The regulation of mitochondrial morphology: Intricate mechanisms and dynamic machinery

Cellular Signalling 23 (2011) 1534–1545

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

Review

The regulation of mitochondrial morphology: Intricate mechanisms and dynamic machinery Catherine S. Palmer a, b, Laura D. Osellame c, d, Diana Stojanovski a, b, Michael T. Ryan a, b,⁎ a

La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne 3086, Australia Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, United Kingdom d UK Parkinson's Disease Consortium, Institute of Neurology University College London, London, WC1N 3BG, United Kingdom b c

a r t i c l e

i n f o

Article history: Received 3 May 2011 Accepted 31 May 2011 Available online 13 June 2011 Keywords: Mitochondria Morphology Fission Fusion Apoptosis Neurodegeneration

a b s t r a c t Mitochondria typically form a reticular network radiating from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites. These mitochondrial networks are regulated through the complex coordination of fission, fusion and distribution events. While a number of key mitochondrial morphology proteins have been identified, the precise mechanisms which govern their activity remain elusive. Moreover, post translational modifications including ubiquitination, phosphorylation and sumoylation of the core machinery are thought to regulate both fusion and division of the network. These proteins can undergo several different modifications depending on cellular signals, environment and energetic demands of the cell. Proteins involved in mitochondrial morphology may also have dual roles in both dynamics and apoptosis, with regulation of these proteins under tight control of the cell to ensure correct function. The absolute reliance of the cell on a functional mitochondrial network is highlighted in neurons, which are particularly vulnerable to any changes in organelle dynamics due to their unique biochemical requirements. Recent evidence suggests that defects in the shape or distribution of mitochondria correlate with the progression of neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's disease. This review focuses on our current understanding of the mitochondrial morphology machinery in cell homeostasis, apoptosis and neurodegeneration, and the post translational modifications that regulate these processes. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . Mitochondrial morphology . . . . . . . . . . . . . 2.1. Mitochondrial fusion machinery . . . . . . . 2.2. Regulation of mitochondrial fusion . . . . . . 2.3. Mitochondrial fission . . . . . . . . . . . . . 2.4. Regulation of mitochondrial fission . . . . . . 3. Mitochondrial morphology and apoptosis . . . . . . 4. Mitochondrial morphology and neurodegeneration . . 4.1. Mitochondrial shape and movement in neurons 4.2. Alzheimer's disease . . . . . . . . . . . . . 4.3. Huntington's disease . . . . . . . . . . . . . 4.4. Parkinson's disease. . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia. Tel.: + 61394792156. E-mail address: [email protected] (M.T. Ryan). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.05.021

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1. Introduction Mitochondria are essential compartments of eukaryotic cells. They supply the cell with ATP through oxidative phosphorylation, synthesise key molecules and buffer calcium gradients. To ensure efficiency of operation and subsequent health of the cell mitochondria must be maintained in optimal condition. As part of this process, mitochondria can be highly dynamic organelles which fuse and divide in response to environmental stimuli, developmental status, and energy requirements of the cell [1–4]. In most cells, mitochondria form a reticular network that radiates from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites [5,6]. However in specialised cells, mitochondrial morphology can be very different. For example, terminally differentiated sperm cells contain a set of fused mitochondria that are wrapped around the midpiece of the flagella where they provide ATP for movement [7,8]. While this represents an extreme example, the differentiating progenitor spermatogonia nevertheless requires a set of molecules to rearrange the mitochondrial network into such a configuration. Other adaptations of the mitochondrial network include the tubular organisation of mitochondria in myotubes, and rearrangements in immune signalling as well as fragmentation of the network during mitosis and apoptosis [9–11]. Variations in mitochondrial morphology are primarily a flux between two extreme states: a reticular network of fused mitochondria and a fragmented arrangement (Fig. 1) [12]. These events are controlled by the regulation of proteins involved in fission and fusion events, and the maintenance of mitochondrial distribution [5,6]. This balance of opposing events affects the inheritance of mitochondrial DNA, transmission of energy, cellular differentiation, metabolite maintenance and calcium homeostasis [13–16]. Mutations in genes encoding proteins directly involved in mitochondrial morphology have been identified in a number of neurodegenerative disorders [17–20]; while a number of other proteins that regulate mitochondrial dynamics are implicated in neurological disorders including Huntington's, Alzheimer's and Parkinson's disease [21–24]. The presence of additional proteins that regulate mitochondrial dynamics in higher eukaryotes has highlighted the complexity of this process. This review will focus on the machinery that governs mitochondrial morphology, and the regulation of these dynamic processes in apoptosis and disease. 2. Mitochondrial morphology 2.1. Mitochondrial fusion machinery Mitochondrial fusion provides a mechanism by which the organelle population is maintained homogeneously and facilitates inter-complementation of mitochondrial DNA [13]. Mitochondria also fuse as part of a cellular stress response [25]. This process requires the

GFP-Fis1

fission

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co-ordination of both inner and outer mitochondrial membrane fusion, ensuring that compartmentalisation is maintained [26–29]. The key proteins involved in mitochondrial fusion are the outer membrane GTPases Mitofusins (Mfn1 and Mfn2, Fzo1 in yeast) [7,30–33] and the inner membrane GTPase Optic atrophy 1 (OPA1, Mgm1 in yeast) [29,34–36] that co-ordinate double membrane fusion with the activity of additional scaffolding proteins (Fig. 2, Table 1). The Mitofusins are essential for mitochondrial fusion, with loss of Mitofusin function causing the mitochondrial network to fragment [13,32]. Mitofusins span the mitochondrial outer membrane twice leading to both a C-terminal coiled-coil domain and an N-terminal GTPase domain being exposed to the cytosol [37,38]. Mitofusins have been suggested to facilitate membrane tethering similar to the action of SNAREs, with both homo and hetero-oligomeric complexes of Mfn1 and Mfn2 formed via the interaction of coiled-coil domains in a GTP dependent manner (Fig. 2A) [13,33,39,40]. While both Mfn1 and Mfn2 are capable of facilitating mitochondrial outer membrane fusion, a growing body of evidence suggests that they share both complimentary and disparate roles in mitochondrial fusion. Mutations in the gene encoding Mfn2 have been shown to cause Charcot–Marie–Tooth disease type 2A (CMT2A), an autosomal dominant neuropathy [41]. In contrast, no Mfn1 deficient patient has been reported to date. Mfn1 and Mfn2 knockout mouse models exhibit embryonic lethality, with Mfn2 knockout resulting in the appearance of defective giant placental cells while Mfn1 null giant placental cells are normal [32]. Conditional knockout mice specifically lacking Mfn2 in the cerebellum exhibit a reduction in dendritic outgrowth and spine formation in Purkinje cells, and decreased cell survival [19]. Mfn1 conditional knockout mice show no significant defect [19]. In addition, mitochondrial fragmentation observed following loss of either Mfn1 or Mfn2 is morphologically distinct [32]. These results suggest differing roles for these proteins in mitochondrial fusion. Indeed, Mfn1 and Mfn2 have different tethering capabilities, with Mfn1 exhibiting higher GTP dependent tethering activity than Mfn2 [33]. Furthermore, Mfn2 is enriched at the endoplasmic reticulum (ER)-mitochondrial interface, and silencing of Mfn2 affects both mitochondrial and ER morphologies [42,43]. The wealth of information gained from these studies has provided new insight into the differing roles of the Mitofusins in mitochondrial fusion; however additional proteins are required to complete fusion of the double membrane. Optical Atrophy 1 (OPA1, Mgm1 in yeast) is a dynamin-like protein associated with the inner mitochondrial membrane and is involved in the maintenance of mitochondrial cristae remodelling and inner membrane fusion (Fig. 2B) [35,36,44]. Mutations in the OPA1 gene have been found to cause autosomal dominant optic atrophy (ODOA) [45–47]. OPA1 is processed into 8 different isoforms and changes in the balance between long (L) and short (S) isoforms affect the fusion of mitochondria [35]. Increased processing of OPA1 leading to greater levels of soluble S-OPA1 isoforms occurs following loss of the

mito-GFP

wildtype

MiD49/51

fusion

Fig. 1. The balance of fission and fusion regulates mitochondrial morphology. Mitochondria typically form a reticular network that is maintained by the co-ordination of fission and fusion machinery. Overexpression of pro-fission mediator GFP-Fis1 in COS7 cells causes mitochondrial fragmentation (left) compared to wild type COS7 controls transfected with matrix targeted GFP (middle). Loss of the pro-fission mediators MiD49 and MiD51 results in dysregulation of mitochondrial fission and unbiased fusion (right). These COS7 cells were co-transfected with matrix-GFP to visualise the mitochondrial network. The balance between a fragmented (left) and fused (right) state forms the wild type reticular network (middle). Scale bar, 20 μm.

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A Bak/Bax Mfn1/2

low GTP

PLD MIB

Outer membrane fusion

Cardiolipin

B Prohibitin S-OPA1

Inner membrane fusion L-OPA1

high GTP

SLP-2

Fig. 2. Co-ordination of mammalian mitochondrial fusion. (A) Outer membrane fusion is mediated by Mfn1 and Mfn2. Mfn1/2 homo and heterodimers assemble in trans and form an organelle tethering complex. Mitofusin binding protein (MIB) negatively regulates mitochondrial fusion by interacting with Mfn1/2. Mitochondrial phospholipase D (mitoPLD) facilitates fusion by hydrolysing cardiolipin to produce phosphatidic acid and subsequently alter membrane curvature. Bax/Bak affects fusion through interaction with Mfn1/2 in response to cellular signals. (B) Inner membrane fusion is governed by OPA1. In the presence of GTP, the OPA1 isoforms oligomerise resulting in fusion of the inner membrane. The balance of the OPA1 isoforms affects membrane stability and remodelling at the inner membrane. OPA1 is stabilised by the scaffold proteins Prohibitin 2 and SLP-2.

mitochondrial membrane potential and/or the induction of apoptosis, resulting in extensive mitochondrial fragmentation and disruption of cristae [35,48,49]. This fragmentation can be rescued by expression of L-OPA1 isoforms [48,49]. Additional components identified in mitochondrial fusion include Prohibitin 2 and stomatin like protein-2 (SLP-2) (Fig. 2B). These proteins have both been proposed to act as scaffolds for OPA1 at the inner mitochondrial membrane. Prohibitin 2 is required for cellular homeostasis and SLP-2 provides protection of mitochondria during cellular stress [25,50]. Deletion of Prohibitin 2 in cells leads to loss of L-OPA1 isoforms and aberrant mitochondrial cristae morphology, which can be rescued by reintroducing L-OPA1, suggesting that Prohibitin 2 is required for stability or maintenance of L-OPA1 [50]. Loss of SLP-2 results in the cleavage of L-OPA1 isoforms and stress induced mitochondrial hyperfusion is blocked, however mitochondrial morphology under normal conditions is not altered [25,51]. SLP-2 has been shown to interact with the prohibitins [52] and Mfn2 [51] and may directly co-ordinate fusion of the inner and outer membranes following stress stimuli. In yeast, the outer mitochondrial membrane protein Ugo1 has been identified to have a role in mitochondrial fusion, with a proposed role in stabilising Fzo1 and Mgm1 to facilitate lipid mixing following mitochondrial tethering [53,54]. No mammalian homologue of Ugo1

has been identified, however given that both OPA1 and Mfn1 are required to facilitate fusion of the mitochondrial double membrane [36]; it is feasible that a functional homologue of Ugo1 mediates the interaction between these two proteins in mammalian cells. 2.2. Regulation of mitochondrial fusion Mitochondria may undergo complete or transient fusion. Transient membrane fusion is followed by a corresponding fission event (termed “kiss-and-run”) [55]. Such events occur with mitochondria tethered to adjacent cytoskeletal tracks and appear important for bioenergetic maintenance [55]. Both forms of mitochondrial fusion require co-ordinated regulation of the fusion machinery. Regulation occurs primarily through the targeted degradation of the core fusion machinery or by proteolytic cleavage events that change activity (Fig. 4). In addition, accessory proteins that modulate the activity of mitochondrial fusion proteins have also been identified (Table 2). Mitofusin binding protein (MIB) is a member of the medium-chain dehydrogenase/reductase family and is mainly found in the cytosol with a small pool at mitochondria [56]. MIB is proposed to negatively regulate Mfn1 activity, with exogenous expression of MIB resulting in extensive mitochondrial fragmentation while knockdown causes mitochondrial

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Table 1 Key proteins of mitochondrial morphology and their post-translational modifications. Protein

Proposed function

Post-translational modification

Reference

Outer membrane fusion Inner membrane fusion Yeast outer membrane fusion Inner membrane scaffold for OPA1 Inner membrane OPA1 scaffold during stress induced hyperfusion

Ubiquitination Isoform balance through protease digestion

[7,26,30–33,37,38,69] [29,34–36,44,47] [53,54] [50] [25]

Mitochondrial fission Fis1 Drp1/Dnm1

Outer membrane receptor for Dnm1/Drp1 Outer membrane fission

Ubiquitination SUMOylation, Ubiquitination, phosphorylation, S-nitrosylation

[78,79,97,101] [79–81,88,103,111–114,116,126]

Mdv1/Caf4 Mff MiD49 and MiD51 MTP18 Endophilin B1 GDAP1

Binds to Fis1 modulating Dnm1 assembly in yeast Recruits Drp1 to mitochondria Recruits Drp1 to mitochondria Inner membrane fission Outer membrane fission Outer membrane fission

Mitochondrial fusion Mfn1 and Mfn2/Fzo1 OPA1/Mgm1 Ugo1 Prohibitin 2 SLP-2

[91,95,98,100] [93,109] [94,130] [104,105] [108] [107]

Phosphorylation PI-3 kinase signalling target

Abbreviations: Mfn, Mitofusin; Fzo1, Fuzzy onion-1; OPA1, Optic atrophy protein 1; SLP-2, stomatin like protein 2; Fis1, mitochondrial fission 1; Drp1/Dnm1, dynamin related protein 1; Mff, Mitochondrial fission factor; MiD49/51, Mitochondrial Dynamics 49 kDa/51 kDa; MTP18, Mitochondrial protein 18 kDa; PI, phosphatidylinositol; GDAP1, Ganglioside-induced differentiation-associated protein 1.

fusion [56]. A direct interaction between Mfn1/2 and MIB has been demonstrated through co-immunoprecipitation studies; however how MIB might regulate Mfn1/2 activity has yet to be determined [56]. Mitochondria have an intimate role in apoptosis, with extensive mitochondrial fragmentation and outer membrane permeabilization being defined steps in the apoptotic signalling cascade. Recent studies have revealed that pro-apoptotic proteins directly influence mitochondrial morphology. Two such proteins are the pro-apoptotic Bcl-2 family members Bax and Bak. While Bax/Bak-induced mitochondrial fission has been extensively described as a precursor to apoptosis, morphological changes to the mitochondrial network are not crucial for cytochrome c release and apoptosis, suggesting that these events are separate [57]. Bak is known to associate with mitochondrial outer membrane proteins to form defined complexes [58] and has been found associated with the mitochondrial fusion mediators Mfn1/2 [59]. It has

been proposed that following apoptotic induction, Bak dissociates from Mfn2 and exhibits enhanced association with Mfn1 [59]. Apoptotically inactive Bax has also been shown to stimulate mitochondrial fusion through interaction with Mfn2 [60,61]. In the absence of Bax/Bak, Mfn2 distribution is altered and no longer forms fusion-competent foci on the mitochondrial outer membrane. This suggests that Bax/Bak expression affects the membrane mobility of Mfn2 [61]. Taken together these data propose a role for Bax/Bak in regulation of mitochondrial fusion in both healthy and apoptotic cells (Fig. 2A). The balance of OPA1 isoforms is regulated by the activity of mitochondrial proteases that target OPA1 at specific cleavage sites upon dissipation of membrane potential, mitochondrial DNA loss or induction of apoptosis [35,62,63]. A number of different proteases have been identified to process OPA1 at different sites, regulating the balance of L- and S-OPA1 isoforms. OPA1 is cleaved by the mitochondrial

Table 2 Proteins that regulate mitochondrial fission and fusion and their molecular targets. Protein

Proposed function

Molecular target

Reference

MIB mitoPLD Lipin1b OMA1 PARL/Pcp1 Yme1 m-AAA protease CaMKIα Calcineurin CDK1/cyclin B PKA MARCH5/MITOL Parkin Mdm30p SENP-5 MAPL Ubc9 Bax Bak Bcl-xL

Negatively regulates Mfn1 activity lipid hydrolysis/PA production DAG production Cleaves OPA1 at S1 under mitochondrial stress Cleaves OPA1 or Mgm1 Cleaves OPA1 isoforms at S2 Cleaves OPA1 Ca2+ dependent phosphorylation Ca2+ dependent dephosphorylation Mitotic phosphorylation cAMP dependent phosphorylation Ubiquitin ligase Ubiquitin ligase Ubiquitin ligase SUMO protease SUMO ligase SUMO ligase Promotes Drp1 SUMOylation during apoptosis, regulates Mfn2 Regulates Mfn1/2 during apoptosis Stimulates Drp1 GTP hydrolysis

Mfn1 Cardiolipin/PC PA OPA1 OPA1/Mgm1 OPA1 OPA1 Drp1 Drp1 Drp1 Drp1 Drp1, Mfn1/2, Fis1 Drp1, Mfn1/2, Fis1 Fzo1 Drp1 Drp1 Drp1 Drp1, Mfn2 Mfn1/2 Drp1

[56] [72,73] [73] [63] [64,65] [49,62] [35] [114] [111] [116] [112,118] [115,122–124] [21,23] [66] [117] [127–129] [125] [60,103] [59] [158]

Abbreviations: Mfn, Mitofusin; OPA1, optic atrophy protein 1; MIB, Mitofusin binding protein; PC, phosphatidylcholine; PA, phosphatidic acid; DAG, diacylglycerol; PARL, presenilinassociated rhomboid-like; CaMKIα, Ca2+/calmodulin-dependent kinase 1α; PKA, cAMP, cyclic AMP; Fzo1, Fuzzy onion-1; Fis1, mitochondrial fission 1; Drp1/Dnm1, dynamin related protein 1; SENP5, sentrin-specific protease 5; MAPL, mitochondrial-anchored protein ligase; SUMO, small ubiquitin-like modifier.

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protease presenilin associated rhomboid-like protease (PARL, Pcp1 in yeast) [64,65]. Following loss of PARL the levels of soluble S-OPA1 isoforms are reduced and mitochondrial cristae become less uniform with wider junction openings that may accelerate cytochrome c release and subsequently increase the cell's susceptibility to undergo apoptosis [64]. Maintenance of OPA1 isoforms also depends on the matrix localised m-AAA proteases AFG3L1 and −2 and paraplegin, and subsequent processing by the peptidase OMA1 following cellular stress stimuli [35,63]. Cleavage of OPA1 is also mediated by the intermembrane space protease Yme1 [49,62]. Regulation of this cleavage of OPA1 by different proteases depends on access to different process sites and cellular stress stimuli. Mdm30 is an F-box motif protein in yeast that serves as a substrate recognition element for Fzo1 [66]. Mdm30 regulates mitochondrial fusion in yeast through targeted ubiquitination of Fzo1, and subsequent degradation by the proteasome [66]. No mammalian homologue of Mdm30 has currently been described, however recent studies have identified Mfn1 and Mfn2 as rapid targets for degradation by the E3 ubiquitin ligase, Parkin [21]. Parkin promotes mitophagy through ubiquitination of mitochondrial proteins [67,68]. Dysfunction of Parkin is a major cause of familial Parkinson's disease, and the ensuing accumulation of damaged mitochondria by defective mitophagy may lead to increased cell death [67,68]. Ubiquitination of Mfn1 and Mfn2 by Parkin may target mitochondria for mitophagy, with targeted degradation of Mfn1/2 segregating damaged mitochondria by blocking mitochondrial fusion [21,69]. Fusion of the mitochondrial double membrane requires not only the tethering activity of Mfn1/2, but also significant remodelling of membrane lipids. Membrane lipid composition can affect the efficiency of fusion, through the formation of membrane microdomains that facilitate protein binding and membrane curvature [70,71]. Mitochondrial PLD (MitoPLD) is a member of the phospholipase D (PLD) family of lipid modifying proteins [72]. MitoPLD promotes mitochondrial fusion through hydrolysis of the lipid cardiolipin (CL) on the outer mitochondrial membrane, resulting in the formation of phosphatidic acid (PA) which facilitates Mfn-mediated fusion [72]. An increase in PA levels leads to a negative-feedback mechanism that involves recruitment of the phosphatase Lipin 1b to mitochondria, conversion of PA to diacylglycerol (DAG) and stimulation of mitochondrial fission [73]. While the signalling of mitochondrial fission with the production of DAG immediately following the stimulation of mitochondrial fusion seems contrary, the activity of MitoPLD may be crucial in the membrane remodelling events that occur during transient membrane fusion [73]. The rapid regulation of mitochondrial morphology during transient membrane fusion utilises the activity of both pro-fusion and pro-fission machineries, resulting in the ‘kiss and run’ phenotype [55]. MitoPLD activity also describes a specialised role for mitochondria in the generation of the lipid signalling molecule PA in germ cells, and the formation of the inter-mitochondrial cement called nuage [73,74]. Loss of MitoPLD in mice results in male infertility due to meiotic arrest during spermatogenesis suggesting a highly specialised role for mitochondrial derived lipid signalling [73,74].

2.3. Mitochondrial fission Mitochondria cannot be created de novo and therefore mitochondrial growth and division are essential for proliferation, and ensure that a full complement of mitochondria is inherited by daughter cells following mitosis [75]. Mitochondrial fission is also required to help clear old or damaged mitochondria from the cell through an autophagic process referred to as mitophagy [76]. Increased or unregulated mitochondrial fission can cause a heterogeneous population of organelles with nonuniform mitochondrial DNA distribution, varied capacity to generate ATP, increased generation of reactive oxygen species and increased susceptibility of cells to undergo apoptosis [77,78].

A key protein involved in scission of mitochondria is Dynamin related protein 1 (Drp1, Dnm1 in yeast) [79–82]. Recent research into the physiological importance of mitochondrial fission has highlighted the crucial role Drp1 plays in both the maintenance of mitochondrial morphology and mitochondrial dynamics [83,84]. A patient with a dominant negative Drp1 allele displayed a broad range of abnormalities including poor brain development, optic atrophy and died at 37 days [85]. The loss of Drp1 in a mouse knockout model results in embryonic lethality, with embryos showing poorly developed liver and cardiac structures, increased apoptosis within the deep neural cortex, and compromised synapse formation [83,84]. These results suggest a role for Drp1 and hence mitochondrial fission in embryonic and neural development. Drp1 belongs to the dynamin family of GTPases which are typically involved in polymerising and constricting membranes leading to scission. Like dynamins, Drp1 is found predominantly in the cytosol and shares structural features common to the dynamin family including a GTPase domain, a GTPase effector domain (GED) and a central domain [79–81,86]. Drp1 assembles at sites of mitochondrial fission into multimeric ring complexes to facilitate scission of the double membrane [87–90]. These rings subsequently constrict following GTP hydrolysis to promote division [91,92]. Unlike dynamin-1, Drp1 lacks a pleckstrin-homology domain that is implicated membrane binding [86]. Instead, membrane receptor proteins are required for the recruitment and assembly of Drp1 at the mitochondrial outer membrane (Fig. 3) [79,91,93–95]. In yeast, Dnm1 recruitment from the cytosol occurs through association with the outer membrane protein Fis1, resulting in the formation of a fission complex and the scission of the dual mitochondrial membranes [79,88,96–98]. This interaction occurs with the addition of the adaptor protein Mdv1, which has a role in co-assembly of Dnm1 into helical structures [91,95]. Fis1 contains four tetratrico-peptide repeats (TPR) that are crucial for interaction with Mdv1, which in turn facilitate Dnm1 binding and assembly [99]. While Mdv1 interacts with Dnm1 and Fis1 to facilitate membrane fission, its function appears to be confined to yeast [98,100]. In mammalian cells a stable interaction between mammalian Drp1 and Fis1 remains controversial. Overexpression of Fis1 initiates mitochondrial fragmentation, while knockdown of Fis1 results in the formation of a highly fused mitochondrial network indicating that fission becomes blocked while fusion events are unopposed [79,97,98,101]. Recently however, targeted knockdown of Fis1 in mammalian cells was found to have little if any effect on mitochondrial morphology, and it was proposed that previous observations made through RNAi studies were due to off-target effects [93]. This conclusion is supported by the finding that Drp1 can still be recruited to the mitochondrial outer membrane following knockdown of Fis1 [102,103], suggesting that other proteins are involved in mammalian mitochondrial fission. Indeed, a number of new proteins have been recently identified with roles in mitochondrial fission particularly in higher eukaryotes (Table 1, Fig. 3), where cells can be highly differentiated and the mitochondrial networks must respond to their specific requirements. Mitochondrial protein 18 kDa (MTP18) was initially identified as a downstream target of phosphatidylinositol 3-kinase activation [104,105]. MTP18 is located at the inner mitochondrial membrane with overexpression of the protein in cultured cells resulting in highly fragmented mitochondria, and its knockdown resulting in mitochondrial fusion and cytochrome c release [104]. MTP18 induced mitochondrial fragmentation is dependent on Drp1 expression, suggesting a role for this protein in Drp1 mediated mitochondrial morphology [104]. Mutations in Ganglioside-induced differentiation-associated protein 1 (GDAP1) result in axonal, demyelinating, and intermediate forms of the peripheral motor and sensory neuropathy Charcot–Marie–Tooth (CMT) disease [106]. GDAP1 is located in the outer mitochondrial membrane with overexpression causing fragmentation of mitochondria, and knockdown resulting in fusion of the mitochondrial network [107]. Endophilin B1 is proposed to act downstream of Drp1 activity during

C.S. Palmer et al. / Cellular Signalling 23 (2011) 1534–1545

Endophilin 1B

1539

Drp1

MiD

Mff

+ GTP MTP18

complex formation

Lipin1b

scission

Fis1 GDAP1

Fig. 3. Mammalian mitochondrial fission. Mitochondrial division occurs when the cytosolic Drp1 is recruited to the mitochondrial outer membrane resulting in organelle constriction. Fis1, Mitochondrial fission factor (Mff) and Mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51) have been proposed to act in recruitment and assembly of Drp1 at the outer mitochondrial membrane. In the presence of GTP, Drp1 forms concentric rings around the scission site. GTP hydrolysis causes constriction of the Drp1 rings and facilitates scission of the mitochondrion. Other proteins involved in mitochondrial fission include Endophilin B1 that is proposed to modulate membrane curvature, and Lipin 1b that converts phosphatidic acid to diacylglycerol (DAG). Intermembrane space MTP18 and outer membrane GDAP1 also have proposed roles in mitochondrial fission.

mitochondrial fission through lipid remodelling of the outer mitochondrial membrane [108]. Endophilin B1 is primarily cytosolic, with a portion associating with mitochondria similar to Drp1 distribution [108]. The mitochondrial pool of Endophilin B1 increases following induction of apoptosis, suggesting that Endophilin B1 activity is increased during apoptotic remodelling of the mitochondrial outer membrane [108]. Proteins with proposed roles in recruiting Drp1 to mitochondria are Mitochondrial fission factor (Mff) [93,109] and Mitochondrial Dynamics proteins of 49 and 51 kDa (MiD49 and MiD51) [94]. Neither Mff nor the MiD proteins are found in yeast, and represent new components of the mammalian mitochondrial fission machinery. Mff is located in the outer mitochondrial membrane and is proposed to have a role in mitochondrial fission independent of Fis1 [93]. Overexpression of Mff results in mitochondrial fission and translocation of the cytosolic pool of Drp1 to mitochondria. Conversely, Mff knockdown by RNAi results in elongation of mitochondria and a reduction in Drp1 at the mitochondrial surface [93]. MiD49 and MiD51 are homologous proteins that share 65% identity and are N-terminally anchored in the mitochondrial outer membrane [94,110]. Both over-expression and knockdown of MiD51 and MiD49 produce morphologically distinct forms of fused mitochondria [94]. Like Mff, overexpression of MiD49/51 results in translocation of the cytosolic pool of Drp1 to the mitochondrial outer membrane, suggesting that these proteins are involved in the recruitment of Drp1 to mitochondria [93,94]. Co-immunoprecipitation experiments have established that both MiD49 and Mff can interact with Drp1; however the precise mechanisms by which they act have yet to be identified [93,94]. Given that fission is a multi-step process, these proteins may have distinct roles in recruitment and retention of Drp1 at mitochondria. It will be important to determine the nature of the interaction between Mff and MiD49/51 with Drp1, and if a direct relationship exists between these effector proteins. 2.4. Regulation of mitochondrial fission Drp1 executes mitochondrial fission by a process that requires its recruitment to the outer mitochondrial membrane, assembly at scission sites, constriction leading to fission and subsequent release back into the

cytosol [79,80,88]. This cycling activity and the formation of fission complexes are proposed to be regulated through a number of posttranslational modifications including phosphorylation, S-nitrosylation, ubiquitylation and sumoylation (Table 2, Fig. 4) [111–117]. The phosphorylation of Drp1 at different positions in the GED domain results in varied effects on Drp1 activity. Phosphorylation of Drp1 at a conserved serine residue (Ser585 in rat, Ser616 in humans) by Cdk1/cyclin B kinase complex occurs during mitosis reportedly increasing fission to ensure efficient segregation of mitochondria to daughter cells [116]. However since Drp1 is not essential for mitochondrial inheritance during cell division, the precise role of Drp1 phosphorylation during mitosis is not clear [83]. Drp1 phosphorylation at Ser637 by cAMP dependent protein kinase A (PKA) inhibits its GTPase activity, and results in impaired mitochondrial fission [112,118]. Calcineurin has been shown to dephosphorylate Drp1 at the same site following increases in intracellular calcium [111]. This in turn was shown to result in accumulation of Drp1 at mitochondria and increased fission. Thus calcium based signalling is involved in the regulation of mitochondrial morphology [111]. In contrast to these findings, it was found that Calcium/calmodulin-dependent protein kinase Iα phosphorylated Drp1 at the same residue (brain isoform: Ser600) and caused an increased association of Drp1 with mitochondria [114]. It is possible that these kinases also regulate other proteins involved in Drp1 recruitment to mitochondria allowing for the variability in results. Nitric oxide acts as a signalling molecule involved in a wide variety of cellular processes and becomes covalently linked to proteins in a process termed S-nitrosylation (SNO). In neuronal cells, nitric oxide treatment results in mitochondrial ultra structural changes along with increased fission events [113,119]. Such changes in mitochondrial morphology are proposed to occur through S-nitrosylation of Drp1 at Cys644, causing an increase in GTPase activity and dimerization of Drp1 [113]. S-nitrosylation of Drp1 and fragmentation of the mitochondrial network have also been observed following cellular expression of the ß-amyloid protein (Aβ), a key mediator of Alzheimer's disease [113,120,121]. The formation of Aβ oligomers is proposed to stimulate nitrosative stress thereby leading to SNO-Drp1 production [113].

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P Drp1 calcineurin

CDK1/cyclinB (mitosis)

CaMKIα,PKA Drp1

Ub Drp1

SNO SUMO MAPL, SENP5, Ubc9, Bax

MiD49/51 P

MARCHV, Parkin

MARCHV, Parkin Fis1

Ub

cleavage by OMA1, PARL, m-AAA

OPA1

Mfn1/2

MARCHV, Parkin Ub

Fig. 4. Post-translational regulation of mammalian mitochondrial morphology. Outer membrane Mitofusins (Mfn1 and Mfn2) are ubiquitinated by the ubiquitin E3 ligases MARCH5 and Parkin. Dynamin related protein 1 (Drp1) and mitochondrial fission 1 (Fis1) are also targets of MARCH5 and Parkin-mediated ubiquitination. Alternate processing of optic atrophy 1 (OPA1) by OMA1, presenilin associated rhomboid-like protease (PARL) and m-AAA peptidases generates different OPA1 isoforms. The balance of these isoforms affects remodelling of the inner membrane. Drp1 activity is also regulated by cyclin dependent kinase 1 (CDK1/cyclinB) and cAMP-dependent protein kinase (PKA) that phosphorylate Drp1, while Ca2+/calmodulin dependent protein kinase 1α (CaMKIα) dephosphorylates Drp1. Drp1 is also sumoylated by mitochondrial-anchored protein ligase (MAPL) with the interaction of the E2 ligase Ubc9. It is desumoylated by sentrinspecific protease 5 (SENP5). Bax has also been found to stimulate Drp1 sumoylation.

The ubiquitin ligase protein MARCH5 (also called MITOL) is proposed to regulate mitochondrial fission via a Drp1 ubiquitindependent switch that negatively regulates the formation of fission complexes [115]. Co-immunoprecipitation experiments uncovered a direct interaction between MARCH5 and Mfn2; and between MARCH5 and ubiquitinated forms of Drp1 and Fis1 [122,123]. A pro-fusion role for MARCH5 was initially suggested as overexpression of MARCH5 in cells led to increased turnover of Fis1 and Drp1, and altered the balance of the mitochondrial network towards fusion [122,123]. However in subsequent studies involving silencing of endogenous MARCH5 or overexpression of dominant-negative MARCH5 E3 ligase mutants in cells, Drp1 distribution and mobility were disrupted (potentially due to stabilisation of mitochondrial fission complexes), resulting in decreased fission events and a shift towards mitochondrial elongation [115,124]. Drp1 and Fis1 have also recently been identified as targets of the E3 ubiquitin ligase Parkin (Fig. 4) [21,23]. Overexpression of GFP-Parkin in cells results in degradation of Drp1, while silencing of Parkin causes an increase in Drp1 protein levels [23]. This degradation of Drp1 and Fis1 by Parkin can be inhibited following chemical inhibition of the proteasome [21,23]. Since a number of outer membrane proteins appear to be turned over in a Parkin-dependent manner the role of Parkin in mitochondrial fission is unclear [21,23]. The potential role of these proteins in Parkinson's disease is discussed below. SenP5 is a SUMO protease that translocates to mitochondria during mitosis, desumoylating Drp1 and increasing the labile pool of Drp1 to ensure a full complement of mitochondria are transmitted to daughter

cells [117]. Sumoylation of Drp1 is also stimulated by translocation of pro-apoptotic Bax to mitochondria resulting in stable Drp1 association with the mitochondrial outer membrane in a process that is independent of Fis1 [103]. However, a Drp1 mutant that cannot be sumoylated is still recruited to mitochondria following apoptotic stimuli, suggesting that SUMO modification of Drp1 may not be specific to apoptosis [125]. The SUMO E2 ligase Ubc9 has been shown to interact with Drp1. This interaction was abolished in the presence of the GTPase inactive mutant Drp1K38E suggesting that SUMO1 modification of Drp1 by Ubc9 requires GTPase active Drp1 [126]. The mitochondrial outer membrane protein MAPL (also known as MULAN) is a SUMO E3 ligase that acts with Ubc9 to sumoylate Drp1, and enhance Drp1 stability and mitochondrial fission [126–129]. The addition of small ubiquitin and SUMO modifiers to Drp1 has been proposed to affect the formation of fission complexes; however the precise role of these proteins in Drp1 regulation has yet to be elucidated. Post-translational modification of proteins involved in mitochondrial morphology as described here has a significant role in the events that regulate fission and fusion. While there is a significant body of research on the post translational modification of key proteins involved in mitochondrial morphology, there are a growing number of newly identified proteins for which there is limited data. For example, MiD51 has been described to undergo phosphorylation [130], however how this modification might affect the fission process is currently unknown (Table 1). These phosphorylated residues are also conserved in MiD49 suggesting that phosphorylation may be important in the function of these proteins. MTP18 mRNA and protein expression has been shown to depend on phosphatidylinositol (PI) 3-kinase activity, establishing that mitochondria are targets of cellular signalling pathways [105], however the pathway regulating MTP18 activity has yet to be described. Adding a further complication is the number of different modifiers that have been identified to act on a single protein, for example Drp1. These modifiers may respond to specific signalling cues exerting tight control over mitochondrial morphology, for example during the cell cycle, following stress, and cellular rearrangements. 3. Mitochondrial morphology and apoptosis Mitochondrial morphological changes are a key step in the cell responding to apoptotic stimuli, and initiation of the signalling cascade. Induction of apoptosis results in cytochrome c release following mitochondrial outer membrane permeabilization, but it also causes extensive fragmentation of the mitochondrial network through fission events [131–134]. A number of components of the fission and fusion machinery including OPA1, Fis1, Drp1, Mfn1 and Mfn2 have been directly implicated in the regulation of apoptosis [78,132,134–137]. Overexpression of Fis1 can directly induce both mitochondrial fragmentation and induction of apoptosis [78]. However Fis1 or mitochondrial fission is not requisite for apoptosis since cytochrome c release is prevented in cells overexpressing Fis1 when pro-apoptotic Bax/Bak are inactivated [78,138]. Similarly, dual silencing of Fis1 and OPA1 can protect cells against apoptotic induction; however this protection is also independent of mitochondrial fragmentation [102]. Recent evidence has shown that during apoptosis, fragmented mitochondria are sensitised to Bax insertion into the outer membrane and this can be suppressed by mitochondrial fusion [139]. The regulation of Drp1 translocation and association with mitochondria following apoptotic signalling have been reported to occur through both phosphorylation and sumoylation of Drp1. As previously mentioned, dephosphorylation of Drp1 by calcineurin at Ser637 following mitochondrial depolarisation results in Drp1 recruitment to mitochondria [140]. Inhibition of calcineurin prevents Drp1 recruitment to mitochondria, cytochrome c release and mitochondrial fragmentation [140]. Phosphorylation of Drp1 by PKA at the same residue results in inhibition of mitochondrial fission and increased resistance to apoptotic stimuli [118]. These results suggest a key role for regulation of Drp1

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phosphorylation in the apoptotic signalling cascade. Depolarised mitochondria may by segregated from the rest of the mitochondrial network following calcineurin dephosphorylation of Drp1, while cAMP mediated phosphorylation of Drp1 by PKA may act to promote cell survival through inhibition of Drp1 activity [118,140]. Drp1 has also been shown to stimulate Bax oligomerization in the presence of cardiolipin following apoptotic stimuli [141]. Bax and Drp1 co-localization occurs at sites of mitochondrial fission upon induction of apoptosis [131,132]. Subsequent to Bax recruitment to mitochondria, Drp1 no longer cycles on and off mitochondria and it becomes locked on the outer membrane following Bax/Bak-dependent sumoylation of Drp1 [103]. Silencing of Drp1 or expression of the GTPase inactive dominant negative mutant Drp1K38A delays apoptosis and cytochrome c release, however it does not prevent Bax translocation to mitochondria. This suggests that Drp1 mediated mitochondrial fragmentation is not a prerequisite for apoptosis [102,131,134]. The pro-fission protein MTP18 has also been implicated in apoptosis, with silencing resulting in induction of apoptosis, cytochrome c release and caspase activation [105]. These results are somewhat contrary to the popular opinion that apoptosis is preceded by mitochondrial fission, as silencing of MTP18 results in fusion of the mitochondrial network [105]. Sensitivity to apoptosis following MTP18 silencing may be a result of loss of inner membrane integrity resulting in cytochrome c release, however this remains to be shown. Mitochondrial dynamics is affected during apoptosis, and the rate of mitochondrial fusion is reduced following the addition of death stimuli, independently of caspase activation [142]. Increased mitochondrial fusion has also been shown to affect cellular sensitivity to apoptotic induction, with overexpression of Mfn1 and Mfn2 in cells exhibiting a protective effect [136]. As discussed previously, the pro-apoptotic proteins Bax and Bak have been described as having roles in the homeostasis of mitochondrial morphology however their activity during apoptosis may include regulation of mitochondrial fusion. Drp1, Mfn2 and Bax have been found to co-localise at mitochondrial fission sites at the initial stages of apoptosis [131], however a direct interaction has yet to be shown. Bak interacts preferentially with Mfn1 during apoptosis, following dissociation from Mfn2 [59]. This interaction may serve to inhibit Mfn1 pro-fusion activity and subsequently promote mitochondrial fission. As OPA1 requires Mfn1 but not Mfn2 to bring about mitochondrial fusion [36], inhibition of Mfn1 by Bak could subsequently result in OPA1 mediated cristae remodelling. OPA1 cleavage occurs during the initiation of apoptosis resulting in cristae remodelling and mobilisation of cytochrome c for release following mitochondrial outer membrane permeabilization [102,137,143]. OPA1 oligomers are proposed to form at cristae junctions, and loss of these oligomers contributes to cristae rearrangement [137,143]. The formation of soluble OPA1 isoforms during apoptosis is regulated by PARL mediated processing and cellular silencing of PARL reduces the pool of soluble intermembrane space OPA1 [64]. Silencing of OPA1 or PARL results in fragmentation of mitochondria, cristae remodelling and increased sensitivity to apoptotic stimuli [64,137,143]. This sensitivity to apoptosis may be due to the proposed anti-apoptotic role of soluble OPA1, or loss/ imbalance of OPA1 isoforms resulting in subsequent destabilisation of the mitochondrial cristae [64,137]. Mitochondria are essential in the intrinsic apoptotic pathway, with recruitment and activation of Bax/Bak at mitochondria, and release of cytochrome c key points of regulation [144]. While mitochondrial fission is observed following apoptotic stimuli, this process can be uncoupled from Bax/Bak oligomerization and subsequent cytochrome c release [102,105]. This separation of Bax translocation from cytochrome c release and organelle fragmentation raises a fascinating question as to where Drp1 mediated mitochondrial fission fits in apoptosis. While silencing/ inhibition of Fis1 and Drp1 have been shown to delay apoptosis and prevent cytochrome c release, apoptosis can ultimately proceed despite loss of the fission machinery or alterations in the mitochondrial network. Recent evidence suggests that changes to the mitochondrial outer

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membrane affect assembly of the apoptotic machinery and that the regulation of mitochondrial fission may facilitate apoptosis through mitochondrial membrane remodelling [141].

4. Mitochondrial morphology and neurodegeneration With a maturing world population, age-related disorders such as Alzheimer's, Huntington's and Parkinson's diseases are a major concern for society. At present approximately 1–2% of the population over 60 are clinically diagnosed with Alzheimer's or Parkinson's diseases [145–147]. In the case of Alzheimer's disease this estimate dramatically increases with age, with 25–45% of people over the age of 85 diagnosed with the disease [147,148]. Mitochondrial dysfunction has been suggested to be a defining factor in such neurodegenerative disorders [149]. The reliance of proper mitochondrial function in neurons is particularly high due to their unique electrophysiological properties and excessive energy demands. As such, any defect in mitochondrial function may be detrimental to neuronal function.

4.1. Mitochondrial shape and movement in neurons Changes in mitochondrial morphology can alter the distribution of mitochondria throughout the cell [18,19,82,150,151]. Efficient transport is required to ensure regeneration of old or damaged organelles through fusion with healthy mitochondria, or alternately target damaged mitochondria for segregation by fission and subsequent degradation. The importance of mitochondrial dynamics has been demonstrated in neuronal cells where mitochondria are positioned at sites of synapse formation and are proposed to regulate synaptic activity [152]. Mitochondria are recruited to the distal region of neurons supplying growth areas with increased ATP in response to the chemo-attractant nerve growth factor [153,154], and to regulate Ca 2+ mediated signalling at synapses [152,155]. Neuronal mitochondria with reduced membrane potential are recycled back to the cell body, and those with high membrane potential radiate to the cell periphery, representing a mechanism to recycle old or damaged organelles [156]. Impairment of mitochondrial dynamics can be detrimental to neuronal cells resulting in loss of synaptic activity and cell death [32,83,152]. Mitochondrial morphology proteins OPA1 and Mfn2 have identified roles in neuronal cell function, with mutations in genes encoding these proteins resulting in autosomal dominant optic atrophy (ADOA) and Charcot–Marie–Tooth disease type 2A (CMT2A) respectively [41,45,46]. These disorders highlight the significant role that mitochondrial dynamics plays in neuronal health. Neuronal maturity affects the expression levels of Mfn1 and Drp1, with higher Mfn1 and lower Drp1 expressions in young neurons, indicating that the regulation of mitochondrial morphology occurs in both time and space within neuronal cells [157]. Complete ablation of Mfn2 is embryonically lethal, while cerebella loss of Mfn2 results in decreased neuronal spine formation and dendritic growth in both development and maturity [19,32]. Expression of the dominant negative mutant Drp1 K38A or wild type OPA1 in dendritic cells decreases mitochondrial number and spine formation, while overexpression of Drp1 increases dendrite mitochondrial density and spine formation [152]. The anti-apoptotic protein Bcl-xL has also been described to increase mitochondrial motility and biomass, and interact directly with Drp1 to enhance GTPase activity and subsequent synaptic formation in neuronal cells [158,159]. The implications of these studies are especially interesting due to the aberrant mitochondrial morphology and distribution observed in patients with neurodegenerative disorders, such as Parkinson's, Alzheimer's, and Huntington's diseases, which can also display a loss in mitochondrial function [24,160–162].

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4.2. Alzheimer's disease Alzheimer's disease (AD) is the most common form of dementia worldwide and is clinically characterised by deterioration in memory, language skills and visual acuity accompanied with behavioural changes and premature death [120,163]. The behavioural and psychiatric presentation of AD is accompanied by a pathology of progressive neuronal and synaptic loss; the formation of amyloid-β (Aβ) plaques and neurofibrillary tangles [120,163]. Mitochondrial dysfunction, altered Ca2+ homeostasis and elevated reactive oxygen species are all features of AD indicating that mitochondria have a clear association with the pathogenesis of neurodegeneration [149,163]. Impaired mitochondrial morphology and distribution in AD neurons have been described, resulting in collapse of the mitochondrial network and a reduction in mitochondrial number in neuronal processes [24,164]. Axonal swelling has been described as an early event in AD, resulting in abnormal accumulation of mitochondria at these sites prior to any obvious clinical signs [165]. This suggests that the decrease in axonal mitochondrial transport may be one of the first in vivo detectable signs of AD. Recent studies have also described a role for the regulation of mitochondrial morphology in AD. Increased production of the signalling molecule nitric oxide occurs in AD cortical neurons and subsequent S-nitrosylation (SNO) of Drp1 results in amplified Drp1 GTPase activity and mitochondrial fission [113,119,166]. This enhanced mitochondrial fission is speculated to be due to the presence of β-amyloid (Aβ) oligomers which cause increased mitochondrial fragmentation in a nitric oxide (NO) dependent manner [113,121]. In addition to increased levels of SNO-Drp1 in AD, the levels of other proteins involved in mitochondrial morphology are also altered. In both fibroblasts and neurons from AD patients, Drp1 protein levels are decreased [24,164]. Reduction of Drp1 protein levels has also been observed following Aβ overexpression [164]. The levels of OPA1, Mfn1 and Mfn2 are also reduced in the AD neuronal cells, while Fis1 expression is increased [24]. This change in the expression levels of mitochondrial morphology proteins is also accompanied by loss of dendritic spine formation in cultured neuronal cells [24]. The altered expression of these proteins also has a significant effect in the distribution of mitochondria [24], and subsequently may have a role in the loss of synaptic activity in AD patients. Changed expression of Drp1, Mfn1, Mfn2, OPA1 and Fis1 in AD patients may also be due to a dysfunctional ubiquitin proteasome system as mutant ubiquitin found in AD has been shown to cause neuritic beading and altered distribution of mitochondria [167]. While recent studies have identified mitochondrial dynamic proteins as targets of K48 linked ubiquitin mediated degradation [21], this has yet to be investigated in AD. 4.3. Huntington's disease Huntington's disease (HD) is a progressive neurodegenerative disorder that is caused by an abnormal polyglutamate expansion in the Huntingtin (Htt) gene, in which the length of the expansion determines the severity and presentation of the disease [168,169]. Symptoms of HD can present at any age and include involuntary movement, rigidity and loss of cognitive function [169,170]. Although the precise mechanisms of the Htt mediated neurotoxicity are not understood, mutant Huntingtin (mHtt) undergoes a conformational change resulting in aggregates that may exert toxic effects in the nucleus, cytoplasm and at mitochondria [170]. Mitochondrial velocity and trafficking in mHtt neurons is diminished both in vivo and in vitro resulting in a loss of bidirectional movement and abnormal dynamics [22,160,161,171]. Interestingly, similar defects in mitochondrial transport were also seen following silencing of endogenous Htt, suggesting that Htt may have a role in mitochondrial trafficking [161]. In fact, the motor proteins kinesin and dynein co-precipitate with mHtt and decreased transport of mitochondria in HD affected cells may be due to mHtt recruitment of trafficking machinery [161]. Expression of mHtt also results in changes to mitochondrial shape, with increased

fragmentation and abnormal cristae arrangement [22,160,172]. Using complimentary models of rat neurons and fibroblasts from individuals with HD, it has been shown that mutant Htt can bind Drp1 and cause increased GTPase activity, oligomerization, and increased dephosphorylation of Drp1 [22,172]. Dephosphorylation of Drp1 occurs following an increase in calcineurin activity, which subsequently increases its translocation to mitochondria [111,172]. mHtt also induces changes in the expression of components of the mitochondrial morphology machinery, with increased levels of pro-fission mediators Drp1 and Fis1, while pro-fusion mediators Mfn1/2 and OPA1 have lower mRNA levels than age-matched, wild type controls [160]. These defects in transport and morphology can be rescued by expression of the GTPase dominant negative Drp1K38A [22], suggesting that Drp1 has a key role in the mitochondrial dysfunction observed in HD. This research provides compelling evidence that Drp1 may be an effective new therapeutic target for HD neurodegeneration. 4.4. Parkinson's disease Parkinson's disease (PD) is characterised by progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain, and the formation of Lewy bodies in the remaining neurons [173]. An individual suffering from PD usually presents with postural instability, tremors at rest and slow movement [174]. Although largely classified as an idiopathic disease, around 10% of patients present as familial cases; inherited forms of the disease of which there are mutations in known PD associated genes [175]. Of these genes, Parkin and Pteninduced kinase 1 (PINK1) have been shown to be important for mitochondrial function and quality control [176–179]. PINK1 is a serine/threonine kinase that localises to the outer mitochondrial membrane, recruiting the E3 ligase Parkin upon dissipation of membrane potential and initiating clearance of damaged mitochondria [179–181]. Initial studies completed in Drosophila melanogaster suggested that PINK1 may play a role in mitochondrial fission as the network appeared fused following expression of PINK1 mutants [182–185]. The mitochondrial network could be rescued by the addition of drp-1 or depletion of Opa-1 or Mfn2 [182–185]. However, the opposite effect is observed in mammalian cells, in which PINK1 depletion causes mitochondrial swelling and fragmentation in conjunction with a decrease in membrane potential [178,186,187]. Mitochondrial fission observed following loss of PINK1 or Parkin function in human cells is proposed to be mediated by dephosphorylation of Drp1 by calcineurin, resulting in the promotion of fission complex formation and fragmentation of mitochondria [188,189]. This fragmentation can be rescued by expression of the dominant negative Drp1K38A or overexpression of fusion proteins Mfn2 and OPA1 [187,188]. However, expression of the dominant negative Drp1 increased cell death, indicating that mitochondrial fission and activation of mitophagy may be cellular protective mechanisms [187]. The E3 ligase activity of Parkin may also be an important regulator of mitochondrial morphology and subsequent clearance of mitochondria through mitophagy. The Mitofusins are rapid targets of Parkin mediated ubiquitination, however they are not crucial for Parkin mediated mitophagy as mitophagy is still present in Mfn null cells [21]. Fis1 and Drp1 have also been identified as targets of Parkin mediated degradation [21,23], suggesting that mitophagy may be a multi-step process starting with degradation of pro-fusion proteins resulting in an imbalance of profission proteins, then subsequent clearance of mitochondria. Given that mitochondrial fragmentation is often a sign of reduced cell viability and precludes apoptosis, it is not unreasonable to suggest that the mitochondrial fragmentation associated with loss of PINK1 and Parkin is due to aberrant mitophagy. 5. Conclusion There are many different roles for mitochondrial morphology proteins in disease, cell survival and inheritance. The identification of

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proteins involved in both the regulation and maintenance of mitochondrial morphology has revealed the crucial role that mitochondrial dynamics plays in the health of the cell. Not surprisingly, defects in mitochondrial fission and fusion result in aberrant cellular function, and in some cases cell death. This loss of function is most apparent in neuronal cells that rely greatly on the correct distribution and function of mitochondria due to high energy demands of the cell. Subsequently, defects in proteins involved in mitochondrial dynamics are implicated in a number of neurodegenerative disorders. While the key proteins involved in mitochondrial fission and fusion have been identified, how these proteins co-ordinate to accomplish remodelling of the double membrane is not well understood. Each of these proteins may function in different stages of mitochondrial fission and fusion highlighting the need for further understanding into the mechanisms that regulate mitochondrial dynamics. There are a growing number of post-translational modifications that have been identified to regulate mitochondrial morphology proteins, however the cellular signals that result in many of these changes are also largely unknown. Further complicating this field is the recent evidence that identifies dual roles for a number of the mitochondrial dynamic proteins in apoptosis, and the involvement of apoptotic proteins in mitochondrial morphology. The field of mitochondrial morphology is relatively young and it is likely that in the coming years many more proteins involved in fission, fusion and distribution will be identified leading to a greater understanding of these complex mechanisms. Acknowledgements This work was supported by grants from the NHMRC and the ARC. LDO is supported by funding from the Wellcome Trust. References [1] D.C. Chan, Annu Rev Cell Dev Biol 22 (2006) 79–99. [2] D.H. Margineantu, W. Gregory Cox, L. Sundell, S.W. Sherwood, J.M. Beechem, R.A. Capaldi, Mitochondrion 1 (5) (2002) 425–435. [3] A.E. Frazier, C. Kiu, D. Stojanovski, N.J. Hoogenraad, M.T. Ryan, Biol Chem 387 (2006) 1551–1558. [4] S. Campello, L. Scorrano, EMBO Rep 11 (9) (2010) 678–684. [5] M. Liesa, M. Palacin, A. Zorzano, Physiol Rev 89 (3) (2009) 799–845. [6] B. Westermann, Nat Rev Mol Cell Biol 11 (12) (2010) 872–884. [7] K.G. Hales, M.T. Fuller, Cell 90 (1) (1997) 121–129. [8] P. Sutovsky, C.S. Navara, G. Schatten, Biol Reprod 55 (6) (1996) 1195–1205. [9] S.A. Detmer, D.C. Chan, Nat Rev Mol Cell Biol 8 (11) (2007) 870–879. [10] A. Quintana, C. Schwindling, A.S. Wenning, U. Becherer, J. Rettig, E.C. Schwarz, M. Hoth, Proc Natl Acad Sci U S A 104 (36) (2007) 14418–14423. [11] M. Spinazzi, S. Cazzola, M. Bortolozzi, A. Baracca, E. Loro, A. Casarin, G. Solaini, G. Sgarbi, G. Casalena, G. Cenacchi, A. Malena, C. Frezza, F. Carrara, C. Angelini, L. Scorrano, L. Salviati, L. Vergani, Hum Mol Genet 17 (21) (2008) 3291–3302. [12] J. Bereiter-Hahn, M. Voth, Microsc Res Tech 27 (3) (1994) 198–219. [13] H. Chen, A. Chomyn, C. Chan, J Biol Chem 280 (28) (2005) 26185–26192. [14] S. Honda, S. Hirose, Biochem Biophys Res Commun 311 (2) (2003) 424–432. [15] K. Nakada, K. Inoue, T. Ono, K. Isobe, A. Ogura, Y.I. Goto, I. Nonaka, J.I. Hayashi, Nat Med 7 (8) (2001) 934–940. [16] H. Chen, D.C. Chan, Hum Mol Genet 14 (2005) R283–R289. [17] M.T. Lin, M.F. Beal, Nature 443 (7113) (2006) 787–795. [18] R.H. Baloh, R.E. Schmidt, A. Pestronk, J. Milbrandt, J Neurosci 27 (2) (2007) 422–430. [19] H. Chen, J.M. McCaffery, D.C. Chan, Cell 130 (3) (2007) 548–562. [20] D.C. Chan, Cell 125 (2006) 1241–1251. [21] N.C. Chan, A.M. Salazar, A.H. Pham, M.J. Sweredoski, N.J. Kolawa, R.L. Graham, S. Hess, D.C. Chan, Hum Mol Genet 20 (9) (2011) 1726–1737. [22] W. Song, J. Chen, A. Petrilli, G. Liot, E. Klinglmayr, Y. Zhou, P. Poquiz, J. Tjong, M.A. Pouladi, M.R. Hayden, E. Masliah, M. Ellisman, I. Rouiller, R. Schwarzenbacher, B. Bossy, G. Perkins, E. Bossy-Wetzel, Nat Med 17 (3) (2011) 377–382. [23] H. Wang, P. Song, L. Du, W. Tian, W. Yue, M. Liu, D. Li, B. Wang, Y. Zhu, C. Cao, J. Zhou, Q. Chen, J Biol Chem 286 (13) (2011) 11649–11658. [24] X. Wang, B. Su, H.G. Lee, X. Li, G. Perry, M.A. Smith, X. Zhu, J Neurosci 29 (28) (2009) 9090–9103. [25] D. Tondera, S. Grandemange, A. Jourdain, M. Karbowski, Y. Mattenberger, S. Herzig, S. Da Cruz, P. Clerc, I. Raschke, C. Merkwirth, S. Ehses, F. Krause, D.C. Chan, C. Alexander, C. Bauer, R. Youle, T. Langer, J.C. Martinou, EMBO J 28 (11) (2009) 1589–1600. [26] S. Meeusen, J.M. McCaffery, J. Nunnari, Science 305 (5691) (2004) 1747–1752. [27] Z. Song, M. Ghochani, J.M. McCaffery, T.G. Frey, D.C. Chan, Mol Biol Cell 20 (15) (2009) 3525–3532.

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