26The parasitic protozoan Toxoplasma gondii becomes encysted in brain and muscle tissue 27 during chronic infection, a stage that was previously thought to be dormant but has been 28 found to be active and associated with physiological effects in the host. Dysregulation of 29 catecholamines in the CNS has previously been observed in chronically-infected animals. In 30 the study described here, the noradrenergic system was suppressed with decreased levels of 31 norepinephrine in brains of infected animals and in infected neuronal cells in vitro. 32 Expression of dopamine β-hydroxylase (DBH), essential for synthesis of norepinephrine 33 from dopamine, was the most differentially-expressed gene in infections in vitro and was 34 down-regulated in infected brain tissue, particularly in the prefrontal cortex and dorsal locus 35 coeruleus/pons region. The down-regulated DBH expression in infected rat 36 catecholaminergic and human neuronal cells corresponded with decreased norepinephrine 37 and increased dopamine. As the DBH suppression was observed in vitro, this effect is not 38 caused by neuroinflammation. Silencing of DBH expression was specific for T. gondii infection 39 and was not observed with CMV infection. The noradrenergic-linked behaviors of sociability 40 and arousal were altered in chronically-infected animals, with a high correlation between 41 DBH expression and infection intensity. These findings together provide a plausible 42 mechanism to explain prior discrepancies in changes to CNS neurotransmitters levels with 43 infection. The suppression of norepinephrine synthesis observed here may, in part, explain 44 behavioural effects of infection, associations with mental illness, and neurological 45 3 consequences of infection such as the loss of coordination and motor impairments associated 46 with human toxoplasmosis. 47 48T. gondii infects warm-blooded animals and is characterised by a transient acute infection 49 wherein vegetative tachyzoite forms rapidly replicate in tissues followed by a persistent 50 chronic infection. Chronic stages of infection can persist for years and potentially the lifetime 51 of the host with the bradyzoite-stage parasites encysted in cells within immunoprivileged 52 tissues, including muscle, eyes, and brain. Several reports have published host behavioral 53 changes with infection. A selective loss of aversion to feline urine and increased motor 54 activity has been observed in rodents, specifically manipulating behavior that will enhance 55 the probability of parasite transmission (1, 2). 56Toxoplasmosis can be a severe disease in immunocompromised individuals and in utero. 57Infection can cause retinochoroiditis and congenital hydrocephalus and cerebral 58 calcifications. T. gondii was recently ranked the second most important food-borne parasite 59 in Europe and is classified as a Neglected Parasitic Infection (CDC, Atlanta) (3). It has also 60 been linked by epidemiological studies to cognitive impairment and major mental illnesses. 61Severe cases are ass...

Although the parasite Toxoplasma gondii is one of the most pervasive neurotropic pathogens in the world, the host-parasite interactions during CNS infection and the consequences of neurological infection are just beginning to be unraveled. The chronic stages of infection have been considered dormant, although several studies have found correlations of infection with an array of host behavioral changes. These may facilitate parasite transmission and impact neurological diseases. During infection, in addition to the presence of the parasites within neurons, host-mediated neuroimmune and hormonal responses to infection are also present. T. gondii induces numerous changes to host neurons during infection and globally alters host neurological signaling pathways, as discussed in this review. Understanding the neurophysiological changes in the host brain is imperative to understanding the parasitic mechanisms and to delineate the effects of this single-celled parasite on health and its contribution to neurological disease.

Although the parasite Toxoplasma gondii is one of the most pervasive neurotropic pathogens in the world, the host-parasite interactions during CNS infection and consequences of neurological infection are just beginning to be unraveled. The chronic stages of infection have been considered dormant, although several studies have found correlations of infection with an array of host behavioral changes. These may facilitate parasite transmission and impact neurological diseases. During infection, in addition to the presence of the parasites within neurons, hostmediated neuroimmune and hormonal responses to infection are also present. T. gondii induces numerous changes to host neurons during infection and globally alters host neurological signaling pathways, as discussed in this review. Understanding the neurophysiological changes in the host brain is imperative to understanding the parasitic mechanisms and to delineate the effects of this single-celled parasite on health and its contribution to neurological disease.

Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro. The mechanism responsible for the NE suppression was found to be downregulation of dopamine ␤-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.

Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function.