The best-characterized mitochondrial transport complex to date uses kinesin heavy chain (KHC, a member of the kinesin-1 family) as its motor, and Miro and milton as its motor adaptors. Miro stands for “mitochondrial Rho” and belongs to the atypical Rho (Ras homolog) family of GTPases (RhoT1/2 in mammals). Miro is anchored to the outer mitochondrial membrane (OMM) via its carboxy-terminus transmembrane domain. Miro binds to milton (trafficking protein, kinase-binding, or TRAK1/2 in mammals), which in turn binds to the carboxy-terminus of KHC ^{25– 27} . Milton was identified in a Drosophila screen for blind flies and was named after the great poet and polemicist John Milton, who was also blind ²⁸ . Together, Miro and milton facilitate anterograde mitochondrial movement along microtubules by connecting mitochondria to KHC ( Figure 1a). When either Miro or milton is mutated in animal models, mitochondria are trapped in the soma and lose the ability to move out into the axons ^{9, 14– 16, 26, 28} .

Miro and milton are not the only adaptors that can link mitochondria and KHC. Syntabulin can bind directly to the OMM and KHC, and disruption of syntabulin function has been shown to inhibit the anterograde transport of mitochondria in neurons ²⁹ . Similarly, disrupting fasciculation and elongation protein zeta-1 (FEZ1), and RAN-binding protein 2 (RANBP2) also affects mitochondrial distribution because of their association with kinesins, possibly KIF3A and KIF5B/C, respectively ^{30– 34} .

Mutations in KHC have been shown to reduce anterograde mitochondrial movement but do not eliminate it entirely, which suggests that other kinesin motors may also play a role in anterograde mitochondrial motility ²¹ . For example, kinesins from the Kinesin-3 family KIF1Bα and KLP6 may interact with KIF1 binding protein (KBP) to transport mitochondria ^{35– 38} . KIF1B can transport mitochondria along microtubules in vitro, and mutations in Klp6 inhibits anterograde mitochondrial motility into neurites; however, the roles of these other kinesins await further clarification.

Dynein is thought to act as the retrograde motor for microtubule-based mitochondrial movement, although far less is known about the mechanisms underlying its action. In contrast to the host of kinesins available for anterograde transport, there is only one identified dynein motor; however, dynein’s larger and more complex structure has made it difficult to study. Dynein has been shown to form a complex with dynactin, and this complex has been shown to also interact with milton/TRAK2 and with Miro ³⁹ , which lends support to dynein’s role in mitochondrial transport ( Figure 1b). Interestingly, dynein movement is also thought to depend on kinesin-1, as mutation in kinesin-1 reduces retrograde movement of mitochondria ²¹ .

A small though not insignificant number of mitochondria are also transported along actin filaments ²² . This is more common in actin-enriched neuronal compartments, like growth cones, dendritic spines, and synaptic boutons. Myosins are actin-based motors, and the myosin Myo19 has been shown to anchor directly to the OMM, and regulate mitochondrial motility ^{24, 40} . Myosins V and VI have also been shown to play a role in mitochondrial motility by opposing microtubule-based mitochondrial transport ²³ , although whether these myosin motors attach directly to mitochondria or require a motor adaptor remains unknown ( Figure 1c). WAVE1 (WASP family verprolin homologous protein 1), which regulates actin polymerization, has been shown to be critical for mitochondrial transport in dendritic spines and filopodia—areas that are actin-rich—and therefore may be involved in the actin-based transport of mitochondria ⁴¹ .

If 30% to 40% of mitochondria are in motion at any given time, then more than half of mitochondria are static. While understanding of how stationary pools of mitochondria are generated is still nascent, one protein, syntaphilin, stands out. Syntaphilin serves as a molecular brake, docking mitochondria by binding to both the mitochondrial surface and to the microtubule ⁴² . Both kinesin-1 and the dynein light chain component LC8 have been shown to regulate this mechanism ^{43, 44} . Intriguingly, a recent study using optogenetics has shown that the mitochondrial dance between mobility and stabilization depends on the balance of forces between motors and anchors, rather than all-or-none switching ⁴⁵ .

The ability of mitochondria to temporarily stop where they are needed is just as important as their ability to move. When cytosolic Ca ²⁺ concentration is elevated, Ca ²⁺ binds to the EF hands of Miro and triggers a transient and instantaneous conformational change in the KHC/milton/Miro complex. This conformational change causes dissociation of either the whole complex from microtubules ⁹ , or KHC from mitochondria ¹⁷ , which arrests movement of mitochondria. When Ca ²⁺ concentration is lowered, Ca ²⁺ is removed from Miro, and mitochondria are reattached to microtubules by the complex and can start moving again. The sensitivity of mitochondrial movement to Ca ²⁺ is likely a means by which mitochondria can be recruited to areas of high metabolic demand or low local ATP, like post-synaptic specializations and growth cones. During glutamate receptor activation, mitochondria are recruited where Ca ²⁺ influx is increased, which confers neurons with resistance to excitotoxicity ^{9, 17} . Interestingly, brain-derived neurotrophic factor (BDNF) has recently been shown to arrest mitochondrial motility via Ca ²⁺ binding to Miro1 in cultured hippocampal neurons ⁴⁶ .

Glucose has recently been shown to influence mitochondrial motility via the KHC/milton/Miro complex. The small sugar UDP-GlcNAc is derived from glucose through the hexamine biosynthetic pathway. UDP-GlcNAc is affixed to milton by O-GlcNAc transferase (OGT), in a process called O-GlcNAcylation ⁴⁷ . Extracellular glucose concentration or OGT activity can modulate mitochondrial motility through O-GlcNAcylation of milton. This mechanism links nutrient availability to mitochondrial distribution, which could be a mechanism by which neurons maintain a balanced metabolic state.

When mitochondria are severely damaged, they undergo mitophagy, a crucial cellular mechanism that eliminates depolarized mitochondria through autophagosomes and lysosomes. Damaged mitochondria must be stopped prior to the initiation of mitophagy. To accomplish this, mitochondrial depolarization activates PINK1 (PTEN-induced putative kinase 1)-mediated phosphorylation of Miro ^{16, 48} , which subsequently triggers Parkin-dependent proteasomal degradation of Miro, thus releasing the mitochondria from its microtubule motors ^{16, 49} . It is likely that stopping mitochondria in this manner is an early step in the quarantine of damaged mitochondria before degradation. In fact, this PINK1-mediated phosphorylation of Miro has been shown to protect dopaminergic neurons in vivo in Drosophila ⁵⁰ . PINK1 and Parkin can also work in concert to remove damaged mitochondria through local mitophagy in distal axons, which would obviate the need for the mitochondria to be transported all the way back to the soma, and instead require the recruitment of autophagosomes to the damaged mitochondria ¹³ . How a cell chooses between transporting a mitochondrion back to the soma or using local mitophagy when it is damaged in the axon remains an outstanding question.

The dynamics of mitochondrial fission and fusion also plays a central role in PINK1/Parkin-mediated mitophagy. For example, mitofusin, a large GTPase that regulates mitochondrial fusion, is a target of the PINK1/Parkin pathway. Degradation of mitofusin prevents mitochondria from being able to fuse, and they instead fragment, a critical step prior to mitophagy ^{51– 55} . An in-depth discussion of the role of mitochondrial dynamics in quality control merits its own review, and an excellent F1000 Faculty Review and two others are recommended in the References section ^{56– 58} .

In humans, milton is encoded by two different genes: TRAK1 and TRAK2. It has been reported that TRAK1 binds to both kinesin-1 and dynein, while TRAK2 predominantly favors dynein ³⁹ . In Drosophila, milton has several splicing variants, one of which (milton-C) does not bind to KHC ²⁶ . These varying forms of milton may play an important role in regulating the KHC/milton/Miro complex.

Another regulator that merits mentioning is HUMMR (hypoxia up-regulated mitochondrial movement regulator), whose expression is induced by hypoxic conditions. HUMMR has been shown to interact with the KHC/milton/Miro complex, and increases the ratio of anterograde to retrograde movement of mitochondria ⁵⁹ . Similarly, a family of proteins encoded by an array of armadillo (Arm) repeat-containing genes has been shown to bind to milton/Miro and regulate mitochondrial motility ⁶⁰ .

It is worthwhile to note that mitochondrial fission and fusion also affect mitochondrial motility. The same mitofusin mentioned previously also binds to milton/Miro, and knockdown of mitofusin 2 has been shown to inhibit mitochondrial motility ⁶¹ . Additionally, transient fusion has been shown to promote mitochondrial movement ⁶² .

Aging itself has been shown to decrease neuronal mitochondrial motility in mice, and several age-dependent neurodegenerative diseases have been linked to mitochondrial motility defects ⁷⁶ . Mutations in the previously mentioned PINK1 and Parkin are both causes of familial Parkinson’s disease (PD) ^{77, 78} . In individuals lacking either functional PINK1 or Parkin, a failure to isolate, stop, and remove the damaged mitochondria may contribute to neuronal cell death. Unpublished work using patients’ samples from our laboratory also suggests that neurodegeneration in non- PINK1/Parkin-related PD cases may arise in a similar manner, and that stopping damaged mitochondrial motility is neuroprotective. This finding highlights the broader implications of mitochondrial motility in neuronal health and pathology.

The pathological forms of amyloid beta and tau, the chief markers of Alzheimer’s disease (AD), have both been shown to inhibit mitochondrial motility in several AD models ^{79– 83} . Superoxide dismutase 1, soluble (SOD1), fused in sarcoma (FUS), C9orf72, and TAR DNA-binding protein 43 (TDP-43) mutations, which cause familial amyotrophic lateral sclerosis (also called Lou Gehrig’s disease), have also been shown to impair mitochondrial transport in mice, flies, and cultured neuronal models ^{84– 91} . Mutant huntingtin protein, with the polyglutamine expansions characteristic of Huntington’s disease etiology, can act to “jam traffic” by mechanical obstruction, and may also bind to milton or even to the mitochondria itself to disrupt mitochondrial motility ^{92– 94} . Mutations in mitofusin 2 causing Charcot-Marie-Tooth disease alter mitochondria movement ⁹⁵ , and finally, mitochondrial motility defects have also been observed in models of hereditary spastic paraplegia, a disease characterized by axonal degeneration ^{96, 97} .

A few psychiatric disorders have also been linked to mitochondrial motility defects. Mutations in disrupted in schizophrenia 1 ( DISC1) may contribute to both schizophrenia and some forms of depression ⁹⁸ . DISC1 complexes with TRAK1/milton and Miro1 to modulate anterograde transport of mitochondria ^{99, 100} , and its interactors NDE1 and GSK3β have recently been shown to associate with TRAK1/milton and similarly play a role in mitochondrial motility ⁶⁸ . DISC1 also interacts with the previously mentioned FEZ1 ¹⁰¹ , which binds to kinesins ^{25, 28} . Among several causes, depression can be attributed to a loss of serotonin ¹⁰² . Interestingly, the application of serotonin to cultured hippocampal neurons has been shown to increase mitochondrial motility ⁶ .