Organelle transport is vital for the development and maintenance of axons, in which the distances between sites of organelle biogenesis, function, and recycling or degradation can be vast. Movement of mitochondria in axons can serve as a general model for how all organelles move: mitochondria are easy to identify, they move along both microtubule and actin tracks, they pause and change direction, and their transport is modulated in response to physiological signals. However, they can be distinguished from other axonal organelles by the complexity of their movement and their unique functions in aerobic metabolism, calcium homeostasis and cell death. Mitochondria are thus of special interest in relating defects in axonal transport to neuropathies and degenerative diseases of the nervous system. Studies of mitochondrial transport in axons are beginning to illuminate fundamental aspects of the distribution mechanism. They use motors of one or more kinesin families, along with cytoplasmic dynein, to translocate along microtubules, and bidirectional movement may be coordinated through interaction between dynein and kinesin-1. Translocation along actin filaments is probably driven by myosin V, but the protein(s) that mediate docking with actin filaments remain unknown. Signaling through the PI 3-kinase pathway has been implicated in regulation of mitochondrial movement and docking in the axon, and additional mitochondrial linker and regulatory proteins, such as Milton and Miro, have recently been described.

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The axonal transport of mitochondria

Mitochondria are one of the major ancient endomembrane systems in eukaryotic cells. Owing to their ability to produce ATP through respiration, they became a driving force in evolution. As an essential step in the process of eukaryotic evolution, the size of the mitochondrial chromosome was drastically reduced, and the behaviour of mitochondria within eukaryotic cells radically changed. Recent advances have revealed how the organelle's behaviour has evolved to allow the accurate transmission of its genome and to become responsive to the needs of the cell and its own dysfunction.

/pdf/mitochondrial-form-and-function-1x51ier51r.pdf

Mitochondrial form and function

PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility

Mitochondrial dysfunction and perturbed degradation of proteins have been implicated in Parkinson's disease (PD) pathogenesis. Mutations in the Parkin and PINK1 genes are a cause of familial PD. PINK1 is a putative kinase associated with mitochondria, and loss of PINK1 expression leads to mitochondrial dysfunction, which increases with time. Parkin is suggested to be downstream of PINK1 and also mediates the removal of damaged mitochondria by macroautophagy (mitophagy). We investigated whether mitochondrial dysfunction in dopaminergic SH-SY5Y cells following decreased PINK1 expression by RNAi may in part be due to the inhibition of mitophagy. Reduced flux through the macroautophagy pathway was found to be coincident with the inhibition of ATP synthesis following 12 days of PINK1 silencing. Overexpression of parkin in these cells restored both autophagic flux and ATP synthesis. Overexpression and RNAi studies also indicated that PINK1 and parkin were required for mitophagy following CCCP-induced mitochondrial damage. The ubiquitination of several mitochondrial proteins, including mitofusin 1 and mitofusin 2, were detected within 3 h of CCCP treatment. These post-translational modifications were reduced following the silencing of parkin or PINK1. The ubiquitination of mitochondrial proteins appears to identify mitochondria for degradation and facilitate mitophagy. PINK1 and parkin are thus required for the removal of damaged mitochondria in dopaminergic cells, and inhibition of this pathway may lead to the accumulation of defective mitochondria which may contribute to PD pathogenesis.

Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy

Mitochondria have a number of essential roles in neuronal function. Their complex mobility patterns within neurons are characterized by frequent changes in direction. Mobile mitochondria can become stationary or pause in regions that have a high metabolic demand and can move again rapidly in response to physiological changes. Defects in mitochondrial transport are implicated in the pathogenesis of several major neurological disorders. Research into the mechanisms that regulate mitochondrial transport is thus an important emerging frontier.

/pdf/mitochondrial-transport-in-neurons-impact-on-synaptic-400ibpyp4x.pdf

Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration

Mitofusins (Mfn1 and Mfn2) are outer mitochondrial membrane proteins involved in regulating mitochondrial dynamics. Mutations in Mfn2 cause Charcot-Marie-Tooth disease (CMT) type 2A, an inherited disease characterized by degeneration of long peripheral axons, but the nature of this tissue selectivity remains unknown. Here, we present evidence that Mfn2 is directly involved in and required for axonal mitochondrial transport, distinct from its role in mitochondrial fusion. Live imaging of neurons cultured from Mfn2 knock-out mice or neurons expressing Mfn2 disease mutants shows that axonal mitochondria spend more time paused and undergo slower anterograde and retrograde movements, indicating an alteration in attachment to microtubule-based transport systems. Furthermore, Mfn2 disruption altered mitochondrial movement selectively, leaving transport of other organelles intact. Importantly, both Mfn1 and Mfn2 interact with mammalian Miro (Miro1/Miro2) and Milton (OIP106/GRIF1) proteins, members of the molecular complex that links mitochondria to kinesin motors. Knockdown of Miro2 in cultured neurons produced transport deficits identical to loss of Mfn2, indicating that both proteins must be present at the outer membrane to mediate axonal mitochondrial transport. In contrast, disruption of mitochondrial fusion via knockdown of the inner mitochondrial membrane protein Opa1 had no effect on mitochondrial motility, indicating that loss of fusion does not inherently alter mitochondrial transport. These experiments identify a role for mitofusins in directly regulating mitochondrial transport and offer important insight into the cell type specificity and molecular mechanisms of axonal degeneration in CMT2A and dominant optic atrophy.

https://www.jneurosci.org/content/jneuro/30/12/4232.full.pdf

Mitofusin 2 Is Necessary for Transport of Axonal Mitochondria and Interacts with the Miro/Milton Complex

Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer's disease. However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood. Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species. By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration. Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction. We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease. These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.

https://www.jneurosci.org/content/jneuro/32/12/4145.full.pdf

Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration.

A single point mutation in peripheral myelin protein 22 (pmp22) of the Trembler-J (TrJ) mouse models the human peripheral neuropathy, Charcot–Marie–Tooth disease type 1 A (CMT1A) An unexplored aspect of this disease is the gradual remodeling of the extracellular matrix in affected nerves To elucidate the mechanism responsible for these changes, the levels of the extracellular matrix molecules laminin, collagen IV, and fibronectin were determined In TrJ nerves, laminin is modestly increased while full-length forms of collagen IV and fibronectin are decreased Matrix metalloproteinases (MMPs) are known to degrade multiple matrix molecules; therefore, nerves were assayed for MMP-2 and MMP-9 proteins In neuropathy nerves, elevated levels of MMP-2 and MMP-9 were detected on western blots, and gelatin zymography confirmed the up-regulation of gelatinalytic activity in affected samples Immunostaining studies revealed an increase in the numbers of MMP-2- and MMP-9-expressing cells in TrJ nerves Cell type-specific immunolabeling showed that infiltrating macrophages are a significant source of both MMP-2 and MMP-9 Finally, the degradation of exogenous collagen IV by TrJ nerve lysates was prevented with a specific MMP inhibitor Together these observations suggest that infiltration by MMP-expressing macrophages contributes to the remodeling of the TrJ nerve matrix

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1471-4159.2002.01200.x

Matrix metalloproteinase mediated degradation of basement membrane proteins in Trembler J neuropathy nerves.

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are two rare genetic disorders that are caused by impairment of the mitochondrial enzyme sulfite oxidase. Sulfite oxidase is catalyzing the terminal reaction of cellular cysteine catabolism, the oxidation of sulfite to sulfate. Absence of sulfite oxidase leads to the accumulation of sulfite, which has been identified as a cellular toxin. However, the molecular pathways leading to the production of sulfite are still not completely understood. In order to identify novel treatment options for both disorders, the understanding of cellular cysteine catabolism - and its alterations upon loss of sulfite oxidase - is of utmost importance. Here we applied a new detection method of sulfite in cellular extracts to dissect the contribution of cytosolic and mitochondrial glutamate oxaloacetate transaminase (GOT) in the transformation of cysteine sulfinic acid to sulfite and pyruvate. We found that the cytosolic isoform GOT1 is primarily responsible for the production of sulfite. Moreover, loss of sulfite oxidase activity results in the accumulation of sulfite, H2S and persulfidated cysteine and glutathione, which is consistent with an increase of SQR protein levels. Surprisingly, none of the known H2S-producing pathways were found to be upregulated under conditions of sulfite toxicity suggesting an alternative route of sulfite-induced shift from oxidative to H2S dependent cysteine catabolism.

The role of glutamate oxaloacetate transaminases in sulfite biosynthesis and H2S metabolism

Objective To define the phenotypic spectrum of isolated sulfite oxidase (ISOD) and molybdenum cofactor deficiency (MoCD), aiming to promote timely diagnosis and assist in future clinical trial design. Methods We analyzed clinical, radiographic, biochemical, and genetic data from 146 patients reported in the literature. Results We stratified patients into 2 phenotypic subgroups based on clinical and radiographic characteristics. In the first (Class I), patients presented early in life (age 1-50 days) with acute onset of neurologic symptoms and development of diffuse brain injury with cystic leukomalacia. Patients in the second subgroup (Class II) presented later in life (age 30 days-23 years) with prominent movement abnormalities and selective injury of the basal ganglia and cerebellum. A significant difference in survival estimates correlated with milder disease severity among Class II patients. Substantial overlap in sulfur-containing metabolite levels prevented discrimination of subgroups based on diagnostic biomarkers, but genotype-phenotype correlations suggested that residual SUOX activity may contribute to milder phenotypes. Conclusions Patients with SUOX and MoCD gravitate toward 1 of 2 distinct clinicoradiographic profiles. Patient stratification may help promote accurate diagnosis, prognostication, and aid in the design of future clinical trials.

https://ng.neurology.org/content/nng/6/4/e486.full.pdf

Albert L. Misko

Papers

Mitofusin 2 Is Necessary for Transport of Axonal Mitochondria and Interacts with the Miro/Milton Complex

Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration.

Matrix metalloproteinase mediated degradation of basement membrane proteins in Trembler J neuropathy nerves.

The role of glutamate oxaloacetate transaminases in sulfite biosynthesis and H2S metabolism

Delineating the phenotypic spectrum of sulfite oxidase and molybdenum cofactor deficiency.