Engagement of Neurotropic Viruses in Fast Axonal Transport: Mechanisms, Potential Role of Host Kinases and Implications for Neuronal Dysfunction

Much remains unknown about mechanisms sustaining the various stages in the life cycle of neurotropic viruses. An understanding of those mechanisms operating before their replication and propagation could advance the development of effective anti-viral strategies. Here, we review our current knowledge of strategies used by neurotropic viruses to undergo bidirectional movement along axons. We discuss how the invasion strategies used by specific viruses might influence their mode of interaction with selected components of the host’s fast axonal transport (FAT) machinery, including specialized membrane-bounded organelles and microtubule-based motor proteins. As part of this discussion, we provide a critical evaluation of various reported interactions among viral and motor proteins and highlight limitations of some in vitro approaches that led to their identification. Based on a large body of evidence documenting activation of host kinases by neurotropic viruses, and on recent work revealing regulation of FAT through phosphorylation-based mechanisms, we posit a potential role of host kinases on the engagement of viruses in retrograde FAT. Finally, we briefly describe recent evidence linking aberrant activation of kinase pathways to deficits in FAT and neuronal degeneration in the context of human neurodegenerative diseases. Based on these findings, we speculate that neurotoxicity elicited by viral infection may involve deregulation of host kinases involved in the regulation of FAT and other cellular processes sustaining neuronal function and survival

HIV Glycoprotein Gp120 Impairs Fast Axonal Transport by Activating Tak1 Signaling Pathways

Sensory neuropathies are the most common neurological complication of HIV. Of these, distal sensory polyneuropathy (DSP) is directly caused by HIV infection and characterized by length-dependent axonal degeneration of dorsal root ganglion (DRG) neurons. Mechanisms for axonal degeneration in DSP remain unclear, but recent experiments revealed that the HIV glycoprotein gp120 is internalized and localized within axons of DRG neurons. Based on these findings, we investigated whether intra-axonal gp120 might impair fast axonal transport (FAT), a cellular process critical for appropriate maintenance of the axonal compartment. Significantly, we found that gp120 severely impaired both anterograde and retrograde FAT. Providing a mechanistic basis for these effects, pharmacological experiments revealed an involvement of various phosphotransferases in this toxic effect, including members of mitogen-activated protein kinase pathways (Tak-1, p38, and c-Jun N-terminal Kinase (JNK)), inhibitor of kappa-B-kinase 2 (IKK2), and PP1. Biochemical experiments and axonal outgrowth assays in cell lines and primary cultures extended these findings. Impairments in neurite outgrowth in DRG neurons by gp120 were rescued using a Tak-1 inhibitor, implicating a Tak-1 mitogen-activated protein kinase pathway in gp120 neurotoxicity. Taken together, these observations indicate that kinase-based impairments in FAT represent a novel mechanism underlying gp120 neurotoxicity consistent with the dying-back degeneration seen in DSP. Targeting gp120-based impairments in FAT with specific kinase inhibitors might provide a novel therapeutic strategy to prevent axonal degeneration in DSP

Pathogenic SOD1 increases neurofilament phosphorylation.

<p>Phosphorylation of squid neurofilaments (NF) in isolated “sister” axoplasms (see Methods) was analyzed using metabolic labeling experiments with ³²P-γ-ATP. (<b>a)</b> Coomassie Blue staining (CB) shows similar levels of perfused WT-SOD1, G93A-SOD1 and total axoplasmic proteins. Immunoblot analysis (WB) with the NFH antibody SMI-31 confirmed the identity of major phosphorylated bands as NF220 and HMW, major NF subunits in squid axoplasm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065235#pone.0065235-Pant1" target="_blank">[30]</a>. Short (S) and long (L) exposure of autoradiograms (³²P) show increased phosphorylation of NF220 and HMW NF subunits in axoplasms perfused with G93A-SOD1, compared to WT-SOD1. <b>(b)</b> Quantitation of squid NF phosphorylation showed ⋍70% increase in G93A-SOD1 treated axoplasms, compared to those treated with WT-SOD1 (p≤0.01 (#) in a paired t-test). <b>(c)</b> In parallel experiments, kinesin-1 was immunoprecipitated from axoplasms labeled with γ-³²P-ATP in the presence of WT-SOD1 or G93A-SOD1. Both heavy (KHC) and light (KLC) chains of conventional kinesin were phosphorylated. <b>(d)</b> The ratio of counts for G93A-SOD1/WT-SOD1 indicates that KHC labeling increased 31% in G93A-SOD1 axoplasms, compared to WT-SOD1 (significant at p≤0.05 by paired t-test, #). KLC phosphorylation increased by 15%, but was not statistically significant (p = 0.123). n = 7.</p

Pseudophosphorylation of kinesin-1 at S175/S176 inhibits movement of kinesin-1.

<p>To determine the effects of modifying S175 and S176 on kinesin-1function, recombinant GFP-tagged kinesin (KHC⁵⁵⁹) was modified to preclude phosphorylation at these sites (S175AS176A) or to mimic phosphorylation (S175ES176E). <b>(a–f)</b> Stage 3 hippocampal neurons were examined 5 h after co-transfection with GFP-tagged KHC⁵⁵⁹ constructs and a tdTomato construct, which diffuses throughout the cell and allows visualization of neurites (<b>b, d, f</b>). Both wild-type kinesin-1 (KHC⁵⁵⁹ WT, <b>a</b>) and a non-phosphorylatable mutant (KHC⁵⁵⁹ S175A/S176A, <b>c</b>) accumulated efficiently at axonal tips (labeled by arrows) with minimal steady-state labeling of cell bodies (arrowheads). In contrast, pseudophosphorylated mutant KHC⁵⁵⁹ S175E/S176E, <b>e</b>) was mainly present in neuronal cell bodies. Quantitative immunofluorescence analysis shows fraction of total KHC⁵⁵⁹ fluorescence at axon tips for all constructs <b>(g)</b>. Far less phosphomimicking KHC⁵⁵⁹ S175E/S176E constructs accumulated at axon tips than KHC⁵⁵⁹ WT or KHC⁵⁵⁹ S175A/S176A (#: p<0.001; <i>n</i>: 27–43 cells per condition). Bars show mean and standard deviation. Scale bar  = 20 µm.</p

Inhibition of anterograde FAT induced by mSOD1 depends on specific MKKK-MKK interactions.

<p>Co-perfusion of G93A-SOD1 with DVD peptide <b>(a)</b>, but not with the Mixed-Lineage Kinase inhibitor CEP11004 <b>(b),</b> prevents inhibition of FAT induced by G93A-SOD1. DVD peptide prevents activation of MKKs by some MKKKs (n =  number of axoplasms) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065235#pone.0065235-Takekawa1" target="_blank">[60]</a>. These data suggest that the activation of p38 and the inhibition of FAT induced by G93A-SOD1 involves activation of one or more MAPKKKs <i>other</i> than MLKs. <b>(c)</b> The DVD peptide also blocks inhibition of FAT by oxidized WT-SOD1 suggesting that FALS mutant SOD1 and misfolded WT-SOD1 activate a common p38 MAPK pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065235#pone.0065235-Bosco2" target="_blank">[23]</a>.</p

Active p38 α directly phosphorylates kinesin-1 at serines 175–176.

<p><b>(a)</b> Recombinant p38α was incubated with γ-³²P-ATP in the presence (+) or absence (–) of a recombinant protein construct comprising the first 584 amino acids of kinesin-1 (KHC⁵⁸⁴). Coomassie blue staining of gels shows the position of KHC⁵⁸⁴ and p38α. Autoradiogram (³²P) shows ³²P incorporation into KHC⁵⁸⁴ (asterisk), as well as autophosphorylation of p38α (arrowhead). <b>(b)</b> Mass spectrometry studies identified a peptide within the motor domain of kinesin-1 (amino acids 174–188) showing unequivocal evidence of phosphorylation by p38α. Tandem mass spectrometry analysis (MS/MS) by collision-induced dissociation further mapped phosphorylation on both Ser175 and Ser176 (grey box). <b>(c)</b> Sequence alignment shows that serines 175 and 176 (grey box) are conserved among human, mouse and squid sequences for kinesin-1.</p

Active p38 α mimics the effects of pathogenic SOD1 on anterograde FAT.

<p>Effects of active, recombinant p38 isoforms on FAT were evaluated using vesicle motility assays in isolated squid axoplasm. P38α and P38β were perfused at a constant specific activity based on <i>in vitro</i> kinase assays with the ATF-2 substrate. <b>(a)</b> Perfusion of active p38α in axoplasm selectively inhibited anterograde FAT, as did pathogenic SOD1 (compare to Fig. 1b-d). (<b>b)</b> Unlike p38α, p38β inhibited both anterograde and retrograde FAT. (<b>c</b>) Quantitation of values obtained between 30-50 minutes shows that p38α most closely mimicked effects of pathogenic SOD1, suggesting this isoform mediates the effects of mSOD1 on FAT in axoplasm (#: difference significant from WT-SOD1 at p<0.01 by t-test).</p