Axonal RNA Transport Research Articles

Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations or deletions in the survival motoneuron 1 (SMN1) gene, resulting in deficiency of the SMN protein that is essential for motoneuron function. Smn depletion in mice disturbs axonal RNA transport and translation, thereby contributing to axon growth impairment, muscle denervation, and motoneuron degeneration. However, the mechanisms whereby Smn loss causes axonal defects remain unclear. RNA localization and translation in axons are controlled by RNA-binding proteins (RBP) and we recently observed that the neuronal RBP Ptbp2 modulates axon growth in motoneurons. Here, we identify Smn as an interactor of Ptbp2 in the cytosolic compartments of motoneurons. We show that the expression level of Ptbp2 is reduced in axons but not in the somata of Smn-depleted motoneurons. This is accompanied by reduced synthesis of the RBP hnRNP R in axons. Re-expression of Ptbp2 in axons compensates for the deficiency of Smn and rescues the defects in axon elongation and growth cone maturation observed in Smn-deficient motoneurons. Our data suggest that Ptbp2 and Smn are components of cytosolic mRNP particles, contributing to the precise spatial and temporal control of protein synthesis within axons and axon terminals.

BDNF elevates the axonal levels of hnRNPs Q and R in cultured rat cortical neurons

Local translation plays important roles in the maintenance and various functions of axons, and dysfunctions of local translation in axons are implicated in various neurological diseases. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA binding proteins with multiple functions in RNA metabolism. Here, we identified 20 hnRNPs in the axons of cultured rat cortical neurons by interrogating published axon mass spectrometric databases with rat protein databases. Among those identified in axons are highly related hnRNPs Q and R. RT-PCR analysis indicated that axons also contained low levels of hnRNPs Q and R mRNAs. We further found that BDNF treatments raised the levels of hnRNPs Q and R proteins in whole neurons and axons. BDNF also increased the level of poly(A) RNA as well as the proportion of poly(A) RNA granules containing hnRNPs Q and R in the axon. However, following severing the connection between the cell bodies and axons, BDNF did not affect the levels of hnRNPs Q and R, the content of poly(A) RNA, or the colocalization of poly(A) RNA and hnRNPs Q and R in the axon any more, although BDNF still stimulated the local translation in severed axons as it did in intact axons. The results are consistent with that BDNF enhances the axonal transport of RNA granules. The results further suggest that hnRNPs Q and R play a role in the mechanism underlying the enhancement of axonal RNA transport by BDNF.

RNA Reigns in Neurons

A workshop entitled "RNA Control of Neuronal Function" was recently held in Kfar Blum, Israel. The main topics discussed at the meeting included neuronal RNA targeting mechanisms and the contributing codes and components, translational control mechanisms in dendrites and axons, and the relevance of these mechanisms for neuronal development, plasticity, and dysfunction.

Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

Transport of Neuronal BC1 RNA in Mauthner Axons

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons <i>in vivo</i>. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 μm/sec. Whereas a BC1 5′ segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3′ segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

Chapter 2.2.1 The magnocellular neurons of the hypothalamo-neurohypophyseal system display remarkable neuropeptidergic phenotypes leading to novel insights in neuronal cell biology

For decades the magnocellular neurons of the hypothalamo-neurophypophyseal system (HNS), in which either vasopressin or oxytocin are produced and released into the bloodstream, have been playing a pivotal role in fundamental discoveries in the nervous system. The primary structure of vasopressin and oxytocin was the first of all neuropeptides to be published, i.e., in the 1950s by the Nobel prize laureate Du Vigneaud. Moreover, many trend-setting discoveries have their origin in the HNS, which abundantly expresses vasopressin and oxytocin, clearly displays its function and is relatively easily to manipulate. Examples are the phenomenon of coexpression of neuropeptides, patch-clamping of nerve endings, axonal transport of RNA, neuroglia interactions and the behavioral effects. An extraordinarily intriguing example is the homozygous Brattleboro rat, which lacks vasopressin by a germ-line mutation, and has disclosed many of the fundamental characteristics of peptidergic neurons, and neurons in general. In this chapter we will discuss a few of them, in particular the recent data on mutations in vasopressin RNA. It is to be expected that the HNS will retain its informative role in the next decades.

The Origin and Regulation of Posterior Pituitary Vasopressin Ribonucleic Acid in Osmotically Stimulated Rats

Abstract Vasopressin ribonucleic acid (VP RNA) accumulates in the posterior lobe of the rat pituitary in response to an osmotic stimulus. The accumulation of posterior pituitary VP RNA can be prevented by stalk transection or the intracerebroventricular injection of colchicine. The hypothalamic and pituitary VP RNAs however, have different structures and are independently regulated. Depletion of serotonin with parachlorophenylalanine blocks the osmotically induced accumulation of both VP and oxytocin RNA in the hypothalamus but does not affect the accumulation of VP RNA in the posterior pituitary gland. In Brattleboro rats, posterior lobe oxytocin RNA but not VP RNA, increases after osmotic stimulation. Although axonal transport of RNA may occur, our results are also consistent with VP RNA synthesis in pituicytes controlled by hypothalamic neuroendocrine factors released by neurohypophyseal terminals in response to osmotic stimuli.

Axonal transport of nucleosides, nucleotides and 4S RNA in the neonatal rat visual system

The axonal migration of RNA, the nucleoside uridine and its nucleotide derivates (NS/NT) were compared in neonatal and young adult rat optic axons. Tritiated uridine was injected into right eyes of developing (1- or 4-day-old) and young adult (40-day-old) rats which were sacrificed at times after injection ranging from 6 h to 20 days. Right and left lateral geniculates were removed and assayed for trichloroacetic acid soluble (NS/NT) and RNA radioactivity. Left minus right geniculate (L-RLG) radioactivity was used as an index of axonally migrating radioactivity. Results showed that uridine and its phosphorylated derivatives were transported along both neonatal and young adult rat optic axons. Greater than 90% of right geniculate (blood-borne) TCA soluble radioactivity was metabolized to volatile substances (probably 3H2O) by three days after injection, leaving approximately 3% of the neonatal and approximately 10% of the adult activity as [3H]NS/NT. In left geniculate fractions (containing transported material) approximately 15% and 40% of total TCA soluble radioactivity was present as [3H]NS/NT in neonates and adults, respectively. Thus, axonal NS/NT appears to be relatively protected from degradation when compared with blood-borne NS/NT. The amount of L-RLG [3H]RNA in the neonates was 10 times higher than in young adults. Peaks of neonatal [3H]RNA occurred at 5 and 10 days after birth, whether injections were made at 1 or 4 days of age indicating that this [3H]RNA may be linked to developmental events. Gel electrophoretic analysis of neonatal geniculate RNA indicated that a small portion of the [3H]RNA in the first peak represented axonally transported 4S RNA. The remainder of the L-RLG [3H]RNA in the neonates was probably due to a rapid and efficient incorporation of axonally transported [3H]NS/NT into extraaxonal geniculate RNA. In contrast, little or no axonal RNA transport could be demonstrated in the young adults.

The effect of inhibition of axonal RNA transport on the restoration of retinotectal projections in regenerating optic nerves of goldfish

The effect of inhibition of axonal RNA transport on the restoration of retinotectal projections in regenerating optic nerves of goldfish

Action of chloroquine on in vivo RNA and protein biosynthesis in the retina and the optic pathway of the rabbit

The in vivo action of chloroquine on RNA and protein metabolism in the optic pathway of the albino or pigmented rabbit was examined. To study the acute effects, chloroquine in a dose of 500 μg was injected into the vitreous body of one eye. The following day the animals were injected into both eyes with [ 3H]uridine or [ 3H]leucine. At various time intervals following the isotope injections the retinal synthesis and the axonal transport of labelled RNA or protein was studied. The results showed no significant difference between the drug-treated and the control side with respect to synthesis and axonal transport of RNA or protein. Nor was any selective effect noticed on the synthesis of different RNA fractions from the retina. In long-term experiments chloroquine (100 mg/kg body weight) was administered via their drinking water 3 days a week for a period of 6 or 8 months. Following an intraocular injection of [ 3H]leucine no significant change in rapid axonal transport could be found in those chronically treated rabbits.

Axonal transport of RNA during regeneration of the optic nerves of goldfish.

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of 3H-uridine has been studied during various stages of optic nerve regeneration. 3H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Five were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, 14C-proline was also injected into the right eye as a marked of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of 3H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microsocpic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be 3H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of 3H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikaprya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.

Development of axonal transport of RNA and its precursors in the optic pathway of the chick embryo

The axonal migration of RNA has been investigated at various developmental stages in the optic pathway of chick embryo after intraocular administration of labeled uridine. The somatofugal movement of soluble RNA precursors has also been studied. An excess of radioactivity in RNA within the optic lobe contralateral to the injected eye relative to that within the ipsilateral lobe was found 3 days after 3H-uridine injection in 13- and 18-day-old embryos. In 9-day-old embryos no asymmetry in radioactive RNA from paired optic lobes developed even 6 days after isotope injection. At all stages no asymmetry was found 6 and 24 hr after isotope administration. These results suggest a slow retinotectal flow of RNA beginning at about Day 13 of incubation. In the newly hatched chick, 3 days after monocular 3H-uridine injection, the asymmetry in label within RNA from paired optic lobes was much greater than was that of embryos. At all stages studied a considerable asymmetry of radioactivity within nucleotides and nucleosides from paired optic lobes also developed 3 days after monocular 3H-uridine injection. However, this was at least as high in 9-day-old embryos as in newly hatched chicks. Intraocular injection of colchicine prior to 3H-uridine blocked the migration of labeled RNA in the newly hatched chick, suggesting such transport to be dependent on the integrity of neuronal microtubules. It is concluded that a significant proximodistal migration of RNA occurs in the optic pathway of chick embryos prior to the onset of electrical activity.

Evidence for axonal transport of RNA in mammalian neurons.

Adult albino rabbits were injected into one eye with (3H) uridine. At various time intervals following the injections the amount of RNA-bound and acid soluble radioactivity was measured in the retrobulbar optic pathway. Evidence was obtained for a relatively slow transport of a small amount of macromolecular RNA along the axons of the retinal ganglion cells down to the nerve terminals.

Effects of excitatory and inhibitory synaptic actions on RNA content of spinal motoneurones

Spinal motoneurones were activated by natural (swimming) and artificial (orthodromic and antidromic stimulation) ways in rats with intact inhibitory mechanisms or after their abolition by tetanus toxin. Postsynaptic inhibition of spinal motoneurones was also evoked in experiments with cats, in which the gigantocellular nucleus of the brain stem reticular formation was electrically stimulated. Cytoplasmic RNA contents of the motoneurones were measured by ultraviolet cytospectrophotometry. The quantity of cytoplasmic RNA in the motoneurones increased with excitatory and decreased with inhibitory synaptic action. The results suggest that these changes were caused by an increase in RNA synthesis during synaptic excitation and by the acceleration of axonal RNA transport and/or of RNA breakdown during postsynaptic inhibition.

Axonal migration of various ribonucleic acid species along the optic tract of the chick.

Abstract—The avian visual system has been used to study the axonal transport of RNA and protein. After monocular injection of radioactive uridine into 1‐day‐old chicks, a considerable amount of labelled RNA migrated along the optic tract to the optic tectum contralateral to the injected eye. This RNA was largely ribosomal, although it was contained in several subcellular fractions. The migration of RNA appeared to be a slow process. However, following monocular injection of radioactive proline, the migration of ribosomal protein was rapid. This discrepancy was resolved by examination of the kinetics of labelling of RNA and protein within the retina after intraocular injection of a mixture of labelled uridine and proline. Cytoplasmic RNA was labelled much more slowly than cytoplasmic protein. This lag in labelling of RNA could account for the delayed arrival of RNA at the contralateral optic lobe and suggests that ribosomes may travel rapidly along the axon.In other experiments, eyes were removed 4 days after the injection of labelled precursors. After a further 14 days, the remaining radioactivity in RNA and protein of contralateral optic lobes was 5–15% of that attributable to migration along the axon in control, unenucleated birds. Thus, the survival of the bulk of migrating macromolecules depends on the integrity of synaptic terminals. This observation suggests that both RNA and protein migrate within the axon rather than extra‐axonally, and that they remain largely within the nerve cells along the axons of which they are transported.