Handbook of Clinical Neurology, Vol. 114 (3rd series)Neuroparasitology and Tropical NeurologyH.H. Garcia, H.B. Tanowitz, and O.H. Del Brutto, Editors© 2013 Elsevier B.V. All rights reserved

Chapter 2

Mechanisms of CNS invasion and damage by parasites

KRISTER KRISTENSSON1*, WILLIAS MASOCHA2, AND MARINA BENTIVOGLIO3

1Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden2Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Kuwait University, Kuwait

3Department of Neurological and Movement Sciences, University of Verona, Verona, Italy

INTRODUCTION

Invasion of the CNS is one of the most devastating com-plications of a parasitic infection in an organism.Although several physical and immunological barriersprovide obstacles to such invasions, parasites haveevolved mechanisms by which they can cross, circum-vent or avoid these barriers to enter the brain and causevarious types of damage related to toxic effects ofparasite-derived molecules or to immune responses tothe infection.

This chapter will give a broad overview on how humanpathogenic parasites belonging to the classes of protozoa,nematoda, trematoda, and cestoda can pass or interactwith various physical barriers to reach the CNS and thiswill be followed by examples of pathogeneticmechanismsof brain dysfunction caused by parasite infections. Thereviewwill, thus, focus on the physical barriers to neuroin-vasion of parasites and will not address the complexity ofthe innate and adaptive immune responses that may con-trol or eliminate pathogens in the organism before theyhave entered the bloodstream and the CNS.

MECHANISMSOF CNS INVASIONBY PARASITES

Themain physical barrier which protects the brain paren-chyma from invasion by parasites circulating in thebloodstream is the blood-brain barrier (BBB). Peripheralnerve fibers are also protected by barriers in the perineu-rium, which surround the nerve fascicles, and the endo-neural blood vessels. Blood vessels in the leptomeningesare more permeable than those in the brain parenchymaand there are regions in the nervous system, i.e., the cho-roid plexus, the brain circumventricular organs (CVOs)

*Correspondence to: Dr. Krister Kristensson, Department of Ne

Sweden. Tel: þ46-8-524 87825, Fax: þ46-8-325 325, E-mail: krister

and peripheral nerve root ganglia, which lack barriers intheir vessels.

Human pathogenic parasites can reach the CNS viathe bloodstream either as free-living or extracellular par-asites, by embolization of eggs, or within red or whiteblood cells when adapted to intracellular life. Some path-ogens can target the areas lacking a BBB and certain par-asites can enter the brain from the nasal cavity throughthe olfactory nerve pathway (Kristensson, 2011).

Before they approach the CNS by the bloodstream,parasites have to enter the host and the first physical bar-rier that they encounter when invading a higher organismis the epithelia in the skin, or in the gastrointestinal orrespiratory tracts. This section will therefore begin witha brief description on how parasites can pass such bar-riers, remain extracellular or become intracellular byinfecting cells such as macrophages/monocytes and thengain access to the bloodstream.

Parasite penetration through cell membranebarriers and adaptation to extra-

or intracellular life

The skin is covered by a layer of keratinized cells, whichcannot support growth of intracellular parasites. A bar-rier to extracellular parasites is further provided by theinterlocked epidermal cells, basement membranes, andmatrix molecules. However, the skin barrier can be pen-etrated by certain protozoa. For instance, trematodeSchistosoma spp., which swim as larvae (cercariae) withelongated tails when released from infected freshwatersnails, attach to the human skin. Recognition, attach-ment, and penetration are stimulated by a series ofchemical signals released from the mammalian skin(Haas et al., 1997, 2008). Penetration through the skin

uroscience, Retzius vag 8, Karolinska Institutet, Stockholm,

.kristensson@ki.se

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is due to proteolytic activity of enzymes secreted fromthe cercarial acetabular glands (Stirewalt, 1973), e.g., ser-ine proteases by S. mansoni (Salter et al., 2000) andcathepsin B by S. japonicum (Dvorak et al., 2008). Afterskin penetration, the cercariae lose their tails and moveas schistomula into the vascular system (Pittella, 1997).Other parasites such as Plasmodium, Trypanosoma bru-cei, and Trypanosoma cruzi can pass the skin of a hostwhen injected into the subcutaneous tissues through thebite of a vector, which thereby inoculates the parasite.

The extracellular environment of the host exposes aninvading parasite to the risk of being rapidly eliminatedby the immune responses. Parasites have adopted a vari-ety of strategies to avoid these dangers, successfullyspread in the host, and allow them to survive and multi-ply. Therefore, evolutionary pressure on parasites hasled to their adaptation to the host environment. Forinstance, T. brucei have adapted by undergoing rapidswitches of their surface molecules, while a large num-ber of other parasites have adapted by invading differenttypes of host cells to avoid rapid detection by theimmune system (Soldati et al., 2004). In adapting tointracellular life, parasites have the additional advantagethat they may be able to traverse or infect epithelial cellsto use respiratory and gastrointestinal pathways as anadditional way to enter an organism. For this purpose,parasites have evolved mechanisms to enter cells, utilizenutrients in the cells for their replication, avoid intracel-lular defense mechanisms, and exit the cells. The strate-gies for such adaptation are highly diverse and differ notonly between species of parasites, but also between par-asites of the same species (Sibley, 2011).

A wealth of recent studies has unraveled how variouspathogens take advantage of cellular signaling pathwaysto promote their entry into a host cell (M€unter et al.,2006). This is also true for signaling pathways activatedby parasites. Mechanisms by which parasites can enter ahost cell have been especially investigated in infectionswith the Apicomplexan parasites Toxoplasma and Plas-modium (Baum et al., 2008). The motility and cell inva-sion of both parasite species are coupled to regulatedsecretion of a series of proteins from the apical poleof the zoites (a specialized elongated and highly mobilestage of the parasite). The tachyzoites (Toxoplasma) andthe sporozoites (Plasmodium) can attach to and moveover cellular surfaces to invade host cells by a processcalled “gliding,” which is powered by an actin�myosinmotor located underneath the parasite membrane. Ajunction is formed between the host cell membraneand the apical tip of the parasite at the site of entry intothe cell, a so-called moving junction, before the parasitesmove into the so-called parasitophorous vacuoles toinfect the host cell (Soldati et al., 2004; Hegge et al.,2010). Polymerization of host cell actins plays a role to

12 K. KRISTEN

anchor these junctions and ensure effective parasiteinvasion (Gonzalez et al., 2009).

In addition to infecting a cell, Plasmodium sporozo-ites, in contrast to Toxoplasma tachyzoites, can traversea cell without multiplying, i.e., they puncture the cellmembrane, glide through the cytoplasm, and exit(Vanderberg et al., 1990; Mota et al., 2001). This maybe a mechanism to escape being degraded by macro-phages at the primary site of infection, the dermis, toenter the bloodstream and reach the liver cells; in thehepatocytes the traversal activities are switched offand cell infection favored (Amino et al., 2008;Gueirard et al., 2010). The parasitophorous vacuoles con-taining Toxoplasma tachyzoites resist fusion with lyso-somes and therefore avoid degradation in monocytesand such cells can spread the parasites through the blood-stream from their primary site of replication in intestinalepithelial cells (Tardieux and Menard, 2008).

In order to gain access to nutrients from the host cellcytosol, many microsporidian parasites may pierce thehost cell plasma membrane and inject the sporoplasmdirectly into the cytosol, while others appear in parasito-phorous vacuoles that contain pores to allow passage ofmolecules from the cytosol (Sibley, 2011). Toxoplasmaparasites may similarly gain access to nutrients fromthe cytosol by forming pores in the parasitophorousvacuole membranes (Sibley, 2011). The kinetoplastidTrypanosoma cruzi escape into the cytosol from vacu-oles and this escape requires the low pH of lysosomes,which is obtained mainly by fusion of the plasmamembrane with lysosomal membranes at a site closeto parasite attachment (Burleigh, 2005; Mott et al.,2009; Albertti et al., 2010).

To favor their own survival within an infected cell,parasites can produce molecules which inhibit host cellsignaling pathways involved in triggering immuneresponses to an infection as well as apoptosis (reviewedby Sibley, 2011).

After parasites have resided and multiplied in a cell,there are a number of mechanisms for their egress(Tardieux and Menard, 2008; Sibley, 2011). Toxoplasmaparasites cause a calcium signaling-dependent release ofa perforin-like molecule that causes the rupture ofboth the parasitophorous vacuolar and plasma mem-branes with release of vital parasites that invade cellsin the neighborhood (Black and Boothroyd, 2000;Kafsack et al., 2009). The exit of Plasmodium parasitesfrom hepatocytes and red blood cells is triggered byactivation of cascades of proteases which rupture firstthe parasitophorous vacuoles and then the cell mem-branes. Trypanosoma cruzi after precisely nine roundsof mitotic divisions in the cytosol differentiate into non-dividing trypanomastigotes that are released by burstingof the cell.

ON ET AL.

MECHANISMS OF CNS INVASION

Vascular route and penetration throughthe blood�brain barrier

Following penetration of the epithelial cell barriers, par-asites can invade the lymphatic or blood vessels to reachthe bloodstream and then spread to the nervous system.Worms may seed eggs into the bloodstream which canreach the CNS as emboli, Plasmodium-infested redblood cells are sequestered in cerebral vessels afterattachment to endothelial cells, while other parasitesmay cross the BBB and enter the brain parenchymaeither free-living or carried by white blood cells.

THE BLOOD�BRAIN BARRIER

The cerebral endothelial cells are linked together by theso-called “tight junctions,” which restrict passage ofmolecules between the blood vessel and brain. The pas-sage of molecules by endocytotic uptake at the luminalside and exocytotic release at the abluminal side of theendothelial cells (transcytosis) is selective for specificmolecules. These two features of the cerebral endothe-lial cells form the basis of the BBB, which prevents non-selective passage of molecules from the blood into theCNS parenchyma (Abbott et al., 2006).

The BBB is notably essential for homeostasis of theCNS internal environment, which is required for stablefunctions of the neuronal networks. This barrier is pre-sent in all vertebrates; perivascular astrocytes form theBBB of ancestral vertebrates, while the endothelial cellbarrier with tight junctions appeared later during evolu-tion (Bundgaard and Abbott, 2008).

Exchange of metabolites across the BBB occursmainly at the level of the capillaries, where astrocytesserve as a bridge between vessels and neurons to formthe so-called neurovascular units (see below). On theother hand, infiltration of white blood cells into the brainparenchyma during inflammation occurs at the level ofpostcapillary venules, which are enclosed by two base-ment membranes. One basement membrane is formedby the endothelial cells, the other by end-feet of astro-cytes apposed to the vessels; the latter parenchymal base-ment membrane is continuous with the pia membrane ofthe CNS surfaces to form the “glia limitans” (Owenset al., 2008; Sorokin, 2010).

The endothelial cells in the blood vessels of the lepto-meninges are also linked by tight junctions, but these ves-sels are more permeable than brain parenchymal vessels(Broadwell and Sofroniew, 1993). These properties mayfacilitate the passage of some pathogens into themeninges.

Concerning the brain structures located outside theBBB, they comprise the choroid plexus, the structureresponsible for cerebrospinal fluid (CSF) formation,

and the CVOs. Endothelial cells of the blood vessels inthe choroid plexus are fenestrated and therefore allowpassage of macromolecules from the bloodstream intothe choroid plexus stroma. However, the epithelial cellsthat line the choroid plexus are linked by tight junctions,which prevent further nonselective passage into the CSF.The endothelial cells in the vessels of the other CVOs (seefurther below) are also fenestrated. These organs arecovered by tanycytes, which inhibit nonselective passageofmolecules into the CSF similarly to choroid plexus epi-thelia. It should also be noted that the ependymal cellswhich line the ventricles are not linked by tight junctionsand this allows diffusion of molecules from the CSF intothe brain parenchyma.

AND DAMAGE BY PARASITES 13

EMBOLIZATION OF SCHISTOSOMA EGGS

Subspecies of Schistosoma, blood flukes, use man andother mammals as definite hosts, and aquatic andamphibious snails as intermediate hosts (Pittella, 1997).The three most important subspecies, i.e., S. mansoni,S. haematobium and S. japonicum, show differencesin the spread of their schistomula, released into thevascular system after invasion through the skin, in thedefinite host. After initially residing in the lung, theyspread either via the bloodstream or directly throughthe diaphragm into the intrahepatic branches of theportal vein, where they mature into schistosomes,mate, and migrate into the inferior mesenteric vein(S. mansoni and S. japonicum) or venules in the pelvicplexus (S. haematobium) (Faust, 1948). At these locationsthe worms lay eggs which can be excreted in the feces orthe urine. However, eggs may also spread to the CNS bythe bloodstream as emboli, which can be sparse and ran-domly distributed, via previously developed shunts oranastomoses from veins to arteries. They may also reachthe epidural venous plexus around the spinal cordthrough retrograde flow in the venules. The eggs aredeposited as emboli to the cerebral vessels (Fig. 2.1A)and secrete antigens such as glycans and glycoproteinsthat elicit an immune response leading to granulomaformation (Schramm and Haas, 2010). The eggs ofS. mansoni and S. haematobium may be retained in ves-sels at a lower spinal level, while the smaller and rounderS. japonicum eggs (70 to 100 mm in length and 40 to70 mm in width) can reach the brain, and typically targetthe cerebral cortex, basal ganglia, and cerebral whitematter (Ross et al., 2012).

In addition, and probably as a main cause of symp-tomatic neuroschistosomiasis, adult worms can migratevia the bloodstream from the mesenteric and pelvicplexus veins to reach meningeal or choroid plexus ves-sels. From such sites they may shed massive amountsof eggs into vessels of confined areas of the spinal cord

Fig. 2.1. Vascular route of neuroinvasion by parasites. Parasites transported by the bloodstream can interact with or cross the

blood�brain barrier into the brain parenchyma. (A) The eggs of Schistosoma (e.g., S. japonicum) lodge within small vessels

as emboli. (B) Erythrocytes infested with Plasmodium parasites attach to endothelial cells of cerebral vessels and to each other

to be sequestered within the vessel. (C) The extracellular parasite Trypanosoma brucei spp. and T cells cross the endothelial cell

layer, the endothelial basementmembrane (BM) and the parenchymalBMof postcapillary venules to invade the brain parenchyma.

In the absence of IFN-g-inducible molecules, the parasites and T cells do not penetrate the parenchymal BM efficiently and they

accumulate within the perivascular space as cuffs. (D) Monocytes carrying Toxoplasma gondii tachyzoites inside parasitophorousvacuoles (PV) cross the BBB into the parenchyma. Once inside the brain parenchyma, astrocytes and neurons are infected and the

parasites are transformed to slowly replicating bradyzoites.

14 K. KRISTENSSON ET AL.

or brain parenchyma followed by granuloma formationsaround the embolized eggs (Pittella, 1997).

INFECTION OF RED BLOOD CELLS

Themalaria parasites do not enter the brain parenchyma,but red blood cells infested withPlasmodiummerozoitescan attach to endothelial cells of cerebral vessels, besequestered in the circulation (Figure 2.1B), and therebyescape from clearance in the spleen (Newton andKrishna, 1998; Combes et al., 2010). Infected and nonin-fected red blood cells also attach to each other to formrosettes (Wahlgren et al., 1989). The attachment betweenthe membranes of red blood cells and endothelial cellsoccurs after binding of aPlasmodium-derivedmolecule,the P. falciparum erythrocyte membrane protein-1(PfEMP-1), expressed on knob-like protrusions on thesurface of the red blood cells (Howard et al., 1990; Suet al., 1995; Jensen et al., 2004): PfEMP-1 binds to a num-ber of molecules, including intercellular adhesion mole-cule (ICAM)-1, induced by inflammation on cerebralendothelial cells (Turner et al., 1994). This is followedby a cascade of endothelial cell activation that may causedisturbances in the functions of the neurovascular units,

lead to breakdown of tight junctions and leakage ofplasma proteins into the perivascular space (Brownet al., 1999) or to apoptosis of endothelial cells with rup-tures of the vascular walls and small ring-shaped bleed-ing in the perivascular parenchyma (Pino et al., 2005).

The protozoaBabesia, which are transmitted by ticks,cause an important disease in livestock and are emerginghuman pathogens (Homer et al., 2000; Elsheikha andKhan, 2010). They also infect red blood cells and altertheir membrane properties tomake them adhere to endo-thelial cell membranes (O’Connor and Allred, 2000;Hutchings et al., 2007; Krause et al., 2007). Cerebralinvolvement is well recognized in cattle (cerebral babesi-osis), but information in humans is very limited(Schetters and Eling, 1999).

MIGRATION OF LARVAE INTO THE CNS

Larvae of helminths can reach the bloodstream andinfect the CNS, but the entry mechanisms remain to beclarified. The embryos of the pork tapeworm, Taeniasolium, are disseminated by the bloodstream after entrythrough themucosa of the intestine. Upon reaching smallblood vessels, they lodge and start to develop into cysts

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(Mahanty and Garcia, 2010), and it is not clear whetherthey cross the BBB or not. In a murine model, disruptedBBB permeability was observed in both meningeal andbrain parenchymal vessels as a consequence of inflamma-tory response to the infection (Alvarez and Teale, 2007).

Toxocara canis larvae hatch in the small intestine,and can reach visceral organs via the portal venous cir-culation. The larvae can also invade the brain in humanscausing eosinophilic meningitis and encephalomyelitis.In experimental mouse models of the infection, larvaeare not randomly distributed in the brain, but show a pre-dilection for the corpus callosum and cerebellum(Dolinsky et al., 1981; Good et al., 2001). Larvae have alsobeen found in the choroid plexus and adjacent brain areasafter oral administration of embryonated eggs; the mech-anisms of brain invasion are still unclear and numbers ofinvading larvae are not correlated with enhanced BBBpermeability (Liao et al., 2008). The preferential localiza-tion of the parasites to the choroid plexus and corpus cal-losum is similar to that of the African trypanosomes(Trypanosoma brucei) in rodent models (see below).

The helminthsAngiostrongylus cantonensis,Gnathos-toma spinigerum, and Baylisascaris procyonis cancause, although rarely, nervous system infections, whichmainly manifest as eosinophilic meningitis (Rosen et al.,1962; Fox et al., 1985; Punyagupta et al., 1990; Lo ReIII and Gluckman, 2003). Larvae of the rat lungwormA. cantonensis spread from rats to snails and slugs thatallow the larvae to develop into an infective form. Inhumans, the larvae spread from the gastrointestinal tract,through the bloodstream, to the CNS where they burrowinto the parenchyma after crossing the vessels by a mech-anism that is still unclear. Most patients recover withoutspecific treatment within 3�6 weeks. Larvae of G. spini-gerum (which have dogs and cats as definitive hosts, andfreshwater fish, frogs, snakes, chicken, or pigs as interme-diate hosts) penetrate the gastricwall andmigrate throughhuman tissues for as long as 10 to 12 years. They migratealong peripheral nerve roots to reach the CNS where theycause necrosis of tissues along their path (Punyaguptaet al., 1990). They are fatal in 7�25% of the cases. Infre-quently, larvae of the raccoon ascarid B. procyonis areingested by humans and tend to invade the CNS via thebloodstream to become manifested as neural larvamigrans (Cunningham et al., 1994). The mechanisms bywhich larvae penetrate the BBB remain to be clarified.

MECHANISMS OF CNS INVASIO

CROSSING THE BLOOD�BRAIN BARRIER BY FREE-LIVING,EXTRACELLULAR PARASITES: AMEBAE AND

AFRICAN TRYPANOSOMES

Free-living, extracellular parasites have developed a vari-ety of mechanisms to cross the BBB and enter the brainparenchyma.

Acanthamoeba are parasites which cause infectionsmainly in immunocompromised individuals, whodevelop a granulomatous encephalitis fatal in more than90% of the cases (Fowler and Carter, 1965;Martinez andVisvesvara, 1997). The mechanisms of spread from theportal of entry through the skin or respiratory tract tothe brain are poorly understood but most likely involvemigration through the BBB via a para- or transcellularroute from the bloodstream (Khan and Siddiqui,2009). Acanthamoeba mannose-binding protein andparasite-derived serine proteases have been suggestedto play a role in this BBB passage (Siddiqui et al., 2011).

Balamuthia amebae can infect immunocompetenthosts and lead almost invariably to death in humans(Matin et al., 2008). Like Acanthamoeba, they probablyspread through the bloodstream and cross the BBB tocause a granulomatous encephalitis. In vitro studies onthe invasion ofB.mandrillaris using human brainmicro-vascular endothelial cells have shown that attachment ofthe parasites to endothelial cells is mediated by agalactose-binding protein on their surface. This bindingmight lead to release of ameba proteases that degradebasement membranes and tight junction proteins tofacilitate passage of the parasite across the BBB(Matin et al., 2006, 2007).

More information concerning parasite neuroinvasionis available for Trypanosoma brucei spp. These extracel-lular parasites cause human African trypanosomiasis,and CNS infection represents the severe evolution ofthe disease. Studies in experimental rodent models ofT. brucei infection have elucidated that brain invasionoccurs in two phases. The parasites first invade thechoroid plexus and the CVOs (Schultzberg et al.,1988), and subsequently cross the BBB through a multi-step process and penetrate into the brain parenchyma(Masocha et al., 2004).

The molecular mechanisms for the passage ofAfrican trypanosomes across the BBB have been sub-jected to several studies. In in vitro paradigms of humanbrain microvascular endothelial cells, the parasite sub-species T. brucei rhodesiense binds to endothelial cellsand passes either trans- or paracellularly (Grab et al.,2004; Nikolskaia et al., 2006a, b). Proteases releasedfrom the parasites may promote their passage by affect-ing BBB permeability (McLaughlin, 1986; Bakalaraet al., 2000; Grab et al., 2009). Under in vivo conditionsother factors are also involved in regulating passage oftrypanosomes across the BBB as observed in rodentmodels. For instance, increased permeability of theBBB is not closely correlated to areas in the brain show-ing trypanosome invasion (Philip et al., 1994) or inflam-matory cell infiltration (Rodgers et al., 2011). In mousemodels of T. brucei brucei infection there is no relation-ship between the levels of parasites in the blood and

AND DAMAGE BY PARASITES 15

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parasite crossing of the BBB, which instead is related toinvasion of T cells into the brain (Masocha et al., 2004).Thus, the trypanosome invasion is similar to the invasionof white blood cells at postcapillary venules, and thisinvasion is at least in part governed by the expressionof a number of proinflammatory cytokines and chemo-kines. The cytokines tumor necrosis factor (TNF)-a andtype 1 and 2 interferons (IFN), and the chemokineCXCL10 are elevated during T. brucei infection and playa permissive role for parasite penetration and accumula-tion in the brain parenchyma (Masocha et al., 2004;Aminet al., 2010, 2012). In this multistep passage across theBBB, T cells (and possibly trypanosomes) attach firstto a cytokine-activated cerebral endothelial cell layer,migrate through it and the endothelial cell basementmembrane, and enter the perivascular space. IFN-g-inducible molecules then facilitate passage of both Tcells and trypanosomes across the parenchymal base-ment membrane into the brain parenchyma (Masochaet al., 2004) (Fig. 2.1C).

16 K. KRISTEN

Fig. 2.2. Naegleria fowleri invasion of the olfactory bulb.

After entering the nasal cavities, the free-living amebaNaegle-ria fowleri attaches to the olfactory epithelium and passes

either through the sustentacular cells or between them after

disruption of the tight junctions that link these cells. They

move along the spaces between the unmyelinated nerve fibers

of the fila olfactoria and traverse the cribriform plate to reach

the subarachnoid space and olfactory bulbs. The parasite

“digests” tissue on its way into the olfactory bulb through

the release of parasite-derived proteases.

PARASITES CARRIED IN WHITE BLOOD CELLS ACROSS

THE BLOOD�BRAIN BARRIER

Parasites carried in the bloodstream within white bloodcells may enter the brain parenchyma by the so-calledtranscytosis across the cerebral endothelial cells (i.e.,within transport vesicles from the luminal to the ablum-inal side of the endothelial cell), or by openings of tightjunctions, the so-called paracellular transmigration.

T. gondii can circulate in the bloodstream as extracel-lular parasites, but have reduced viability in this form(Unno et al., 2008) which therefore does not seem to con-tribute to brain invasion. These parasites can insteadspread to the CNS via the bloodstream through infectedmonocytes, which carry the rapidly replicating tachy-zoite forms of T. gondii and can cross the BBB by a leu-kocyte extravasation process that is much enhanced ininfected as compared to noninfected animals (Courretet al., 2006; Lachenmaier et al., 2011). After enteringthe brain parenchyma T. gondii released frommonocytes(see above) can infect microglia, astrocytes, and neurons(Fig. 2.1D). Their replication is markedly slowed down inastrocytes and neurons, in which the parasites can persistin bradyzoite form. The replication of the parasites in neu-rons may be controlled by factors released from the sur-rounding activated astrocytes (Dr€ogem€uller et al., 2008).Microglial cells may on the one hand inhibit growth of theparasites (L€uder et al., 1999), but on the other hand con-tribute to their spread within the brain parenchyma sincethey, similar to macrophages, show hypermotility wheninfected (Dellacasa-Lindberg et al., 2011).

The intracellular parasite Trypanosoma cruzi (whichmainly infects neurons of autonomic ganglia) can spread

to the CNS in immunosuppressed individuals, e.g., HIV-infected patients, and infect astrocytes and microglia(Rocha et al., 1994; da Silva et al., 2010).Most likely theseparasites spread within infected cells in the bloodstream,but the target blood cell and the mechanisms for BBBcrossing during HIV infection have not been investi-gated. In immunocompromised hosts, the microspori-dium Encephalitozoon cuniculi may also spread to theCNS from the bloodstream and enter glial cells in thebrain (Mertens et al., 1997; Wasson and Peper, 2000).

ON ET AL.

Olfactory route for ameba penetration

The free-living amebaNaegleria fowleri thrives in warmfreshwater and can contaminate swimming pools. Afterinhalation, the ameba can attach to the olfactory epithe-lium possibly via a Nfa1 protein expressed in their foodpockets (Shin et al., 2001; Cho et al., 2003). They maypass transcellularly through the sustentacular cells orsecrete proteases or phospholipases that degrade thetight junctions between these cells, pass the basementmembrane and move along the spaces between theunmyelinated nerve fibers of the fila olfactoria; theythus traverse the cribriform plate to reach the subarach-noid space and olfactory bulbs (Martinez and Visvesvara,1997). Most likely, through further release of proteases(Serrano-Luna et al., 2007), the parasites digest the olfac-tory bulb and spread into the brain parenchyma to cause agranulomatous meningoencephalitis (Fig. 2.2). Becausethis parasite rapidly digests the brain parenchyma, it has

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been called “the brain eating ameba.” In experimental ani-mals the ameba Balamuthia mandrillaris can similarlyspread along the olfactory route following intranasalinstillation (Kiderlen and Laube, 2004) although inhumans this ameba reaches the brain as a blood-borneinfection (Schuster and Visvesvara, 2004; see above).

MECHANISMS OF CNS INVASIO

MECHANISMSOF CNS STRUCTURALAND/OR FUNCTIONAL DAMAGE

BY PARASITES

Parasites can cause damage or dysfunctions in the ner-vous system by diverse mechanisms, with clinical mani-festations related to the targeted brain areas.Disturbances in nervous system function can even occurwhen parasites have not crossed the BBB. Examples ofsuch events are provided by functional disruption of neu-rovascular units by Plasmodium-infested red blood cellsand by localization of Trypanosoma brucei to CVOs.

Cytopathic effects by parasites in thebrain parenchyma

After entering the brain parenchyma, parasites can causedamage by the release of toxic molecules and/or by eli-citing an immune response. For example, as mentionedabove, amebae and larvae of certain helminths cansecrete proteases that degrade the brain parenchymacausing localized necrosis. In infections caused by otherparasites such as Toxoplasma gondii and Taenia solium,neural dysfunction is at least in part related to theimmune response they trigger in the brain.

Despite the brain “immune privilege,” immuneresponses can, in fact, be elicited in the parenchyma(Carson et al., 2006; Niederkorn, 2006). Growth of para-sites, e.g., Toxoplasma, in neurons can be controlled byrelease of inflammatory cytokines, such as IFN-g, andactivation of astrocytes (Dr€ogem€uller et al., 2008), whilecysticerci of the extracellular Taenia solium may releasefrom their cysts molecules that inhibit immune responses(Wang et al., 2008). An inflammatory immune response istriggered only when the larvae die, with release of cyto-kines such as TNF-a and appearance of clinical signs,which in this infection are characterized by seizures.

Although pathogenetic mechanisms of seizures dur-ing brain infections have not yet been investigateddirectly, it has been demonstrated that inflammatorycytokines may affect synaptic functions and induce sei-zures (Vezzani et al., 2011a). For instance, IFN-g cancause downregulation of GABAergic neuron activitythat can lead to increased activity in some neuronalnetworks (Vikman et al., 2003), long-term exposure toTNF-a can cause seizure-like activities (Savin et al.,2009), and interleukin-1/Toll-like receptor signaling

can mediate increased excitability and lowered seizurethresholds (Vezzani et al., 2011b).

AND DAMAGE BY PARASITES 17

Dysfunctions in the neurovascular units

Asmentioned above, parasites can cause disturbances ofneural function even without crossing the BBB. It shouldbe considered, in this respect, that cerebral capillaries arefunctionally coupled to synapses by astrocytic processeswhich ensheath the synapses and cover the vessel walls,thus forming a bridge between them (Fig. 2.1). Thereby,astrocytes form nonoverlapping territorial microdo-mains, neurovascular units, in which the activities inneural circuits are integrated with the local bloodflow (Iadecola and Nedergaard, 2007; McCarty, 2009;Verkhratsky, 2010). As one arm of the neurovascularcoupling, increased synaptic activity induces Ca2þ

signals in the covering astrocytic processes and thesesignals can propagate to the astrocytic end-feet at thearteriolae triggering the release of vasoactive sub-stances. In the other direction, astrocytes transfer tro-phic and metabolic support molecules to neurons fromthe blood circulation.

Cytokines released during inflammation can modifythe permeability of the BBB (Abbott, 2000; Hawkinsand Davis, 2005). This causes a vasogenic edema, whichmay perturb neuron functions and affect neuron survival(Holmin and Mathiesen, 2000; Zlokovic, 2005; Iadecola,2010). The relationship between perturbed synapses,immune activation of endothelial cells, and bridgingastrocytes in the absence of increased BBB permeabilityis not clear.

Plasmodium is an example of a parasite that does notenter the brain but causes severe dysfunctions in cerebralmalaria. The infection causes a strong inflammatoryresponse with immune activation of cerebral endothelialcells to which infested red blood cells attach. The seques-tered red blood cells may disturb blood flow through thebrain, but also contribute to dysfunctions in the neuro-vascular units caused by cytokine-induced increase invascular permeability and disrupted signaling betweenthe endothelial cells and synapses (Brown et al., 1999;Rogerson et al., 2004; Combes et al., 2010).

Parasite invasion of circumventricularorgans and neuronal dysfunction

The CVOs, specialized structures identified in the brainof vertebrates including humans, are located in strategicpositions along the surface of the brain ventricles(McKinley et al., 2004). They comprise the subfornicalorgan, vascular organ of the lamina terminalis, medianeminence, neurohypophysis, subcommissural organ,located at various positions in the wall of the third

Table 2.1

Portals of entry into the host, cell invasion, and routes for spread to the CNS of selected human parasites

Parasite Mode of entry Host cell interactionRoute of spreadto CNS BBB interaction

Schistosoma spp. Skin, larvalpenetration

Extracellular worms Bloodstream asmigrating worms

or seeding of eggs

Egg embolization

Plasmodium spp. Skin, mosquito bite Traversal:macrophages,hepatocytes

PV: erythrocytes,hepatocytes

Bloodstream inerythrocytes

Infected erythrocytesattach toendothelia

Babesia Skin, tick bite Erythrocytes Bloodstream Infected erythrocytes

attach toendothelia

Taenia solium Intestine, larval

penetration

Extracellular larvae Bloodstream Lodge in small

cerebral vesselsToxocara canis Intestine Extracellular larvae Bloodstream Choroid plexus

Cross BBB

Angiostrongyluscantonensis

Gnathostomaspinigerum

Intestine

Intestine

Extracellular larvae

Extracellular larvae

Bloodstream

Along peripheralnerve roots

Cross BBB

Unclear

Acanthamoeba Respiratory tract,skin

Free-living ameba Bloodstream Cross BBB

Balamuthiamandrillaris

Respiratory tract,

skin

Free-living ameba Bloodstream Degrade and cross

BBBTrypanosoma brucei Skin, tsetse fly bite Extracellular

parasitesBloodstream Choroid plexus,

CVOs

Cross BBB similarto T cells

Toxoplasma gondii Intestinal epithelia PV: all karyotic cells Bloodstream inmonocytes

Cross in infectedmonocytes

Trypanosoma cruzi Skin, bug bite Escape from PV tocytosol: variouscells incl. muscle,

autonomicneurons,macrophages, glia

Bloodstream Cross BBB, probablywithin monocytes

Encephalitozooncuniculi

Respiratory,intestinal tracts

PV: various cells incl.macrophages, glia

Bloodstream Unclear

Naegleria fowleri Nasal cavity Free-living ameba Olfactory route None

PV, parasitophorous vacuole.

18 K. KRISTENSSON ET AL.

ventricle, the area postrema located in the wall of thefourth ventricle. The CVOs are therefore located in prox-imity to many brain regions, especially the diencephalon.

Highly vascularized structures, the CVOs have pecu-liar vascular arrangements and capillary loops thatalmost reach the ependymal surface, and many of thecapillary beds are surrounded by extensive perivascularspace. As mentioned previously, a BBB is absent due tofenestrations in the capillary endothelium. Some CVOs,including the median eminence, are endowed with

neurosecretory functions, allowing delivery of sub-stances synthesized in the brain to the periphery viathe circulation (McKinley et al., 2004). Interestingly,the CVOs are interconnected, through afferent andefferent projections, with a number of brain circuits,as ascertained in experimental studies in rodents(Oldfield and McKinley, 1995).

Parasites can reach the CVOs via the bloodstream evenwithout invading the brain parenchyma and/or prior toneuroinvasion. From the CVOs parasites can potentially

N

affect neuronal functions at distinct brain sites, throughhumoral parasite�host interaction and/or axonal retro-grade signaling.

A paradigmatic example (and the only one that hasbeen up to now experimentally investigated) is providedby Trypanosoma brucei (Kristensson et al., 2010). Asshown in experimental T. brucei brucei infections, theseparasites reside for some time within the CVOs beforecrossing the BBB. African trypanosomiasis causes char-acteristic disturbances of sleep structure and sleep-wakealternation in both humans and rats, in the absence ofovert neurodegenerative events or other neuronaldamage. The pathogenesis of the distinct clinical signsof African trypanosome infection remains to be fullyclarified. However, a dialog between the host andparasites located in the CVOs is very likely to be impli-cated in this dysfunction even prior to parasite neuroin-vasion. This dialog can occur via factors released byparasites and molecules implicated in the host brainimmune response to the presence of parasites in theCVOs. In particular, these mechanisms could involvethe arcuate nucleus�median eminence complex, whichis connected with diencephalic neuronal cell groups thatsubserve the regulation of sleep/wakefulness andcircadian rhythms (Buijs et al., 2006; Kristenssonet al., 2010).

MECHANISMS OF CNS INVASIO

CONCLUDINGREMARKSANDFUTUREDIRECTIONS

Brain infections by parasites, in spite of their frequencyworldwide and high incidence, are a neglected area ofresearch in basic neuroscience. This is due at least in partto the fact that these infections are most prevalent inlow-income countries. Nevertheless, as indicated bythe present review, nervous system involvement is theleading sign of several parasitic diseases, and there areimportant gaps in knowledge on mechanisms the patho-gens employ to target the nervous system and causedysfunctions.

With the exception of some amebae that can use theolfactory route, parasites spread to the nervous systemvia the bloodstream in humans (Table 2.1). A deeper under-standing is needed on how human host factors influencethe entry, development, replication, and spread of para-sites (The malERA Consultative Group on Basic Scienceand Enabling Technologies, 2011). The importance ofknowledge of host�parasite interactions at the molecularlevel is also evident from the newdrug discovery paradigmthat aims to identify and target host factors that affectinvasion, growth, and spread of parasites in a host, insteadof focusing only on drug targets in the parasites. Thisendeavor is partly spurred by the increasingmicrobial drug

resistance that reduces the number of drug targets avail-able in the parasites (Schwegmann andBrombacher, 2008).

Neuroscience research should therefore contribute tobetter understanding of how parasites interact with theBBB and on mechanisms of parasite traversal and target-ing of various regions in the nervous system. Consideringthe bidirectional flow of information between cerebralsmall vessels and synapses mediated by astrocytic pro-cesses, analysis is needed on the effects of inflammatoryreactions in cerebral endothelia on synaptic functions inneurovascular units. Studies on the indirect effectsexerted on diencephalic neurons by parasites targetingCVOs could give insight on how parasites cause distur-bances in state-dependent behavior of the host.

Last but not least, neuroscience may provide anunderstanding of mechanisms by which functional dis-turbances appear as a late sequel to a parasite infec-tion; this knowledge could help devise preventivestrategies. More in general, progress in integratedbasic research on parasite- and host-derived factorsthat influence nervous system infections has a greatpotential for new strategies of prevention and/or man-agement of these most dreadful complications of par-asite infections.

AND DAMAGE BY PARASITES 19

ACKNOWLEDGMENTS

The studies have been supported by grants from the USNIH/Fogarty (1R21NS064888-01A1), from the WellcomeTrust (WT089992MA) and the Swedish Research Coun-cil (04480).

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