Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture.

The molecular mechanisms underlying the axon arborization of mammalian neurons are poorly understood but are critical for the establishment of functional neural circuits. We identified a pathway defined by two kinases, LKB1 and NUAK1, required for cortical axon branching in vivo. Conditional deletion of LKB1 after axon specification or knockdown of NUAK1 drastically reduced axon branching in vivo, whereas their overexpression was sufficient to increase axon branching. The LKB1-NUAK1 pathway controls mitochondria immobilization in axons. Using manipulation of Syntaphilin, a protein necessary and sufficient to arrest mitochondrial transport specifically in the axon, we demonstrate that the LKB1-NUAK1 kinase pathway regulates axon branching by promoting mitochondria immobilization. Finally, we show that LKB1 and NUAK1 are necessary and sufficient to immobilize mitochondria specifically at nascent presynaptic sites. Our results unravel a link between presynaptic mitochondrial capture and axon branching.

Correction: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

[This corrects the article DOI: 10.1371/journal.pbio.1002516.].

Pleiotropic Mitochondria: The Influence of Mitochondria on Neuronal Development and Disease.

Mitochondria play many important biological roles, including ATP production, lipid biogenesis, ROS regulation, and calcium clearance. In neurons, the mitochondrion is an essential organelle for metabolism and calcium homeostasis. Moreover, mitochondria are extremely dynamic and able to divide, fuse, and move along microtubule tracks to ensure their distribution to the neuronal periphery. Mitochondrial dysfunction and altered mitochondrial dynamics are observed in a wide range of conditions, from impaired neuronal development to various neurodegenerative diseases. Novel imaging techniques and genetic tools provide unprecedented access to the physiological roles of mitochondria by visualizing mitochondrial trafficking, morphological dynamics, ATP generation, and ultrastructure. Recent studies using these new techniques have unveiled the influence of mitochondria on axon branching, synaptic function, calcium regulation with the ER, glial cell function, neurogenesis, and neuronal repair. This review provides an overview of the crucial roles played by mitochondria in the CNS in physiological and pathophysiological conditions.

LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

A role for myosin VI in the localization of axonal proteins.

In neurons polarized trafficking of vesicle-bound membrane proteins gives rise to the distinct molecular composition and functional properties of axons and dendrites. Despite their central role in shaping neuronal form and function, surprisingly little is known about the molecular processes that mediate polarized targeting of neuronal proteins. Recently, the plus-end-directed motor Myosin Va was shown to play a critical role in targeting of transmembrane proteins to dendrites; however, the role of myosin motors in axonal targeting is unknown. Here we show that Myosin VI, a minus-end-directed motor, plays a vital role in the enrichment of proteins on the surface of axons. Engineering non-neuronal proteins to interact with Myosin VI causes them to become highly concentrated at the axonal surface in dissociated rat cortical neurons. Furthermore, disruption of either Myosin VI function or expression leads to aberrant dendritic localization of axonal proteins. Myosin VI mediates the enrichment of proteins on the axonal surface at least in part by stimulating dendrite-specific endocytosis, a mechanism that has been shown to underlie the localization of many axonal proteins. In addition, a version of Channelrhodopsin 2 that was engineered to bind to Myosin VI is concentrated at the surface of the axon of cortical neurons in mice in vivo, suggesting that it could be a useful tool for probing circuit structure and function. Together, our results indicate that myosins help shape the polarized distributions of both axonal and dendritic proteins.

Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

Activity-dependent compartmentalization of dendritic mitochondria morphology through local regulation of fusion-fission balance in neurons in vivo.

Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly between the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Ca2+ and Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a signaling pathway underlying the subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise and activity-dependent regulation of mitochondria fission/fusion balance.

Metformin treatment results in distinctive skeletal muscle mitochondrial remodeling in rats with different intrinsic aerobic capacities.

The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand the potential context specificity of metformin treatment on skeletal muscle, we used a rat model (high-capacity runner/low-capacity runner [HCR/LCR]) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (intermyofibrillar vs. subsarcolemmal). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.

Metformin treatment results in distinctive skeletal muscle mitochondrial remodeling in rats with different intrinsic aerobic capacities.

The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand potential context specificity of metformin treatment on skeletal muscle, we used a rat model (HCR/LCR) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (IMF vs SS). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.

Exercise and mitochondrial remodeling to prevent age-related neurodegeneration.

Healthy brain activity requires precise ion and energy management creating a strong reliance on mitochondrial function. Age-related neurodegeneration leads to a decline in mitochondrial function and increased oxidative stress, with associated declines in mitochondrial mass, respiration capacity, and respiration efficiency. The interdependent processes of mitochondrial protein turnover and mitochondrial dynamics, known together as mitochondrial remodeling, play essential roles in mitochondrial health and therefore brain function. This mini-review describes the role of mitochondria in neurodegeneration and brain health, current practices for assessing both aspects of mitochondrial remodeling, and how exercise mitigates the adverse effects of aging in the brain. Exercise training elicits functional adaptations to improve brain health, and current literature strongly suggests that mitochondrial remodeling plays a vital role in these positive adaptations. Despite substantial implications that the two aspects of mitochondrial remodeling are interdependent, very few investigations have simultaneously measured mitochondrial dynamics and protein synthesis. An improved understanding of the partnership between mitochondrial protein turnover and mitochondrial dynamics will provide a better understanding of their role in both brain health and disease, as well as how they induce protection following exercise.

Activity-dependent subcellular compartmentalization of dendritic mitochondria structure in CA1 pyramidal neurons.

Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly in the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a new activity-dependent molecular mechanism underlying the extreme subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise regulation of mitochondria fission/fusion balance.

Neuronal deletion of MnSOD in mice leads to demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Neuronal oxidative stress has been implicated in aging and neurodegenerative disease. Here we investigated the impact of elevated oxidative stress induced in mouse spinal cord by deletion of Mn-Superoxide dismutase (MnSOD) using a neuron specific Cre recombinase in Sod2 floxed mice (i-mn-Sod2 KO). Sod2 deletion in spinal cord neurons was associated with mitochondrial alterations and peroxide generation. Phenotypically, i-mn-Sod2 KO mice experienced hindlimb paralysis and clasping behavior associated with extensive demyelination and reduced nerve conduction velocity, axonal degeneration, enhanced blood brain barrier permeability, elevated inflammatory cytokines, microglia activation, infiltration of neutrophils and necroptosis in spinal cord. In contrast, spinal cord motor neuron number, innervation of neuromuscular junctions, muscle mass, and contractile function were not altered. Overall, our findings show that loss of MnSOD in spinal cord promotes a phenotype of demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion.

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, α-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.

Aß42 oligomers trigger synaptic loss through CAMKK2-AMPK-dependent effectors coordinating mitochondrial fission and mitophagy.

During the early stages of Alzheimer's disease (AD) in both mouse models and human patients, soluble forms of Amyloid-ß 1-42 oligomers (Aß42o) trigger loss of excitatory synapses (synaptotoxicity) in cortical and hippocampal pyramidal neurons (PNs) prior to the formation of insoluble amyloid plaques. In a transgenic AD mouse model, we observed a spatially restricted structural remodeling of mitochondria in the apical tufts of CA1 PNs dendrites corresponding to the dendritic domain where the earliest synaptic loss is detected in vivo. We also observed AMPK over-activation as well as increased fragmentation and loss of mitochondrial biomass in Ngn2-induced neurons derived from a new APPSwe/Swe knockin human ES cell line. We demonstrate that Aß42o-dependent over-activation of the CAMKK2-AMPK kinase dyad mediates synaptic loss through coordinated phosphorylation of MFF-dependent mitochondrial fission and ULK2-dependent mitophagy. Our results uncover a unifying stress-response pathway causally linking Aß42o-dependent structural remodeling of dendritic mitochondria to synaptic loss.

Necroptosis increases with age in the brain and contributes to age-related neuroinflammation.

Chronic inflammation of the central nervous system (CNS), termed neuroinflammation, is a hallmark of aging and a proposed mediator of cognitive decline associated with aging. Neuroinflammation is characterized by the persistent activation of microglia, the innate immune cells of the CNS, with damage-associated molecular patterns (DAMPs) being one of the well-known activators of microglia. Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we hypothesized that an age-associated increase in necroptosis contributes to increased neuroinflammation with age. The marker of necroptosis, phosphorylated form of MLKL (P-MLKL), and kinases in the necroptosis pathway (RIPK1, RIPK3, and MLKL) showed a region-specific increase in the brain with age, specifically in the cortex layer V and the CA3 region of the hippocampus of mice. Similarly, MLKL-oligomers, which cause membrane binding and permeabilization, were significantly increased in the cortex and hippocampus of old mice relative to young mice. Nearly 70 to 80% of P-MLKL immunoreactivity was localized to neurons and less than 10% was localized to microglia, whereas no P-MLKL was detected in astrocytes. P-MLKL expression in neurons was detected in the soma, not in the processes. Blocking necroptosis using Mlkl-/- mice reduced markers of neuroinflammation (Iba-1 and GFAP) in the brains of old mice, and short-term treatment with the necroptosis inhibitor, necrostatin-1s, reduced expression of proinflammatory cytokines, IL-6 and IL-1ß, in the hippocampus of old mice. Thus, our data demonstrate for the first time that brain necroptosis increases with age and contributes to age-related neuroinflammation in mice.

Mitochondrial biogenesis in developing astrocytes regulates astrocyte maturation and synapse formation.

The mechanisms controlling the post-natal maturation of astrocytes play a crucial role in ensuring correct synaptogenesis. We show that mitochondrial biogenesis in developing astrocytes is necessary for coordinating post-natal astrocyte maturation and synaptogenesis. The astrocytic mitochondrial biogenesis depends on the transient upregulation of metabolic regulator peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α), which is controlled by metabotropic glutamate receptor 5 (mGluR5). At tissue level, the loss or downregulation of astrocytic PGC-1α sustains astrocyte proliferation, dampens astrocyte morphogenesis, and impairs the formation and function of neighboring synapses, whereas its genetic re-expression is sufficient to restore the mitochondria compartment and correct astroglial and synaptic defects. Our findings show that the developmental enhancement of mitochondrial biogenesis in astrocytes is a critical mechanism controlling astrocyte maturation and supporting synaptogenesis, thus suggesting that astrocytic mitochondria may be a therapeutic target in the case of neurodevelopmental and psychiatric disorders characterized by impaired synaptogenesis.

Emerging roles of mitochondria in synaptic transmission and neurodegeneration.

Mitochondria play numerous critical physiological functions in neurons including ATP production, Ca2+ regulation, lipid synthesis, ROS signaling, and the ability to trigger apoptosis. Recently developed technologies, including in vivo 2-photon imaging in awake behaving mice revealed that unlike in the peripheral nervous system (PNS), mitochondrial transport decreases strikingly along the axons of adult neurons of the central nervous system (CNS). Furthermore, the improvements of genetically-encoded biosensors have enabled precise monitoring of the spatial and temporal impact of mitochondria on Ca2+, ATP and ROS homeostasis in a compartment-specific manner. Here, we discuss recent findings that begin to unravel novel physiological and pathophysiological properties of neuronal mitochondria at synapses. We also suggest new directions in the exploration of mitochondrial function in synaptic transmission, plasticity and neurodegeneration.

MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size.

Neurons display extreme degrees of polarization, including compartment-specific organelle morphology. In cortical, long-range projecting, pyramidal neurons (PNs), dendritic mitochondria are long and tubular whereas axonal mitochondria display uniformly short length. Here we explored the functional significance of maintaining small mitochondria for axonal development in vitro and in vivo. We report that the Drp1 'receptor' Mitochondrial fission factor (MFF) is required for determining the size of mitochondria entering the axon and then for maintenance of their size along the distal portions of the axon without affecting their trafficking properties, presynaptic capture, membrane potential or ability to generate ATP. Strikingly, this increase in presynaptic mitochondrial size upon MFF downregulation augments their capacity for Ca2+ ([Ca2+]m) uptake during neurotransmission, leading to reduced presynaptic [Ca2+]c accumulation, decreased presynaptic release and terminal axon branching. Our results uncover a novel mechanism controlling neurotransmitter release and axon branching through fission-dependent regulation of presynaptic mitochondrial size.

Haploinsufficiency of autism spectrum disorder candidate gene NUAK1 impairs cortical development and behavior in mice.

Recently, numerous rare de novo mutations have been identified in patients diagnosed with autism spectrum disorders (ASD). However, despite the predicted loss-of-function nature of some of these de novo mutations, the affected individuals are heterozygous carriers, which would suggest that most of these candidate genes are haploinsufficient and/or lead to expression of dominant-negative forms of the protein. Here, we tested this hypothesis with the candidate ASD gene Nuak1 that we previously identified for its role in the development of cortical connectivity. We report that Nuak1 is haploinsufficient in mice with regard to its function in cortical development. Furthermore Nuak1+/- mice show a combination of abnormal behavioral traits ranging from defective spatial memory consolidation, defects in social novelty (but not social preference) and abnormal sensorimotor gating. Overall, our results demonstrate that Nuak1 haploinsufficiency leads to defects in the development of cortical connectivity and a complex array of behavorial deficits.

Virus-Enabled Optimization and Delivery of the Genetic Machinery for Efficient Unnatural Amino Acid Mutagenesis in Mammalian Cells and Tissues.

Unnatural amino acid (UAA) mutagenesis of recombinant proteins in live mammalian cells requires coexpression of the mutant target, as well as an engineered tRNA/aminoacyl-tRNA synthetase pair. The ability to readily determine the optimal relative expression levels of these three genetic components for efficient expression of the UAA-modified target is highly desirable, but remains challenging to accomplish. Here we report a facile strategy to achieve this by taking advantage of the efficient gene-delivery by a baculovirus vector, which enables systematic variation of the expression level of each genetic component in a population-wide manner. Insights gained from this study led to the design of an optimal expression system, which can be delivered into mammalian cells by a single baculovirus vector to provide significantly improved UAA incorporation efficiency at a low virus load. Furthermore, this optimized baculovirus vector was shown to enable efficient UAA mutagenesis of proteins expressed in mouse brain tissue.