A quantitative study of retrograde axonal transport in motor and sensory neurons

We used 3H N-succinimidyl propionate to covalently label in vivo proteins of the rat sciatic nerve, and studied the accumulation of radioactively labeled proteins in the cell bodies of the ipsilateral dorsal root ganglion and ventral horn of spinal cord to assess retrograde axonal transport in sensory and motor neurons respectively. In each case the early accumulation of a small amount of radioactively labeled protein is followed by the later accumulation of a larger amount, which subsequently declines to lower levels. The differences between accumulation in the motor neuron and sensory neuron are discussed. Quantitative assessment of retrograde axonal transport will allow future determination of alterations in that transport after nerve injury and in toxic states, which will help elucidate the role of retrogradely transported proteins in neuronal cell biology.     

In sympathetic but not sensory neurones, phosphoinositide-3 kinase is important for NGF-dependent survival and the retrograde transport of I-βNGF

The way in which the same ligands and receptors have different functional effects in different cell types must depend on subtle differences in the second messenger cascades. Sensory and sympathetic neurones both retrogradely transport nerve growth factor (NGF) and depend on NGF for their developmental survival. NGF binding to the high affinity tyrosine kinase (TrkA) receptors initiates second messenger signalling cascades, one of which includes the activation of phosphoinositide-3 kinase (P13-kinase). We demonstrate that 100-fold higher concentrations of the P13-kinase inhibitor. Wortmannin, are required to inhibit the survival effects and retrograde axonal transport of NGF in sensory neurones than in sympathetic neurones. Similarly, although less potently than Wortmannin, the P13-kinase inhibitor LY294002 required a 10-fold higher concentration to inhibit the survival effects of NGF in sensory than in sympathetic neurones. Inhibitors of other second messengers, including staurosporine, pertussis and cholera toxins, failed to have an effect on the transport of the NGF receptor complex in both cell types. Also, Wortmannin did not affect the structural integrity of the sympathetic nerve terminals. As P13-kinase is present in both neuronal populations, this suggests that the Wortmannin sensitive isoform of P13-kinase (p110) is essential in sympathetic neurones both for survival and for NGF-TrkA receptor complex trafficking. As sensory neurones also depend on NGF for their developmental survival and endocytose and retrogradely transport the NGF-TrkA receptor complex, this population of neurones may either recruit a different isoform of P13-kinase or utilize P13-kinase independent signalling pathways for these cellular functions.     

Binding, uptake and retrograde axonal transport of herpes virus suis in sympathetic neurons

Newborn rat dissociated sympathetic neurons were grown in a chamber culture system, where a Teflon ring sealed with silicon grease separated the axonal plexus from the corresponding nerve cell bodies. The binding of 35S-labeled herpes virus suis (HVS) to the neurites was partially inhibited by an excess of unlabeled HVS as well as by concanavalin A, indicating the presence of specific binding sites for the virus. Specific binding was a prerequisite for the subsequent uptake and retrograde transport of HVS to the nerve cell bodies. Predominantly free nucleocapsids were detected by electron microscopy in the axons at the time of retrograde transport, both in culture and in vivo, suggesting the possibility that nucleocapsids without lipid membrane and not contained in cellular membrane compartments can be transported by retrograde axonal transport.     

Rapid retrograde transport of proteins in sensory neurons in rats

Twenty-four hours following the injection of N-succinimidyl[2,3-3H]propionate into rat sciatic nerve, labeled protein appeared in the ipsilateral dorsal root ganglia. Autoradiography showed that the labeled proteins were found only in neuronal cell bodies. Gel electrophoresis showed a distinct pattern of rapidly retrogradely transported proteins were accumulating in the DRG cells. This is the first demonstration of the rapid retrograde transport of endogenous axonal proteins in mammalian peripheral nerve.     

Uptake and retrograde axonal transport of protein tracers in hypoglossal neurons

Summary Protein tracers (albumin labelled with Evan's blue and horseradish peroxidase) accumulate in the cytoplasm of hypoglossal neurons in the brain stem following injection into the tongue of rats and mice resulting apparently from axonal uptake and subsequent retrograde transport from the periphery. The present study was performed to follow the fate of the protein tracers and to find out if they induce any signs of neuronal necrosis.The proteins remained in the nerve cell bodies for about 6–11 days as revealed by fluorescence microscopy and light microscopical enzyme histochemistry after the injection into the tongue. Thereafter the proteins disappeared from the neurons presumably as a result of lysosomal degradation. The incorporation of foreign proteins did not produce as observed by light and electron microscopy any signs of cell degeneration.     

A quantitative analysis of the retrograde axonal transport of 4 different fluorescent dyes in peripheral sensory and motor neurons and lack of anterograde transport in the corticospinal system

Many fluorescent dye compounds are transported by axons in retrograde and anterograde directions. In the present study the uptake and retrograde axonal transport of 4 chemically related fluorescent dyes was evaluated in the peripheral nervous system of adult mice. Anterograde transport was studied in the corticospinal tract of adult rats. In addition to confirming the previously reported intra-axonal transport of Rhodamine-B-isothiocyanate, we report the transport of Rhodamine-X-isothiocyanate. Sulforhodamine-101-acid chloride and Lissamine rhodamine-B-sulfonyl chloride. By using the fluorescence intensity of the labeled motor and sensory neurons as well as cell counts of fluorescently labeled motor neurons and percent of labeled dorsal root ganglia (DRG) cells, we were able to quantitate the amount of retrograde transport of a given fluorescent compound. The two dyes with isothiocyanate groups available for conjugation were transported in higher amounts compared to the dyes containing sulfonyl chloride groups. No anterograde transport in the corticospinal system was observed. We conclude that the 4 dyes described are useful for retrograde neuroanatomical tracing experiments. We describe methods for quantifying the amount of retrograde transport by peripheral motor and sensory neurons.     

Muscle sensory neurons in the spinal ganglia in the rat determined by the retrograde transport of horseradish peroxidase

The method of retrograde transport of horseradish peroxidase (HRP) was used to identify muscle sensory neurons in the spinal ganglia in the rat. Experiments were conducted on 25 albino rats. Injections of 0.06 to 0.08 ml 2 to 20% Sigma type VIHRP were made unilaterally into anterior tibial muscle. Cells of origin of muscle receptors and motor endings in the same area where HRP was administered were demonstrated. The labeled cells, medium to large, were found in fourth and fifth lumbar ganglia ipsilateral to the site of injection. Simultaneously, labeled neurons were also found in the ipsilateral ventral horn of the same cord segments as the labeled sensory ganglia.     

Signalling events regulating the retrograde axonal transport of I−βNerve growth factor in vivo

The molecular mechanisms regulating the retrograde axonal transport of nerve growth factor (NGF) are currently unknown. This study identifies some of the signalling events involved. The phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin (1 nmol/eye) irreversibly inhibits the amount of ¹²⁵I−βNGF retrogradely transported in both sensory and sympathetic neurons. Another PI3-kinase inhibitor LY294002 (100 nmol/eye) also inhibited ¹²⁵I−βNGF retrograde transport in sensory neurons. The pp70^S6K inhibitor rapamycin (1 μmol/eye) had the same effect, inhibiting ¹²⁵I−βNGF transport only in sensory neurons. The cPLA₂ inhibitor AACOCF₃ (10 nmol/eye) had no effect on ¹²⁵I−βNGF transport in either sensory or sympathetic neurons. The TrkA receptor tyrosine kinase inhibitor AG-879 (10 nmol/eye) reduced ¹²⁵I−βNGF transport by approximately 50% in both sensory and sympathetic neurons. Cytochalasin D (2 nmol/eye), a disruptor of actin filaments and the dynein ATPase inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) both inhibited ¹²⁵I−βNGF retrograde transport. These results demonstrate that in vivo TrkA tyrosine kinase activity, actin filaments and dynein are involved in the retrograde transport of NGF. In addition, different PI3-kinase isoforms may be recruited within different neuronal populations to regulate the retrograde transport of NGF. Potentially, these isoforms could activate alternative signalling pathways, such as pp70^S6K in sensory neurons.     

Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

The phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway is considered important for cellsurvival and has been shown to mediate various anti-apoptotic biological effects. This ...     

The Janus role of c-Jun: cell death versus survival and regeneration of neonatal sympathetic and sensory neurons

We investigated the functional outcome of c-Jun activation in sympathetic and sensory neurons of neonatal rat superior cervical ganglion (SCG) and dorsal root ganglion (DRG), respectively. Distinctly different roles of c-Jun activation have been suggested for these two types of neurons. In dissociated sympathetic neurons, c-Jun has been demonstrated to promote apoptosis, whereas in sensory neurons it stimulates axonal outgrowth. In organ-cultured ganglia, we found that c-Jun was activated within 24 h of explantation in both types of neurons, and that the JNK inhibitor SP600125 could mitigate this response. In both types of neurons, c-Jun activation was also reduced by NGF treatment. Inhibition of c-Jun activation did not affect the viability of sympathetic neurons, whereas the number of apoptotic sensory neurons increased. Furthermore, inhibition of c-Jun reduced axonal outgrowth from both SCG and DRG. Thus, in organ culture, c-Jun activation may be required for axonal outgrowth and, at least in sensory neurons, it promotes survival. The role of ATF3, a neuronal marker of injury and a c-Jun dimerization partner, was also examined. We found an ATF3 induction in both SCG and DRG neurons, a response, which was reduced by JNK inhibition. The reduction of ATF3 upon JNK inhibition was much larger in DRG than in SCG, a result which might account for the higher number of apoptotic neurons in JNK inhibitor exposed DRG. Taken together, and contrary to our expectations, neonatal sympathetic and sensory neurons seem to respond to axonal injury similarly with respect to c-Jun activation, and in no case was this activation pro-apoptotic.     

Regulatory role of monoamine neurotransmitters in astrocytic NT-3 synthesis

Astrocytes actively control neuronal activity and synaptic transmission and by producing various neurotrophic factors represent an important local cellular source of trophic support in the normal and diseased brain. Our present study showed the ability of astrocytes to synthesize neurotrophin-3 (NT-3) and the active involvement of the monoamine neurotransmitters noradrenaline, adrenaline, dopamine, and serotonin, as well as basic intracellular second messenger systems, in the regulation of NT-3 production in neonatal rat cortical astrocytes.     

Membrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways

It has been well established that the NGF-mediated survival of sympathetic neurons in culture occurs through the phosphatidylinositol (PI) 3-kinase/Akt-dependent pathway. In contrast, the mechanism by which membrane depolarization promotes neuronal survival independently of NGF remains unresolved. Here we show that LY294002, a specific inhibitor of PI 3-kinase, induced cell death of sympathetic neurons under depolarizing conditions with elevated K(+) (IC(50)= approximately 30 microM). Interestingly, lower concentrations of this agent (< or =10 microM) were sufficient to suppress Akt phosphorylation at Ser-473, a putative downstream target of PI 3-kinase, under these conditions. We also show that KN-62, a specific inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) suppressed depolarization-mediated survival in a does-dependent manner (IC(50)= approximately 2 microM) that paralleled attenuation of sustained levels of intracellular Ca(2+) evoked by depolarization. This IC(50) value is greater than that for CaMKII ( approximately 0.8 microM). These findings led us to hypothesize that depolarization-mediated survival occurs through both the PI 3-kinase/Akt and the CaMKII pathways. Indeed, combined treatment with LY294002 (25 microM) and KN-62 (0.5 microM) dramatically abolished depolarization-mediated survival, whereas each alone did not significantly attenuate it. Under these conditions, KN-62 neither impaired sustained levels of intracellular Ca(2+), nor inhibited the phosphorylation of Akt. It is thus likely that PI 3-kinase and CaMKII independently promote the membrane depolarization-mediated survival of sympathetic neurons in culture.     

α-Bungarotoxin binding sites on sensory neurones and their axonal transport in sensory afferents

The autoradiographic localization of [¹²⁵I]α-bungarotoxin binding sites on primary sensory fibres was investigated. Nicotinic α-bungarotoxin binding sites were localized to a small sub-population of large dorsal root ganglion cells in the rat, monkey, cat and human dorsal root ganglia. Ligation of the sciatic nerve or dorsal root in the rat resulted in an anterograde accumulation of binding sites proximal to the dorsal root ganglion, and a small retrograde accumulation. Unilateral dorsal root section in the rat produced a loss of toxin binding sites mainly within lamina III of the dorsal horn. These results suggest that nicotinic α-bungarotoxin binding sites manufactured in large dorsal root ganglion cell bodies are transported both centrally to the spinal cord and also peripherally.     

Evidence for the role of PI(3) -kinase-AKT-eNOS signalling pathway in secondary inflammatory process after spinal cord compression injury in mice

Endothelial nitric oxide synthase (eNOS) is a dynamic enzyme tightly controlled by co‐ and post‐translational lipid modifications, phosphorylation and regulated by protein–protein interactions. In this study we have pharmacologically modulated the activation of eNOS, at different post‐translational levels, to assess the role of eNOS‐derived NO and regulatory mechanisms in tissue damage associated with spinal cord injury (SCI). SC trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four‐level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, and production of inflammatory mediators, tissue damage and apoptosis. LY294002, an inhibitor of phosphatidylinositol 3‐kinase that initiates Akt‐catalysed phosphorylation of eNOS on Ser1179, was administered 1 h before the induction of SCI; 24 h after SCI sections were taken for histological examination and for biochemical studies. In this study we clearly demonstrated that pre‐treatment with LY294002 reversed the increased activation of eNOS and Akt observed following SCI, and developed a severe trauma characterized by oedema, tissue damage and apoptosis (measured by TUNEL staining, Bax, Bcl‐2 and Fas‐L expression). Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity. Overall these results suggest that activation of the Akt pathway in SC tissue subject to SCI is a protective event, triggered in order to protect the injured tissue through a fine tuning of the endothelial NO pathway.     

Phosphatidylinositol 3‐kinase/protein kinase Cδ activation induces close homolog of adhesion molecule L1 (CHL1) expression in cultured astrocytes

Upregulation of expression of the close homolog of adhesion molecule L1 (CHL1) by reactive astrocytes in the glial scar reduces axonal regeneration and inhibits functional recovery after spinal cord injury (SCI). Here, we investigate the molecular mechanisms underlying upregulation of CHL1 expression by analyzing the signal transduction pathways in vitro. We show that astrogliosis stimulated by bacterial lipopolysaccharide (LPS) upregulates CHL1 expression in primary cultures of mouse cerebral astrocytes, coinciding with elevated protein synthesis and translocation of protein kinase δ (PKCδ) from cytosol to the membrane fraction. Blocking PKCδ activity pharmacologically and genetically attenuates LPS‐induced elevation of CHL1 protein expression through a phosphatidylinositol 3‐kinase (PI3K) dependent pathway. LPS induces extracellular signal‐regulated kinases (ERK1/2) phosphorylation through PKCδ and blockade of ERK1/2 activation abolishes upregulation of CHL1 expression. LPS‐triggered upregulation of CHL1 expression mediated through translocation of nuclear factor κB (NF‐κB) to the nucleus is blocked by a specific NF‐κB inhibitor and by inhibition of PI3K, PKCδ, and ERK1/2 activities, implicating NF‐κB as a downstream target for upregulation of CHL1 expression. Furthermore, the LPS‐mediated upregulation of CHL1 expression by reactive astrocytes is inhibitory for hippocampal neurite outgrowth in cocultures. Although the LPS‐triggered NO‐guanylate cyclase‐cGMP pathway upregulates glial fibrillary acid protein expression in cultured astrocytes, we did not observe this pathway to mediate LPS‐induced upregulation of CHL1 expression. Our results indicate that elevated CHL1 expression by reactive astrocytes requires activation of PI3K/PKCδ‐dependent pathways and suggest that reduction of PI3K/PKCδ activity represents a therapeutic target to downregulate CHL1 expression and thus benefit axonal regeneration after SCI. © 2009 Wiley‐Liss, Inc.     

Streptozotocin-induced diabetes reduces retrograde axonal transport in the afferent and efferent vagus nerve

Diabetes-induced alterations in nerve function include reductions in the retrograde axonal transport of neurotrophins. A decreased axonal accumulation of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of streptozotocin (STZ)-induced diabetic rats was previously shown. In the current study, no changes in the NGF and NT-3 protein or mRNA levels in the stomach or atrium, two vagally innervated organs, were noted after 16 or 24 weeks of diabetes. Moreover, the amounts of neurotrophin receptor (p75, TrkA, TrkC) mRNAs in the vagus nerve and vagal afferent nodose ganglion were not reduced in diabetic rats. These data suggest that neither diminished access to target-derived neurotrophins nor the loss of relevant neurotrophin receptors accounts for the diabetes-induced alteration in the retrograde axonal transport of neurotrophins. To assess whether diabetes causes a defect in axonal transport that may not be specific to neurotrophin transport, we studied the ability of a neuronal tracer (FluoroGold, FG) to be retrogradely transported by vagal neurons of control and diabetic rats. After vagal target tissue (stomach) injections of FG, the numbers of FG-labeled afferent and efferent vagal neurons were counted in the nodose ganglion and in the dorsal motor nucleus of the vagus, respectively. After 24 weeks of diabetes, FG was retrogradely transported to more than 50% fewer afferent and efferent vagal neurons in the STZ-diabetic compared to control rats. The diabetes-induced deficit in retrograde axonal transport of FG is likely to reflect alterations in basic axonal transport mechanisms in both the afferent and efferent vagus nerve that contribute to the previously observed reductions in neurotrophin transport.     

Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress

Cyclin-dependent kinase 5 (cdk5) phosphorylates the high molecular weight neurofilament (NF) protein. Overexpression of cdk5 inhibits NF axonal transport and induces perikaryal accumulation of disordered phospho-NF cables. Experimental and clinical motor neuron disease is characterized by oxidative stress, increased cdk5 activity, and accumulation of phospho-NFs within perikarya or proximal axons. Because oxidative stress increases cdk5 activity in experimental motor neuron disease, we examined whether oxidative stress induced cdk5-mediated NF phosphorylation. Treatment of cultured neuronal cells with hydrogen peroxide inhibited axonal transport of green fluorescent protein-tagged NF subunits and induced perikaryal accumulation of NF phosphoepitopes normally confined to axons. These effects were prevented by treatment with the cdk5 inhibitor roscovitine or transfection with a construct expressing the endogenous cdk5 inhibitor peptide. These findings indicate that oxidative stress can compromise NF dynamics via hyperactivation of cdk5 and suggest that antioxidants may alleviate multiple aspects of neuropathology in motor neuron disease.     

Striatal afferent connections in the turtle (Chrysemys picta) as revealed by retrograde axonal transport of horseradish peroxidase.

Horseradish peroxidase (HRP, 30% solution, 0.1-0.3 mul, 72 h) was injected unilaterally into the basal striatum (STR) and the dorsal ventricular ridge (DVR) of adult turtles (Chrysemys picta) in order to demonstrate the cells of origin of some afferents to these telencephalic structures. After selective STR injection, HRP-labeled cells were visualized in the dorsal thalamus and midbrain tegmentum, ipsilaterally. At thalamic level, HRP-positive neurons were located around nucleus rotundus, i.e., mainly within nuclei dorsomedialis anterior, dorsolateralis anterior and less abundantly in nuclei ventralis and reuniens. At midbrain level, a large population of labeled neurons was disclosed within the ventrolateral portion of rostral tegmentum. Other HRP-positive neuronal somata were found scattered throughout the lateral portion of the caudal midbrain tegmentum. In addition, labeled axons were visualized in both peduncles of the lateral forebrain bundle (LFB) after STR injection. The HRP-positive fibers of the dorsal peduncle of the LFB were followed up to the ipsilateral labeled thalamic cells where they appear to arise, whereas the HRP-containing axons of the ventral peduncle were traced down to the lateral midbrain tegmentum where they appear to arborize. Most of the HRP injections into the DVR were confined to the mediodorsal quadrant of the rostral half of the DVR. In such a case, a very large number of HRP-positive cells were disclosed within all thalamic nuclei surrounding nucleus rotundus, ipsilaterally. In addition, numerous labeled neurons were also found in nucleus rotundus itself and within nucleus reuniens. No HRP-positive cells were disclosed caudally to the meso-diencephalic junction after DVR injection.