Mitochondrial trafficking and morphology in healthy and injured neurons

Progress in Neurobiology 80 (2006) 241–268 www.elsevier.com/locate/pneurobio

Mitochondrial trafficking and morphology in healthy and injured neurons Diane T.W. Chang a, Ian J. Reynolds b,* a

Medical Scientist Training Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, United States b Merck Research Laboratories, 770 Sumneytown Pike, Mail Stop WP42-229, West Point, PA 19486, United States Received 12 May 2006; received in revised form 14 September 2006; accepted 18 September 2006

Abstract Mitochondria are the primary generators of ATP and are important regulators of intracellular calcium homeostasis. These organelles are dynamically transported along lengthy neuronal processes, presumably for appropriate distribution to cellular regions of high metabolic demand and elevated intracellular calcium, such as synapses. The removal of damaged mitochondria that produce harmful reactive oxygen species and promote apoptosis is also thought to be mediated by transport of mitochondria to autophagosomes. Mitochondrial trafficking is therefore important for maintaining neuronal and mitochondrial health while cessation of movement may lead to neuronal and mitochondrial dysfunction. Mitochondrial morphology is also dynamic and is remodeled during neuronal injury and disease. Recent studies reveal different manifestations and mechanisms of impaired mitochondrial movement and altered morphology in injured neurons. These are likely to cause varied courses toward neuronal degeneration and death. The goal of this review is to provide an appreciation of the full range of mitochondrial function, morphology and trafficking, and the critical role these parameters play in neuronal physiology and pathophysiology. # 2006 Published by Elsevier Ltd. Keywords: Mitochondria; Transport; Excitotoxicity; Neurodegeneration; Huntington’s disease; Fission; Fusion

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial function and dysfunction . . . . . . . . . 2.1. Importance of mitochondria in brain . . . . . . . 2.2. Consequences of mitochondrial injury. . . . . . 2.2.1. Energy deprivation and cell death. . . 2.2.2. Dysregulation of [Ca2+]i homeostasis 2.2.3. ROS production . . . . . . . . . . . . . . . 2.2.4. Mitochondrial release of apoptogenic Mitochondrial life cycle in neurons . . . . . . . . . . . . 3.1. Evolution and biogenesis of mitochondria . . . 3.2. Mitochondrial autophagy . . . . . . . . . . . . . . .

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Abbreviations: Cm, mitochondrial membrane potential; Ab, amyloid beta; AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; ANT, adenine nucleotide translocator; APP, amyloid precursor protein; ATP, adenosine triphosphate; ER/SR, endoplasmic/sarcoplasmic reticulum; ETC, electron transport chain; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HD, Huntington’s disease; Htt, Huntingtin; KHC, kinesin heavy chain; KIF, kinesin superfamily protein; LHON, Leber’s hereditary optic neuropathy; MAP, microtubule associated protein; MAPK, mitogen activated protein kinase; MELAS, mitochondrial myopathy with encephalopathy, lactic acidosis and stroke; MERRF, myoclonic epilepsy and ragged red fibers; mPTP, mitochondrial permeability transition pore; mtDNA, mitochondrial DNA; NFT, neurofibrillary tangle; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PD, Parkinson’s disease; Pi, inorganic phosphate; Pi 3-kinase, phosphoinositol 3-kinase; ROS, reactive oxygen species; SOD1, Cu/Zn superoxide dismutase; TTX, tetrodotoxin; VDAC, voltagedependent anion channel * Corresponding author. Tel.: +1 215 652 4377; fax: +1 215 993 0797. E-mail address: [email protected] (I.J. Reynolds). 0301-0082/$ – see front matter # 2006 Published by Elsevier Ltd. doi:10.1016/j.pneurobio.2006.09.003

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Mitochondrial morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Regulation of morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Functional implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Altered morphology in neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Significance of mitochondrial transport: focus on synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How mitochondria move. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Patterns of mitochondrial movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Mechanisms of mitochondrial movement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Mechanisms of mitochondrial docking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adopting a reductionist approach to studying mitochondrial trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Subdividing the mitochondrial pool for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1. Nonspecific mitochondrial measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2. Measuring regional subpopulations of mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.3. Studying trafficking of an individual mitochondrion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Studying cellular targets and modulators of mitochondrial movement . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1. Measuring mitochondrial trafficking to a single cue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2. Studying mitochondrial movement responses to global pharmacologic treatments . . . . . . . . . . 7.2.3. Local pharmacologic manipulations as a future approach to studying mitochondrial movement . Relationship between mitochondrial movement and mitochondrial and cellular injury . . . . . . . . . . . . . . . . . . Manifestations and mechanisms of mitochondrial movement impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Whole-cell impairments in mitochondrial movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Axon- or dendrite-specific impairments in mitochondrial movement . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Discrete sites of impaired mitochondrial movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. Functional heterogeneity of individual mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complex mitochondrial movement impairments in neuronal injury and disease . . . . . . . . . . . . . . . . . . . . . . . 10.1. Glutamate excitotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Zinc-mediated neuronal injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Neuronal trauma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5. Huntington’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6. Other neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Mitochondria in most cells are highly dynamic organelles that fuse, divide, move and replicate, and are associated with a finite lifetime that is typically shorter than the life of the host cell. In this review, we will focus on these dynamics in the context of neurons. In most respects we anticipate that the basic functional properties of mitochondria in neurons resemble those in other cell types. However, the challenges posed by having to move organelles over extreme distances, and also to maintain cellular homeostasis in very large and highly active cells are unique to neurons, making mitochondrial function in these cells of particular interest. Mitochondrial dynamics in neurons is a fundamental but highly complex property with broad implications for normal and abnormal cellular and mitochondrial function. Mitochondrial movement is intimately tied with the functional status of cells and of the organelles themselves (Malaiyandi et al., 2005; Reynolds and Santos, 2005; Rintoul et al., 2003a,b; Stout et al., 1998). Additionally, the consequences of dynamic changes in mitochondrial morphology under physiological and injurious conditions require further study (Frank et al., 2001; Rintoul et al., 2003a; Szabadkai et al., 2004; Yu et al., 2006). In this review,

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we discuss normal mitochondrial physiology, mitochondrial dysfunction in neuronal injury and disease, the importance of mitochondrial trafficking and morphology in neuronal health, mechanisms that regulate mitochondrial movement and morphology, and current and future approaches to studying mitochondrial trafficking. Since appropriate mitochondrial delivery is critical to neuronal health, it is not surprising that impaired mitochondrial trafficking can contribute to pathophysiology. Recent studies implicate aberrant mitochondrial movement in multiple neuronal injuries and diseases (Chang et al., 2006a,b; Ebneth et al., 1998; Malaiyandi et al., 2005; Piccioni et al., 2002; Rintoul et al., 2003a; Sasaki et al., 2005). However, it is becoming clear that trafficking impairment can have diverse manifestations that likely contribute to different patterns of neurodegeneration and cell death. In the second part of this review, we discuss causes and consequences of impaired mitochondrial movement, and manifestations and mechanisms of heterogeneous movement abnormalities in neurons. Lastly, we consider what the future will uncover about mitochondrial trafficking and how that knowledge can be harnessed to develop pharmacologic therapies for neuronal injury and disease.

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2. Mitochondrial function and dysfunction 2.1. Importance of mitochondria in brain Mitochondria are vital organelles for cell survival. They are the primary generators of cellular energy and they accomplish this with great efficiency. By coupling electron transport to the generation of proton gradients for oxidative phosphorylation, mitochondria produce approximately 15 times more ATP from glucose than the glycolytic pathway in eukaryotic cells. Cells of highly metabolic tissues such as muscle, liver and brain, are therefore particularly dependent on mitochondria. Mitochondria also localize to regions of high ATP consumption in cells. They are densely arranged in the clefts between cardiac muscle myofibrils that utilize ATP for contraction and are coiled around the base of sperm flagella to power motility, implying that mitochondrial distribution serves a functional purpose (Cardullo and Baltz, 1991; Segretain et al., 1981). The central nervous system has an intense demand for mitochondria; the human brain consumes 20% of resting metabolic energy while only comprising 2% of total body mass (Silver and Erecinska, 1998). Mitochondria produce over 95% of ATP utilized by the brain (Erecinska and Silver, 1994). The generation, processing and transmission of neural impulses rely heavily on Na+, K+ and Ca2+ ion gradients across the plasma membrane. In fact, 50–60% of total brain ATP is used to maintain these gradients, especially through Na+/K+ pumps (Erecinska and Silver, 1994). Within neurons, mitochondria are again distributed to regions of high metabolic demand, including synapses, nodes of Ranvier and myelination/ demyelination interfaces (Berthold et al., 1993; Bristow et al., 2002; Kageyama and Wong-Riley, 1982; Rowland et al., 2000). In addition to the generation of cellular energy, mitochondria also play an important role in regulating calcium homeostasis (Babcock et al., 1997; Budd and Nicholls, 1996b; Jouaville et al., 1995; Werth and Thayer, 1994). Calcium serves as a regulator of kinases, phosphatases, proteases, transcription factors and ion channels as well as an intracellular messenger for membrane excitability, exocytosis, vesicle trafficking, muscle contraction, cell proliferation, fertilization, metabolism, crosstalk between signaling pathways, and apoptosis (Carafoli et al., 2001). Additionally, Ca2+-sensitive dehydrogenases can regulate oxidative phosphorylation and ATP synthesis during times of high cellular demand (McCormack and Denton, 1980). These diverse Ca2+-mediated processes, which occur over the course of microseconds to hours, are highly dependent on the spatiotemporal distribution of [Ca2+]i (Berridge et al., 2003, 2000). Microdomains of high [Ca2+]i have been identified near Ca2+ channels on the plasma membrane and endoplasmic reticulum (Brini, 2003). Mitochondria play an important role in regulating [Ca2+]i, in concert with the sarco-endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase and Na+/Ca2+ exchanger (Saris and Carafoli, 2005). In particular, mitochondrial Ca2+ uptake becomes important at [Ca2+]i above 400– 500 nM (Nicholls and Scott, 1980). Mitochondria are able to

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buffer [Ca2+]i by virtue of their negatively charged mitochondrial membrane potential (cm) and low [Ca2+]mito relative to the [Ca2+]i. Uptake of Ca2+ into the mitochondrial matrix is therefore electrochemically favored, and is largely accomplished through the Ca2+-uniporter (Crompton et al., 1978). Mitochondrial Ca2+ sequestration is reversible, with Ca2+ efflux back into the cytosol occurring through the mitochondrial Na+/ Ca2+ exchanger (Crompton and Heid, 1978; Crompton et al., 1978). Ca2+ release from mitochondria can prolong elevated [Ca2+]i, mediating important physiological functions such as exocytosis and post-tetanic potentiation at presynaptic terminals (Nicholls, 1978; Tang and Zucker, 1997). 2.2. Consequences of mitochondrial injury 2.2.1. Energy deprivation and cell death One of the most important consequences of extensive disruption of mitochondrial function is loss of oxidative ATP production. This causes gross bioenergetic failure and necrotic cell death when insufficiently compensated for by glycolytic ATP synthesis, such as during severe anoxic and ischemic injuries (Ankarcrona et al., 1995). ATP levels are an important determinant of whether cells die by apoptosis or necrosis, as ATP is required for events preceding apoptotic cell death such as apoptosome formation and caspase activation. Indeed, cells can be rescued from hypoxiainduced necrosis when supplemented with glycolytic substrates (Anundi et al., 1987; Leist et al., 1997). ATP regeneration during reperfusion of ischemic tissues is therefore likely to promote apoptotic cell death over necrotic cell death. Additionally, cells induced to undergo apoptosis instead die by necrosis when ATP synthase is inhibited (Formigli et al., 2000; Nieminen et al., 1994). Prolonged bioenergetic defects can also result from impaired electron transport chain complexes, compromised Cm and reduced ATP production. Such defects characterize many neurodegenerative diseases. Perhaps the clearest example is sporadic Parkinson’s disease (PD) caused by complex I deficiency (Schapira et al., 1989). In Alzheimer’s disease (AD), mutated amyloid precursor protein (APP) targets and causes injury to mitochondria, and amyloid-b (Ab) impairs respiration, depolarizes mitochondria and promotes cytochrome c release and apoptosis (Anandatheerthavarada et al., 2003; Canevari et al., 1999; Gibson et al., 1998; Morais Cardoso et al., 2002; Paker et al., 1994; Sheu et al., 1985). Evidence also exists for defects in complexes II, III and IV and reduced ATP production in Huntington’s disease (HD) (Beal et al., 1993; Benchoua et al., 2006; Browne et al., 1997; Ludolph et al., 1991; Milakovic and Johnson, 2005). Studies in amyotrophic lateral sclerosis (ALS) tissues and Cu/Zn superoxide dismutase (SOD1) mutant models of ALS reveal depolarized cm, reduced mitochondrial mass, decreased activity of the mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase enzyme and increased toxicity with mitochondrial toxins (Borthwick et al., 1999; Carri et al., 1997; Kaal et al., 2000; Wiedemann et al., 2002).

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2.2.2. Dysregulation of [Ca2+]i homeostasis Consequences of insufficient mitochondrial Ca2+ sequestration in cells include not only disruption of normal [Ca2+]i cycling needed to maintain appropriate Ca2+-dependent signaling pathways and enzyme activities, but also pathological accumulation of [Ca2+]i. Ca2+ influx into cells is an important event in ischemic injury, glutamate excitotoxicity, epilepsy and trauma (Nishizawa, 2001; Wolf et al., 2001). Our laboratory and others previously demonstrated the importance of mitochondrial Ca2+ uptake rather than just elevated [Ca2+]i in mediating excitotoxic cell death of neurons after glutamate treatment (Budd and Nicholls, 1996a; Stout et al., 1998). Mitochondria become overloaded at [Ca2+]i of 1–3 mM, and consequences include cm depolarization, impaired ATP synthesis, generation of reactive oxygen species (ROS), permeability transition and cell death (Murphy et al., 1999; Nicholls and Crompton, 1980). Although [Ca2+]mito may be the most important determinant for excitotoxic cell death, grossly elevated [Ca2+]i also disrupts cells by activating: (1) Ca2+sensitive proteases such as calpain that cause cytoskeletal breakdown, (2) phospholipases that disrupt membranes, activate the arachidonic acid cascade and lead to ROS production, (3) endonucleases that cause DNA degradation and (4) Ca2+-binding proteins such as calmodulin which activates nitric oxide synthase for generation of reactive nitrogen species (Farooqui et al., 2004; Sattler and Tymianski, 2000). Chronic conditions of mild mitochondrial injury such as those found in neurodegenerative diseases may impart a different type of disrupted Ca2+ homeostasis in cells and in mitochondria. Damaged mitochondria often have reduced ATP synthesis, which is exacerbated by the cm depolarization that accompanies normal physiological Ca2+ cycling between the cytosol and mitochondria (Jouaville et al., 1999). Consequent compromise of ATP-dependent mechanisms for Ca2+ extrusion from the cell can cause prolonged periods of harmful elevations in [Ca2+]i and [Ca2+]mito. Another possible outcome of damaged mitochondria and their reduced ability to uptake Ca2+ is the inability of Ca2+-sensitive proteins in mitochondria to upregulate ATP synthesis appropriately during times of high demand (Brini, 2003). Impaired mitochondrial Ca2+ regulation has been demonstrated in AD, ALS and HD (Panov et al., 2002; Sheehan et al., 1997; Siklos et al., 1998). In summary, mitochondrial injury can disrupt Ca2+ homeostasis in the cytosolic and mitochondrial compartments, leading to separate but interrelated cellular perturbations. 2.2.3. ROS production Mitochondria are important normal generators of ROS for cell signaling, but they can also be important contributors to oxidative damage. The electron transport chain is a key generator of ROS in cells (Andreyev et al., 2005; Duchen, 2004). High-throughput electron transport down the respiratory chain inevitably results in the escape of unpaired electrons, largely at complexes I and III (St-Pierre et al., 2002). Reaction of electrons with O2 yields the highly reactive superoxide anion, which can convert to other ROS such as hydrogen

peroxide and hydroxyl radicals. Mitochondria have a high capacity for scavenging ROS by enzymes such as superoxide dismutase, catalase and glutathione peroxidase. However, when ROS generation surpasses these protective mechanisms, multiple destructive redox reactions may ensue: (1) lipid peroxidation that compromises the integrity of membranes, including mitochondrial membranes, (2) protein oxidation that disrupts enzymes and structural proteins, and (3) oxidative damage to DNA, including mtDNA (Andersen, 2004). ROS are therefore harmful to both cells and mitochondria. Mitochondrial defects can increase ROS production as well as reduce ROS removal. Electron leak is heightened during complex I inhibition by rotenone, cm hyperpolarization, and complex IV poisoning by cyanide (Sipos et al., 2003; Turrens and Boveris, 1980; Votyakova and Reynolds, 2001). On the other hand, when mitochondrial permeability transition is activated in pathological conditions as discussed in the next section, loss of glutathione from the matrix can severely compromise ROS removal (Savage et al., 1991). ROS production is promoted during glutamate excitotoxicity and reperfusion after ischemic injury (Du et al., 1998; Lafon-Cazal et al., 1993; Reynolds and Hastings, 1995; Schild et al., 1997). Additionally, chronic oxidative stress is implicated in many neurodegenerative conditions, including Parkinson’s disease (PD), AD, Friedreich’s ataxia, ALS and aging (Ferrante et al., 1997; Foury and Cazzalini, 1997; Jauslin et al., 2003; Kirkwood and Austad, 2000; Shaw et al., 1995; Smith et al., 1992). Modest inhibition of electron transport chain complex I, an effect mimicked by systemic administration of low concentrations of rotenone, was suggested to be a pathogenic cause of sporadic PD (Betarbet et al., 2000; Parker et al., 1989; Schapira et al., 1989). Instead of impairing mitochondrial respiration, chronic rotenone exposure in vitro was shown to reduce glutathione levels in cells, cause oxidative damage to proteins and DNA, and increase cell vulnerability to ROSinduced caspase-dependent cell death (Sherer et al., 2002) Ongoing oxidative stress also characterizes AD. Pathologic hallmarks of the disease are extracellular Ab plaques and intracellular neurofibrillary tangles (NFT) composed of the axonal MAP, tau. ROS damages mitochondria, generates mutations in mtDNA, increases production of Ab from APP and promotes plaque formation, and increases tau aggregation into paired helical filaments (Dyrks et al., 1992; Frederikse et al., 1996; Mecocci et al., 1994; Schweers et al., 1995). 2.2.4. Mitochondrial release of apoptogenic proteins Apoptosis is a highly regulated process of cell killing that is characterized by cell shrinkage, caspase activation, DNA fragmentation, chromatin condensation and nuclear fragmentation. Proteins released from mitochondria into the cytosol are important inducers of apoptosis. Cytochrome c is one such protein that forms an apoptosome complex in association with apoptosis protease-activating factor 1, ATP or dATP, and procaspase-9 (Cain et al., 2000; Li et al., 1997; Liu et al., 1996). ATP-dependent activation of procaspase-9 then initiates a proteolytic caspase cascade that leads to cell death (Li et al., 1997). The mitochondrial intermembrane space proteins

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Smac/DIABLO and Omi/HtrA2 are also released and promote apoptosis by antagonizing inhibitors of caspases (Du et al., 2000; Suzuki et al., 2001; van Loo et al., 2002; Verhagen et al., 2000). Additionally, apoptosis inducing factor and endonuclease G are mitochondrial proteins that mediate caspaseindependent DNA fragmentation and chromatin condensation (Li et al., 2001; Susin et al., 1999). Thus, mitochondria harness a cohort of proteins that can lead to the demise of cells. The release of proapoptotic proteins from mitochondria is a critical event that probably involves permeabilization of mitochondrial membranes. Various mechanisms for achieving permeability have been suggested although the specific sequence of events and regulations thereof remain unclear. One proposal involves activation of the mitochondrial permeability transition pore (mPTP), a large conductance channel of solutes up to 1.5 kDa that spans the outer and inner mitochondrial membranes. Although mPTP is well described in isolated mitochondria, its involvement in neuronal injury remains controversial (Reynolds, 1999). The pore is composed of the inner membrane adenine nucleotide translocator (ANT), the outer membrane voltage-dependent anion channel (VDAC), and matrix cyclophilin D. mPTP can be opened in pathological conditions of high [Ca2+]mito, high levels of inorganic phosphate (Pi), oxidative stress, ATP depletion and cm depolarization (Crompton, 1999). Cyclophilin D was recently shown to mediate mitochondrial permeability transition and death of non-neuronal cells induced by Ca2+ overload and reactive oxygen species (Baines et al., 2005; Nakagawa et al., 2005). The influx of water into the matrix following mPTP activation causes swelling and outer membrane rupture, which would facilitate apoptogen release. A second possibility is that proapoptotic proteins Bax and tBid interact with ANT and VDAC to cause mitochondrial permeability transition (Marzo et al., 1998; Rostovtseva et al., 2004). Lastly, activated Bax or Bak may directly form channels that permit release of apoptogenic mitochondrial proteins (Kuwana et al., 2002; Wei et al., 2000). Ultimately, permeability transition of mitochondrial membranes leads to cm depolarization, uncoupling of oxidative phosphorylation, mitochondrial swelling, ATP depletion and cell death.

proteins, approximately 850 in total, are encoded by nuclear DNA and imported into the appropriate mitochondrial compartments (Neupert, 1997; Pfanner, 1998). Mitochondria have a limited lifespan, but our observations indicate that the number and function of mitochondria seem well preserved through the healthy lifetime of neurons (Chang and Reynolds, 2006; Gross et al., 1969; Menzies and Gold, 1971). Although de novo synthesis of mitochondria has not been shown explicitly, mtDNA clearly replicates and repairs in vertebrates (Shadel and Clayton, 1997). Mitochondrial number can also increase from fission of preexisting mitochondria in dividing cells and post-mitotic neurons, which would preserve total mitochondrial mass unlike true mitochondrial biogenesis. Mitochondria seem to proliferate in response to increased energetic demands of cells, such as in resting skeletal muscle after prolonged contractile stimulation (Hood, 2001). However, mitochondria also proliferate in myopathies associated with mtDNA mutations (Aure et al., 2006). A reasonable proposition is that mitochondrial biogenesis occurs near the cell body in close proximity to the transcriptional and translational machinery for nuclear DNA on which mtDNA replication depends. In support of this, mtDNA replication has been shown to occur perinuclearly in mammalian cells and distribute peripherally over time (Davis and Clayton, 1996). Furthermore, 90% of mitochondria with relatively high membrane potentials move anterogradely toward growth cones in dorsal root ganglia neurons, implying that healthy mitochondria are trafficked away from their origins near the cell body (Miller and Sheetz, 2004). An important consideration therefore is how the appropriate distribution of mitochondria can be achieved in cells, and particularly neurons that have extensive neuronal processes. It should be noted, however, that very little is known for certain about mitochondrial biogenesis and where it occurs in neurons. Evidence for protein synthesis in axons and dendrites opens the possibility that mitochondrial biogenesis can also occur remotely from the cell body (Glanzer and Eberwine, 2003; Piper and Holt, 2004).

3. Mitochondrial life cycle in neurons

Rat brain mitochondria are reported to have a half-life of 24– 30 days, in contrast to 9–10 days for liver mitochondria (Gross et al., 1969; Menzies and Gold, 1971). Mitochondria represent an interesting paradox because while they provide critical functions for cell survival, their dysfunction can be a key determinant of cell death. It is therefore imperative for cells to regulate the proper removal of damaged mitochondria, presumably when they are functionally impaired or have accumulated mtDNA mutations that generate ROS. This is accomplished through autophagy, a process in which autophagosomes engulf cytosolic contents and then fuse with lysosomes for proteolytic degradation (Kelekar, 2006). Autophagy can be targeted directly toward mitochondria, which is mediated by the outer mitochondrial membrane protein Uth1p in yeast cells (Kissova et al., 2004). Mitochondrial autophagy occurs in cells when apoptosis is induced, in

3.1. Evolution and biogenesis of mitochondria Mitochondria in eukaryotic organisms are thought to be the result of engulfment of aerobically respiring prokaryotic organisms 1.5  109 years ago. A symbiotic relationship was then forged whereby mitochondria were able to rely on the host cell for transcription and protein synthesis and the host adopted efficient energy production (Gray, 1993). Therefore, while present-day mitochondria still have their own circular doublestranded genomes and protein synthetic machinery, evolution probably removed a fair amount of redundancy. In fact, mammalian mtDNA encodes only 37 genes: 22 tRNAs, 2 rRNAs and 13 structural proteins for oxidative phosphorylation (Garesse and Vallejo, 2001). All remaining mitochondrial

3.2. Mitochondrial autophagy

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hepatocytes with mitochondrial permeability transition pore formation, and in yeast with depolarized mitochondria (Elmore et al., 2001; Priault et al., 2005; Xue et al., 2001). Much like the biogenesis of mitochondria, the degradation of mitochondria is hypothesized to occur near the cell soma where lysosomes are more prevalent. Indeed, 80% of low-potential mitochondria in dorsal root ganglia neurons move retrogradely, implying less healthy mitochondria may return to the cell body for repair or removal (Miller and Sheetz, 2004). Again, this raises the challenge of retrieving distant mitochondria from axons and dendrites for processing in the cell soma. Electron microscopy indicates that autophagic vacuoles also exist in distal axons of cultured sympathetic neurons, and undergo regulated retrograde transport back to the lysosomal compartment (Hollenbeck, 1993). Importantly, there is still no evidence excluding mitochondrial autophagy in neuronal processes. 4. Mitochondrial morphology Mitochondrial morphology is diverse and dynamic, varying between different cell types, within individual cells, and under different cellular environments. We observed changes in mitochondrial morphology as cortical neurons develop synaptic connections, and between axons and dendrites of mature neurons (Figs. 1 and 2A and B). Popov et al. (2005) reported similar findings of filamentous dendritic mitochondria compared to discrete axonal mitochondria in hippocampal slices prepared from rats and ground squirrels. Mitochondria undergo fission and fusion with each other and can elongate or shorten under normal physiological conditions as well as during injurious stimuli. Studies reveal instances where mitochondria are electrically isolated from each other, where they form large interconnected filamentous networks, and where both morphologies are found in a single cell (Collins and Bootman, 2003; Skulachev, 2001). Mediators and mechanisms of mitochondrial fusion and fission are currently being identified. Pharmacologic manipulations are also revealing how mitochondrial morphology may relate to mitochondrial function and cell survival. However, it is important to mention that although we have demonstrated endogenous expression of mitochondrial fusion and fission proteins in cultured cortical neurons, much of our current knowledge about mitochondrial morphology is derived

from studies in non-neuronal cells in vitro (Chang and Reynolds, 2006). 4.1. Regulation of morphology Mitochondrial fission and fusion are directly responsible for altering mitochondrial morphology in a rapid and dynamic manner in addition to changing the number of independently operating organelles. Fission and fusion are complex processes that involve separate reactions of the inner and outer mitochondrial membranes. A host of proteins are known to mediate these reactions. Dynamin-related GTPases mediate division of the outer mitochondrial membrane (Pitts et al., 1999; Smirnova et al., 2001). Drp1 and its homologues can assemble into spiral structures thought to wrap around mitochondrial scission sites. Studies in yeast indicate that outer membrane fission is accomplished in conjunction with other proteins, including Mdv1/Fis2/Gag3, Dnm1, and Fis1/Mdv2, that either directly form or help to assemble a division apparatus (Mozdy et al., 2000; Tieu and Nunnari, 2000). Very little is known about the nature of inner membrane division, which is a separate process thought to be mediated by inner mitochondrial membrane proteins. Electron microscopy revealed that septation of the inner membrane divides the mitochondrial matrix before the outer membrane divides (Larsen, 1970). Inner membrane fission is often but not always coupled to outer membrane division (Twig et al., 2006). Mitochondrial fusion also involves reactions between the outer and inner membranes that are mediated by mitochondrial membrane proteins. In mammals, the outer membrane GTPases, mfn1 and mfn2, and the inner membrane dynamin-related protein, OPA1, interact to effect fusion events (Chen et al., 2003; Cipolat et al., 2004; Legros et al., 2002). Notably, OPA1 is also implicated in mitochondrial fission (Griparic et al., 2004; Misaka et al., 2002). It is still not known how fusion of the separate membranes is coordinated. Interesting evidence that inhibition of glycolysis and cm depolarization preferentially impair fusion of the inner membrane over the outer membrane supports the idea that fusion of the two membranes is separate and differentially regulated (Malka et al., 2005).

Fig. 1. Mitochondrial movement and morphology differ between synaptically immature and mature neurons. (A and B) Transfection of cortical neurons with mitoeYFP revealed a different mitochondrial morphology and distribution in 5 DIV neurons compared to 14 DIV neurons. Scale bar, 25 mm. (C) Mitochondria in mature neurons were significantly longer than in immature neurons (adapted from Chang and Reynolds, 2006).

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Fig. 2. Mitochondrial morphology, distribution and movement differ between axons and dendrites. (A) Primary cortical neuron transfected with mito-CFP (red), presynaptic label synaptophysin-eYFP (green) and postsynaptic label PSD-95-mRFP (blue). (B) Mitochondrial length was significantly shorter in axons than in dendrites. (C) Mitochondrial flux, measured as the number of mitochondria passing a given point on a neuronal process over time, was greater in axons than dendrites and was increased in all processes by 1 h treatment with 200 nM TTX. Asterisk (*) represents comparisons between control and TTX treatment and + represents comparisons between axons and dendrites (adapted from Chang et al., 2006a). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

4.2. Functional implications There is very little evidence directly relating mitochondrial morphology to mitochondrial function. Experiments in yeast indicate that mitochondria fuse after conjugation and the integrity of mtDNA relies on exchanges that occur when mitochondria fuse and divide (Guan et al., 1993; Nunnari et al., 1997; Shepard and Yaffe, 1999). For example, when fission and fusion are blocked, mitochondrial morphology appears normal but the organelles become less capable of respiration and lose their mtDNA more rapidly than wildtype mitochondria (Okamoto and Shaw, 2005). Furthermore, the respiratory defects of mutation-harboring mammalian mitochondria were protected by fusion and subsequent mtDNA complementation with wildtype mitochondria (Nakada et al., 2001; Ono et al., 2001). Complementation and possible recombination and repair of mtDNA between organelles is important because mtDNA is vulnerable to mutations from mitochondriallygenerated ROS and mitochondria have inefficient mtDNA repair mechanisms (Linnane et al., 1989). Beyond the role of mitochondrial fusion in mtDNA exchange, little is known about why mitochondria fuse, divide, elongate and shorten. There is much speculation about the impact of different mitochondrial morphologies and these theories are discussed below. Different mitochondrial morphologies and the ability to rapidly alter morphology during normal physiological conditions are thought to confer certain advantages to the organelles. For example, mitochondrial morphology might influence energy distribution. ATP dispersal over a more extensive cytoplasmic area could be facilitated by increasing mitochondrial length and thus reducing diffusion distance. By the same reasoning, longer mitochondria may sequester ions over a given surface area more effectively. This might be disadvantageous with regard to Ca2+, of which mitochondrial uptake can be cytotoxic (Stout et al.,

1998; Szabadkai et al., 2004). Another bioenergetic advantage can be derived from the electric cable equation, which predicts that the energy loss associated with respiration is minimized over longer mitochondrial lengths (Skulachev, 2001). Therefore, mitochondria-requiring sites that are in close proximity to each other might be efficiently served by longer mitochondria. This could be an adaptive response since mitochondria in yeast and plant cells transform from isolated organelles into extended networks under conditions of hypoxia and impaired energy production (Bereiter-Hahn, 1990; Foissner, 1983). On the other hand, it is thought, though not experimentally proven, that discrete mitochondrial bodies harbor unique benefits over long filamentous mitochondrial structures. Mitochondrial mass is limited in cells, so division of mitochondria may be an effective method of generating ‘‘new’’ organelles (Posakony et al., 1977). Mitochondrial fission may also be an important response for addressing different energetic demands in post-mitotic cells, as newly divided mitochondria have been anecdotally reported to adopt different motility patterns from each other. It is possible that smaller cargos might require fewer motor proteins, and thus less ATP to power motility. Discrete mitochondria could additionally have the advantage of being electrochemically protected from mitochondrial damage or depolarization, which would spread through an extensive mitochondrial network (Szabadkai et al., 2004). Thus, injury-induced mitochondrial division may be a protective mechanism. Fission of the inner but not outer mitochondrial membrane was shown to mediate electrical dissociation within a mitochondrial network (Twig et al., 2006). It has also been suggested that condensed mitochondria have heightened metabolic activity because they have a more electron dense matrix (Hackenbrock, 1966). A recent study interestingly demonstrated that mitochondrial fission under high glucose conditions increased respiration rates (Yu et al.,

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2006). Many of these studies were performed in non-neuronal cells. If different morphologies prove to confer functional advantages to mitochondria in neurons, then a balance is probably achieved between mitochondrial morphology, the functional consequences of altered morphology, and the demands of the cellular environment. 4.3. Altered morphology in neurotoxicity Not only is mitochondrial morphology regulated for the maintenance of normal cell health, but it also undergoes remodeling during mitochondrial and cellular injury and cell death. The morphology change typically takes the form of shortening or fragmenting. Experiments with uncouplers and inhibitors of electron transport and ATP synthase in fibroblasts suggest that intact oxidative phosphorylation is necessary to maintain mitochondrial networks (Skulachev, 2001). While the spread of mitochondrial injury through extensive organellar networks might be limited by dividing mitochondria into isolated entities, Drp1 seems to mediate fission of even punctate mitochondria during programmed cell death (Karbowski et al., 2002; Szabadkai et al., 2004). Remodeling of mitochondrial morphology is therefore likely to have both protective and toxic outcomes. Our laboratory has a special interest in the role of mitochondria in glutamate excitotoxicity. We previously showed that mitochondria uptake large Ca2+ loads after acute excitotoxic glutamate exposure, and that this uptake by mitochondria rather than just elevated [Ca2+]i has direct consequences for cell death (Stout et al., 1998; White and Reynolds, 1997). However, our investigation of the effects of moderately excitotoxic glutamate treatment on mitochondrial movement cessation and morphologic remodeling revealed a dependence on [Ca2+]i (Rintoul et al., 2003a). We found that remodeling of mitochondrial morphology, which consisted of shortening and rounding of the organelles, was specific to elevated Ca2+ influx through Nmethyl-D-aspartate (NMDA) receptors separately from cm depolarization and inhibition of mitochondrial ATP synthesis. Glutamate treatment actually caused swollen varicosities to form

in the cytosol, which could very well influence mitochondrial morphology (Fig. 3). Shortening of mitochondrial length was also demonstrated in cortical neurons under hypoxic conditions (Zanelli et al., 2006). It is possible that mitochondrial fragmentation contributes to the punctate morphology observed in these experiments, but limitations in imaging resolution precludes the differentiation of true membrane fission from independent rounding of closely apposed mitochondria. In contrast to glutamate-induced morphologic remodeling which appears to preserve the continuity of individual mitochondria, induction of apoptosis by staurosporine causes mitochondria to fragment. Frank et al. (2001) demonstrated Drp1 mobilization from the cytosol to mitochondria when apoptosis was induced in COS-7 and HeLa cells. The proapoptotic protein Bax localized to discrete mitochondrial foci containing Drp1 during the initial stages of apoptosis (Karbowski et al., 2002). In fact, a dominant negative mutant of Drp1 prevented not only mitochondrial fission, but also cm depolarization, cytochrome c release and cell death. Mitochondrial fission is thought to be related to mitochondrial outer membrane permeabilization and release of proteins involved in the cell death cascade (Perfettini et al., 2005). Recently, release of the mitochondrial inner membrane space protein DDT/TIMM8a into the cytosol was shown to mediate redistribution of Drp1 onto mitochondria during programmed cell death in HeLa cells (Arnoult et al., 2005). Similar findings were reported in yeast, where the antiapoptotic proteins Bcl-2 and Bcl-xL inhibited Dnm1-mediated mitochondrial fission and cell death (Fannjiang et al., 2004). The Bcl-2 related protein in C. elegans, CED-9, was also shown to interact with mfn-2 and promote mitochondrial fusion in mammalian cells, suggesting that the role of Bcl-2 family proteins in mitochondrial morphology dynamics is evolutionarily conserved (Delivani et al., 2006). 5. Significance of mitochondrial transport: focus on synapses Mitochondrial movement has been described for many years (Lewis and Lewis, 1914), but is assisted these days by

Fig. 3. Changes in mitochondrial morphology during excitotoxic and apoptotic stimuli. A neuron co-transfected with mito-eYFP (green) and cytosolic eCFP (yellow) is shown before (A) and after (B) a 5 min treatment with 30 mM glutamate/1 mM glycine. Mitochondrial rounding and cytosolic varicosities are seen after glutamate treatment. Inset: magnified region (adapted from Rintoul et al., 2003a). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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fluorescent labels for mitochondria in live cells, time-lapse microscopy and genetic mutants with abnormal trafficking patterns. These techniques are used to study patterns of mitochondrial movement, mechanisms of mitochondrial transport, and perhaps most importantly, cellular and mitochondrial signals that dynamically govern mitochondrial trafficking. Anterograde and retrograde trafficking of mitochondria are thought to support active processes throughout the cellular lifetime as well as the organellar lifetime. Mitochondrial movement facilitates fusion and fission events between individual organelles that likely maintain mitochondrial health and homeostasis, as well as retrieval of damaged mitochondria for degradation by autophagy. It is important to note that our understanding of mitochondrial trafficking is largely derived from in vitro studies that may present experimental artifacts related to cellular bioenergetics. Recent reports using Drosophila containing a mitochondriallytargeted GFP transgene provide the first evidence that mitochondrial movement patterns and mediators initially described in vitro are recapitulated in vivo (Glater et al., 2006; Pilling et al., 2006). While mitochondrial transport in mature neurons is of great interest as it relates to neurodegeneration and aging, it is also important to consider the relevance of mitochondrial transport in neurodevelopment. Neurons develop from undifferentiated neuroblasts that lack any sort of axonal or dendritic specializations. Mitochondria are therefore located in the cell soma. Neuronal morphology then changes dramatically as neuronal processes rapidly elongate, an axon is designated, axonal and dendritic branches form and remodel, and synaptic connections assemble and mature (Antonini and Stryker, 1993; Ramoa et al., 1988). These are highly dynamic processes marked by an intense need for ATP. Indeed, mitochondria have been shown to localize to growth cones of actively growing axons by chemoattraction to nerve growth factor (NGF) (Chada and Hollenbeck, 2003, 2004; Morris and Hollenbeck, 1993). As neurons continue to grow, energetic demands extend even greater distances from the cell body. Thus, appropriate trafficking of mitochondria is a necessary task beginning in the earliest moments of neuronal development that becomes increasingly important as development proceeds.

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Neurons are highly specialized cell types and they have a great capacity to support complex neurotransmission communications with other cells. They represent a particular anatomical and physiological challenge for mitochondrial trafficking because: (i) neuronal processes are highly branched and extensive, as long as several meters in some cases, and (ii) sites with large demands for ATP and local [Ca2+]i regulation, such as synapses, are distributed throughout the length of axons and dendrites. Furthermore, synaptic connections are highly plastic, undergoing continuous, spontaneous and directed remodeling to modulate information processing (Hayashi and Majewska, 2005; Konur and Yuste, 2004). Remodeling of presynaptic terminals and postsynaptic sites involves not only local cytoskeletal rearrangements and protein synthesis, but also recruitment of vesicles and receptors as well as removal and degradation of synaptic proteins (Bradshaw et al., 2003; Rao and Craig, 1997; Zito et al., 2004). Neuronal processes therefore present many sites of dynamically changing demand for mitochondria, and these sites are likely regulators of mitochondrial trafficking. Understanding how mitochondria are trafficked to synapses in healthy neurons will help us deduce how disruptions of trafficking lead to synaptic dysfunction in aging, neuronal injury and neurodegeneration. Our studies of mitochondrial dynamics during the development of synaptic connections in cortical neurons revealed reduced mitochondrial movement and elongated morphology in mature neurons compared to developing neurons (Fig. 1) (Chang and Reynolds, 2006). We then demonstrated that synapses are targets for long-term mitochondrial localization and dynamic recruitment of moving mitochondria in spontaneously firing neurons (Fig. 4) (Chang et al., 2006a). This coincides with recent observations that moving mitochondria pause at active vesicle release sites in spontaneously active respiratory neurons (Mironov, 2006). While the mitochondrial localization at synapses we measured was unchanged by inhibition of synaptic activity by tetrodotoxin (TTX), it increased in dendritic synapses and decreased in axonal synapses during overactivity by veratridine. In fact, we and others demonstrated that mitochondrial movement increases during synaptic inactivity, presumably because mitochondria have fewer associations with firing synapses (Fig. 2C) (Li et al.,

Fig. 4. Mitochondrial localization to synapses are modulated by changes in synaptic activity. (A) Mitochondria localized preferentially to presynaptic synaptophysin clusters (white bars) in untreated and TTX-treated cells but not in veratridine-treated cells. Black bars represent the probability that mitochodria were located in axonal sites void of synaptophysin clusters. (B) Mitochondria localized preferentially to postsynaptic PSD-95 clusters (white bars) and veratridine treatment increased this localization frequency relative to untreated cells. Black bars represent the probability that mitochodria were located in dendritic sites void of PSD-95 clusters. Asterisk (*) represents comparisons between synaptic sites and nonsynaptic sites in the same cells, and + represents comparisons between untreated cells and pharmacologically treated cells (adapted from Chang et al., 2006a).

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2004; Mironov, 2006). High synaptic density in dendrites also correlated with reduced mitochondrial movement and longer morphology compared to axons. Therefore, mitochondrial trafficking and morphology patterns are probably optimized to best provide for changing energetic demands as synaptic connections are formed, and also to support different synaptic processes in axons and dendrites of individual neurons. Several recent studies also examined the importance of mitochondrial delivery for synaptic function. Stowers et al. (2002) identified Milton as a novel protein in Drosophila that associates with kinesin heavy chain (KHC) to mediate axonal transport of mitochondria for normal photoreceptor neurotransmission. Miro, a Rho-GTPase located on the outer mitochondrial membrane, was recently shown to mediate the interaction between Milton and KHC (Fransson et al., 2003; Glater et al., 2006). Mutation of Drosophila miro (dMiro) caused redistribution of mitochondria in Drosophila motor neurons from axons and presynaptic terminals to neuronal cell bodies (Guo et al., 2005). Reintroducing dMiro presynaptically caused mitochondrial accumulation in distal boutons, implying that dMiro mediates anterograde mitochondrial transport. Functional studies then showed that mitochondrial localization to presynaptic terminals was required for adequate neurotransmitter release and mitochondrial Ca2+-buffering at neuromuscular junctions (NMJ) during prolonged stimulation. Therefore, it seems mitochondria may play a more important role in supporting synapses with heightened activity rather than general synaptic function. In support of this, Verstreken et al. (2005) mutated a Drp1 ortholog protein to demonstrate that proper mitochondrial distribution is needed for mobilization of reserve pool vesicles in Drosophila NMJs. In contrast to the recycling vesicle pool, which is mobilized and continuously recycled during moderate physiological stimulation, the reserve vesicle pool is typically recruited during high stimulation (Rizzoli and Betz, 2005). Mutant axons contained elongated mitochondria, few of which localized to boutons. Prolonged stimulation evoked excitatory junctional potentials with reduced amplitude that recovered with exogenous ATP. Studies with the membrane probe FM143 then demonstrated that mitochondrial ATP supply was

required for normal recycling and/or mobilization of vesicles in the reserve pool but not recycling pool. Finally, an investigation by Li et al. (2004) examining mitochondrial localization at dendritic spines provides an interesting postsynaptic comparison to the presynaptic studies described above. After intense stimulation of cultured hippocampal neurons and slices, new dendritic protrusions formed, mitochondria extended into protrusions, and mitochondria occupied a smaller fraction of the dendrite shaft and moved at slower velocities. This was suggested to be caused by Drp1mediated mitochondrial fission and redistribution out of the dendrite shaft into spines undergoing morphogenesis. The finding that only a small fraction of protrusions seemed to recruit mitochondria further implies that mitochondria are dynamically trafficked to subsets of synapses with specific activity characteristics. Popov et al. (2005) proposed that their finding of mitochondria penetrating CA3 thorny excrescences but not CA1 spines is related to differing rates of Ca2+ diffusion from the dendritic shaft into protrusions in different hippocampal regions. The next logical step is to identify distinguishing functional qualities of synaptic sites that recruit mitochondria. Our findings suggest at least three populations of mitochondria may exist with different motility characteristics: (1) relatively stationary mitochondria, (2) mobile mitochondria that pause on the order of seconds and (3) mobile mitochondria that pause on the order of minutes (Fig. 5) (Chang et al., 2006a). Two recent studies described differences in Cm and Ca2+-sensitivity between synaptic and nonsynaptic mitochondria in Xenopus nerve–muscle co-cultures and isolated from rat cortices, respectively (Brown et al., 2006; Lee and Peng, 2006). An exciting theory is that populations of mitochondria with different functional capacities within a single neuron can be trafficked to specific locations such as synapses. 6. How mitochondria move 6.1. Patterns of mitochondrial movement Mitochondrial movement in neurons is highly diverse and complex (vide infra; Chang et al., 2006a; Chang and Reynolds,

Fig. 5. Populations of mitochondria with different movement patterns can be observed. Mitochondria that were relatively immobile tended to remain stationary at synaptic and nonsynaptic sites for >15 min. Relatively mobile mitochondria exhibited saltatory movement, making stops at synaptic and nonsynaptic sites for shorter (s) or longer time periods (min) (adapted from Chang et al., 2006a).

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2006; Ligon and Steward, 2000a; Morris and Hollenbeck, 1995, 1993; Overly et al., 1996). Some mitochondria appear stationary whereas others are motile. Motile mitochondria not only move with different speeds and in different directions, but they exhibit saltatory movement, making stops along their trajectory. These stops are also variable. Mitochondria can stop for seconds, minutes, or much longer time periods. They often stop where other mitochondria already reside, but can also pause along processes where no mitochondria are present. Moving mitochondria spontaneously change direction, sometimes appearing to shuttle between several specific locations and other times continuing to travel consistently in the new direction. Not only are mitochondrial movement patterns highly variable between axons, dendrites and among individual mitochondria in a single neuron, but also in different types of neurons at different developmental stages. Hollenbeck and Saxton (2005) provide a recent review of mitochondrial transport in axons. The intra- and intercellular diversity of mitochondrial movement patterns complicates quantitative description. Automated data analysis is often unfeasible because it cannot account for the variability in movement patterns. Consequently, some of the most detailed data analysis has been performed manually, which is labor intensive and open to subjectivity. Studies in cultured hippocampal neurons consistently indicate that a greater fraction of mitochondria move in axons than in dendrites (Ligon and Steward, 2000a; Overly et al., 1996). Our observations suggest the same to be true in cultured cortical neurons (Fig. 2C). Mean mitochondrial speeds are reported to be similar in the anterograde and retrograde direction, but while the probability of moving in either direction is equal in axons, it is increased in the anterograde direction in dendrites (Li et al., 2004; Ligon and Steward, 2000a; Overly et al., 1996). 6.2. Mechanisms of mitochondrial movement Three major protein groups are involved in transporting mitochondria in neurons: (1) cytoskeletal proteins, (2) molecular motors and (3) a host of adaptor and scaffolding proteins that mediate interactions between motors, cargos and the cytoskeleton. Several proteins outside of these classes have also been implicated as modulators of movement, including plekstrin homology domain proteins and transiently expressed developmental proteins (De Vos et al., 2003; Gross et al., 2003). Mitochondria are transported on microtubules and actin microfilaments. How exactly these cytoskeletal substrates mediate the dynamics of saltatory mitochondrial movement is not clear, as different studies often report different effects of cytoskeletal disruption. For example, depolymerization of microtubules with nocodazole or vinblastine results in fewer moving mitochondria and slower velocities while bidirectionality is preserved. When actin filaments are depolymerized or disorganized by latrunculin B, cytochalasin D or E, the results include fewer moving mitochondria, increased velocity bidirectionally, or no effect (Ligon and Steward, 2000b; Morris and Hollenbeck, 1995). In neurons, microtubules are likely to be tracks for transport over long distances while actin

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microfilaments mediate travel over short distances. Since mitochondrial transport on microtubules is bidirectional, the polarity orientation of tubulin filaments may also affect mitochondrial trafficking patterns. In axons, microtubules are uniformly arranged with (+)-ends directed toward the growth cones. Distal dendritic segments display the same orientation, but proximal segments contain microtubules with (+)-ends oriented in both directions (Baas et al., 1989). Different microtubule-associated proteins (MAPs) are distributed in axons, dendrites or throughout cells, and help stabilize and promote oriented microtubule polymerization. Regulating MAP phosphorylation, which causes microtubules to disassemble, might be another method of modulating mitochondrial trafficking (Ebneth et al., 1999). Kinesins and cytoplasmic dynein are ATPases that transport mitochondria toward (+)- and ( )-ends of microtubules, respectively. Generally, kinesin superfamily proteins (KIFs) consist of: (i) a globular motor domain containing microtubuleand ATP-binding sequences, and (ii) a cargo-binding domain that imparts selectivity to different isoforms. Each KIF has an intrinsic velocity, ranging from 0.2 to 1.5 mm/s (Hirokawa and Takemura, 2005). Monomeric KIF1Ba and homodimeric KIF5 transport mitochondria in axons and dendrites (Kanai et al., 2000; Nangaku et al., 1994; Tanaka et al., 1998). Cytoplasmic dynein has a globular head from which a stalk and stem extend. The former binds microtubules and the latter binds cargo through association with the dynactin protein complex. Dynein transports organelles retrogradely although it can also localize to those that are transported anterogradely (Hirokawa et al., 1990). Phosphorylation states, conformational changes, and abundances of motor proteins and MAPs are thought to regulate aspects of motor activity, directionality and association with cargo (Reilein et al., 2001; Sheetz, 1999). The axonal MAPs, MAP1B and tau, are thought to mediate mitochondrial movement (Jime´nez-Mateos et al., 2006; Trinczek et al., 1999). Furthermore, motor proteins on mitochondria are distributed in discrete clusters that seem to generate force independently of each other, suggesting that differential regulation of individual motor protein clusters may mediate changes in velocity and direction (Martz et al., 1984). Interestingly, motor proteins are fully functional at ATP concentrations below normal [ATP]i, suggesting that transport of mitochondria is an efficient method of energy dispersal in cells (Erecinska and Silver, 1989; Visscher et al., 1999). The interaction between motor proteins and their cargos are mediated by adaptor and scaffolding proteins. Milton and syntabulin are implicated as scaffolding proteins for mitochondria to KHC (Cai et al., 2005; Stowers et al., 2002). When expression of these proteins is reduced, mitochondria are abnormally distributed as expected from disrupted anterograde transport. APLIP1 is another example of a scaffolding protein that may interact with both kinesin and dynein to affect retrograde transport of mitochondria (Horiuchi et al., 2005). In summary, complex mitochondrial movement patterns are likely mediated by complex and dynamic interactions between a cohort of regulated motor proteins, adaptor proteins, and cytoskeletal elements.

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6.3. Mechanisms of mitochondrial docking It is important to consider not only what drives mitochondria to move, but also what signals them to stop and how these stops are mechanically executed. A large fraction of mitochondria are stationary for extended time periods and moving mitochondria often make stops along their trajectory. It can be speculated that some pauses are incidental based on the structure and length of cytoskeletal filaments. However, many stops are likely to occur for functional reasons. The clearest evidence for a chemoattractive signal for mitochondria in neurons is nerve growth factor (NGF) (Chada and Hollenbeck, 2003, 2004). Time-lapse microscopy demonstrated more mitochondria moving toward and being retained at the focus of NGF-coated beads compared to control beads. Furthermore, these ‘‘docking’’ events involved phosphoinositol 3-kinase-(PI 3-kinase) mediated signaling and intact actin microfilaments, neither of which were necessary for mitochondrial movement. One resulting theory is that phosphorylation events regulate dislodgement of mitochondria carried by kinesin or dynein from microtubule tracks in exchange for a longer-term static interaction of actin with myosin or other docking proteins (Reynolds and Rintoul, 2004). In support of this, a recent study in Xenopus spinal neurons demonstrated mitochondrial clustering at beads coated with growth factors that was inhibited by disrupting actin filaments but not microtubules (Lee and Peng, 2006). In a more correlational study, Li et al. (2004) demonstrated mitochondrial localization at dendritic spines undergoing morphogenesis over 30 min to hour-long time intervals after hippocampal neurons were depolarized. The high concentration of actin in dendritic spines suggests that this may be another actin-mediated docking event. Interactions between mitochondria and neurofilaments, promoted by neurofilament phosphorylation, may also mediate mitochondrial docking (Leterrier et al., 1994; Wagner et al., 2003). 7. Adopting a reductionist approach to studying mitochondrial trafficking Mitochondrial movement in neurons is extremely diverse with velocity, direction changes and pauses during movement varying between individual organelles, different neuronal processes, and different cells. There is also likely to be variability in the regulatory influences on movement between individual mitochondria such that: (i) they may be targeted to different cues in cells, (ii) these cues may have various distributions in different neuronal processes, (iii) the characteristics of motor and adaptor proteins that mediate movement may differ between organelles and may be subject to different functional modifications, and (iv) cytoskeletal arrangements and interactions with MAPs can vary between neuronal processes and among different regions of a single process. This complexity makes it difficult to study any single variable in a controlled manner. One experimental approach is to identify trafficking cues by observing where mitochondria stop and go. Another method focuses on a cellular signal and attempts to determine how mitochondria move relative to that

signal. The results from one approach can then be tested by the other to confirm the identity and effects of a physiological signal on normal mitochondrial trafficking. 7.1. Subdividing the mitochondrial pool for analysis 7.1.1. Nonspecific mitochondrial measurements Trafficking of the mitochondrial pool can either be studied in totality or in partiality. The former involves nonspecifically measuring movement of any or all mitochondria in entire cells. This is not so much of an issue in cell lines with relatively indiscriminate distributions of cellular contents, but polarized neurons provide a different challenge. Sampling from the whole mitochondrial pool usually has little or no regard for the anatomic regions or activities of neuronal processes being examined, and sometimes even with processes from more than one cell being imaged at a time. Most experiments are done in this manner, as it is simpler, yields data rapidly, and in many cases, pharmacologic treatments seem to affect mitochondrial movement quite uniformly in cells (Rintoul et al., 2003a; Vanden Berghe et al., 2004; Verstreken et al., 2005; Yi et al., 2004). Yet true differences in mitochondrial movement in various cellular regions after drug application can easily be overlooked when those regions comprise a small fraction, if any, of the imaging field. For example, we recently uncovered a dendrite-specific remodeling of mitochondrial morphology and a preservation of mitochondrial movement in distal axons after acute glutamate exposure that was previously concealed by the all-inclusive imaging approach (Fig. 6A and B). 7.1.2. Measuring regional subpopulations of mitochondria The next step toward achieving specificity of measurements is distinguishing between anatomic regions of neurons, such as axons and dendrites or proximal and distal segments of processes. The axon–dendrite comparison has been made in previous reports characterizing mitochondrial movement patterns in hippocampal neurons (Ligon and Steward, 2000a; Overly et al., 1996). A differentiation between proximal and distal dendrites, where microtubule polarities are known to differ, was performed as well (Overly et al., 1996). Additionally, mitochondrial movement was compared in proximal and distal axons to determine the effect of growth cone proximity on trafficking (Morris and Hollenbeck, 1993). We also examined the differential regulation of mitochondrial morphology and movement relative to synapses on axons compared to dendrites (Figs. 2 and 4). These studies have been important in describing mitochondrial movement activities on a smaller spatial scale, but still suffer from lack of specificity because mitochondria with varying movement patterns almost always occupy a given region. In terms of studying the mitochondrial pool, the finest detail can be achieved by tracking the movement of individual mitochondria. 7.1.3. Studying trafficking of an individual mitochondrion It is important to understand how the variable movement patterns of single mitochondria contribute to the diversity observed in the whole mitochondrial pool. A promising

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Fig. 6. Dendritic mitochondria are more susceptible to movement impairment and morphological remodeling than axonal mitochondria after acute excitotoxic glutamate treatment, neurotoxic zinc exposure, and uncoupling of oxidative phosphorylation. (A) Mitochondrial length shortened in dendrites but not axons after glutamate treatment. No recovery was observed after a 10 min wash. (B) Mitochondrial movement (measured as average event count/average number of mitochondrial pixels) in dendrites and proximal axon segments decreased significantly after glutamate treatment, but was unaffected in distal axon segments. (C) Mitochondrial movement in axons but not dendrites significantly recovered 20 min after exposure to 10 min 3 mM ZnCl2/20 mM Na-pyrithione treatment followed by chelation with 5 min 25 mM TPEN. (D) Mitochondrial movement in axons but not dendrites significantly recovered 15 min after depolarization with 5 min 750 nM FCCP (adapted from Chang et al., 2006a).

approach for tracking individual mitochondria is time-lapse imaging of neurons transfected with a photoactivatable fluorescent protein targeted to mitochondria (Karbowski et al., 2004; Rintoul et al., 2003b; Twig et al., 2006). Focal photoactivation would label a discrete group of mitochondria, which could be analyzed individually once they moved away from each other. This method would provide greater resolution than fluorescent lipophilic cationic dyes that label all mitochondria on a coverslip, or transfection with mitochondrially-targeted fluorescent plasmids that label all mitochondria in a transfected cell. A fundamental question that remains unanswered is if mitochondria with different motility properties serve different trafficking functions, or if all mitochondria are subject to the same movement and recruitment cues in cells. For instance, if a particular site on a neuronal process requires ATP, does it indiscriminately recruit passing mitochondria until the demand is met, or does it recruit faster or slower moving mitochondria depending on the expected duration of demand? By the same token, are mitochondria that travel with high speed more likely to serve sites with transient demands, or is their speed caused by targeting to a distant destination where they will become stationary? The idea of a destination also raises the question of whether individual mitochondria are targeted to specific sites, or if they continuously circulate along neuronal processes and respond to recruitment signals as they are encountered. Observations by Chada and Hollenbeck (2004) over a 10 min interval where many but not all mitochondria moving past an NGF-coated bead stopped, and almost half of these mitochondria continued to move away from the bead after pausing suggest that: (i) almost all moving mitochondria can respond to a strong

recruitment stimulus, (ii) mitochondrial residence time at a single recruitment signal varies between individual organelles, and (iii) some mitochondria may not veer from their intended trajectory or may not recognize certain recruitment signals. Separately tracking the movement trajectories of individual mitochondria could very well reveal that organelles with certain velocities also exhibit different trafficking patterns. Mitochondria display various speeds and movement patterns, but it is unclear whether distinct populations of mitochondria really exist. For example, an oversimplified scheme may categorize mitochondria as: (i) relatively stationary, (ii) slow movers or (iii) fast movers. Frequency distributions of mitochondrial velocities indicate that velocities exist on a continuum up to approximately 1 mm/s, and are found in more discrete distributions at velocities greater than 1 mm/s (Ligon and Steward, 2000a,b; Morris and Hollenbeck, 1995). This agrees with the thought that structural properties of individual motor proteins dictate that protein’s power stroke, and there must be a finite number of motor protein isoforms associated with mitochondria (Trinczek et al., 1999). Alternatively, a combination of differentially regulated isoforms may transport a single organelle, thus allowing for marked velocity changes during its trajectory. Collecting movement data from single mitochondria could easily reveal if this is the case. Furthermore, correlating velocity with the frequency with which individual mitochondria stop, the time duration for which they stop, and whether stopping points are distributed at similar intervals for different organelles would provide valuable information about whether mitochondria are equally responsive to cellular cues or if different mitochondria respond to different cues, perhaps because of different mobility characteristics.

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7.2. Studying cellular targets and modulators of mitochondrial movement 7.2.1. Measuring mitochondrial trafficking to a single cue An alternative to deriving cellular cues from mitochondrial movement patterns is to select some cellular signal and study mitochondrial trafficking in response to changes in that signal. This was accomplished quite elegantly by Chada and Hollenbeck (2004), who plotted incoming and outgoing mitochondrial movements in the vicinity of an NGF-coated bead placed next to an axon. In contrast to control beads which mitochondria moved past with relatively continuous motion, more mitochondria moved toward NGF beads and tended to remain stationary after arrival. This resulted in a net accumulation of mitochondria specifically around the NGF beads, which was not observed with endosomes, lysosomes or vesicles. Their work provides an example of a single chemical target to which mitochondria are recruited. The experimental system used in the NGF studies offers several conveniences to the investigation of mitochondrial movement (Chada and Hollenbeck, 2003, 2004). First, large focal concentrations of NGF are not distributed throughout neuronal processes, but are instead found specifically at growth cones of developing axons where NGF promotes elongation and sprouting of collateral branches (Diamond et al., 1992; Gallo and Letourneau, 1998; Letourneau, 1978). This allowed the investigators to largely avoid the effects of confounding mitochondrial trafficking to high physiological NGF concentrations by placing experimental beads distant from the growth cone. Another advantage is their use of young, developing neurons that were still elongating their axons. Assembly of synapses, which influence mitochondrial movement and can act as confounding ‘‘docking’’ signals, is minimal at this time. Difficulties arise when studying mature neurons with many potential mitochondrial recruitment cues distributed throughout neuronal processes. 7.2.2. Studying mitochondrial movement responses to global pharmacologic treatments The adoption of a reductionist approach to studying mitochondrial trafficking is also relevant to pharmacologic manipulations. Describing normal mitochondrial movement patterns is necessary, but rarely enough to answer mechanistic questions about movement regulation. Studying the response of mitochondrial movement to pharmacologic alterations in cellular ion concentrations, neuronal activity and mitochondrial activity can elucidate some aspects of movement regulation. Many past studies applied drugs universally to cells on a coverslip through pre-incubation and/or perfusion. This is more physiologically relevant in some cases than in others. For example, in a model of ischemic excitotoxicity during which glutamate release is known to increase profoundly and cause elevated Ca2+ entry into cells, global perfusion of neurons with glutamate seems appropriate (Rintoul et al., 2003a). During ischemia, glutamate release from cells occurs at presynaptic terminals as well as by reverse transport of glutamate through glutamate transporters (Kimura et al., 1998; Rossi et al., 2000;

Takizawa et al., 1995). Presumably, drug perfusion might activate some glutamate receptors that would not normally encounter pathophysiologically released glutamate, but this is probably a minor concern given the substantially overactivated glutamatergic signaling that occurs during excitotoxicity. In another study, Yi et al. (2004) examined the effects of [Ca2+]i on mitochondrial movement by systemic application of vasopressin to mobilize Ca2+ from endoplasmic/sarcoplasmic reticulum (ER/SR) stores in the H9c2 cardiac cell line. This resulted in increased [Ca2+]i that was approximately simultaneous with reduced mitochondrial motility. Limits in spatial resolution precluded detection of microdomains of more elevated [Ca2+]i that would be expected near ER. Although cessation of mitochondrial movement occurred throughout entire cells in this study, [Ca2+]i microdomains are still highly regarded as potential targets for mitochondrial trafficking. Previous examinations of intact mitochondrial function in the form of cm or ATP synthesis as a requirement for normal mitochondrial movement have also employed global drug perfusion. Use of uncouplers such as carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP) indicates that cm depolarization inhibits mitochondrial movement (Rintoul et al., 2003a; Vanden Berghe et al., 2004). However, by applying the drug to entire coverslips, certain confounding factors are exacerbated. Uncoupling oxidative phosphorylation dissipates cm and the protonmotive force for ATP synthase, but also depletes cellular ATP by reverse flow of protons through ATP synthase (Nicholls and Ward, 2000). Mitochondrial movement could therefore be impaired by lack of ATP rather than just cm depolarization, as shown in response to the ATP synthase inhibitor oligomycin (Rintoul et al., 2003a). Interestingly, mitochondrial movement stops with oligomycin treatment even though ATP continues to be provided through glycolysis and molecular motors function at very low ATP concentrations (Visscher et al., 1999). Another consideration is that FCCP is a protonophore, which is not necessarily selective for mitochondrial membranes and it can disrupt ion homeostasis directly or indirectly through effects on cm and mitochondrial ATP synthesis (Nicholls, 2006; Rottenberg and Wu, 1998). Undesired effects include plasma membrane depolarization, Ca2+ entry through voltagegated calcium channels, elevated [Na+]i, blocked K+ conductance and cytoplasmic acidification (Buckler and VaughanJones, 1998; Tretter et al., 1998). We have observed a small increase in [Ca2+]i after FCCP treatment, but at levels lower than expected to impair mitochondrial movement (Chang and Reynolds, 2006). In summary, while investigations of mitochondrial function yield some interesting suggestions about movement regulation, the global perfusion of drugs produces physiological changes in whole cells that can also influence movement indirectly. Investigations of mitochondrial trafficking responses to synaptic activity have also employed global pharmacologic treatments. Modulation of voltage-gated Na+ channels to achieve synaptic silence and overactivity is a reasonable approach to gaining a global view of mitochondrial trafficking in entire cells when all synaptic sites are presumed to be

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behaving similarly. The case is simpler when considering synaptic silence by TTX treatment because action potential propagation is blocked and cell–cell communication ceases as evidenced by dissipation of synchronous [Ca2+]i transients in cultures (Chang et al., 2006a). Global silencing of synaptic activity was actually ideal for revealing an overall increase in nonspecific mitochondrial movement that might be obfuscated if fewer mitochondria or smaller regions of neuronal processes were studied (Chang et al., 2006a; Li et al., 2004). The examination of mitochondrial movement during global synaptic overactivity is more complicated. In our studies, cells were perfused with veratridine, an inhibitor of Na+ channel inactivation, which produced high-amplitude, recoverable [Ca2+]i transients as neurons fired spontaneously (Chang et al., 2006a). Presumably, this causes larger or prolonged neurotransmitter release that leads to postsynaptic overactivation. Our experience perfusing excitatory agonists, depolarizing KCl solutions, and antagonists of inhibitory GABA receptors produced some unfavorable effects, including prolonged Ca2+ influx and subsets of neurons that responded atypically. Despite potential pitfalls in heightening synaptic activity with global pharmacologic treatments, such methods have demonstrated mitochondrial recruitment to postsynaptic densities and dendritic spines (Chang et al., 2006a; Li et al., 2004).

receptor types, (ii) determining how local mitochondrial ATP depletion and/or impairment of mitochondrial Ca2+ uptake influence synaptic firing during physiological and long term stimulations, (iii) investigating whether local inhibition of mitochondrial function causes recruitment of normally functioning mitochondria and (iv) studying whether mitochondria are recruited when synapses are stimulated to undergo short or long-term plasticity and what Ca2+-buffering and ATPgenerating roles they play once recruited. In summary, the ease of pharmacologic manipulation of whole neurons has contributed a great deal over a short time period to our understanding of mitochondrial movement. However, the yield of such methodology is diminishing. There is a real difficulty in achieving specificity of results when drugs can have a host of undesired effects on whole cells that alter movement. The diversity in mitochondrial populations and physiological responses to changes in whole neurons also complicate data interpretation. Further examination of mitochondrial movement will benefit substantially from more sophisticated, local manipulations and mitochondrial measurements.

7.2.3. Local pharmacologic manipulations as a future approach to studying mitochondrial movement Adapting pharmacologic modulations to a smaller scale will permit more detailed examination of targets for mitochondrial trafficking, how the functional status of mitochondria affects motility, and the functional role that mitochondria play at specific neuronal locations. For example, instead of perfusing drugs over entire coverslips, a micro-perfusion system can be utilized to apply drugs locally over a small segment of neuronal process or over a small population of mitochondria. With regard to the studies described above, local application of: (1) a Ca2+mobilizing drug, Ca2+ ionophore or an NMDA receptor agonist could reveal mitochondrial recruitment to regions of elevated [Ca2+]i requiring mitochondrial buffering, (2) an uncoupler could effectively depolarize a small subset of mitochondria without causing substantial ATP loss or ion homeostasis disruption, and (3) global TTX treatment combined with local application of a postsynaptic receptor agonist or depolarizing agent could selectively activate synapses on a very small and measurable segment of neuronal process. Measuring mitochondrial trafficking under conditions of locally controlled synaptic activity, assisted by electrophysiological techniques and FM dyes that label vesicle release sites, provides an exciting opportunity to study the role of mitochondria at different types of synapses with different activities and plasticities. The function of mitochondria at these different synaptic sites as Ca2+-buffers or ATP synthesizers can be explored in detail with the combined use of locally applied modulators of cm and mitochondrial ATP synthesis as well as fluorescent calcium- and cm-indicators. Some interesting experiments include: (i) measuring mitochondrial trafficking to excitatory or inhibitory synapses containing different

The close relationship between mitochondrial movement, mitochondrial health and cell health dictates that impairment of any one of these properties can negatively impact the other two properties (Fig. 7). As discussed in Sections 6.2 and 7.2.2, mitochondrial movement in neurons is sensitive to changes in mitochondrial functional status, as well as to changes in the cellular environment such as shifts in ion concentrations and alterations in cytoskeletal structure. Depolarization of cm or inhibition of ATP synthase causes mitochondria to stop moving (Rintoul et al., 2003a). Therefore, cm seems important for mitochondrial movement, perhaps because it is a direct requirement for mitochondrial ATP synthesis. Other drugs that impair respiration, including nitric oxide (NO) and rotenone, also impair mitochondrial movement (Reynolds and Santos, 2005; Rintoul et al., 2006; Zanelli et al., 2006). These studies suggest that mitochondrial movement can be compromised under a variety of altered physiologic states that occur in neuronal pathology. Different injury pathways may also converge on mitochondrial movement in individual neurons. Impaired mitochondrial transport probably has multiple consequences that increase in severity with the duration of impaired transport. Poor ATP distribution and inefficient Ca2+-sequestration are likely to be initial outcomes. Since mitochondria are found throughout neuronal processes, these effects would probably be limited by the diffusion of ATP and Ca2+ and the ability of other ATP sources and Ca2+-regulators to compensate for ineffective mitochondrial delivery. The cellular processes that are most likely to be at a disadvantage are those that require mitochondria in a rapid and dynamic manner. These processes, which remain to be identified but probably include specific synaptic sites undergoing morpho-

8. Relationship between mitochondrial movement and mitochondrial and cellular injury

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Fig. 7. Relationship between impaired mitochondrial movement, mitochondrial dysfunction and neuronal injury and disease.

genesis or potentiation, are likely to actively recruit mitochondria under normal conditions (Li et al., 2004; Tang and Zucker, 1997; Yang et al., 2003). Prolonged impairment of mitochondrial movement may also impede fusion-mediated mtDNA complementation and autophagic degradation of damaged mitochondria (Nakada et al., 2001; Ono et al., 2001; Priault et al., 2005). As a result, mtDNA mutations can accumulate and cause electron transport defects and ROS production. This would lead to further mtDNA mutation, lipid peroxidation, protein oxidation, and ultimately cell death (Andersen, 2004). The course of neuronal dysfunction and cell death is likely impacted by the timeframes over which mitochondrial movement is impaired and recovers, and any concurrent cellular and mitochondrial defects. Neuronal insults can generate courses of mitochondrial movement inhibition that are slow and progressive, rapid and irreversibe, or rapid and reversible (Malaiyandi et al., 2005; Reynolds and Santos, 2005; Rintoul et al., 2006, 2003a). Initial consequences such as inappropriate ATP distribution and Ca2+ sequestration would have more severe consequences in cells that are already injured and have compromised mitochondrial function. In turn, reduced ATP supply and disrupted [Ca2+]i homeostasis compromise the ability of mitochondria to maintain cm. Worsening mitochondrial damage and increased ROS production can lead to a cascade of further mitochondrial damage coupled to inappropriate repair or removal of the damaged organelles and activation of cell death pathways. Neurotoxicity could be acute if mitochondria stopped moving abruptly, as in the case glutamate excitotoxicity or delayed if mitochondria stopped moving gradually, as expected in neurodegenerative diseases (Chang et al., 2006b; Rintoul et al., 2003a; Trushina et al., 2004). Impaired mitochondrial movement has the capacity to be a highly debilitating pathophysiological process that activates a vicious cycle of injury to cells and to mitochondria.

9. Manifestations and mechanisms of mitochondrial movement impairment Cessation of mitochondrial movement can have diverse manifestations in a single injured or diseased neuron. In some cases, all mitochondria may be equally exposed to pathologic conditions that impair movement. In others, sources of injury may localize to specific neuronal regions, producing spatially restricted transport defects. Variability in axonal and dendritic physiology and in proteins and signals that mediate transport of individual mitochondria can also contribute to heterogeneous manifestations of movement cessation. In this section, we discuss how different spatiotemporal distributions of impaired mitochondrial transport can develop and influence neuronal pathology. 9.1. Whole-cell impairments in mitochondrial movement Common features of AD, ALS and HD are cm depolarization, respiratory defects and abnormal [Ca2+]i homeostasis, all of which can impair mitochondrial movement (Section 2). It is reasonable to predict that prolonged disease courses, coupled with ongoing disruptions such as oxidative stress and mtDNA damage, may predispose the vast mitochondrial population to injury. This is supported by our finding that mitochondrial movement is indiscriminately impaired in neurons treated with rotenone, the complex I inhibitor implicated in PD (Parker et al., 1989; Reynolds and Santos, 2005; Schapira et al., 1989). In animal models, oxidative stress rather than mitochondrial respiration defects is thought to be the primary effecter of rotenonemediated neuronal death (Betarbet et al., 2000; Sherer et al., 2002). Disrupted trafficking of all mitochondria in chronically diseased neurons may very well be caused by mitochondrial dysfunction, however, as well as by interactions between abnormal proteins and cytoskeletal or motor protein constituents (Gunawardena and Goldstein, 2001; Trushina et al., 2004).

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Chronic mild mitochondrial dysfunction can gradually reduce mitochondrial transport and lead to slow development of synaptic and cellular dysfunction corresponding to the protracted course of neurodegenerative diseases. This is supported by findings of only modest disruption in vesicle cycling at synaptic terminals when mitochondrial ATP synthesis is blocked, with the greatest defects in mobilization of the reserve pool, which is not typically recruited at physiological stimulation levels (Verstreken et al., 2005). Furthermore, if mitochondrial movement stops ‘‘in place’’ as our experiments show, then mitochondria that presumably still retain some functional capacity would continue to be well distributed in neuronal processes and support cell demands statically. The overall consequence of persistent and progressive mitochondrial movement impairment would probably be pre- and postsynaptic dysfunction and degeneration of axons and dendrites. This would likely proceed in a dying back degenerative pattern initiating in distal neuronal process segments, especially those of axons that are less likely to receive ATP from the large mitochondrial population near the cell body. Neurons contacting the diseased cells would then degenerate, probably with postsynaptic neurons degenerating before presynaptic neurons in correspondence with the earlier axonal degeneration of the injured neurons. In summary, impaired mitochondrial trafficking can be an early initiator of synaptic dysfunction and neuritic degeneration that may take years to cause substantial neuron loss and clinical symptoms in progressive diseases. 9.2. Axon- or dendrite-specific impairments in mitochondrial movement One of our most interesting findings is that mitochondrial movement can be impaired differentially in axons and dendrites after neuronal insults relevant to acute injuries such as ischemia and trauma. Movement impairment can be characterized as temporary, reversible cessation during the insult or irrecoverable persistence of cessation even after removal of the insult. Both situations were demonstrated preferentially in dendritic mitochondria, which were: (i) more vulnerable to movement cessation and showed slower recovery after glutamate and FCCP treatments, and (ii) irreversibly impaired in movement after zinc/chelation treatment compared to axonal mitochondria (Fig. 6B–D) (Chang et al., 2006a; Malaiyandi et al., 2005; Rintoul et al., 2003a). Additionally, glutamate-induced mitochondrial shortening was only observed in dendrites (Fig. 6A). Studies on neuritic degeneration often focus on axons rather than dendrites. Since mitochondria are more fully distributed through dendrites (Fig. 2A), it might be predicted that ATP can be spatially dispersed over a reasonable area even as mitochondrial movement and function become compromised. The most significant consequence of impaired mitochondrial trafficking in dendrites might therefore be a failure of dynamic recruitment of mobile mitochondria into excitatory spines (Chang et al., 2006a; Li et al., 2004). This may be especially relevant as remodeling of postsynaptic structures might occur

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during intense release of neuromodulators of excitatory synapses, including glutamate and zinc, into the extracellular space during ischemic or epileptic events (Benveniste et al., 1984; Bresink et al., 1996; Lobner and Lipton, 1990; Paoletti et al., 2000). Postsynaptic dysfunction might therefore be an early consequence of selectively impaired mitochondrial movement or slow recovery of movement in dendrites during excitotoxic injury. Preferential degeneration of dendrites is a possible later outcome. Axons and dendrites differ anatomically and functionally, so compartmental differences in protein distributions, signaling mechanisms and cytoskeletal arrangements could cause mitochondrial movement to stop or recover in different manners. Studies of motor proteins in C. elegans and mammals suggest that different kinesins can be selective for axons or dendrites (Setou et al., 2004). This allows cargos such as synaptic vesicles or neurotransmitter receptors to be transported to the appropriate neuronal processes. If the same targeting rules apply to mitochondrial cargos, then molecular motor isoforms in axons and dendrites probably differ and their differential regulations could affect movement cessation and subsequent recovery. Any compartmental differences in cytoskeletal properties that mediate mitochondrial movement and docking may also affect how rapidly mitochondria stop moving or recover after insults that disrupt microtubules or actin filaments. Examples of cytoskeletal properties to consider are microtubule assembly and disassembly processes, volume of cytoskeletal substrates, activities of microtubule-severing proteins, and phosphorylation states of MAPs that are often distributed preferentially in axons or dendrites (Baas et al., 2005; Baas and Qiang, 2005). Movement recovery may also be delayed in dendrites if the relatively longer mitochondria in these processes require reformation of longer microtubule tracks for transport. 9.3. Discrete sites of impaired mitochondrial movement Mitochondrial transport can be inhibited in neurodegenerative diseases where abnormal proteins form insoluble aggregates in neuronal processes. Discrete sites of blocked transport represent a unique pathology where the mitochondrial pool as a whole probably retains normal function, but gradual titration of organelles out of the pool reduces the availability of recruitable mitochondria and the efficiency with which ATP can be dispersed and [Ca2+]i can be buffered. Since mitochondrial movement is dynamic and can probably compensate for reduced mitochondrial number quite effectively, synaptic dysfunction and neuritic degeneration likely develop over very long time periods. Cytosolic aggregates that cause transport defects are part of the pathology of multiple diseases, although it is not always clear if the aggregates are acting as physical roadblocks, if they impair trafficking more broadly by interactions with transportrelated proteins and chaperones, or if they associate directly with mitochondria to cause mitochondrial injury and immobilization (Anandatheerthavarada et al., 2003; Canevari et al., 1999; Chang et al., 2006b; Gunawardena et al., 2003;

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Gutekunst et al., 1998; Kong and Xu, 1998; Trushina et al., 2004). The conclusion that transport is inhibited often relies on fixed images revealing particle accumulations rather than dynamic particle tracking that would provide qualitative and quantitative information about the defect. However, recent studies do confirm distinct aberrations in mitochondrial motility in Huntington’s disease, as discussed in Section 10.5 (Chang et al., 2006b; Trushina et al., 2004). It is less straightforward to predict how neuronal processes will degenerate in diseases where insoluble protein aggregates form indiscriminately in a cell. One possibility is that blockage and accumulation of mitochondria by aggregates in distal processes will be more severe because those regions receive less total mitochondrial traffic that could compensate for reduced mitochondrial number. Alternatively, it can be predicted that mitochondrial movement would be most significantly impaired where aggregates are largest and where titration of organelles is facilitated by high mitochondrial traffic. The most mitochondrial traffic converges on proximal branches of axons and dendrites and the large caliber of these segments probably also permits larger aggregates to form (Fig. 8C). Severe blockages in proximal segments of axons and dendrites could facilitate degeneration of all downstream branches. Impaired transport and titration of other organelles, synaptic vesicles, receptors, chaperones and proteasomes can also contribute to cellular dysfunction and degeneration. 9.4. Functional heterogeneity of individual mitochondria The mitochondrial population is diverse at least with regard to trafficking and morphology, so it makes sense for functional variation to also exist between individual organelles. Mitochondrial lifespan is limited so there will inevitably be a subset of unhealthy mitochondria in a cell, even if they are being transported for removal (Miller and Sheetz, 2004). Among healthy mitochondria, functional heterogeneity in cm may result from different numbers of oxidative phosphorylation enzymes or ion pumps that maintain appropriate gradients across mitochondrial membranes. Functional heterogeneity of mitochondria could influence how the movement of a given organelle will be affected by injurious stimuli. Since cm-

depolarization reduces movement, mitochondria with inherently lower cm might reach the threshold for movement cessation more rapidly when faced with depolarizing insults including elevated [Ca2+]i (Rintoul et al., 2003a; Vanden Berghe et al., 2004). Similarly, mitochondria that can regenerate cm quickly could regain motility soon after an insult is removed. Variation in the capacities of individual organelles to safely uptake Ca2+ before frank cm depolarization and activation of membrane permeability transition is another important consideration. In order to sequester large amounts of cytosolic Ca2+ without dangerously increasing the matrix Ca2+ load, the mitochondrial matrix contains Pi that precipitate Ca2+ into insoluble calcium salts (David, 1999). Thus, mitochondrial heterogeneity in Ca2+ uptake can theoretically be achieved by differences in cm, numbers of Ca2+ uniporter, and concentrations of Pi in the matrix. Indeed, we have seen that individual mitochondria isolated from rat brain exhibit heterogeneity in Ca2+-induced cm depolarization, although the mechanism for this has yet to be determined (Vergun et al., 2003). Additionally, differences have been reported in the Ca2+-sensitivity of mitochondrial permeability transition in different brain regions and in the Ca2+ uptake capacity of synaptic and nonsynaptic mitochondria (Brown et al., 2006; Friberg et al., 1999). Mitochondrial morphology also has important implications for movement because it can impact the volume of mitochondrial compartments, the ion concentrations within them, and consequently the ability of enzymes to maintain cm and efficiently generate ATP. Given the dynamic fission and fusion events and the interchange of membranes, enzymes and mtDNA that occur between mitochondria, it is doubtful that functional homogeneity can be conserved between all mitochondria. However, it is interesting to consider that if mitochondria of similar morphology are functionally similar, then the stereotypically shorter axonal mitochondria and longer dendritic mitochondria could contribute to a general difference in mitochondrial movement in neuronal processes (Chang et al., 2006a; Popov et al., 2005). Additionally, mitochondria are exposed to different local demands for Ca2+ uptake in cells that could impact the energetic state of individual mitochondria (Park et al., 2001). A last possibility independent of

Fig. 8. Mitochondria accumulated around and were immobilized by mutant Htt aggregates. (A) Mutant GFP-Httexon1-103Q (red) expressed diffusely throughout the cytosol did not affect mitochondrial trafficking. (B) Mutant GFP-Httexon1-103Q aggregates in neuronal processes colocalized with mito-mRFP-labeled mitochondria (green). (C) Large mitochondrial accumulation surrounding a GFP-Httexon1-103Q aggregate in a proximal neuronal process. Scale bar, 10 mm (adapted from Chang et al., 2006b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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mitochondrial function per se, is that molecular motor isoforms, which are known to be differentially modulated by phosphorylation and protein interactions, may be variably distributed on individual mitochondria (Reilein et al., 2001; Sheetz, 1999). This may account for different motility responses between different mitochondria, and may also explain the axonal and dendritic difference in mitochondrial movement if each compartment employs separate motor isoforms. It is important to note that while the end result of many of our pharmacologic manipulations is homogenous cessation of mitochondrial movement in the affected compartment, individual organelles that remain mobile for longer time periods can often be identified. Even more striking is the heterogeneity of movement recovery after drug washout. Sometimes a sole moving mitochondrion can be seen in a given field; other times a small population of mitochondria regain motility. These observations suggest that the mitochondria that regain movement earliest tend to travel with high velocity. It is interesting to speculate that some functional properties of fast-moving mitochondria predisposes them to rapid movement recovery, or that certain mitochondrial recruitment sites might develop after drug treatment to which the small number of mitochondria that have regained motility are rapidly targeted. 10. Complex mitochondrial movement impairments in neuronal injury and disease Axonal transport is impaired in multiple neurodegenerative conditions and it is clear that mitochondria are affected cargos. Possible causes of impaired transport in neurons include mitochondrial injury, [Ca2+]i disturbance, oxidative stress, mutations in motor proteins, defects in proteins that regulate or interact with motor proteins, and nonspecific disease processes such as protein aggregation that may block transport. Mitochondria represent a special cargo because transport is related to mitochondrial function, which is often compromised in pathologic conditions. In this section, we use specific examples of neuronal injury and disease to highlight how different mechanisms interact to impair mitochondrial movement and contribute to pathology. 10.1. Glutamate excitotoxicity Elevated glutamate release has been demonstrated during ischemic events and traumatic brain injury (Benveniste et al., 1984). Neuronal vulnerability to glutamate excitotoxicity and disrupted Ca2+ homeostasis are also features of neurodegenerative conditions such as Huntington’s and Alzheimer’s diseases (Mattson et al., 1992; Panov et al., 2002; Sheehan et al., 1997; Zeron et al., 2001). Activation of ionotropic NMDA receptors on dendrites mediates neuroxtoxic Ca2+ influx into cells during ischemia (Lobner and Lipton, 1990; Nishizawa, 2001). NMDA receptors are also known to mediate [Ca2+]i elevation in hippocampal neurons during seizures, acquired epilepsy and excitotoxic susceptibility during alcohol withdrawal and neurodegeneration (Mattson et al., 1992; Nagy,

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2004; Raza et al., 2004; Zeron et al., 2001). In these cases, it can be predicted that cessation of mitochondrial movement will be most severe in the dendritic compartment where Ca2+ entry points are located, with axonal involvement dependent on the ability of neurons to spatially buffer the elevated [Ca2+]i. Indeed, our results confirmed this regionally selective effect on mitochondrial movement after acute exposures to glutamate concentrations just sufficient to cause excitotoxic cell death (Fig. 6A and B). We found that mitochondrial movement in axons and dendrites is equally susceptible to impairment by elevated [Ca2+]i (Chang et al., 2006a). However, after acute glutamate treatment, mitochondrial movement stopped in all dendrites with recovery over hours and in proximal axons with recovery over minutes, while movement continued normally in distal axons (Chang et al., 2006a; Rintoul et al., 2003a). Yet 30– 50% of treated neurons proceed to die by delayed excitotoxicity. We showed that mitochondrial movement could be affected by cytosolic remodeling caused by elevated Ca2+ influx (Fig. 3), mitochondrial Ca2+ uptake, cm depolarization and inhibition of ATP synthesis (Fig. 3). Prolonged cessation of movement and mitochondrial dysfunction in dendrites may complicate recovery of cytoskeletal disruptions, [Ca2+]i homeostasis and synaptic function. Indeed, loss of dendritic spines after severe ischemia and recovery of spine structure after reperfusion may be mediated in part by effects on mitochondrial movement (Zhang et al., 2005). Selective impairments in mitochondrial movement may therefore play a pathophysiological role in dendritic degeneration observed in injuries involving Ca2+ influx through glutamate receptors (Lin et al., 1997). 10.2. Zinc-mediated neuronal injury Altered zinc homeostasis is implicated in ischemia, seizures, brain trauma, aging and chronic neurodegenerative diseases (Mocchegiani et al., 2005). We previously described the ability of neurotoxic zinc concentrations to inhibit movement while maintaining intact cm, in contrast to excitotoxic glutamate concentrations that stop movement while depolarizing cm (Malaiyandi et al., 2005; Rintoul et al., 2003a). We demonstrated that the effects of zinc were mediated by a rapidly activated PI 3-kinase dependent signaling cascade. The idea that irreversible cessation of mitochondrial movement in dendrites leads to degeneration and cell death is supported by our finding that chelation of zinc after a neurotoxic exposure selectively restores axonal mitochondrial movement but does not provide any protection against neurotoxicity (Fig. 6C) (Chang et al., 2006a; Malaiyandi et al., 2005). If impaired dendritic mitochondrial movement and subsequent dendritic degeneration significantly mediate zinc-induced toxicity, then findings in other experiments demonstrating chelationmediated protection from cell death may have used milder zinc exposures that permitted recovery of mitochondrial movement in dendrites (Canzoniero et al., 2003). Separate signaling pathways or faster activation of the same pathway may account for the ability of zinc to cause irreversible cessation of mitochondrial movement in dendrites but

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chelation-dependent recovery of movement in axons during a single exposure. Zinc-mediated inhibition of mitochondrial movement and toxicity depend on the concentration and duration of zinc treatment (Malaiyandi et al., 2005). Impaired movement after less severe zinc exposure can be reversed by chelation in axons and dendrites, but increased exposure activates a PI 3-kinase-mediated signaling cascade of movement cessation that can no longer be reversed by chelation in dendrites (Chang et al., 2006a; Malaiyandi et al., 2005). Axons may receive a lower effective zinc exposure if they contain: (1) improved Zn2+-buffering by more highly available Zn2+binding proteins such as metallothioneins, (2) enhanced efflux of cytoplasmic Zn2+ into the extracellular space or into cellular compartments by metal transporters and (3) high capacity zinc storage vesicles in presynaptic terminals (Aschner et al., 1997; Cole et al., 1999; Liuzzi and Cousins, 2004; Palmiter et al., 1996). These possibilities suggest that the [Zn2+]i needed to activate downstream pathways mediating irreversible movement inhibition can eventually develop in axons once Zn2+clearance mechanisms are saturated. An alternate hypothesis is that zinc signaling actually differs in axons and dendrites so that either PI 3-kinase-mediated inhibition of movement is never activated in axons or downstream effecters are effectively neutralized perhaps by increased phosphatase activity. It can be speculated that PI 3-kinase activation in dendrites may mediate phosphorylation of neurofilaments, which increase mitochondrial binding and immobilization (Wagner et al., 2003). 10.3. Neuronal trauma An example of a localized source of pathologic Ca2+ influx is that which occurs during axon shearing and stretch injury after head trauma. In these situations, axonal deformation triggers Na+ influx through voltage-gated Na+ channels, followed by inward flow of Ca2+ through Na+/Ca2+ exchangers and voltage-gated Ca2+ channels. Influx of Na+ and Ca2+ may be potentiated by Ca2+-mediated proteolysis of the Na+ channel a subunit that prevents its inactivation (Iwata et al., 2004; Wolf et al., 2001). Altered [Ca2+]i homeostasis and prolonged elevations in [Ca2+]i result in secondary damage to axons from downstream signaling cascades and mitochondrial failure (Weber et al., 1999). Mitochondrial movement can be predicted to stop at the site of axonal injury and surrounding regions of elevated [Ca2+]i. Clearance of [Ca2+]i may be further compromised by the inability of healthy mitochondria to move to the damaged axon segment. Therefore, impaired mitochondrial transport in axons may be an important contributor to axonal degeneration after traumatic injury. 10.4. Alzheimer’s disease AD is the most common form of dementia and is characterized clinically by progressive memory loss, language disturbance, visuospatial impairment, and behavioral and psychiatric symptoms. As discussed in Section 2, mitochondrial dysfunction, altered Ca2+ homeostasis and oxidative stress are all features of AD neurons that can impair mitochondrial

movement. These chronic disruptions may be distributed throughout neuronal processes, especially since they develop over long time periods. The severity of mitochondrial movement impairment by these mechanisms may therefore show temporal worsening, but not necessarily spatial discrimination. However, other pathologic processes in AD may produce regionally selective impairment in mitochondrial movement. Axonal swellings that potentially block transport were observed before any pathology developed in AD mouse models and human AD brains (Stokin et al., 2005). Neurofibrillary tangles and altered APP expression are associated with axonal transport defects (Fig. 9A and B) (Ebneth et al., 1998; Gunawardena and Goldstein, 2001; Torroja et al., 1999). APP can actually target mitochondria, reducing cytochrome oxidase activity and ATP levels (Anandatheerthavarada et al., 2003). Overexpression of the axonal MAP, tau, blocks motor protein binding sites on microtubules and reduces axonal transport of mitochondria, vesicles and endoplasmic reticulum (Ebneth et al., 1998). Tau hyperphosphorylation is thought to be a protective measure taken by the cell to dislodge the protein from microtubules. However, hyperphosphorylated tau forms neurofibrillary tangles in axons that can further act as physical roadblocks for transport. The dislodgement of tau also exposes microtubules to severing proteins that can ultimately result in axonal degeneration (Baas and Qiang, 2005). Therefore, unlike the downstream dendritic degeneration we predict occurs during acute neuronal injury, cessation of mitochondrial movement is more likely to cause axonal degeneration in AD. 10.5. Huntington’s disease HD is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the Huntingtin (Htt) gene, and characterized by uncontrolled movement, dementia, emotional disturbance and premature death. Mutant Htt protein containing an expanded polyglutamine tract forms intranuclear and intracytoplasmic aggregates (Cooper et al., 1998; Hazeki et al., 1999). Our examination of cortical neurons transfected with mutant Htt demonstrate reduced mitochondrial trafficking specifically to sites of extranuclear aggregates and impeded passage of moving mitochondria by aggregates (Chang et al., 2006b). This resulted in discrete regions of mitochondrial immobilization and accumulation around aggregates that manifested before any other mitochondrial or cellular defects (Fig. 8). We found no preference for aggregate formation in axons over dendrites or for increased vulnerability of mitochondrial transport to blockage in any specific region. The degree of movement impairment may therefore rely on qualities such as aggregate size, time required for aggregate formation, and amount of mitochondrial traffic in the vicinity of the aggregate. Trushina et al. (2004) also described early aberrations in mitochondrial motility in HD striatal neurons, but with different manifestations and mechanisms. They found nearly all mitochondria moved with slower velocity because mutant Htt sequestered wildtype Htt and trafficking proteins. Later in the course of HD, impaired mitochondrial movement

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Fig. 9. Mitochondrial trafficking defects are implicated in neurodegenerative diseases. (A and B) Mitochondria are rounder and cluster near the microtubule organizing center in CHO cells stably transfected with tau40 (A) compared to the cytoplasmic distribution seen in mock-transfected cells (B). Scale bar, 20 mm (adapted from Ebneth et al., 1998). (C–E) Mitochondria (green, arrows) accumulate around neuritic aggregates in immortalized motoneuronal cells expressing androgen receptors with elongated polyglutamine tracts (GFP-AR.Q48, blue). Inset: magnified neurite (adapted from Piccioni et al., 2002, published online as doi:10.1096/fj.01-1035fje). (F) Transgenic mouse with a G93A mutant SOD1 gene displays an increased number of rounded mitochondria in the axon hillock and initial axonal segments of an anterior horn cell. Scale bar, 1 mm (adapted from Sasaki et al., 2005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

and a gradual decline in the number of functional mitochondria could lead to mitochondrial dysfunction, insufficient ATP, [Ca2+]i dysregulation and excitotoxicity, ROS release and cell death (Section 2). If trafficking defects affect mitochondria more universally in striatal neurons than in cortical neurons, then harmful consequences may develop more rapidly and lead to selective striatal neurodegeneration. 10.6. Other neurodegenerative diseases There is a well-documented role for mitochondrial movement impairment in the injury and disease conditions discussed above. Although not as thoroughly described, reports suggest

that mitochondrial trafficking abnormalities also occur in many other neurodegenerative conditions. Impaired mitochondrial movement is implicated in Parkinson’s disease by our recent findings in cortical neurons treated chronically with subtoxic rotenone concentrations (Reynolds and Santos, 2005). Formation of neurofilament ‘‘torpedoes’’ and a corresponding reduction of organelles were found in Purkinje cell axons of a patient with the polyglutamine disease spinocerebellar ataxia type 6 (Yang et al., 2000). In spinobulbar muscular atrophy, androgen receptors with a mutant polyglutamine repeat aggregate in neurites and colocalize with mitochondria (Fig. 9C and D) (Piccioni et al., 2002). Increased numbers of mitochondria, especially with a rounded morphology, were

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observed in axon hillocks and initial axonal segments of anterior horn cells in human and mouse models for ALS (Fig. 9E) (Sasaki and Iwata, 1996; Sasaki et al., 2005). These mitochondrial collections were thought to be caused by neurofilament accumulations near the cell soma impeding axonal transport of mitochondria and ultimately causing axonal degeneration (Collard et al., 1995). Further research will clarify the role of transport defects in these diseases and demonstrate just how common mitochondrial trafficking impairments are in neuropathology. 11. Conclusions While a lot is known about mitochondrial physiology and cell survival and death mechanisms in neurons, we are only beginning to understand mitochondrial trafficking. Despite all the complexities, research in the last decade produced detailed descriptions of movement patterns, identified potential targets and functional roles for mitochondrial trafficking, and implicated disrupted mitochondrial transport in neuronal injury and degeneration. Efficient progress is largely contributed by the growing appreciation for the importance of mitochondrial movement and the interdisciplinary enthusiasm in studying mitochondrial function in neurons, cellular trafficking mechanisms, and mitochondrial dysfunction in disease. Reducing the scale of pharmacologic and imaging techniques and employing electrophysiologic methodology will provide unprecedented resolution of trafficking mechanisms among individual mitochondria. We will uncover the dynamics and functional purposes of mitochondrial recruitment to sites of neurotransmission during physiological synaptic activity and short and long term plasticity. These findings will demonstrate the significance of mitochondrial trafficking in learning and memory, as well as how synaptic transmission and plasticity can fail when mitochondrial movement is impaired. Elucidating these physiological and pathophysiological properties would be relevant to any neurodegenerative disease characterized by synaptic dysfunction and especially to AD where memory loss is an early and devastating clinical feature. Understanding how normal mitochondrial trafficking supports cellular function will therefore reveal how abnormal trafficking causes cellular dysfunction and neuritic degeneration in injury and disease. Studies often focus on where and why mitochondria are transported to specific locations, but much is still unknown about the mediators of the physical trafficking of mitochondria. This includes the motive properties of individual mitochondriatransporting motor protein isoforms, the identities of scaffolding proteins that associate motor proteins with mitochondria, and how mitochondrial motility patterns can be modulated by regulating scaffolding proteins, motors and other accessory proteins. More detailed analysis will reveal whether certain mitochondrial populations, for example those in axons or dendrites or those with shorter or longer morphology, tend to associate with certain scaffolds and motors that dictate motile capabilities. In terms of the saltatory properties of mitochondrial movement, chemical and physical cues that recruit

mitochondria need to be clarified, as do signals, mediators and regulatory events required for physical docking of mitochondria on the cytoskeleton. Understanding how mitochondria are trafficked and how mediators are regulated will provide insight into how dysregulation during injury alters mitochondrial movement. Just as importantly, it will provide specific targets for pharmacologic manipulations to restore impaired mitochondrial movement. The relationship between impaired mitochondrial movement and neuronal injury and disease is difficult to elucidate because concurrent and protracted pathophysiological processes occur in neurological conditions. It is necessary to determine the circumstances in which impaired mitochondrial movement is a cause of further pathology versus an effect of a more significant precipitating injury. Our examination of mutant Htt aggregates offers an example of a subtle trafficking defect that precedes other mitochondrial, synaptic or cellular disruptions. However, the story is rarely so simple. Cessation of mitochondrial movement is highly sensitive to many stimuli present in disease states such as elevated [Ca2+]i, oxidative stress, inhibition of electron transport and uncoupling of oxidative phosphorylation. In most cases, mitochondrial movement can therefore be predicted to stop as a result of some other pathophysiological effecter. Important questions that need to be addressed are at what point in the injury process mitochondrial trafficking is compromised, the functional consequences of impaired movement, and the time required for irreversible cellular damage and expression of a diseased phenotype. Understanding these properties will permit the design of pharmacologic interventions targeted to preserving mitochondrial movement and indicate when these interventions should be implemented in different disease processes. Acknowledgement This work was supported by NIH grant NS049560. References Anandatheerthavarada, H.K., Biswas, G., Robin, M.-A., Avadhani, N.G., 2003. Mitochondrial targeting and a novel transmembrane arrest of Alzheimer’s amyloid precursor protein impairs mitochondrial function in neuronal cells. J. Cell Biol. 161, 41–54. Andersen, J.K., 2004. Oxidative stress in neurodegeneration: cause or consequence? Nat. Rev. Neurosci. 5, S18–S25. Andreyev, A.Y., Kushnareva, Y.E., Starkov, A.A., 2005. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc.) 70, 200–214. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., Nicotera, P., 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961–973. Antonini, A., Stryker, M.P., 1993. Rapid remodeling of axonal arbors in the visual cortex. Science 260, 1819–1821. Anundi, I., King, J., Owen, D.A., Schneider, H., Lemasters, J.J., Thurman, R.G., 1987. Fructose prevents hypoxic cell death in liver. Am. J. Physiol. Gastrointest. Liver Physiol. 253, G390–G396. Arnoult, D., Rismanchi, N., Grodet, A., Roberts, R.G., Seeburg, D.P., Estaquier, J., Sheng, M., Blackstone, C., 2005. Bax/Bak-dependent release of DDP/ TIMM8a promotes Drp1-mediated mitochondrial fission and mitoptosis during programmed cell death. Curr. Biol. 15, 2112–2118.

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