and ROS production. Our data indicate that autophagy aims to clear out exhausted mitochondria, as determined by enhanced localization of p62 and Lysotracker-red to mitochondria. In addition, we newly demonstrate that NGF differentiation is accompanied by increased mitochondrial remodeling involving higher levels of fission (P-Drp1) and fusion proteins (Opa1 and Mfn2), as well as induction of Sirt3 and the transcription factors mtTFA and PPARγ, which regulate mitochondria biogenesis and metabolism to sustain increased mitochondrial mass, potential, and bioenergetics. Overall, our data indicate a new NGF-dependent mechanism involving mitophagy and extensive mitochondrial remodeling, which plays a key role in both neurogenesis and nerve regeneration.
Highlights
Cell differentiation is a complex process that requires modifications of biochemical and morphological properties to meet novel specialized functions
Timecourse studies showed that LC3-II levels do not change at short time-points, but start to increase in PC12-615 exposed to nerve growth factor (NGF) (10 ng/ml) for 12 h (Fig. 1c) and remain twofold higher than CTR for 24–120 h, showing a trend that correlates with their initial neurite extension and the post-mitotic state (Fig. 1c and Supplementary Fig. S1D, G)
We newly report that NGF-induced differentiation requires the activation of mitophagy through mechanisms that are dependent upon altered energy homeostasis and requirement of mitochondrial remodeling
Summary
Cell differentiation is a complex process that requires modifications of biochemical and morphological properties to meet novel specialized functions. Mitochondria play a crucial role during neurogenesis and in post-mitotic neurons by supplying the energy requested for growth cone activity, axonal growth, and synaptic function[11]. Several studies found that neuronal differentiation is accompanied by metabolic reprogramming to meet the increased energy demand. This is achieved by fostering glucose and glutamine metabolism[12,13], as well as the oxidative phosphorylation[14,15], leading to higher generation of ROS and the need to increase mitochondrial biogenesis[12] and quality control by mitophagy[13]
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