Table of Contents

COVER

TITLE PAGE

CONTRIBUTORS

PREFACE

SECTION I: MITOCHONDRIAL STRUCTURE AND ION CHANNELS

1 MITOCHONDRIAL PERMEABILITY TRANSITION: A LOOK FROM A DIFFERENT ANGLE

1.1 REGULATION OF INTRACELLULAR CALCIUM IN NEURONS

1.2 CALCIUM OVERLOAD AND MITOCHONDRIAL PERMEABILITY TRANSITION

1.3 THE MITOCHONDRIAL TRANSITION PORE

ACKNOWLEDGMENTS

REFERENCES

2 THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE, THE c-SUBUNIT OF THE F₁Fo ATP SYNTHASE, CELLULAR DEVELOPMENT, AND SYNAPTIC EFFICIENCY

2.1 INTRODUCTION

2.2 MITOCHONDRIA AT THE CENTER OF CELL METABOLISM AND CELL DEATH

2.3 MITOCHONDRIAL INNER MEMBRANE LEAK: REGULATOR OF METABOLIC RATE AND UNCOUPLING

2.4 MITOCHONDRIAL INNER MEMBRANE CHANNELS AND EXCHANGERS ARE NECESSARY FOR Ca²⁺ CYCLING AND CELLULAR Ca²⁺ DYNAMICS

2.5 MITOCHONDRIAL INNER AND OUTER MEMBRANE CHANNEL ACTIVITY REGULATES Ca²⁺ RE-RELEASE FROM MITOCHONDRIA AFTER BUFFERING

2.6 BCL-2 FAMILY PROTEINS REGULATE PATHOLOGICAL OUTER MITOCHONDRIAL MEMBRANE PERMEABILIZATION (MOMP)

2.7 PATHOLOGICAL INNER MEMBRANE DEPOLARIZATION: MITOCHONDRIAL PERMEABILITY TRANSITION

2.8 THE QUEST FOR AN INNER MEMBRANE Ca²⁺-SENSITIVE UNCOUPLING CHANNEL: THE PT PORE

2.9 THE mPTP: A MOLECULAR DEFINITION

2.10 CLOSING OF THE mPTP MAY ENHANCE MITOCHONDRIAL METABOLIC PLASTICITY AND REGULATE SYNAPTIC PROPERTIES IN HIPPOCAMPAL NEURONS

2.11 mPTP OPENING CORRELATES WITH CELL DEATH IN ACUTE ISCHEMIA, ROS DAMAGE, OR GLUTAMATE EXCITOTOXICITY

2.12 PRO-APOPTOTIC PROTEOLYTIC CLEAVAGE FRAGMENT OF Bcl-xL CAUSES LARGE CONDUCTANCE MITOCHONDRIAL ION CHANNEL ACTIVITY CORRELATED WITH HYPOXIC SYNAPTIC FAILURE: OUTER MITOCHONDRIAL CHANNEL MEMBRANE ACTIVITY ALONE OR mPTP?

2.13 SYNAPTIC RESPONSES DECLINE DURING LONG-TERM DEPRESSION IN AsSOCIATION WITH BCL-2 FAMILY-REGULATED MITOCHONDRIAL CHANNEL ACTIVITY

2.14 SYNAPSE LOSS DURING NEURODEGENERATIVE DISEASE MAY REQUIRE MITOCHONDRIAL CHANNEL ACTIVITY

2.15 CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

3 MITOCHONDRIAL CHANNELS IN NEURODEGENERATION

3.1 INTRODUCTION

3.2 Mitochondrial Channels in the Healthy Neuron

3.3 MITOCHONDRIAL CHANNELS IN THE DYING CELL

3.4 MITOCHONDRIAL CHANNELS IN NEURODEGENERATIVE DISEASES

3.5 CONCLUSIONS

REFERENCES

SECTION II: CONTROL OF MITOCHONDRIAL SIGNALING NETWORKS

4 MITOCHONDRIAL Ca2+ TRANSPORT IN THE CONTROL OF NEURONAL FUNCTIONS: MOLECULAR AND CELLULAR MECHANISMS

4.1 INTRODUCTION

4.2 PHYSIOLOGICAL AND PHARMACOLOGICAL CHARACTERISTICS OF MITOCHONDRIAL Ca²⁺ TRANSPORT IN NEURONS

4.3 MOLECULAR COMPONENTS OF MITOCHONDRIAL Ca²⁺ TRANSPORT IN NEURONS

4.4 MITOCHONDRIAL Ca²⁺ SIGNALING AND NEURONAL EXCITABILITY

4.5 MITOCHONDRIAL Ca²⁺ CYCLING IN THE REGULATION OF SYNAPTIC TRANSMISSION

4.6 MITOCHONDRIAL Ca²⁺ TRANSPORT AND THE REGULATION OF GENE EXPRESSION IN NEURONS

4.7 FUTURE DIRECTIONS

ACKNOWLEDGMENTS

REFERENCES

5 AMP-ACTIVATED PROTEIN KINASE (AMPK) AS A CELLULAR ENERGY SENSOR AND THERAPEUTIC TARGET FOR NEUROPROTECTION

5.1 INTRODUCTION

5.2 CONCLUSION AND FUTURE PERSPECTIVES

REFERENCES

6 HDAC6: A MOLECULE WITH MULTIPLE FUNCTIONS IN NEURODEGENERATIVE DISEASES

6.1 INTRODUCTION

6.2 MOLECULAR PROPERTIES OF HDAC6

6.3 HDAC6 AND NEURODEGENERATIVE DISEASES

6.4 PERSPECTIVES

REFERENCES

7 NEURONAL MITOCHONDRIAL TRANSPORT

7.1 INTRODUCTION

7.2 COMPLEX MOTILITY PATTERNS OF AXONAL MITOCHONDRIA

7.3 MECHANISMS OF MITOCHONDRIAL TRANSPORT

7.4 MECHANISMS OF AXONAL MITOCHONDRIAL ANCHORING

7.5 REGULATION OF MITOCHONDRIAL TRANSPORT BY SYNAPTIC ACTIVITY

7.6 MITOCHONDRIAL TRANSPORT AND SYNAPTIC TRANSMISSION

7.7 MITOCHONDRIAL TRANSPORT AND PRESYNAPTIC VARIABILITY

7.8 MITOCHONDRIAL TRANSPORT AND AXONAL BRANCHING

7.9 MITOCHONDRIAL TRANSPORT AND MITOPHAGY

7.10 CONCLUSIONS AND NEW CHALLENGES

ACKNOWLEDGMENTS

REFERENCES

8 MITOCHONDRIA IN CONTROL OF HYPOTHALAMIC METABOLIC CIRCUITS

8.1 INTRODUCTION

8.2 YIN-YANG RELATIONSHIP BETWEEN COMPONENTS OF HYPOTHALAMIC FEEDING AND SATIETY CIRCUITS

8.3 MITOCHONDRIA AND THEIR DYNAMICS

8.4 METABOLIC PRINCIPLES OF HUNGER AND SATIETY PROMOTION: MITOCHONDRIA IN SUPPORT OF FAT VERSUS GLUCOSE UTILIZATION

8.5 MITOCHONDRIA DYNAMICS AND CELLULAR ENERGETICS

8.6 MITOCHONDRIAL DYSFUNCTION AND METABOLIC DISORDERS

8.7 CONCLUSIONS

REFERENCES

9 MITOCHONDRIA ANCHORED AT THE SYNAPSE

9.1 INTRODUCTION

9.2 CALIBRATED POSITIONING OF MITOCHONDRIA

9.3 MITOCHONDRIA AND CRISTA STRUCTURE

9.4 ADHERING JUNCTIONS AND LINKAGES TO THE CYTOSKELETON

9.5 LINKAGES OF THE OMM TO THE MITOCHONDRIAL PLAQUE AND RETICULATED MEMBRANE

9.6 FUNCTIONS OF THE ORGANELLE COMPLEX

9.7 MACs AND FILAMENTOUS CONTACTS: A CONTINUUM OF STRUCTURE?

ACKNOWLEDGMENTS

REFERENCES

SECTION III: DEFECTIVE MITOCHONDRIAL DYNAMICS AND MITOPHAGY

10 NEURONAL MITOCHONDRIA ARE DIFFERENT: RELEVANCE TO NEURODEGENERATIVE DISEASE

10.1 INTRODUCTION

10.2 MITOCHONDRIAL DYNAMICS IN NEURONS AND NEURODEGENERATIVE DISEASE

10.3 TRIGGERING MITOPHAGY IN NEURONS VERSUS OTHER CELL TYPES

10.4 BCL-xL: THE GUARDIAN OF MITOCHONDRIA

ACKNOWLEDGMENTS

REFERENCES

11 PINK1 AS A SENSOR FOR MITOCHONDRIAL FUNCTION: DUAL ROLES

11.1 INTRODUCTION

11.2 PINK1 PROMOTES MITOCHONDRIAL FUNCTION

11.3 HEALTHY MITOCHONDRIA IMPORT AND PROCESS PINK1

11.4 ACCUMULATION OF FULL LENGTH-PINK1 AS A SENSOR OF MITOCHONDRIAL DYSFUNCTION

11.5 CYTOSOLIC PINK1 AS A SENSOR FOR MITOCHONDRIAL FUNCTION

11.6 PINK1 AND MITOCHONDRIAL DYNAMICS

11.7 DUAL ROLES FOR PINK1 AS A SENSOR OF MITOCHONDRIAL FUNCTION AND DYSFUNCTION

REFERENCES

12 A GET-TOGETHER TO TEAR IT APART: THE MITOCHONDRION MEETS THE CELLULAR TURNOVER MACHINERY

12.1 MITOCHONDRIAL QUALITY CONTROL IN NEURODEGENERATION

12.2 AN OVERVIEW OF THE UBIQUITIN-PROTEASOME SYSTEM

12.3 ACTIVITIES OF THE CYTOSOLIC PROTEASOME AT THE OUTER MITOCHONDRIAL MEMBRANE

12.4 THE TURNOVER OF WHOLE MITOCHONDRIA BY MITOPHAGY

12.5 PROTEASOMES AND PHAGOPHORES CONVERGE IN THE PINK1/PARKIN PATHWAY

12.6 IMPLICATIONS OF PINK1-/PARKIN-DEPENDENT MITOPHAGY IN THE BRAIN AND IN PD

12.7 EMERGING MITOCHONDRIAL QUALITY CONTROL MECHANISMS

REFERENCES

13 MITOCHONDRIAL INVOLVEMENT IN NEURODEGENERATIVE DEMENTIA

13.1 INTRODUCTION

13.2 MITOCHONDRIAL DYSFUNCTION IN ALZHEIMER DISEASE

13.3 MITOCHONDRIAL DYSFUNCTION, BIOENERGETIC DEFICITS, AND OXIDATIVE STRESS IN AD

13.4 MITOCHONDRIAL FRAGMENTATION IN AD

13.5 SYNAPTIC MITOCHONDRIA IN AD

13.6 MITOCHONDRIAL DYSFUNCTION AND CATIONIC DYSHOMEOSTASIS IN AD

13.7 MITOCHONDRIAL DYSFUNCTION IN DLB

13.8 LRRK2 MUTATIONS, MITOCHONDRIA AND DLB

13.9 AKINETIC CRISIS IN SYNUCLEINOPATHIES IS LINKED TO GENETIC MUTATIONS INVOLVING MITOCHONDRIAL PROTEINS

13.10 CONCLUSIONS

REFERENCES

SECTION IV: MITOCHONDRIA-TARGETED THERAPEUTICS AND MODEL SYSTEMS

14 NEURONAL MITOCHONDRIA AS A TARGET FOR THE DISCOVERY AND DEVELOPMENT OF NEW THERAPEUTICS

14.1 NEURODEGENERATIVE DISORDERS AND THE STATUS OF DRUG DISCOVERY

14.2 MITOCHONDRIA AS TARGETS FOR THE DEVELOPMENT OF NEW NDD THERAPIES

14.3 THE EFFECTS OF DEXPRAMIPEXOLE ON MITOCHONDRIAL CONDUCTANCES: AN EXAMPLE OF AN APPROACH FOR ALS AND OTHER NDDs

14.4 WHAT IS THE FUTURE OF A MITOCHONDRIAL APPROACH FOR NDD THERAPY?

ACKNOWLEDGMENTS

REFERENCES

15 MITOCHONDRIA AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE

15.1 INTRODUCTION

15.2 MITOCHONDRIAL ABNORMALITIES AND DYSFUNCTION IN ALZHEIMER’S DISEASE

15.3 MITOCHONDRIA AS A DRUG TARGET

15.4 CONCLUSIONS

REFERENCES

16 MITOCHONDRIA IN PARKINSON’S DISEASE

16.1 INTRODUCTION

16.2 ROLE OF MITOCHONDRIA IN SPORADIC PD

16.3 MITOCHONDRIAL DYSFUNCTION IN MONOGENIC PD

16.4 CONCLUSIONS

REFERENCES

17 THERAPEUTIC TARGETING OF NEURONAL MITOCHONDRIA IN BRAIN INJURY

17.1 INTRODUCTION

17.2 MITOCHONDRIA BIOENERGETICS

17.3 TRAUMATIC BRAIN INJURY

17.4 PHARMACEUTICAL INTERVENTIONS

17.5 CONCLUSION

REFERENCES

18 THE USE OF FIBROBLASTS FROM PATIENTS WITH INHERITED MITOCHONDRIAL DISORDERS FOR PATHOMECHANISTIC STUDIES AND EVALUATION OF THERAPIES

18.1 INTRODUCTION

18.2 PATHOMECHANISTIC STUDIES OF MITOCHONDRIAL DISORDERS IN PATIENTS’ FIBROBLASTS

18.3 EVALUATION OF THERAPEUTIC OPTIONS USING PATIENT DERIVED FIBROBLASTS

18.4 CONCLUSION

ACKNOWLEDGMENTS

REFERENCES

INDEX

END USER LICENSE AGREEMENT

List of Tables

Chapter 03

TABLE 3.1 Biophysical properties of mitochondrial channels. Otherwise stated, all parameters were measured in mitochondrial membrane preparations or mitoplasts in 0.15 M KCl.

TABLE 3.2 Possible involvement of mitochondrial channels in neurodegenerative diseases.

Chapter 15

TABLE 15.1 List of current mitochondria-targeted neuroprotective compounds for Alzheimer’s disease.

TABLE 15.2 List of potential mitochondria-targeted neuroprotective compounds.

List of Illustrations

Chapter 01

FIGURE 1.1 Glutamate-induced changes in cytosolic Ca²⁺ ([Ca²⁺]c) and mitochondrial membrane potential in rat cultured hippocampal neurons. Protection with NIM811. Neurons (12–14 days in vitro [12–14 DIV]) were coloaded with 2.6 μM Fura-2FF-AM and 1.7 μM Rhodamine 123 (Molecular Probes, Eugene, OR) to monitor changes in cytosolic Ca²⁺ and mitochondrial membrane potential, respectively. In a and c, the individual (thin grey traces obtained from individual neurons) and average Fura-2FF (F₃₄₀/F₃₈₀, thick black traces) fluorescence signals are shown. In b and d, the individual (thin grey traces obtained from individual neurons) and average Rhodamine 123 (Rh123, F/F₀, thick black traces) fluorescence signals are shown. Where indicated, neurons were treated with 25 μM glutamate (Glu, plus 10 μM glycine) for 30 seconds. In c and d, neurons were preincubated for 45 minutes with 3 μM NIM811. NIM811 (3 μM) was also present in the bath solution during the experiment.

FIGURE 1.2 ⁴⁵Ca accumulation in rat cultured hippocampal neurons exposed to glutamate. NIM811 increases ⁴⁵Ca accumulation in mitochondria in situ. Neurons (12-14 days in vitro [DIV]) were incubated in the standard bath solution supplemented with 5 μCi of ⁴⁵Ca. ⁴⁵Ca accumulation was measured at 5 minutes after application of 25 μM glutamate (Glu) plus 10 μM glycine, as indicated. Nonspecific ⁴⁵Ca binding was measured in the presence of 10 μM MK801 (an inhibitor of NMDA receptor) and 10 μM CNQX (an inhibitor of AMPA/kainate receptors). Mitochondrial ⁴⁵Ca accumulation was evaluated by subtracting the amount of ⁴⁵Ca accumulated in neurons in the presence of 1 μM FCCP and 1 μM rotenone (Rot) from the amount of ⁴⁵Ca accumulated in the absence of inhibitors. This value was taken as 100%. Where indicated, neurons were preincubated for 45 minutes with 3 μM NIM811 before glutamate exposure. NIM811 (3 μM) was also present in the bath solution during the experiment. *p<0.05 comparing neurons with and without NIM811 treatment; **p<0.01 comparing neurons with and without mitochondrial inhibitors and antagonists of glutamate receptors; #p<0.01 comparing NIM811-treated neurons incubated with and without mitochondrial inhibitors and antagonists of glutamate receptors. Data are mean±SEM, N=3.

FIGURE 1.3 Oligomycin failed to inhibit Ca²⁺-induced permeability transition in isolated nonsynaptic brain mitochondria. Isolated Percoll gradient-purified rat brain mitochondria were incubated at 37° C under continuous stirring in the incubation medium containing 210 mM mannitol, 50 mM sucrose, 3 mM succinate, 3 mM glutamate, 3 mM KH₂PO₄, 0.5 mM MgCl₂, 10 mM EGTA, 0.1% BSA (free from fatty acids), 10 mM Hepes, pH 7.4. In a and b, mitochondrial swelling (thick traces) was evaluated by following changes in light scattering of mitochondrial suspension at 525 nm with an incident light beam under 180° C in a 0.3-ml chamber at 37° C and continuous stirring. Simultaneously, Δψ was monitored by following the distribution of tetraphenylphosphonium cation (TPP⁺) between the external medium (initially 1.8 μM TPP⁺-Cl) and the mitochondrial matrix with a TPP⁺-sensitive electrode (Kamo et al., 1979). Where indicated, 2.4 µmol Ca²⁺/mg protein and 1 μM oligomycin were added. In c, recording of mitochondrial oxygen consumption, with oligomycin inhibiting ADP-stimulated respiration, is shown as a positive control. Additions: ADP, 300 μM; oligomycin, 1 μM; 2,4-dinitrophenol (DNP), 60 μM. Mitochondrial respiration was recorded in the 0.3-ml incubation chamber equipped with a Clark-type oxygen electrode and a tightly closed lid. The slope of the O₂ electrode trace corresponds to the respiration rate.

FIGURE 1.4 Ca²⁺ uptake capacity of isolated nonsynaptic brain mitochondria. The effects of oligomycin (Oligo), adenosine diphosphate (ADP), carboxyatractyloside (CAT), bovine serum albumin (BSA), and cyclosporin A (CsA). Mitochondria were incubated at 37° C in 0.3-ml thermostatically regulated chamber under continuous stirring. Ca²⁺ uptake by mitochondria was followed by measuring Ca²⁺ concentration in the incubation medium ([Ca²⁺]ext) with a miniature, custom-made Ca²⁺-selective electrode. The standard incubation medium contained 125 mM KCl, 0.5 mM MgCl₂, 3 mM KH₂PO₄, 10 mM Hepes, pH 7.4, 10 μM EGTA, 3 mM succinate, and 3 mM glutamate. Where indicated, 10 μM Ca²⁺ pulses were applied. In a, mitochondria were incubated in the standard incubation medium without other additions. In b, the incubation medium was supplemented with 1 μM oligomycin (Oligo); in c, with 1 μM oligomycin and 100 μM ADP; in d, with 1 μM oligomycin, 100 μM ADP, and 20 μM carboxyatractyloside (CAT), in e, with 1 μM oligomycin, 100 μM ADP, and 0.1% BSA (free from fatty acids), in f, with 1 μM oligomycin, 100 μM ADP, 0.1% BSA (free from fatty acids), and 1 μM cyclosporin A (CsA). Time bar in f is applicable to all panels.

Chapter 02

FIGURE 2.1 The current-voltage relationship (I-V) of proteoliposome recordings of the purified c-subunit mimics that of mitoplast recordings of mPTP. a–c, Graphs showing examples of the different I-V relationships for the mPTP found in different mitoplast (isolated inner mitochondrial membrane) recordingsd–e, Graphs showing examples of the different I-V relationships found in different proteoliposome recordings of the purified c-subunitShown in e are a low and high conductance mode of the channel (Alavian et al., 2014a). Portions of this figure reprinted with the kind permission of Springer Science and Business Media.

FIGURE 2.2 Model of physiological mPTP activity. β subunit, which binds Bcl-xL and ATP, blocks c-subunit conductance. CsA, inhibitor of CypD, blocks c-subunit conductance. Both Bcl-xL and CsA bind to F1 components during pore closure. The pore diameter also decreases during pore closure. The channel can switch reversibly between these open and closed states.

FIGURE 2.3 Model of pathophysiological mPTP activity. Ca²⁺ induces separation of F₁ from FO during mPTP opening. The pore diameter also expands during Ca²⁺-induced increases in mPTP conductance.

FIGURE 2.4 Proposed role of Bcl-xL in mitochondrial metabolism during synaptic plasticity. Events at the synapse after stimuli that produce long-term changes in synaptic responsiveness. (1) A change in level of activity produces a change in Ca²⁺ influx through NMDA receptors. (2) Ca²⁺ binds to calmodulin, which forms a complex with Drp1 and Bcl-xL. (3) The Drp1-Bcl-xL-complex translocates to mitochondria in a calmodulin-dependent manner. At the same time, Ca²⁺ is being taken up into mitochondria, TCA cycle is stimulated, mitochondria buffer and re-release Ca²⁺. (4) Bcl-xL-Drp1 complex enhances mitochondrial positioning to postsynaptic sites (close to the postsynaptic density). (5) Bcl-xL enhances the efficiency of production and release of ATP over the long term. ΔN Bcl-xL decreases the efficiency of ATP production. (6) Changes in phosphorylation of downstream targets occurs, producing long-lasting changes in synaptic structure and function, including, for example, the expression level of postsynaptic glutamate receptors.

Chapter 03

FIGURE 3.1 Mitochondrial ion channels by location and function. The pie chart represents the spatial and functional distribution of ion channels in the outer (grey circle) and inner (shadowed cristae) mitochondrial membranes. Detailed information and references are compiled in the text.

Chapter 04

FIGURE 4.1 Mitochondrial Ca²⁺ signaling in neurons. Ca²⁺ entering neurons via voltage-gated Ca²⁺ channels (VGCC), glutamate NMDA receptors (NMDA-R), or other Ca²⁺-permeable channels (e.g., TRP channels) is rapidly taken up by mitochondria. This Ca²⁺ uptake is mediated by the mitochondrial Ca²⁺ uniporter. Within mitochondria, Ca²⁺ stimulates ATP synthesis through activation of several Ca²⁺-activated dehydrogenases (Denton, 2009; Glancy and Balaban, 2012). Free Ca²⁺ within the matrix is buffered into Ca-phosphate complexes. Ca²⁺ is released from mitochondria back into the cytosol via at least three mechanisms, Na⁺/Ca²⁺ exchanger, H⁺/Ca²⁺ exchanger, and mitochondrial permeability transition pore (mPTP). Under physiological conditions, Ca²⁺ mobilization from mitochondria is mediated primarily by the mitochondrial Na⁺/Ca²⁺ exchanger. The Ca²⁺ released from mitochondria is ultimately extruded from neurons via the plasma membrane Ca²⁺-ATPases (PMCA) and Na⁺/Ca²⁺ exchangers (NCX). Ca²⁺ uptake and release by mitochondria reduces the amplitude of increase in cytosolic Ca²⁺ concentration ([Ca²⁺]i), and prolongs the duration of [Ca²⁺]i elevation. By shaping [Ca²⁺]i, mitochondrial Ca²⁺ cycling modulates activity of many neuronal Ca²⁺ targets and influences numerous Ca²⁺-dependent functions such as excitability, synaptic transmission, and gene regulation. Under pathological conditions, mitochondrial overload with Ca²⁺ triggers opening of the mPTP, a large channel permeable for Ca²⁺ and small proteins including the pro-apoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Opening of the mPTP triggers a cascade of toxic events (e.g., mitochondrial depolarization, collapse of ATP synthesis, increase in reactive oxygen species (ROS) production, Ca²⁺ deregulation, activation of caspases, and the Ca²⁺-dependent protease calpain), ultimately leading to neuronal demise. The deregulation of mitochondrial Ca²⁺ handling is implicated in neuronal toxicity in stroke, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), and other neurodegenerative disorders (Bezprozvanny, 2009; Mattson, Gleichmann, and Cheng, 2008; Pivovarova and Andrews, 2010).

FIGURE 4.2 Mitochondrial Ca²⁺ cycling regulates amplitude and duration of [Ca²⁺]i responses in neurons. Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]i) were studied in cultured dorsal root ganglion (DRG) sensory neurons using the Ca²⁺-sensitive indicator Fura-2 as previously described (Kim and Usachev, 2009; Kim et al., 2014; Schnizler et al., 2008; Usachev et al., 2002). a, Superimposition of [Ca²⁺]i responses to the TRPV1 agonist capsaicin (1 μM; 30 s) recorded in control (black) or in the presence of the electron transport inhibitor antimycin (1 μM) and ATP-synthase inhibitor oligomycin (2 μM) (grey trace). Both traces were obtained from the same cell. Inhibition of mitochondrial Ca²⁺ transport by the antimycin with oligomycin treatment increased the amplitude and reduced the duration of capsaicin-evoked [Ca²⁺]i elevation (compare grey and black traces). b, [Ca²⁺]i transients were evoked by trains of action potentials (8 Hz; arrows) of increased duration using extracellular field stimulation. The number of action potentials is shown under the traces. Increasing the strength of the stimulation led to a gradual appearance and prolongation of the [Ca²⁺]i plateau phase mediated by Ca²⁺ release from mitochondria.

FIGURE 4.3 Comparison of depolarization-induced mitochondrial Ca²⁺ signals between dorsal root ganglion (DRG) and hippocampal neurons. Ca²⁺ concentration was simultaneously monitored in the cytosol ([Ca²⁺]i) and mitochondria ([Ca²⁺]mt) of DRG (a) and hippocampal (b) neurons using Fura2 (black) and genetic mitochondrial Ca²⁺ indicator mito-LAR-GECO1.2 (grey), respectively, as recently described (Wu et al., 2014). Depolarization was induced by applying extracellular solution containing 50 mM KCl (30 s).

FIGURE 4.4 Molecular components of mitochondrial Ca²⁺ transport in neurons. The upper panel shows the main components of the mitochondrial Ca²⁺ transport machinery in the inner mitochondrial membrane. The table summarizes the information about the expression and function of the mitochondrial Ca²⁺ transporters in neurons, including the data from knockdown (KD) and knockout (KO) studies, as well as a recent report on the loss-of-function MICU1 mutations in humans.

Chapter 06

FIGURE 6.1 Classes of human histone deacetylase (HDAC) family members and their protein structures, cellular localizations, and possible connections to neurodegenerative diseases. HDACs consist of four classes (I, II, III, and VII) according to their sequence identities and similarities to the yeast orthologues. Members of Class III HDACs are not included here because of their unique enzyme kinetics. The light blue-filled rectangle boxes indicate deacetylase domains. The 14-3-3 chaperone binding motifs and the myocyte enhancer factor 2 (MEF2) binding motifs are shown in grey and dark squares, respectively. Class IIb members possess an additional ubiquitin-binding domain (dark grey) or a leucine rich domain (light grey). Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) indicate that the corresponding HDACs may function in these neurodegenerative diseases. – shows that no relevant studies are available; ND* (neurodegeneration), known functions in neurodegeneration but not specified.

FIGURE 6.2 Protein domains and functions of HDAC6. HDAC6 possesses two deacetylase domains (light grey rectangles) and an ubiquitin-binding zinc (BUZ) finger domain (cross rule rectangle). There are also two nuclear export signal (NES) domains (middle grey rectangles) and a unique Ser-Glu-containing (SE14) region (dark grey rectangle). Functions of HDAC6 are summarized as indicated in the grey boxes: transcriptional regulation, signal transduction, cell motility, and cell survival. Grey terms indicate the substrates of HDAC6, whereas the proteins whose names are in black represent the interacting partners of HDAC6.

FIGURE 6.3 Schematic processes of HDAC6-mediated autophagic elimination of proteotoxicity. Proteotoxicity elimination requires HDAC6 at multi-levels. Step 1, genetic or environmental causes lead to accumulated misfolded proteins that is prone to aggregate into oligomer species. These oligomer species, if not eliminated, are toxic to the cells leading to eventually cell deaths. Step 2, the toxic oligomers with markers such as specific ubiquitinations can be recognized by the ubiquitin-binding domain of HDAC6. Recruitment of HDAC6 to the oligomers facilitates the formation of high weight protein aggregates. Step 3, the ubiquitin-positive protein aggregates can be transported to the microtubule organizing center (MTOC) by HDAC6-mediated microtubule (MT) network. Step 4, HDAC6 promotes the fusion of protein aggregates-containing autophagosomes and lysosomes, which is essential for the eventual clearance of protein aggregates.

FIGURE 6.4 Schematic processes of HDAC6-mediated clearance of damaged mitochondria. HDAC6 is required for the autophagic clearance of dysfunctional mitochondria. Step 1, the damaged mitochondria that are resulted from causative mutations or environmental factors are accumulated in the cytoplasm, leading to cellular toxicity. Step 2, HDAC6 is recruited to the damaged mitochondria by specific ubiquitin signals and the recruitment of HDAC6 to the dysfunctional mitochondria enhances the autophagic clearance (mitophagy). Step 3, autophagic activities are necessary for the degradation of damaged mitochondria. It is unknown whether HDAC6 is also involved in the fusion of the damaged mitochondria-containing autophagosomes with the lysosomes.

Chapter 07

FIGURE 7.1 Mitochondrial trafficking and anchoring in neurons. Neurons require specialized mechanisms for delivery and distribution of mitochondria in distal regions to maintain energy and Ca²⁺ homeostasis. This figure highlights mitochondrial transport to the presynaptic bouton (a) and the axon terminal (b). Long-range microtubule (MT)-based transport of mitochondria depends on MT polarity. In axons, MT plus-ends (+) are directed toward the terminal and minus-ends (–) are oriented toward the soma. KIF5 motors drive anterograde axonal transport to distal synapses while dynein motors drive retrograde transport to the soma. Trak motor adaptors mediate both anterograde and retrograde mitochondrial transport (van Spronsen et al., 2013). Myosin motors drives short-range mitochondrial movement along actin filaments within presynaptic terminals. Mitochondria can be anchored to MTs via a docking interaction with syntaphilin (Kang et al., 2008) or through an unknown actin-based docking receptor (Chada and Hollenbeck, 2004). Docking mechanisms ensure proper distribution of neuronal mitochondria along axons and at synapses.

FIGURE 7.2 Regulation of mitochondrial transport by synaptic activity. a, KIF5-driven mitochondrial transport is mediated by the Miro-Milton/TRAK complexes. b, Syntabulin, RanBP2, or FEZ1 function as alternative KIF5 adaptors driving anterograde mitochondrial transport. c and d, Two alternative models of the role of the Miro-Ca²⁺ pathway in regulating mitochondrial transport. Miro has 4 Ca²⁺-binding EF-hand motifs. The KIF5 C-terminal cargo-binding domain binds to the Miro-Trak adaptor complex. The binding of Ca²⁺ to the EF-hands of Miro causes the motor domain to detach from MTs, preventing motor-MT engagement (c) (Wang and Schwarz, 2009). Alternatively, binding of Ca²⁺ releases KIF5 from mitochondria (d) (MacAskill et al., 2009a). The influx of Ca²⁺ triggered by synaptic activity stops motile mitochondria at activated synapses. e, The syntaphilin-mediated “engine-switch and brake” model. A Miro-Ca²⁺ sensing pathway initiates the switch of KIF5 binding from Miro-Trak to syntaphilin, immobilizing axonal mitochondria by inhibiting the motor ATPase activity. Thus, syntaphilin switches off the “KIF5 motor engine” by sensing a “Stop Sign” (increased Ca²⁺ levels) and places a brake on mitochondria, anchoring them to the MTs. Stationary KIF5-loaded mitochondria remain bound to MT tracks, whereas the KIF5 ATPase is in an inactive state (Chen and Sheng, 2013).

FIGURE 7.3 Motile axonal mitochondria contribute to presynaptic variation. Mitochondria docked within presynaptic terminals provide stable adenosine triphosphate (ATP) supplies that power neurotransmission (left panel). In the absence of mitochondria within the presynaptic bouton (right panel), there is no stable ATP supply; motile mitochondria passing through this bouton can temporarily supply ATP, altering synaptic energy levels and causing fluctuation of ATP-dependent synaptic functions. Mitochondrial transport is a primary source of the variability in synaptic vesicle release and amplitude of excitatory postsynaptic currents (EPSCs) (Sun et al., 2013).

FIGURE 7.4 Coordination of mitochondrial transport and mitophagy. Chronically depolarizing mitochondrial membrane potential (Δψm) with uncoupling reagents induces the accumulation of Parkin-tagged mitochondria in the soma and proximal regions of processes. The restriction of dysfunctional mitochondria to these regions is the result of enhanced retrograde and reduced anterograde transport (Cai et al., 2012). This spatial organization enables damaged mitochondria in distal axons to be eliminated through the autophagy-lysosomal pathway in the soma, where mature lysosomes are primarily localized. Dysfunctional mitochondria can also recruit Parkin in axonal terminals when anchored by syntaphilin (Cai et al., 2012) or immobilized by degradation of Miro (Chan et al., 2011; Liu et al., 2012; Sarraf et al., 2013; Wang et al., 2011; Weihofen et al., 2009; Yoshli et al., 2011). The predominant retrograde transport of autophagic vacuoles including those engulfing mitochondria from distal axons to the proximal regions supports the hypothesis that dysfunctional mitochondria immobilized in distal regions may be also degraded in the soma (Maday et al., 2012).

Chapter 08

FIGURE 8.1 Melanocortin system in the arcuate nucleus of the hypothalamus: control of energy homeostasis. The primary neurons of the melanocortin system are the orexigenic NPY/AgRP/GABA neurons and the anorexigenic pro-opiomelanocortin (POMC) neurons. Both sets of neurons send projections to the paraventricular nucleus (PVN). Additionally, the NPY/AgRP neurons send inhibitory projections to the neighboring POMC cells.

FIGURE 8.2 Regulation of pro-opiomelanocortin (POMC) and NPY/AgRP neuron activation. The firing of POMC neurons during elevated glucose levels (satiety) relies on cellular glucose metabolism and signaling pathways activated by leptin and insulin receptors (top). It appears that a fundamental signaling event that affects action potential generation in these cells is glucose-derived reactive oxygen species (ROS). The same event, however, hyperpolarizes NPY/AgRP neurons and suppresses their firing. During low glucose levels, fatty acid metabolism is increased (bottom). Active phosphorylated AMP-activated protein kinase (AMPK) phosphorylates ACC, leading to a decrease in its enzymatic activity. This latter event decreases levels of malonyl-CoA, releasing CPT1 to uptake fatty acids inside the mitochondria. Fatty acid metabolism, promoted by UCP2 activity, reduces intracellular ROS levels leading to the decreased activity of POMC neurons but elevated firing of NPY/AgRP neurons.

FIGURE 8.3 Feeding states and mitochondrial dynamics in AgRP and pro-opiomelanocortin (POMC) neurons. The activity of POMC neurons promotes satiety associated with glucose utilization, elevated reactive oxygen species (ROS) levels, increase in mitochondrial fusion and contact with the ER. By contrast, the activity of NPY/AgRP neurons during fasting or caloric restriction promotes fat utilization and hunger associated with low ROS levels and increase mitochondrial fission.

Chapter 10

FIGURE 10.1 Mitochondrial organelles in primary neurons differ from those in cancer cell lines by their propensity to avoid Parkin-mediated mitophagy. This mechanism potentially preserves essential mitochondrial functions and delays neurodegeneration as long as possible. Agents that trigger the collapse of mitochondrial inner membrane potential are useful in vitro models and likely mimic in part physiological damage to mitochondria. Depolarization (loss of ΔΨ) induced by ionophores/protonophores such as CCCP/FCCP potently induce trigger Parkin translocation to mitochondria and mitophagy in HeLa cells but not in neurons. In contrast, transient exposure to the excitatory neurotransmitter glutamate, which potently and rapidly induces mitochondrial depolarization in neurons, induces Parkin translocation to mitochondria but only a fraction of the mitochondria in each neuron. Furthermore, subsequent mitophagy does not occur unless neurons are treated with the antioxidant NAC. NAC-triggered mitophagy is NMDA receptor-dependent and Ca²⁺-flux dependent in this model.

Chapter 11

FIGURE 11.1 PINK1 is processed by multiple mitochondrial proteases. PINK1 is cleaved by PARL at A103. Cleavage by m-AAA generates a product that is approximately the same size as the PARL processed fragment (Greene et al., 2012a). Cleavage sites for m-AAA and ClpXP have not been identified. The MTS of PINK1 contains two predicted MPP cleavage motifs (Sim et al., 2012). The first would cleave PINK1 at amino acids G10 and L18, the second at F69 and L77. However, it remains unclear whether MPP is involved in PINK1 processing (Becker et al., 2012; Kato et al., 2013).

FIGURE 11.2 PINK1 as a sensor of mitochondrial health and damage. (a) In healthy mitochondria, PINK1 is imported through the TOM/TIM complexes and processed by several mitochondrial proteases before being released back into the cytosol. This cytosolic pool of PINK1 may serve as a signal to the cell that the integrity of its mitochondrial network remains intact. Processed, cytosolic PINK1 has been implicated in suppressing autophagy/mitophagy, promoting neurite extension, enhancing mitochondrial trafficking into dendrites, and activating pro-survival and differentiation signaling pathways. (b) When mitochondria lose their membrane potential, full-length PINK1 accumulates on the OMM. As a result, axonal transport of mitochondria is arrested and the damaged mitochondrion becomes a target for Parkin-mediated degradation.

Chapter 14

FIGURE 14.1 Effects of Dexpramipexole (DEX) in isolated mitochondrial preparations and stressed cells. a, Following treatment of rats in vivo with a proteasome inhibitor (Z-lle-Glu(OtBu)-Ala-Leu-al; PSI), isolated brain-derived mitochondria exhibited large-conductance channel activity (organelle-attached patch clamp). Application of DEX produced a concentration-dependent and reversible inhibition of this activity; in the example shown the holding potential was +80 mV, and the channel(s) was nearly constantly in the open state. DEX at 200 nM produced a greater than 50% reduction in the open probability. b, In brain-derived submitochondrial vesicles (SMVs), application of DEX produced a concentration dependent inhibition of membrane conductances (not shown). Similar to brain-derived mitochondria isolated from rats that were pretreated with PSI, the effects were potent (EC₅₀ ~100 nM for mitochondria, ~110 nM in SMVs). Treatment of these SMVs with urea (shown), resulted in complete elimination of current inhibition by DEX (200 nM, which was >EC₅₀), but no effect on the inhibition produced by ATP (1.0 mM). This indicated that some non-membrane bound component expressed on the inner membrane of the (inside-out) complex-V enriched SMVs, which was denatured by urea treatment, was required for current inhibition by DEX but not ATP. This conclusion was supported by the experiments shown in c, wherein [¹⁴C]DEX binding in SMVs was significantly reduced by urea treatment. d, Treatment of SH-SY5Y cells in culture with the proteasome inhibitor PSI stresses cells, resulting in cell compromise and death. Pretreatment of these cells for 24 hours with DEX at the indicated concentrations, before 24 hour treatment with PSI (at 150 or 650 nM), resulted in significant reductions in the levels of cell death. ** P<0.05; *** P<0.001; in b and c, t-test, in d, Bonferroni-corrected t-tests following significant ANOVA.

FIGURE 14.2 Effects of dexpramipexole in a phase 2 study in human subjects with amyotrophic lateral sclerosis. a, The bar chart demonstrates the differential effect of the highest dose of DEX (300 mg/day) versus placebo on measures of the loss of function, as indicated by ALSFRS-R decline, in the fine motor, gross motor, bulbar and respiratory subdomains measured during the course of Part 1 in the phase 2 study of safety and efficacy in subjects with ALS. b, Kaplan-Meier estimates of the probability of survival in part 2 of the study. Censored patients were those that discontinued for any reason other than death, P = 0.07, log rank test. c, Mean (± SEM) values from a joint rank analysis (see text) of death and change from baseline in ALSFRS-R scores in survivors comparing treatment groups in part 2 of the study; there was no placebo group in part 2.

Chapter 15

FIGURE 15.1 Current and potential pharmacological targets in mitochondria. Current and potential therapeutic target are labeled with numbers. (1) Mitochondria-targeted antioxidants aim to abolish oxidative stress; (2) manipulation of mitochondrial membrane potential and (3) modulation of Ca²⁺ signaling pathways to attenuate Ca²⁺ overload in mitochondria; (4) inhibition of opening of the mitochondrial permeability transition pore; (5) enhancement of mitochondrial metabolism, and (6) manipulation of mitochondrial dynamics (fission, fusion, and transport). ΔΨm, mitochondrial membrane potential; ANT, adenine nucleotide translocase; CypD, cyclophilin D; ETC, electron transport chain; mPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel.

Chapter 18

FIGURE 18.1 Representation of the pathomechanistic role of mitochondrial respiratory chain defects and subsequent mitochondrial and cellular dysfunction in the development of mitochondrial disorders. Dashed lines represent secondary events. mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation.

FIGURE 18.2 Summary of some therapeutic strategies for improving mitochondrial respiratory chain function in mitochondrial disorders. Dashed lines represent secondary events. OXPHOS, oxidative phosphorylation.

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Wiley Series on Neuropharmacology
Valentin K. Gribkoff and Leonard K. Kaczmarek, Series Editors

The Functions, Disease-Related Dysfunctions, and Therapeutic Targeting of Neuronal Mitochondria

Edited by Valentin K. Gribkoff, Elizabeth A. Jonas, and J. Marie Hardwick

THE FUNCTIONS, DISEASE-RELATED DYSFUNCTIONS, AND THERAPEUTIC TARGETING OF NEURONAL MITOCHONDRIA

Edited by

VALENTIN K. GRIBKOFF

Department of Internal Medicine, Section of Endocrinology

Yale University School of Medicine

New Haven, Connecticut, USA

ELIZABETH A. JONAS

Department of Internal Medicine, Section of Endocrinology

Yale University School of Medicine

New Haven, Connecticut, USA

J. MARIE HARDWICK

Department of Molecular Microbiology and Immunology, and Dept. of Neurology

Johns Hopkins University Bloomberg Schools of Public Health and Medicine

Baltimore, Maryland, USA

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