N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

AbstractRecent studies have shown that DNA N6-methyladenine (N6-mA) modification is emerging to be a novel and important epigenetic regulator of mammalian gene transcription. Several studies demonstrated DNA N6-mA in human or rodents was regulated by methyltransferase N6AMT1 and demethylase ALKBH1. Moreover, studies in mouse brain or human glioblastoma cells showed that reduced level of N6-mA or higher level of ALKBH1 was correlated with up regulated levels of genes associated with neuronal development. We thus investigated the functional roles of ALKBH1 in sensory axon regeneration. Our results showed that ALKBH1 regulated the level of N6-mA in sensory neurons, and upon peripheral nerve injury ALKBH1 was up regulated in mouse sensory neurons. Functionally, knocking down ALKBH1 in sensory neurons resulted in reduced axon regeneration in vitro and in vivo, which could be rescued by simultaneously knocking down N6AMT1. Moreover, knocking down ALKBH1 led to decreased levels of many neurodevelopment regulatory genes, including neuritin that is well known to enhance axon growth and regeneration. Our study not only revealed a novel physiological function of DNA N6-mA, but also identified a new epigenetic mechanism regulating mammalian axon regeneration.Significance StatementThe study demonstrated that DNA N6-methyladenine (N6-mA) modification played important roles in regulation of sensory axon regeneration, likely through controlling the expression of neurodevelopmental associated genes. The results will add new evidence about the physiological function of DNA N6-mA and its regulatory demethylase ALKBH1 in neurons.

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Depolarization and electrical stimulation enhance in vitro and in vivo sensory axon growth after spinal cord injury

Experimental Neurology ◽

10.1016/j.expneurol.2017.11.011 ◽

2018 ◽

Vol 300 ◽

pp. 247-258 ◽

Cited By ~ 12

Author(s):

Ioana Goganau ◽

Beatrice Sandner ◽

Norbert Weidner ◽

Karim Fouad ◽

Armin Blesch

Keyword(s):

Spinal Cord ◽

Spinal Cord Injury ◽

Electrical Stimulation ◽

Axon Growth ◽

Sensory Axon ◽

Cord Injury

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Injury-free priming: induction of Primary Afferent Collateral Sprouting in uninjured sensory neurons in vivo primes them for enhanced axon outgrowth in vitro

10.1101/463935 ◽

2018 ◽

Author(s):

Sara Soleman ◽

Jeffrey C. Petruska ◽

Lawrence D.F. Moon

Keyword(s):

Axon Regeneration ◽

Axonal Injury ◽

Axon Growth ◽

Drg Neurons ◽

Primary Afferent ◽

Collateral Sprouting ◽

Axon Elongation ◽

Defined Media

AbstractPrior “conditioning” nerve lesions can prime DRG neurons for enhanced axon regeneration. Here, we tested the hypothesis that adult DRG neurons can be primed for axon elongation in vitro without axonal injury by prior induction of Primary Afferent Collateral Sprouting (PACS) in vivo. Thoracic cutaneous nerves (T9, T10, T12, T13 but not T11) were transected to create zones of denervated skin. Neurons from the uninjured T11 DRG underwent PACS within the skin, as demonstrated by the expansion of its zones responsive to pinch up to 14 days. At 7 or 14 days after induction of collateral sprouting, DRG neurons were dissociated and cultured for 18 hours in defined media lacking neurotrophins and growth factors. Neurons from the uninjured T11 DRG had longer mean neurite lengths than neurons from naïve DRG. A larger proportion of neurons from the uninjured T11 DRG showed an elongating or arborizing phenotype than neurons from naïve DRG. Transcriptomic analysis of the uninjured T11 DRG and denervated/reinnervated skin reveal regulation of receptor/ligand systems and regulators of growth during collateral sprouting. For example, the glial cell-derived neurotrophic family ligands Artemin and Persephin were upregulated in denervated skin after 7 and/or 14 days. We suggest that extracellular cues in denervated skin modify the intrinsic growth program of uninjured DRG neurons that enhances their ability to elongate or arborize even after explantation. Collectively, these data confirm that induction of collateral sprouting does not induce an injury response yet primes many of these uninjured neurons for in vitro axon growth.

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Inhibition of PTEN activity promotes IB4-positive sensory neuronal axon growth

10.1101/2020.04.02.019042 ◽

2020 ◽

Author(s):

Li-Yu Zhou ◽

Feng Han ◽

Shi-Bin Qi ◽

Jin-Jin Ma ◽

Yan-Xia Ma ◽

...

Keyword(s):

Nervous System ◽

Sensory Neurons ◽

Strong Evidence ◽

Axon Regeneration ◽

Axon Growth ◽

Clinical Problem ◽

Sensory Axon ◽

Nerve Injuries ◽

Critical Process ◽

Common Clinical Problem

AbstractTraumatic nerve injuries have become a common clinical problem, and axon regeneration is a critical process in the successful functional recovery of the injured nervous system. In this study, we found that peripheral axotomy reduce total PTEN expression in adult sensory neurons, however, it did not alter the expression level of PTEN in IB4-positive sensory neurons. Additionally, our results indicate that the artificial inhibition of PTEN markedly promotes adult sensory axon regeneration, including IB4-positive neuronal axon growth. Thus, our results provide strong evidence that PTEN is a prominent repressor of adult sensory axon regeneration, especially in IB4-positive neurons.

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MicroRNA-210 promotes sensory axon regeneration of adult mice in vivo and in vitro

Neuroscience Letters ◽

10.1016/j.neulet.2016.04.034 ◽

2016 ◽

Vol 622 ◽

pp. 61-66 ◽

Cited By ~ 22

Author(s):

Yi-Wen Hu ◽

Jing-Jing Jiang ◽

Yan-Gao ◽

Rui-Ying Wang ◽

Guan-Jun Tu

Keyword(s):

Axon Regeneration ◽

Sensory Axon ◽

Adult Mice

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Chronic neuronal activation increases dynamic microtubules to enhance functional axon regeneration after dorsal root crush injury

Nature Communications ◽

10.1038/s41467-020-19914-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Di Wu ◽

Ying Jin ◽

Tatiana M. Shapiro ◽

Abhishek Hinduja ◽

Peter W. Baas ◽

...

Keyword(s):

Dorsal Root ◽

Axon Regeneration ◽

Axon Growth ◽

Crush Injury ◽

Neuronal Activation ◽

Drg Neurons ◽

Root Entry Zone ◽

The Status

AbstractAfter a dorsal root crush injury, centrally-projecting sensory axons fail to regenerate across the dorsal root entry zone (DREZ) to extend into the spinal cord. We find that chemogenetic activation of adult dorsal root ganglion (DRG) neurons improves axon growth on an in vitro model of the inhibitory environment after injury. Moreover, repeated bouts of daily chemogenetic activation of adult DRG neurons for 12 weeks post-crush in vivo enhances axon regeneration across a chondroitinase-digested DREZ into spinal gray matter, where the regenerating axons form functional synapses and mediate behavioral recovery in a sensorimotor task. Neuronal activation-mediated axon extension is dependent upon changes in the status of tubulin post-translational modifications indicative of highly dynamic microtubules (as opposed to stable microtubules) within the distal axon, illuminating a novel mechanism underlying stimulation-mediated axon growth. We have identified an effective combinatory strategy to promote functionally-relevant axon regeneration of adult neurons into the CNS after injury.

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Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

10.1101/2021.05.24.445420 ◽

2021 ◽

Author(s):

Zubair Ahmed ◽

Sharif Alhajlah ◽

Adam Thompson

Keyword(s):

Spinal Cord ◽

Optic Nerve ◽

Neurite Outgrowth ◽

Functional Recovery ◽

Axon Regeneration ◽

Axon Growth ◽

Dorsal Root Ganglion Neurons ◽

Ganglion Neurons

CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-66. Here, we focused on RTN3 as its contribution in CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury.

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Receptive Properties of Embryonic Chick Sensory Neurons Innervating Skin

Journal of Neurophysiology ◽

10.1152/jn.1997.78.5.2560 ◽

1997 ◽

Vol 78 (5) ◽

pp. 2560-2568 ◽

Cited By ~ 24

Author(s):

Martin Koltzenburg ◽

Gary R. Lewin

Keyword(s):

Sensory Neurons ◽

Axon Growth ◽

Embryonic Chick ◽

Sensory Afferents ◽

Noxious Heat ◽

C Fibers ◽

Afferent Fibers ◽

Mechanical Thresholds

Koltzenburg, Martin and Gary R. Lewin. Receptive properties of embryonic chick sensory neurons innervating skin. J. Neurophysiol. 78: 2560–2568, 1997. We describe a new in vitro skin-nerve preparation from chick embryos that allows detailed study of the functional properties of developing sensory neurons innervating skin. Functionally single sensory afferents were isolated by recording from their axons in microdissected filaments of the cutaneous femoralis medialis nerve, which innervates skin of the thigh. A total of 157 single neurons were characterized from embryos [embryonic days 17–21 ( E17–E21), n = 115] and hatchlings up to 3 wk old ( n = 42). Neurons were initially classified on the basis of their conduction velocity; those conducting below 1.0 m/s were being classified as C fibers and faster conducting fibers as A fibers. The proportions of A and C fibers encountered in embryonic and hatchling preparations were not very different, indicating that myelination and axon growth proceeds quite slowly over the period studied. Afferent fibers that could subserve nociceptive and nonnociceptive functions were identified in the time period studied. Subpopulations of low-threshold myelinated afferent units exhibited rapidly or slowly adapting discharges to constant force stimuli and could have tactile functions. Many afferent fibers responded to noxious heat and were excited and sensitized by exposure to inflammatory mediators, suggesting that they are nociceptors. The behavior of these units changed in several respects over the period studied. The discharge of C fibers to noxious heat increased with age as did their mechanical thresholds. A substantial population of heat-responsive neurons (34% of the A fibers) present in embryos were not encountered in hatchling chicks. This indicates that substantial changes in the physiological response properties of sensory afferents occur after hatching. We conclude that this new preparation can be used for quantitative assessment of the receptive properties of developing sensory neurons and has considerable potential for the investigation of factors, such as neurotrophins, that specify and influence the functional phenotype of sensory neurons during embryonic development in vivo.

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Overexpression of Reticulon 3 Enhances CNS Axon Regeneration and Functional Recovery after Traumatic Injury

Cells ◽

10.3390/cells10082015 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2015

Author(s):

Sharif Alhajlah ◽

Adam M Thompson ◽

Zubair Ahmed

Keyword(s):

Spinal Cord ◽

Optic Nerve ◽

Neurite Outgrowth ◽

Functional Recovery ◽

Axon Regeneration ◽

Axon Growth ◽

Dorsal Root Ganglion Neurons ◽

Ganglion Neurons

CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-A. Here, we focused on RTN3 as its contribution to CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a well-characterised model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin, however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00579-3 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Haoting Sun ◽

Chaoqun Wang ◽

Beiyuan Hu ◽

Xiaomei Gao ◽

Tiantian Zou ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cancer Progression ◽

Calcium Binding ◽

Tumor Metastasis ◽

Metastatic Potential ◽

Functional Roles ◽

Stat3 Phosphorylation ◽

Hcc Cells

AbstractIntercellular cross-talk plays important roles in cancer progression and metastasis. Yet how these cancer cells interact with each other is still largely unknown. Exosomes released by tumor cells have been proved to be effective cell-to-cell signal mediators. We explored the functional roles of exosomes in metastasis and the potential prognostic values for hepatocellular carcinoma (HCC). Exosomes were extracted from HCC cells of different metastatic potentials. The metastatic effects of exosomes derived from highly metastatic HCC cells (HMH) were evaluated both in vitro and in vivo. Exosomal proteins were identified with iTRAQ mass spectrum and verified in cell lines, xenograft tumor samples, and functional analyses. Exosomes released by HMH significantly enhanced the in vitro invasion and in vivo metastasis of low metastatic HCC cells (LMH). S100 calcium-binding protein A4 (S100A4) was identified as a functional factor in exosomes derived from HMH. S100A4rich exosomes significantly promoted tumor metastasis both in vitro and in vivo compared with S100A4low exosomes or controls. Moreover, exosomal S100A4 could induce expression of osteopontin (OPN), along with other tumor metastasis/stemness-related genes. Exosomal S100A4 activated OPN transcription via STAT3 phosphorylation. HCC patients with high exosomal S100A4 in plasma also had a poorer prognosis. In conclusion, exosomes from HMH could promote the metastatic potential of LMH, and exosomal S100A4 is a key enhancer for HCC metastasis, activating STAT3 phosphorylation and up-regulating OPN expression. This suggested exosomal S100A4 to be a novel prognostic marker and therapeutic target for HCC metastasis.

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