Myeloid cell-associated aromatic amino acid metabolism facilitates CNS myelin regeneration.

Regulation of myeloid cell activity is critical for successful myelin regeneration (remyelination) in demyelinating diseases, such as multiple sclerosis (MS). Here, we show aromatic alpha-keto acids (AKAs) generated from the amino acid oxidase, interleukin-4 induced 1 (IL4I1), promote efficient remyelination in mouse models of MS. During remyelination, myeloid cells upregulated the expression of IL4I1. Conditionally knocking out IL4I1 in myeloid cells impaired remyelination efficiency. Mice lacking IL4I1 expression exhibited a reduction in the AKAs, phenylpyruvate, indole-3-pyruvate, and 4-hydroxyphenylpyruvate, in remyelinating lesions. Decreased AKA levels were also observed in people with MS, particularly in the progressive phase when remyelination is impaired. Oral administration of AKAs modulated myeloid cell-associated inflammation, promoted oligodendrocyte maturation, and enhanced remyelination in mice with focal demyelinated lesions. Transcriptomic analysis revealed AKA treatment induced a shift in metabolic pathways in myeloid cells and upregulated aryl hydrocarbon receptor activity in lesions. Our results suggest myeloid cell-associated aromatic amino acid metabolism via IL4I1 produces AKAs in demyelinated lesions to enable efficient remyelination. Increasing AKA levels or targeting related pathways may serve as a strategy to facilitate the regeneration of myelin in inflammatory demyelinating conditions.

ER and Golgi trafficking in axons, dendrites, and glial processes.

Both neurons and glia in mammalian brains are highly ramified. Neurons form complex neural networks using axons and dendrites. Axons are long with few branches and form pre-synaptic boutons that connect to target neurons and effector tissues. Dendrites are shorter, highly branched, and form post-synaptic boutons. Astrocyte processes contact synapses and blood vessels in order to regulate neuronal activity and blood flow, respectively. Oligodendrocyte processes extend toward axons to make myelin sheaths. Microglia processes dynamically survey their environments. Here, we describe the local secretory system (ER and Golgi) in neuronal and glial processes. We focus on Golgi outpost functions in acentrosomal microtubule nucleation, cargo trafficking, and protein glycosylation. Thus, satellite ER and Golgi are critical for local structure and function in neurons and glia.

Emerging cellular themes in leukodystrophies.

Leukodystrophies are a broad spectrum of neurological disorders that are characterized primarily by deficiencies in myelin formation. Clinical manifestations of leukodystrophies usually appear during childhood and common symptoms include lack of motor coordination, difficulty with or loss of ambulation, issues with vision and/or hearing, cognitive decline, regression in speech skills, and even seizures. Many cases of leukodystrophy can be attributed to genetic mutations, but they have diverse inheritance patterns (e.g., autosomal recessive, autosomal dominant, or X-linked) and some arise from de novo mutations. In this review, we provide an updated overview of 35 types of leukodystrophies and focus on cellular mechanisms that may underlie these disorders. We find common themes in specialized functions in oligodendrocytes, which are specialized producers of membranes and myelin lipids. These mechanisms include myelin protein defects, lipid processing and peroxisome dysfunction, transcriptional and translational dysregulation, disruptions in cytoskeletal organization, and cell junction defects. In addition, non-cell-autonomous factors in astrocytes and microglia, such as autoimmune reactivity, and intercellular communication, may also play a role in leukodystrophy onset. We hope that highlighting these themes in cellular dysfunction in leukodystrophies may yield conceptual insights on future therapeutic approaches.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

mRNA Transport and Local Translation in Glia.

Though mRNA transport and local translation are extensively studied in neurons, emerging evidence supports that these cellular processes are also abundant in non-neuronal glial cells. Here, we explore mechanisms of mRNA transport and local translation in oligodendrocytes, astrocytes, microglia, radial glia, and their functions in development, structure, and intercellular interactions.

Microtubule organization and dynamics in oligodendrocytes, astrocytes, and microglia.

Though much is known about microtubule organization and microtubule-based transport in neurons, the development and function of microtubules in glia are more enigmatic. In this review, we provide an overview of the literature on microtubules in ramified brain cells, including oligodendrocytes, astrocytes, and microglia. We focus on normal cell biology-how structure relates to function in these cells. In oligodendrocytes, microtubules are important for extension of processes that contact axons and for elongating the myelin sheath. Recent studies demonstrate that new microtubules can form outside of the oligodendrocyte cell body off of Golgi outpost organelles. In astrocytes and microglia, changes in cell shape and ramification can be influenced by neighboring cells and the extracellular milieu. Finally, we highlight key papers implicating glial microtubule defects in neurological injury and disease and discuss how microtubules may contribute to invasiveness in gliomas. Thus, future research on the mechanisms underlying microtubule organization in normal glial cell function may yield valuable insights on neurological disease pathology.

Golgi Outposts Nucleate Microtubules in Cells with Specialized Shapes.

Classically, animal cells nucleate or form new microtubules off the perinuclear centrosome. In recent years, the Golgi outpost has emerged as a satellite organelle that can function as an acentrosomal microtubule-organizing center (MTOC), nucleating new microtubules at distances far from the nucleus or cell body. Golgi outposts can nucleate new microtubules in specialized cells with unique cytoarchitectures, including Drosophila neurons, mouse muscle cells, and rodent oligodendrocytes. This review compares and contrasts topics of functional relevance, including Golgi outpost heterogeneity, formation and transport, as well as regulation of microtubule polarity and branching. Golgi outposts have also been implicated in the pathology of diseases including muscular dystrophy, and neurodegenerative diseases, such as Parkinson's disease (PD). Since Golgi outposts are relatively understudied, many outstanding questions regarding their function and roles in disease remain.

Fear Deficits in Hypomyelinated Tppp Knock-Out Mice.

Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to facilitate efficient electrical conduction. These myelin sheaths contain lamellar microtubules that enable vesicular transport into the inner sheath. Mechanistically, oligodendrocytes rely on Golgi outpost organelles and the associated protein tubulin polymerization promoting protein (TPPP) to nucleate or form new microtubules outside of the cell body. Consequently, elongation of lamellar microtubules is defective in Tppp knock-out (KO) mice, which have thinner and shorter myelin sheaths. We now explore the behavioral phenotypes of Tppp KO mice using a number of different assays. In open-field assays, Tppp KO mice display similar activity levels and movement patterns as wild-type mice, indicating that they do not display anxiety behavior. However, Tppp KO mice lack fear responses by two types of assays, traditional fear-conditioning assays and looming fear assays, which test for innate fear responses. Deficits in fear conditioning, which is a memory-dependent task, as well as in spatial memory tests, support possible short-term memory defects in Tppp KO mice. Together, our experiments indicate a connection between CNS myelination and behavioral deficits.

The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for Myelination.

Oligodendrocytes extend elaborate microtubule arbors that contact up to 50 axon segments per cell, then spiral around myelin sheaths, penetrating from outer to inner layers. However, how they establish this complex cytoarchitecture is unclear. Here, we show that oligodendrocytes contain Golgi outposts, an organelle that can function as an acentrosomal microtubule-organizing center (MTOC). We identify a specific marker for Golgi outposts-TPPP (tubulin polymerization promoting protein)-that we use to purify this organelle and characterize its proteome. In in vitro cell-free assays, recombinant TPPP nucleates microtubules. Primary oligodendrocytes from Tppp knockout (KO) mice have aberrant microtubule branching, mixed microtubule polarity, and shorter myelin sheaths when cultured on 3-dimensional (3D) microfibers. Tppp KO mice exhibit hypomyelination with shorter, thinner myelin sheaths and motor coordination deficits. Together, our data demonstrate that microtubule nucleation outside the cell body at Golgi outposts by TPPP is critical for elongation of the myelin sheath.

Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo.

Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mbp mRNA transport in zebrafish, it is not entirely clear how mbp transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in actr10, which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the actr10 mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mbp mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mbp mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged Mbp mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests Mbp mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates Mbp mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mbp mRNA transport and myelination in vivo.

CNS myelin wrapping is driven by actin disassembly.

Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility.

MAPK8IP1/JIP1 regulates the trafficking of autophagosomes in neurons.

Autophagy is a spatially regulated process in axons; autophagosomes form preferentially in the distal axon tip then move actively and processively toward the cell body. Despite the primarily unidirectional transport observed in live-cell imaging experiments, both anterograde-directed KIF5/kinesin-1 motors and retrograde-directed dynein motors are tightly associated with axonal autophagosomes. Here, we discuss our recent work identifying the scaffolding protein MAPK8IP1/JIP1 (mitogen-activated protein kinase 8 interacting protein 1) as a key regulator of autophagosome transport in neurons. MAPK8IP1 tightly coordinates motor activity to ensure the fidelity of retrograde autophagosome transport in the axon.

Integrated regulation of motor-driven organelle transport by scaffolding proteins.

Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment.

LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes.

Autophagy is essential for maintaining cellular homeostasis in neurons, where autophagosomes undergo robust unidirectional retrograde transport along axons. We find that the motor scaffolding protein JIP1 binds directly to the autophagosome adaptor LC3 via a conserved LIR motif. This interaction is required for the initial exit of autophagosomes from the distal axon, for sustained retrograde transport along the midaxon, and for autophagosomal maturation in the proximal axon. JIP1 binds directly to the dynein activator dynactin but also binds to and activates kinesin-1 in a phosphorylation-dependent manner. Following JIP1 depletion, phosphodeficient JIP1-S421A rescues retrograde transport, while phosphomimetic JIP1-S421D aberrantly activates anterograde transport. During normal autophagosome transport, residue S421 of JIP1 may be maintained in a dephosphorylated state by autophagosome-associated MKP1 phosphatase. Moreover, binding of LC3 to JIP1 competitively disrupts JIP1-mediated activation of kinesin. Thus, dual mechanisms prevent aberrant activation of kinesin to ensure robust retrograde transport of autophagosomes along the axon.

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150(Glued) to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.

Retrograde axonal transport: pathways to cell death?

Active transport along the axon is crucial to the neuron. Motor-driven transport supplies the distal synapse with newly synthesized proteins and lipids, and clears damaged or misfolded proteins. Microtubule motors also drive long-distance signaling along the axon via signaling endosomes. Although positive signaling initiated by neurotrophic factors has been well-studied, recent research has focused on stress-signaling along the axon. Here, the connections between axonal transport alterations and neurodegeneration are discussed, including evidence for defective transport of vesicles, mitochondria, degradative organelles, and signaling endosomes in models of amyotrophic lateral sclerosis, Huntington's, Parkinson's and Alzheimer's disease. Defects in transport are sufficient to induce neurodegeneration, but recent progress suggests that changes in retrograde signaling pathways correlate with rapidly progressive neuronal cell death.