Publications by authors named "Riki Kawaguchi"

49 Publications

Selective axonal translation of prenylated Cdc42 mRNA isoform supports axon growth.

J Cell Sci 2021 Mar 5. Epub 2021 Mar 5.

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA

The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic gene is alternatively spliced to generate mRNAs with two different 3'UTRs that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CAAX and CCAX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases during PNS axon regeneration. Functional studies indicate that increases axon length and this requires axonal targeting of the mRNA and an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, the has no effect on axon growth but it selectively increased dendrite length. Together, these data show that alternative splicing of the gene product generates an axon growth promoting locally synthesized prenyl-Cdc42 protein.
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http://dx.doi.org/10.1242/jcs.251967DOI Listing
March 2021

miR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy.

Hum Mol Genet 2021 Mar;30(1):103-118

Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.
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http://dx.doi.org/10.1093/hmg/ddaa252DOI Listing
March 2021

Analysis of the immune response to sciatic nerve injury identifies efferocytosis as a key mechanism of nerve debridement.

Elife 2020 12 2;9. Epub 2020 Dec 2.

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, United States.

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C monocytes infiltrate the nerve first and rapidly give way to Ly6C inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient () mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.
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http://dx.doi.org/10.7554/eLife.60223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735761PMC
December 2020

The effect of Rbfox2 modulation on retinal transcriptome and visual function.

Sci Rep 2020 11 12;10(1):19683. Epub 2020 Nov 12.

Stein Eye Institute, University of California, Los Angeles, 100 Stein Plaza, Los Angeles, CA, 90095, USA.

Rbfox proteins regulate alternative splicing, mRNA stability and translation. These proteins are involved in neurogenesis and have been associated with various neurological conditions. Here, we analyzed Rbfox2 expression in adult and developing mouse retinas and the effect of its downregulation on visual function and retinal transcriptome. In adult rodents, Rbfox2 is expressed in all retinal ganglion cell (RGC) subtypes, horizontal cells, as well as GABAergic amacrine cells (ACs). Among GABAergic AC subtypes, Rbfox2 was colocalized with cholinergic starburst ACs, NPY (neuropeptide Y)- and EBF1 (early B-cell factor 1)-positive ACs. In differentiating retinal cells, Rbfox2 expression was observed as early as E12 and, unlike Rbfox1, which changes its subcellular localization from cytoplasmic to predominantly nuclear at around P0, Rbfox2 remains nuclear throughout retinal development. Rbfox2 knockout in adult animals had no detectable effect on retinal gross morphology. However, the visual cliff test revealed a significant abnormality in the depth perception of Rbfox2-deficient animals. Gene set enrichment analysis identified genes regulating the RNA metabolic process as a top enriched class of genes in Rbfox2-deficient retinas. Pathway analysis of the top 100 differentially expressed genes has identified Rbfox2-regulated genes associated with circadian rhythm and entrainment, glutamatergic/cholinergic/dopaminergic synaptic function, calcium and PI3K-AKT signaling.
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http://dx.doi.org/10.1038/s41598-020-76879-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665016PMC
November 2020

A Ca-Dependent Switch Activates Axonal Casein Kinase 2α Translation and Drives G3BP1 Granule Disassembly for Axon Regeneration.

Curr Biol 2020 Dec 15;30(24):4882-4895.e6. Epub 2020 Oct 15.

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA. Electronic address:

The main limitation on axon regeneration in the peripheral nervous system (PNS) is the slow rate of regrowth. We recently reported that nerve regeneration can be accelerated by axonal G3BP1 granule disassembly, releasing axonal mRNAs for local translation to support axon growth. Here, we show that G3BP1 phosphorylation by casein kinase 2α (CK2α) triggers G3BP1 granule disassembly in injured axons. CK2α activity is temporally and spatially regulated by local translation of Csnk2a1 mRNA in axons after injury, but this requires local translation of mTor mRNA and buffering of the elevated axonal Ca that occurs after axotomy. CK2α's appearance in axons after PNS nerve injury correlates with disassembly of axonal G3BP1 granules as well as increased phospho-G3BP1 and axon growth, although depletion of Csnk2a1 mRNA from PNS axons decreases regeneration and increases G3BP1 granules. Phosphomimetic G3BP1 shows remarkably decreased RNA binding in dorsal root ganglion (DRG) neurons compared with wild-type and non-phosphorylatable G3BP1; combined with other studies, this suggests that CK2α-dependent G3BP1 phosphorylation on Ser 149 after axotomy releases axonal mRNAs for translation. Translation of axonal mRNAs encoding some injury-associated proteins is known to be increased with Ca elevations, and using a dual fluorescence recovery after photobleaching (FRAP) reporter assay for axonal translation, we see that translational specificity switches from injury-associated protein mRNA translation to CK2α translation with endoplasmic reticulum (ER) Ca release versus cytoplasmic Ca chelation. Our results point to axoplasmic Ca concentrations as a determinant for the temporal specificity of sequential translational activation of different axonal mRNAs as severed axons transition from injury to regenerative growth.
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http://dx.doi.org/10.1016/j.cub.2020.09.043DOI Listing
December 2020

Microglia-organized scar-free spinal cord repair in neonatal mice.

Nature 2020 11 7;587(7835):613-618. Epub 2020 Oct 7.

F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA.

Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal-but not adult-microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system.
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http://dx.doi.org/10.1038/s41586-020-2795-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704837PMC
November 2020

Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury.

Neuron 2020 10 17;108(1):128-144.e9. Epub 2020 Aug 17.

Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115, USA; F.M. Kirby Neurobiology Center, Boston Children's Hospital, 3 Blackfan Cir., Boston, MA 02115, USA. Electronic address:

Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.
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http://dx.doi.org/10.1016/j.neuron.2020.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590250PMC
October 2020

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Mol Cell 2020 08 26;79(3):521-534.e15. Epub 2020 Jun 26.

Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Computer and Data Sciences, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address:

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
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http://dx.doi.org/10.1016/j.molcel.2020.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415676PMC
August 2020

Injured adult neurons regress to an embryonic transcriptional growth state.

Nature 2020 05 15;581(7806):77-82. Epub 2020 Apr 15.

Department of Neurosciences, University of California San Diego, La Jolla, CA, USA.

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.
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http://dx.doi.org/10.1038/s41586-020-2200-5DOI Listing
May 2020

Regulatory mechanism for the transmembrane receptor that mediates bidirectional vitamin A transport.

Proc Natl Acad Sci U S A 2020 05 16;117(18):9857-9864. Epub 2020 Apr 16.

Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095;

Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its transport is regulated is poorly understood. STRA6 is a multitransmembrane domain cell-surface receptor and mediates vitamin A uptake from plasma retinol binding protein (RBP). STRA6 can mediate both cellular vitamin A influx and efflux, but what regulates these opposing activities is unknown. To answer this question, we purified and identified STRA6-associated proteins in a native mammalian cell type that takes up vitamin A through STRA6 using mass spectrometry. We found that the major protein repeatedly identified as STRA6-associated protein is calmodulin, consistent with the cryogenic electron microscopy (cryo-EM) study of zebrafish STRA6 associated with calmodulin. Using radioactivity-based, high-performance liquid chromatography (HPLC)-based and real-time fluorescence techniques, we found that calmodulin profoundly affects STRA6's vitamin A transport activity. Increased calcium/calmodulin promotes cellular vitamin A efflux and suppresses vitamin A influx through STRA6. Further mechanistic studies revealed that calmodulin enhances the binding of apo-RBP to STRA6, and this enhancement is much more pronounced for apo-RBP than holo-RBP. This study revealed that calmodulin regulates STRA6's vitamin A influx or efflux activity by modulating its preferential interaction with apo-RBP or holo-RBP. This molecular mechanism of regulating vitamin A transport may point to new directions to treat human diseases associated with insufficient or excessive vitamin A uptake.
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http://dx.doi.org/10.1073/pnas.1918540117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211970PMC
May 2020

Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map.

Nat Neurosci 2020 04 16;23(4):500-509. Epub 2020 Mar 16.

Department of Paediatrics, Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.
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http://dx.doi.org/10.1038/s41593-020-0602-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116562PMC
April 2020

DYNLRB1 is essential for dynein mediated transport and neuronal survival.

Neurobiol Dis 2020 07 20;140:104816. Epub 2020 Feb 20.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.

The cytoplasmic dynein motor complex transports essential signals and organelles from the cell periphery to the perinuclear region, hence is critical for the survival and function of highly polarized cells such as neurons. Dynein Light Chain Roadblock-Type 1 (DYNLRB1) is thought to be an accessory subunit required for specific cargos, but here we show that it is essential for general dynein-mediated transport and sensory neuron survival. Homozygous Dynlrb1 null mice are not viable and die during early embryonic development. Furthermore, heterozygous or adult knockdown animals display reduced neuronal growth, and selective depletion of Dynlrb1 in proprioceptive neurons compromises their survival. Conditional depletion of Dynlrb1 in sensory neurons causes deficits in several signaling pathways, including β-catenin subcellular localization, and a severe impairment in the axonal transport of both lysosomes and retrograde signaling endosomes. Hence, DYNLRB1 is an essential component of the dynein complex, and given dynein's critical functions in neuronal physiology, DYNLRB1 could have a prominent role in the etiology of human neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.nbd.2020.104816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273200PMC
July 2020

White Matter Stroke Induces a Unique Oligo-Astrocyte Niche That Inhibits Recovery.

J Neurosci 2019 11 7;39(47):9343-9359. Epub 2019 Oct 7.

Department of Neurology, David Geffen School of Medicine at UCLA Health Sciences, Los Angeles, California 90095, and

Subcortical white matter stroke is a common stroke subtype. White matter stroke stimulates adjacent oligodendrocyte progenitor cells (OPCs) to divide and migrate to the lesion, but stroke OPCs have only a limited differentiation into mature oligodendrocytes. To understand the molecular systems that are active in OPC responses in white matter stroke, OPCs were virally labeled and laser-captured in the region of partial damage adjacent to the infarct in male mice. RNAseq indicates two distinct OPC transcriptomes associated with the proliferative and limited-regeneration phases of OPCs after stroke. Molecular pathways related to nuclear receptor activation, ECM turnover, and lipid biosynthesis are activated during proliferative OPC phases after stroke; inflammatory and growth factor signaling is activated in the later stage of limited OPC differentiation. Within ECM proteins, Matrilin-2 is induced early after stroke and then rapidly downregulated. Prediction of upstream regulators of the OPC stroke transcriptome identifies several candidate molecules, including Inhibin A-a negative regulator of Matrilin-2. Inhibin A is induced in reactive astrocytes after stroke, including in humans. In functional assays, Matrilin-2 induces OPC differentiation, and Inhibin A inhibits OPC Matrilin-2 expression and inhibits OPC differentiation. , Matrilin-2 promotes motor recovery after white matter stroke, and promotes OPC differentiation and ultrastructural evidence of remyelination. These studies show that white matter stroke induces an initial proliferative and reparative response in OPCs, but this is blocked by a local cellular niche where reactive astrocytes secrete Inhibin A, downregulating Matrilin-2 and blocking myelin repair and recovery. Stroke in the cerebral white matter of the brain is common. The biology of damage and recovery in this stroke subtype are not well defined. These studies use cell-specific RNA sequencing and gain-of-function studies to show that white matter stroke induces a glial signaling niche, present in both humans and mice, between reactive astrocytes and oligodendrocyte progenitor cells. Astrocyte secretion of Inhibin A and downregulation of oligodendrocyte precursor production of Matrilin-2 limit OPC differentiation, tissue repair, and recovery in this disease.
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http://dx.doi.org/10.1523/JNEUROSCI.0103-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6867825PMC
November 2019

Longitudinal RNA-Seq analysis of acute and chronic neurogenic skeletal muscle atrophy.

Sci Data 2019 09 24;6(1):179. Epub 2019 Sep 24.

Department of Neurology, Neuromuscular Division, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.

Skeletal muscle is a highly adaptable tissue capable of changes in size, contractility, and metabolism according to functional demands. Atrophy is a decline in mass and strength caused by pathologic loss of myofibrillar proteins, and can result from disuse, aging, or denervation caused by injury or peripheral nerve disorders. We provide a high-quality longitudinal RNA-Seq dataset of skeletal muscle from a cohort of adult C57BL/6J male mice subjected to tibial nerve denervation for 0 (baseline), 1, 3, 7, 14, 30, or 90 days. Using an unbiased genomics approach to identify gene expression changes across the entire longitudinal course of muscle atrophy affords the opportunity to (1) establish acute responses to denervation, (2) detect pathways that mediate rapid loss of muscle mass within the first week after denervation, and (3) capture the molecular phenotype of chronically atrophied muscle at a stage when it is largely resistant to recovery.
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http://dx.doi.org/10.1038/s41597-019-0185-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760191PMC
September 2019

Regeneration Enhances Metastasis: A Novel Role for Neurovascular Signaling in Promoting Melanoma Brain Metastasis.

Front Neurosci 2019 9;13:297. Epub 2019 Apr 9.

Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States.

Neural repair after stroke involves initiation of a cellular proliferative program in the form of angiogenesis, neurogenesis, and molecular growth signals in the surrounding tissue elements. This cellular environment constitutes a niche in which regeneration of new blood vessels and new neurons leads to partial tissue repair after stroke. Cancer metastasis has similar proliferative cellular events in the brain and other organs. Do cancer and CNS tissue repair share similar cellular processes? In this study, we identify a novel role of the regenerative neurovascular niche induced by stroke in promoting brain melanoma metastasis through enhancing cellular interactions with surrounding niche components. Repair-mediated neurovascular signaling induces metastatic cells to express genes crucial to metastasis. Mimicking stroke-like conditions displays an enhancement of metastatic migration potential and allows for the determination of cell-specific signals produced by the regenerative neurovascular niche. Comparative analysis of both and expression profiles reveals a major contribution of endothelial cells in mediating melanoma metastasis. These results point to a previously undiscovered role of the regenerative neurovascular niche in shaping the tumor microenvironment and brain metastatic landscape.
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http://dx.doi.org/10.3389/fnins.2019.00297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465799PMC
April 2019

Required growth facilitators propel axon regeneration across complete spinal cord injury.

Nature 2018 09 29;561(7723):396-400. Epub 2018 Aug 29.

Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury, but efficient reversal of this regrowth failure remains elusive. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity, (ii) growth-supportive substrate and (iii) chemoattraction-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor delivered via spatially and temporally controlled release from biomaterial depots, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.
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http://dx.doi.org/10.1038/s41586-018-0467-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151128PMC
September 2018

hnRNPs Interacting with mRNA Localization Motifs Define Axonal RNA Regulons.

Mol Cell Proteomics 2018 11 23;17(11):2091-2106. Epub 2018 Jul 23.

From the ‡Department of Biological Sciences, University of South Carolina, Columbia, SC, 29208;

mRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNA-protein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. Here we used proteomics to identify RBPs interacting with the axonal localizing motifs of , , , and mRNAs, revealing many novel RBPs in axons. Interestingly, no RBP is shared between all four RNA motifs, suggesting graded and overlapping specificities of RBP-mRNA pairings. A systematic assessment of axonal mRNAs interacting with hnRNP H1, hnRNP F, and hnRNP K, proteins that bound with high specificity to and , revealed that axonal mRNAs segregate into axon growth-associated RNA regulons based on hnRNP interactions. Axotomy increases axonal transport of hnRNPs H1, F, and K, depletion of these hnRNPs decreases axon growth and reduces axonal mRNA levels and axonal protein synthesis. Thus, subcellular hnRNP-interacting RNA regulons support neuronal growth and regeneration.
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http://dx.doi.org/10.1074/mcp.RA118.000603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210225PMC
November 2018

Adult rat myelin enhances axonal outgrowth from neural stem cells.

Sci Transl Med 2018 05;10(442)

Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA.

Axon regeneration after spinal cord injury (SCI) is attenuated by growth inhibitory molecules associated with myelin. We report that rat myelin stimulated the growth of axons emerging from rat neural progenitor cells (NPCs) transplanted into sites of SCI in adult rat recipients. When plated on a myelin substrate, neurite outgrowth from rat NPCs and from human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) was enhanced threefold. In vivo, rat NPCs and human iPSC-derived NSCs extended greater numbers of axons through adult central nervous system white matter than through gray matter and preferentially associated with rat host myelin. Mechanistic investigations excluded Nogo receptor signaling as a mediator of stem cell-derived axon growth in response to myelin. Transcriptomic screens of rodent NPCs identified the cell adhesion molecule neuronal growth regulator 1 (Negr1) as one mediator of permissive axon-myelin interactions. The stimulatory effect of myelin-associated proteins on rodent NPCs was developmentally regulated and involved direct activation of the extracellular signal-regulated kinase (ERK). The stimulatory effects of myelin on NPC/NSC axon outgrowth should be investigated further and could potentially be exploited for neural repair after SCI.
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http://dx.doi.org/10.1126/scitranslmed.aal2563DOI Listing
May 2018

Translatome Regulation in Neuronal Injury and Axon Regrowth.

eNeuro 2018 Mar-Apr;5(2). Epub 2018 May 10.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.

Transcriptional events leading to outgrowth of neuronal axons have been intensively studied, but the role of translational regulation in this process is not well understood. Here, we use translatome analyses by ribosome pull-down and protein synthesis characterization by metabolic isotopic labeling to study nerve injury and axon outgrowth proteomes in rodent dorsal root ganglia (DRGs) and sensory neurons. We identify over 1600 gene products that are primarily translationally regulated in DRG neurons after nerve injury, many of which contain a 5'UTR cytosine-enriched regulator of translation (CERT) motif, implicating the translation initiation factor Eif4e in the injury response. We further identified approximately 200 proteins that undergo robust de novo synthesis in the initial stages of axon growth. ApoE is one of the highly synthesized proteins in neurons, and its receptor binding inhibition or knockout affects axon outgrowth. These findings provide a resource for future analyses of the role of translational regulation in neuronal injury responses and axon extension.
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http://dx.doi.org/10.1523/ENEURO.0276-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944006PMC
January 2019

Activity-Dependent Regulation of Alternative Cleavage and Polyadenylation During Hippocampal Long-Term Potentiation.

Sci Rep 2017 12 12;7(1):17377. Epub 2017 Dec 12.

Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.

Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3' untranslated regions (3'UTRs) and/or coding sequences. Changes in the 3'UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3' end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3'UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3'UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.
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http://dx.doi.org/10.1038/s41598-017-17407-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727029PMC
December 2017

Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others.

Neuron 2017 Jun;94(6):1112-1120.e4

F.M. Kirby Neurobiology Center, Children's Hospital, and Department of Neurology, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA. Electronic address:

At least 30 types of retinal ganglion cells (RGCs) send distinct messages through the optic nerve to the brain. Available strategies of promoting axon regeneration act on only some of these types. Here we tested the hypothesis that overexpressing developmentally important transcription factors in adult RGCs could reprogram them to a "youthful" growth-competent state and promote regeneration of other types. From a screen of transcription factors, we identified Sox11 as one that could induce substantial axon regeneration. Transcriptome profiling indicated that Sox11 activates genes involved in cytoskeletal remodeling and axon growth. Remarkably, α-RGCs, which preferentially regenerate following treatments such as Pten deletion, were killed by Sox11 overexpression. Thus, Sox11 promotes regeneration of non-α-RGCs, which are refractory to Pten deletion-induced regeneration. We conclude that Sox11 can reprogram adult RGCs to a growth-competent state, suggesting that different growth-promoting interventions promote regeneration in distinct neuronal types.
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http://dx.doi.org/10.1016/j.neuron.2017.05.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519288PMC
June 2017

Mapping Gene Expression in Excitatory Neurons during Hippocampal Late-Phase Long-Term Potentiation.

Front Mol Neurosci 2017 22;10:39. Epub 2017 Feb 22.

Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, University of California, Los AngelesLos Angeles, CA, USA; Department of Biological Chemistry, University of California, Los AngelesLos Angeles, CA, USA.

The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation.
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http://dx.doi.org/10.3389/fnmol.2017.00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319997PMC
February 2017

Identification of an Efficient Gene Expression Panel for Glioblastoma Classification.

PLoS One 2016 17;11(11):e0164649. Epub 2016 Nov 17.

Semel Institute for Neuroscience & Human Behavior and Department of Psychiatry and Biobehavioral Sciences, University of California Los Angeles, Los Angeles, California 90095, United States of America.

We present here a novel genetic algorithm-based random forest (GARF) modeling technique that enables a reduction in the complexity of large gene disease signatures to highly accurate, greatly simplified gene panels. When applied to 803 glioblastoma multiforme samples, this method allowed the 840-gene Verhaak et al. gene panel (the standard in the field) to be reduced to a 48-gene classifier, while retaining 90.91% classification accuracy, and outperforming the best available alternative methods. Additionally, using this approach we produced a 32-gene panel which allows for better consistency between RNA-seq and microarray-based classifications, improving cross-platform classification retention from 69.67% to 86.07%. A webpage producing these classifications is available at http://simplegbm.semel.ucla.edu.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113897PMC
June 2017

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

Mol Neurobiol 2017 08 8;54(6):4584-4596. Epub 2016 Jul 8.

Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, 635 Charles E. Young Dr. South, Rm 415, Los Angeles, CA, 90095, USA.

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.
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http://dx.doi.org/10.1007/s12035-016-9974-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5219956PMC
August 2017

Astrocyte scar formation aids central nervous system axon regeneration.

Nature 2016 Apr 30;532(7598):195-200. Epub 2016 Mar 30.

Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1763, USA.

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.
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http://dx.doi.org/10.1038/nature17623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5243141PMC
April 2016

GDF10 is a signal for axonal sprouting and functional recovery after stroke.

Nat Neurosci 2015 Dec 26;18(12):1737-45. Epub 2015 Oct 26.

Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.

Stroke produces a limited process of neural repair. Axonal sprouting in cortex adjacent to the infarct is part of this recovery process, but the signal that initiates axonal sprouting is not known. Growth and differentiation factor 10 (GDF10) is induced in peri-infarct neurons in mice, non-human primates and humans. GDF10 promotes axonal outgrowth in vitro in mouse, rat and human neurons through TGFβRI and TGFβRII signaling. Using pharmacogenetic gain- and loss-of-function studies, we found that GDF10 produced axonal sprouting and enhanced functional recovery after stroke; knocking down GDF10 blocked axonal sprouting and reduced recovery. RNA sequencing from peri-infarct cortical neurons revealed that GDF10 downregulated PTEN, upregulated PI3 kinase signaling and induced specific axonal guidance molecules. Using unsupervised genome-wide association analysis of the GDF10 transcriptome, we found that it was not related to neurodevelopment, but may partially overlap with other CNS injury patterns. Thus, GDF10 is a stroke-induced signal for axonal sprouting and functional recovery.
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http://dx.doi.org/10.1038/nn.4146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790086PMC
December 2015

Vitamin A Transport Mechanism of the Multitransmembrane Cell-Surface Receptor STRA6.

Membranes (Basel) 2015 Aug 28;5(3):425-53. Epub 2015 Aug 28.

Department of Physiology, Jules Stein Eye Institute, and Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.

Vitamin A has biological functions as diverse as sensing light for vision, regulating stem cell differentiation, maintaining epithelial integrity, promoting immune competency, regulating learning and memory, and acting as a key developmental morphogen. Vitamin A derivatives have also been used in treating human diseases. If vitamin A is considered a drug that everyone needs to take to survive, evolution has come up with a natural drug delivery system that combines sustained release with precise and controlled delivery to the cells or tissues that depend on it. This "drug delivery system" is mediated by plasma retinol binding protein (RBP), the principle and specific vitamin A carrier protein in the blood, and STRA6, the cell-surface receptor for RBP that mediates cellular vitamin A uptake. The mechanism by which the RBP receptor absorbs vitamin A from the blood is distinct from other known cellular uptake mechanisms. This review summarizes recent progress in elucidating the fundamental molecular mechanism mediated by the RBP receptor and multiple newly discovered catalytic activities of this receptor, and compares this transport system with retinoid transport independent of RBP/STRA6. How to target this new type of transmembrane receptor using small molecules in treating diseases is also discussed.
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http://dx.doi.org/10.3390/membranes5030425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4584289PMC
August 2015

Identification of PLXDC1 and PLXDC2 as the transmembrane receptors for the multifunctional factor PEDF.

Elife 2014 Dec 23;3:e05401. Epub 2014 Dec 23.

Department of Physiology, Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States.

Pigment Epithelium Derived Factor (PEDF) is a secreted factor that has broad biological activities. It was first identified as a neurotrophic factor and later as the most potent natural antiangiogenic factor, a stem cell niche factor, and an inhibitor of cancer cell growth. Numerous animal models demonstrated its therapeutic value in treating blinding diseases and diverse cancer types. A long-standing challenge is to reveal how PEDF acts on its target cells and the identities of the cell-surface receptors responsible for its activities. Here we report the identification of transmembrane proteins PLXDC1 and PLXDC2 as cell-surface receptors for PEDF. Using distinct cellular models, we demonstrate their cell type-specific receptor activities through loss of function and gain of function studies. Our experiments suggest that PEDF receptors form homooligomers under basal conditions, and PEDF dissociates the homooligomer to activate the receptors. Mutations in the intracellular domain can have profound effects on receptor activities.
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http://dx.doi.org/10.7554/eLife.05401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303762PMC
December 2014

How free retinol behaves differently from rbp-bound retinol in RBP receptor-mediated vitamin A uptake.

Mol Cell Biol 2014 Jun;34(11):2108-10

Department of Physiology, Jules Stein Eye Institute, and Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

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http://dx.doi.org/10.1128/MCB.01426-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019051PMC
June 2014

Apo-RBP, holo-RBP, and insulin resistance.

Mol Cell Biol 2014 Jun;34(11):2105-6

Department of Physiology, Jules Stein Eye Institute, and Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

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http://dx.doi.org/10.1128/MCB.01425-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019056PMC
June 2014