InteractiveFly: GeneBrief

Mitochondrial Rho: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

EMS-induced mutations have been identified in Drosophila Miro, an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in Miro mutant muscles and neurons are abnormally distributed. Instead of being transported into axons and dendrites, mitochondria accumulate in parallel rows in neuronal somata. Mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, but neurotransmitter release and acute Ca²⁺ buffering is impaired only during prolonged stimulation. Neuronal, but not muscular, expression of Miro in Miro mutants restores viability, the transport of mitochondria to NMJs, the structure of synaptic boutons, the organization of presynaptic microtubules, and the size of postsynaptic muscles. In addition, gain of Miro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, these findings suggest that Miro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals (Guo, 2005).

Mitochondria are critical for aerobic respiration, Ca²⁺ homeostasis, and apoptosis. The activity and subcellular distribution of mitochondria are not static, but adaptable to physiological stresses and changes in the metabolic demands of the cell. Control of mitochondrial distribution is believed to be especially important for neurons because of their high metabolic demands and their complex polar morphology (e.g., axons, dendrites, and synapses). Neuronal mitochondria are enriched in regions of intense energy consumption, including mobile growth cones, nodes of Ranvier, and synaptic terminals. For example, synaptic terminals may consume up to 10% of the total energy required for neuronal signaling. Consistently, loss of mitochondria from photoreceptor terminals is associated with blindness and a failure of synaptic transmission in the Drosophila mutant milton (Stowers, 2002). In cultured hippocampal neurons, the number of dendritic mitochondria correlates with the number and plasticity of dendritic spines and synapses. In addition, mitochondrial transport responds specifically to growth cone activity and nerve growth factor signaling. Together, these studies underscore the functional significance of controlling the subcellular targeting of mitochondria (Guo, 2005).

The molecular mechanisms that control the subcellular distribution of mitochondria involve long-distance transport along microtubules (MTs), which provide polar tracks for plus end-directed kinesin and minus end-directed dynein motor proteins. Mitochondria, like vesicles, display bidirectional motion where the cargo stops, starts, and often changes direction (Welte, 2004; Vale, 2003; Hollenbeck, 1996; Hollenbeck, 2005). Assuming that mitochondria are simultaneously attached to two opposing motors (De Vos, 2003), then net movement in one direction may be determined by the motor with the overall highest activity or, alternatively, only one of the motors may be engaged with the MT track (Welte, 2004). However, the molecular mechanisms that achieve specificity, directionality, and temporal control of mitochondrial transport in response to intracellular signals remain poorly understood. A better understanding is highly desirable, as vulnerabilities of transport systems to genetic and/or environmental insults often result in human neurological or neurodegenerative diseases (Guo, 2005 and references therein).

The mitochondrial Rho-GTPase (Miro) protein family may have the potential to link cellular signaling pathways with mitochondrial dynamics and function. Orthologs of human Miro (hMiro1 and 2) are all characterized by the presence of two different GTPase domains and two Ca²⁺ binding EF hand domains (Frederick, 2004; Fransson, 2003). Overexpression of constitutively active human Miro in COS7 cells causes perinuclear mitochondrial aggregates and increased apoptosis (Fransson, 2003). Yeast cells lacking Miro (Gem1p) reveal a collapse of the tubular mitochondrial network that is not caused by defects in mitochondrial fission and/or fusion. Genetic studies further indicate that both GTPase domains and both EF-hand motifs are required for Gem1p function (Guo, 2005).

A genetic screen was carried out for mutations that affect synaptic structure and function in Drosophila. From this screen, lethal mutations were identified in Drosophila Miro (Miro). Molecular and genetic analyses suggest that Miro is required for anterograde axonal transport of mitochondria and their proper subcellular distribution. Although Miro mutant motor terminals are structurally deformed and chronically lack mitochondria, they can sustain neurotransmitter release at basic levels, but fatigue during high-frequency stimulation. While presynaptic Ca²⁺ resting levels are elevated, abnormal accumulations of Ca²⁺ occur only during prolonged periods of repetitive stimulation (Guo, 2005).

Thus Miro is essential for the proper distribution of mitochondria into dendrites and axon terminals. Although the true cause for the slim body, the smaller muscle size, the progressive deterioration of locomotion, and the premature death of Miro mutant larvae remains to be established, it is remarkable that all of these deficiencies have an exclusively neuronal origin, since they are rescued by neuronal expression of normal Miro (Guo, 2005).

Motor nerve terminals of Miro null mutants lack mitochondria, but contain relatively undisturbed numbers of synaptic vesicles. Instead of being transported, mitochondria accumulate like 'strings of pearls' in neuronal somata, which indicates a traffic jam of mitochondria that are connected to microtubles but cannot be transported into axons and dendrites. This defect is unlikely to be caused by a structural or functional deficit of mitochondria, since they exhibit neither an ultrastructural defect nor a reduced mitochondrial membrane potential. The altered microtuble organization is also unlikely to cause the defect. Since both mitochondria and vesicles employ kinesin motors for anterograde transport, one would expect that a defect of microtubles affects mitochondrial and vesicular transport in qualitatively and quantitatively similar ways. However, neither the loss nor the gain of Miro activity affects transport mechanisms of these organelles in the same way. Hence, it is unlikely that the altered organization of microtubles is the primary cause of the defect in mitochondrial transport (Guo, 2005).

The mitochondrial accumulations of Miro mutants are similar to the perinuclear accumulations of microtuble-associated mitochondria in mouse mutants of the mitochondria-associated kinesin heavy chain Kif5B (Tanaka, 1998). Mutations in Drosophila Milton, a potential adaptor protein that links mitochondria to kinesin motors, also causes mitochondrial accumulations in photoreceptor somata and a loss of mitochondria at photoreceptor terminals (Gorska-Andrzejak, 2003; Stowers, 2002). Accordingly, the mitochondrial clusters in Miro mutants are consistent with an impairment of the anterograde transport machinery (Guo, 2005).

The effects of Miro overexpression further support a role in regulating mitochondrial transport. While expression of normal Miro protein in Miro mutants restored the transport of mitochondria out of somata into axons, it also caused an abnormal accumulation of mitochondria in terminal boutons of NMJs. This new defect is apparently induced by a gain of activity since similar alterations were also observed upon overexpression of Miro in otherwise wild-type flies. Hence, loss and gain of Miro activity consistently alter the subcellular distribution of mitochondria in neurons in opposite ways: while loss of Miro arrests mitochondrial transport in cell bodies, gain of Miro activity accumulates mitochondria at terminal synaptic boutons of motor axons. Three possibilities may explain the gain-of-function phenotype: excessive anterograde movement, a failure to terminate anterograde movement, or a failure in identifying appropriate subcellular target sites. Although this study cannot distinguish among these possibilities, the opposing effects induced by loss and gain of Miro activity consistently suggest that Miro is required for anterograde transport of mitochondria to ensure a normal subcellular distribution. Such a role is consistent with the suggestion that small GTPases, but not heterotrimeric G proteins, regulate organelle transport along axonal microtubles (Guo, 2005).

Assuming that Miro proteins, like other small GTPases, provide 'signaling nodes' that integrate signals to coordinate multiple downstream events, Miro proteins may provide an interface between cellular signaling pathways and mitochondrial transport to control the subcellular distribution of mitochondria. Such a signal-integrating role is supported by a structural analysis of yeast Miro, showing that both GTPase domains and both Ca²⁺ binding EF-hands are required for Gem1p function (Frederick, 2004). Accordingly, guanine nucleotide exchange and hydrolysis factors or Ca²⁺ binding could potentially modulate Miro activity and mitochondrial mobility at 'turnaround' or stationary 'target zones'. However, further work will be required to resolve how mitochondrial transport might be mediated by an independent or cooperative action of Miro GTPase and EF-hand domains (Guo, 2005).

The subcellular distribution of mitochondria in neurons is assumed to be important for neuronal physiology, but direct evidence is scarce. This study has uncovered a connection between presynaptic mitochondria and the structure of NMJs. The loss of Miro activity results in the loss of presynaptic mitochondria, an increased number of synaptic boutons, and an altered bouton structure, suggesting that presynaptic mitochondria and/or mitochondrial proteins are important for structurally organizing NMJs. However, presynaptic mitochondria at NMJs are not required for the formation of new synaptic boutons, which contrasts with the role that has been suggested (Li, 2004) for mitochondria in dendritic spine formation (Guo, 2005).

Li (2004) manipulated the GTPases Drp1 (dynamin-related protein 1) and Opa1 (optic atrophy), both of which alter the morphology and distribution of mitochondria by controlling mitochondrial fission and fusion. Manipulations that decreased the number of mitochondria in dendrites of cultured hippocampal cells reduced the number of synapses and dendritic spines. Reciprocally, increasing dendritic mitochondrial content or activity caused an increase in the number of synapses and dendritic spines, suggesting that dendritic mitochondria are rate limiting for the support of synapses (Li, 2004). The contrasting lack of any correlation between synapse number and the number of presynaptic mitochondria at Miro mutant NMJs may indicate different roles of pre- and postsynaptic mitochondria or differences between NMJs and central synapses. There also may be a critical difference between a complete absence and a reduction of mitochondria, since reduced numbers of mitochondria in hippocampal dendrites do not affect dendritic patterns (Li, 2004), while the loss of mitochondria at Miro mutant NMJs caused significant presynaptic structural changes. Consistently, loss of Drp1 function in Drosophila neither alters the structure nor increases the number of synaptic boutons at NMJs (see Verstreken, 2005), although the number of mitochondria is much reduced (Guo, 2005).

The abnormal structure of synaptic boutons at Miro mutant NMJs may be linked to the abnormal organization of presynaptic microtubles, as indicated by the loss of microtuble loops and bundles. The cause of the abnormal presynaptic microtuble organization at Miro mutant NMJs remains unknown, but chronic ATP depletion may be excluded because missing microtubles were also observed in Miro mutant muscles in which the general prevalence of abnormally clustered mitochondria is unlikely to result in areas of ATP depletion (Guo, 2005).

Motor nerve terminals of Miro mutants, with their chronic absence of mitochondria, provide interesting insights into the role of presynaptic mitochondria in synaptic function. The high energy costs of synaptic transmission, arising mostly from the ATP dependence of synaptic vesicle exo- and endocytosis, ion pumps, transporters, and transmitter metabolism, suggest that synaptic function requires the continuous presence of mitochondria. Although the presynaptic defects of Miro mutant nerve terminals support this notion, it is still surprising how well these synapses can adjust to the chronic lack of mitochondria. Since larval Miro motor terminals can maintain basic synaptic function for at least several days, the question arises of how these terminals are supplied with ATP. Potentially, diffusion of ATP from the cell body through the motor axon, together with local glycolysis, could substitute for oxidative ATP synthesis by presynaptic mitochondria (Guo, 2005).

Motor nerve terminals that chronically lack mitochondria provide a unique opportunity for studying the role of mitochondrial Ca²⁺ uptake in transmitter release. Acute pharmacological inactivation of mitochondria at NMJs of frog, lizard, and mouse suggest that mitochondrial Ca²⁺ uptake critically limits the accumulation of presynaptic Ca²⁺ during repetitive stimulation, thereby preventing desynchronization of evoked release (David, 2003 and Talbot, 2003). Recordings of presynaptic Ca²⁺ and transmitter release from Miro NMJs reveal remarkable differences between nerve terminals that acutely or chronically lack mitochondrial function. Chronic absence of mitochondria causes nanomolar increases in presynaptic Ca²⁺ levels during comparable repetitive stimulation, but not micromolar increases as reported for acute manipulations (David, 2003, Talbot, 2003, Suzuki, 2002; David, 1999; Tang, 1997). Since mitochondrial Ca²⁺ uptake occurs at Drosophila motor nerve terminals, it is suggested that mitochondrial Ca²⁺ uptake for Ca²⁺ homeostasis at these nerve terminals is either not required or easily compensated by other mechanisms. Assuming that the latter occurs, then it is surprising how powerful and effective these compensatory mechanisms are. A likely candidate for compensation is the endoplasmic reticulum, which interacts with mitochondria, exchanges Ca²⁺ with mitochondria, and can act as a Ca²⁺ sink (Rizzuto, 2000). Alternatively, Na⁺/Ca²⁺ exchange or membrane Ca²⁺ ATPase activities may be altered (Guo, 2005).

This study did not reveal a large, activity-dependent increase in presynaptic Ca²⁺ levels that correlated with the desynchronization of transmitter release, which contrasts with other studies in which mitochondria were acutely inactivated (David, 2003; Talbot, 2003). Consequently, the desynchronization of transmitter release in Miro mutants may be due either to the abnormal synaptic structure or the lack of mitochondrial ATP production impairing mobilization of synaptic vesicles in the reserve pool (Verstreken, 2005). In conclusion, the results reveal that the chronic loss of presynaptic mitochondria at Drosophila NMJs has severe consequences for presynaptic structure and neurotransmitter release, but unexpectedly mild consequences for presynaptic Ca²⁺ homeostasis (Guo, 2005).

Vimar is a novel regulator of mitochondrial fission through Miro

As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF). However, the GEF for Miro has not been identified. While studying mitochondrial morphology in Drosophila, it was incidentally observed that the loss of vimar, a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro in vitro, it was speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF) vimar mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF) Miro transgene; whereas a GOF vimar transgene enhanced Miro function. In addition, vimar lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, mitochondrial fission was found to play a functional role in high-calcium induced necrosis, and a LOF vimar mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miro's effect on mitochondrial morphology is altered upon binding with calcium. In addition, a PINK1 mutant, which induced mitochondrial enlargement and had been considered as a Drosophila model of Parkinson's disease (PD), caused fly muscle defects, and the loss of vimar could rescue these defects. Furthermore, it was found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional (Ding, 2016).

Mitochondrial fission, fusion and transport play important roles for the function of this organelle. The balance between fusion and fission controls mitochondrial morphology, which is mediated by series of large dynamin-related GTPases. Among these GTPases, mitofusin1/mitofusin2 (MFN1/MFN2) and optic atrophy protein1 (OPA1) are the core components that are responsible for mitochondrial fusion, whereas dynamin-related protein 1 (Drp1) is the core component that is responsible for mitochondrial fission. In addition to these GTPases in dynamin-related family, mitochondrial Rho (Miro), an atypical member of the Rho small GTPase family, has a well-known function of transporting the mitochondria along microtubules. Miro also regulates mitochondrial morphology via inhibition of fission under physiological Ca2+ conditions, although the mechanism is not that clear. Large GTPases such as dynamin-like GTPase family members hydrolyze GTP and exchange GTP and GDP without the assistance from other regulators. However, members of the small GTPase family often require other proteins to help release their tightly bound GDP or enhance their low GTPase activities. These proteins are referred to as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. To date, most small GTPases require unique GEFs or GAPs (Ding, 2016).

An understanding of the regulation of mitochondrial dynamics may help address many human diseases. For instance, mutations in OPA1 or MFN2 result in dominant optic atrophy or Charcot-Marie-Tooth neuropathy type 2A. Abnormal mitochondrial fission also promotes aging and cell death. In necroptosis, the formation of the necrosome promotes mitochondrial fission through dephosphorylation of Drp1. In neuronal excitotoxicity, calcium ions are overloaded, resulting in reduced levels of the MFN2 protein, which enhances mitochondrial fission and leads to neuronal necrosis In addition, other components such as Miro may participate in this process. Miro has two EF hand motifs that bind calcium; thus, Miro can couple calcium increase with reduced mitochondrial motility to meet the locally increased energy demands. Interestingly, Miro also promotes fission in the presence of excess calcium, which is distinct from its inhibitory role in fission under normal calcium concentrations. It is unclear whether Miro plays a functional role in neuronal necrosis (Ding, 2016).

The mitochondrial morphology represents a transient balance between mitochondrial fusion and fission. Using a systematic genetic screen in yeast covering approximately 88% of genes, 117 genes that regulate mitochondrial morphology were identified. Similarly, a screen of 719 genes that are predicted to encode mitochondrial proteins in worms demonstrated that more than 80% of these genes regulate mitochondrial morphology. Although many genes may regulate mitochondrial morphology, their relationships to the core mitochondrial fusion and fission components are unclear (Ding, 2016).

In studying mitochondrial morphology, it was accidently discovered that the loss of vimar (visceral mesodermal armadillo-repeats), which encodes an atypical GEF, promoted mitochondrial fission in Drosophila flight muscle cells. Furthermore, it was found that vimar was capable of interacting with Miro in vitro. Genetically, vimar required normal GDP- or GTP-bound activity of Miro to affect mitochondrial morphology, suggesting vimar is likely the Miro GEF. In addition, it was found that the Miro/Vimar complex suppressed mitochondrial fission during necrosis and mitochondrial fusion in PINK1 mutant model of Parkinson's disease (PD), making vimar a potential drug target (Ding, 2016).

Mitochondrial function can be assessed by the enzymatic activity of citrate synthase (CS), the first enzyme in the Krebs cycle that converts acetyl-CoA and oxaloacetate to citrate. In cultured Drosophila S2 cells, vimar knock down by RNAi resulted in reduced CS activity, indicating that vimar may positively regulate mitochondrial function. Because mitochondrial fission has generally been associated with reduced mitochondrial respiration, the decreased CS activity may be a result of mitochondrial fission. Consistent with this notion, the results demonstrated that the LOF of vimar promoted mitochondrial fission. In addition, a GOF vimar transgene had a minimal effect on mitochondrial morphology, indicating that vimar activity might be saturated under normal physiological conditions (Ding, 2016).

Because Vimar has been predicted to be a GEF, it was hypothesized that Vimar may regulate mitochondrial morphology by affecting a small GTPase, which requires a GEF to help with the GTP/GDP exchange process. Interestingly, Miro is one such small GTPase that is known to play important roles in mitochondrial fission and transport. It is proposed that Vimar and Miro may function as a complex. First, a fraction of the Vimar protein was localized to the mitochondria, possibly indicating a functional role on mitochondria. Interestingly, the mitochondrial localization of Vimar seems not dependent on Miro, because LOF Miro did not affect the mitochondrial fraction of Vimar. This indicates that Vimar may directly bind with mitochondria or through other scaffolding proteins. Second, Vimar and Miro could physically interact with each other, at least in vitro. Their interaction seems not affected by the GTPase activity of Miro, because the constitutively GDP- or GTP-bound Miro mutants did not affect their interactions. Third, vimar genetically interacted with Miro. This included the result demonstrating that the LOF vimar mutant reduced the effect of Miro on mitochondrial fission inhibition and the GOF vimar transgene had the opposite effect. Moreover, in the constitutive GFP-bound or GDP-bound Miro mutants, the effect of the GOF or LOF vimar was abolished. Therefore, Vimar requires the normal GDP/GTP binding activity of Miro to function. It is also known that Miro1 overexpression increase mitochondrial size partially by suppression of the Drp1 function. Consistently, increased mitochondrial fission in the LOF of Miro or vimar was abolished by loss of Drp1, suggesting the Miro/vimar complex depends on Drp1 to regulate mitochondrial morphology (Ding, 2016).

Familial PD caused by mutations in PINK1 or Parkin results in a series of mitochondrial dysfunctions, particularly the failure to eliminate damaged mitochondria through mitophagy. In these PINK1 or Parkin mutants, the key proteins involved in mitochondrial fusion and fission, such as Marf/Mitofusin and Miro, accumulate. In the PINK1 mutant flies, the flight muscle is damaged, resulting in wing posture defects. Similarly, it was observed that Miro overexpression in the flight muscle resulted in a strong wing posture defect. This result may explain the wing posture defect in the PINK1 mutant, in which the levels of the Miro protein are increased. This study demonstrated that the LOF of vimar could rescue the wing defect in the PINK1 mutant, consistent with the hypothesis that vimar functions through Miro (Ding, 2016).

When the intracellular calcium level is high, Miro switches from promoting mitochondrial fission inhibition to enhancing mitochondrial fission. The mechanism for this switch is unclear, although alterations of Drp1 function could be one possibility. Interestingly, Gem1, the yeast homolog of Miro GTPase, has been reported to function as a negative regulator for ER-mitochondria contacts, where Drp1 aggregates and cleaves mitochondria into smaller units. This may serve as the mechanism for Miro to regulate mitochondrial morphology via Drp1. In addition to affect mitochondrial fission, Miro also regulates mitochondrial transport in a calcium dependent manner. For mitochondrial transport, Miro forms protein complexes with Milton, a kinesin adaptor, and with motor proteins, such as kinesin and dynein. In high calcium conditions, Miro alters its binding patterns and results in reduced transport activity. Based on these reports, it is proposed that the Miro/Vimar complex acts together to affect mitochondrial morphology: at normal condition, Miro/Vimar inhibits fission via Drp1; at high calcium state, Ca²⁺ bound Miro switches its function to promote fission. Indeed, Vimar responds to the calcium change in the same way as Miro. In addition, the data demonstrated that knocking down RAP1GDS1 and Miro1 increased mitochondrial fission and could rescue calcium overload induced necrosis, similar to the loss of Vimar or Miro in Drosophila. These data support the hypothesis that RAP1GDS1 is the mammalian homolog of Vimar, supporting a previous prediction (Ding, 2016).

Mitochondrial fission plays important role in apoptosis by promoting mitochondrial outer-membrane permeabilization (MOMP) to release cytochrome c from the mitochondria. The use of the Drp1 inhibitor mdivi to block fission has been shown to be an effective treatment for stroke, and the function of mitochondrial fission on necrotic cell death has been well documented. The uncertainty lies in the lack of genetic evidence and downstream mechanism of mitochondrial fission in necrosis. The current data demonstrated that mitochondrial fragmentation occurs in necrotic neurons, and the LOF Drp1 and vimar mutants both suppressed neuronal necrosis (Ding, 2016).

Much evidence suggests that the mitochondrial fusion and fission defects are directly linked to many human diseases, and strategies that target the Miro/vimar complex may affect a broad spectrum of diseases. For instance, mutations in the fragile X mental retardation 1 (FMR1) gene, which result from expansion of trinucleotide repeat in the 5' untranslated region, often cause enhanced mitochondrial fission and mental retardation syndrome. Likewise, aberrant mitochondrial fusion was observed in a Drosophila Alzheimer's disease model induced by the ectopic expression of a human tau mutant (tau^R406W). In this case, the tau mutant may promote excessive actin stabilization to decrease Drp1 recruitment to the mitochondria, which results in excessive mitochondrial fusion and neurodegeneration. Due to the dual function of the Miro/Vimar complex in high-Ca²⁺ induced necrosis and PINK1 mutant induced PD, a drug to target this complex may benefit both disease states. As a modulator, it may be safer to target Vimar/ RAP1GDS1 (Ding, 2016).

REGULATION

PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. This study reports that two Parkinson's disease (PD) proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 was shown to act upstream of Parkin, but the mechanism corresponding to this relationship has been unknown. This study proposes that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome (Wang, 2011).

Although genetic and cell biological data have placed PINK1 upstream of Parkin in a pathway that regulates mitochondrial morphology and degradation, the relationship of the two enzymes has been obscure. One model proposes that Parkin is a PINK1 substrate activated by phosphorylation, but others have failed to find this phosphorylation. Findings in this study indicate an alternative model: PINK1 and Parkin bind to the same target and its phosphorylation by PINK1 allows Parkin, presumably acting as an ubiquitin-ligase, to designate that protein for removal from the mitochondrial membrane and proteasomal degradation. Indeed, hMiro1 and hMiro2 were shown to be among a list of proteins down-regulated by Parkin overexpression and CCCP. The ability of Parkin to bring about Miro degradation is consistent with its ability to ubiquitinate mitofusin and thereby to cause its degradation through the sequential action of p97/VPC and the proteasome. Interestingly, Miro and mitofusin interact with one another and their shared interaction with Parkin suggests coordinated regulation (Wang, 2011).

Mitochondrial motility is especially critical to neurons where it may take days for a mitochondrion to move between the cell body and a distant axonal or dendritic ending. The need for mitochondria to undergo turnover, as well as their redistribution to balance changes in local energy demand, make mitochondrial movement an important on-going and regulated process. The mitochondrion-specific adaptor proteins, Miro and Milton, are control points for this motility. Damaged mitochondria in cell lines selectively recruit Parkin and are in turn targeted for mitophagy. In contrast to an earlier report, it was found that this recruitment also occurs in axons; when highly expressed, YFP-Parkin is observed on mitochondria without depolarization (consistent with its ability to arrest mitochondrial motility upon overexpression), but with lower expression levels it is recruited to mitochondria by treatment with Antimycin A. Parkin recruitment is initiated by the depolarization-induced stabilization of PINK1 on the mitochondrial surface and PINK1 is also upstream of Parkin in regulating mitochondrial morphology. This relationship also holds for mitochondrial motility. PINK1 arrests mitochondrial motility in wildtype but not Parkin−/− mice or Parkin RNAi flies. Mitochondrial depolarization with CCCP causes the degradation of Miro in a Parkin-dependent manner. Similarly, PINK1 expression causes the degradation of Miro in Parkin expressing cells, but not in Parkin-lacking HeLa cells (Wang, 2011).

In previous genetic studies of PINK1 and Parkin, differences are noted between mice and Drosophila. Drosophila loss of function mutants exhibit profound defects in mitochondrial morphology that are seen in knockout mice only when neurons are additionally stressed. Differences were also observed in this study between Drosophila and murine models. In both, PINK1 or Parkin overexpression arrests mitochondria and in both Parkin is required downstream of PINK1. However, in Drosophila neurons, RNAi knockdown of PINK1 or Parkin increases mitochondrial motility whereas differences of motility in murine Parkin−/− neurons are not statistically significant. These differences may reflect a difference in how the species employ the pathway: in mammals, it may be strictly reserved for the response to mitochondrial depolarization whereas in the fly, whose short lifespan may make mitochondrial damage less critical, it may contribute to the ongoing turnover of proteins that participate in mitochondrial dynamics (Wang, 2011)

The ability of Parkin overexpression to alter mitochondrial motility in the presence of PINK1 RNAi or mitochondrial morphology in a PINK1 null background indicates that, although PINK1 can stimulate Parkin function, Parkin can act independently as well. Results from this study do not show if Parkin is effective because of residual PINK1 in the RNAi-expressing cells, because other kinases can also activate Miro as a Parkin substrate, or because elevated levels of Parkin can lead to Miro degradation even in the absence of a phosphorylation. Thus, PINK1 is likely to enhance Parkin function but probably is not required (Wang, 2011).

The observation that two PD-associated genes encode regulators of mitochondrial motility is consistent with other findings linking misregulation of mitochondrial dynamics to neurodegeneration. Changes in mitochondrial distribution, transport, and dynamics are implicated in Charcot-Marie-Tooth, Amyotrophic Lateral Sclerosis, Alzheimer’s and Huntington’s diseases. These findings underscore the importance of mitochondrial dynamics for supplying distal regions with sufficient energy and Ca2+-buffering capacity, compensating for changes in energy demand, refreshing older mitochondria through fusion with newly-synthesized mitochondria, and clearing damaged mitochondria (Wang, 2011).

Clarification of the relationship of PINK1 and Parkin supports the view that PD is a mitochondrial disorder. In the etiology of PD, the regulation of Miro levels may be significant. Either through a specific sorting pathway or as a consequence of the random reassortment of mitochondrial proteins that occur with repeated fusion and fission, some organelles or fragments of the organelle will arise in which the burden of dysfunctional proteins is sufficient to compromise the membrane potential. The resulting stabilization of PINK1 on the surface and targeting of Miro, mitofusin, and other proteins for Parkin action and degradation, will bring about the sequestration and eventual engulfment of that dysfunctional organelle. Sequestration and mitophagy thereby prevent further cellular damage due to reactive oxygen species and enable the cellular complement of mitochondria to be replenished by healthier organelles. The greater the stresses on mitochondria, the more acute the need for this clearance pathway. The heightened sensitivity of the dopaminergic neurons in the substantia nigra to disruption of this ubiquitous pathway may therefore reflect exceptional challenges for mitochondria in these cells. Those stresses may include the susceptibility of dopamine to oxidation and high rates of Ca2+ influx. When this quality control mechanism is defective in patients carrying mutations in either gene, damaged mitochondria will retain Miro and mitofusin, and therefore may move about in the neuron and, through fusion reactions, reintroduce damaged components to otherwise healthy organelles rather than undergo mitophagy (Wang, 2011).

PINK1-mediated phosphorylation of Miro inhibits synaptic growth and protects dopaminergic neurons. in Drosophila

Mutations in the mitochondrial Ser/Thr kinase PINK1 cause Parkinson's disease (see Drosophila as a Model for Human Diseases: Parkinson's disease). One of the substrates of PINK1 is the outer mitochondrial membrane protein Miro, which regulates mitochondrial transport. This study uncovered novel physiological functions of PINK1-mediated phosphorylation of Miro, using Drosophila as a model. Endogenous Drosophila Miro (DMiro) was replaced with transgenically expressed wildtype, or mutant DMiro predicted to resist PINK1-mediated phosphorylation. The expression of phospho-resistant DMiro in a DMiro null mutant background was found to phenocopy a subset of phenotypes of PINK1 null. Specifically, phospho-resistant DMiro increased mitochondrial movement and synaptic growth at larval neuromuscular junctions, and decreased the number of dopaminergic neurons in adult brains. Therefore, PINK1 may inhibit synaptic growth and protect dopaminergic neurons by phosphorylating DMiro. Furthermore, muscle degeneration, swollen mitochondria and locomotor defects found in PINK1 null flies were not observed in phospho-resistant DMiro flies. Thus, this study established an in vivo platform to define functional consequences of PINK1-mediated phosphorylation of its substrates (Tsai, 2014).

Mutations in the Ser/Thr kinase PINK1 (PTEN-induced Putative Kinase 1) cause Parkinson's disease (PD), one of the most common neurodegenerative disorders. Emerging evidence suggests that PINK1 functions upstream of another PD-associated protein, the E3 ubiquitin ligase Parkin, to clear damaged mitochondria via mitophagy. How PINK1 primes cytosolic Parkin for mitophagy remains unclear, although PINK1-mediated phosphorylation of Parkin or ubiquitin may be involved. Previous work has shown that PINK1, in cooperation with Parkin, also regulates mitochondrial trafficking by controlling turn-over of Miro (Wang, 2011), an outer mitochondrial membrane (OMM) protein that anchors the kinesin and dynein motors to mitochondria. Work in cultured cells has demonstrated that mitochondrial depolarization or damage stabilizes PINK1 on the OMM. Concomitantly, PINK1 phosphorylates Miro, which then activates proteasomal degradation of Miro in a Parkin-dependent manner and arrests mitochondrial transport. This may serve as a critical step in quarantining damaged mitochondria prior to their degradation via mitophagy. However, the physiological significance of PINK1-mediated phosphorylation of Miro in vivo has not yet been determined (Tsai, 2014).

Recent studies have shown that Mitofusin, another OMM protein, is also a common substrate for both PINK1 and Parkin. Mitofusin facilitates mitochondrial fusion, and mitochondrial damage rapidly degrades Mitofusion causing mitochondria to fragment prior to mitophagy. PINK1 also phosphorylates the anti-apoptotic protein Bcl-xL on the OMM of depolarized mitochondria, not to regulate mitophagy, but to prevent cell death. In addition to the PINK1 OMM substrates Miro, Mitofusin and Bcl-xL, PINK1 mediates phosphorylation of the mitochondrial chaperon TRAP1 and the Serine protease HtrA2, which are both located in the mitochondrial inter-membrane space. This wide range of the potential substrates of PINK1 suggests that it may have multiple cellular functions (Tsai, 2014).

The consensus target sequence for phosphorylation by PINK1 has remained elusive. To date, Miro is the only PINK1 mitochondrial substrate whose phosphorylation residues have been determined (b4 (Wang, 2011). Two Drosophila Miro (DMiro) peptides with a high degree of similarity to the human sequence were identified as potential targets of PINK1-mediated phosphorylation in vitro. This study has determined the critical role of PINK1 phosphorylation sites on DMiro for maintaining neuronal homeostasis and protecting dopaminergic (DA) neurons in vivo (Tsai, 2014). <>Although several phosphorylation substrates of the mitochondrial Ser/Thr kinase PINK1 have been identified, the precise functional consequences of PINK1-mediated phosphorylation in vivo remain unclear. This study report that in Drosophila PINK1 may inhibit mitochondrial movement and synaptic growth at larval NMJs, and protect DA neurons in adult brains, by phosphorylating the atypical GTPase DMiro (Tsai, 2014).

Drosophila is a robust genetic and cellular tool for modeling human neurodegenerative diseases. Loss of PINK1 in Drosophila mimics many aspects of PD pathology, including a severe loss of DA neurons, which is a hallmark of PD. However, few of the molecular and cellular mechanisms underlying the behavioral and cellular phenotypes of PINK1 null mutant flies have been clearly defined. This study identifies that DMiro^{S182A,S324A,T325A}, which is predicted to resist PINK1-mediated phosphorylation, causes increased mitochondrial movement, synaptic overgrowth, and loss of DA neurons. All three of these defects are also observed in PINK1 null mutant flies. Hence, this work suggests that Miro is a crucial substrate for causing these phenotypes by mutant PINK1. This study opens a new door to fully dissect PINK1 functions by studying its individual substrates. Since PINK1-related hereditary PD shares symptomatic and pathological similarities with the majority of idiopathic PD, such work will advance understanding of the cellular and molecular underpinnings of PD's destructive path (Tsai, 2014).

Extensive studies using cell cultures have established a critical role for PINK1 in damage-induced mitophagy. PINK1/Parkin-dependent regulation of mitochondrial transport by controlling Miro protein levels on mitochondria is likely a key step prior to initiating mitophagy in cultured neurons. This study show that PINK1-mediated phosphorylation of DMiro is required for normal mitochondrial movement in axon terminals, synaptic growth, and the neuroprotection of DA neurons. Importantly, loss of PINK1-mediated phosphorylation of DMiro has no significant effect on the mitochondrial membrane potential, excluding the possibility that the observed phenotypic effects are due to an impairment of mitophagy and an accumulation of damaged mitochondria. Accordingly, under these conditions PINK1-mediated phosphorylation of DMiro may not be required for mitophagy. However, this does not necessarily contradict its mitophagic role; rather, this represents circumstances under which its mitophagic role is dispensable. It is tempting to speculate that an efficient regulation of mitophagy is more critical in aging neurons (Tsai, 2014).

These studies identify a conserved site in human and Drosophila Miro, MiroSer156/DMiroSer182, to be a main residue for PINK1-mediated phosphorylation. Additional conserved sites were found in DMiro that may have a cooperative role. Future studies determining their functions in mammalian systems are warranted to confirm if a similar regulatory mechanism is at play. This study suggests that these PINK1 phosphorylation sites in DMiro are not absolutely required for the subsequent Parkin-dependent degradation of DMiro, because when harsh treatment of mitochondrial uncoupler CCCP is applied, the phospho-resistant DMiro^{S182A,S324A,T325A} is degraded. The failure of DMiro^{S182A,S324A,T325A} to prevent degradation under this condition might be due to PINK1-mediated phosphorylation on other sites that promote DMiro degradation, or due to activation of additional mechanisms. In two recent studies, Miro^S156A is significantly degraded by co-expression of PINK1 and Parkin in addition to CCCP treatment in Hela cells, or by overexpression of Parkin together with Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, another mitochondrial uncoupler) treatment in SH-SY5Y cells; whereas in a previous study, Miro^S156A is resistant to degradation when only PINK1 or Parkin is individually expressed in HEK293T cells (Wang, 2011). This again suggests that if the PINK1/Parkin pathway is overwhelmingly activated, mutating the few known PINK1-mediated phosphorylation residues in Miro is not sufficient to prevent its degradation (Tsai, 2014).

Why is mitochondrial motility increased in "DMiro^null, da > DMiro^{S182A,S324A,T325A}"? DMiro^{S182A,S324A,T325A} is resistant to PINK1/Parkin-mediated degradation, which may lead to more DMiro^{S182A,S324A,T325A} accumulation on mitochondria. Unexpectedly, DMiro^{S182A,S324A,T325A} protein level in "DMiro^null, da > DMiro^{S182A,S324A,T325A}" is not significantly upregulated as compared with DMiro^wildtype in "DMiro^null, da > DMiro^wildtype" using fly whole body lysates. It is likely that PINK1/Parkin-dependent degradation of Miro only occurs in certain cell types, at certain subcellular locations, on certain populations of mitochondria, or under certain circumstances, and thus it is hard to detect a dramatic change using whole body lysates or without overexpression of PINK1/Parkin. Future mechanistic study is needed to test these hypotheses, such as detecting Miro subcellular localization and expression levels in different cell types, in different developmental stages, and with different mitochondrial stresses (Tsai, 2014).

This work highlights the importance of a precise control of mitochondrial movement for neuronal health. Anterograde mitochondrial transport in axons is mediated by a conserved motor/adaptor complex, which includes the motor kinesin heavy chain (KHC), the adaptor protein Milton and the mitochondrial membrane anchor Miro. In the current model, Miro binds to Milton, which in turn binds to KHC recruiting mitochondria to the motors and microtubules. In addition to the transmembrane domain inserted into the OMM, Miro features a pair of EF-hands and two GTPase domains. Miro was also recently found to be a substrate of the Ser/Thr kinase PINK1 and of the E3 ubiquitin ligase Parkin, both mutated in PD. Thus, mitochondrial transport can be regulated by multiple signals upstream of Miro and the motor complex maintaining energy and Ca²⁺ homeostasis in neuronal processes and terminals. For example, loss of PINK1-mediated phosphorylation of DMiro increases local mitochondrial movement at NMJs. In turn, this may disrupt synaptic homeostasis leading to synaptic overgrowth by mechanisms yet to be identified. Similarly, the loss of DA neurons in adult brains could well be a consequence of impaired synaptic homeostasis together with an accumulation of dysfunctional mitochondria. Local signals that regulate mitochondrial transport through Miro must be crucial to supporting neuronal functions. This study elucidates a fundamental biological mechanism demanded by a healthy neuron (Tsai, 2014).

EFFECTS OF MUTATION

Genetic screens were employed to identify previously unknown genes required for axonal and synaptic function in Drosophila. To bypass the inefficiency of lethal F3-screening methods, advantage was taken of the EGUF/Hid method, which produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation. Since 'blindness' is caused by a loss of either phototransduction, nerve excitation, or synaptic function, a screen was carried out for 'blind' flies by assaying the phototactic behavioral response of Drosophila, using the 'counter-current apparatus' (Benzer, 1967). Targeting chromosome 3R, ~13,000 EMS-mutagenized F1 flies were screened and 102 mutations were recovered that disrupt phototaxis, but do not grossly affect eye morphology. Genetic complementation analysis revealed a total of 53 lethal complementation groups, which were further characterized by their genetic map position and their synaptic defects at larval NMJs (Guo, 2005).

The lethal mutation B682 was selected for further analysis because it caused an unusual activity-dependent defect in synaptic transmission and a lack of mitochondria at nerve terminals. Deletion mapping limited the B682 locus to a region containing 17 mostly predicted genes at chromosomal position 95D7-11. An independent genetic screen had also identified four lethal EMS-induced alleles that mapped to 95D7-11 and caused defects in synaptic transmission (Babcock, 2003). Further genetic analysis revealed that B682 failed to complement the lethality of all four alleles, suggesting that all five alleles disrupt the same gene (Guo, 2005).

Identification of the mutated gene was facilitated by genetic fine mapping of the B682 mutation using site-specific male recombination. The data placed the B682 locus between two P insertion sites, containing three predicted genes. Further genetic analysis excluded one flanking gene, leaving only two viable candidates: CG5977, the fly homolog of human Spastin and CG5410, the fly ortholog of human Miro (Guo, 2005).

Sequencing of the spastin and Miro genes in all five alleles, using genomic DNA and RT-PCR-derived cDNA, revealed that four of the alleles (B682, sd23, sd26, and sd32) contained mutations in Drosophila miro (Miro), but not in the spastin gene. One allele, sd10, exhibited mutations in both genes and is molecularly a double mutant. The Miro and dspastin genes are transcribed in opposite directions, but are separated by only ~306 bp. To exclude effects of Miro mutations on spastin expression, spastin RNA expression was examined by RT-PCR. Only the predicted double mutant sd10 showed reduced spastin RNA expression. Together, the molecular and genetic data consistently suggest that the alleles B682, sd23, sd26, and sd32 are single gene mutations of the Miro gene. The premature truncation of Miro protein in B682 and sd32 within the first GTPase domain suggests that both alleles are presumably null mutations. Consistently, phenotypes of homozygous B682 and sd32 mutants are indistinguishable from those of transheterozygous combinations involving the deletion Df(3R)mbc-R (Guo, 2005).

Miro is an essential gene; all examined Miro alleles are recessive larval to early-pupal lethals. Homozygous Miro mutant larvae are slim and exhibit abnormal locomotion. Control larvae crawl steadily in a given direction, showing rhythmic muscle contractions that run over the entire body. In contrast, homozygous Miro larvae tend to wiggle on the spot and gain little distance. Posterior segments of Miro larvae appear to be excessively sluggish and sometimes even paralyzed, while anterior segments are often excessively bent, as if to compensate for failing muscle contractions in the more posterior segments. This abnormal crawling behavior indicates a progressive weakness of the larval body-wall musculature that originates at posterior segments and eventually leads to paralysis (Guo, 2005).

To test whether Miro mutations affect mitochondria, live mitochondria were imaged by visualizing mitochondria selectively in neurons or muscles, using a modified GFP that specifically labels mitochondria (MitoGFP). Neuronal expression of MitoGFP revealed an abnormal distribution of neuronal mitochondria in ventral ganglia of Miro mutant larvae. In contrast to controls, MitoGFP fluorescence in Miro mutants was enriched in neuronal somata, but reduced in the neuropil, reduced in segmental nerves close to the ganglia, and absent in more distal nerve regions. At larval NMJs, presynaptic MitoGFP fluorescence was, in general, not detectable; only rarely mutant NMJs showed one or two punctae of GFP fluorescence while almost all synaptic boutons of control NMJs were labeled. The absence of presynaptic mitochondria at larval NMJs of Miro mutants was confirmed by live dye staining using MitoTracker dyes and by an ultrastructural analysis. Together, these results demonstrate that Miro mutant mitochondria are retained in neuronal cell bodies and are not properly distributed into neuronal processes (Guo, 2005).

Muscle-specific expression of MitoGFP reveals a highly regular and stereotypical pattern of mitochondria in control muscles, which is reminiscent of the pattern revealed by actin-phalloidin stainings. While the distribution of actin is normal in Miro mutant muscles, the distribution of mitochondria is severely disrupted: mitochondria were found in small clusters that were distributed over the entire muscle. The abnormal mitochondrial clusters in mutant muscles were not associated with nuclei, which contrasts with the abnormal, perinuclear mitochondrial clusters in mutant neurons (Guo, 2005).

An ultrastructural analysis provided further insights into the nature of the mitochondrial accumulations in neuronal somata. Sections of proximal larval nerves showed reduced numbers of mitochondria in axons and in the processes of glial cells of Miro mutants in comparison to control larvae. Reduced numbers of mitochondria were also found in the neuropil of Miro mutant ventral ganglia. In contrast, neuronal somata of Miro mutants exhibited increased numbers of mitochondria, while control neuronal somata contained few mitochondria. Specifically, Miro mutant somata showed large, organized clusters of mitochondria, which were variable in size and close to Golgi apparati. Within these clusters, mitochondria were neatly lined up in parallel rows, like 'strings of pearls'. In many cases, the mitochondria within a string showed a similar orientation of their cristae. The occurrence of organized mitochondrial clusters in Miro mutants has two implications: (1) the 'string of pearls' appearance suggests that the clustered mitochondria are associated with the cytoskeleton, presumably with MTs, and (2) it suggests that a MT-associated 'roadblock' or impaired anterograde motor activity may arrest anterograde mitochondrial transport (Guo, 2005).

Structural and/or functional changes in mitochondria often cause a redistribution of mitochondria and could also account for the defect in mitochondrial transport of Miro mutants. However, EM analysis revealed no noticeable ultrastructural defects of mitochondria in Miro mutants. In addition, mitochondria in cultured neurons from both Miro mutant and control ganglia showed similar intensities of MitoTracker staining. Finally, labeling of mitochondria with JC-1 dye showed no abnormalities in the membrane potential of Miro mutant mitochondria. Hence, it is unlikely that structural or functional changes of mitochondria underlie the abnormal transport of mitochondria in Miro mutants (Guo, 2005).

To test whether the arrest of mitochondrial transport in Miro mutants correlates with a defect in vesicular transport, larval nerves were examined for the presence of abnormal accumulations of synaptic vesicles, as such accumulations are typically associated with mutations affecting axonal transport. Immunostainings of Miro mutant larval nerves revealed a significant accumulation of immunopositive punctae for the synaptic vesicle protein cysteine string protein (CSP) and synaptotagmin. However, quantification of synaptic vesicle staining at NMJs revealed no significant differences from controls, suggesting that the defect in vesicular transport is weak and does not limit vesicle supply. Similarly, immunostainings with mAb nc82, a presynaptic marker for synapses, also showed an accumulation of discrete nc82-positive punctae in Miro mutant nerves, while the number of synapses at NMJs was not reduced. Since nc82-positive punctae are very small and sparsely distributed in control nerves, the numerous large punctae in mutant nerves suggest an abnormal accumulation of transported cargo vesicles. While these data suggest that vesicular axonal transport is impaired in Miro mutants, these impairments are also qualitatively and quantitatively different from the defects in mitochondrial transport. Hence, it seems unlikely that both transport defects are caused by the impairment of a common mechanism (Guo, 2005).

Consistent with the smaller body size of Miro mutant larvae, the length of larval body-wall muscles was reduced in homozygous Miro mutants. However, the normalized length of the NMJ innervating the smaller muscle (NMJ/muscle ratio) of Miro mutants was generally increased to ~121% of that in controls. In addition, Miro mutant NMJs exhibit a general increase in the number of boutons to ~148% of that in controls after normalizing to the NMJ length, indicating synaptic overgrowth (Guo, 2005).

Close examination of Miro mutant synaptic boutons reveals an abnormal structure. The average volume of synaptic boutons is reduced, and the distance between individual boutons is shortened such that they were often aggregated in cauliflower-like clusters. Dextran-conjugated dye fillings or double immunostainings revealed individual compartments within these clusters, containing synaptic vesicles and synapses. An ultrastructural 3-D reconstruction from serial sections of synaptic boutons confirmed the smaller size of Miro boutons, their tendency to form densely packed clusters, and the absence of mitochondria. As already indicated by immunostainings, each compartment or bouton within a cluster contained its own set of synapses and synaptic vesicles (Guo, 2005).

To determine whether the altered bouton structure correlates with an abnormal microtubule cytoskeleton, presynaptic microtubules were examined, using anti-HRP and anti-acetylated tubulin double stainings. Control NMJs showed robust presynaptic microtubule bundles extending through the entire NMJ, and large synaptic boutons exhibited easily identifiable microtubule loops. In Miro mutants the number of microtubule loops was significantly reduced at all examined NMJs. In contrast to controls, presynaptic microtubule bundles at Miro mutant NMJs also often fail to extend into the last synaptic bouton of axonal branches. Hence, the altered presynaptic microtubule cytoskeleton of Miro mutant NMJs may contribute to the abnormal synaptic bouton structure (Guo, 2005).

The abnormal microtubule organization could be caused by the loss of Miro activity, the loss of other mitochondrial proteins, or by a depletion of ATP due to the loss of presynaptic mitochondria. To address this, microtubles were examined in Miro mutant muscles where a steep ATP gradient seems unlikely because mitochondrial clusters are uniformly distributed throughout the entire muscle. Microtubles surrounding cell nuclei appear more pronounced in Miro mutant muscles than in those of controls. However, quantification of microtubles surrounding individual nuclei showed no differences, while quantification of peripheral microtubles (arbitrarily defined as microtubles midway between neighboring nuclei) showed a significant reduction of microtuble filament density in Miro mutants, suggesting that microtuble organization in the muscle is altered under conditions in which ATP depletion is unlikely. In contrast, phalloidin staining of actin in muscles was normal in Miro mutants, indicating that Miro mutations selectively affect the microtuble, but not the actin cytoskeleton (Guo, 2005).

It seemed likely that mitochondrial transport requires Miro cell-autonomously. However, the structural defects of Miro nerve terminals may have been caused by the loss of pre- or postsynaptic Miro. To resolve this, normal Miro protein was selectively expressed in either neurons or muscles of Miro mutants, using two independent transgenes, which produced similar results. Remarkably, neuronal, but not muscular, expression of Miro rescues the lethality of Miro mutants, suggesting that Miro activity is essential only for neuronal, but not for non-neuronal, function. In addition, neuronal, but not muscular, expression of Miro restores normal larval body shape and muscle size. Neuronal expression does not rescue the abnormal mitochondrial distribution and/or microtuble organization in Miro mutant muscles. Hence, the smaller body and muscle size of Miro mutant larvae is not caused by muscle abnormalities, but by an abnormally functioning nervous system compromising the function of NMJs or neurosecretory terminals (Guo, 2005).

Neuronal expression of Miro in Miro mutants restores vesicular and mitochondrial transport in neurons such that mitochondria are again present at synaptic terminals of NMJs and in the neuropil of ventral ganglia. Presynaptic expression also restores normal synaptic bouton structure and reverses the absence of presynaptic microtuble loops at NMJs. However, postsynaptic expression of Miro in Miro mutant muscles neither reverses the defects in mitochondrial transport of the presynaptic neuron nor affects the abnormal structure of synaptic boutons and presynaptic microtubles. Together, these data suggest that Miro is required presynaptically to supply synaptic terminals with mitochondria and ensure normal structure of the NMJ (Guo, 2005).

While presynaptic expression of Miro restores mitochondrial transport to Miro mutant nerve terminals, it does not lead to a normal distribution of mitochondria; mitochondria accumulate abnormally in the most distal synaptic bouton of each terminal branch and are present in reduced numbers in most of the remaining boutons. The abnormal accumulation was also observed for DiOC₂(5)-labeled mitochondria, confirming structural mitochondrial integrity. Overexpression of Miro in control neurons showed a similar, excessive accumulation of mitochondria in the terminal bouton of axonal branches, suggesting that this new phenotype is caused by a gain of Miro activity. This gain-of-function phenotype is not observed for synaptic and cargo vesicles since neither synaptic vesicles nor synapses were abnormally distributed. Hence, reducing or increasing Miro activity alters the subcellular distribution of mitochondria such that abnormal mitochondrial accumulations are switched from the cell body to the end of the axon, respectively. Accordingly, it is concluded that Miro specifically controls the activity of anterograde mitochondrial transport (Guo, 2005).

All Miro mutant alleles were identified by the loss of phototaxis of EGUF-induced mosaic flies; this phenotype is apparently caused by a defect in synaptic transmission, as indicated by the activity-dependent loss of Off transients in electroretinogram recordings from Miro mutant eyes (Babcock, 2003). To examine in more detail the effects of the chronic loss of mitochondria on synaptic physiology, evoked excitatory junctional potentials (EJPs) and miniature excitatory junctional potentials (mEJPs) were recorded from larval NMJs. Less than half of all Miro mutant muscles examined showed decreased resting potentials and/or a high rate of spontaneous quantal release. The remaining muscles exhibited a normal resting potential, deviating by no more than ± 5 mV from control, and a normal frequency of spontaneous release. Since the resting potential was normal for the majority of Miro muscles, partially depolarized muscles were excluded from the subsequent analysis, because their defects were likely secondary and degenerative (Guo, 2005).

Evoked EJP amplitudes of Miro mutants were normal at a low stimulation frequency of 0.2 Hz. Even during prolonged stimulation at 1 Hz for 5 min, EJP amplitudes were normal. However, during stimulation at 5 Hz, EJP amplitudes showed a steadily progressing fatigue after 90 s, and by 300 s, no more EJPs were elicited. Consistently, higher stimulation frequencies caused a progressively earlier onset of fatigue; at 10 Hz, EJPs became depressed within 10 s, and after 15-20 s, almost all stimuli failed to elicit EJPs. The activity-dependent fatigue of evoked release in Miro was not due to a failure of action potentials because electrotonically elicited EJPs showed a similar fatigue. A similar activity-dependent fatigue of evoked release was observed for transheterozygous combinations of B682 with other Miro alleles and a deletion uncovering the locus. Presynaptic expression of Miro at Miro-null mutant NMJs restored the activity-dependent fatigue of evoked release (Guo, 2005).

An activity-dependent failure of EJP amplitudes often indicates a depletion of releasable synaptic vesicles due to a defect in vesicle trafficking and/or endocytosis. However, the frequency of mEJPs at Miro mutant NMJs increased up to 5-fold during high-frequency stimulation and persisted for extended periods after stimulation. The abnormal increase in mEJP frequency was activity dependent. Prolonged stimulation at 1 Hz over 5 min did not cause an increase in the frequency of unitary quantal events at Miro mutant NMJs, while stimulation ≥5 Hz increased mEJP frequency. Hence, the activity-dependent increase in mEJP frequencies makes it unlikely that a defect in vesicle recycling causes the fatigue of evoked release. Interestingly, similar activity-dependent effects on transmitter release are observed upon acute pharmacological inhibition of mitochondrial function and are correlated with a severe loss of presynaptic Ca²⁺ clearance (Guo, 2005).

To test whether the activity-dependent defects in transmitter release of Miro mutants are linked to an impaired presynaptic Ca²⁺ homeostasis, presynaptic Ca²⁺ concentrations were examined at Miro NMJs. The resting [Ca²⁺]_i was significantly elevated more than two fold in Miro mutant terminals. However, during stimulation at 80 Hz for 5 s, the peak cytosolic [Ca²⁺]_i and the time course of [Ca²⁺]_i decay in mutant terminals were indistinguishable from those in controls. This suggests that the chronic lack of mitochondria does not affect nerve-evoked Ca²⁺ levels during brief periods of stimulation. Nerve-evoked Ca²⁺ levels during longer periods of stimulation (10 Hz/30 s) were initially similar between control and Miro but progressively increased during stimulation to a significantly larger degree in Miro mutants, suggesting that Ca²⁺ buffering during extended periods of high activity is impaired (Guo, 2005).

Assuming that mitochondria in control terminals accumulate a significant amount of Ca²⁺ in response to nerve stimulation and subsequently slowly release it into the cytosol, one might expect that post-tetanic Ca²⁺ decay might be altered in the mutants, but this was not the case. In both mutants and controls, 3 s after stimulation ended, Ca²⁺ levels had fallen to ~8% of the plateau level achieved at the end of the stimulation train, indicating that cytosolic Ca²⁺ clearance is effectively no different between strains. Hence, these results suggest that cytosolic Ca²⁺ handling is impaired only during prolonged repetitive stimulation in Miro mutants, while fast Ca²⁺ clearance after stimulation is not affected. Hence, it is unlikely for two reasons that an abnormal accumulation of Ca²⁺ during 10 Hz stimulation causes the desynchronization of transmitter release in Miro mutant terminals: (1) stimulated Ca²⁺ levels only reached ~140 nM after 30 s of 10 Hz stimulation, which is in the physiological range for these terminals, and (2) Ca²⁺ resting levels in Miro mutant terminals before and after stimulation were indistinguishable (Guo, 2005).

The analysis of presynaptic Ca²⁺ dynamics in nerve terminals that chronically lack mitochondria suggests only a minor role for mitochondria as presynaptic Ca²⁺ sinks, which is much in contrast to the suggested dominant role of mitochondrial Ca²⁺ sinks at other NMJs. However, the current results could be misleading for two reasons: (1) mitochondria at larval Drosophila motor terminals may not sequester Ca²⁺ during stimulation; (2) other mechanisms may compensate for the chronic lack of mitochondrial Ca²⁺ sinks. To test the first possibility, mitochondrial Ca²⁺ uptake at presynaptic terminals of wild-type NMJs was examined by employing the Ca²⁺ indicator dihydrorhod-2 AM (rhod-2), which accumulates specifically in mitochondria (Guo, 2005).

At rest, rhod-2 produced little measurable fluorescence, presumably due to low Ca²⁺ levels in mitochondria since rhod-2 fluorescence below 100 nM Ca²⁺ is effectively invisible in vitro. Upon high-frequency nerve stimulation (80 Hz, 2 s), bright fluorescent punctae appeared that were within synaptic boutons and had the same size and distribution as live-stained mitochondria. The activity-dependent increase in the intensity of rhod-2 fluorescence was dependent on the onset, but not on the offset, of nerve stimulation; rhod-2 fluorescence faded slowly after stimulation had ceased and became invisible only after ~5 to 10 min. The slow decay of rhod-2 fluorescence is consistent with a slow release of mitochondrial Ca²⁺ and contrasts with the exceedingly rapid time course of decay of cytosolic Ca²⁺ indicators, like rhodamine-dextran or transgenically expressed G-CaMP. Hence, it is concluded that presynaptic mitochondria at Drosophila NMJs can sequester Ca²⁺ in response to high-frequency nerve stimulation. However, as suggested by the relatively normal Ca²⁺ homeostasis at Miro terminals lacking mitochondria, their significance as short-term Ca²⁺ sinks at these nerve terminals is either very limited or easily compensated by other mechanisms (Guo, 2005).

Miro's N-Terminal GTPase domain is required for transport of mitochondria into axons and dendrites

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. This study shows that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, the loss-of-function mutations dMiro^T25N and dMiro^T460N were used to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiro^T25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiro^T25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiro^T25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiro^T460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiro^T460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, this study showed that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, this analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state (Babic, 2015).

EVOLUTIONARY HOMOLOGS

Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells, Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics (Frederick, 2004).

The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Reported in this study is the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrial Rho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early in evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induce an aggregation of the mitochondrial network and results in an increases apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis (Fransson, 2003).

Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho-GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double-knockout mouse embryos and single- and double-knockout embryonic fibroblasts, this study demonstrated the essential and non-redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double-knockout cells. In contrast, this study shows that TRAK2-mediated retrograde mitochondrial transport is Miro1-dependent. Interestingly, Miro was found to be critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin-dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double-knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine-tune actin- and tubulin-dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation (Lopez-Domenech, 2018).

REFERENCES

Search PubMed for articles about Drosophila Mitochondrial Rho

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Babic, M., Russo, G. J., Wellington, A. J., Sangston, R. M., Gonzalez, M. and Zinsmaier, K. E. (2015). Miro's N-Terminal GTPase domain is required for transport of mitochondria into axons and dendrites. J Neurosci 35: 5754-5771. PubMed ID: 25855186

Benzer, S. (1967). Behavioral mutants of Drosophila isolated by countercurrent distribution. Proc. Natl. Acad. Sci. 58: 1112-1119.

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Ding, L., Lei, Y., Han, Y., Li, Y., Ji, X. and Liu, L. (2016). Vimar is a novel regulator of mitochondrial fission through Miro. PLoS Genet 12: e1006359. PubMed ID: 27716788

Fransson, A., Ruusala A. and Aspenstrom, P. (2003). Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis. J. Biol. Chem. 278: 6495-6502. 12482879

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Guo, X., et al. (2005). The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses. Neuron 47: 379-393. 16055062

Hollenbeck, P. J. (1996). The pattern and mechanisms of mitochondrial transport in axons. Front. Biosci. 1: d91-d102. 9159217

Hollenbeck, P. J. and Saxton, W. M. (2005). The axonal transport of mitochondria. J Cell Sci. 118(Pt 23): 5411-9. 16306220

Li, Z., Okamoto, K., Hayashi, Y. and Sheng, M. (2004). The importance of dentritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119: 873-887. 15607982

Lopez-Domenech, G., Covill-Cooke, C., Ivankovic, D., Halff, E. F., Sheehan, D. F., Norkett, R., Birsa, N. and Kittler, J. T. (2018). Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution. EMBO J 37(3):321-336. PubMed ID: 29311115

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date revised: 25 April 2024

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