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Transmissible neurodegenerative prion diseases are characterized by the conversion of the cellular prion protein (PrPC) to misfolded isoforms denoted as prions or PrP^(Sc). Although the conversion can occur in the test tube containing recombinant prion protein or cell lysates, efficient prion formation depends on the integrity of intact cell functions. Since neurons are main targets for prion replication, we asked whether their most specialized function, i.e. synaptic plasticity, could be a factor by which PrP^(Sc) formation can be modulated. Immortalized gonadotropin-releasing hormone cells infected with the Rocky Mountain Laboratory prion strain were treated with L-type calcium channels (LTCCs) and NMDA receptors (NMDARs) stimulators or inhibitors. Western blotting was used to monitor the effects on PrP^(Sc) formation in relation to ERK signalling. Infected cells showed enhanced levels of phosphorylated ERK (pERK) compared with uninfected cells. Exposure of infected cells to the LTCC agonist Bay K8644 enhanced pERK and PrP^(Sc) levels. Although treatment with an LTCC blocker (nimodipine) or an NMDAR competitive antagonist (D-AP5) had no effects, their combination reduced both pERK and PrP^(Sc) levels. Treatment with the non-competitive NMDAR channel blocker MK-801 markedly reduced pERK and PrP^(Sc) levels. Our study shows that changes in LTCCs and NMDARs activities can modulate PrP^(Sc) formation through ERK signalling. During synaptic plasticity, while ERK signalling promotes long-term potentiation accompanied by expansion of post-synaptic lipid rafts, other NMDA receptor-depending signalling pathways, p38-JNK, have opposing effects. Our findings indicate that contrasting intracellular signals of synaptic plasticity can influence time-dependent prion conversion.

In this review we describe how Trypanosoma brucei brucei, a rodent pathogenic strain of African trypanosomes, can invade the nervous system, first by localization to the choroid plexus, the circumventricular organs (CVOs) and peripheral ganglia, which have fenestrated vessels, followed by crossing of the blood-brain barrier (BBB) into the white matter, hypothalamus, thalamus and basal ganglia. White blood cells (WBCs) pave the way for the trypanosome neuroinvasion. Experiments with immune deficient mice show that the invasion of WBCs is initiated by the toll-like receptor 9, followed by an augmentation phase that depends on the cytokine IFN-γ and the chemokine CXCL10. Nitric oxide (NO) derived from iNOS then prevents a break-down of the BBB and non-regulated passage of cells. This chain of events is relevant for design of better diagnostic tools to distinguish the different stages of the disease as well as for better understanding of the pathogenesis of the nervous system dysfunctions, which include circadian rhythm changes with sleep pattern disruption, pain syndromes, movement disorders and mental disturbances including dementia.

The global public health concern is heightened over the increasing number of emerging viruses, i.e., newly discovered or previously known that have expanded into new geographical zones. These viruses challenge the health-care systems in sub-Saharan Africa (SSA) countries from which several of them have originated and been transmitted by insects worldwide. Some of these viruses are neuroinvasive, but have been relatively neglected by neuroscientists. They may provide experiments by nature to give a time window for exposure to a new virus within sizeable, previously non-infected human populations, which, for instance, enables studies on potential long-term or late-onset effects on the developing nervous system. Here, we briefly summarize studies on the developing brain by West Nile, Zika, and Chikungunya viruses, which are mosquito-borne and have spread worldwide out of SSA. They can all be neuroinvasive, but their effects vary from malformations caused by prenatal infections to cognitive disturbances following perinatal or later infections. We also highlight Ebola virus, which can leave surviving children with psychiatric disturbances and cause persistent infections in the non-human primate brain. Greater awareness within the neuroscience community is needed to emphasize the menace evoked by these emerging viruses to the developing brain. In particular, frontline neuroscience research should include neuropediatric follow-up studies in the field on long-term or late-onset cognitive and behavior disturbances or neuropsychiatric disorders. Studies on pathogenetic mechanisms for viral-induced perturbations of brain maturation should be extended to the vulnerable periods when neurocircuit formations are at peaks during infancy and early childhood.

PrPSc forms micrometer long amyloidic strings that can congregate in clusters and webs at the surface of living cells., Mammalian prions refold host glycosylphosphatidylinositol-anchored PrPC into β-sheet–rich PrPSc. PrPSc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrPSc rather than on its truncated PrP27-30 product. We show that N-terminal PrPSc epitopes are exposed in their physiological context and visualize, for the first time, PrPSc in living cells. PrPSc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrPSc amyloids.

Peroxiredoxins contribute to protection of some bacteria against reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with ROIs and RNIs during infection. In this study, we investigated the involvement of the 2-Cys peroxiredoxin (Prx) homologue in L. monocytogenes in the protection against ROIs and RNIs and in virulence through the construction of an in-frame prx deletion mutant. The Δprx mutant had increased sensitivity to hydrogen peroxide and cumene hydroperoxide compared to the wild-type strain. The mutant also exhibited an increased susceptibility to the nitric oxide-generating compound S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor. Furthermore, a diminished virulence of the Δprx mutant relative to the wild-type was observed in C57BL/6 mice, but not in inducible nitric oxide synthase-deficient mice. The results suggest that Prx protects L. monocytogenes against oxidative and nitrosative stress in vitro and in vivo and that the prx-encoded polypeptide thereby is involved in L. monocytogenes virulence.

Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority (www.prionpriority.eu) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper (www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies. © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC.

The blood-brain barrier (BBB) is a structural and functional barrier that protects the central nervous system (CNS) from invasion by blood-borne pathogens including parasites. However, some intracellular and extracellular parasites can traverse the BBB during the course of infection and cause neurological disturbances and/or damage which are at times fatal. The means by which parasites cross the BBB and how the immune system controls the parasites within the brain are still unclear. In this review we present the current understanding of the processes utilized by two human neuropathogenic parasites, Trypanosoma brucei spp and Toxoplasma gondii, to go across the BBB and consequences of CNS invasion. We also describe briefly other parasites that can invade the brain and how they interact with or circumvent the BBB. The roles played by parasite-derived and host-derived molecules during parasitic and white blood cell invasion of the brain are discussed.

Infections with subspecies of the extracellular parasite Trypanosoma brucei cause African trypanosomiasis, a disease that affects both humans (sleeping sickness) and animals. During the early stage of human African trypanosomiasis the parasites invade the hemolymphatic system, and during the late meningoencephalitic stage severe signs of nervous system involvement are observed [1–3]. In a mouse model of the disease, T. brucei brucei penetrate the blood-brain barrier (BBB) at a late stage and can enter the brain parenchyma [4]. Within the CNS, activation of white blood cell infiltrates and resident cells probably leads to the nervous system disease [5]. The innate immune system has evolved several strategies of self-nonself discrimination that are based on the recognition of molecular patterns demarcating infectious nonself. Different Toll-like receptors (TLRs), by recognizing diverse pathogen-associated molecular patterns of microbes, activate innate immunity and may initiate the subsequent development of adaptive immunity. TLR agonists stimulate the secretion of proinflammatory cytokines and type I interferons (IFN-α/β) that are involved not only in protection against infections but also in infection-mediated pathology. The development of adaptive immune responses is at least in part mediated through the ability of the innate receptor signaling to activate and stimulate the migration of antigen-presenting cells into the lymph nodes [6]. The binding of TLRs (except TLR3) by their corresponding ligands results in the recruitment of the intracellular adaptor molecule MyD88. Myd88–/– animals are highly susceptible to infection with a wide variety of different pathogens, including infection with T. brucei brucei [7–9]. We have shown elsewhere that the T-cell–derived cytokine IFN-γ, as well as the IFN-inducible chemokine CXCL10, promote the penetration of T cells and parasites in the brain [4, 10], suggesting also that parasites follow T cells during their brain invasion across the BBB. Despite the accepted view that signaling from specific innate immune receptors are required to activate and determine the quality of T-cell responses, the role of innate immunity in T-cell–mediated central nervous system (CNS) diseases, such as sleeping sickness, is poorly understood. In the current study, we investigated whether signals emanating from TLR control the accumulation of T cells and parasites in the brain parenchyma. We demonstrate that TLR signaling regulates the penetration of T cells and parasites across the BBB and controls survival of the latter in the brain parenchyma through distinct mechanisms.

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.

The extracellular parasite Trypanosoma brucei causes human African trypanosomiasis (HAT), also known as sleeping sickness. Trypanosomes are transmitted by tsetse flies and HAT occurs in foci in sub-Saharan Africa. The disease, which is invariably lethal if untreated, evolves in a first hemo-lymphatic stage, progressing to a second meningo-encephalitic stage when the parasites cross the blood-brain barrier. At first, trypanosomes are restricted to circumventricular organs and choroid plexus in the brain outside the blood-brain barrier, and to dorsal root ganglia. Later, parasites cross the blood-brain barrier at post-capillary venules, through a multi-step process similar to that of lymphocytes. Accumulation of parasites in the brain is regulated by cytokines and chemokines. Trypanosomes can alter neuronal function and the most prominent manifestation is represented by sleep alterations. These are characterized, in HAT and experimental rodent infections, by disruption of the sleep-wake 24h cycle and internal sleep structure. Trypanosome infections alter also some, but not all, other endogenous biological rhythms. A number of neural pathways and molecules may be involved in such effects. Trypanosomes secrete prostaglandins including the somnogenic PGD2, and they interact with the host's immune system to cause release of pro-inflammatory cytokines. From the sites of early localization of parasites in the brain and meninges, such molecules could affect adjacent brain areas implicated in sleep-wakefulness regulation, including the suprachiasmatic nucleus and its downstream targets, to cause the changes characteristic of the disease. This raises challenging issues on the effects of cytokines on synaptic functions potentially involved in sleep-wakefulness alterations.

African sleeping sickness is characterized by alterations in rhythmic functions. It is not known if the disease affects the expression of clock genes, which are the molecular basis for rhythm generation. We used a chronic rat model of experimental sleeping sickness, caused by the extracellular parasite Trypanosoma brucei brucei (Tb brucei), to study the effects on clock gene expression. In tissue explants of pituitary glands from Period1- luciferase ( Per1-luc) transgenic rats infected with Tb brucei, the period of Per1-luc expression was significantly shorter. In explants containing the suprachiasmatic nuclei (SCN), the Per1-luc rhythms were flat in 21% of the tissues. We also examined the relative expression of Per1, Clock, and Bmal1 mRNA in the SCN, pineal gland, and spleen from control and infected rats using qPCR. Both Clock and Bmal1 mRNA expression was reduced in the pineal gland and spleen following Tb brucei infection. Infected rats were periodic both in core body temperature and in locomotor activity; however, early after infection, we observed a significant decline in the amplitude of the locomotor activity rhythm. In addition, both activity and body temperature rhythms exhibited decreased regularity and “robustness.” In conclusion, although experimental trypanosome infection has previously been shown to cause functional disturbances in SCN neurons, only 21% of the SCN explants had disturbed Per1-luc rhythms. However, our data show that the infection overall alters molecular clock function in peripheral clocks including the pituitary gland, pineal gland, and spleen.

Prions are infectious agents resulting from the conversion of a normal cellular protein, PrPC, to a misfolded species, PrPSc. Iatrogenic transmission of prions is known from surgical procedures involving stainless steel materials. Here, it was shown that stainless steel containing nickel and molybdenum binds PrPSc more efficiently and transmits infection to cells in culture to a higher degree than if these elements are not present. Furthermore, both nickel and molybdenum alone adsorbed PrPSc, and nickel powder could be used to extract PrPSc from dilute solutions, thus providing a simple approach to concentration of PrPSc. The fact that nickel and molybdenum in steel alloys increased the binding affinity, and bound infectivity, of PrPSc is an important issue to consider in the manufacture of surgical instruments and abattoir tools.

The ultrastructural and histochemical properties of the classical, eosinophilic inclusion bodies in neurones from mice infected with herpes simplex virus were examined. Ultrastructurally the inclusion body was found to consist of finely fibrillary material with a few interspersed virus nucleocapsids. Histochemically they contained small amounts of DNA as demonstrated with the fluorescent Feulgen method. Furthermore they contained strongly basic proteins, probably rich in arginine, responsible for the characteristic eosinophilia. The origin of the inclusions is discussed and it is considered more likely that the proteins and DNA are specified by the virus rather than by the host cell.

Epidemiological studies suggest that early life infections may contribute to the development of psychiatric disorders characterized by cognitive deficits. Here, we studied the effects of a neonatal influenza A/WSN/33 virus infection on locomotor activity, working memory and emotional behavior in adult mice. In addition to wild type mice, immunodeficient (Tap1(-/-)) mice lacking functional CD8(+) T cells, were included in the study to model the potential influence of a genetic deficit relating to virus clearance. Three to four months after the infection, infected Tap1(-/-) mice, but not wild type mice, exhibited deficits in working memory as well as increased rearing activity and anxiety. In the medial prefrontal cortices of these infected Tap1(-/-) mice reduced levels of type III Nrg1 transcripts were observed supporting a role for neuregulin 1 signaling in neuronal circuits involved in working memory. Virus replication, distribution or clearance did not differ between the two genotypes. The lack of CD8(+) T cells, however, appeared to contribute to a more pronounced glia response in Tap1(-/-) than in wild type mice. Thus, the present study suggest that the risk of developing deficits in cognitive and emotional behavior following a CNS infection during brain development is influenced by genetic variation in genes involved in the immune response.

The effect of tumor necrosis factor-alpha (TNF-alpha) on excitability and synaptic function was analyzed in slice preparations of the suprachiasmatic nuclei (SCN), the major mammalian circadian pacemaker. TNF-alpha caused a rapid increase in the spontaneous firing rate in most SCN neurons examined that was paralleled by an increase of inhibitory postsynaptic currents. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester abolished these effects. No effect of TNF-alpha was found on miniature synaptic currents. The lack of effect on miniature synaptic currents indicates that TNF-alpha primarily affects neuronal membrane properties to cause the changes in spontaneous firing. TNF-alpha, levels of which show circadian variation in the brain and increase during inflammatory conditions and aging, may thus through nitric oxide induction modulate SCN electrical output to affect downstream circadian rhythms.

Trypanosoma brucei subspecies invade the brain parenchyma at late stages of human and experimental rodent infections. In this study, we compared the outcome of infection with T. b. brucei in MHC-matched (H-2b) C57BL/6 (B6) and 129Sv/Ev (Sv-129). Sv-129 showed higher parasitaemia and lower specific IgM (but not IgG) antibody levels than B6 mice. The number of trypanosomes, CD4+ and CD8+ T cells in the brain parenchyma was higher in B6 mice. B6 mice lost weight and showed higher cumulative mortality when compared with Sv-129 mice. Higher levels of IL-1beta, IL-6, IL-10, TNF-alpha, IFN-gamma, ICAM-1 and E-selectin, but low levels of TGF-beta mRNA were present in brains of B6 when compared with Sv-129-infected mice. Thus, host genetics differentially determine the invasion of T. b. brucei into the brain parenchyma, which is paralleled by the severity of inflammation in the brain and course of the disease, but not by parasitaemia nor by antibody titres.

Brain-derived neurotrophic factor, which activates the extracellular regulated kinase (ERK) pathway, increases formation of prions in scrapie-infected gonadotropin-releasing hormone (GT1-1) cells. This indicates that conversion of the cellular prion protein PrP(C) to its pathogenic isoform, PrP(Sc), can be regulated by physiological stimuli acting on specific signal transduction pathways. In the present study, we examined the involvement of different mitogen-activated protein (MAP) kinase cascades and the cAMP-PKA pathway in formation of proteinase K-resistant PrP(Sc) (rPrP(Sc)). Long-term depolarization of GT1-1 cells infected with the Rocky Mountain Laboratory strain of scrapie increased the formation of rPrP(Sc). This effect was associated to ERK activation and was blocked by the MAPK/ERK kinase (MEK) inhibitor U0126. Treatment with forskolin caused a similar increase in rPrP(Sc) formation that was prevented by the protein kinase A (PKA) inhibitor H89. Both depolarization and forskolin treatment were accompanied by increased phosphorylation of the S6 ribosomal protein, while phosphorylation of histone H3 occurred only after forskolin treatment. Inhibitors of p38- and c-Jun NH(2)-terminal kinase (JNK) promoted the formation of rPrP(Sc), in contrast to the clearance of rPrP(Sc) produced by inhibitors of the ERK pathway. Thus, the ERK and the p38-JNK MAP kinase pathways appear to exert opposing effects on rPrP(Sc) formation, suggesting that balances between these intracellular signaling cascades may regulate replication of prions.

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.

Infections during early life have been suggested to play a role in the etiology of schizophrenia. Most studies have focused on fetal life; few have explored risk associated with infection during childhood. The results of these have been inconsistent. The present study aims to investigate whether there is an increased risk of schizophrenia and other nonaffective psychoses associated with viral or bacterial CNS infections during childhood and, if so, which specific agents are involved.A national cohort consisting of 1.2 million children born between 1973 and 1985 was followed up by using Swedish national registers to retrieve data on hospital admissions for CNS infections at 0-12 years of age (bacterial: N=2,435, viral: N=6,550) as well as admissions for nonaffective psychotic illnesses from the 14th birthday (N=2,269).There was a slightly increased risk of nonaffective psychotic illness associated with viral CNS infections, as well as schizophrenia. There was no evidence of increased risk in relation to bacterial infections. When divided into specific agents, exposures to mumps virus or cytomegalovirus were associated with subsequent psychoses.Serious viral CNS infections during childhood appear to be associated with the later development of schizophrenia and nonaffective psychoses. The association with specific viruses suggests that the risk is related to infectious agents with a propensity to invade the brain parenchyma.

An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009-2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep-wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep-wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.

Spread of pathogens to the nervous system is a serious complication of infections. In addition to infections with well-known microbes in the Western world, and the World Health Organization priorities of HIV/AIDS, malaria, and tuberculosis, several neglected tropical infectious diseases target the nervous system and have high mortality rates. Infections can cause cognitive and behavioral disturbances as well as late-onset epilepsy in survivors. The specialized environment in the brain dampens immune responses to avoid harmful effects on the non-renewable nervous tissue. Some microbes can therefore evade efficient elimination, and interactions between microbes and nervous tissues have evolved that favor either one of them or both. Microbes are useful tools to study the structure and function of the nervous system. In return, neuroscience can disclose mechanisms of neurodegeneration and brain dysfunctions from studies of the interplay among pathogens, nervous tissues, and immune responses that could lead to better management of brain disorders.

Recent incidences of direct passage of highly pathogenic avian influenza A virus strains of the H5N1 and H7N7 subtypes from birds to man have become a major public concern. Although presence of virus in the human brain has not yet been reported in deceased patients, these avian influenza subtypes have the propensity to invade the brain along cranial nerves to target brainstem and diencephalic nuclei following intranasal instillation in mice and ferrets. The associations between influenza and psychiatric disturbances in past epidemics are here commented upon, and the potentials of influenza to cause nervous system dysfunction in experimental infections with a mouse-neuroadapted WSN/33 strain of the virus are reviewed. This virus strain is closely related to the Spanish flu virus, which is characterized as a uniquely high-virulence strain of the H1N1 subtype. The Spanish flu virus has recently been reconstructed in the laboratory and it passed once, most likely, directly from birds to humans to cause the severe 1918-1919 pandemic.

The aging process brings about a switch to a low-grade chronic inflammatory condition in the periphery and brain, a condition which may prime brain cells, including those of the hypothalamic suprachiasmatic nucleus (SCN). Little information is available, however, on the responses of the SCN to neuroinflammation and immune-related challenges, and such responses have not been hitherto investigated during aging. We here provide an overview of these issues and summarize data we obtained in the study of the SCN of young and aged mice. In particular, we analyzed: i) the electrophysiological properties of the SCN core (the retino-recipient region) in tissue slices; ii) expression and day/night variation of transcripts encoding the receptors for the cytokines interferon-gamma and tumor necrosis factor-alpha, as well as the expression of transcripts encoding the proteins "suppressors of cytokine signaling" SOCS1 and SOCS3, by means of quantitative real-time polymerase chain reaction; levels of mRNAs were correlated with neuronal activation, revealed by Fos induction, elicited in the SCN by intracerebroventricular injections of a mixture of interferon-gamma and tumor necrosis factor-alpha during the daytime and nighttime; and iii) response of astrocytes and microglia in the SCN to the same paradigm of cytokine administration. Marked changes of all the above-mentioned parameters were found in the aged SCN, indicating that the circadian pacemaker is a target of the aging process. In addition, the findings indicate that neurons and glial cells of the biological clock are sensitive to inflammatory signals, and that the response to such signals is altered during senescence.

Prions represent a unique class of infectious agents in which the normal cellular prion protein (PrPC) is converted to an abnormal isoform (PrPSc), which accumulates in the brain and constitutes the major, if not the only, component of the infectious particle. Factors that still remain to be identified may facilitate the conversion of PrPCto PrPSc. In the present study, we first demonstrated that a growth factor of the neurotrophin family, brain-derived neurotrophic factor (BDNF), stimulates the formation of PrPScin a gonadotropin-releasing hormone-secreting neuronal cell line (GT1-1 cells) infected with the Rocky Mountain Laboratory (RML) strain of scrapie as determined by Western blot analysis. We then observed that the prion-infected cells can be cleared from PrPScby treatment with three inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene and 2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one, as well as α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile, which passes the blood–brain barrier], a component of one of the intracellular signaling pathways activated by BDNF. The MEK1/2 inhibitors were also efficient in clearing PrPScfrom prion-infected GT1-1 cells stimulated to accumulate high levels of PrPScby enhanced serum concentrations in the medium or by the use of a serum-free neuron-specific neurobasal medium. PrPScdid not reappear in the cultures within 5 weeks after completion of treatment. We conclude that inhibitors of the MEK1/2 pathway can efficiently and probably irreversibly clear PrPScfrom prion-infected cells. The MEK pathway may therefore be a suitable target for therapeutic intervention in prion diseases.

In this study, we tested the hypothesis that exposure to a maternal infection during fetal life can lead to the appearance of alterations in the brain later in life. C57BL/6 mice were infected intranasally with influenza A/WSN/33 virus on day 14 of gestation. The levels of transcripts encoding neuroleukin and fibroblast growth factor 5 were significantly elevated in the brains of the virus-exposed offspring at 90 and 280 days of age, but not at earlier time-points. For neuroleukin, this difference could also be observed at the protein level. Thus, a maternal influenza A virus infection can give rise to alterations in gene expression in the brain that become apparent only after a prepubertal latency period.

Certain viruses can infect neurons and cause persistent infections with restricted expression of viral proteins. To study the consequences of such viral proteins on synaptic functions, the effects of two influenza A virus proteins, the nonstructural protein 1 (NS1) and the nucleoprotein (NP), were analyzed in cultures of rat hippocampal neurons. Transduction of the NS1 and NP proteins into the neurons was performed by applying the 11-amino acid peptide transduction domain (PTD) of human immunodeficiency virus (HIV) TAT coupled to the viral proteins. Neurons exposed to the NS1 and NP fusion proteins (NS1-PTD and NP-PTD, respectively) for 4 h were immunopositive for these proteins as diffuse cytoplasmic and nuclear distribution. After exposure for 48 h to NP-PTD, a punctate pattern of the immunolabel appeared in dendritic spinelike processes. Electrophysiologically, a reduction in both the frequency of spontaneous excitatory synaptic activity and in the amplitude of the miniature excitatory postsynaptic currents were recorded after exposing the hippocampal neurons to NP-PTD between 17 and 22 days in culture. These changes may reflect disturbances in postsynaptic functions. No such alterations in synaptic activities were recorded after exposure to NS1-PTD or to green fluorescent protein-PTD, which was used as a control. Based on these findings the authors hypothesize that the viral NP, by its localization to dendritic spinelike structures, interferes with the expression or anchoring of postsynaptic glutamate receptors and thereby disturbs synaptic functions. Thus a persistent viral infection in the brain may be associated with functional disturbances at the synaptic level.

The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA , two genes, cheY and cheA , encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA. cheY , and cheA to elucidate their role in the biology of infection with L. monocytogenes . The Δ cheY , Δ cheA , and double-mutant Δ cheYA mutants, but not Δ flaA mutant, were motile in liquid media. However, the Δ cheA mutant had impaired swarming and the Δ cheY and Δ cheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The Δ flaA , Δ cheY , Δ cheA , and Δ cheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the Δ flaA and Δ cheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the Δ flaA or Δ cheYA mutant. In summary, we show that listerial flaA. cheY , and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.

Certain disorders of the nervous system may have their origin in disturbances in the development of synaptic connections and network structure that may not become overt until later in life. As inflammatory cytokines can influence synaptic activity in neuronal cultures, we analysed whether cytokine exposure during synaptogenesis can lead to imbalances in a neuronal network. Short-term application of interferon-gamma (IFN-gamma), but not tumour necrosis factor-alpha, during peak synaptogenesis (but not before or after) in Sprague-Dawley rat hippocampal cultures, caused both a decrease in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) and an increase in the frequency of spontaneous inhibitory postsynaptic currents (IPSCs). These effects were only detected in recordings made weeks later. This was not due to a depression of glutamatergic synapses or to a change in the relative number of neurons containing glutamic acid decarboxylase (GAD). There was an increase in the average amplitude of miniature IPSCs, and in GAD-expressing neurons the amplitude of miniature EPSCs were larger as well as the responses to glutamate. This indicates that IFN-gamma-treatment induced increased inhibition via postsynaptic changes. These effects of IFN-gamma treatment were not observed when neuronal nitric oxide synthase was inhibited. Our study therefore shows that exposure to IFN-gamma during a restricted period of development, which coincides with the peak of excitatory synaptogenesis, can cause progressive changes in synaptic activity in the network. Thus, cytokine exposure at a critical period of development may constitute a 'hit-and-run' mechanism for certain nervous system disorders that become manifest after a latency period.

The nervous system is an «immune-privileged» site and can provide a reservoir to harbor as persistent or latent infections certain microbes that find their way to the brain. From an evolutionary standpoint, such infections are characterized at most times by low levels of the infectious agent in the systemic domain, except when multiplication has just taken place. Hence the ability for transmission of the pathogens from animals to Man will be determined by the availability of microbes to be transferred by a vector (e.g. in trypanosomiasis), or the amount of infective forms of the microbes shed into an environment (e.g. in toxoplasmosis). Using African trypanosomes, toxoplasma,Listeria and influenza A virus as examples, mechanisms by which microbes can spread and be targeted to and within the brain to cause various types of nervous system dysfunctions is reviewed. Newly revealed potentials of certain cytokines to stimulate neurons to control the growth, and even kill, microbes in their cell bodies is also described.

Dysfunction of subcortical serotoninergic neurons has been implicated in some behaviour disturbances. The serotoninergic neurons in the dorsal and median raphe project widely in the brain. They innervate the olfactory bulbs and can be targets for exogenous agents attacking the olfactory epithelium and bulbs. We report here an injury to the serotoninergic neurons after intranasal infection in 12-day-old rats with a temperature-sensitive mutant of vesicular stomatitis virus. The brain infection was focal and transient. Viral antigens could no longer be detected 13–15 days after infection. In spite of this the animals, as adults, had a severe serotonin depletion in the cerebral cortex and hippocampus, and showed abnormal locomotor and explorative behaviour as well as learning deficits. The neocortex was histologically intact and parameters related to other neurotransmitters such as dopamine, noradrenaline, GABA and acetylcholine showed no marked changes. A relatively selective damage to serotoninergic nuclei as a result of virus neuroinvasion through a natural portal of entry, may constitute a new pathogenetic mechanism for cortical dysfunction and behavioural deficits.

A temperature-sensitive mutant of vesicular stomatitis virus was inoculated intranasally into infant Sprague Dawley rats aged 9 to 17 days. Rats receiving the virus at 9 days of age had an extensive spread of infection throughout the brain and the animals died after a few days. Rats inoculated at day 11 postnatally survived and the infection was limited to the olfactory pathways, hypothalamus, diagonal bands and the anterior raphe nuclei. Stereological measurements showed that the volume of infected neurons constituted 67 ± 10% of the total neuronal volume in the dorsal raphe nucleus. Double-labelling experiments revealed that both 5-hydroxytryptamine- and substance P-immunoreactive neurons contained the virus antigen. The motor stimulant effect of amphetamine was studied at 3 months post infection. The increase in amphetamine-induced frequency and duration of rearing was significantly attenuated in infected rats and the amphetamine-induced locomotion was slightly reduced.