Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the expression in the hypoglossal nucleus after its axotomy (original) (raw)
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Molecular Brain Research, 2002
ADP-ribosylation factors (ARFs) play important roles in vesicular trafficking and cytoskeletal regulation and its activation depends on guanine nucleotide-exchange proteins (GEPs). By way of in situ hybridization histochemistry, the localization of mRNAs for subfamily members of low-molecular-weight ARF-GEPs in the rat brain was studied at embryonic and postnatal stages. In the embryonic brain, the gene expression for msec7-1 was distinct in the ventricular zone while that for msec7-1,-3 and EFA6 in the mantle zone. In early postnatal brain, the expression for msec7-1,-2,-3 and EFA6 was seen widely in various loci of the gray matter with different intensity, and the expression of msec7-1 and-2 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for the four ARF-GEPs decreased more or less in most gray matter and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression of EFA6 mRNA was also evident in the molecular layer of the hippocampus and dentate gyrus. No obvious gene expression for cytohesin-4 and ARF-GEP100 was detected in the brain at any stages of development. The present findings suggest that ARF-GEPs are differentially involved in some processes essential to neuronal differentiation and maturation in association with ARFs.
Journal of Neuroscience, 2012
Nerve growth factor (NGF) induces collateral branching along sensory axons by promoting the formation of axonal filopodia dependent on the actin-nucleating Arp2/3 complex. This study shows that chicken embryonic sensory axons contain mRNAs for the actin-nucleating Arp2/3 complex activator WAVE1 and the complex stabilizer cortactin. NGF increases the axonal levels of WAVE1 and cortactin through localized protein synthesis even in axons isolated from the cell body. Inhibition of protein synthesis in severed axons impairs NGFinduced branching, the formation of axonal filopodia, and the initiation of Arp2/3-dependent axonal actin patches, which serve as precursors to the emergence of filopodia. Overexpression of WAVE1 or cortactin in axons not treated with NGF increased the rate of actin patch formation and the frequency of the emergence of filopodia from actin patches, respectively. Antisense inhibition of cortactin mRNA translation in isolated axons blocked NGF-induced filopodia. NGF also activated the Rac1 GTPase, which drives WAVE1 activity, in a protein synthesis-independent manner. Similarly, inhibition of protein synthesis did not impair the effects of NGF on the axonal microtubule cytoskeleton during branching. The effects of NGF on Rac1 activity and increases in axonal levels of WAVE1 and cortactin were both dependent on phosphoinositide 3-kinase (PI3K) signaling. Collectively, the data indicate that NGF promotes sensory axon branching through regulation of the actin cytoskeleton using both canonical signaling mechanisms and intra-axonal protein synthesis downstream of PI3K signaling. Finally, we present experimental evidence of axonal mRNA translation in sensory axons in the living embryonic spinal cord.
Biochemical and Biophysical Research Communications, 2007
To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.
ARF6 and Rab11 as intrinsic regulators of axon regeneration
Small GTPases, 2018
Adult central nervous system (CNS) axons do not regenerate after injury because of extrinsic inhibitory factors, and a low intrinsic capacity for axon growth. Developing CNS neurons have a better regenerative ability, but lose this with maturity. This mini-review summarises recent findings which suggest one reason for regenerative failure is the selective distribution of growth machinery away from axons as CNS neurons mature. These studies demonstrate roles for the small GTPases ARF6 and Rab11 as intrinsic regulators of polarised transport and axon regeneration. ARF6 activation prevents the axonal transport of integrins in Rab11 endosomes in mature CNS axons. Decreasing ARF6 activation permits axonal transport, and increases regenerative ability. The findings suggest new targets for promoting axon regeneration after CNS injury.
Developmental Biology, 1987
Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45-52) and that hippocampal NGF and mRNANGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389-1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNANGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNANGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNANGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNANGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.
Molecular Brain Research, 1992
The hypoglossal nerve is a useful model system for analysis of gene expression in injured motoneurons. In particular, we sought to determine whether the increased appearance of the low affinity nerve growth factor receptor (p75 NGFr) observed immunocytochemically following nerve injury can be directly correlated to increased levels of the p75 NGFr mRNA. The present study also examined the relative effects of nerve crush versus nerve transection on the expression of p75 NGFr mRNA. In sham-operated or intact animals, p75 NGFr mRNA is detected rarely and then only at levels slightly higher than background. Following unilateral transection or crush of the rat hypoglossal nerve, the levels of p75 NGFr mRNA increase in a time dependent fashion that parallels the appearance of the protein as reported previously. Moreover, this increase in p75 NGFr mRNA following transection is dependent on a signal from the injured site, since blockage of axonal transport with vincristine also blocks the increased p75 rqGFr mRNA levels. When comparing the effect of nerve crush to nerve transection, we observed that the intensity of the response was greater in the crush paradigm versus that observed following transection. The duration of the response following nerve crush was shorter than that observed following transection of the nerve. The increase in p75 NGFr mRNA after crush was most robust 4 days postlesion and appeared more robust primarily due to a 90-150% increased number of motoneurons expressing p75 NGFr mRNA when compared to nerve transection. These data suggest that nerve crush is more effective than nerve transection in eliciting increased p75 NGF~ mRNA levels.
Transcriptional insights on the regenerative mechanics of axotomized neurons in vitro
2012
One of the striking features of the injured central nervous system (CNS) is the failure of severed axons to adequately regenerate to restore loss of function. This was initially believed to be caused by an intrinsic inability of injured axons to sprout regenerative processes. However, the seminal studies of Albert Aguayo and others using peripheral or cellular tissue grafts transplanted into the lesioned spinal cord have clearly demonstrated that the environment of the injured CNS is a critical determinant of whether injured axons can regenerate . The molecular determinates of the inhibitory CNS environment are now well-understood, with major players being myelin-associated molecules (such as nogo, myelin-associated glycoprotein) and chondroitin sulphate proteoglycans .
ARF6 Regulates Neuron Differentiation through Glucosylceramide Synthase
PLOS ONE, 2013
The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels.
The Journal of Neuroscience, 1997
Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regenerationassociated genes such as GAP-43 and T␣1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors. In situ hybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment. Moreover, BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and T␣1-tubulin mRNA and maintained the level of trkB expression. Vehicle, NGF, or NT-3 treatment had no significant effect on cell size or GAP-43 and T␣1-tubulin expression. In a separate experiment, infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft. Thus, in BDNF-treated animals, the prevention of neuronal atrophy and the stimulation GAP-43 and T␣1-tubulin expression is correlated with an increased regenerative capacity of axotomized RSNs.