J. Bradley Zuchero

All Publications

• Schwann cell-secreted PGE2 promotes sensory neuron excitability during development. Cell Kantarci, H., Elvira, P. D., Thottumkara, A. P., O'Connell, E. M., Iyer, M., Donovan, L. J., Dugan, M. Q., Ambiel, N., Granados, A., Zeng, H., Saw, N. L., Brosius Lutz, A., Sloan, S. A., Gray, E. E., Tran, K. V., Vichare, A., Yeh, A. K., Münch, A. E., Huber, M., Agrawal, A., Morri, M., Zhong, H., Shamloo, M., Anderson, T. A., Tawfik, V. L., Du Bois, J., Zuchero, J. B. 2024

  Abstract

  Electrical excitability-the ability to fire and propagate action potentials-is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage-gated sodium channels, and to fire action potential trains. Inactivating this signaling pathway in Schwann cells impairs somatosensory neuron maturation, causing multimodal sensory defects that persist into adulthood. Collectively, our studies uncover a neurodevelopmental role for prostaglandin E2 distinct from its established role in inflammation, revealing a cell non-autonomous mechanism by which glia regulate neuronal excitability to enable the development of normal sensory functions.

  View details for DOI 10.1016/j.cell.2024.07.033

  View details for PubMedID 39142281

• Translating Molecular Approaches to Oligodendrocyte-Mediated Neurological Circuit Modulation. Brain sciences Song, J., Saglam, A., Zuchero, J. B., Buch, V. P. 2024; 14 (7)

  Abstract

  The central nervous system (CNS) exhibits remarkable adaptability throughout life, enabled by intricate interactions between neurons and glial cells, in particular, oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs). This adaptability is pivotal for learning and memory, with OLs and OPCs playing a crucial role in neural circuit development, synaptic modulation, and myelination dynamics. Myelination by OLs not only supports axonal conduction but also undergoes adaptive modifications in response to neuronal activity, which is vital for cognitive processing and memory functions. This review discusses how these cellular interactions and myelin dynamics are implicated in various neurocircuit diseases and disorders such as epilepsy, gliomas, and psychiatric conditions, focusing on how maladaptive changes contribute to disease pathology and influence clinical outcomes. It also covers the potential for new diagnostics and therapeutic approaches, including pharmacological strategies and emerging biomarkers in oligodendrocyte functions and myelination processes. The evidence supports a fundamental role for myelin plasticity and oligodendrocyte functionality in synchronizing neural activity and high-level cognitive functions, offering promising avenues for targeted interventions in CNS disorders.

  View details for DOI 10.3390/brainsci14070648

  View details for PubMedID 39061389

  View details for PubMedCentralID PMC11275066

• Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies. bioRxiv : the preprint server for biology Markusson, S., Raasakka, A., Schröder, M., Sograte-Idrissi, S., Rahimi, A. M., Asadpour, O., Körner, H., Lodygin, D., Eichel-Vogel, M. A., Chowdhury, R., Sutinen, A., Muruganandam, G., Iyer, M., Cooper, M. H., Weigel, M. K., Ambiel, N., Werner, H. B., Zuchero, J. B., Opazo, F., Kursula, P. 2024

  Abstract

  2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognizing the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibers and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional biology of myelination, including high-resolution imaging of nerve tissue.

  View details for DOI 10.1101/2024.05.25.595513

  View details for PubMedID 38826303

  View details for PubMedCentralID PMC11142274

• pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis. Journal of cell science O'Shaughnessy, E. C., Lam, M., Ryken, S. E., Wiesner, T., Lukasik, K., Zuchero, J. B., Leterrier, C., Adalsteinsson, D., Gupton, S. L. 2024

  Abstract

  Exocytosis is a fundamental process used by eukaryotes to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate exocytosis in neuronal morphogenesis, previously we developed computational tools with a graphical user interface to enable the automatic detection and analysis of exocytic events from fluorescence timelapse images. Though these tools were useful, we found the code was brittle and not easily adapted to different experimental conditions. Here we developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. We show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons.

  View details for DOI 10.1242/jcs.261828

  View details for PubMedID 38690758

• Optical Control of G-Actin with a Photoswitchable Latrunculin. Journal of the American Chemical Society Vepřek, N. A., Cooper, M. H., Laprell, L., Yang, E. J., Folkerts, S., Bao, R., Boczkowska, M., Palmer, N. J., Dominguez, R., Oertner, T. G., Pon, L. A., Zuchero, J. B., Trauner, D. H. 2024

  Abstract

  Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

  View details for DOI 10.1021/jacs.3c10776

  View details for PubMedID 38511265

• SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination. Proceedings of the National Academy of Sciences of the United States of America Iram, T., Garcia, M. A., Amand, J., Kaur, A., Atkins, M., Iyer, M., Lam, M., Ambiel, N., Jorgens, D. M., Keller, A., Wyss-Coray, T., Kern, F., Zuchero, J. B. 2024; 121 (12): e2307250121

  Abstract

  Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

  View details for DOI 10.1073/pnas.2307250121

  View details for PubMedID 38483990

• Oligodendrocyte calcium signaling promotes actin-dependent myelin sheath extension. Nature communications Iyer, M., Kantarci, H., Cooper, M. H., Ambiel, N., Novak, S. W., Andrade, L. R., Lam, M., Jones, G., Münch, A. E., Yu, X., Khakh, B. S., Manor, U., Zuchero, J. B. 2024; 15 (1): 265

  Abstract

  Myelin is essential for rapid nerve signaling and is increasingly found to play important roles in learning and in diverse diseases of the CNS. Morphological parameters of myelin such as sheath length are thought to precisely tune conduction velocity, but the mechanisms controlling sheath morphology are poorly understood. Local calcium signaling has been observed in nascent myelin sheaths and can be modulated by neuronal activity. However, the role of calcium signaling in sheath formation remains incompletely understood. Here, we use genetic tools to attenuate oligodendrocyte calcium signaling during myelination in the developing mouse CNS. Surprisingly, genetic calcium attenuation does not grossly affect the number of myelinated axons or myelin thickness. Instead, calcium attenuation causes myelination defects resulting in shorter, dysmorphic sheaths. Mechanistically, calcium attenuation reduces actin filaments in oligodendrocytes, and an intact actin cytoskeleton is necessary and sufficient to achieve accurate myelin morphology. Together, our work reveals a cellular mechanism required for accurate CNS myelin formation and may provide mechanistic insight into how oligodendrocytes respond to neuronal activity to sculpt and refine myelin sheaths.

  View details for DOI 10.1038/s41467-023-44238-3

  View details for PubMedID 38177161

  View details for PubMedCentralID 9651929

• Focused ultrasound-induced inhibition of peripheral nerve fibers in an animal model of acute pain. Regional anesthesia and pain medicine Anderson, T. A., Pacharinsak, C., Vilches-Moure, J., Kantarci, H., Zuchero, J. B., Butts-Pauly, K., Yeomans, D. 2023

  Abstract

  Moderate-to-severe acute pain is prevalent in many healthcare settings and associated with adverse outcomes. Peripheral nerve blockade using traditional needle-based and local anesthetic-based techniques improves pain outcomes for some patient populations but has shortcomings limiting use. These limitations include its invasiveness, potential for local anesthetic systemic toxicity, risk of infection with an indwelling catheter, and relatively short duration of blockade compared with the period of pain after major injuries. Focused ultrasound is capable of inhibiting the peripheral nervous system and has potential as a pain management tool. However, investigations of its effect on peripheral nerve nociceptive fibers in animal models of acute pain are lacking. In an in vivo acute pain model, we investigated focused ultrasound's effects on behavior and peripheral nerve structure.Focused ultrasound was applied directly to the sciatic nerve of rats just prior to a hindpaw incision; three control groups (focused ultrasound sham only, hindpaw incision only, focused ultrasound sham+hindpaw incision) were also included. For all four groups (intervention and controls), behavioral testing (thermal and mechanical hyperalgesia, hindpaw extension and flexion) took place for 4 weeks. Structural changes to peripheral nerves of non-focused ultrasound controls and after focused ultrasound application were assessed on days 0 and 14 using light microscopy and transmission electron microscopy.Compared with controls, after focused ultrasound application, animals had (1) increased mechanical nociceptive thresholds for 2 weeks; (2) sustained increase in thermal nociceptive thresholds for ≥4 weeks; (3) a decrease in hindpaw motor response for 0.5 weeks; and (4) a decrease in hindpaw plantar sensation for 2 weeks. At 14 days after focused ultrasound application, alterations to myelin sheaths and nerve fiber ultrastructure were observed both by light and electron microscopy.Focused ultrasound, using a distinct parameter set, reversibly inhibits A-delta peripheral nerve nociceptive, motor, and non-nociceptive sensory fiber-mediated behaviors, has a prolonged effect on C nociceptive fiber-mediated behavior, and alters nerve structure. Focused ultrasound may have potential as a peripheral nerve blockade technique for acute pain management. However, further investigation is required to determine C fiber inhibition duration and the significance of nerve structural changes.

  View details for DOI 10.1136/rapm-2022-104060

  View details for PubMedID 36822815

• A class of anti-inflammatory lipids decrease with aging in the central nervous system. Nature chemical biology Tan, D., Konduri, S., Erikci Ertunc, M., Zhang, P., Wang, J., Chang, T., Pinto, A. F., Rocha, A., Donaldson, C. J., Vaughan, J. M., Ludwig, R. G., Willey, E., Iyer, M., Gray, P. C., Maher, P., Allen, N. J., Zuchero, J. B., Dillin, A., Mori, M. A., Kohama, S. G., Siegel, D., Saghatelian, A. 2022

  Abstract

  Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-kappaB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

  View details for DOI 10.1038/s41589-022-01165-6

  View details for PubMedID 36266352

• CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes. Nature communications Lam, M., Takeo, K., Almeida, R. G., Cooper, M. H., Wu, K., Iyer, M., Kantarci, H., Zuchero, J. B. 2022; 13 (1): 5583

  Abstract

  Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is not fully understood. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease.

  View details for DOI 10.1038/s41467-022-33200-4

  View details for PubMedID 36151203

• Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17. Nature Iram, T., Kern, F., Kaur, A., Myneni, S., Morningstar, A. R., Shin, H., Garcia, M. A., Yerra, L., Palovics, R., Yang, A. C., Hahn, O., Lu, N., Shuken, S. R., Haney, M. S., Lehallier, B., Iyer, M., Luo, J., Zetterberg, H., Keller, A., Zuchero, J. B., Wyss-Coray, T. 2022

  Abstract

  Recent understanding of how the systemic environment shapes the brain throughout life has led to numerous intervention strategies to slow brain ageing1-3. Cerebrospinal fluid (CSF) makes up the immediate environment of brain cells, providing them with nourishing compounds4,5. We discovered that infusing young CSF directly into aged brains improves memory function. Unbiased transcriptome analysis of the hippocampus identified oligodendrocytes to be most responsive to this rejuvenated CSF environment. We further showed that young CSF boosts oligodendrocyte progenitor cell (OPC) proliferation and differentiation in the aged hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, we identified serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, as a mediator of OPC proliferation following exposure to young CSF. With age, SRF expression decreases in hippocampal OPCs, and the pathway is induced by acute injection with young CSF. We screened for potential SRF activators in CSF and found that fibroblast growth factor 17 (Fgf17) infusion is sufficient to induce OPC proliferation and long-term memory consolidation in aged mice while Fgf17 blockade impairs cognition in young mice. These findings demonstrate the rejuvenating power of young CSF and identify Fgf17 as a key target to restore oligodendrocyte function in the ageing brain.

  View details for DOI 10.1038/s41586-022-04722-0

  View details for PubMedID 35545674

• Clonally Expanded B Cells in Multiple Sclerosis Bind EBV EBNA1 and GlialCAM. Nature Lanz, T. V., Brewer, R. C., Ho, P. P., Moon, J. S., Jude, K. M., Fernandez, D., Fernandes, R. A., Gomez, A. M., Nadj, G. S., Bartley, C. M., Schubert, R. D., Hawes, I. A., Vazquez, S. E., Iyer, M., Zuchero, J. B., Teegen, B., Dunn, J. E., Lock, C. B., Kipp, L. B., Cotham, V. C., Ueberheide, B. M., Aftab, B. T., Anderson, M. S., DeRisi, J. L., Wilson, M. R., Bashford-Rogers, R. J., Platten, M., Garcia, K. C., Steinman, L., Robinson, W. H. 2022

  Abstract

  Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal fluid (CSF) of MS patients contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies.

  View details for DOI 10.1038/s41586-022-04432-7

  View details for PubMedID 35073561

• Modeling myelin: A toolkit for exploring myelin's mysteries in vitro. Developmental cell Cooper, M. H., Zuchero, J. B. 2021; 56 (9): 1215–17

  Abstract

  In the myelin field, there is a lack of reliable in vitro tools to study myelination, especially using human cells. In this issue of Developmental Cell, James et al. present a guide to generating human iPSC-derived "myelinoids"-3D models of myelination that reliably achieve mature myelin formation.

  View details for DOI 10.1016/j.devcel.2021.04.015

  View details for PubMedID 33945781

• Label-free optical detection of bioelectric potentials using electrochromic thin films. Proceedings of the National Academy of Sciences of the United States of America Alfonso, F. S., Zhou, Y., Liu, E., McGuire, A. F., Yang, Y., Kantarci, H., Li, D., Copenhaver, E., Zuchero, J. B., Muller, H., Cui, B. 2020

  Abstract

  Understanding how a network of interconnected neurons receives, stores, and processes information in the human brain is one of the outstanding scientific challenges of our time. The ability to reliably detect neuroelectric activities is essential to addressing this challenge. Optical recording using voltage-sensitive fluorescent probes has provided unprecedented flexibility for choosing regions of interest in recording neuronal activities. However, when recording at a high frame rate such as 500 to 1,000 Hz, fluorescence-based voltage sensors often suffer from photobleaching and phototoxicity, which limit the recording duration. Here, we report an approach called electrochromic optical recording (ECORE) that achieves label-free optical recording of spontaneous neuroelectrical activities. ECORE utilizes the electrochromism of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) thin films, whose optical absorption can be modulated by an applied voltage. Being based on optical reflection instead of fluorescence, ECORE offers the flexibility of an optical probe without suffering from photobleaching or phototoxicity. Using ECORE, we optically recorded spontaneous action potentials in cardiomyocytes, cultured hippocampal and dorsal root ganglion neurons, and brain slices. With minimal perturbation to cells, ECORE allows long-term optical recording over multiple days.

  View details for DOI 10.1073/pnas.2002352117

  View details for PubMedID 32632007

• How Support of Early Career Researchers Can Reset Science in the Post-COVID19 World. Cell Gibson, E. M., Bennett, F. C., Gillespie, S. M., Guler, A. D., Gutmann, D. H., Halpern, C. H., Kucenas, S. C., Kushida, C. A., Lemieux, M., Liddelow, S., Macauley, S. L., Li, Q., Quinn, M. A., Roberts, L. W., Saligrama, N., Taylor, K. R., Venkatesh, H. S., Yalcin, B., Zuchero, J. B. 2020

  Abstract

  The COVID19 crisis has magnified the issues plaguing academic science, but it has also provided the scientific establishment with an unprecedented opportunity to reset. Shoring up the foundation of academic science will require a concerted effort between funding agencies, universities, and the public to rethink how we support scientists, with a special emphasis on early career researchers.

  View details for DOI 10.1016/j.cell.2020.05.045

  View details for PubMedID 32533917

• Anchors Away: Glia-Neuron Adhesion Regulates Myelin Targeting and Growth DEVELOPMENTAL CELL Garcia, M. A., Zuchero, J. 2019; 51 (6): 659–61

  View details for DOI 10.1016/j.devcel.2019.11.018

  View details for Web of Science ID 000502848800001

• Anchors Away: Glia-Neuron Adhesion Regulates Myelin Targeting and Growth. Developmental cell Garcia, M. A., Zuchero, J. B. 2019; 51 (6): 659-661

  Abstract

  Myelination in the CNS requires oligodendrocytes to first select correct axonal targets and then extend their membranes around and along these axons. In this issue of Developmental Cell, Klingseisen et al. (2019) find that the adhesion protein Neurofascin is required in oligodendrocytes for both target selection and myelin growth.

  View details for DOI 10.1016/j.devcel.2019.11.018

  View details for PubMedID 31951538

• A Tribute to Ben Barres: Remembrances from Barres Lab Members DEVELOPMENTAL CELL Eroglu, C., Emery, B., Stevens, B., Daneman, R., Allen, N. J., Ullian, E. M., Zhang, Y., Zuchero, J., Foo, L. C., Agalliu, D., Bennett, M. 2018; 44 (4): 415–19

  View details for Web of Science ID 000426150700007

  View details for PubMedID 29486193

• Schwann cells use TAM receptor-mediated phagocytosis in addition to autophagy to clear myelin in a mouse model of nerve injury. Proceedings of the National Academy of Sciences of the United States of America Brosius Lutz, A., Chung, W. S., Sloan, S. A., Carson, G. A., Zhou, L., Lovelett, E., Posada, S., Zuchero, J. B., Barres, B. A. 2017; 114 (38): E8072-E8080

  Abstract

  Ineffective myelin debris clearance is a major factor contributing to the poor regenerative ability of the central nervous system. In stark contrast, rapid clearance of myelin debris from the injured peripheral nervous system (PNS) is one of the keys to this system's remarkable regenerative capacity, but the molecular mechanisms driving PNS myelin clearance are incompletely understood. We set out to discover new pathways of PNS myelin clearance to identify novel strategies for activating myelin clearance in the injured central nervous system, where myelin debris is not cleared efficiently. Here we show that Schwann cells, the myelinating glia of the PNS, collaborate with hematogenous macrophages to clear myelin debris using TAM (Tyro3, Axl, Mer) receptor-mediated phagocytosis as well as autophagy. In a mouse model of PNS nerve crush injury, Schwann cells up-regulate TAM phagocytic receptors Axl and Mertk following PNS injury, and Schwann cells lacking both of these phagocytic receptors exhibit significantly impaired myelin phagocytosis both in vitro and in vivo. Autophagy-deficient Schwann cells also display reductions in myelin clearance after mouse nerve crush injury, as has been recently shown following nerve transection. These findings add a mechanism, Axl/Mertk-mediated myelin clearance, to the repertoire of cellular machinery used to clear myelin in the injured PNS. Given recent evidence that astrocytes express Axl and Mertk and have previously unrecognized phagocytic potential, this pathway may be a promising avenue for activating myelin clearance after CNS injury.

  View details for DOI 10.1073/pnas.1710566114

  View details for PubMedID 28874532

  View details for PubMedCentralID PMC5617301

• DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells NATURE METHODS Harterink, M., da Silva, M. E., Will, L., Turan, J., Ibrahim, A., Lang, A. E., van Battum, E. Y., Pasterkamp, R. J., Kapitein, L. C., Kudryashov, D., Barres, B. A., Hoogenraad, C. C., Zuchero, J. B. 2017; 14 (5): 479-?

  Abstract

  The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively induce actin disassembly in eukaryotic cells. We demonstrate that DeActs are universal tools for studying the actin cytoskeleton in single cells in culture, tissues, and multicellular organisms including various neurodevelopmental model systems.

  View details for DOI 10.1038/NMETH.4257

  View details for Web of Science ID 000400253800011

  View details for PubMedID 28394337

• Glia in mammalian development and disease. Development Zuchero, J. B., Barres, B. A. 2015; 142 (22): 3805-3809

  Abstract

  Glia account for more than half of the cells in the mammalian nervous system, and the past few decades have witnessed a flood of studies that detail novel functions for glia in nervous system development, plasticity and disease. Here, and in the accompanying poster, we review the origins of glia and discuss their diverse roles during development, in the adult nervous system and in the context of disease.

  View details for DOI 10.1242/dev.129304

  View details for PubMedID 26577203

  View details for PubMedCentralID PMC4712885

• CNS Myelin Wrapping Is Driven by Actin Disassembly DEVELOPMENTAL CELL Zuchero, J. B., Fu, M., Sloan, S. A., Ibrahim, A., Olson, A., Zaremba, A., Dugas, J. C., Wienbar, S., Caprariello, A. V., Kantor, C., Leonoudakus, D., Lariosa-Willingham, K., Kronenberg, G., Gertz, K., Soderling, S. H., Miller, R. H., Barres, B. A. 2015; 34 (2): 152-167

  Abstract

  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.

  View details for DOI 10.1016/j.devcel.2015.06.011

  View details for Web of Science ID 000358599400007

  View details for PubMedCentralID PMC4519368

• Purification and culture of dorsal root ganglion neurons. Cold Spring Harbor protocols Zuchero, J. B. 2014; 2014 (8): pdb top073965-?

  Abstract

  Dorsal root ganglion neurons (DRGs) are sensory neurons that reside in ganglions on the dorsal root of the spinal cord. Here we introduce a method for the acute, prospective purification and culture of DRGs from rodents in a serum-free, defined medium, in the absence of glial cells. This immunopanning-based method facilitates the study of DRG biology and function.

  View details for DOI 10.1101/pdb.top073965

  View details for PubMedID 25086024

• Purification of dorsal root ganglion neurons from rat by immunopanning. Cold Spring Harbor protocols Zuchero, J. B. 2014; 2014 (8): pdb prot074948-?

  Abstract

  Dorsal root ganglion neurons (DRGs) are sensory neurons that facilitate somatosensation and have been used to study neurite outgrowth, regeneration, and degeneration and PNS and CNS myelination. Studies of DRGs have relied on cell isolation strategies that generally involve extended culture in the presence of antimitotic agents or other cytotoxic treatments that target dividing cells. The surviving cells typically are dependent on serum for growth. Other methods, involving purification of DRGs based on their large size, produce low yield. In contrast, the immunopanning-based method described here for prospective isolation of DRGs from rodents allows for rapid purification in the absence of antimitotic agents and serum. These DRG cultures take place in a defined medium. They are free of Schwann cells and other glia and thus can be used to study the role of glia in the biology of DRG neurons.

  View details for DOI 10.1101/pdb.prot074948

  View details for PubMedID 25086011

• Intrinsic and extrinsic control of oligodendrocyte development. Current opinion in neurobiology Zuchero, J. B., Barres, B. A. 2013; 23 (6): 914-920

  Abstract

  Oligodendrocytes (OLs) are the myelinating glia of the central nervous system. Myelin is essential for the rapid propagation of action potentials as well as for metabolic support of axons, and its loss in demyelinating diseases like multiple sclerosis has profound pathological consequences. The many steps in the development of OLs - from the specification of oligodendrocyte precursor cells (OPCs) during embryonic development to their differentiation into OLs that myelinate axons - are under tight regulation. Here we discuss recent advances in understanding how these steps of OL development are controlled intrinsically by transcription factors and chromatin remodeling and extrinsically by signaling molecules and neuronal activity. We also discuss how knowledge of these pathways is now allowing us to take steps toward generating patient-specific OPCs for disease modeling and myelin repair.

  View details for DOI 10.1016/j.conb.2013.06.005

  View details for PubMedID 23831087

• Cytoplasmic actin: purification and single molecule assembly assays. Methods in molecular biology (Clifton, N.J.) Hansen, S. D., Zuchero, J. B., Mullins, R. D. 2013; 1046: 145-170

  Abstract

  The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy.

  View details for DOI 10.1007/978-1-62703-538-5_9

  View details for PubMedID 23868587

  View details for PubMedCentralID PMC4013826

• Actin binding to WH2 domains regulates nuclear import of the multifunctional actin regulator JMY MOLECULAR BIOLOGY OF THE CELL Zuchero, J. B., Belin, B., Mullins, R. D. 2012; 23 (5): 853-863

  Abstract

  Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. In response to DNA damage, JMY accumulates in the nucleus and promotes p53-dependent apoptosis. JMY's actin-regulatory activity relies on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate filaments directly and also promote nucleation activity of the Arp2/3 complex. In addition to these activities, we find that the WH2 cluster overlaps an atypical, bipartite nuclear localization sequence (NLS) and controls JMY's subcellular localization. Actin monomers bound to the WH2 domains block binding of importins to the NLS and prevent nuclear import of JMY. Mutations that impair actin binding, or cellular perturbations that induce actin filament assembly and decrease the concentration of monomeric actin in the cytoplasm, cause JMY to accumulate in the nucleus. DNA damage induces both cytoplasmic actin polymerization and nuclear import of JMY, and we find that damage-induced nuclear localization of JMY requires both the WH2/NLS region and importin β. On the basis of our results, we propose that actin assembly regulates nuclear import of JMY in response to DNA damage.

  View details for DOI 10.1091/mbc.E11-12-0992

  View details for Web of Science ID 000300936800011

  View details for PubMedID 22262458

  View details for PubMedCentralID PMC3290644

• Between the sheets: a molecular sieve makes myelin membranes. Developmental cell Zuchero, J. B., Barres, B. A. 2011; 21 (3): 385-386

  Abstract

  Myelin is a lipid-rich, spiraled membrane structure that allows for rapid propagation of action potentials through axons. In this issue, Aggarwal et al. (2011) present evidence that myelin basic protein, essential for myelination by oligodendrocytes, regulates the biosynthesis of myelin membranes by restricting diffusion of membrane-bound proteins into compact myelin.

  View details for DOI 10.1016/j.devcel.2011.08.023

  View details for PubMedID 21920305

• Hts/Adducin Controls Synaptic Elaboration and Elimination NEURON Pielage, J., Bulat, V., Zuchero, J. B., Fetter, R. D., Davis, G. W. 2011; 69 (6): 1114-1131

  Abstract

  Neural development requires both synapse elaboration and elimination, yet relatively little is known about how these opposing activities are coordinated. Here, we provide evidence Hts/Adducin can serve this function. We show that Drosophila Hts/Adducin is enriched both pre- and postsynaptically at the NMJ. We then demonstrate that presynaptic Hts/Adducin is necessary and sufficient to control two opposing processes associated with synapse remodeling: (1) synapse stabilization as determined by light level and ultrastructural and electrophysiological assays and (2) the elaboration of actin-based, filopodia-like protrusions that drive synaptogenesis and growth. Synapse remodeling is sensitive to Hts/Adducin levels, and we provide evidence that the synaptic localization of Hts/Adducin is controlled via phosphorylation. Mechanistically, Drosophila Hts/Adducin protein has actin-capping activity. We propose that phosphorylation-dependent regulation of Hts/Adducin controls the level, localization, and activity of Hts/Adducin, influencing actin-based synapse elaboration and spectrin-based synapse stabilization. Hts/Adducin may define a mechanism to switch between synapse stability and dynamics.

  View details for DOI 10.1016/j.neuron.2011.02.007

  View details for Web of Science ID 000288886900009

  View details for PubMedID 21435557

  View details for PubMedCentralID PMC3073818

• p53-cofactor JMY is a multifunctional actin nucleation factor NATURE CELL BIOLOGY Zuchero, J. B., Coutts, A. S., Quinlan, M. E., La Thangue, N. B., Mullins, R. D. 2009; 11 (4): 451-U198

  Abstract

  Many cellular structures are assembled from networks of actin filaments, and the architecture of these networks depends on the mechanism by which the filaments are formed. Several classes of proteins are known to assemble new filaments, including the Arp2/3 complex, which creates branched filament networks, and Spire, which creates unbranched filaments. We find that JMY, a vertebrate protein first identified as a transcriptional co-activator of p53, combines these two nucleating activities by both activating Arp2/3 and assembling filaments directly using a Spire-like mechanism. Increased levels of JMY expression enhance motility, whereas loss of JMY slows cell migration. When slowly migrating HL-60 cells are differentiated into highly motile neutrophil-like cells, JMY moves from the nucleus to the cytoplasm and is concentrated at the leading edge. Thus, JMY represents a new class of multifunctional actin assembly factor whose activity is regulated, at least in part, by sequestration in the nucleus.

  View details for DOI 10.1038/ncb1852

  View details for Web of Science ID 000265264900015

  View details for PubMedID 19287377

  View details for PubMedCentralID PMC2763628

• In vitro actin assembly assays and purification from Acanthamoeba. Methods in molecular biology (Clifton, N.J.) Zuchero, J. B. 2007; 370: 213-226

  Abstract

  The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility and endocytosis. Actin polymerization is tightly regulated by various cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. The pyrene actin assembly assay is a powerful tool for determining the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleators or capping factors. In addition, fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how these proteins influence actin filament superstructure.

  View details for PubMedID 17416997