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september 2010 volume 8 no. 9 www.nature.com/reviews MICROBIOLOGY Nervous travellers The complex journey of viruses and toxins Protein export from malaria parasites Hostile takeover REVIEWS A hitchhiker’s guide to the nervous system: the complex journey of viruses and toxins Sara Salinas*‡, Giampietro Schiavo§ and Eric J. Kremer*‡ Abstract | To reach the central nervous system (CNS), pathogens have to circumvent the wall of tightly sealed endothelial cells that compose the blood–brain barrier. Neuronal projections that connect to peripheral cells and organs are the Achilles heels in CNS isolation. Some viruses and bacterial toxins interact with membrane receptors that are present at nerve terminals to enter the axoplasm. Pathogens can then be mistaken for cargo and recruit trafficking components, allowing them to undergo long-range axonal transport to neuronal cell bodies. In this Review, we highlight the strategies used by pathogens to exploit axonal transport during CNS invasion. Tetanus A spastic paralysis resulting from the inhibition of neurotransmitter release at the level of inhibitory interneurons. This inhibition is caused by intoxication with TeNT (a protein toxin produced by Clostridium tetani), which is taken up at neuromuscular junctions. *Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, 34293 Montpellier Cedex 5, France. ‡ Universités de Montpellier I & II, 34090 Montpellier, France. § Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, London WC2A 3LY, UK. e-mails: [email protected]; Giampietro.Schiavo@ cancer.org.uk; [email protected] doi:10.1038/nrmicro2395 Corrected 13 August 2010 In most cases, pathogen access to the central nervous system (CNS) can be prevented by a neurovascular fil‑ tering system made up of firmly sealed endothelial cells that create a physical barrier known as the blood–brain barrier (BBB; see BOX 1). Nonetheless, numerous patho‑ gens find their way into the CNS. Some viruses, such as HIV‑1, can cross the BBB using cells of the immune system1 (BOX 1), whereas other neurotropic pathogens can reach the CNS using long‑range axonal transport. Indeed, an Achilles heel in the protection of the CNS is the existence of neuronal projections that cross the BBB and functionally connect peripheral organs and tissues with the soma of neurons. Molecules present at nerve terminals can serve as receptors for some pathogens, leading to neuronal uptake and subsequent transport of these organisms. In many cases, neuronal infection as a result of micro‑ bial agents using axonal transport causes impaired neuro‑ nal homeostasis, leading to a range of severe pathologies. For example, tetanus, caused by tetanus toxin (TeNT; also known as TetX), and botulism, caused by botulinum toxins (BoNTs; also known as Bot proteins), are neu‑ rological disorders that result from the impairment of neurotransmission2. Rabies virus (RABV)‑induced neu‑ rodegeneration is another example of the damage that microbial agents can cause3. Theiler’s murine encephalo‑ myelitis virus (TMEV) causes demyelination and is used as a model for multiple sclerosis in rodents4, whereas Borna disease virus (BDV) infections induce defects in synaptogenesis5 as well as behavioural changes (TABLE 1). Because inflammation is frequent in patients with age‑related neurological diseases, a link between viral infections of the CNS and neurodegenerative disorders has been proposed6,7; for example, Parkinson’s disease may be linked to infection with influenza virus or West Nile virus (WNV)8,9. Recent advances in cell biology and cell imaging have led to a better understanding of the mechanisms under‑ lying the entry and transport of several neurotropic path‑ ogens. In this Review, we focus on the different strategies used by viruses and bacterial toxins to reach the CNS by long‑range axonal transport. The mechanisms of entry through the BBB, such as the use of haematopoietic cells to invade the CNS, have been reviewed elsewhere10. Neuronal architecture and axonal trafficking Polarization is an essential aspect of neuron biology, as it allows neuronal networks to receive, integrate and transmit vectorial signals. During development, a specific neurite becomes an axon and the remaining projections become dendrites11. Not surprisingly, the neuronal architecture creates challenges when it comes to delivering signals or cargoes over long distances12. To ensure spatial and temporal control of cargo pro‑ gression along the secretory and endocytic pathways, cytoskeletal tracks and motors must be regulated 13 (FIG. 1) . Motors move cargo in an ATP‑dependent directional process: cytoplasmic dynein is responsi‑ ble for axonal retrograde transport from the synapses to the cell body, whereas members of the kinesin family ensure the delivery of cargo to nerve endings through axonal anterograde transport13 (FIG. 1). Dynein and kinesins NATuRE REVIEWS | Microbiology VoluME 8 | SEPTEMBER 2010 | 645 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Box 1 | The blood–brain barrier and neurotropic pathogens The blood–brain barrier (BBB) is a neurovascular filtering system that allows the brain to be supplied with nutrients such as oxygen and glucose while being protected from potentially toxic molecules that are present in the blood. Under ‘normal’ physiological conditions, the BBB does not allow agents such as bacteria to invade the central nervous system (CNS). Only small, non-polar molecules can diffuse through the BBB, and chemical backbones of such molecules therefore represent chemical templates for many of the drugs that are designed to cross this barrier. The BBB is composed of microvascular endothelial cells that are firmly sealed with tight junctions, creating a physical barrier between the bloodstream and the CNS. Other cell types, such as astrocytes, microglia, pericytes and neurons, provide molecular support to the BBB and form a functional neurovascular unit. The ‘immune-privileged’ status of the CNS does not mean that it is free from immune surveillance. Macrophages and leukocytes can actively migrate through the BBB and may, paradoxically, serve as Trojan Horses for pathogen entry into the CNS. For example, HIV is often associated with neurological dysfunctions that result from the migration of HIV-infected leukocytes through the BBB followed by viral spreading. Ultimately, HIV will also lead to impairment of the BBB. Similarly, measles virus is thought to use lymphoid cells to cross the BBB. Botulism A flacid paralysis resulting from the inhibition of acetylcholine release at neuromuscular junctions, mediated by BoNTs. These toxins act by cleaving the synaptic components that mediate fusion of synaptic vesicles to the plasma membrane. Synaptogenesis Formation of synapses in the central and peripheral nervous systems. This process initiates early in development and continues throughout adulthood. Axonal retrograde transport Traffic of molecules and organelles from nerve terminals to cell bodies. This mechanism is mainly microtubuledependent and involves the molecular motor cytoplasmic dynein. Axonal anterograde transport Traffic of molecules and organelles from cell bodies to nerve terminals. This mechanism is mainly microtubule-dependent and involves molecular motors of the kinesin family. Neurotrophin One of a family of growth factors involved in neuronal growth, differentiation and survival. A classical example is NGF, which is involved in the survival of specific neurons during development. coordinate the transport of several organelles (for exam‑ ple, mitochondria and endoplasmic reticulum), under‑ lining the complex interactions between motor proteins during intracellular transport 14,15. Axonal transport is also modulated by microtubule‑interacting proteins and neuronal subcompartments such as the axon initial segment (FIG. 1), which regulates the targeting of cargo to the axons16. Moreover, to ensure fast and restricted control of protein synthesis in rapidly expanding areas such as growth cones or during synaptic plasticity and injury responses, mRNAs and microRNAs are spe‑ cifically transported in neurites, allowing neurons to synthesize proteins on demand locally17–19. Axonal retrograde transport also allows peripheral signals to be translated into nuclear responses. For example, receptors that are activated by target‑derived neurotrophins during development create ‘signalling endosomes’, which contain neurotrophin receptor complexes as well as downstream‑activated molecules such as extracellular signal‑regulated kinase 5 (ERK5; also known as MAPK7) and the cyclic AMP‑responsive element‑binding (CREB) transcription factors20. The delivery of signalling organelles to the soma ensures the survival of neurons that have reached a physiological target, whereas cells that do not connect with appropri‑ ate targets undergo apoptosis21. During adulthood, this mechanism is maintained in specific neuronal popula‑ tions that rely on neurotrophins for survival. It is there‑ fore not surprising that environmental factors or genetic mutations resulting in impaired axonal transport lead to neuronal death and that disrupted transport is associ‑ ated with several neurodegenerative disorders, including Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and hereditary spastic paraplegia14,22. Neurotropic viruses and toxins Given the essential role of axonal transport, it seems logi‑ cal that viruses and bacterial toxins would take advantage of this pathway to access the CNS. owing to their impact on human health, poliovirus23, RABV24, alphaherpes‑ viruses25 and TeNT26 are among the best characterized pathogenic agents that undergo axonal transport. In addition, axonal transport is used by WNV27,28, TMEV29, measles virus30, BDV31, human enterovirus 71 (REF. 32) and influenza A virus H5N1 (REF. 9), among others (TABLE 1). Moreover, BoNT type A (BoNT/A), which was thought to act mainly at neuromuscular junctions (NMJs) and other peripheral nerve terminals, also undergoes retrograde transport in motor neurons, unlike the other BoNTs 33 (TABLE 1) . Interestingly, WNV27 and canine distemper virus34 can enter the CNS through both axonal transport and the BBB, giving rise to distinct neuronal pathologies. other microbial agents — such as measles virus30 — exploit the axonal transport route after passing the BBB. Numerous studies on axonal transport of patho‑ gens have used direct intravitreous, intramuscular or intrasciatic injections of the pathogen; for example, this has been carried out for WNV28, TMEV29 and several adenoviruses35. These sites of delivery may differ from the physiological route of pathogen entry, bypassing some endogenous internalization mechanisms or forc‑ ing ‘unnatural’ tropism. Nonetheless, these approaches have helped to dissect the molecular mechanisms behind the transport of these pathogens and have opened new avenues for their use in gene therapy and neuronal‑ network tracing. Indeed, viral vectors can be delivered into almost any site of the CNS, making them promising tools for gene therapy of the CNS36 as well as for address‑ ing questions about more complex cognitive behav‑ iours (BOX 2). Because of their transynaptic spreading, herpesviruses and rabies viruses have also been used to map transneuronal circuits37,38. Several laboratories use viruses and toxins to characterize factors that are also involved in the regulation of axonal dynamics and that are defective in neurodegenerative diseases. Finally, the advent of ‘cellular microbiology’ in the mid 1990s39 instigated the study of intracellular traffick‑ ing of viruses, bacteria and virulence factors. To date, most studies have focused on epithelial‑like cells40, irre‑ spective of the cellular tropism of the infectious agent. Several seminal studies have identified key cellular proteins and their functions in pathogen trafficking. Interestingly, some of these ubiquitous cellular players also have specialized roles in axonal transport. Entering the central nervous system Binding to nerve terminals is a crucial step in the neu‑ ronal uptake of some microbial agents, but the pathways of entry can be quite different. RABV41 and alphaherpes‑ viruses25 must replicate and spread in non‑neuronal cells before entering peripheral nerves, possibly to boost the chances of a viral particle accessing the CNS. other pathogens and virulence factors, such as BDV 42 and clostridial neurotoxins, can enter neurons directly using endocytic and intracellular trafficking pathways coupled to the axonal machinery (see below). Binding the axonal membrane. To reach the CNS, viruses and toxins can enter different types of nerve endings, such as sensory‑nerve endings and NMJs (after dermal or muscle wounds, respectively). NMJs are spe‑ cialized synapses that connect motor neurons to muscles, 646 | SEPTEMBER 2010 | VoluME 8 www.nature.com/reviews/micro © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | The toolkit of neurotropic viruses and toxins Agent Viral family Entry into the cNS Pathology Herpesvirus Herpesviridae Sensory endings Oral and genital herpes, encephalitis, and keratitis 25 Poliovirus Picornaviridae BBB and NMJs Paralysis 23 Rabies virus Rhabdoviridae NMJs Encephalitis 24 Measles virus Paramyxoviridae BBB and peripheral nerves? Encephalitis 30 West Nile virus Flaviviridae BBB and peripheral nerves Encephalitis and flacid paralysis 27 Bornavirus Bornaviridae Nose and olfactory neuroepithelia Behavioural changes 31 Influenza A virus H5N1 Orthomyxoviridae Peripheral nerves Enterovirus 71 Picornaviridae Peripheral nerves Encephalitis and flacid paralysis Theiler’s murine encephalomyelitis virus Picornaviridae Transfer to oligodendrocytes after axonal transport Inflammation, demyelination and axonal damage Adenovirus Adenoviridae NMJs and occular infections? Brain tumours and encephalitis Tetanus toxin (a clostridial neurotoxin) N/A NMJs Tetanus (spastic paralysis) Botulinum toxin A (a clostridial neurotoxin) N/A NMJs Botulism (flaccid paralysis) gp120 (an HIV envelope protein) and Tat (the HIV transactivator protein) N/A BBB (through HIV-infected leukocytes) Dementia Encephalitis refs 9 33 4 45 2, 26 2 101, 102 BBB, blood–brain barrier; CNS, central nervous system; NMJ, neuromuscular junction. Neuromuscular junction Specialized synapse that connects motor neurons to muscle fibres. Axon terminals contact muscle fibres through motor end plates, which are specialized regions that are responsible for the transmission of electrical signals. Lipid raft Microdomain of the plasma membrane that is enriched in cholesterol and sphingolipids. Certain classes of GPI-anchored and transmembrane proteins acting as virus and toxin receptors seem to be concentrated in these structures. forming a functional motor unit (FIG. 1). NMJs have been intensively studied as a model for both synaptic connec‑ tion and pathogen invasion of the CNS. uptake of patho‑ gens or their agents at NMJs has been described in mice and non‑human primates for poliovirus23,43, RABV44 and clostridial neurotoxins2. Moreover, intramuscular injec‑ tion of canine adenovirus type 2 (CAdV‑2; also known as CAV‑2)35,45 in leg muscles gives rise to preferential motor neuron transduction. Although RABV transport was reported in motor and sensory neurons, data from rodents and primates suggest that NMJs may be the main point of RABV entry 46. This is probably due to the presence of RABV receptors — the neural cell adhe‑ sion molecules (NCAMs) and nicotinic acetylcholine receptors (nAChRs) — at NMJs46. By contrast, sensory‑ nerve endings are the main gateway for herpesvirus infection of the CNS25. Receptors for many pathogens seem to be con‑ centrated at synapses. Synapses are regions with high membrane dynamics owing to the exo‑endocytosis of synaptic vesicles that occurs during stimulation (FIG. 1). Exocytosis of synaptic vesicles and granules occurs mainly at active zones, which typically occupy the centre of synapses47. By contrast, synaptic vesicle endocytosis is thought to occur mainly outside active zones47 and may regulate internalization of the receptors that pathogens exploit to reach the CNS. Specialized regions of the syn‑ apse also concentrate specific molecules. For example, lipid rafts contain several lipids and proteins that interact with viruses (such as cholesterol, which interacts with pseudorabies virus48) or with bacterial toxins (such as polysialogangliosides, which interact with BoNTs and TeNT49). In addition to binding polysialogangliosides, BoNTs and TeNT bind other synaptic‑vesicle proteins, including synaptotagmins, bound by BoNT/B and BoNT/G, and synaptic vesicle 2 proteins (SV2s), bound by BoNT/A, BoNT/E and BoNT/F2 (FIG. 1) (see below). Given the high rate of fusion and recycling of these organelles and their high concentration at nerve termi‑ nals, it is easy to understand why membrane proteins associated with synaptic vesicles might be preferential targets for neurotropic viruses and toxins. In addition to synaptic vesicle components, other classes of molecules found at nerve endings are recog‑ nized by infectious agents. For example, highly con‑ served cell adhesion proteins act as pathogen receptors in numerous species (FIG. 1). This is illustrated by the broad host range of alphaherpesviruses50 and RABV24. Furthermore, members of the same protein family can act as receptors for different viruses. CD155, a member of the immunoglobulin superfamily, serves as a recep‑ tor for poliovirus, and nectin 1 (also known as PVRl1) and nectin 2 (also known as PVRl2), which also belong to the immunoglobulin superfamily, are receptors for members of the Herpesviridae family 51. The interaction of herpesvirus glycoprotein D with nectin 1 initiates viral uptake and is consistent with preferential targeting of the virus to dorsal root ganglia neurons, because nectin 1 NATuRE REVIEWS | Microbiology VoluME 8 | SEPTEMBER 2010 | 647 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Dendrite Somatodendritic compartment Nucleus Axon initial segment Microtubules Axon Motor neuron Dynein Kinesin Retrograde Anterograde – Dynactin Node of Ranvier + Myelin Synaptic vesicle Nectin 1 CD155 NCAM G SV2 CAR Muscle nAChR Herpesvirus Glycoprotein D RABV Sensory-nerve ending (DRG axon) TeNT BoNT Poliovirus CAdV-2 Neuromuscular junction Figure 1 | Neuronal architecture and axonal transport. Neurons are the most Nature Reviews | Microbiology polarized cells of the body, with extensions (axons) that exceed a metre in length in large animals. They are the functional cellular unit of the nervous system. Axons can be enclosed in myelin sheaths (consisting of several layers of cellular membranes originating from oligodendrocytes or Schwann cells), which provide enhanced propagation of electrical signals. Intracellular transport has an essential role in the distribution of neuronal proteins and organelles. Microtubules are polymers of α-tubulin and β-tubulin and form major cytoskeletal tracks that are involved in intracellular transport. Microtubules display mixed polarity in proximal dendrites and are unipolar in axons and distal dendrites. Cytoplasmic dynein is the motor responsible for retrograde transport of cargo from nerve terminals to the cell body, whereas members of the kinesin family move cargo in the opposite direction. Specific subcompartments, such as the axonal initial segment, regulate polarized transport. Neuromuscular junctions and sensory-nerve endings contain membrane molecules that can serve as receptors for viruses and toxins. Some adenoviruses bind the coxsackievirus and adenovirus receptor (CAR; also known as CXADR) at neuromuscular junctions to enter the central nervous system. Similarly, poliovirus binds CD155, and rabies virus (RABV) binds neural cell adhesion molecules (NCAMs) and nicotinic acetylcholine receptors (nAChRs). Some botulinum toxins (BoNTs; also known as Bot proteins) bind synaptic vesicle 2 (SV2) proteins to enter neurons. Sensory-nerve endings of dorsal root ganglia (DRG) neurons contain nectin 1 (also known as PVRL1), a surface protein recognized by glycoprotein D of alphaherpesviruses. CAdV-2, canine adenovirus type 2 (also known as CAV-2); G, ganglioside; TeNT, tetanus toxin (also known as TetX). is present at sensory‑nerve endings but not at NMJs52,53 (FIG. 1). Similarly, binding of poliovirus to NMJ‑localized CD155 may be the first step in the neuronal spread of this virus23,54. The coxsackievirus and adenovirus recep‑ tor (CAR; also known as CXADR), another member of the immunoglobulin superfamily, and NCAM are found at NMJs and act as receptors for adenoviruses35,45 and RABV55, respectively (FIG. 1). In addition, molecules involved in neuronal homeostasis, such as the neuro‑ trophin receptor p75NTR (also known as NGFR), which is recognized by RABV glycoprotein G56, may be ideal targets for pathogen recognition and uptake. Some receptors are involved in transcytosis, a mecha‑ nism that allows the transfer or targeting of ligands from axon terminals to the somatodendritic compartment and then to other juxtaposed cells. This axodendritic path‑ way is mirrored by a somatodendritic‑to‑axonal target‑ ing mechanism that is used by some newly synthesized proteins, such as neuronal‑glial cell adhesion molecule (NgCAM) and tropomyosin‑related kinase B (TrkB; also known as NTRK2)57,58. Both proteins are targeted to axons following ligand‑independent internalization or recycling at the somatodendritic membrane57,58. It is tempting to speculate that similar mechanisms exist to target axonal receptors back to the cell body. Pathogens using these molecules as receptors may exploit this proc‑ ess for their transport to the soma. For example, CAR seems to be constitutively transported in the sciatic nerve without exogenous ligand45 and so may be an ideal means for some adenoviruses and for coxsackievirus B to reach the CNS and cause pathogenesis59,60. Entering neurons through endosomes. Receptor‑ mediated entry provides pathogens with an efficient way of exploiting the regulatory mechanisms that control endocytosis and trafficking. For the most part, as men‑ tioned above, pathogen internalization and sorting have been characterized in epithelial‑like cells61, in which dis‑ tinct endocytic mechanisms have been described. These mechanisms include clathrin‑dependent and clathrin‑ independent uptake, caveolae‑dependent internaliza‑ tion, phagocytosis and macropinocytosis62. In these cells, endocytic vesicles can be retrogradely trans‑ ported to the trans‑Golgi network or recycled to allow internalized molecules to be redirected to the plasma membrane, or they can deliver cargo to lysosomes for degradation62. Although these vesicle transport pathways are mostly conserved in neurons, some notable exceptions exist. For example, the tethering factor early‑endosome antigen 1 (EEA1) is absent in axons, suggesting a dif‑ ference in the mechanisms of early‑endosome sort‑ ing in epithelial‑like cells and axons63. Furthermore, synaptic vesicles are distinct from the early‑endocytic structures found in fibroblasts and vary depending on whether the vesicles are recruited to the readily releasable or the reserve pool64. The contribution of these pools to the uptake and intracellular traffic of pathogens is still unclear. As synaptic vesicles mainly undergo cycles of local exo‑endocytosis, a mechanistic understand‑ ing of the link between these organelles, their trans‑ synaptic shuttling 65 and their long‑range axonal transport needs to be addressed. For example, BoNT/A, which interacts with SV2 proteins to enter NMJs (FIG. 1), is transported to the soma of motor neurons33. Whether the transported BoNT/A is still in the lumen of a syn‑ aptic vesicle, sorted to another endocytic organelle or transported directly in the axoplasm is unknown2. 648 | SEPTEMBER 2010 | VoluME 8 www.nature.com/reviews/micro © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Box 2 | Viral vectors for gene transfer in the central nervous system There are more genes expressed in the mammalian central nervous system (CNS) than in any other tissue, creating a formidable temporal and spatial complexity that confounds functional and therapeutic questions. The choice of gene transfer vector, which can be derived from pathogens of various origins, depends on many parameters, including the required duration of expression, the cell type to be targeted and the cloning capacity of the vector36. Four major CNS vector platforms are briefly described below. Adenoviruses Adenoviruses are non-enveloped DNA viruses. Adenovirus vectors have cloning capacities of ~30 kb, can be purified into pure high titres (>1013 physical particles per millilitre) and lead to long-term expression (in the order of years) in vivo without integration. In vivo, human adenovirus serotype 5 vectors can transduce ependymal cells, oligodendrocytes and neurons, but they preferentially transduce astrocytes in the brain parenchyma. Limited retrograde transport has been reported. Vectors derived from canine adenovirus type 2 preferentially transduce neurons, and axonal retrograde transport of these vectors is particularly efficient. Adeno-associated viruses Adeno-associated viruses (AAVs) are non-enveloped single-stranded-DNA viruses of the family Parvoviridae. Most serotypes have a cloning capacity of ~5 kb. Human and non-human primate serotypes transduce a wide range of cells in the CNS, including neurons. The ability to be transported by axonal retrograde transport varies depending on the serotype. The duration of transgene expression also seems to vary depending on the serotype, the target cells and the host. The serotypes that preferentially transduce neurons can lead to long-term expression in vivo. Herpes simplex virus Herpes simplex virus type 1 (HSV-1) is an enveloped DNA virus. Replication-incompetent vectors (~108 infectious particles per millilitre) efficiently express transgenes in central and peripheral neurons. Amplicon vectors have a theoretical cloning capacity of 150 kb and efficiently express transgenes in central and peripheral neurons. The enormous potential of HSV vectors has been slow to come to fruition, because of their immunogenicity and cytotoxicity. lentiviruses Lentiviruses are enveloped retroviruses belonging to the family Retroviridae. They have a cloning capacity of ~8 kb and can readily be pseudotyped with surface glycoproteins from numerous other viruses. Pseudotyping with envelops from vesicular stomatitis virus leads to a preferential infection of neurons in the rodent brain. Lentiviruses pseudotyped with rabies virus protein G undergo efficient retrograde transport in neurons after intramuscular injections. Specific endocytic pathways at nerve terminals can provide direct access to axonal retrograde transport. The binding fragment of TeNT (TeNT HC)66 (BOX 3) is internalized along with CAdV‑2 and CAR45 in clathrin‑ coated vesicles and progresses from Rab5‑positive to Rab7‑positive endocytic compartments, which undergo axonal transport (see below). Similarly, poliovirus and CD155 are co‑internalized in endocytic organelles that become coupled to the axonal transport machinery 43,67, whereas p75NTR, which is used by RABV, can be taken up through a similar pathway involving clathrin68. Microtubule-organizing centre Site of microtubule nucleation in eukaryotic cells; it organizes flagella, ciliae and spindle poles, and it is closely associated with the Golgi apparatus. Direct membrane fusion and endosomal escape. The internalization of pathogens does not always involve entry or progression through the endocytic pathway. Some enveloped viruses, such as alphaherpesviruses, undergo pH‑independent fusion with the plasma membrane25,69 and enter the cytoplasm ‘naked’. In the axoplasm, the exposed tegument proteins may recruit adaptor proteins and motors to be transported along microtubules. other viruses, such as RABV, undergo pH‑dependent fusion with the membrane at pH <6.4 (REF. 70). This observation implies that certain viruses escape endocytic organelles at later stages of transport, when the pH is optimal for their fusion (see below). Indeed, neuronal transport of fully enveloped RABV in endosomes has been described71. Non‑enveloped viruses can also trigger endosomal lysis. In epithelial cells, some adenoviruses rapidly escape early endosomes after acidi‑ fication and are then transported to the microtubuleorganizing centre by direct recruitment of dynein72. It is likely that the exit point from the endosomal pathway is an important difference between viral transport in epithelial cells and neurons, because most of the axonal transport of CAdV‑2 occurred in intact endosomes45. However, this does not exclude the possibility that a minority of virions escape axonal endosomes and traf‑ fic by interacting directly with motors, as suggested by studies in epithelial cells73,74. Long-range axonal transport of pathogens Why is motor‑based axonal transport a key mecha‑ nism for CNS invasion by pathogens? The large dis‑ tances separating cell bodies from nerve terminals mean that neither viruses nor endogenous molecules can rely on passive diffusion. Indeed, it was calcu‑ lated that viral transport through diffusion would take hundreds of years per centimetre of cytoplasm75. By contrast, molecular motors ensure the transport of cargoes at speeds exceeding several micrometres per second (equivalent to 3–10 millimetres per hour)14. It is likely that neurotropic viruses and toxins were therefore selected, in evolutionary terms, owing to their ability to take advantage of the axonal transport machinery. Recruitment of motors. Members of the Herpesviridae family — for example, herpes simplex virus (HSV) and pseudorabies virus — are classical examples of pathogens that can directly recruit molecular motors. Early studies showed the crucial role of microtubules in herpesvirus transport76, and live‑cell imaging in the giant squid axon NATuRE REVIEWS | Microbiology VoluME 8 | SEPTEMBER 2010 | 649 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Box 3 | Clostridium spp. toxins Tetanus toxin (TeNT; also known as TetX) and botulinum toxins (BoNTs; also known as Bot proteins) are the most potent neurotoxins affecting humans. They are produced by several Clostridium species and cause spastic (TeNT) and flaccid (BoNTs) paralysis. The high toxicity is due to their high-affinity binding to neuronal membranes and their specific cleavage of synaptic SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein (SNAP) receptor) proteins. TeNT and the seven serotypes of BoNT (types A–G) share sequence and structure homologies. They are each synthesized as a single-chain, inactive polypeptide of 150 kDa, which is cleaved by cellular proteases to give rise to a 100 kDa heavy chain (H chain) and a 50 kDa light chain (L chain). The H chain is functionally divided in two 50 kDa parts, the amino-terminal (HN) fragment, which is involved in pH-dependent membrane translocation, and the carboxy-terminal (HC) fragment, which is responsible for neuronal binding. The L chain bears an endopeptidase activity specific for members of the SNARE family, which are key regulators of the fusion of synaptic vesicles with the plasma membrane. allowed one of the first visualizations of viral axonal transport77,78. Several HSV‑1 and pseudorabies virus pro‑ teins can interact with dynein or dynactin25. Incoming capsids are associated with the dynein–dynactin com‑ plex, and their transport is blocked by destabilization of this complex, triggered by dynamitin overexpression79. Several alphaherpesvirus proteins associate with mem‑ bers of the dynein complex: pul34 interacts with cyto‑ plasmic dynein 1 intermediate chain 1 (REF. 80), whereas the viral helicase pul9 contains the dynein light chain DlC8 (also known as DYNll1) binding site, KSTQT, and associates with DlC8 in vitro81. However, the rel‑ evance of these interactions for the retrograde transport of herpesviruses is unclear, because these viral proteins are not found in mature virions25. A more relevant find‑ ing, in the context of transported virions, is the demon‑ stration that HSV‑1 capsid protein pul35 (also known as VP26) interacts with dynein axonemal light chain 4 (RP3; also known as DNAl4) and dynein light chain TCTEX1 (also known as DYNlT1)82. Interestingly, cap‑ sids lacking pul35 or most of the tegument proteins retained their retrograde transport capacity83,84, suggest‑ ing that other tegument proteins associated with entering capsids, such as pul25, pul36 and pul37 (REFS 25,85), may mediate retrograde transport. Dynein is not the only motor recruited by alphaherpes‑ viruses: the HSV‑1 tegument protein uS11 can bind conventional kinesin heavy chain (KIF5B) in vitro 86. Whereas retrograde transport is the key process in CNS invasion by herpesviruses, bidirectional transport was observed during both entry and egress in sensory neu‑ rons87,88. Not surprisingly, the average speed of retrograde traffic is higher during viral entry, whereas anterograde transport becomes dominant during viral egress, sug‑ gesting an efficient regulation of motor coordination and/or recruitment (see below). Although the mecha‑ nism underlying this phenomenon is still unclear, it may be due to a difference in the capsid composition dur‑ ing these two phases25,88,89. Interestingly, DlC8‑binding domains are also present in other viral components81, such as RABV phosphoprotein81,90. However, depend‑ ing on the immune status of mice, mutations in this domain of the viral proteins had contrasting effects on the pathogenicity of the virus24,91. A study using RABV with a deletion in the DlC8‑binding domain of phos‑ phoprotein showed that this mutant had normal CNS access, but early transcription of viral genes in neurons was strongly impaired, suggesting a role for DlC8 during neuronal replication of the virus rather than in axonal transport 92. Moreover, RABV protein G, which binds to p75NTR (REF. 56), induced axonal retrograde transport of pseudotyped lentivirus93; it is therefore unlikely that the interaction between RABV phosphoprotein and host DlC8 has a crucial role in the neuronal transport of the virus. Linking receptors to motor complexes. one of the sim‑ plest ways for a pathogen to access the CNS is to bind a synaptic receptor that can be internalized and directly recruit motors. The poliovirus protein CD155 has a TCTEX1‑binding site that allows the protein to recruit the dynein–dynactin complex and results in the trans‑ port of endosomes containing the virus54,94. In PC12 cells, direct interaction between TCTEX1 and the cyto‑ plasmic domain of CD155, as well as transport studies with CD155 mutants that cannot bind TCTEX1, sug‑ gested that this interaction is required for the retrograde transport of poliovirus and its receptor 54. However, a recent study showed that some CD155‑independent transport of poliovirus may occur in mice expressing human CD155, suggesting that parallel trafficking routes may be exploited by pathogens in vivo67. Pathogens hitchhiking on long-range vesicular transport pathways. Endocytic and exocytic vesicles are constantly shuttled in axons and therefore provide a continuous source of membranes and signals. Some pathogens can access these organelles to spread in the CNS. For example, TeNT exploits the vesicular pathway used by neurotrophins such as β‑nerve growth factor (NGF) and brain‑derived neurotrophic factor (BDNF), and their receptors p75NTR and TrkB, to reach the cell body 66,95 (FIG. 2). These endocytic structures are also responsible for the receptor‑dependent axonal trans‑ port of poliovirus67 and CAdV‑245 (FIG. 2). Importantly, these axonal endosomes have a lumenal pH of close to neutral45,96, which allows cargo to be transported over long distances in a protective environment, precluding pH‑induced conformational changes and avoiding deg‑ radation. This finding could also explain why CAdV‑2 is found in the lumen of endosomes in axons but in the cytoplasm of epithelial cells, in which the pH drop that occurs in early endosomes is linked to viral escape72,97. RABV could also use neutral endosomes to be trans‑ ported over long distances, as the pH required for viral fusion with the membrane is pH <6.4 (REF. 70). Neutral axonal endosomes could therefore provide a multi‑ functional transport pathway for ligands with distinct somatodendritic fates. Pathogenic proteins and axonal transport. Endogenous pathogenic proteins also traffic in the CNS. The glycosyl phosphatidylinositol‑anchored prion protein (PrP) can switch from a non‑pathogenic, protease‑sensitive form (PrPC) to a pathogenic, protease‑resistant, aggregated 650 | SEPTEMBER 2010 | VoluME 8 www.nature.com/reviews/micro © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS p75NTR TrkB CD155 Herpesviridae BDNF Egress Entry Dynein Kinesin CAdV-2 RABV or Poliovirus TeNT CAR + G ? Dynactin Microtubules – Retrograde Mitochondria Anterograde + Figure 2 | Axonal transport of viruses and toxins. Microbial agents exploit several of the mechanisms used by endogenous organelles such as mitochondria and endocytic vesicles to be transported in the nervous system. Despite Nature Reviews | Microbiology some unresolved controversies, studies addressing the transport of members of the Herpesviridae family are shedding light on how the viruses can access and spread in the central nervous system after entry at peripheral nerve endings. Viral proteins — in particular, tegument components — can bind cytoplasmic dynein and play a part during axonal transport or virus assembly. Viral egress may involve fully formed, enveloped virions, or it may take place with non-enveloped particles and separate capsids (consisting of tegument proteins and viral membranes), with the assembly of virions occurring in growth cones. Tetanus toxin (TeNT; also known as TetX) enters a dynein-dependent vesicular pathway that is regulated by the small GTPase Rab7 and used by neurotrophins and their receptors; the question mark indicates the possible involvement of kinesin in this pathway. Poliovirus, canine adenovirus type 2 (CAdV-2; also known as CAV-2) and their respective receptors, CD155 and coxsackievirus and adenovirus receptor (CAR; also known as CXADR), are also found in these multifunctional transport vesicles. Rabies virus (RABV) is transported in axonal vesicles, although some of its glycoproteins bind dynein, suggesting a direct recruitment of motors to ‘naked’ virions. However, mutations in the dynein-binding sites of RABV did not abrogate axonal retrograde transport of this virus. BDNF, brain-derived neurotrophic factor; G, ganglioside; TrkB, tropomyosin-related kinase B (also known as NTRK2). form (PrPSc) that will spread in the CNS and, ultimately, cause neurodegeneration98. Prions undergo retrograde transport in axons after the injection of PrPSc‑containing brain extracts into the tongue muscles of hamsters99. Anterograde movement is also detected on intracerebral injection99, suggesting a complex interaction between prions and motors of different polarities. Finally, direct impairment of axonal transport in motor neurons was also observed after inoculation of muscles with PrPSc (REF. 100). Similarly to PrPSc, viral proteins can be transported in axons independently of fully assembled viruses. Although HIV is often found in the brains of patients with AIDS, the virus itself does not seem to infect neurons6. AIDS‑ associated dementia may be due, at least in part, to two HIV proteins, gp120 and Tat, which trigger neuronal apopto‑ sis101 (TABLE 1). Axonal transport is involved in the spread of gp120 and Tat and their associated neurotoxic effects. The envelope protein gp120 undergoes microtubule‑ dependent axonal retrograde transport in the rat CNS and is responsible for neurotoxic activity 102,103, and Tat‑ induced apoptosis was detected at distal sites following injection of Tat‑producing astrocytes104. To date, the molecular mechanisms underlying the transport of Tat and gp120 remain largely uncharacterized. Transport efficiency and redundancy. From an evolution‑ ary perspective, natural selection favours microbial agents that can use several pathways to reach their final destina‑ tion. Viruses engaging the trafficking machinery in mul‑ tiple ways (such as poliovirus, which can be transported in either a CD155‑dependent or CD155‑independent manner 67) will have an advantage in CNS invasion and propagation. In the same vein, HSV and pseudorabies virus contain several proteins that bind directly to the dynein complex and, as a result, mutations in individual viral components have only a mild effect on retrograde spread of the pathogens24,83. This redundancy is probably one of the main mechanisms allowing these and other viruses to be transported efficiently in the CNS25. NATuRE REVIEWS | Microbiology VoluME 8 | SEPTEMBER 2010 | 651 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Different pathogens also recruit motors with a dis‑ tinct polarity to ensure their efficient axonal transport. Alphaherpesviruses and CAdV‑2 undergo bidirectional transport with a bias for the retrograde direction dur‑ ing their first phase of infection45,88. Inhibition of either dynein or kinesin‑mediated trafficking led to a strong reduction of the overall transport of CAdV‑2, suggesting a b TeNT transcytosis Axon TeNT VAMP2 SV c Measles virus synaptic microfusion Microfusion formation MV HSV NK1 HSV episome heterochromatin d Rabies virus trans-synaptic spread RABV Figure 3 | Somatodendritic sorting of microbial agents. After microorganisms Reviews | Microbiology reach the neuronal cell body, they can have distinct fates. Nature Cell-to-cell transfer is called transcytosis and can be due to different molecular mechanisms, such as local synaptic fusion events, or release of material from the presynaptic compartment into the synaptic cleft and subsequent uptake by endocytosis at postsynaptic sites. a | Viruses of the Herpesviridae family, such as herpes simplex virus (HSV), can package their DNA genome into a chromatin-like structure and establish latency in the nucleus. b | Tetanus toxin (TeNT; also known as TetX) is transcytosed from the somatodendritic compartment of motor neurons in the spinal cord to adjacent inhibitory neurons, where a portion of its heavy chain (BOX 3) triggers the cytoplasmic translocation of the active subunit (the light chain). In the cytoplasm of nerve terminals, the endopeptidase activity of the light chain cleaves VAMP2 (also known as synaptobrevin 2), which is a SNARE (soluble NSF (N-ethylmaleimidesensitive factor) attachment protein (SNAP) receptor) protein, and so inhibits the fusion of synaptic vesicles (SVs) with the plasma membrane, preventing neurotransmitter release. c | Measles virus (MV) continues its journey in a new neuron by trans-synaptic spread, possibly involving microfusion events requiring the specific interaction of the MV glycoprotein F with neurokinin 1 (NK1; also known as TACR1). d | Rabies virus (RABV) is released in the synaptic lumen and re-enters at the postsynaptic level to disseminate in the central nervous system. a possible coordination between these two motors45. Interestingly, motors of different polarities are found on organelles undergoing bidirectional transport, such as mitochondria. How the coordination between these large complexes is regulated is only starting to emerge105. In this light, viruses and toxins may be ideal tools to understand the regulation between different motor complexes. Reaching the neuronal cell body The last step in the transfer of cargo from an axon to the cell body is still poorly understood. In epithelial‑like cells, numerous viruses that are targeted to the nucleus transit through the MToC40. Although the MToC may be a potential sorting platform for axonally‑transported cargoes, no clear role has been reported for this struc‑ ture. Pathogens follow different intracellular fates after they have arrived in the soma (FIG. 3). In the case of DNA viruses, the nucleus is their final destination. Alphaherpesviruses establish latency by ‘silencing’ the expression of their episomal DNA, partly owing to their genome being packaged into chromatin‑like structures106 (FIG. 3a). Periodic reactivation of latent HSV leads to DNA replication and de novo viral‑protein synthesis, resulting in new viruses travelling along axons back to the primary infection site. The mechanisms responsible for antero‑ grade transport and egress of herpesviruses remain con‑ troversial25. live‑cell imaging with fluorescently labelled pseudorabies viruses and electron microscopy analyses showed anterograde transport of fully formed viruses (with teguments and an envelop)107–109, whereas other studies found mostly unenveloped capsids in axons110,111 (FIG. 2). In the unenveloped or ‘subassembly’ transport model, assembly of virus particles with a full envelope would only occur at distal axonal sites before release25. However, recent ultrastructural data suggest that the anterograde transport of pseudorabies virus occurs in vesicles109. Interestingly, glycoprotein E, which is needed for replication in epithelial cells and subsequent retro‑ grade transport of HSV‑1 in neurons112, is also crucial for the anterograde spread of HSV‑1 (REF. 113). In contrast to DNA viruses, other viruses can repli‑ cate in the cytoplasm. RABV, an RNA virus, uses protein aggregates called Negri bodies, which contain the innate immune response receptor Toll‑like receptor 3 (TlR3), as a viral factory to enhance its replication in neuronal cells114. Whether axoplasmic replication of RABV can also occur remains to be seen. Transcytosis. Some toxins (for example, TeNT) and viruses (for example, HSV and RABV) undergo transcy‑ tosis and continue their journey in connecting neurons (FIG. 3b–d). In this case, the mechanisms used by infectious agents seem to differ: neuron‑to‑neuron spread can occur across synapses, as has been shown for measles virus30, WNV27 and RABV3, or by direct cell‑to‑cell transfer, as is the case for pseudorabies virus115. However, the molecular mechanisms involved in transneuronal transfer remain mostly uncharacterized. It is tempting to speculate that viruses which bind to receptors undergoing transcytosis may be efficiently transferred to adjacent neurons. Data 652 | SEPTEMBER 2010 | VoluME 8 www.nature.com/reviews/micro © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Microfusion event Local membrane fusion triggered by the interaction between certain viral proteins and surface receptors. addressing the trans‑synaptic spread of measles virus shed light on how viruses can force this cellular cross‑ ing: in this example, synaptic transfer depends on the interaction between viral proteins and neurokinin 1 (also known as TACR1), leading to microfusion events30 (FIG. 3c). In another model system, it was shown that TMEV was transferred to myelin sheets of oligodendrocytes after axonal transport in the optic nerve to allow persistent infection29. In mice expressing mutants of myelin com‑ ponents, TMEV could not induce persistent infection after intravitreous injections. Clostridial neurotoxins can also reach secondary neurons (BOX 3; FIG. 3b). TeNT is transcytosed from motor neurons to the synapses of inhibitory neurons, where it cleaves the synaptic protein VAMP2 (also known as synaptobrevin 2), thus inhibiting neurotransmitter release2. on intramuscular injection of high doses of BoNT/A, this toxin undergoes a similar transcytosis process after axonal retrograde transport in motor neurons33. Conclusions and perspectives understanding the function of neuronal networks remains one of the exciting challenges of modern neuro‑ biology. Many pathogens undergo axonal transport to reach and damage the CNS, but we are only just start‑ ing to understand the mechanisms responsible for their neuronal targeting. Many questions concerning axonal transport of viruses and pathogenic proteins remain. For example, there is increasing evidence that local translation is a key process for ensuring a fast response to acute stimuli in axons, such as injury or local growth. local translation of importin‑α is required for the retro‑ grade transport of signalling complexes containing nuclear localization signals18. These targeting motifs are found in many viral proteins (for example, pul36 of alphaherpesviruses) and numerous endosomal proteins that are involved in the axonal transport of pathogens. Therefore, signals that are activated during the entry of infectious agents at nerve terminals might stimulate local translation, leading to enhanced axonal transport. Along these lines, little is known about the signalling molecules that may be activated during entry and may regulate axonal transport of pathogens. Whether these 1. 2. 3. 4. 5. 6. 7. Resnick, L. et al. 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Acknowledgments We thank the members of the G.S. and E.J.K. laboratories and J. Schwarz for constructive comments. Work in the G.S. laboratory is supported by Cancer Research UK, the Motor Neurone Disease Association, the Jean Coubrough Charitable Trust and funding from the European Community’s 7th Framework Programme (FP7/2007–2013; grant 222992 — BrainCAV). The E.J.K. laboratory is supported by BrainCAV, the French Agence National de la Recherche, the Region Languedoc Roussillon, the Fondation de France and the Association Française contre les Myopathies. E.J.K. and S.S. are Institut National de la Santé et de la Recherche Médicale (INSERM) fellows. Competing interests statement The authors declare no competing financial interests. DATABASES Entrez Genome: http://www.ncbi.nlm.nih.gov/genome BDV | canine distemper virus | CAdV-2 | RABV Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ genomeprj HIV-1 | WNV UniProtKB: http://www.uniprot.org BDNF | BoNT/A | CAR | CD155 | glycoprotein D | nectin 1 | p75NTR | TeNT | TrkB FURTHER INFORMATION Eric J. Kremer’s homepage: http://www.igmm.cnrs.fr/spip. php?rubrique35 Giampietro Schiavo’s homepage: http://london-researchinstitute.co.uk/research/loc/london/lifch/schiavog/?view=L RI&source=research_portfolio All liNkS ArE ActiVE iN tHE oNliNE Pdf VoluME 8 | SEPTEMBER 2010 | 655 © 2010 Macmillan Publishers Limited. All rights reserved