Toxoplasma gondii Actively Inhibits Neuronal Function in Chronically Infected Mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii–infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca2+) imaging studies revealed that tachyzoites actively manipulated Ca2+ signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca2+ uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca2+ stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host

Response to letter to the editor from Elinder and Nordberg concerning Byber et al. 2016. Cadmium or cadmium compounds and chronic kidney disease in workers and the general population: a systematic review, Crit Rev Toxicol. 46(3):191-240. DOI: 0.3109/10408444.2015.1076375.

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Cadmium or cadmium compounds and chronic kidney disease in workers and the general population: a systematic review

Background: Cadmium (Cd) is abundantly documented as a metal mainly affecting tubular function both in workers and in the general population indirectly exposed via the environment. Results from epidemiological studies linking Cd exposure and risk of progression to chronic kidney disease (CKD) are, however, conflicting. Objectives: To perform a systematic review of the association between Cd exposure and CKD. Methods: A systematic appraisal of publications found in MEDLINE (1946–2014), EMBASE (1974–2012) and an in-house database (1986–2013) was conducted. Additional studies were searched for by contacting experts and checking reference lists. Search terms used key and text words. No language restriction was applied. Cohort, case–control and case-series with follow-up including individual and objective assessment of occupational or environmental exposure were eligible. Studies were selected and data extracted by two independent reviewers using predefined forms. Study characteristics and results were extracted to structured tables. Synthesis was qualitative and results appraised with causality criteria. Results: Thirty-four exposed groups, totaling more than 3000 participants, were eligible. Overall, results disclosed no convincing evidence supporting a risk of progression to CKD in populations exposed to Cd. Lack of information about methods, risk of bias and heterogeneity were identified as limitations and precluded conducting a meta-analysis. Publication bias did not appear as a major problem. Conclusions: This qualitative systematic review does not support the contention that human exposure to Cd leads to progressive CKD. Abbreviations: AQC: analytical quality control; A1MG: a1-microglobulin; B2MG: b2-microglobulin; Cd: cadmium; Cd-B: cadmium in blood; Cd-kidney: Cd in kidney; Cd-liver: Cd in liver; Cd-U: cadmium in urine; CI: confidence interval; ClC5: chloride channel 5; CKD: chronic kidney disease; CKD-EPI: CKD Epidemiology Collaboration; CKDu: chronic kidney disease of unknown origin; ESRD: end-stage renal disease (kidney failure treated by dialysis or transplantation); GFR: glomerular filtration rate; eGFR: estimated glomerular filtration rate; GM: geometric mean; GSD: geometric standard deviation; HMW: high-molecular-weight; ICD: International Classification of Diseases; KDIGO: Kidney Disease Improving Global Outcomes; LMW: low-molecular-weight; MDRD: Modification of Diet in Renal Disease; mGFR: measured GFR; NA: non-applicable; NAG: N-acetyl-b-D-glucosaminidase; NI: no information; ND: not done; ns: non-significant; P: plasma; RBP: retinol-binding protein; RRT: renal replacement therapy; S: serum; SD: standard deviation; SMR: standardized mortality ratio; U: urin

The number of intracerebral T. gondii cysts declines over time in the total brain but not in brain regions important for behaviour.

<p>The total number of <i>T. gondii</i> cysts was evaluated on complete <i>T. gondii</i>-immunostained brain section series at day 30 (n = 4) and 60 p.i. (n = 5). (<i>A</i>) The number significantly declined from day 30 to 60 p.i. (p<0.05). (<i>B</i>) However, the number of <i>T. gondii</i> cysts significantly declined only in the indicated (*) brain regions (p<0.05) but not in the remaining brain regions.</p

Disturbed Ca²⁺ response in glutamate-stimulated, tachyzoite infected cortical neurons in vitro.

<p>(<i>A–C</i>) Tachyzoites infect dendrites (<i>A</i>), soma (<i>B</i>), and axons (<i>C</i>) of cultivated cortical neurons as revealed by double immunofluorescence anti-<i>T. gondii</i> (red fluorescent) and anti-class III β-tubulin staining (green fluorescent). <i>(D)</i> Live cell Ca2⁺ imaging of non-infected control and tachyzoite-infected neurons upon stimulation with glutamate. The time course of Ca2⁺ response is shown as three serial images at 0, 500, and 1000 seconds. Red arrow exhibits a hyper-responsive infected cell while the white arrows demonstrate hypo-responsive infected cells. A counter immunofluorescence staining of the infected culture was done with anti-<i>T. gondii</i> showing the presence of toxoplasma tachyzoits in the imaged neurons. <i>(E)</i> A graphical representation of calcium responses elicited by controls and infected neurons; control (n = 6), infected hyper-responsive (n = 7), infected hypo-responsive (n = 7). Data are represented as mean ± SEM. (<i>F</i>) Neurons infected with heat killed <i>T. gondii</i> in-vitro showed no changes in calcium responses upon glutamate stimulation. (<i>G</i>) Thapsigargin induces a huge calcium response in the uninfected controls (n = 8) which does not regress due to the inability to reuptake calcium in the endoplasmic stores, but the infected neurons (n = 9) show only a weak signalling upon thapsigargin-stimulation indicating a depletion of intracellular calcium stores.</p

Loss of fear against cat urine in chronically T. gondii-infected mice.

<p>(<i>A</i>) Compared to non-infected BALB/c mice, no increase in the time spent in cat urine corner was evident in mice at day 30 p.i. (control: n = 6, infected: n = 6). In contrast, at day 60 p.i., infected mice spent a significantly longer time in the cat urine corner as compared to non-infected control mice (p<0.05; control: n = 8, infected: n = 9). (<i>B</i>) The relative occupancy of the cat urine corner (ratio of time spent only in the cat urine corner to the total time spent in both cat and rabbit urine corner) was significantly increased at day 60 (p<0.05) but not at day 30 p.i. as compared to non-infected control mice. (<i>C, E</i>) Compared to non-infected BALB/c mice, no difference in the number of visits or the distance travelled within the cat and rabbit urine corner were evident at day 30 p.i. (control: n = 6, infected: n = 6). However at day 60 p.i., the number of visits to and the distance travelled within the rabbit urine corner were decreased (p<0.05; control: n = 8, infected n = 9). (<i>D, F</i>) The relative visits to the cat urine corner and the relative distance travelled within the cat urine corner were significantly increased in the infected mice at day 60 (p<0.05 for both parameters) but not at day 30 p.i. as compared to non-infected control mice.</p

T. gondii cysts reside in all major compartments of neurons.

<p>(<i>A–F</i>) <i>T. gondii</i> cysts were identified in dendrites (<i>A, B</i>) (<i>striatum, day 30 i.p.</i>), somata (<i>C, D</i>) pyramide cell in cortex, day 30 i.p), and axons <i>(E, F)</i> (<i>striatum, day 60</i> i.p.) in <i>T. gondii</i> immunostained brain sections. <i>T. gondii</i> antigen was also located outside intraneuronal cysts in dendrites (<i>A–D, G</i>), axons (<i>E–G</i>), and somata (<i>A–G</i>). In (<i>G</i>) (cortex, day 60 i.p.), the small arrow points to a dendrite and the large arrow to an axon of a pyramidal neuron which was stained Golgi-like by the <i>T. gondii</i> antibody.</p

The majority of cyst-infected neurons are functionally silenced in chronic TE.

<p>Thallium uptake in <i>T. gondii</i> infected mice 30 d p.i. <i>(A, B)</i> and 60 d p.i. <i>(C, D)</i>. Tl⁺AMG shows T1⁺ positive, functionally active, cyst-infected neurons (white arrows) at day 30 p.i. <i>(A)</i> and day 60 p.i. <i>(C)</i> as well as Tl⁺ negative cyst-infected neurons at day 30 p.i. <i>(B)</i> and 60 p.i. <i>(D)</i>. The upper-left insets in <i>(A–D)</i> show an overview of the Tl⁺ stained sections and the area (rectangle) containing the illustrated cysts. In <i>(A, D)</i>, c depicts the cytoplasm of the infected neurons and Φ the cysts. In <i>(A)</i>, lower-left inset, the tissue section has been stained with Tl⁺ and thereafter counterstained with hemalum to show the intact cell wall of the neuron and cyst (blue arrow). Similarly, in <i>(D)</i> lower-left inset shows the Nissl-counterstained neuron and cyst (blue arrow). (<i>E</i>) A quantitative analysis of Tl⁺ positive and negative neurons was performed at day 30 and 60 p.i. from three mice per experimental group. The total number of cysts as well as the number of Tl⁺ negative cysts per brain were determined on 50 corresponding sections from various brain regions of each mouse. Data show a significant increase of Tl⁺ negative cyst-infected neurons at day 60 compared to day 30 p.i. (p<0.05).</p