microbes' roadmap to neurons

A wide variety of microbes threatens the nervous system. They can cause lethal infections or affect the function of the nervous system if not fully overcome. In low-income tropical countries, bacterial meningitis and parasitic diseases such as cerebral malaria, African trypanosomiasis and neurocysticercosis have a large death toll. Various neurovirulent arthropod-borne viruses spread progressively over all inhabited continents from their sites of origin (TABLE 1), and infections with rabies, measles and polio still cause nervous system diseases despite control programmes1. A change in environment or lifestyle can cause viruses from animal reservoirs to cross into humans (for example, HIV from primates and, recently, lethal henipaviruses from bats) or lead to a re-emergence of pathogens that were under con-trol in the human populations (for example, African trypanosomes)1.

CNS neurons are located behind the blood–brain barrier (BBB) in an immunologically ‘privileged’ environ ment that protects them from some of the harmful effects of the immune response to a pathogen2. In particular, infected neurons are not readily killed by cytotoxic T cells, as most of them do not normally express major histocompatibility complex (MHC) class I molecules. As a result, antigen-specific T cells cannot recognize the presence of infected cells and trigger their death. In addition, the nervous system expresses high levels of immuno suppressive molecules, which reduce the toxic-ity of inflammation2. Neurons therefore provide an ideal site for microbes to ‘hide’ from the immune system to ensure their survival.

Different microbes can target different regions in the nervous system depending on the route of invasion, on the distribution of cellular receptors for the microbes and on the molecular and metabolic environment in neurons

and glia, which can favour the replication of the pathogen to different degrees. Some pathogens preferentially affect neuronal populations, whereas others infect myelin-forming cells (for example, lepra bacteria infect Schwann cells and polyomaviruses infect oligodendroglia) to cause demyelination. Here, we focus on selected human pathogens to illustrate various mechanisms of neuroinvasion and briefly discuss their effects on neu-ronal functions. Recent discoveries of mechanisms for pathogen entry and the movement of macromolecules and white blood cells (WBCs) into the nervous system, which microbes may use for propagation, have sparked a renewed interest in this field.

The routes of pathogen invasion include: entry through epithelial linings of the body into the blood-stream, where pathogens can circulate as free particles or be carried by red blood cells (RBCs) or WBCs; entry through the BBB and barriers between the blood and the cerebrospinal fluid (CSF) in ventricular and subarachnoid spaces; propagation along axons; and entry through the olfactory system. The influence of pathogens on host behaviour is also briefly discussed, in view of the current interest in microbes and their role in the pathogenesis of nervous system dysfunctions.

Microbe entry via the BBBViruses replicate intracellularly using the synthetic machinery of a cell, whereas bacteria, parasites and fungi grow either intra- or extracellularly or switch between the two environments. Pathogens can cross the BBB either on their own or carried by WBCs, and intracel-lular pathogens may target subpopulations of neurons and glial cells. Malaria parasites in RBCs do not pass the BBB, but attach to cerebral vessel walls to cause cerebral malaria.

Department of Neuroscience, Retzius väg 8, Karolinska Institutet, Stockholm SE‑171 77, Sweden.e‑mail: [email protected]:10.1038/nrn3029Published online 18 May 2011

NeurovirulentRefers to microbes that can replicate in the nervous system and cause functional disturbances.

Blood–brain barrierA structure that protects the brain from non-selective passage of molecules and toxins in the blood while still allowing essential metabolites to cross. It is composed of tightly linked endothelial cells surrounded by astrocyte cell projections that are termed astrocytic endfeet.

Subarachnoid spacesThe space filled with cerebrospinal fluid between the arachnoid and the pia mater.

Microbes’ roadmap to neuronsKrister Kristensson

Abstract | The nervous system is protected by barriers that restrict the invasion of pathogens. Nevertheless, mechanisms have evolved by which microbes can pass these barriers, enter and exit neurons and target various regions of the nervous system. In the brain, immune responses to pathogens are generally not robust, so microbes can hide and survive or, conversely, cause severe uncontrolled infections. Depending on their sites of entry and the regions that they target, microbes can cause diverse nervous system dysfunctions and even influence host behaviour to their own advantage. This Review discusses routes by which microbes can reach the nervous system and cause persistent or life-threatening infections.

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Tight junctionsStructures apposed to the plasma membrane of adjacent endothelial cells. They consist of a network of sealing strands that prevent passage of extracellular fluids; ZO1 is a tight junction protein that is often used as a marker.

Passage of pathogens across endothelial cells. The first hurdle that a blood-borne pathogen approaching the brain encounters is the cerebral endothelial cells. These cells show a paucity of transcellular endocytosis (trans-cytosis) and they are interconnected by tight junctions; this forms the basis of the BBB that prevents unselective passage of macromolecules into the parenchyma (FIG. 1). Some pathogens can cause disruption of the BBB, which enables their passage into the brain. This may result in severe haemorrhagic encephalitis, as occurs, for exam-ple, following infections with adenovirus type 1 in sus-ceptible mice3 and Nipah virus in humans4. Nipah virus

is a highly pathogenic paramyxovirus that emerged in 1998 in Malaysian and Singaporean pig farmers4. The bacterium Bacillus anthracis, which causes a mostly fatal haemorrhagic meningoencephalitis, can attach to cerebral endothelia by a surface layer factor (BslA) encoded by a virulence plasmid (pXO1)5. This fac-tor also contributes to optimal activity of the anthrax toxin, which disrupts tight junctions, as reflected in altered distribution of vascular endothelial cadherin6 and downregulation of the tight junction protein ZO1 (REF. 5.) The intracellular signalling pathways that trigger this dysfunction are not known, but the anthrax

Table 1 | Human pathogens discussed in this Review

Pathogen Vertebrate host or reservoir (vector)

Geographical distribution

Estimated annual incidence

Entry points to CNS

Japanese encephalitis virus (JEV)

Pigs, water birds (mosquitoes) From Japan to South East Asia, Northern Australia and India

50,000 cases with disease, 15,000 deaths

Blood-borne, peripheral nerves

West Nile virus (WNV) >200 species of birds (mosquitoes)

From Uganda to the Middle East, Eastern Europe and North America

>1 million infected in the United States. <1 in 100 have CNS disease

Blood-borne, peripheral nerves

Chikungunya virus (CHIKV)

Primates, humans (mosquitoes) From sub-Saharan Africa to Indian Ocean islands, India, Italy and France

>6 million infected worldwide. Rare CNS disease

Blood-borne, peripheral nerves?

Tick-borne encephalitis virus (TBEV)

Wild rodents (ticks) From Russia to Northern/Central Europe and Japan

>10,000 cases with disease Unclear. Peripheral nerves?

Rabies Any warm-blooded animal, but domestic dogs are the principal host and major vector

High incidence in India, China and sub-Saharan Africa

55,000 deaths worldwide Peripheral nerves

Herpes simplex virus (HSV)

Humans are the sole reservoir Worldwide 70–90% of the world population

Peripheral nerves

HIV Humans are the host and reservoir Worldwide distribution with the highest incidence in sub-Saharan Africa

CNS disease in 10–30% of ~33 million patients with AIDS

Blood-borne (monocytes)

Human T-lymphotropic virus type 1 (HTLV-1)

Humans are the host and reservoir Endemic in Japan, South America, sub-Saharan Africa and the Caribbean

10–20 million infected. 0.3–4% at risk for HAM/TSP

Blood-borne (T cells)

Plasmodium falciparum Humans (mosquitoes) Sub-Saharan Africa, South East Asia, Latin America

Cerebral malaria affects 1 in 1,000 children with malaria

Infested red blood cells

Trypanosoma brucei gambiense and T. brucei rhodesiense

Humans, pigs, wild game, cattle (tsetse fly)

Sub-Saharan Africa Decreased to 20,000 cases in 2008

Blood-borne

Naegleria fowleri Humans (free-living organisms; no vector, swimming pools are transmission reservoirs)

Worldwide (Australia, Asia, Europe, USA) but very low incidence

>100 cases described in total

Olfactory route

Taenia solium Humans definite hosts; pigs intermediate hosts

Latin America, sub-Saharan Africa, India, China and South East Asia

30% of patients with epilepsy in endemic countries

Blood-borne

Toxoplasma gondii Cats definite hosts; rodents, birds and humans intermediate hosts

Worldwide distribution 30–65% of the world population. Decreasing

Blood-borne (monocytyes)

Neisseria meningitidis Humans (human carriers) Worldwide distribution with the highest incidence in sub-Saharan Africa

>30,000 deaths worldwide Blood-borne (meningeal vessels)

Listeria monocytogenes Ruminants (such as sheep), poultry, humans. Found in food supplies, including poultry and dairy products and salmon

Worldwide (perinatal and non-perinatal infections)

1 in 106 people develop meningoencephalitis and 1 in 107 people develop brainstem infections

Blood-borne (monocytes), trigeminal nerve

HAM/TSP, human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis.

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LeptomeningesThe CNS is enclosed by three membranes — the outermost dura mater and the innermost pia mater with the arachnoid in between. The pia mater and the arachnoid are collectively called the leptomeninges.

toxin can bind to endothelial cells and specifically enhance nuclear translocation of interferon regulatory factor 1 (IRF1) and nuclear factor-κB (NF-κB), which regulates endothelial inflammatory responses6.

Other pathogens may cross the BBB without causing its immediate destruction. For instance, a neuroviru-lent strain of simian immunodeficiency virus (SIV; the

monkey equivalent of HIV) can replicate efficiently in macaque brain endothelial cells7. These cells lack the main receptor for the virus, CD4, which indicates that there is a CD4-independent pathway for virus entry into the brain7. This pathway may involve proteogly-cans expressed on endothelial cells, as they can bind the HIV envelope protein gp120 (REF. 8) and human

Figure 1 | Nervous system entry sites for microbes. a | The brain is protected by three membranes — the outermost dura mater and the innermost pia mater with the arachnoid membrane in between. The pia mater and the arachnoid membrane are collectively called the leptomeninges. The endothelial cells of blood vessels supplying the leptomeninges (c, the vessel to the right) are joined by tight junctions, but they are more permeable to proteins in the serum than parenchymal vessels33. They express different adhesion molecules from the brain parenchymal vessels, such as P-selectin100 (which contributes to the rolling of leukocytes on endothelial cells), and they are surrounded by a leptomeningeal basement membrane, which may be more permeable than the parenchymal basement membrane. This may facilitate microbe invasion into the cerebrospinal fluid (CSF) in the subarachnoid space. b | The endothelial cells of blood vessels in the choroid plexus, circumventricular organs and peripheral nerve root ganglia have fenestrations — that is, openings in the endothelial cells that permit the passage of macromolecules as well as microbes, such as bacteria, African trypanosomes and viruses (and virus-infected white blood cells (WBCs)) into the stroma. Unlike ependymal cells and the pia mater, the epithelia of the choroid plexus prevent further passage into the CSF in the ventricles. c | The endothelial cells of brain parenchymal vessels (the vessel to the left) are interconnected by tight junctions, which prevent the passage of macromolecules and most microbes. However, certain viruses, Toxoplasma spp. and trypanosomes have evolved special strategies for crossing tight junctions (in some cases by infecting or following WBCs). The malaria parasite Plasmodium falciparum, contained in red blood corpuscles, attaches to the endothelial cells, but does not cross. d | Peripheral nerve fibres are enclosed by a perineurium that forms a diffusion barrier of cells linked by tight junctions; blood vessels in the endoneurium are also equipped with tight junctions. However, axon terminals of motor neurons and several classes of sensory neurons that innervate the skin end openly at the periphery, through which macromolecules and certain pathogens can be taken up and transferred by retrograde axonal transport to the nerve cell bodies in the spinal cord or in peripheral root ganglia. From there they can propagate into the CNS by anterograde axonal transport after replication in the peripheral nerve cell bodies.

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MonocytesMononuclear cells that are derived from the bone marrow and circulate in the bloodstream. They pass into the body tissues, where they can differentiate into various types of macrophages.

Perivascular cuffsAreas surrounding an inflamed blood vessel that contain inflammatory lymphocytes and are delimited by endothelial basement membrane on one side and parenchymal basement membrane on the other side.

T-lymphotropic virus type 1 (HTLV-1)9. In addition, viruses such as HIV8 and Japanese encephalitis virus (JEV)10 can be carried through brain endothelial cells by transcytosis without undergoing replication in the cells. Transcytosis is regulated by the pericytes, which are located within the endothelial basement membrane; pericyte-deleted mice show enhanced transcytosis across the BBB that can be arrested by the tyrosine kinase inhib-itor imatinib11. Mechanisms that regulate transcytosis are of obvious interest for understanding how viruses enter into the brain; however, the contribution of the passage of cell-free viruses across endothelia in relation to other mechanisms for entry in vivo (such as cell-associated entry (see below)) is not clear8,12.

Passage of pathogens in infected WBCs. An alternative way of crossing the BBB without breaking its integrity is the use of WBCs as carriers — a so-called ‘Trojan horse’ mechanism. Under normal circumstances, WBCs traverse the BBB and patrol the brain parenchyma. During inflammation, this traffic is greatly increased. WBCs activated by inflammatory molecules released during the systemic infection cross the BBB by a multi-step process that involves attachment to and invasion through the postcapillary venule wall and the surround-ing endothelial and parenchymal basement membranes, which differ in their laminin composition and perme-ability for WBCs13 (FIG. 2). Laminin is one of the four major components of basement membranes and can self-assemble to form networks of various densities14. Neuroinvasion of WBCs during inflammation is also regulated by interactions between chemo kine receptors expressed on the WBCs and chemokines produced in the brain.

Several neurovirulent viruses, such as herpes, measles and HIV/SIV, replicate in monocytes and/or T cells and can be carried into the brain parenchyma by WBCs. HIV-infected monocytes contribute more to neuro invasion than infected CD4+ T cells15. Expression of chemokine (C-C motif ) ligand 2 (CCL2), which recruits monocytes, is increased in endothelial cells and astrocytes upon contact with HIV-infected cells16, and the appearance of SIV in rhesus monkey brains coincides with increased levels of CCL2 in the CSF17. After entering the brain, the monocytes carrying the viruses infect perivascular macrophages and microglial cells, but not neurons; instead, neuron function is dis-turbed by the release of inflammatory molecules and/or viral proteins by neighbouring cells15. HTLV-1-infected CD4+ T cells cross into the spinal cord after the func-tion of the BBB is disrupted; this disruption might be caused by an initial infection of the endothelial cells by the virus9. The contribution of measles virus-infected WBCs to nervous system invasion is unclear, as they have a reduced capacity to cross endothelial cell barriers12.

In a similar way to viruses, the bacterium Listeria monocytogenes18 and the parasite Toxoplasma gondii19,20 can invade the brain within infected monocytes follow-ing uptake at the gastrointestinal tract. In vitro, cell-free Toxoplasma spp. parasites can also breach intercellular

junctions and therefore transmigrate between endothe-lial cells or pass by transcytosis, but such free parasites have a low viability in vivo and it is not clear whether they cross the BBB on their own20. After entering neu-rons, the replication of Toxoplasma spp. parasites slows down markedly, and they may persist in these cells as so-called ‘bradyzoites’ for the lifespan of the host. Passage across the BBB by the extracellular parasite Trypanosoma brucei, which causes human African trypanosomiasis, or sleeping sickness, occurs in parallel with that of WBCs at a late stage of the disease. The passage of WBCs may pave the way for the free parasites to follow into the brain parenchyma through openings in the endothelial cell layer and basement membrane barriers21 (FIG. 2). The invasion of this parasite is not dependent on their number in the blood but on expression of inflammatory molecules in the brain. Mice lacking the genes encod-ing the cytokine interferon-γ (IFNγ) and its receptor showed enhanced numbers of trypanosomes in the blood, which together with T cells passed the endothe-lial, but not the parenchymal, basement membranes to form perivascular cuffs; these mice ultimately die owing to an overwhelming systemic infection21. Mice deficient in the IFN-inducible chemokine (C-X-C motif) ligand 10 (CXCL10), or its receptor chemokine (C-X-C motif) receptor 3 (CXCR3), also showed reduced T cell and trypanosome neuroinvasion, but no accumulation as cuffs around blood vessels. This indicates that differ-ent IFNγ-regulated gene products are involved in the multistep cerebral invasion of T cells and trypanosomes — some gene products open the parenchymal basement membrane for passage (FIG. 2), whereas others induce the production of chemokines22.

West Nile virus (WNV) provides another example of a pathogen for which the level in the blood is not cor-related to its neuroinvasion. The viral load in the periph-ery is increased, but encephalitis is mitigated in Toll-like receptor 3 (Tlr3)–/– mice infected with WNV23 (BOX 1). In such mice, release of tumour necrosis factor (TNF)23 and activation of matrix metalloproteinase 9 (MMP9)24 are reduced, and this impedes the passage of infected WBCs, and possibly also of free viruses, across the BBB (FIG. 2). In the brain this virus preferentially infects neurons in the basal ganglia and thalami as well as in the brainstem and cerebellum, and in humans it causes what is known as ‘West Nile neuroinvasive disease’ (REF. 25).

Trapping of parasites in cerebral blood vessels. Neuronal functions can be severely disturbed even if the patho-gen has not crossed the BBB, as seen in cerebral malaria. The malaria parasite Plasmodium falciparum is car-ried in RBCs that, upon infection, express a parasite-derived protein (PfEMP1) that binds to molecules on cerebral endothelial cells (such as intercellular adhesion molecule 1 (ICAM1), which is induced by TNF26, and heparan sulphate, which is also present on RBCs27). The adhesion of RBCs to the endothelial cells is mediated by a number of constitutive and inducible surface antigens in the blood vessels26, and rosette formation between par-asite-infested RBCs is promoted by a number of serum factors28. Blood flow through cerebral capillaries can be

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α α

βα

α

α β

Neurovascular unitsMicrodomains in the brain formed by astrocytes that integrate neural circuitry with local blood flow. Astrocytic processes form a bridge between cerebral vessels and synapses, and both are covered by astrocytic membranes.

obstructed by sequestration, which may be ameliorated by treatment with depolymerized heparin in rat and nonhuman primate models27. Recently, an association between the release of so-called microparticles — small cell-membrane-derived vesicles that bud mainly from RBCs, platelets, WBCs and endothelial cells — and cer-ebral malaria has emerged26. Microparticle release is dra-matically increased in humans with cerebral malaria26. After binding to TLR4 on macrophages and to cerebral endothelial cells, these particles stimulate the secretion of pro-inflammatory cytokines29, which may perturb the BBB integrity and cause dysfunctions in the neurovascular units. Administration of a low-molecular-weight thiol,

pantethine, interrupts overproduction of microparticles and prevents signs of cerebral malaria in a mouse model26. This compound has limited side effects in humans and may be experimentally evaluated as an adjunctive ther-apy26. However, opinions differ concerning how close murine models of cerebral malaria are to the human disease. Unlike human autopsy materials, the mouse models show more inflammation than sequestration of parasite-infested RBCs.

Similar pathogen sequestration is observed with the embryos of the pork tapeworm Taenia solium. After reaching the bloodstream from the intestinal mucosa, they get stuck in microvessels in the brain and form

Figure 2 | White blood cells as carriers of microbes. Following infection by viruses, white blood cells (WBCs) can pass from the lumen of a blood vessel in the brain parenchyma either by transcellular migration over endothelial cells or by opening tight junctions14. After crossing a permissible endothelial basement membrane, they may remain between this and the parenchymal basement membrane until the latter is opened, allowing WBCs carrying the virus particles to pass between astrocytic endfeet (blue) into the brain parenchyma to infect neurons and/or glial cells. a | As a first step in crossing the blood–brain barrier, WBCs are systemically activated to express members of the integrin family — LFA1 (also known as β2 integrin) and VL4 — to attach to adherence molecules expressed on endothelial cells. After a tethering with matching receptors (intercellular adhesion molecule 1 (ICAM1), ICAM2 and vascular cell adhesion protein 1 (VCAM1)). WBCs can firmly attach to the endothelial cells by recognizing chemokines induced in the endothelium by inflammation13. b | After passing through the endothelial cell layer by opening tight junctions, the next hurdle for the WBCs is to cross the endothelial basement membrane, which is derived from the endothelial cells. The composition of the laminins of the endothelial basement membrane determines whether T cells, and to a lesser extent monocytes, can pass. The ubiquitous α4 laminin subunit forms loose networks that allow the transmigration of WBCs across cerebral endothelial basement membranes, whereas networks containing α5 laminin are tight and more resistant to cell infiltration. c | Last, WBCs have to cross the parenchymal basement membrane, which is derived from the astrocytic endfeet abutting the vascular walls. The parenchymal basement membrane contains α1 and α2 laminin subunits, which are resistant to WBC infiltration. The transmigration of WBCs into the parenchyma requires focal activation of matrix metalloproteinase 2 (MMP2) and MMP9, which selectively cleave a dystroglycan receptor (β-DG) that anchors the astrocytic endfeet to the basement membrane14. If the MMPs are not activated, WBCs can be seen trapped as cuffs around the vessels13. JAM, junctional adhesion molecule.

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SeizuresTransient abnormal, excessive or synchronous neuronal activity in the brain with clinical manifestations ranging from tonic spasm and clonic contractions of the muscles and convulsions to brief loss of awareness or psychic symptoms. Epilepsy signifies recurrent unprovoked seizures.

viable cysts in which the parasite develops30. These cysts are usually asymptomatic until they degenerate owing to normal attrition 3–5 years after the initial infection. Viable cysts secrete a factor that suppresses immune responses mediated by the cytokine osteopontin31. Therefore, the influx of WBCs across the BBB32 mainly occurs around degenerating cysts. The inflammatory reaction that ensues could trigger seizures, which are a key sign of disease30.

Blood–CSF barriersPathogens can also cross barriers between the blood and the CSF in subarachnoid and ventricular spaces (FIG. 1) and thereby indirectly cause neuronal dysfunctions.

Passage of pathogens into subarachnoidal spaces. Although equipped with tight junctions between their endothelial cells, meningeal vessels are more permeable to macromolecules than those in the brain parenchyma33, and this may facilitate the passage of pathogens. Several bacteria and fungi can rapidly grow in the CSF of sub-arachnoid spaces and cause meningitis. These include the bacteria Streptococcus pneumoniae, Neisseria men-ingitides and Haemophilus influenzae, and the fungus Cryptococcus neoformans, which can pass by transcytosis across brain endothelial cells in culture34. A molecular mimicry mechanism has been suggested for the binding of bacteria to the endothelial cells. Pneumococcal bacte-ria express a surface phosphorylcholine (PCho), which mimics the chemokine platelet activating factor (PAF) and uses the interaction with the PAF receptor to cross endothelial cells by transcytosis35. PCho binding to the

PAF receptor also triggers a neuron-specific signalling pathway that results in cell death, but endothelial and lung epithelial cells survive36. H. influenzae and N. men-ingitides bacteria also express PCho, suggesting that the mechanisms for invasion have converged for these three different bacteria36. However, there are multiple other mechanisms through which bacteria can cross the brain endothelial cells; recently, N. meningitides was found to stimulate a β2-adrenoceptor–β-arrestin pathway that led to cytoskeletal reorganization and the formation of interendothelial gaps through which the bacteria could penetrate37.

Although bacteria are most likely to reach the sub-arachnoid space by crossing the meningeal vessels from the bloodstream, alternative mechanisms and sites of entry could be considered. As pneumococcal bacteria colonize the nasal cavity, they might reach the sub-arachnoid space through the olfactory route (discussed below). Indeed, bacteria have been found in the brain in the absence of bacteria in the blood38.

Viruses can pass the meningeal vessels by similar mechanisms to those described for the BBB and repli-cate in meningeal fibroblasts. Infections in the meninges may be combined with an infection in the brain — that is, meningoencephalitis — but several viral infections are restricted to the meninges and then cause a usually mild and transient infection.

Passage of pathogens into ventricular spaces. Bacteria are not generally associated with infections in the ventricles in humans, but in vitro experiments with Streptococcus suis revealed transcytosis across the polarized choroid plexus epithelial cells from the basolateral surface (facing the stroma of the plexus) to the apical surface (facing the CSF)39. This transfer may be regulated by the bacterial capsule, which triggers the phosphatidylinositol 3-kinase signalling pathway, paving the way for cellular uptake and transcytosis of the bacteria, probably by modulat-ing endosomal trafficking39. Transcriptional activation of TLR2 and expression of inflammatory cytokines in the choroid plexus early after infection make it a potential entry site for these bacteria in vivo40.

The choroid plexus may also be an entry site for viruses. For instance, WBCs infected with canine dis-temper virus accumulate in the choroid plexus early during infection, before viruses are seeded into the ventricles to infect abutting ependymal cells and, subse-quently, brain parenchymal cells41. Lymphocytic chori-omeningitis virus (LCMV) antigens also appear first in choroid plexus epithelial cells and later in microglial cells and macrophages in the white matter around the ven-tricles42. During this infection, the chemokine CXCL10 has a crucial role in attracting and retaining CD8+ T cells in the brain parenchyma. This chemokine is first pro-duced in cells in the meninges and choroid plexus, and then in astrocytes around the ventricles as a biphasic response to the infection43. These findings suggest that the initially infected microglial cells produce IFNα and IFNβ, which induces a limited production of CXCL10 that attracts virus-specific CD8+ T cells to enter the site of infection in the brain (BOX 1). Following recognition

Box 1 | The innate immune response and Toll-like receptors

The early, innate immune response recognizes a vast number of pathogens on the basis of their molecular structures, which are evolutionarily conserved and shared between different species of pathogens99. There are four different classes of pattern recognition receptor families99. The best characterized is the family of transmembrane Toll- like receptors (TLRs), which are of particular interest in the present context as they recognize invading pathogens at the cell surface or after uptake by endolysosomes. Ten TLRs have been identified in humans and 12 in mice. On the cell surface, TLR2 and TLR4 sense lipoproteins and lipopolysaccharides, respectively, derived from bacteria, parasites or viral envelopes. In endolysosomes, TLR3 senses double-stranded RNA viruses, TLR7 (human TLR8) detects single-stranded RNA viruses and TLR9 recognizes unmethylated DNA with CpG motifs derived from degraded bacteria, viruses and parasites.

After stimulation with the pathogen-derived ligands, complexes of intracellular signalling molecules that translocate information to the nucleus are recruited; nuclear translocation of nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs) drive the expression of pro-inflammatory cytokines and interferon-α and -β (IFNα and IFNβ) genes, respectively99. The release of these effector molecules influences the invasion of the CNS by microbes. For instance, the pro-inflammatory cytokine tumour necrosis factor (TNF) can induce the expression of adhesion molecules on cerebral endothelial cells. It may also activate matrix metalloproteinase 2 (MMP2) and MMP9, which cleave dystroglycan receptors, leading to the opening of parenchymal basement membrane and the passage of white blood cells, which may carry pathogens13,14. IFNα and IFNβ can reduce viral replication in the CNS by setting neighbouring cells into an antiviral state — that is, metabolically resistant to viral replication. It also recruits antigen-specific T cells into the tissue by initiating expression of chemokine (C-X-C motif) ligand 10 (CXCL10), which attracts and retains T cells expressing the CXCL10 receptor, chemokine (C-X-C motif) receptor 3 (CXCR3)43. If such T cells recognize an antigen, they will produce IFNγ, which augments the expression of CXCL10 to induce a full-blown inflammatory response.

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of virus-infected cells, the T cells produce IFNγ, which triggers further CXCL10 production and a full-blown inflammatory response. In this case, the invading T cells, and not the virus per se, are the major cause of neuronal dysfunctions43. When the lethal inflammatory response to LCMV is inhibited, as in immune-deficient mice, infected cortical neurons are observed in the surviving mice, probably because the weak inflammatory response allows enough time for the virus to spread from the initial target cells to the neurons44.

The choroid plexus has also been found to be a target for viral infections in humans, as seen in post-mortem tissues from patients with AIDS in which monocytes carrying HIV appear in the choroid plexus45.

Another striking example of early targeting of the ven-tricle spaces during an infection is provided by T. brucei parasites. These extracellular parasites first localize to the choroid plexus and circumventricular organs, where they induce an inflammatory response, and they only cross the BBB together with WBCs at a late stage of the disease (discussed above)21. From the inflamed regions, pro-inflammatory cytokines may be released into ven-tricular CSF and then diffuse into brain parenchyma, triggering the second wave of invasion21. Because the white matter and diencephalic structures are adjacent to the ventricles, this course of events could explain why trypanosome brain invasion targets mainly these areas.

Pathogen propagation along axonsPeripheral nerve fibres are shielded from microbial attacks by diffusion barriers in the perineurium and endoneural vessels (FIG. 1). However, unprotected axonal endings can take up macromolecules, which then undergo retrograde transport to the nerve cell bodies46, providing entry points for a number of microbes.

Axonal uptake and transport of viruses. Viruses can be broadly divided into enveloped and non-enveloped viruses; the former acquire their envelope by bud-ding through membranes of the host cell before being released, whereas the latter escape by lysis of the cell. For infection, viruses bind to a cellular receptor, after which non-enveloped viruses (such as polioviruses and adenoviruses) and some enveloped viruses (such as rabies virus) can be incorporated by clathrin-, caveolar- or raft-mediated endocytosis, by macropinocytosis or by novel endocytic mechanisms47. Clathrin-mediated uptake is the most likely neuronal entry mechanism for the viruses described in this Review. The viral particles release their capsids into the cytosol after penetrating the vesicle membrane in the acid environment of late endo-somes. Other enveloped viruses (such as herpesviruses) release their capsids directly into the cell after fusion of the envelopes with the plasma membrane48 (FIG. 3a). Endocytic uptake gives the virus the advantage of leaving no trace on the plasma membrane for immunodetec-tion; recently, herpes simplex virus (HSV) has also been found to be incorporated by this mechanism49.

Endosomes at axon terminals can either be recycled to the cell surface or be sorted for retrograde axonal transport to the nerve cell bodies. For the endocytosed

Figure 3 | Axon-terminal uptake and transport of pathogens. Axon terminals in the periphery end openly and are not protected by perineurial or endoneurial barriers. a | An enveloped virus may be incorporated by endocytosis or fuse its envelope with the cell membrane to release the naked capsid into the axoplasm. A non-enveloped virus is incorporated by endocytosis. Early endosomes are decorated on their cytosolic surfaces by the small GTPase Rab5, which is replaced by Rab7 as they mature into late endosomes (not shown)101. Endosomes marked by Rab7 become destined for retrograde transport to the nerve cell bodies102, which contain the cellular synthetic machinery necessary for viral replication. Such endosomes are transported in association with the microtubular transport system. The mechanoenzyme dynein is a huge complex of proteins that moves along microtubules in the retrograde direction by hydrolysing ATP. Dynactin is an associated protein that regulates dynein activity and binding capacity for the endosomal cargoes to which a number of adaptor proteins, including Rab7, are associated depending on the nature of the cargoes103. Naked viral capsids may associate with the same transport system, but mechanisms for this are still unclear. Newly formed viral particles or components in the nerve cell body can be carried by anterograde transport along microtubules associated with kinesin family members (KIFs)103. Virus envelopes and capsids can be transported separately from each other and assemble to form complete viral particles upon reaching the axon terminals or they may assemble within vesicles close to the cell body and be transported together. At the axon terminal, such viral particles may be released by exocytosis. b | Listeria monocytogenes may reach axon terminals through an initial cycle of replication in macrophages or dendritic cells (colony-stimulating factor 1-dependent cells). After uptake and lysis of the phagosome membrane, the bacteria multiply in the cytosol of the cell. The new bacteria can polymerize actin asymmetrically, producing actin tails, which propels them through the cytoplasm and into an adjacent non-phagocytic cell (for example, an axon terminal) in a receptor-independent manner18. Following L. monocytogenes-induced lyses of the enclosing two membranes, the bacteria are free in the axoplasm61 to acquire a new actin tail to propel them towards the cell body.

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Circumventricular organsExcept for area portrema in the fourth ventricle, these organs are all located along the wall of the third ventricle and include the subfornicular organ, the laminar terminalis, the subcommisural organ, the median eminence and the neurohypophysis. They have fenestrated, leaky vessels whereby, for instance, axons projecting from hypothalamic neurons can secrete products into the bloodstream, but they can also serve as chemoreceptors to regulate homeostatic functions.

PerineuriumLayers of flattened cells that enclose fascicles of peripheral nerve fibres. The cells are joined by tight junctions, which prevent the diffusion of macromolecules into the enclosed nerve fibres.

Endoneural vesselsVessels within the endoneurium (all connective tissue elements and spaces enclosed by the perineurium) that have tight junctions between adjacent endothelial cells to prevent the diffusion of macromolecules from the blood into the endoneural spaces.

Capsids(From the Latin capsa, meaning box.) Protein shells that directly package viral DNA or RNA. The term nucleocapsid is sometimes used to describe the shell and its nucleic acid content.

Peripheral gangliaGanglia of nerve cell bodies in the peripheral nervous system encompassing dorsal root ganglia and cranial ganglia of the somatic division as well as ganglia of the autonomic division.

viruses to survive and replicate, they have to associate with the retrograde transport machinery in axons50 to reach the nerve cell bodies that contain the synthetic machinery. This sorting may be determined at an early stage, after binding of ligands at the cell surface51.

Rabies virus is an example of an enveloped RNA virus that is carried in endosomes by retrograde axonal transport, as visualized by live tracking52. After initial replication in skeletal muscle cells, the virus can spread to axon terminals at neuromuscular junctions and be transported to motor neurons in the spinal cord for further replication and spread in the CNS53. The virus can also propagate within sensory and autonomic nerve fibres and reach salivary glands to be released into the saliva ready to infect the next individual53. During retro-grade axonal transport, viruses such as canine serotype 2 adenoviruses in C57Bl/6 mice may be contained within Rab7-positive endosomes, which are kept non-acidic to prevent release of the viruses into the axoplasm before they reach the nerve cell bodies54. Although transport of viruses is mainly linked to the fast microtubule transport system, a slower retrograde transport may also occur. For instance, in mice, human poliomyelitis virus not only shows rapid retrograde axonal transport that is depend-ent on the poliomyelitis virus receptor and is mediated by fast dynein-dependent transport of endocytosed viral particles, but also a slower retrograde transport that is receptor independent; the mechanisms for this transport are not clear55.

Herpesviruses are enveloped DNA viruses that mostly fuse with the plasma membrane of host cells, releasing their nucleocapsids into the cytosol. They can spread by retrograde axonal transport46 and establish latent infections in peripheral ganglia. Numerous struc-tural features of herpesviruses are required for invasion of the nervous system. From the initial site of infection in epithelial cells, transfer of HSV to axons is achieved by expression of a viral envelope component, glycopro-tein E56. This protein mediates cell-to-cell spread of the virus either through its cytoplasmic tail domain, which targets viruses to cell junctions (the sites for cell-to-cell spread), or its ectodomain, which may interact with a ligand at these junctions56. In the case of the porcine pathogen Suid herpesvirus 1 (pseudorabies), the deubiq-uitinase domain of the tegument protein pUL36, which surrounds the viral capsid57, is required for the initial axonal invasion but not for the subsequent transport or replication.

After release into the axoplasm, herpesvirus capsids may attach to dynein light chains for retrograde trans-port along microtubules by their outer capsid protein VP26 (REF. 48). However, when VP26 was deleted in mice, no effect on the translocation of the virus from the eye to the trigeminal ganglia was observed58, suggest-ing that either other capsid proteins are involved or that viral particles can be transported in endosomes after the alternative endocytic uptake.

Following successful invasion of nerve terminals, retrograde transport and replication in the body of a sen-sory neuron, newly replicated HSV may move into the CNS by anterograde transport in the central axon branch

(in experimental animals) or move back to peripheral nerve terminals. Such newly formed herpesviruses may be transported in vesicles as enveloped complete viral particles or as separate elements (capsids and envelope proteins) that assemble on reaching the axon termi-nals59,60 (FIG. 3a). Anterograde, but not retrograde, axonal transport of capsids is facilitated by two viral protein kinases encoded by loci US3 and UL13 in the genome61. Deciphering the molecular mechanisms of intraneuro-nal sorting and transport of herpesvirus components should provide fundamental pathogenetic insights into these viral infections62 and offer clues on how to block the virus spread and why reactivated latent herpesvirus infections in human trigeminal ganglia spread along peripheral and not central branches of the axons to cause recurrent blisters in the skin instead of lethal brainstem encephalitis.

Axonal uptake and transport of bacteria. L. mono-cytogenes can pass within monocytes across the BBB (as indicated above)18. It is also one of the few, if not the only, bacteria that can infect neurons. L. monocy-togenes can spread via the trigeminal nerve and cause severe brainstem encephalitis, which is often lethal in humans and manifests as ‘circling disease’ in sheep18. Following injection into the snout in mice, macrophages and dendritic cells are infected. Dendritic cells transfer the pathogen to the lymph nodes to elicit an adaptive immune response. In mice depleted of these cells, an overwhelming systemic infection occurs but, paradoxi-cally, the invasion of the bacteria along the trigeminal nerve is impeded, indicating that dendritic cells and/or macrophages have a role in transferring L. monocytogenes to axons63 (FIG. 3b).

Olfactory route to the brainOlfactory neurons are unique in that they come into direct contact with the external environment and can provide an entrance for pathogens to the brain. In ani-mal models these neurons can be infected by a number of viruses, such as herpesviruses, Borna disease virus (BDV), paramyxoviruses, morbilliviruses and rhabdovi-ruses. Such pathogens may then be transported along axons to the olfactory bulb, from where they can target the limbic system by anterograde axonal transport or the monoaminergic brainstem neurons (which project to the olfactory bulbs) by retrograde transport64 (FIG. 4). For instance, infection of suckling mice with a mutant rhab-dovirus that targets serotonergic raphe neurons in the brainstem causes life-long selective serotonin depletion in the brain64. In some studies, the olfactory pathways have been the predominant route for neuroinvasion of viruses that are otherwise associated with a blood-borne spread: for example, WNV65,66 and JEV67. Chikungunya virus (CHIKV) can also spread along olfactory pathways in mice, but the relevance of this mode of entry for the brain infections it causes in humans is not clear68.

Several bacteria colonize the nasal cavity and could potentially cross epithelial barriers into the subarach-noid space, which extends into the cribriform plate at the base of the skull, and cause meningitis. Certain amoebae

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have been shown to gain access to the brain in this way. For instance, the human-pathogenic Naegleria fowleri (‘the brain-eating amoeba’) and Balamuthia mandrillaris (which was recently isolated from baboons) can infect the brain after nasal instillation by passing through the olfactory epithelium and migrating through the cri-briform plate into the olfactory bulb to cause a severe necrotizing meningoencephalitis69. The mechanisms of neuroinvasion and tissue destruction by amoebae are poorly understood. Proteins such as Nfa1 (which is located in the food-cups of these amoebae70) and proteases with an activity pattern that differs between pathogenic and non-pathogenic Naegleria strains71 may have a role in these processes.

Microbe control and nervous system dysfunctionSome microbes that have entered the CNS can cause severe and lethal infections, whereas others may remain as persistent infections or be eliminated by the immune system (even in this immunologically privileged environment). Persistent infections may cause functional disturbances of the nervous system and behaviour changes of the host, which may benefit some pathogens.

Control and persistence of microbes in neurons. Spread of an infection along peripheral nerves can be impeded at the level of peripheral ganglia, which, in contrast to the CNS and peripheral nerves, lack a barrier between the blood and the parenchyma46. In this respect, natural killer cells and antigen-specific T cells that have passed fenes-trated blood vessels in the ganglia may serve a sentinel function to control an infection and prevent its further spread into the CNS72. For instance, IFNγ released from lymphocytes can kill L. monocytogenes63 and suppress early HSV gene expression within preserved peripheral neurons73. This enables HSV to persist in the neurons, even for the lifespan of the host, but poses the virus with a problem: when the host dies the virus also perishes unless it spreads to another host in advance. This may be a reason why latent HSV infections can be reactivated by various stimuli, such as stress73, whereupon new viruses move to the periphery along axons by anterograde transport to increase the chances of transmission to another indi-vidual. Sorting of the virus in the axons to the periphery instead of to the brainstem is obviously beneficial both for the pathogen and the host.

Although electrically silent hippocampal neurons treated with IFNγ can express antigen-presenting MHC

Figure 4 | Olfactory route of neuroinvasion. Olfactory neurons are unique because their dendrites come into direct contact with the external environment and their axons are in direct contact with the brain (the olfactory bulb). Viral particles can infect olfactory neurons and be transported along axons to the olfactory bulb. From here, some viruses (for example, measles) may spread via anterograde transport to reach the limbic system104, whereas others (for example, the rhabdovirus vesicular stomatitis virus (VSV)) may be taken up by axon terminals and spread via retrograde transport to neurons that project to the olfactory bulbs (such as serotonergic raphe neurons). By targeting different regions in the brain, various behavioural disturbances can be induced in the infected host animal. Bacteria (such as pneumococci) often colonize the nasal cavity. In a similar way to the amoebae Naegleria fowleri, they may potentially pass the olfactory mucosa and, via the holes of the cribriform plate, reach the subarachnoid space and cause meningitis.

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γ

class I molecules74, it is not clear whether this occurs in vivo to clear a CNS infection. However, antigen- specific T cells may recognize viral components released from infected neurons that are taken up and processed by MHC class I-expressing microglial cells. Upon recog-nition, the T cells secrete IFNγ, which, in some but not all nerve cell populations, can clear viral RNA without killing the neurons through a so-called non-cytolytic clearance mechanism75. The effector molecule may be IFNγ itself or IFNγ-induced release of an antimicro-bial molecule, such as nitric oxide75 (FIG. 5). If a balance between viral synthesis and degradation is established, RNA viruses may persist for long periods of time (even years) in the nervous system76. Persistence of Toxoplasma spp. parasites in CNS neurons is promoted through an interaction between IFNγ and factors derived from hypertrophic astrocytes surrounding parasite-associated lesions77.

Another mechanism through which microbes may survive in neurons is by inducing an upregulation of immunosuppressive molecules such as human leukocyte antigen (HLA)-G1, thus protecting the cells from attacks by infiltrating lymphocytes. This is observed in rabies infections of human neural cells78.

Endogenous retroviruses have developed a strategy for persistence by integrating into germ lines. Through subsequent expansions during evolution, human endogenous retrovirus (HERV) elements today make up approximately 8% of our genome79. One of the very few human retroviral elements that has retained its

ability to encode proteins is the member of the HERV-W family in the ERVWE1 locus encoding syncytin 1, which is highly expressed in the human placenta80. In rats, retroelements seem to target mRNA transcripts to dendrites81, but no functional roles have been identified in the human brain.

Pathogens and neuronal function. Some viruses kill the infected neuron, whereas others may cause non-cytolytic infections. Even if the neuron is structurally preserved, the infection may interfere with neuronal signalling pathways and disturb synaptic activity. For instance, phosphorylation of the BDV phosphopro-tein (BDV-P) by protein kinase C (PKC) is required for dissemination of the virus82. During replication of wild-type BDV, neuronal PKC signalling and activity-modulated synaptic functions are disturbed, whereas cells infected with a recombinant BDV with a mutated PKC phosphorylation site on BDV-P show normal phos-phorylation of endogenous PKC substrates (including synaptosomal-associated protein 25 (SNAP25), which modulates neuronal excitability) and synaptic activity82. This suggests that BDV-P can act as a decoy substrate to interfere with PKC signalling in neurons82. Neuronal dysfunctions can also be caused indirectly by T cell-derived cytokines that aim to control the infection, as cytokines are known to affect ion channels and stimu-late or inhibit synaptic activity in various neuronal net-works83. As pointed out above, neuronal dysfunctions in cerebral malaria and HIV infections may result from such indirect effects.

Pathogens and host behaviour. Depending on the regions of the nervous system that are infected, microbes can make a host elicit various types of behav-iours. Examples of this are aggressive, biting behaviour in rabid dogs and behavioural disturbances in rodents infected with Toxoplasma spp. which increase their risk of exposure to predators84,85; both pathogens benefit from these changes, as they increase the chances for transmission. These two infections show marked differ-ences in their topographical localizations in the brain: rabies localizes to the brainstem, thalamic and hip-pocampal neurons86 and Toxoplasma localizes to more widespread areas85, including the prefrontal cortex87. The pathogenesis of the neuronal dysfunctions and the clinical–pathological correlations of these infections remain to be determined.

Tick-borne encephalitis virus (TBEV), which infects motor neurons in the human spinal cord and causes flaccid paralysis, has also been associated with a form of epilepsy called ‘epilepsia partialis continua’ (REF. 88), which is characterized by frequent focal (hands and face) seizures that can last for days or years. This type of epi-lepsy, which is associated with infections with a Siberian variant of TBEV88, is linked with the motorcortex, sug-gesting that the virus can propagate to this area of the brain. Seizures appear not only during acute infections, but often up to 5–6 years after recovery from bacterial meningitis89. The high prevalence of epilepsy in Latin America, India and African countries is partly ascribed

Figure 5 | Non-cytolytic control of viral replication. Viral components may be released from infected neurons (right) by exocytosis or cytolysis and be taken up by microglia, in which they are degraded into peptides. Viral peptides may be presented by major histocompatibility complex (MHC) class I molecules at the surface of microglia and be recognized by the T cell receptor on an antigen-specific T cell. The activated T cells secrete interferon-γ (IFNγ) that can interfere directly with replication of certain viruses (for example, herpes simplex virus) or by inducing release of antimicrobial molecules (for example, nitric oxide) from microglia. Growth of Toxoplasma parasites (bottom right in the neuron) may also be controlled by IFNγ from T cells, although a role for astrocyte-derived gp130 has also been described77 (see main text).

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to infections with pneumococcal bacteria89, viruses90, P. falciparum91, T. gondii92 and T. solium (which causes cysticercosis)93. Although, the release of cytokines may be involved in facilitating seizures during acute infec-tions94, few studies have addressed the mechanisms underlying the subsequent late-onset seizures95.

Infections can also be associated with prominent changes in sleep–wake regulation, as in von Economo encephalitis (encephalitis lethargica) and human African trypanosomiasis (sleeping sickness), or with insomnia, as in patients infected with HIV21. In fact, von Economo pioneered the work of hypothalamic sleep–wake regulation by describing lesions in the anterior hypothalamic area as “sleep-promoting” and in the posterior hypothalamic area as “wake-promot-ing” in a post-mortem study of the brains of deceased patients with encephalitis lethargica. Human African trypanosomiasis is characterized by a disruption of sleep–wake alternation and sleep organization, indicat-ing functional alterations in hypothalamic nerve cell groups implicated in the regulation of sleep, wake and circadian rhythms. The release of parasite-derived pro-staglandins or cytokines, such as TNF and IFNγ, from trypanosomes or T cells located in the circumventricu-lar organs, including the median eminence–arcuate nucleus complex, may be responsible for affecting the function of these nerve cell groups21.

Another example of a common pathogen-induced neuronal dysfunction relates to pain transmission. For example, infection by varicella zoster virus (human herpesvirus type 3) frequently results in post-herpetic neuralgia. Herpesviruses localize to dorsal root ganglia neurons, where their replication is controlled by IFNγ. Interestingly, IFNγ can elicit neuropathic pain through a nitric oxide-dependent mechanism that downregu-lates glutamate receptors on GABAergic neurons in the spinal cord posterior horns, thus reducing synap-tic inhibition and potentially contributing to the pain syndrome96.

ConclusionThe microbes discussed in this Review can cause dev-astating damage to the nervous system and have a large death toll in low-income countries. In spite of this, they are relatively neglected, in particular by the neuroscience community. Survivors may often be left with neurological handicaps or cognitive dysfunctions that have a strong negative impact on socio-economic development. With the increased mobility of people, infections can rapidly spread into new areas, and changes in the environment will foster the emergence of novel microbial mutants. Thus, there is a need not only to continue surveillance and identification of microbes but also to develop new prevention and treatment strategies.

With regards to those microbes that affect the nerv-ous system, several specific questions arise: how do microbes enter the CNS? How do they cause neuro-nal dysfunction? And how do they interact with the immune response in the brain? As described, a number of strategies have evolved by which microbes can cross the BBB and blood–CSF barrier, or bypass these barriers

by using the axonal transport machinery in peripheral nerve fibres, to infect neurons. New antiviral approaches that inhibit the functions in a host cell that are criti-cal for virus replication are being sought. Such com-pounds are less likely to generate resistant viruses and could block the spread of more than one pathogen47. Experimental paradigms in which transcytosis across the BBB and axonal uptake and transport are system-atically manipulated could settle the question on the relative importance of these entry mechanisms and consequently lead to the design of adjunctive therapies that prevent neuroinvasion. Knowledge on molecular mechanisms for selective passage of different types of WBCs across the BBB will shed light on the ‘Trojan horse’ mechanism for pathogen neuroinvasion and the diverse signalling mechanisms that regulate their entry and subsequent growth.

Better knowledge of the timing for neuroinvasion of pathogens and the identification of clinical markers for this event are important to optimize post-exposure immune therapy against neurovirulent viruses, such as the rabies virus53, and for deciding whether to use drugs that penetrate the BBB, which can be very toxic. For instance, in sleeping sickness, relatively non-toxic drugs are efficient at an early stage of the disease, but arsenic compounds that can have lethal side effects are still in use for treating the late encephalitic stage21.

The complexity of events related to neuropatho-genicity of microbes cannot be underestimated. As pointed out, delicately balanced virus–cell interactions may lead to the persistence of RNA viruses in various neuronal networks in the CNS. It would be interesting to find out whether such interactions during critical stages of nervous system development can cause permanent or progressive changes in neuronal differentiation, function or lifespan. If the balance is tilted over time to clearance of the pathogen, we may be met with the challenging situation of addressing neuronal dysfunction in neu-ronal networks that no longer harbour any trace of the causative agent.

Another interesting area to study is the mechanisms underlying the behavioural disturbances or epilepsy that can appear late after infection. Whether late-onset seizures are associated with mossy fibre sprout-ing in the hippocampus (as in models of epilepsy after stroke97), with homeostatic upregulation of excitatory synapses (which can develop over time after trauma to the neocortex98) or with something else remains to be determined.

Collaborations between neuroscientists and micro-biologists will help to further our understanding of the pathogenesis of these prevalent but too-often-neglected diseases of the nervous system. As microbes have explored all possible mechanisms to interact with cells, they could also continue to unravel basic neurobiologi-cal features, as they have in the past (for example, the existence of a retrograde axonal transport was predicted from studies mapping the propagation of HSV in the nervous system46, and the BBB was discovered following attempts to treat African trypanosome infections in mice with trypan dyes21).

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With the advent of high-resolution imaging and intra-cellular tracing techniques, it is now possible to follow the multistep replication and propagation of microbes, and by applying functional genomics, proteomics and metabolomics to diverse types of infections, we can start to unravel the mechanisms by which defined pathogens cause dysfunctions in the nervous system. In the near future, studies on the interactions between viral elements

and regulatory mechanisms in neurons may disclose how dysfunctions in neuronal networks can emerge as a consequence of early-life infections. Exciting results are also likely to emerge from further research into how pathogens exploit cellular mechanisms to spread and target the nervous system; such research may reveal host-directed targets for adjunctive therapies that could prevent such infections.

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AcknowledgementsThe studies have been supported by grants from the Swedish Research Council (04480), European Commission (222887) and the US National Institutes of Health/Fogarty (5 R21 NS064888-02).

Competing interests statementThe author declares no competing financial interests.

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