Deletion of guanine nucleotide binding protein alpha z subunit in mice induces a gene dose dependent tolerance to morphine.

The mechanism underlying the development of tolerance to morphine is still incompletely understood. Morphine binds to opioid receptors, which in turn activates downstream second messenger cascades through heterotrimeric guanine nucleotide binding proteins (G proteins). In this paper, we show that G(z), a member of the inhibitory G protein family, plays an important role in mediating the analgesic and lethality effects of morphine after tolerance development. We blocked signaling through the G(z) second messenger cascade by genetic ablation of the alpha subunit of the G protein in mice. The Galpha(z) knockout mouse develops significantly increased tolerance to morphine, which depends on Galpha(z) gene dosage. Further experiments demonstrate that the enhanced morphine tolerance is not caused by pharmacokinetic and behavioural learning mechanisms. The results suggest that G(z) signaling pathways are involved in transducing the analgesic and lethality effects of morphine following chronic morphine treatment.

Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components.

Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine beta-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

What is the importance of multivesicular bodies in retrograde axonal transport in vivo?

Neurons with long axons have a unique problem in generating signaling cascades that are able to reach the nucleus after receptor activation by neurotrophins at the nerve terminal. The straightforward concept of receptor binding and local generation of 2nd second messenger cascades is too simplistic. In this review we will outline a mechanism that would enable the complex signals generated at the nerve terminal to be conveyed intact to the cell body. There are three different sites in the neuron where 2nd messenger proteins can interact with the signaling complex and be activated. Signaling cascades are initiated both at the nerve terminal and at the cell body when 2nd messengers are recruited to the plasma membrane by activated receptors. After receptor-mediated endocytosis, 2nd messenger molecules continue to be recruited to the internalized vesicle; however, the mix of proteins differs in the nerve terminal and in the cell body. At the nerve terminal the activated pathways result in the formation of the neurotrophin signaling endosome, which includes molecules to be retrogradely transported to the cell body. When the retrograde neurotrophin signaling endosome reaches the cell body, it can recruit additional 2nd messenger molecules to finally generate the unique signal derived from the nerve terminal. We propose that the multivesicular body observed in vivo functions as an endosome carrier vehicle or retrosome. This retrosome enables the mix of signaling molecules recruited at the terminal to be transported intact to the cell body. This will allow the cell body to receive a snapshot of the events occurring at the nerve terminal at the time the retrosome is formed.

Anterograde and retrograde transport of active extracellular signal-related kinase 1 (ERK1) in the ligated rat sciatic nerve.

Neurons are one of the most polarized cells and often the nerve terminals may be located long distances from the cell body, thus signal transduction in neurons unlike other cells may need to be conducted over large distances. The mitogen-activated protein/extracellular signal-regulated kinases (MAP kinases or ERKs) regulate a diverse array of functions and in neurons, the ERK signalling pathways appear to have an important role in activity-dependent regulation of neuronal function. Using the ligated rat sciatic nerve as an experimental model we previously showed that the ERK1/2, MAP/ERK kinase (MEK1/2) and the p110 catalytic subunit of PI3-kinase are transported in the rat sciatic nerve. We have extended these findings to determine if these proteins are transported in the active state using antibodies that specifically detect the active form of ERK1/2, MEK1/2 and AKT which is activated downstream of PI3-kinase. We show significant accumulation of active ERK1 on the proximal and distal sides of a nerve ligation after 16 h. Active ERK2 also appeared to be accumulating at the ligature, however this did not reach statistical significance. In contrast there was not any significant accumulation of active MEK1/2 or active AKT. A component of both active ERK1 and active ERK2 is present in between the two ligations suggesting they are also present in the surrounding Schwann cells and are activated in response to nerve injury. Taken together our results suggest that a component of the accumulation of active ERK1 on the distal and proximal side of the nerve ligations results from transport in the anterograde and retrograde direction in the rat sciatic nerve.

Alterations in ciliary neurotrophic factor signaling in rapsyn deficient mice.

Rapsyn is a key molecule involved in the formation of postsynaptic specializations at the neuromuscular junction, in its absence there are both pre- and post-synaptic deficits including failure to cluster acetylcholine receptors. Recently we have documented increases in both nerve-muscle branching and numbers of motoneurons, suggesting alterations in skeletal muscle derived trophic support for motoneurons. The aim of the present study was to evaluate the contribution of target derived trophic factors to increases in motoneuron branching and number, in rapsyn deficient mice that had their postsynaptic specializations disrupted. We have used reverse transcription-polymerase chain reaction and Western blot to document the expression of known trophic factors and their receptors in muscle, during the period of synapse formation in rapsyn deficient mouse embryos. We found that the mRNA levels for ciliary neurotrophic factor (CNTF) was decreased in the rapsyn deficient muscles compared with litter mate controls although those for NGF, BDNF, NT-3 and TGF-beta2 did not differ. We found that both the mRNA and the protein expression for suppressor of cytokine signaling 3 (SOCS3) decreased although janus kinase 2 (JAK2) did not change in the rapsyn deficient muscles compared with litter mate controls. These results suggest that failure to form postsynaptic specializations in rapsyn deficient mice has altered the CNTF cytokine signaling pathway within skeletal muscle, the target for motoneurons. This alteration may in part, account for the increased muscle nerve branching and motoneuron survival seen in rapsyn deficient mice.

Targeted disruption of the mouse Gz-alpha gene: a role for Gz in platelet function?

Gz is one of nine G proteins identified in platelets and its role in these cells is unknown. Our laboratory has generated a mouse deficient in the Gz-alpha gene in the hope of determining its in vivo function. Bleeding times from the tail tip of Gzalpha deficient mice was significantly longer than wild type mice. Platelet aggregation and ATP secretion did not differ between wild type and Gzalpha deficient mice. When mice were presented with a thromboembolism challenge no differences were observed in the survival or mortality of wild type or Gzalpha deficient mice, however a strain difference was observed. Ignoring the genetic background of a mutant mouse might lead to a misinterpretation of results and thus it is absolutely critical to take the genetic background into account when assessing any aspect of a mutant mouse.

Transport of endosomal early antigen 1 in the rat sciatic nerve and location in cultured neurons.

Early endosomal antigen 1 (EEA1) is known to be a marker of early endosomes and in cultured hippocampal neurons it preferentially localizes to the dendritic but not the axonal compartment. We show in cultured dorsal root ganglia and superior cervical ganglia neurons that EEA1 localizes to the cell bodies and the neurites of both sensory and sympathetic neurons. We then show in vivo using a ligated rat sciatic nerve that EEA1 significantly accumulates on the proximal side and not on the distal side of the ligation. This suggests that EEA1 is transported in the anterograde direction in axons either as part of the homeostatic process or to the nerve ligation site in response to nerve injury.

A one-step quantitative reverse transcription polymerase chain reaction procedure.

Our laboratory has developed a one-step quantitative reverse transcription polymerase chain reaction (RT-PCR) procedure in which the reverse transcriptase enzyme and Taq DNA polymerase are combined in the one tube and a single, non-interrupted, thermal cycling program is performed. In the past, RT-PCR has been carried out with two separate steps: (1) reverse transcription of RNA to generate a cDNA pool and (2) polymerase chain reaction amplification of the cDNA. The two-step method can affect the accuracy of the procedure as the total number of manipulations is greater, thereby allowing a greater chance for pipetting errors. Quantitation by our method is achieved in a single reaction by the use of a competitive internal standard that is identical in sequence to the target RNA except for a deletion of 107 base pairs and uses identical primers and cycling conditions. Using this method, we have been able to quantify the amount of message of a G protein (G(zalpha)), in small amounts of tissue, such as dorsal root ganglia, from embryonic as well as postnatal mice.

Prolonged recycling of internalized neurotrophins in the nerve terminal.

BACKGROUND: Neurons require contact with their target tissue in order to survive and make correct connections. The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. However, the mechanism by which the neurotrophin signal is propagated from axon terminal to cell body remains unclear. METHODS: Retrograde axonal transport was examined using the transport of I(125)-labeled neurotrophins from the eye to sympathetic and sensory ganglia. The phenomena was further studied by adding rhodamine-labeled nerve growth factor (NGF) to cultures of dissociated sympathetic ganglia and the movement of organelles followed with the aid of video microscopy. RESULTS: I(125)-labeled neurotrophins were transported from the eye to the sympathetic and sensory ganglia. A 100-fold excess of unlabeled neurotrophin, administered up to 4 h after the labeled material, completely prevented accumulation of labeled neurotrophin in the ganglia. The effect was specific for the labeled neurotrophin as administration of a high concentration of a different neurotrophin failed to inhibit the transport. In dissociated cultures, we found rapid binding of label, to surface membrane receptors, followed by an accumulation of labeled vesicles in the growth cone. Incubation of these cultures with unlabeled NGF led to a rapid loss of label in the growth cones. CONCLUSIONS: These results suggest that there is a pool of internalized neurotrophin, in vesicles in the nerve terminal, which is in rapid equilibrium with the external environment. It is from this pool that a small fraction of the neurotrophin-containing vesicles is targeted for retrograde transport. Potential models for this system are presented.

PtdIns 4-kinasebeta and neuronal calcium sensor-1 co-localize but may not directly associate in mammalian neurons.

It was recently demonstrated that the yeast homologue of phosphatidylinositol 4-kinasebeta PIK1 is directly associated with frq1, the yeast homologue of mammalian neuronal calcium sensor-1 (NCS-1) (Hendricks et al., [1999] Nat. Cell Biol. 1:234- 241). This was a novel finding and suggests that a calcium binding protein activates and regulates PtdIns 4-kinasebeta. This finding had not been shown in mammalian cells and both PtdIns 4-kinasebeta and NCS-1 have been shown to have important roles in the regulation of exocytotic release associated with neurotransmission. The aims of this study were to determine if PtdIns 4-kinasebeta and NCS-1 directly associate in mammalian neural tissues. We show that the immunostaining pattern for PtdIns 4-kinasebeta and NCS-1 is co-localized throughout the neurites of newborn cultured dorsal root ganglia (DRG) neurons but not in E13 DRG neurons. We then provide biochemical evidence that PtdIns 4-kinasebeta may not be in physical association with NCS-1 in mammalian nervous tissue unlike that previously reported in yeast.

Molecular mechanisms regulating the retrograde axonal transport of neurotrophins.

Neurotrophins are released from target tissues following neural innervation and bind to specific receptors situated on the nerve terminal plasma membrane. The neurotrophin-receptor complex undergoes retrograde axonal transport towards the cell soma, where it signals to the nucleus. This process allows neurotrophins to perform their numerous functions, which include the promotion of neuronal survival and the outgrowth of axons towards certain target tissues. The molecular events controlling each of the components of retrograde axonal transport are beginning to become defined. There is good evidence for the participation of phosphatidylinositol 3-kinase, phosphatidylinositol 4-kinase and the actin cytoskeleton in neurotrophin retrograde axonal transport in vivo. It also appears that the retrograde motor protein dynein mediates the retrograde axonal transport in vivo of neurotrophins such as nerve growth factor. This review discusses the role of the neurotrophin receptors in binding and axonal transport, the endocytic processes required for neurotrophin internalization, the targeting and trafficking of neurotrophins, and the propagation of neurotrophin-induced signals along the axon.

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal.

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.

Transforming growth factor-beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury.

The survival of motoneurons is dependent on them receiving continual trophic support from muscle fibres and various other cell types. Numerous putative survival factors have been identified and a set of criteria established by which these candidates can be assessed. These criteria include the need for the factor and its receptors to be in appropriate locations and for the factor or its second message to be retrogradely transported. In this paper, we demonstrate that a multifunctional cytokine, transforming growth factor-beta 2, appears to meet these criteria. The locations of the transforming growth factor-beta 2 and its receptors in the neuromuscular system were determined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Motoneurons were shown to synthesize the three proteins involved in transforming growth factor-beta 2 signalling (types I and II transforming growth factor-beta receptor and betaglycan) and to transport them anterogradely, where they were inserted into the axonal membrane and nerve terminal. Transforming growth factor-beta 2 was detected in the synaptic portions of muscle fibres, motoneurons and in injured nerves, indicating that motoneurons may be exposed to multiple and potentially redundant sources of transforming growth factor-beta 2. Double-ligation experiments were used to demonstrate that motoneurons transport transforming growth factor-beta 2 up and down their axons. The anterograde transport of both transforming growth factor-beta 2 and its receptors, coupled with the fact that most of a motoneuron's mitochondria are located in the axon, raises the issue of whether the repression of the initiation of apoptosis is restricted to the cell body or occurs along the entire length of a neuron.

Development and characterization of human and mouse specific antibodies to CuZn-superoxide dismutase (SOD1).

Mutations in the copper/zinc superoxide dismutase (SOD1) gene are associated with 15-20% of the familial forms of motor neuron disease. Mice where a transgene has been incorporated that encodes for the human SOD1 mutation develop a form of motor neurone disease that closely resembles human forms of this disease. We have produced and characterized species-specific antibodies to epitopes in the SOD1 protein, amino acids 25-37, a region that distinguishes between the human and the mouse species of SOD1. The antisera generated were unable to immunoprecipitate the mouse or the human forms of SOD1 from tissue extracts unless the homodimeric complex of SOD1 was denatured. As SOD1 exists as a homodimeric complex in the cytoplasm of cells, this suggests that amino acids in position, 25-37 are close to the dimeric interface of SOD1.

Axonal transport of neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta in the adult rat sciatic nerve.

Neuronal calcium sensor-1 (NCS-1) and its putative substrate phosphatidylinositol 4-kinase beta (PtdIns 4-kinase beta) both indirectly regulate synaptic vesicle exocytosis and are located in DRG neurites. In this study we have tested whether NCS-1 and PtdIns 4-kinase beta are transported in axons using the analysis of double ligation approach in the adult rat sciatic nerve. We show that NCS-1 accumulates on both the distal and proximal side of the nerve ligation indicating that this protein undergoes bidirectional transport in axons. In contrast, PtdIns 4-kinase beta accumulated on the distal side which suggests that it undergoes retrograde axonal transport and unlike NCS-1 was also present in non-neuronal cells.

Hypertolerance to morphine in G(z alpha)-deficient mice.

Our laboratory has generated a mouse deficient in the alpha (alpha) subunit of the G protein, G(z), (G(z alpha)) gene and we have examined the involvement of G(z alpha) in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by the response times to heat or cold tail flick times in a water bath at 50 degrees C or -5 degrees C and supraspinal analgesia was tested by the times for paw licking and jumping from a plate at 52 degrees C or 0.5 degrees C. Tolerance to morphine was induced in wild type and G(z alpha)-deficient mice over a 5 day period and the behavioral tests were performed daily. The tail flick reaction times to both hot and cold stimuli did not differ between the wild type and G(z alpha)-deficient mice. Analysis of the reaction times from the hot and cold plate tests showed the G(z alpha)-deficient mice developed tolerance to morphine to a greater degree and at a faster rate than wild type mice. Opioid binding assays were performed on synaptic membranes prepared from naive and morphine tolerant wild type and G(z alpha)-deficient brains. No changes in the affinity of morphine for its receptor or in the density of mu and delta opioid receptors were found between the two groups of mice in the naive or morphine tolerant state. This indicates that the absence of G(z alpha) does not affect opioid receptor affinity or receptor up or down regulation. Our results suggest that the presence of G(z alpha) delays the development of morphine tolerance and represents a possible therapeutic target for improving the clinical use of morphine.

Solh, the mouse homologue of the Drosophila melanogaster small optic lobes gene: organization, chromosomal mapping, and localization of gene product to the olfactory bulb.

The Drosophila melanogaster small optic lobes gene (sol) is required for normal development of the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein products of sol and its human homologue SOLH contain zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal domain of unknown function. Long-distance PCR was used to amplify genomic DNA for Solh, the mouse homologue of sol, following the identification of mouse Solh expressed sequence tags. The nucleotide sequence of the Solh coding region (6.0 kb) was determined. The predicted Solh protein of 1095 amino acid residues shows 89% identity (93% similarity) to the human homologue. Solh was localized by in situ hybridization to band A3.3 on mouse Chromosome 17, in a region of maintained homology with human 16p13.3. Antipeptide antibodies were prepared and verified by demonstration of specific reactivity with recombinant human SOLH protein prepared by in vitro transcription/translation and expression in insect cells using the baculovirus system. The antibodies were used to show that the Solh protein localizes to the olfactory bulb in mouse and rat brain, suggesting that it could have an analogous role in development of sensory system neurons in Drosophila and in mammals.

The regulation of the retrograde axonal transport of (125)I-beta nerve growth factor is independent of calcium.

Calcium has been shown to play a major role in the regulation of endocytosis and exocytosis of synaptic vesicles and retrograde axonal transport of proteins. The role of calcium in the regulation of neurotrophin retrograde axonal transport is unknown. This study aimed to determine if calcium plays a role in the uptake and retrograde axonal transport of (125)I-beta nerve growth factor ((125)I-betaNGF) within sympathetic neurons innervating the iris by comparing it with (125)I-anti-dopamine beta hydroxylase (anti-DBH). The nonspecific voltage-sensitive calcium channel (VSCC) antagonists, cadmium (200 nmol/eye) and nickel (100 nmol/eye) reduced the amount of (125)I-anti-DBH retrograde axonal transport by 90 and 70%, respectively. In contrast, cadmium (200 nmol/eye) had no effect on (125)I-betaNGF retrograde axonal transport, while nickel (100 nmol/eye) caused a significant increase in the amount transported to the ganglia. The L-type VSCC antagonist nifedipine (10 nmol/eye) and N-type VSCC antagonist omega-conotoxin (1.5 nmol/eye) both had no effect on (125)I-anti-DBH retrograde axonal transport which suggests that these types of calcium channels are not involved in the exocytosis/endocytosis of anti-DBH containing vesicles. Thapsigargin (0.2 nmol/eye), an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPases also significantly inhibited (125)I-anti-DBH transport but had no effect on (125)I-betaNGF retrograde transport. This suggests that (125)I-anti-DBH and (125)I-betaNGF are internalized into different vesicle types and that the endocytosis and retrograde axonal transport of (125)I-betaNGF are not dependent upon calcium.

Evidence for phosphatidylinositol 4-kinase and actin involvement in the regulation of 125I-beta-nerve growth factor retrograde axonal transport.

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled betaNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I-betaNGF retrograde transport. PAO (20 nM to 200 microM) also inhibited NGF-dependent survival of both sympathetic and sensory neuronal populations. F-actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-betaNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-betaNGF retrograde axonal transport in vivo.