Correct mitochondrial dynamics are essential to neuronal function. These dynamics include mitochondrial trafficking and quality-control systems that maintain a precisely distributed and healthy mitochondrial network, so that local energy demands or Ca2+-buffering requirements within the intricate architecture of the neuron can be met. Mitochondria make use of molecular machinery that couples these organelles to microtubule-based transport via kinesin and dynein motors, facilitating the required long-range movements. These motors in turn are associated with a variety of adaptor proteins allowing additional regulation of the complex dynamics demonstrated by these organelles. Over recent years, a number of new motor and adaptor proteins have been added to a growing list of components implicated in mitochondrial trafficking and distribution. Yet, there are major questions that remain to be addressed about the regulation of mitochondrial transport complexes. One of the core components of this machinery, the mitochondrial Rho GTPases Miro1 (mitochondrial Rho 1) and Miro2 have received special attention due to their Ca2+-sensing and GTPase abilities, marking Miro an exceptional candidate for co-ordinating mitochondrial dynamics and intracellular signalling pathways. In the present paper, we discuss the wealth of literature regarding Miro-mediated mitochondrial transport in neurons and recently highlighted involvement of Miro proteins in mitochondrial turnover, emerging as a key process affected in neurodegeneration.

Introduction

The brain is only 2% of the body's weight, but it uses 20% of the body's resting energy production. This high level of energy consumption is mainly used to reverse the ion fluxes that underlie action potential signalling and synaptic transmission [1]. Mitochondria are highly efficient in utilizing oxygen and substrates mainly derived from glucose, to produce energy for cell function in the form of ATP, and are key organelles for the generation of brain energy. As most neurons are large in size (e.g. a motor neuron axon can be up to a metre long in humans), precluding rapid diffusion of ATP between distant regions of the cell, mechanisms must be in place to spatially match energy production to local energy usage. Thus mitochondria must be transported and localized close to areas of high energy usage such as the sites of the ion influxes that underpin synaptic and action potential signalling. There are several locations in neurons which are thought to have a particularly high energy demand and where mitochondria have also been found to be enriched, including axonal domains such as the axon initial segment, nodes of Ranvier, growth cones and presynaptic terminals [2]. Similarly, in neuronal dendrites, mitochondria are enriched at the postsynaptic domains [3,4]. In addition to energy provision, mitochondria can also buffer and sequester intracellular Ca2+ and thus their localization can influence the dynamics of intracellular Ca2+ signalling with important implications for neural communication and plasticity. Interestingly, the mitochondrial position in these locations can be dynamically controlled on rapid timescales by neuronal activity and neuromodulatory signalling pathways to allow for the rapid redistribution and recruitment of mitochondria to subcellular areas that have increased energy, Ca2+ buffering or signalling requirements.

Motor-protein-dependent mitochondrial transport in neurons

Mitochondria in eukaryotic cells are part of an organellar network with a highly complex distribution and morphology which has been documented for over 100 years [5]. Mitochondria can be found as individual organelles with a wide variety of morphologies and can also be part of an extended reticular network [4].

Using live-cell imaging to study mitochondrial transport has revealed that, in neurons, the mitochondrial network is highly dynamic [2,4], with mitochondria exhibiting rapid bidirectional transport, characterized by stopping and starting in addition to fission and fusion events. Most of this transport in neurons is mediated by microtubule motor proteins, which transport mitochondria over long distances. In contrast, the actin cytoskeleton may be more important for short-range movements of mitochondria and their anchoring [68]. Neuronal mitochondrial transport velocities have mostly been shown to fall in the range 0.3–1.0 μm/s with rates observed in vivo comparable with those of some neuronal culture studies [3,911]. Thus mitochondrial transport along microtubules by motor proteins is a key mechanism for the delivery of mitochondria throughout the cell to provide a localized ATP supply and Ca2+-buffering capacity. In addition, damaged mitochondria may need to be transported to the cell soma from distal sites for their repair or degradation by mitophagic processes (i.e. mitochondrial autophagy). The mechanisms that regulate cross-talk between trafficking and mitochondrial degradation and repair, however, are only just beginning to be elucidated.

Mitochondrial transport by kinesin and dynein motors

Kinesin superfamily (KIF) microtubule motor proteins are a large family, containing at least 45 different genes in human and mouse [12]. Microtubule plus-end-directed mitochondrial transport (i.e. anterograde in the axon) in neurons is mediated primarily by the KIF5 motors which have been found to be enriched on brain mitochondria using biochemical and imaging approaches [9,1316]. In mammalian neuronal cells, roles for KIF1B and Klp6 have also been proposed in the transport of mitochondria to the plus ends of microtubules [17,18]. However, much less is known about how transport mediated by these motors may cross-talk with KIF5-dependent trafficking. Whereas there is only one KIF5 isoform in Drosophila [19], there are three forms of KIF5 (KIF5A, KIF5B and KIF5C) in mammals. KIF5B is present in almost all cell types, and KIF5A and KIF5C are predominantly expressed in the nervous system [7,20]. Mutating KIF5 in Drosophila neurons disrupts anterograde axonal mitochondrial transport, whereas deletion of KIF5B blocks transport of mitochondria to the periphery in non-neuronal mouse cells, resulting in their perinuclear clustering [21]. Expression of dominant-negative KIF5 constructs disrupts mitochondrial trafficking in axons of rat hippocampal neurons [9,1316,22]. In dendrites, the role of kinesins is less clear. KIF5 function-blocking antibodies prevent bidirectional mitochondrial trafficking along mixed polarity microtubules in dendrites [9], but, in other studies, expression of KIF5 dominant-negatives had little effect on mitochondrial transport in primary proximal dendrites [23]. Several adaptors including syntabulin, FEZ1 (fasciculation and elongation protein ζ-1), RanBP2 (Ran-binding protein 2), TRAKs (trafficking protein, kinesin-binding) and Miro (mitochondrial Rho) have been implicated as adaptors coupling mitochondria to the kinesin-dependent transport pathway [4,22,24]. The kinesin adaptor FEZ1 was shown to be important for mitochondrial transport in PC12 neuroblastoma cells and hippocampal neurons [25,26], while syntabulin partially co-localizes to the OMM (outer mitochondrial membrane) and interacts directly with KIF5 isoforms [22]. Depletion of syntabulin blocks the trafficking of both mitochondria and axonal transport vesicles and results in depletion of presynaptic vesicle cluster density and disruption of synaptic function. Genetic screens in Drosophila identified the kinesin binding protein Milton (the fruitfly orthologue of TRAK proteins) and the atypical GTPase dMiro (Drosophila Miro) as being crucial for anterograde transport of mitochondria along axons in fruitflies [27,28]. Miro and the mammalian Milton/TRAK orthologues have emerged as key conserved regulators of mitochondrial trafficking in neurons and are discussed in further detail below [9,16,28,29].

Retrograde (minus-end-directed) axonal transport of mitochondria is primarily mediated by dynein motors [7,13,30]. In contrast with the multiple kinesins, this complex is composed of a cytoplasmic dynein heavy chain 1 (DYNC1H1) dimer, containing the motor domain and large numbers of accessory proteins, including light chains and dynactin (a multisubunit complex necessary for dynein activity) that allow selectivity between dynein motors and different cargoes [30,31]. Dynactin components (e.g. p50 and p150Glued) in addition to dynein, have been detected on purified mitochondria. Dynein has been localized to both anterogradely and retrogradely moving axonal mitochondria and mutating dynein heavy chain or p150Glued inhibits axonal mitochondrial trafficking [13,14,3234]. Several adaptors have been proposed to link mitochondria to dynein-dependent retrograde transport pathways, including the dynein light chain Tctex1, which can support dynein-mediated transport [35] and can interact with VDACs (voltage-dependent anion channels) localized on the OMM, and APLIP1 [APP (amyloid precursor protein)-like protein-interacting protein 1], a Drosophila homologue of mammalian JIP [JNK (c-Jun N-terminal kinase)-interacting protein] that can activate kinesin-dependent transport of cargo in mammalian cells [33,36,37]. Recently, dynein was also found to interact with the TRAK adaptors, raising the possibility that the kinesin adaptor Miro is also the adaptor that recruits dynein motors to these organelles to co-ordinate dynein-mediated mitochondrial transport. This suggests that Miro proteins may be important for regulatory cross-talk between dynein and kinesin transport [23,38].

Miro protein complexes and mitochondrial dynamics

Miro1 and Miro2 were originally identified in mammals as atypical Rho-like small GTPases [39]. They are an evolutionarily conserved family of proteins, unique in their domain organization comprising two GTPase domains flanking two EF-hand Ca2+-binding domains and a C-terminal transmembrane domain which localizes the protein to the OMM [3941] (Figure 1A). In Drosophila, dMiro was found to interact with the kinesin adaptor Milton by yeast two-hybrid analysis implicating Miro in mitochondrial trafficking [42]. In agreement with this, deletion of dMiro in Drosophila was shown to reduce the number of moving mitochondria in axons [28,38], and Miro proteins were found to form protein complexes with Milton and kinesin motors [43].

Miro and the mitochondrial transport machinery

Figure 1
Miro and the mitochondrial transport machinery

(A) Schematic diagram of Miro functional domains. At the N-terminus, two GTPase domains flank the two Ca2+-binding EF-hand domains, whereas the mitochondrial transmembrane domain (TM) is located at the C-terminus of the protein. (B) Mitochondrial transport along the microtubules is mediated by the interaction between the mitochondrial Miro proteins and the microtubule motor proteins. Specifically, Miro binds the kinesin motors and the TRAK adaptor molecules directly, whereas dynein-dependent transport is mediated by the interaction between the dynactin subunit p150 and TRAK. The TRAK adaptors also bind OGT.

Figure 1
Miro and the mitochondrial transport machinery

(A) Schematic diagram of Miro functional domains. At the N-terminus, two GTPase domains flank the two Ca2+-binding EF-hand domains, whereas the mitochondrial transmembrane domain (TM) is located at the C-terminus of the protein. (B) Mitochondrial transport along the microtubules is mediated by the interaction between the mitochondrial Miro proteins and the microtubule motor proteins. Specifically, Miro binds the kinesin motors and the TRAK adaptor molecules directly, whereas dynein-dependent transport is mediated by the interaction between the dynactin subunit p150 and TRAK. The TRAK adaptors also bind OGT.

Milton has two mammalian orthologues, TRAK1 and TRAK2 {also known as OIP106 [OGT (O-GlcNAc transferase)-interacting protein 106 kDa] and OIP98/Grif-1 respectively} [44,45], which can form complexes with mammalian Miro1 and Miro2 and with microtubule motors (Figure 1B). Depletion of Miro1 or TRAKs in cultured hippocampal neurons significantly reduces the number of moving mitochondria in both axons and dendrites [9,23,28,29,46].

Similarities and differences have both emerged between fruitfly Milton and the mammalian TRAKs. In Drosophila, Milton acts as an essential bridging molecule necessary to link Miro to kinesin motors [16,43]. Roles for Milton other than mitochondrial trafficking have not yet been identified, and Milton fruitfly mutants do not appear to have defects in the trafficking of other cargo such as synaptic vesicles [27]. Thus Milton appears to be a highly specific mitochondrial trafficking factor. The TRAK proteins exhibit 31.8% and 34.2% similarity to Milton for human TRAK1 and TRAK2. They have been localized to endosomal compartments (in addition to mitochondria) and also associate with several ion channels including GABAA (γ-aminobutyric acid A) receptors and K+ channels, and therefore appear to have evolved other cargo transport roles within the cell [4749]. Regardless of other potential roles, there is very strong evidence that the TRAK proteins are important for mitochondrial trafficking [9,45,50]. Although the TRAK proteins were initially confirmed to interact directly with KIF5 motors in a similar fashion to what was observed for Milton [44], recent work has also demonstrated that they can additionally interact with dynein motors [presumably via an interaction with p150Glued and the TRAK N-terminal domain which is homologous with the N-terminus of HAP1 (huntingtin-associated protein 1), another p150Glued-binding protein] [23] (Figure 1B). In the same study, it was shown that the two adaptors of the family display different localizations (TRAK1 being more axonal, TRAK2 more dendritic) and affinities for the two different motors (kinesin/dynein) suggesting that differential association capabilities may control the polarized transport of mitochondria into the different compartments of the neuron. Interestingly, it was also shown that a conformational change in TRAK2 was responsible for its preferential association with dynein, precluding the interaction with kinesin. In this context, Miro emerges as an interesting candidate in regulating the polarized transport of mitochondria either by regulating the differential recruitment of TRAK1 or TRAK2 into the motor complex or by controlling its molecular conformation, and thus interacting affinities. In any case, whether TRAKs are essential for bridging Miro to kinesin and dynein motors in mammals or rather act as accessory regulatory factors for the co-ordination of kinesin and dynein transport remains unclear. The mammalian Miro1 has been shown to bind directly to KIF5 in addition to the TRAKs [9] (Figure 1B) and thus suggests the possibility of coexistence of different combinations of molecular complexes with different capabilities and functions.

In addition to motors, TRAK1 and TRAK2 have also been found in complexes with the enzyme OGT [51] (Figure 1B), which catalyses O-linked glycosylation of serine and threonine residues with the post-translational modification O-GlcNAc [52]. Recent proteomic studies [53] suggest that TRAKs are modified by O-GlcNAc and exploring the consequences of this modification for regulation of the mitochondrial trafficking complex further is an important goal. Since the levels of O-GlcNAc can be linked to glucose levels, and therefore energy levels, this suggests an exciting possible link between mitochondrial trafficking and the availability of mitochondrial substrates [52]. TRAKs could also potentially recruit OGT to Miro or kinesins to modify them with O-GlcNAc and regulate their function, although neither Miro nor KIF5 have as yet emerged as being O-GlcNAc-modified from proteomic studies of the O-GlcNAc-modified proteome.

As well as interacting with the TRAK proteins, Miro has more recently been found to interact with protein components of the mitochondrial fusion machinery. Miro1/Miro2 (and TRAKs) interact with the Mfns (mitofusins) [54], dynamin-related GTPases expressed on the OMM, critical for regulating mitochondrial fusion. Mitochondrial trafficking assays in Mfn2-knockout neurons show a decrease in motility which cannot be rescued by Miro2 overexpression. Therefore Mfn2 and Miro2 act co-operatively to regulate mitochondrial trafficking. Moreover, several studies showed that overexpressing Miro1 increased mitochondrial length in neurons [29]. Interestingly, this effect on mitochondrial morphology was enhanced by the EF-hand mutant and was reproduced with the constitutively active form of the Miro1 GTPase domain I (Miro1-V13) [29,55]. Altogether, these results suggest that Miro is also likely to be playing an important role in co-ordinating cross-talk between mitochondrial fission and fusion mechanisms and trafficking.

A newly reported mitochondrial trafficking complex protein, Alex3, was recently shown to regulate mitochondrial trafficking in axons and to interact with Miro and TRAK2 in a Ca2+-dependent manner. Alex3 belongs to a family of proteins (Armc10/Armcx) exclusive to Eutherians that is composed of up to seven members sharing a common C-terminal domain with six armadillo repeats. Although the exact mechanism of Alex3 function is not yet known, the identification of this family potentially provides a new level of regulation of the mitochondrial transport machinery and points to an increased regulatory complexity of mitochondrial dynamics in the most evolved mammals [56].

Miro signalling domains and the control of mitochondrial trafficking

Miro proteins were also identified as the key Ca2+ sensors for Ca2+-dependent mitochondrial stopping, in both axons and dendrites. This stopping is dependent on the EF-hand domains which bind Ca2+ upon a neural activity-dependent Ca2+ rise [9,16,29]. Although there is agreement that Miro proteins act as key Ca2+-dependent sensors for mitochondrial stopping, several molecular mechanisms have been proposed for the uncoupling of mitochondria from the microtubule transport pathway by Miro-mediated Ca2+-sensing (Figure 2). In one model, under low Ca2+ conditions, Miro interacts with KIF5 via the adaptor Milton. An increase in Ca2+ causes the KIF5 motor domain to dissociate from microtubules and interact with Miro on the mitochondrion, thereby dissociating the motors from microtubules (Figure 2A). In an alternative mechanism, it is proposed that Miro1 can bind directly to KIF5, and this interaction is inhibited by increased Ca2+ levels, thereby directly uncoupling Miro and mitochondria from the motor transport pathway [9] (Figure 2B). Recently, the axonal tethering protein SNPH (syntaphilin) [57] was also proposed to play a key role in Miro and Ca2+-dependent mitochondrial arrest in axons in a revised ‘engine-switch and brake’ model [58]. In this model, in axons upon Ca2+-binding, Miro releases KIF5 motors allowing KIF5 to interact with microtubule-bound SNPH (Figure 2C). In this model, SNPH therefore acts both to sense Ca2+-dependent stopping and as a tether by anchoring mitochondria to the mictrotubule track, keeping both the KIF5 motor and stationary mitochondria bound to the microtubules.

Models of Ca2+-dependent mitochondrial arrest

Figure 2
Models of Ca2+-dependent mitochondrial arrest

According to the first model (A), Ca2+ binding to Miro EF-hand domains promotes the detachment of the kinesin motors from the microtubules and the interaction of their motor domains with Miro, causing mitochondrial arrest [16]. In the second model (B), Ca2+ binding to Miro causes the release from the kinesin motors, determining the detachment of the mitochondrion from the microtubule TRAKs [9]. A third model (C) shows both the detachment of kinesin from Miro and its subsequent interaction with the mitochondrial tether SNPH [58].

Figure 2
Models of Ca2+-dependent mitochondrial arrest

According to the first model (A), Ca2+ binding to Miro EF-hand domains promotes the detachment of the kinesin motors from the microtubules and the interaction of their motor domains with Miro, causing mitochondrial arrest [16]. In the second model (B), Ca2+ binding to Miro causes the release from the kinesin motors, determining the detachment of the mitochondrion from the microtubule TRAKs [9]. A third model (C) shows both the detachment of kinesin from Miro and its subsequent interaction with the mitochondrial tether SNPH [58].

Another mechanism has been proposed by which Ca2+ can regulate different aspects of mitochondrial transport. It is known that when intracellular Ca2+ is elevated, mitochondria are able to buffer cytoplasmic Ca2+ by its uptake through the MCU (mitochondrial calcium uniporter) [59,60]. This effect has been shown to be inversely related to the mitochondrial velocity in axons, thus providing a molecular mechanism by which mitochondrial transport and localization can also be regulated by intramitochondrial Ca2+ concentration [61]. Interestingly, two different studies have shown that the mitochondrial matrix Ca2+ concentration is affected by the level of Miro proteins at the OMM, although the importance of the Ca2+-sensing abilities of Miro is not yet clear [29,61]. Together, these data suggest a molecular link between the motor machineries and the complexes that regulate mitochondrial Ca2+ uptake and could further pinpoint a regulatory pathway to match mitochondrial bioenergetic efficiency to transport state as elevated intramitochondrial Ca2+ concentration (associated with slower movement or stopping) is related to an increase in ATP production [62,63].

Less is known about the role of Miro GTPase domains. Consitutively active Miro1 causes apoptosis and alters mitochondrial morphology in non-neuronal cells [39]. In vertebrates, TRAK2 can be recruited to Miro1 and mitochondria dependent on the GTPase state of Miro [55], suggesting that the GTPase state of Miro might also have important regulatory roles in mitochondrial trafficking, similar to the mechanisms of action of Rab GTPases, where GTP or GDP binding affects downstream signalling and binding partners [64].

Miro, autophagy and pathology

Damaged mitochondria that cannot sustain the cell's metabolic requirements need to be selectively isolated by a mitochondrial quality-control system and subsequently cleared by mitophagy. PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1] (PINK1 or PARK6 gene) and parkin (PARK2 gene) are key components of this signalling apparatus, and loss-of-function mutations in PINK1 and parkin have been associated with rare recessive forms of PD (Parkinson's disease) [65]. Upon mitochondrial damage, PINK1 accumulates in its full-length form on the OMM and recruits parkin, an E3 ubiquitin ligase, from the cytosol. Parkin then ubiquitinates various substrates on the OMM [VDAC1, Drp1 (dynamin-related protein 1), Mfns, TOM20 (translocase of outer mitochondrial membrane 20) and TOM40], a crucial step in initiating mitophagy [6670]. Interestingly, Miro was found to interact with PINK1 and parkin [7173], and to be ubiquitinated upon mitochondrial damage [74,75], raising the possibility that Miro-mediated transport pathways and PD-associated disruption of mitochondrial turnover are linked. Whether PINK1 accumulation on the OMM is sufficient to trigger the signalling pathway that leads to the ubiquitination of specific mitochondrial substrates or the kinase needs to be activated, activate parkin or prime by phosphorylation the mitochondrial proteins that will be then ubiquitinated, or a combination of these possibilities, is currently under debate. Miro was found to be subjected to a PINK1-dependent phosphorylation on Ser156, a step essential for its parkin-dependent degradation [72]; however, a more recent study was unable to find a role for the phosphorylation of this site for protein stability [74] and, in agreement with this, a recent study has found parkin to be the only substrate for PINK1-mediated phosphorylation [76]. The regulation of Miro stability and turnover by PINK1 and parkin could act to isolate damaged organelles from the functional mitochondrial network [71,72] and promote their transport to the cell soma and degradation [74].

In addition to a potential role in PD, Miro and TRAKs may also be important in other disorders of altered mitochondrial trafficking and function. Miro1 and Miro2 interact with HUMMR (hypoxia up-regulated mitochondrial movement regulator), a mitochondrial protein expressed in neurons and markedly induced by HIF-1α (hypoxia-inducible factor 1α) and which, biases mitochondria transport in the anterograde direction [77]. Moreover, Miro Ca2+ sensing was also recently proposed to play a role in altered Ca2+ homoeostasis and mitochondrial trafficking caused by a mutation in the motor neuron disease susceptibility gene VAPB [VAMP (vesicle-associated membrane protein)-associated protein B] [78,79]. Knockdown of Milton and Miro in Drosophila has also been shown to increase levels of tau phosphorylation at Ser262, known to cause its detachment from microtubules, via an increase in kinase activity of PAR-1 [80]. This proposes a role for defects in Miro-mediated mitochondrial trafficking in Alzheimer's disease-related neurodegeneration.

Conclusion

In the present article, we have discussed the necessity for strict and correct regulation of mitochondrial dynamics, and the mechanisms by which Miro is involved in this process. However, there are still major mechanistic questions that will need to be addressed in the near future. The co-ordination of kinesin and dynein transport, the many ways by which Ca2+ may influence this transport, and the involvement of other signals in such regulation are critical to understand how mitochondria are transported, distributed, maintained in good health and, most importantly, how all these processes are integrated within a cellular context. In this regard, Miro proteins are situated in an excellent position. The further characterization of the various binding partners of Miro will contribute to our better understanding of how the control of mitochondrial dynamics is linked to the major intracellular signalling pathways. Moreover, the structural features of Miro itself, the possession of GTPase and Ca2+-sensing domains, place this protein in the very centre of a signalling network addressed to co-ordinate a mitochondrial response to support any cellular process that may require a specific supply of energy or a particular Ca2+-buffering capacity.

5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders: A joint Biochemical Society/European Society for Neurochemistry Focused Meeting held at the University of Bath, U.K., 23–26 June 2013. Organized and Edited by Marcus Rattray (University of Bradford, U.K.) and Rob Williams (University of Bath, U.K.).

Abbreviations

     
  • dMiro

    Drosophila Miro

  •  
  • Mfn

    mitofusin

  •  
  • Miro

    mitochondrial Rho

  •  
  • OGT

    O-GlcNAc transferase

  •  
  • OIP

    OGT-interacting protein

  •  
  • OMM

    outer mitochondrial membrane

  •  
  • PD

    Parkinson’s disease

  •  
  • PINK1

    PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1

  •  
  • SNPH

    syntaphilin

  •  
  • TOM

    translocase of outer mitochondrial membrane

  •  
  • TRAK

    trafficking protein, kinesin-binding

  •  
  • VDAC

    voltage-dependent anion channel

Funding

J.T.K. is the recipient of a Lister Institute for Preventive Medicine award and an European Research Council starting grant (Fuelling Synapses). Grants have also been awarded by the Medical Research Council and the Wellcome Trust.

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