Mitochondria are ubiquitous intracellular organelles enclosed by a double membrane structure. The primary function of mitochondria is the production of cellular energy in the form of adenosine triphosphate (ATP). Mitochondrial shape is maintained by two opposing forces: fission and fusion (Chan 2006). In healthy neurons, fission and fusion balance equally; imbalances between these processes lead to abnormal mitochondrial function (Westermann 2002; Chan 2006). Fission and fusion are controlled by evolutionarily conserved, large GTPases belonging to the dynamin family.
Mitochondrial fission is controlled by dynamin-related protein 1 (Drp1) and the protein mitochondrial fission 1 (Fis1) (Knott et al. 2008). Although Drp1 mainly localizes within the cytoplasm, a small amount of Drp1 can translocate to the outer mitochondrial membrane where it promotes mitochondrial fragmentation (Smirnova et al. 2001). Fis1 is localized to the outer mitochondrial membrane (Yoon et al. 2003; Chang and Blackstone 2010). Drp1 and Fis1 are activated by increased free radical production in the mitochondria, which is critical for mitochondrial fission.
Mitochondrial fusion is also regulated by three other GTPase proteins: two outer-membrane-localized proteins, Mfn1 and Mfn2 (mitofusin 1 and 2), and the inner-membrane-localized protein Opa1 (optic atrophy 1) (Cerveny et al. 2007; Detmer and Chan 2007; Benard and Karbowski 2009). The C-terminus of Mfn2 mediates oligomerization between Mfn molecules from adjacent mitochondria and facilitates mitochondrial fusion (Ishihara et al. 2004; Zuchner et al. 2004).
Another important protein involved in mitochondrial dynamics is PTEN-induced novel kinase 1 (PINK1), which was originally reported to be associated with autosomal recessive parkinsonism (Hardy et al. 2006). Although debate still exists on the exact function of PINK1, recent studies have demonstrated the ability of PINK1 to alter the balance between mitochondrial fission and fusion (Exner et al. 2007; Yang et al. 2008; Dagda et al. 2009; Sandebring et al. 2009). Our previous research experience demonstrated that PINK1 exerts an overall fusion effect. Strategies impairing the function of Pink1, either by knocking down of endogenous Pink1 or over-expression of mutant forms of PINK1 induced severe mitochondrial fragmentation in N27 cells. By over-expressing dominant negative forms of Drp1 or mfn2 to inhibit fission or enhance fusion, we successfully reversed this morphological change (Cui et al. 2010).
The majority of literature on the influence of mitochondrial dynamics in pathologies of the central nervous system is associated with neuronal cell death in age-related neurodegenerative diseases. It is suggested that impaired regulation of mitochondrial dynamics towards fission would induce essential neuronal apoptosis (Otera and Mihara 2012). Although severe energy crisis occurs in the pathology of acute brain injury, such as ischemic stroke, support for the role of mitochondrial dynamics in these pathologies is strikingly rare. Only recently, evidence showed that mitochondrial fission may be associated with neuronal cell death induced by glutamate toxicity and oxygen–glucose deprivation (OGD), as inhibition of mitochondrial fission protected neurons from glutamate toxicity and ischemic damage (Grohm et al. 2012). Although, as mentioned above, the function of Pink1 in the regulation of mitochondrial dynamics is not fully characterized, Pink1 is mainly considered to be a neuroprotective protein. PINK1 over-expression confers resistance to staurosporine, MPP+, and rotenone toxicity (Petit et al. 2005; Haque et al. 2008; Sandebring et al. 2009). Based on our previous finding that Pink1 is a mitochondrial fusion protein, in this study we aimed to characterize the role of Pink1 in acute cerebral ischemia and its association with mitochondrial dynamics.
We demonstrated that Pink1 deficiency sensitizes primary rat neurons to ischemic damage in vitro. Over-expression of human PINK1 could prevent mitochondrial fission, bioenergetic defects, loss of mitochondrial membrane potential (MMP), and cell death induced by OGD. PINK1 blocked mitochondrial fission induced by ischemic damage, by preventing translocation of Drp1 from the cytosol to the mitochondria. Our results indicate that a classic Parkinson's disease gene can also exert an important protective function during the response of the brain to stroke, which is likely to be mediated through the capacity of PINK1 to regulate mitochondrial dynamics.
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Mitochondria are dynamic organelles that continually undergo the opposing processes of fusion and fission to maintain their distinct morphology. The balance between fission and fusion events regulates mitochondrial morphology. Mitochondrial shape corresponds to metabolic status (Rossignol et al. 2004) and the health of the cell (Youle and Karbowski 2005). Recently, several neurodegenerative diseases, including PD, have been linked to perturbations of mitochondrial dynamics (Han et al. 2011). Disruption of the PD-associated genes PINK1, Parkin, DJ-1 or LRRK2 results in mitochondrial defects and aberrant mitochondrial morphology (Greene et al. 2003; Clark et al. 2006; Park et al. 2006; Irrcher et al. 2010; Krebiehl et al. 2010; Wang et al. 2012). Although the importance of mitochondrial dynamics in neurodegenerative diseases has been widely addressed, the importance of mitochondrial dynamics in acute brain injuries, such as stroke, remains poorly understood. A recent study showed that inhibition of the mitochondrial fission protein Drp1 using small molecules (Mdivi compounds) prevented cell death after glutamate toxicity and OGD (Grohm et al. 2012), indicating that perturbations of mitochondrial dynamics may participate in OGD-mediated cell damage.
In this study, we provide evidence that the OGD/reoxygenation procedure induced excessive fission in primary cultured neurons. After OGD, the cells displayed severe mitochondrial fragmentation, with a dramatic collapse in MMP and reduced neuronal ATP levels. Moreover, we demonstrated that OGD dramatically increased the mitochondrial Drp1 level by recruiting Drp1 from the cytosol to the mitochondria; OGD had no significant influence on the expression of other fission/fusion proteins. Overall, these changes tipped the balance of the mitochondrial fission/fusion machinery towards excessive fission.
As a major energetic crisis in the central nervous system, stroke undoubtedly has tremendous impact on mitochondrial function. Although the precise role of mitochondrial dynamics in the pathology of stroke is not clear, there are clues from the DJ-1 study that mitochondrial fission may deteriorate stroke damage. Loss of DJ-1 increased the sensitivity to excitotoxicity and ischemia (Aleyasin et al. 2007). Recently, DJ-1 is implicated to be involved in the regulation of mitochondrial dynamics (Irrcher et al. 2010; Krebiehl et al. 2010; Thomas et al. 2011). Over-expression of various pathogenic DJ-1 mutants induces mitochondrial fragmentation in neurons. It has been demonstrated that PINK1 acts upstream of or in parallel to DJ-1 to maintain proper mitochondrial morphology and function (Thomas et al. 2011). Although controversy remains regarding the exact function of PINK1, several recent studies suggest that PINK1 may alter the balance between fusion and fission, according to the intimate interaction between Pink1 and DJ-1 (Cookson 2010; Thomas et al. 2011). Therefore, it is of great interest to explore the role of PINK1 in cerebral ischemia.
Using lentiviral mediated siRNA to knockdown endogenous Pink1, we demonstrated that loss of function in Pink1 increased the sensitivity of primary neurons to ischemic damage. In contrast, over-expression of wild-type PINK1 remarkably rescued neurons from OGD injury by improving mitochondrial morphology and bioenergetic production. These findings support the suggestion that PINK1 plays a neuroprotective role in ischemic stroke.
The exact mechanisms by which PINK1 protects neurons from ischemic damage remained unclear. To this end, we over-expressed human wild-type PINK1 and two recessive mutants (PINK1-W437X and PINK1-K219M) in primary cultured cortical neurons. Mutations that impair kinase activity (the PD-associated mutation W437X and the artificial kinase-dead construct K219M) failed to protect the neurons from ischemic damage, indicating that kinase activity might be required for the neuroprotective function of Pink1.
Mitochondrial morphology is determined by the balance between mitochondrial fission and fusion and it was linked to the maintenance of proper mitochondrial functions. There has been controversy over whether the PINK1 modulates mitochondrial fusion or fission. In Drosophila, PINK1 loss of function results in swelling or enlargement of mitochondria, and these defective phenotypes of the dPINK1-null flies are strongly suppressed by the over-expression of Drp1 (Deng et al. 2008; Poole et al. 2008; Yang et al. 2008; Park et al. 2009). However, PINK1 deficient mammalian cells have a fragmented and truncated mitochondrial morphology, which would suggest an imbalance towards fission (Exner et al. 2007; Cui et al. 2010). In our study, we found that the influence of PINK1 on mitochondrial morphology was consistent with changes in mitochondrial fission/fusion proteins after OGD. OGD led to the recruitment of a large amount of Drp1 from the cytosol to the mitochondria, and induced mitochondrial fragmentation and dysfunction. Over-expression of PINK1 inhibited the translocation of Drp1 and shifted mitochondrial fission back towards the normal balance between fusion and fission, thereby preventing neuronal dysfunction and death in response to OGD.
To further corroborate the mechanisms by which PINK1 exerts a neuroprotective effect under ischemic conditions, endogenous rat Pink1 was knocked down using siRNA. Not surprisingly, the opposite effects were detected. Thus, these results suggested that PINK1 may prevent neurons from ischemic damage through maintaining proper mitochondrial functions by attenuating Drp1 translocation and reversing the mitochondrial fission induced by ischemia. To confirm this hypothesis, we manipulated the fission/fusion process by inhibiting Drp1 activity using the small molecule Mdivi-1. Inhibition of the fission process attenuated the effects of Pink1 knockdown on neuronal cell death and ATP generation after OGD. These observations demonstrated the protective effects of PINK1 against OGD injury are mediated, at least in part, through the mitochondrial fission/fusion pathway. Because of the fact that PINK1 is normally considered an autosomal recessive gene in PD, this is a novel and significant finding as the involvement of PINK1 in the pathology of stroke has rarely been explored.
Taken together, our findings provide evidence to support a neuroprotective role of PINK1 in mammalian neuronal models of ischemic injury. Given the pathological changes which occur in the mitochondria after stroke, PINK1 may be a promising new target for the prevention of stroke damage.