Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons

Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.Mitochondria are essential organelles for cell function and viability, and are central to several processes such as energy production, metabolism, calcium buffering, and life/death decisions.¹ Neurons have a high and constant demand for mitochondrial metabolism, to maintain their functions, and contain many mitochondria throughout the cytoplasm, distributed to axons, presynaptic terminals, and dendritic shafts. Mitochondria are highly dynamic organelles that continuously move and change shape. Their morphology is governed by the dynamic equilibrium between fusion and fission processes, both of which are mediated by evolutionarily conserved members of the dynamin family of large GTPases.² Fusion between the outer mitochondrial membranes (OMMs) is mediated by membrane-anchored mitofusins (Mfn1 and Mfn2), whereas that between inner mitochondrial membranes is controlled by optic atrophy 1.³ Mitochondrial fission is regulated by dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1).⁴ Drp1 is predominantly expressed in the cytoplasm and is recruited to mitochondria, where it associates with Fis1 to form a complex that constricts the inner and outer membranes, allowing mitochondria to divide.^{5, 6}Mitochondrial dynamics are crucial to the maintenance of mitochondrial function and neuron survival, as evidenced by findings that pathological imbalances between fusion and fission events develop in many neurodegenerative disorders and brain trauma.^{7, 8} Moreover, mitochondrial fission regulates organelle shape and mediates mitochondria-dependent cell death.⁹ The release of proapoptotic factors, such as cytochrome c (with consequent formation of the apoptosome and caspase activation), from depolarized mitochondria into the cytosol is a significant event in the induction of apoptosis and is associated with Drp1-mediated fragmentation of the mitochondrial network.¹⁰The elimination of dysfunctional mitochondria is therefore a key process with regard to the viability of neurons (and other cell types). Damaged mitochondria that accelerate cell death are removed through autophagy, an evolutionarily conserved lysosome-mediated degradation pathway that maintains the balance between organelle biogenesis, protein synthesis, and degradation of cellular components.¹¹Mitochondria can be selectively degraded by autophagy – a pathway known as mitophagy.¹² Priming of damaged mitochondria can involve several mechanisms, one of which is triggered by Parkin, a cytosolic E3 ubiquitin ligase that is mutated in familial forms of Parkinson''s disease (PD).¹³ Parkin recruitment to impaired mitochondria requires the kinase activity of PINK1 (PTEN-induced putative kinase 1),^{14, 15, 16} a serine/threonine kinase that is also mutated in other autosomal recessive forms of PD.¹⁷ PINK1 levels are very low in polarized mitochondria, to prevent mitophagy of healthy mitochondria; in contrast, when mitochondria are depolarized, full-length PINK1 accumulates rapidly at damaged organelles, crossing the OMM and acting as cellular sensors of damaged mitochondria.¹⁵ PINK1 then recruits Parkin to the mitochondrial surface, where it ubiquitinates several OMM proteins, which in turn recruit other proteins to initiate mitophagy.¹⁸ Mfn1 is a direct substrate of Parkin and its degradation has been suggested to prevent the fusion of damaged and healthy mitochondria.¹⁹ Drp1-dependent fission of mitochondria is also a crucial event that effects their degradation through mitophagy and the inhibition of fission specifically prevents mitochondrial autophagy.²⁰In this study, we examined mitochondrial function and its relationship with autophagy machinery in an in vivo model of acute focal CNS (central nervous system) lesion, focusing on remote changes that are induced by hemicerebellectomy (HCb). Unilateral HCb is a suitable model in which axonal damage-induced neuronal death mechanisms can be studied.^{21, 22} In this model, neuronal degeneration is caused by target deprivation and axonal damage of contralateral precerebellar nuclei of the inferior olive and pontine nuclei. Remote damage is a multifactorial phenomenon in which many components become active in specific time frames²¹ and is significant in determining the overall clinical outcome in many CNS pathologies, including spinal cord injury and traumatic brain injury.^{23, 24, 25}Recently, we demonstrated that autophagy is activated in axotomized neurons after HCb, subsequent to cytochrome c release from mitochondria.²⁶ Further, rapamycin-enhanced autophagy has neuroprotective effects, reducing neuronal death and improving functional recovery. In this study, we examined mitochondrial dynamics and function in axotomized neurons after HCb in mice and analysed the effects of rapamycin on selective elimination of damaged mitochondria.