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Journal of Virology, May 2009, p. 4995-5004, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02225-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Receptor-Dependent and -Independent Axonal Retrograde Transport of Poliovirus in Motor Neurons ^,

Seii Ohka,^1* Mai Sakai,¹ Stephanie Bohnert,² Hiroko Igarashi,¹ Katrin Deinhardt,^2, Giampietro Schiavo,² and Akio Nomoto¹

Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,¹ Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom²

Received 22 October 2008/ Accepted 18 February 2009

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Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic (Tg) mice carrying the human PV receptor (hPVR/CD155) gene. We have previously demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerves of hPVR-Tg mice and that intramuscularly inoculated PV causes paralytic disease in an hPVR-dependent manner. Here we showed that hPVR-independent axonal transport of PV was observed in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Using primary motor neurons (MNs) isolated from these mice or rats, we demonstrated that the axonal transport of PV requires several kinetically different motor machineries and that fast transport relies on a system involving cytoplasmic dynein. Unexpectedly, the hPVR-independent axonal transport of PV was not observed in cultured MNs. Thus, PV transport machineries in cultured MNs and in vivo differ in their hPVR requirements. These results suggest that the axonal trafficking of PV is carried out by several distinct pathways and that MNs in culture and in the sciatic nerve in situ are intrinsically different in the uptake and axonal transport of PV.

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In humans, paralytic poliomyelitis results from the invasion of the central nervous system by circulating poliovirus (PV), probably via the blood-brain barrier. This conclusion is supported by the finding that circulating PV after intravenous inoculation in mice appears to cross the blood-brain barrier at a high rate in a human PV receptor (hPVR/CD155)-independent manner (44). After the virus enters the central nervous system, it replicates in neurons, especially in motor neurons (MNs), inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, a neuron-specific pathway has been reported in humans (31), monkeys (18), and PV-sensitive transgenic (Tg) mice carrying the hPVR gene (34, 37). This neuron-specific pathway appears to be important in causing "provocation poliomyelitis," which is triggered by injuries after PV ingestion (11). Using differentiated PC12 cells and a PV-sensitive Tg mouse line, we have shown that intramuscularly inoculated PV is taken up by endocytosis at synapses.

hPVR is a member of the immunoglobulin (Ig) superfamily, with three linked extracellular Ig-like domains, followed by a membrane-spanning domain and a cytoplasmic domain. Two membrane-bound forms ( and ) and two secreted forms (β and ) of hPVR derived by alternative splicing are likely to be expressed in human cells (23). Membrane-bound hPVRs are considered to play important roles in the early steps of infection, such as the binding of the virus to the cell surface, its entry into the cell, and the uncoating of the virus. The N-terminal Ig-like domain harbors the sites for PV binding, and anti-hPVR monoclonal antibodies (MAbs) directed against this region block PV infection (9, 24, 39).

hPVR has the ability to alter the conformation of PV from the 160S intact infectious particle to a 135S particle from which the viral capsid protein VP4 is missing (2, 29). PV-related materials recovered from the sciatic nerves of PV-sensitive Tg mice after intramuscular inoculation with PV were mainly composed of intact 160S virions. The amount of 160S particles recovered was greatly reduced by coinjection with MAb p286, which specifically recognizes hPVR (34). Thus, most of the intramuscularly inoculated PV is incorporated into the sciatic nerves of PV-sensitive Tg mice as intact particles in an hPVR-dependent manner. This surprising finding might be due to either of two alternative, yet not mutually exclusive, possibilities: (i) a small number of PVRs bound per virion does not result in a conformational change in the viral capsid with a loss of VP4, but it is sufficient to induce endocytosis of the virus on the cell surface, or (ii) a cellular inhibitor(s) of PV uncoating may exist in the endocytic pathway responsible for PV uptake and transport in Tg mice (34).

This mouse strain also allowed us to demonstrate that PV inoculated into the calf was incorporated into the sciatic nerve and retrogradely transported through the axons as intact virion particles. Furthermore, PV dissemination via the neural pathway has been found to rely on a fast retrograde axonal transport system and was inhibited by MAb p286 (34). Moreover, the efficient direct interaction of the hPVR cytoplasmic domain with Tctex-1, a light chain of cytoplasmic dynein (21), has been suggested to play an important role in retrograde transport, together with microtubule integrity (33). Cytoplasmic dynein, a minus-end-directed microtubule-based motor complex (13, 14, 17, 43), is implicated in the transport of early and late endosomes, lysosomes, synaptic vesicles, and endoplasmic reticulum along microtubules (1, 8, 13, 14, 17, 43). Notwithstanding the recent progress in the understanding of PV trafficking, the molecular determinants of the axonal transport of PV in MNs have not yet been elucidated.

Despite the importance of axonal retrograde transport in health and disease, the direct visualization of retrograde transport and its quantitative analysis have been hampered by the lack of a reliable assay for living MNs. Such an assay was established in MNs by using a nontoxic fluorescent fragment of tetanus toxin (TeNT H_C), which binds to MNs and is retrogradely transported (28). Here, we applied this assay to the visualization of PV in living MNs.

We employed hPVR-Tg and non-Tg mice, together with cultured MNs isolated from these mice, to clarify the mechanisms of axonal retrograde transport of PV. Experiments involving cultured MNs showed that the entry and axonal transport of PV are strictly hPVR dependent. However, hPVR-independent axonal transport of PV can be observed in non-Tg as well as in hPVR-Tg mice, suggesting that multiple axonal transport routes for PV are present in vivo.

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Viruses and cells. The virulent Mahoney strain [PV1(M)OM] and the attenuated Sabin 1 strain [PV1(Sab)IC-0] of type 1 PV, derived from the infectious cDNA clones pOM1 (40) and pVS(1)IC-0(T) (22), respectively, were used in this study.

African green monkey kidney cells were grown in Dulbecco modified Eagle medium supplemented with 5% newborn calf serum and were used for the preparation of viruses, transfection experiments with the RNAs transcribed from infectious cDNA clones, and plaque assays. Suspension-cultured HeLa S3 cells were maintained in RPMI 1640 medium supplemented with 5% newborn calf serum and were used for preparation of [³⁵S]methionine-labeled or fluorescently labeled virus.

Rodents. IQI mice and their hPVR-Tg mouse line, IQI-PVRTg21 (25, 26), in the hemizygous stage were used as an animal model for studying the mechanisms of PV transport by the neural pathway. All mice were 6 to 10 weeks of age. For the preparation of MNs in culture (3, 12), embryonic day 14 (E14) Sprague-Dawley rat embryos or E13 IQI-PVRTg21 or C57BL/6-PVRTg21 mouse embryos were used. Mice and rats were treated according to the guidelines for the care and use of laboratory animals of the University of Tokyo. All mice and rats used were free from specific pathogens.

Antibodies. For PV immunohistochemistry in the sciatic nerve, 22.6 µg of mouse anti-hPVR MAb p286 (34) was intramuscularly coinjected with a virus suspension. In the case of coinjection with [³⁵S]methionine-labeled virus, as much as 42.2 µg of the MAb was added to a virus suspension. For competition studies in living MNs, 5.5 µg of the MAb was added to 100 µl of Neurobasal medium without phenol red (Invitrogen). For the detection of PV, a rabbit anti-PV type 1 hyperimmune serum, followed by a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody, was used.

Intramuscular inoculation. The precise method for intramuscular inoculation has been described previously (34). Briefly, after the mice had been anesthetized with an intraperitoneal inoculation of 300 to 400 µl of ketamine (10 mg/ml) and xylazine (0.2 mg/ml) in saline, 5 µl of PV was intramuscularly inoculated into each of four points on the left calf of each Tg mouse with a Hamilton microsyringe. A total of 20 µl of the virus suspension containing 2.7 x 10⁷ PFU of PV was used for immunohistochemistry. In the case of inoculation with [³⁵S]methionine-labeled virus, the nerve was ligated tightly with silk thread near the junction of the thighbone and pelvis as described previously (15, 16), and 5 µl of the labeled virus was inoculated into each of four points on the left calf of each mouse. A total of 20 µl of the virus suspension contained 5 x 10⁶ PFU (0.37 µCi).

Immunohistochemistry. The sciatic nerve was removed at a distance of ca. 2 cm from the inoculation points and was embedded. Sections, fixed with acetone-methanol (3:2) at room temperature, were reacted with rabbit hyperimmune serum against PV type 1 at 37°C for 2 h and were then treated with goat anti-rabbit IgG conjugated with fluorescein isothiocyanate at 37°C for 2 h, as previously described (34). The sections were mounted with 80% (vol/vol) glycerol and were analyzed by confocal laser scanning microscopy (Carl Zeiss MicroImaging).

Sucrose density gradient centrifugation. One and one-half hours after the intramuscular inoculation with [³⁵S]methionine-labeled PV, a portion of the sciatic nerve spanning the ligation and a point 5 mm from the site of PV injection was homogenized at 4°C in phosphate-buffered saline (8 g of NaCl, 0.2 g of KCl, 1.15 g of Na₂HPO₄, and 0.2 g of KH₂PO₄ per liter) containing 1% Nonidet P-40 and 0.1% bovine serum albumin fraction V (Sigma-Aldrich Co.). After centrifugation to remove cellular debris, the supernatant was applied to a 15-to-30% sucrose density gradient (34) and centrifuged at 39,000 rpm for 2 h at 4°C in a Beckman SW41 rotor. The radioactivity of each fraction was measured in a liquid scintillation counter.

Purification of radiolabeled PV. [³⁵S]methionine-labeled Mahoney virus was grown in HeLa S3 cells in suspension in a methionine-free medium containing [³⁵S]methionine (44). Virus in cytoplasmic extracts of infected cells was purified by using DEAE-Sepharose CL-6B (GE Healthcare Bio-Sciences KK), followed by two cycles of CsCl equilibrium centrifugation (44). The specific radioactivity of purified radiolabeled Mahoney virus was calculated to be 7.3 x 10^–8 µCi/PFU.

Fluorescent labeling of PV. PV was purified by a protocol described previously (20). HeLa S3 cells were infected with Mahoney or Sabin 1 virus at a multiplicity of infection (MOI) of 10. The cells were harvested at 7 h postinfection for Mahoney virus or at 8 h postinfection for Sabin 1 virus, and the virus was purified from cytoplasmic extracts of the infected cells by using DEAE-Sepharose CL-6B, followed by sucrose density gradient and CsCl equilibrium centrifugation. Purified virus was desalted by gel filtration on a PD-10 column (GE Healthcare Bio-Sciences KK) equilibrated with PBS(–) (per liter, 8.00 g NaCl, 1.15 g Na₂HPO₄, 0.20 g KCl, 0.10 g MgCl₂·6H₂O, 0.20 g KH₂PO₄ [pH 7.4]). The PV concentration was determined by measuring the absorbance at 260 nm; 1.0 optical density unit was regarded as equivalent to 9.4 x 10¹² virions. Virus labeling was based on a protocol kindly provided by Lucas Pelkmans (35) and described previously (32). Briefly, PV (0.4 mg at 0.4 mg/ml) was labeled with 0.39 µl of Alexa Fluor-succinimidyl ester (10 mg/ml in dimethyl sulfoxide) according to the manufacturer's instructions (Invitrogen). The labeled virus was repurified on NAP5 columns (GE Healthcare Bio-Sciences KK), dialyzed against PBS(–), and stored at –80°C. The labeling ratio was 14 mol of dye per mol of virus. The specific infectivity of the labeled virus was not affected.

Retrograde transport assay in living cultured rodent MNs. Five or six days after being plated on glass bottom dishes (MatTek Corporation) (3, 12), spinal cord MNs were incubated with 7 x 10⁵ PFU/well of Alexa Fluor 555-labeled strain Sabin 1 at 35.5°C or with 6 x 10⁷ PFU/well of Alexa Fluor 488-labeled strain Mahoney at 37°C for 15 min; then they were washed and imaged by confocal laser scanning microscopy (Carl Zeiss MicroImaging). For the competition assay, MNs were incubated with or without 4.7 x 10⁸ PFU/well of unlabeled PV or an anti-hPVR MAb at 37°C for 5 min; labeled PV and 667 µg/ml of tetramethyl rhodamine (TMR)-conjugated dextran (molecular weight, 3,000; Invitrogen) was then added, and MNs were incubated at 37°C for 15 min and then washed and observed by confocal laser scanning microscopy. Selected samples were incubated with 40 nM Alexa Fluor 555-labeled TeNT H_C (7) in complete medium for 30 min at 37°C, followed by three washes. Cells were placed in a humidified chamber maintained at 37°C, and after 20 min, images were acquired by confocal laser scanning microscopy or wide-field fluorescent microscopy using a Hamamatsu C4742-95 Orcal cooled charge-coupled device camera (Hamamatsu Photonic Systems) controlled by Kinetic Acquisition Manager 2000 software (Andor Technology).

Microinjection. MNs were injected with 0.05 mg/ml of plasmid pPVR-GFP, encoding hPVR-green fluorescent protein (GFP) (33), or plasmid pPVRM-GFP, encoding a mutant form of hPVR (33), between days 5 and 6 in vitro. Eight hours after the injection, MNs were incubated with Alexa Fluor 555-labeled TeNT H_C or PV.

Tracking and data quantification. Vesicle tracking was performed on time lapse sequences as previously described (28). Speed and average speed values were determined by measuring the distance covered by each carrier between two consecutive frames and that between the initial and final tracking points, respectively. Eight-bit images were assembled into Audio Video Interleave movies with Imaris (Bitplane).

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PV shows a wide range of retrograde transport speeds in axons of hPVR-Tg mice. Previously, we reported that the in vivo transport speed of the majority of PV in hPVR-Tg mice is ca. 16 cm/day (ca. 1.9 µm/s) and comprises rates of axonal transport in the range of ca. 4 to ca. 32 cm/day (ca. 0.5 to ca. 3.7 µm/s) (34). To investigate the hPVR-independent transport of PV, the virus was coinjected with the anti-hPVR MAb p286 into the left calves of hPVR-Tg mice, and the rate of viral transport in the sciatic nerve was examined by an immunohistochemical approach (34). Transverse sections of the sciatic nerve were cut 2 cm from the injection point at various times after intramuscular inoculation with the virus and were examined for PV antigens. When 42.2 µg of MAb p286 was coinjected with the virus into hPVR-Tg mice, it took 12 h for the intramuscularly inoculated virus to reach the transection point (Table 1). This dose of MAb was sufficient to significantly diminish the incorporation of the virus into the sciatic nerve in hPVR-Tg mice (34), causing a reduction in the combined rate of PV transport, which also includes the kinetics of PV internalization, in hPVR-Tg mice to ca. 4 cm/day (ca. 0.5 µm/s). This result indicates that the slowest PV transport seen in hPVR-Tg mice in the absence of MAb p286 is likely to be hPVR independent, and the faster PV transport (more than 4 to ca. 32 cm/day; equivalent to more than 0.5 to ca. 3.7 µm/s) is likely to be hPVR dependent. Furthermore, our findings suggest that different transport mechanisms are involved in the axonal transport of intramuscularly inoculated PV in hPVR-Tg mice.

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TABLE 1. PV antigen observed inside axons at 2 cm from the point of injection

PV shows low rates of retrograde transport in the sciatic nerves of non-Tg mice. To investigate hPVR-independent PV transport, the virus was injected into the left calves of non-Tg mice and was detected by immunohistochemical analysis of transverse sections of the sciatic nerve (Table 1). The viral antigen was detected in the sciatic nerve when the sections were prepared from non-Tg mice 12 h postinjection (p.i.), whereas no axons, or only a few, showing PV antigens were detected at 6 h p.i., indicating that the rate of PV transport in non-Tg mice was ca. 4 cm/day (ca. 0.5 µm/s). These results confirm that the rate of hPVR-independent PV axonal transport is lower than that of hPVR-dependent transport in vivo. Moreover, these data show that PV is present inside axons of the sciatic nerve in non-Tg mice (data not shown).

In order to compare these transport rates with those of other molecules undergoing retrograde transport in vivo, the transport of wheat germ agglutinin (WGA) was analyzed by immunohistochemistry. WGA is a lectin that has high affinity for N-acetylglucosamine and N-acetylneuraminic acid moieties present on surface proteins (30) and is efficiently endocytosed (5, 10, 42). It has been shown that most WGA inoculated intramuscularly (into the flexor forelimb muscle) is transported retrogradely to the motor neuron cell bodies located in the ventral horn of the spinal cord in mice (4). As in the experiments described above, transverse sections of the sciatic nerve were examined for WGA antigens by immunohistochemistry (Table 2). WGA was able to reach the transection point in 1.5 h both in hPVR-Tg and in non-Tg mice and was detected at all the time points examined. These results indicate that the rate of WGA transport ranges from ca. 4 to ca. 32 cm/day (from ca. 0.5 to ca. 3.7 µm/s) and that WGA transport is independent of the presence of hPVR, as shown by the results for the MAb p286-WGA coinjection experiments reported in Table 2.

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TABLE 2. WGA antigen observed inside axons at 2 cm from the point of injection

PV was incorporated into the sciatic nerves of non-Tg mice as infectious particles. We have already shown that intramuscularly inoculated PV was incorporated into the sciatic nerves of PVR-Tg21 mice as infectious particles and that the extent of incorporation was reduced by MAb p286 (34). To investigate whether PV is also incorporated into the sciatic nerves of non-Tg mice as infectious particles, [³⁵S]methionine-labeled PV was injected intramuscularly into the lower thighs of non-Tg mice, and the presence of the virus in the sciatic nerve was examined. The mice inoculated with [³⁵S]methionine-labeled virus were sacrificed at 1.5 h p.i., and the sciatic nerves were homogenized and analyzed by sucrose density gradient centrifugation as described in Materials and Methods. As shown in Fig. 1A, the majority of PV-related materials in the sciatic nerve had a sedimentation coefficient of 160S, which corresponds to the intact virion particle (2, 29). The notion that intact virion particles are transported through the axon is strengthened by our finding that a virus suspension recovered from the sciatic nerve was found to be infectious for cultured primate cells (data not shown). This 160S peak was not reduced when 42.2 µg of MAb p286 was coinjected with the virus (Fig. 1B). However, this dose of MAb was sufficient to greatly reduce the incorporation of the virus into the sciatic nerve in hPVR-Tg mice (34). These results suggest that PV can be endocytosed into the neuromuscular junction (NMJ) and transported in vivo in the sciatic nerves of non-Tg mice in an hPVR-independent manner. Intramuscular inoculation of 1 x 10⁶ PFU of the Mahoney strain did not cause neurological symptoms in non-Tg mice, although it led to the initial signs of paralysis in the inoculated limbs 48 h after injection into Tg mice (34). These data indicate that the axonal retrograde transport of PV in non-Tg mice may not result in detectable virus replication and MN death.

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FIG. 1. Analysis of virus-related transport in the sciatic nerves of non-Tg mice. Non-Tg mice with sciatic nerve ligations were inoculated intramuscularly with 5 x 106 PFU of [35S]methionine-labeled Mahoney virus, either alone (A) or mixed with the anti-hPVR MAb p286 (B). Radioactivity was recovered from the sciatic nerves and analyzed by sucrose density gradient centrifugation as described in Materials and Methods. Filled, shaded, and open arrowheads indicate 160S, 135S, and 80S, respectively.

Axonal transport of PV in cultured rat MNs relies on hPVRs. To investigate the molecular mechanisms controlling the axonal transport of PV, we used primary MNs isolated from rat embryos. Differentiated rat pheochromocytoma (PC12) cells have been shown to internalize fluorescently labeled PV in a manner dependent on hPVR-GFP expression (33). MNs microinjected with an hPVR-GFP-expressing plasmid were incubated with Alexa Fluor 555-labeled PV and analyzed by time lapse confocal laser scanning microscopy. hPVR-GFP is targeted to both the axonal and somatodendritic compartments in cultured rat MNs (data not shown). In these cells, fluorescently labeled PV was cotransported with hPVR-GFP in axonal retrograde carriers (Fig. 2A; see also movie S1 in the supplemental material). In contrast, MNs not expressing hPVR-GFP did not internalize or transport labeled PV (Fig. 2C). When unconjugated Alexa Fluor 555 was added to the cells in the same molar amount as that used for the labeled virus, no fluorescently labeled carriers were observed (data not shown). Taken together, these results demonstrate that the entry and axonal transport of PV are specific and rely totally on hPVR expression in cultured rat MNs.

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FIG. 2. Axonal transport of hPVRs and PV in cultured rat MNs. MNs expressing hPVR-GFP (A), hPVRM-GFP (B), or no hPVR (C) were incubated with Alexa Fluor 555-labeled PV and imaged by time lapse confocal microscopy. Carriers containing both hPVRs and PV are shown (arrowheads) in the time series provided. Green shows hPVRs, whereas red indicates the labeled PV. BF, bright-field images. The MN soma is located on the right. Bar, 10 µm.

For the next step, hPVRM-GFP (33) was expressed in cultured rat MNs, and the transport of Alexa Fluor 555-labeled PV was monitored. In the hPVRM mutant, the two basic residues of the Tctex-1 binding motif in the cytoplasmic domain of hPVR, KXXR, were replaced by two alanines (AXXA). hPVRM shows a lower affinity to cytoplasmic dynein (33) than hPVR. Interestingly, labeled PV was retrogradely transported together with hPVRM-GFP, as observed above for hPVR-GFP-expressing MNs (Fig. 2B; see also movie S2 in the supplemental material). These results suggest that the axonal retrograde transport of PV in rat MNs is independent of the ability of its receptor to recruit the dynein motor complex on these axonal transport carriers.

In MNs expressing hPVR-GFP or hPVRM-GFP, we observed axonal vesicles containing hPVRs that did not carry PV even after preincubation of labeled PV with MNs. Interestingly, these organelles displayed bidirectional transport (data not shown), strongly suggesting that in rat MNs, the directionality of the carriers containing only hPVRs differ from that of those containing both hPVRs and its ligand. It is possible that vesicles containing only hPVR carry newly synthesized hPVR along the secretory pathway or that they represent endosomes internalized prior to, or independently of, PV addition. As shown in Fig. 2C, we were not able to observe any PV-positive endosome lacking hPVRs. This observation indicates that PV is transported together with hPVR in cultured rat MNs.

Expression of hPVRM-GFP impairs fast retrograde transport in rat MNs. To assess whether rat MNs in culture expressing hPVR-GFP or hPVRM-GFP show identical axonal transport characteristics, we examined the kinetics of PV transport under these conditions. As shown in Fig. 3, the transport kinetics were reproducibly different. Displacement graphs of representative examples of the carriers are shown in Fig. 3A and B. In cells expressing hPVR-GFP, two speed components that differ in their average transport velocity were estimated. The fast speed component was 1.4 µm/s, whereas the slow component was centered between 0.4 µm/s and 1.0 µm/s (Fig. 3C). In MNs expressing hPVRM-GFP, the relative frequency of the fast speed component was greatly reduced, while that of the slow speed component was increased compared to those observed in MNs expressing hPVR-GFP. These results suggest that the high-affinity interaction of hPVR with cytoplasmic dynein is required to ensure fast retrograde transport of axonal carriers containing both hPVR and PV in cultured rat MNs.

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FIG. 3. Kinetic analysis of axonal transport vesicles containing both hPVRs and PV. The transport kinetics of PV were analyzed in rat MNs expressing hPVR-GFP or hPVRM-GFP. Only vesicles containing both hPVRs and PV were analyzed. (A and B) Representative examples of displacement graphs for vesicles containing hPVR-GFP (A) or hPVRM-GFP (B). (C) The speed distribution of these carriers in MNs expressing hPVR-GFP (pink squares) or hPVRM-GFP (green circles) is shown. The total number of movements was 134, and 22 independent carriers were observed, in two independent experiments for hPVR-GFP. The total number of movements was 321, and 57 independent carriers were observed, in two independent experiments for hPVRM-GFP.

TeNT H_C and hPVRs share retrograde transport carriers in rat MNs. To correlate the axonal transport features of hPVR with an established retrograde transport cargo in rat MNs, we compared the dynamics of hPVR-GFP and hPVRM-GFP with that of the fluorescently labeled binding fragment of tetanus toxin (TeNT H_C). TeNT H_C enters the nervous system at the NMJ, where it is internalized and retrogradely transported along the MN axons, following a pathway shared with neurotrophins and their receptors (7). Rat MNs were incubated with TeNT H_C and, after washing, were directly imaged. As reported previously, TeNT H_C underwent fast retrograde axonal transport following established kinetics (Fig. 4). Interestingly, in MNs expressing hPVR-GFP, both hPVR-GFP and TeNT H_C colocalized in axonal carriers that were transported retrogradely (data not shown). Similarly, simultaneous transport of hPVRM-GFP and TeNT H_C was observed (data not shown). These results suggest that hPVRs are cotransported in axonal carriers containing not only PV but also TeNT H_C. With regard to the transport kinetics, the speed distribution profile of carriers containing both hPVR-GFP and TeNT H_C was not dramatically different from that of organelles containing TeNT H_C alone in terms of the positions of the average speed components, although the slower component was slightly increased in hPVR-GFP-containing carriers (Fig. 4). In contrast, carriers containing both hPVRM-GFP and TeNT H_C (Fig. 4) displayed much slower kinetics than hPVR-GFP-containing organelles, with the slower speed component contributing to the majority of the speed profile of these organelles (Fig. 4). These results suggest that expression in rat MNs of an hPVR mutant with an impaired Tctex-1 binding site influences the transport kinetics of TeNT H_C in this system similarly to that of PV. These findings indicate that hPVR plays a functional role in the traffic of TeNT H_C-positive carriers, and they raise the possibility that hPVR is a main dynein-interacting protein with a primary role in axonal retrograde transport.

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FIG. 4. Kinetic analysis of axonal transport vesicles containing both hPVRs and TeNT HC. Shown are speed distribution profiles of carriers containing TeNT HC in control rat MNs (orange triangles) or MNs expressing hPVR-GFP (pink squares) or hPVRM-GFP (green circles). Only vesicles containing both hPVRs and TeNT HC were analyzed in MNs expressing hPVRs. For the control, the total number of movements was 482, and 78 independent carriers were observed; for hPVR-GFP, the total number of movements was 149, and 38 independent carriers were observed; for hPVRM-GFP, the total number of movements was 123, and 33 independent carriers were observed.

Axonal retrograde transport of PV is dependent on hPVR in hPVR-Tg mice. To assess whether PV can be transported in isolated MNs of hPVR-Tg mice, the transport of a fluorescently labeled virus was tested in MNs isolated from this mouse strain. Alexa Fluor 488-labeled PV was added to hPVR-Tg mouse-derived MNs in culture and was imaged by confocal laser scanning microscopy. As previously observed in rat MNs expressing hPVR-GFP, the untagged hPVR showed a nonpolarized distribution in spinal MNs isolated from hPVR-Tg mice (data not shown). As expected, fluorescent PV was retrogradely transported in the axons of these MNs in culture. This result indicates that the axonal transport pathway for PV observed in hPVR-Tg mice is preserved in isolated MNs. When Alexa Fluor 488-labeled PV was added to spinal MNs isolated from hPVR-Tg mice together with fluorescent dextran (TMR-dextran) as a fluid-phase endosomal marker, distinct populations of vesicles were observed. Whereas one of these vesicle pools contained both fluorescently labeled PV and TMR-dextran (Fig. 5A; see also movie S3 in the supplemental material), a second class of vesicles labeled by fluorescently labeled PV contained undetectable levels of dextran (Fig. 5B; see also movie S4 in the supplemental material). Both types of PV-containing axonal carriers undergo retrograde transport (Fig. 5). These results are in agreement with the PV transport characteristics observed in differentiated PC12 cells (33) and suggest that PV and dextran are incorporated into the same vesicles and cotransported retrogradely in cultured MNs of hPVR-Tg mice. As expected, vesicles containing TMR-conjugated dextran with undetectable or insignificant amounts of labeled PV were also present. Interestingly, these vesicles undergo bidirectional transport (data not shown). The number of carriers for each vesicle pool was as follows: 45 carriers containing both fluorescent PV and dextran, 19 carriers containing PV with undetectable amounts of dextran, and 28 organelles containing dextran with undetectable or insignificant amounts of PV (92 carriers in total in two independent experiments). From these observations, some vesicles containing only fluorescently labeled PV or only TMR-conjugated dextran might exist in cultured MNs.

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FIG. 5. The transport mechanism of PV in isolated MNs derived from hPVR-Tg mice is hPVR specific. (A) Axonal carriers containing both PV and dextran are retrogradely transported in MNs isolated from hPVR-Tg mice. MNs derived from hPVR-Tg mice were incubated with Alexa Fluor 488-labeled PV and TMR-conjugated dextran, and their axonal transport was imaged as described in Materials and Methods (arrowheads). (B) Vesicles containing PV and undetectable levels of dextran were also retrogradely transported in cultured MNs of hPVR-Tg mice (arrowheads). (C) Axonal transport of Alexa Fluor 488-labeled PV was not observed in the presence of unlabeled PV. MNs isolated from hPVR-Tg mice were incubated with Alexa Fluor 488-labeled PV and TMR-conjugated dextran in the presence of unlabeled PV prior to imaging. Arrowheads indicate PV-negative, TMR-positive transported organelles. (D) Axonal transport of Alexa Fluor 488-labeled PV was not observed in the presence of an anti-hPVR MAb. MNs isolated from hPVR-Tg mice were incubated with Alexa Fluor 488-labeled PV and TMR-conjugated dextran in the presence of an anti-hPVR MAb prior to imaging. Arrowheads indicate PV-negative, TMR-positive transported organelles. PV appears green, while dextran appears red. The rightmost panels are the merged images. Cell bodies are located on the right. Bar, 10 µm.

To investigate the specificity of transport of the labeled virus, competition experiments with unlabeled PV were performed. The transport of Alexa Fluor 488-labeled PV strain Mahoney and TMR-conjugated dextran was assessed in MNs derived from hPVR-Tg mice upon incubation with 4.7 x 10⁸ PFU of unlabeled PV strain Mahoney (Fig. 5C). In contrast with the results obtained in the absence of unlabeled PV (Fig. 5A), no transport of labeled PV was detected under these conditions, whereas the transport of TMR-conjugated dextran was unmodified (Fig. 5C; see also movie S5 in the supplemental material). Furthermore, the transport of Alexa Fluor 488-labeled PV, but not that of TMR-conjugated dextran, was blocked by the addition of an anti-hPVR MAb to MNs isolated from hPVR-Tg mice (Fig. 5D; see also movie S6 in the supplemental material). These results indicate that the axonal transport of the fluorescently labeled virus in cultured MNs derived from hPVR-Tg mice is specific and is strictly hPVR dependent.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we employed murine and rat MNs in culture as well as intact sciatic nerves in situ to provide insights into the mechanisms of uptake and axonal retrograde transport of PV. Differentiated MNs in culture are considered to reflect the native characteristics of MNs in vivo. We have recently developed a method to fluorescently label PV virions without altering their biological properties (32). In addition, microinjected hPVR-GFP was successfully expressed in rat MNs. These techniques allowed us to observe fluorescently labeled PV and hPVR-GFP in living MNs and to analyze the characteristics of the axonal retrograde transport of PV.

Using differentiated PC12 cells, we previously showed that hPVRM vesicles loaded with PV move bidirectionally, whereas PV-containing hPVR carriers move strictly retrogradely (33). These data do not overlap with the transport kinetics of hPVRM- and hPVR-containing vesicles observed in rat MNs in this study. This discrepancy might be due to differences in the cells analyzed, as, for example, with the mixed polarity seen in PC12 neurites. MNs in culture are considered to be superior to differentiated PC12 cells in reflecting the original characteristics of MNs in vivo.

Our results with MNs isolated from hPVR-Tg mice show that some vesicles containing only fluorescently labeled PV or only TMR-conjugated dextran might exist in cultured MNs. It is possible that TMR-conjugated dextran can be sorted into different vesicles from labeled PV after the endocytosis in cultured MNs. It is also possible that the vesicle is so small as to allow very little TMR-conjugated dextran in the residual luminal space.

Based on our previous report, the velocity of PV transport in hPVR-Tg mice ranges from ca. 4 to ca. 32 cm/day (from ca. 0.5 to ca. 3.7 µm/s) (34). This closely matches the rate observed in this work for hPVR-dependent PV transport. In contrast, the rates of hPVR-independent uptake and transport of PV in non-Tg mice, and in hPVR-Tg mice in the presence of MAb p286, are comparable to each other and much lower (ca. 4 cm/day; equivalent to ca. 0.5 µm/s). WGA was transported in hPVR-Tg or non-Tg mice at rates ranging from ca. 0.5 to ca. 3.7 µm/s, similar to those of hPVR-dependent PV transport. These results strongly suggest that different systems for PV transport do coexist in the axons of hPVR-Tg mice. In sharp contrast, no hPVR-independent PV transport was observed in cultured MNs under our experimental conditions. Although the reason is presently unknown, this discrepancy may derive from the different conditions occurring in vivo and in primary MNs in culture. At present, we cannot estimate with sufficient precision the MOI for intramuscular inoculation with PV. The actual MOI in vivo might be much higher than that in primary culture because of the anatomical structure of the NMJ, resulting in a high yield of hPVR-independent incorporation of PV in vivo. Alternatively, a still unidentified PV receptor may be expressed at NMJs in vivo, causing a delay in PV uptake or its targeting to an alternative endocytic route engaged only upon stimulation, a condition not investigated in isolated MNs.

TeNT H_C was transported at rates as high as 3.6 µm/s in cultured MNs expressing hPVR or hPVRM, and its speed distribution curves (Fig. 4 and data not shown) fit well with those previously reported (3). Interestingly, the highest rate of TeNT transport has been shown to be 7.5 mm/h (ca. 2.1 µm/s) in vivo (41). According to these results, the rate of TeNT transport in isolated rat MNs correlates well with that observed in vivo. The fastest component of PV transport in cultured rat MNs expressing hPVR-GFP was ca. 2.6 µm/s, although it is a small population. The rate of the PV transport observed in the cultured MNs may correlate with the hPVR-dependent PV velocity in vivo (>0.5 to ca. 3.7 µm/s). The rate of the slow component of the hPVR-independent PV transport seen in vivo (ca. 1.7 mm/h; ca. 0.5 µm/s) (Fig. 6Ac and Bc) is similar to that of the slow component seen in cultured MNs (0.5 µm/s) (Fig. 6Cb). This agreement seems to be a coincidence, since it is unlikely that the mechanisms for hPVR-dependent and hPVR-independent transport are identical. However, it might be possible that the slow component in cultured MNs requires hPVR during endocytosis, whereas its transport may be hPVR independent (Fig. 6Cb). In the presence of an anti-hPVR MAb in hPVR-Tg mice, hPVR-independent transport of PV was observed. Based on this observation, it is therefore possible that similar hPVR-independent transport occurs in hPVR-Tg mice even in the absence of the anti-hPVR MAb and that some of the PV-containing vesicles in hPVR-Tg mice exhibit hPVR-independent slow transport (Fig. 6Ab).

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FIG. 6. Mechanisms for hPVR-dependent and -independent transport of PV in MNs. (A) In hPVR-Tg mice, PV is endocytosed after binding to hPVR (in green). Most of the hPVR-containing vesicles are retrogradely transported in an hPVR-dependent manner (orange membrane carrier). The transport of these vesicles shows fast kinetics. In vivo, hPVR-independent endocytosis and transport of PV also occur (purple membrane organelle), possibly mediated by a still unidentified PV receptor expressed at NMJs. Alternatively, hPVR-independent endocytosis may be promoted by synaptic activity, which occurs in vivo but was not tested in isolated MNs. PV-containing vesicles with or without hPVR can be retrogradely transported in an hPVR-independent manner with slow kinetics. (B) In non-Tg mice, PV is endocytosed and transported with slow kinetics in an hPVR-independent manner. (C) In isolated MNs, only hPVR-dependent PV endocytosis is detected. The hPVR-dependent transport of axonal carriers shows fast kinetics. It might be possible that the slow component in cultured MNs requires hPVR during endocytosis, whereas its transport may be hPVR independent. (D) No endocytosis of PV is detected in control cultured MNs lacking hPVR. The green solid arrows denote hPVR-dependent fast retrograde transport (Aa and Ca), whereas the orange arrows indicate slow retrograde transport (Ab, Ac, Bc, and Cb).

It has been reported that the rate of transport of horseradish peroxidase-conjugated WGA in hamster facial MNs distributes continuously from 1.0 to 1.4 mm/h (0.3 to 0.4 µm/s) to 8.3 to 10.9 mm/h (2.3 to 3.0 µm/s) (19). Although the probes used in this study are different, the rate of WGA transport we observed in the sciatic nerve (from ca. 0.5 to 3.7 µm/s) seems appropriate. It has been reported that retrograde transport of WGA exhibits biphasic kinetics in pike olfactory nerve c-fibers (6). Nevertheless, the biphasic peaks overlapped with each other, and the rate of the WGA transport distributed continuously. Since WGA binds glycoproteins ubiquitously distributed on the cell surface, it is likely that WGA is transported by multiple classes of axonal organelles with overlapping kinetic properties. Compared with WGA-positive carriers, the hPVR-independent transport of PV exhibited only slow kinetics. This result strengthens the possibility that the hPVR-independent uptake of PV at the NMJ is specific and that the unknown PV receptor mentioned above is involved in the hPVR-independent transport of PV in vivo.

Lalli et al. reported a dramatic decrease in the fast (1.15-µm/s) speed component of TeNT H_C transport in MNs treated with the cytoplasmic dynein inhibitor erythro-9-[3-2-(hydroxynonyl)] adenine (EHNA) (36, 38), whereas the intermediate (0.53-µm/s) component was unaffected (27). Similarly, we have observed a decrease in the fast component (1.4 µm/s) of PV transport in cultured MNs expressing hPVRM, which has a reduced affinity with the dynein complex. Taken together, these findings suggest that the high affinity of hPVR with cytoplasmic dynein contributes to the fast component of the axonal retrograde transport of PV in cultured MNs. Although it does not bind PV, the murine homologue of hPVR may play an indirect role in axonal transport of the virus in non-Tg mice, for example, by associating with hPVRM, by competing for dynein recruitment or influencing its endosomal sorting. In this regard, the loss-of-function phenotype for PV transport observed for hPVRM may be enhanced by performing these experiments in a murine homologue of an hPVR-null background.

Residual low levels of PV transport may be present in isolated MNs not expressing hPVR, but they may be undetectable by our experimental system. A possible explanation is that the individual PV virions may be less concentrated in axons lacking hPVR and thus less likely to be observed by direct fluorescence. This seems unlikely, however, given the rather high ratio of incorporation of Alexa Fluor dye into the fluorescent PV (14 mol of dye per mol of virus; see Materials and Methods). Another possibility is that the hPVR-independent route of PV uptake and transport is much less efficient than the hPVR-dependent route, resulting in a very low frequency of transport events in isolated MNs.

 
We thank A. Ohmura for breeding the mice and E. Suzuki for help in the preparation of the manuscript.

This work was supported in part by Grants-in-Aid for Advanced Medical Science Research by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT); a Grant-in-Aid for Scientific Research on Priority Areas; a Grant-in-Aid for Scientific Research (S); a Grant-in-Aid for Young Scientists (B); Human Frontier Science Program, Special Coordination Funds for Promoting Science and Technology; a contracted research allowance, "Research and Development in a New Converting Field Based on Nanotechnology and Materials Science," from MEXT; the Industrial Technology Research Grant Program in 2002 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan; The Naito Foundation; a grant from the Ministry of Health, Labor and Welfare of Japan; and Cancer Research UK.

 
* Corresponding author. Mailing address: Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-5841-3407. Fax: 81-3-5841-3374. E-mail: seii{at}m.u-tokyo.ac.jp

Published ahead of print on 25 February 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, May 2009, p. 4995-5004, Vol. 83, No. 10
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