key: cord-0277968-y1kxyv0q
authors: Spirhanzlova, Petra; Sébillot, Anthony; Vancamp, Pieter; Gothié, Jean-David; Mével, Sébastien Le; Leemans, Michelle; Wejaphikul, Karn; Meima, Marcel; Mughal, Bilal B.; Butruille, Lucile; Roques, Pierre; Remaud, Sylvie; Fini, Jean-Baptiste; Demeneix, Barbara A.
title: ZIKA virus effects on neuroprogenitors are exacerbated by the main pyriproxyfen metabolite via thyroid hormone signaling disruption
date: 2020-11-03
journal: bioRxiv
DOI: 10.1101/2020.11.03.366468
sha: 5e04ec0ae72574416130295e746e209779fd4841
doc_id: 277968
cord_uid: y1kxyv0q

North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. TH represses MSI1. Being a suspected TH disruptor, we hypothesized that co-exposure to the main metabolite of PPF, 4’-OH-PPF, would exacerbate ZIKV effects through increased MSI1 expression. This was tested using in vitro mouse neurospheres and an in vivo TH signaling reporter model, Xenopus laevis. TH signaling was decreased by 4’-OH-PPF in both models. In mouse-derived neurospheres the metabolite reduced neuroprogenitor proliferation as well as markers of neuronal differentiation. The results demonstrated that 4’-OH-PPF significantly induced MSI1 at both the mRNA and protein level, as well as Fasn mRNA. Other TH target genes were also significantly modified. Importantly, several key genes implicated in neuroprogenitor fate and commitment were not dysregulated by 4’-OH-PPF alone, but were in combination with ZIKV infection. These included the neuroprogenitor markers Nestin, Egfr, Gfap, Dlx2 and Dcx. Unexpectedly, 4’-OH-PPF decreased ZIKV replication, although only at the fourth and last day of incubation, and RNA copy numbers stayed within the same order of magnitude. However, intracellular RNA content of neuroprogenitors was significantly decreased in the combined presence of the PPF metabolite and ZIKV. We conclude that 4’-OH-PPF interferes with TH action in vivo and in vitro, inhibiting neuroprogenitor proliferation. In the presence of ZIKV, TH signaling pathways crucial for cortical development are significantly impacted. This provides another example of viral effects that are exacerbated by drug or pesticide use. Significance statement In 2015, an increase in children born with unusually small heads (microcephaly) in North-Eastern Brazil was linked to infection with the ZIKA virus. An insecticide with thyroid hormone disruptive properties was used in the same areas. We investigated whether simultaneous exposure to the insecticide could increase viral susceptibility. The main metabolite 4’-OH-PPF dysregulated thyroid hormone signaling pathways crucial for brain development in both models used. Neural stem cells proliferated less and contained more Musashi-1, a protein the virus needs to replicate. Infecting stem cells pre-exposed to the endocrine disruptor did not amplify viral replication, but aggravated expression of genes implicated in brain development. Our results suggest the insecticide is particularly deleterious to brain development in areas with ZIKA virus prevalence.

Multiple lines of evidence link infection by the ZIKA virus (ZIKV) to congenital Zika syndrome 83 and the increased cases of neonatal microcephaly in Central and South America starting from early 84 2015 (1) (2) (3) (4) (5) . Experiments on human brain organoids and neurospheres showed that neural stem cells 85 (NSCs) are a direct target of ZIKV (6). ZIKV reduced proliferation, altered fate choice, increased cell 86 death and decreased neuronal cell layer volumes (7, 8) . ZIKV infection in mice led to smaller brains 87 with thinner cortical layers and associated neurological disabilities by interfering with the cellular 88 processes required for normal brain development (9).

Remarkably, a few regions in North-Eastern Brazil showed a much higher prevalence of ZIKV-90 associated microcephaly than others, prompting the question as to whether other factors could be linked 91 to this regional effect (10, 11). It has been suggested that the use of the pesticide pyriproxyfen (PPF), 92 a juvenile hormone analog, approved worldwide for use against household insects and in agriculture 93 (12, 13) , could be implicated in the rise of microcephaly cases (3, (14) (15) (16) . High amounts of PPF were 94 used in the same northeastern region where most ZIKV cases associated with microcephaly were found 95 (17, 18) . This could be yet another example of drug or pesticide exacerbating the effects of viral infection 96 (19, 20) . Even though a small cohort (< 80 cases) did not find a link between PPF and congenital 97 microcephaly (3), it remains to be experimentally investigated whether or not the pesticide could be 98 implicated in the microcephaly outbreak (15).

PPF was first introduced into Brazilian drinking water in late 2014 to control the Aedes aegypti 100 mosquito population, the vector for the dengue and the ZIKA viruses. The WHO recommended that 101 daily intake of PPF should not exceed 0.3 mg/L for an average adult, and advised that the concentration 102 of PPF in drinking water containers should be lower than 0.01 mg/L (13). The main pathway of PPF 103 metabolism is hydroxylation at the 4'-position, producing principally 4'-OH-PPF in both vertebrates and 104 invertebrates (13, (21) (22) (23) . PPF is documented to be rapidly taken up and metabolized, resulting in low 105 bio-concentrations of the parent compound circulating in the organism (21). We therefore hypothesized 106 that 4'-OH-PPF rather than PPF could be the main compound acting in vivo. We decided thus to carry 107 out our experiments using the PPF main metabolite 4'-OH-PPF.

In 2017, Chavali et al. found that the RNA binding protein Musashi-1 (MSI1) is indispensable 109 for ZIKV replication. MSI1 is an important translational regulator in NSCs in both vertebrates and 110 invertebrates (24). Interestingly, we previously showed that this gene is downregulated in vivo in the 111 murine subventricular zone (SVZ) by T3/thyroid hormone (TH) receptor α (TRα), when progenitors 112 commit to the neuroblast lineage (25). THs regulate many brain developmental processes including cell 113 proliferation, migration and differentiation to acquire the characteristic brain cyto-architecture (26, 27) .

In severe cases of hypothyroidism, disruption of these processes also leads to smaller brains and 115 aberrant cortical layer formation (28). Considering that PPF was previously shown to interfere with 116 thyroid-responsive endpoints in the amphibian metamorphosis assay and the male and female pubertal 117 rat assays (29), we questioned if co-exposure to the two factors -4'-OH-PPF and ZIKV -promoted 118 increased viral replication.

We tested whether 4'-OH-PPF exposure could inhibit TH action, by increasing MSI1 expression 120 and as such perturb NSC properties and replication. We used two TH model systems, Xenopus laevis 121 5 tadpoles and adult mouse SVZ neuroprogenitors. In Xenopus laevis, we studied TH-disruptive effects 122 in vivo using the XETA-assay, msi1 expression as well as various morphometric and behavioral 123 endpoints (30, 31 132 tadpoles were exposed to an environmentally-relevant dose range of 4'-OH-PPF in the presence or 133 absence of 5 nM T3 using the XETA assay. Control tadpole brains showed low basal fluorescence under 134 physiological conditions (Fig 1A, left panel) . As expected, adding 5 nM T3 strongly increased eGFP 135 transcription, resulting in increased fluorescent activity (Fig 1A, middle panel) . When simultaneously 136 exposing to 4'-OH-PPF and T3, the fluorescent signal strongly decreased (Fig 1A, right panel) . Testing 137 the fluorescent signal in each condition with a specific concentration of 4'-OH-PPF + T3 showed the 138 fluorescence in brains to be significantly reduced (Fig 1B, C , 0.05<p<0.0001). Further, even the lowest 139 concentration (10 -7 nM 4'-OH-PPF) reached significance (p<0.05) and the highest concentration, 140 representing environmentally relevant levels had the strongest effects (Fig 1C, p<0 .0001). These 141 results suggest that 4'-OH-PPF exhibits TH-disrupting properties in vivo, and that short-term (72 h) 142 exposure negatively impacts TR-mediated gene expression.

Experiments in JEG3 cells also showed TRα1-dependent luciferase expression to be affected 144 at the highest dose used (3 mg/L 4'-OH-PPF) (Fig S1A, p<0 .05). Similarly, applying the TR antagonist 145 NH3 to the same cell line inhibited TRα1-dependent luciferase expression at the two highest 146 concentrations tested (Fig S1B, p<0 .05). This reemphasizes that high doses of the metabolite 147 antagonize TR-dependent transcriptional activity.

We assessed whether 4'-OH-PPF affected tadpole behavior. Non-exposed tadpoles were 149 active during the light periods and mostly inactive in the dark. Tadpoles exposed to 4'-OH-PPF exhibited 150 reduced mobility compared to controls, reaching significance at the two highest concentrations (10 -1 151 and 3x10 -1 mg/L) (Fig S2A, B , p<0.05).

Next, total head size and brain areas of tadpoles were measured and brain/head ratios 153 calculated. Tadpoles exposed to 3x10 -1 mg/L 4'-OH-PPF had significantly smaller heads compared to 154 controls (Fig S2C) , corresponding to increased brain/head ratios (Fig S2D) . However, the overall brain 155 size of these tadpoles was not affected (Fig S2E) . The lowest concentration of 10 -5 mg/L 4'-OH-PPF 156 significantly increased the width of forebrain and midbrain junction (Fig S2F, p<0 .0001) and the width 6 Next, we studied the consequences of a 24 h exposure to 4'-OH-PPF on musashi-1 (msi1) 162 expression and its encoded protein in the brains of NF45 Xenopus laevis tadpoles in vivo. T3 163 significantly reduced msi1 expression (Fig 1D, p<0 .05) as well as the encoded protein (Fig 1E, p<0 .05).

At both concentrations used, 4'-OH-PPF increased msi1 expression (p<0.05) in the presence of 5 nM 165 T3 (Fig 1D, p<0 .05), and significantly increased Msi1 protein levels at concentrations of 10 -2 mg/L (Fig   166   1E , F, p<0.05). Moreover, Msi1 protein levels were predominantly upregulated in regions bordering the 167 ventricular system, where most NSCs reside producing the cells of the central nervous system (Fig 1F) .

Hence, short exposure to 4'-OH-PPF at varying concentrations, and in presence of T3, increases Msi- 

We next addressed whether 4'-OH-PPF ± 10 nM T3 altered the in vitro proliferation of primary 173 neurospheres grown from adult mouse SVZ-NSCs, analyzing diameters and numbers of neurospheres 174 (Fig 2A) . After seven days, exposure to 10 -1 and 3x10 -1 mg/L 4'-OH-PPF reduced the average 175 neurosphere diameter by 27.9% and 47.7% respectively, an effect also seen by adding T3 (Fig 2B,   176 p<0.0001). Importantly, as neurospheres are 3D structures, the overall decrease in volume and cell 177 number was greater. A diameter reduction of 28% corresponds to a volume decrease of 62.7% (ctrl: 

OH-PPF ± 10 nM T3 induced a dose-dependent increase in Msi1 expression (Fig 2D) . Without adding 189 T3, the highest doses of 4'-OH-PPF (range: 10 -1 -3.10 -1 mg/L) induced Msi1 expression 4.5-fold to 9.4-190 fold compared to controls (p<0.05). With the addition of T3, the induction was respectively 2.7-fold to 

Based on the previous findings, we set up a protocol for testing whether ZIKV infection was 200 modulated by 4'-OH-PPF. To establish the exposure conditions and the ZIKV infection period, we 7 carried out a preliminary experiment on murine neurospheres (Fig S3) to determine the temporal Msi1 202 expression patterns during and after 4'-OH-PPF exposure. We found that exposing neurospheres to 4'-203 OH-PPF 10 -1 mg/L for 2 days before and 4 days after dissociation slightly but significantly induced Msi1 204 as well as the strongly T3-responsive gene Krüppel-like factor 9 (Klf9) (Fig S3, Msi1 , Klf9: p<0.01).

Based on this knowledge, we then tested ZIKV infection. NSCs proliferated for a week and 206 were exposed to 4'-OH-PPF for the last 5 days. We then dissociated the neurospheres and infected 207 them with ZIKV for 4 days ± 4'-OH-PPF (Fig 3A) . We measured ZIKV replication in two independent 208 experiments, where we quantified RNA copy number (copies/mL) in the supernatant by RT-PCR each 

We then analyzed the gene expression profile of well-known TH target genes at day 4 after 214 ZIKV infection in the four conditions. These were with or without ZIKV and with or without 4'-OH-PPF. 

The same observation applies to Klf9 expression (Fig 3E) . However, the presence of ZIKV in 

ZIKV infection was proposed as the causative factor in the sudden microcephaly outbreak in 239 South and Central America in 2015. Data from human cultured NSCs, brain organoids and animal 240 models indicate that these neuropathies predominantly resulted from a ZIKV-associated decrease in 241 NSC proliferation, cell-cycle dysregulation, altered fate choice and increased apoptosis during brain 242 development (7, 32-37). In the present study, we investigated whether there could be a connection 243 between the pesticide PPF and the microcephaly outbreak in certain regions in North-Eastern Brazil 244 where prevalence and use was much higher as compared to other regions (14, 15). PPF could increase 245 virus susceptibility, aggravating the adverse effects ZIKV has on brain development.

Our hypothesis was based on two ideas. First, THs are key to evolutionary expansion of brain 247 size and complexity, a primary characteristic of humans (38). These hormones regulate NSC pool 248 expansion, progenitor multiplication, fate choice, migration and differentiation, important processes 249 underlying normal brain development (27). Amongst the genes specifically implicated in human brain 250 evolution are TH-regulated genes such as transthyretin (TTR) (38) and sonic hedgehog (SHH) (39).

Many genes involved in neuroprogenitor proliferation have also undergone human-specific acceleration 252 (39). One such gene is adenlyate-cyclase-activating-polypetide 1 (ADCYAP1), which is also a TH- 

A central idea to our hypothesis was that the main metabolite of PPF, 4'-OH-PPF, might affect 258 TH signaling and thereby increase expression of the negatively TH-regulated gene Msi1. As its encoded 259 protein MSI1 is a crucial mediator of viral replication, the metabolite could exacerbate ZIKV replication.

We tested our hypothesis in two models: Xenopus laevis tadpoles and in vitro mouse neurospheres.

Lastly, we infected NSCs pre-exposed to 4'-OH-PPF with ZIKV to evaluate viral replication and effects 262 on the TH signaling pathway.

The results from the XETA assay confirmed our initial hypothesis that the lowest 266 environmentally relevant concentrations of 4'-OH-PPF exhibited anti-thyroid activity in the tadpole brain.

Furthermore, we found that major brain compartments developed disproportionately, suggesting brain 268 development was perturbed. This could also explain the observed decreased tadpole mobility.

We then extended these findings with an in vitro gene expression study on mouse NSCs. The 

We found that NSC proliferation was strongly decreased following 4'-OH-PPF exposure. Other 277 genes include a tendency to increased Dio2 expression and significantly decreased expression of the 278 stem cell marker Sox2, as well as Sox10, both T3-negatively regulated genes. The latter directs NSC 279 fate towards the oligodendroglial lineage (44). These observations could indicate decreased T3 levels 280 in NSCs suggesting that they are in a local hypothyroid state. The results on JEG3 cells corroborated 281 this concept, namely that TR-dependent processes were disrupted. In addition, we analyzed the 282 regulatory element upstream of human MSI1 gene using an algorithm-based search for binding sites 283 (NHR scan). We found that the human sequence contained a number of DR4 type response elements, 284 i.e. potential TR binding sites (45-47), as did the mouse. 

This corresponds to what is found in other vertebrates (24). Further, exposing mouse neuroprogenitors 299 to 4'-OH-PPF for 6 days increased levels of Msi1 and Klf9, another T3-inducible gene that promotes 300 neuronal development (53, 54) (Fig S3) . Msi1 is also a key negatively TH-regulated gene in NSCs (25), 301 and MSI1 is crucial for ZIKV replication in NSCs, interacting with ZIKV RNA (24).

We found that 4'-OH-PPF alters NSC proliferation and the growth of mouse neurospheres, and 303 significantly decreased neuroprogenitor intracellular RNA content with and without ZIKV infection.

However, the greatest reduction in intracellular content was seen when the virus and 4'-OH-PPF were 305 both present. Similar results were found in a study using human IPC-based neurospheres, the diameter 306 of which reduced 2-fold following ZIKV infection (8). Our results not only confirmed that 4'-OH-PPF 307 reduced neurosphere numbers, but also had cumulative effects with the virus reflected by the increased 308 numbers of dysregulated genes. We conclude that ZIKV combined with 4'-OH-PPF strongly affects the 

Most importantly, most key TH target genes were not dysregulated by 4'-OH-PPF alone, but 314 they were when combined with ZIKV infection. These included Gfap, Egfr, Nestin, Ng2, Dlx2 and Dcx, 315 which are all implicated in NSC self-renewal and neuronal and oligodendroglial commitment, processes 316 underlying normal corticogenesis. These findings offer a plausible mechanism to explain the potential 317 increase of susceptibility to ZIKV in regions where intensive insecticide use was seen.

However, we did not find increased numbers of viral particles per cell, although they remained 319 in the same order of magnitude. However, our neurosphere system has the constraint of being neither 320 a human or a primate-based system, nor an in vivo mouse system, for which one could develop a robust 321 4'-OH-PPF pre-exposure protocol that can be potentially extrapolated to humans. Further, the fetal dose 322 of 4'-OH-PPF nor materno-fetal transmission cannot be perfectly replicated. Nevertheless, work on 323 human neuroprogenitors does show that ZIKV infection attenuates proliferation (7), a result that we also 324 found on mouse neurospheres. 

Luciferase activity was measured in cell lysates as previously described (60) 

Xenopus laevis tadpoles were placed into each well of a 6-well plate. Each well corresponded to one 362 exposure group. Eight mL of previously prepared final exposure solution was added per well. Tadpoles 363 were exposed for 72 h at 23 °C in dark with daily renewal at the same hour for the XETA assay. For 364 brain gene expression analysis, exposure was limited to 24 h. Tadpoles were exposed to 4'-OH-PPF ± In a first experiment, 10 4 cells/well in 100 µL/well were exposed in 96-well plates (12 wells/group). On 456 day 0, 10 nM T3 (Sigma -CAS 55-06-1) together with 10 -2 , 10 -1 , or 3x10 -1 mg/L 4'-OH-PPF was applied 457 for seven days in a 5% CO2, 20% O2 environment at 37 °C. Thirty µL/well of the growth medium was 458 supplemented every two days. At day 7, brightfield images of neurospheres were taken (ZEISS AXIO 459 Zoom V16 macroscope; 21 images/well) and the projected areas of neurospheres were measured using 460 the ImageJ software. Then, after 5 min centrifugation at 1000 rcf, cell pellets were frozen at −20 °C until 461 RNA extraction.

In a second experiment, 3×10 5 cells in 5 ml of medium were exposed with 0.01% DMSO ± 4'-OH-PPF 

One-way ANOVA was done in the case of parametric distribution and followed by a Tukey's or Dunnett's 507 multiple comparison post-test. Two-way ANOVA followed by Bonferroni's multiple comparisons test was 508 used to analyse ZIKV replication. A Kruskal-Wallis test with Mann-Whitney multiple comparison post-509 tests was used for non-parametric data. For comparison of two groups, a t-test (parametric) or a Mann-510 Whitney U test (non-parametric) was used. Data are presented as scatter plots depicting mean ± 511 standard error of mean (SEM) or standard deviation (SD). P values <0.05 were thereby considered as 512 statistically significant. Table 1 : Specific Taqman probes used for qPCR experiments.

Taqman Probe

Fasn Mm00662319_m1

Klf9 Mm00495172_m1

Dio2 Mm00515664_m1

Gfap Mm01253033_m1

Sox2 Mm03053810_s1

Nestin Mm01223404_g1

Egfr Mm01187858_m1

Sox10 Mm00569909_m1

Dlx2 Mm00438427_m1

Gadph Mm99999915_g1

Hprt Mm00446968_m1

ActB Mm00607939_s1 

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WHO), "Pyriproxyfen in Drinking-water: Use for Vector Control in Drinking-water 559 Sources and Containers

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A Possible Link Between 567 Pyriproxyfen and Microcephaly

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Thyroid hormones and brain development

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Identifying reference chemicals for thyroid bioactivity screening

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Genetic basis of human brain evolution. Trends 611 Neurosci

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Musashi-1 gene expression and its 673 encoded protein in Xenopus laevis. (A) Tg(thibz:eGFP) tadpoles exposed for 72 h to (from left to 674 right) vehicle control, 5 nM T3 and 5 nM T3 + 3x10 -1 mg/L 4'-OH-PPF. Scale bar: 500 μm. (B, C) 72 h 675 exposure of NF45 Tg(thibz:eGFP) tadpoles to 4'-OH-PPF with (B) or without 5 nM T3 (C). Values 676 normalized to control group or T3-treated group, respectively (3 independent experiments; n=15 per 677 experiment). (D) msi1 gene expression

2 ; 10 -1 mg/L) together with 5 nM T3 (3 independent experiments, n=5 per experiment)

Msi1 protein expression in dissected brains from NF46 tadpoles exposed for 24 h to 4'-OH-PPF

Protein levels were quantified by measuring fluorescence intensity 681 from dorsal views (2 independent experiments with 4-10 tadpoles per experiment). -Data were 682 analyzed by a One-way ANOVA with Dunnett's post-hoc test (Mean ± SEMs

Representative pictures of dorsal views of NF46 Xenopus tadpoles exposed to 5 nM T3 or T3

Msi1 levels are visualized as a heat 686 map representing integrated density with arbitrary values from 0-256. Dark blue areas -no or low 687 expression of Msi1; yellow/white -highest expression of Msi1); fb: forebrain, mb: midbrain

Figure 2. 4'-OH-PPF exposure decreases NSCs proliferation and enhances Fasn expression 694 levels, as well as Msi1 mRNA and its encoded protein. (A) Schematic timeline illustrating the 695 experimental design for the neurosphere assay. (B) Graphs showing neurosphere diameter in presence 696 of 10 nM T3 ± 3x10 -1 mg/L 4'-OH-PPF 10 -1 versus controls without added T3

0001). (C) Graphs 698 showing the average neurosphere number in presence of 10 nM T3 ± different doses of 4

mg/L versus controls without added T3 (n=12 wells/group

D-E) Graphs showing Msi1 and Fasn mRNA expression 701 levels in presence of 10 nM T3 ± different doses of 4'-OH-PPF (n=3-6 wells per group, Kruskal-Wallis 702 test: Fasn, p=0.0019, Msi1, p=0.0009, followed by Mann-Whitney post-tests: Fasn *: p=0.0238, Msi1*: 703 p=0.0238). (F) Representative images of immunostained MSI1 (green) protein in cultured DAPI-positive 704 adult mouse NSCs (blue) following exposure

Scale bars: 10 µm. (G) MSI1 levels were analyzed 707 by quantifying the integrative density of the green signal of MSI1+ cells, (H) and by measuring the 708 relative proportion of MSI1+ cells in the total DAPI+ cell population (n=3 replicates/groups, n=10 709 images/replicates, Mann-Whitney test ****: p<0.0001)

DIV: day in vitro, EGF: epidermal growth factor, FGF: fibroblast growth factor, NPCs: neural progenitor 711 cells, NSCs: neural stem cells, OPCs: oligodendrocyte progenitor cells, SVZ: subventricular zone

Figure 3: 4'-OH-PPF exposure does not alter ZIKV replication in neurospheres

A) Schematic timeline of the exposure 716 and infection protocol. (B) ZIKV virus RNA copy number (copies/ml) at 0 and 4 days post-infection, 717 showing the exponential increase over 4 days. Exposure to 4'-OH-PPF led to small but significant 718 number of ZIKV particles at day 4, but no other day

Graph showing neuroprogenitor intracellular RNA content (n= 6-12

Graphs showing expression profiles of TH-target and neuroprogenitor marker genes in all four 723 conditions

tests: *: p<0.05, **: p<0.01. For all graphs: n=7: Ctrl

n=12: ZIKV ± 4'-OH-PPF 10 -1 mg/L. Graphs 726 depict means ± SDs. Ctrl: control, EGF: epidermal growth factor, FGF: fibroblast growth factor, NSCs: 727 neural stem cells, SVZ: subventricular zone, ZIKV: Zika virus

Key TH-target genes and those involved in NSC processes are modified by ZIKV 760 infection with and without 4'-OH-PPF. (A) Scheme showing neuro-and gliogenesis in adult the 761 subventricular zone and associated biomarkers during NSC commitment. (B-I) Graphs showing gene 762 expression in NSCs: n=7: Ctrl

Graphs depict means ± SDs. aNSC: activated neural stem cell, qNSC: quiescent neural 766 stem cell, NPC: neural progenitor cells

The transcriptional activity of TRα1 stimulated by 1 nM 771 T3 was significantly reduced under increasing concentrations of 4'-OH-PPF (A) and NH3 (B), the latter 772 being a strong TH antagonist. The data are presented as mean ± SEM of two independent experiments 773 performed in triplicate (One-way ANOVA p<0.05

A-B) Total distance travelled by tadpoles exposed to 10 -1 g/mL 4'-OH-PPF (A) or 779 3x10 -1 g/mL 4'-OH-PPF (B) during a 10 minute trial, with distance travelled recorded every 10 s. Dark 780 backgrounds represent unlit periods, white backgrounds represent light periods. Values were 781 normalized to the first 10 s period of the control group (Pool of 3 independent experiments; n = 12 per 782 experiment, Kruskal-Wallis, mean ± SEM, * p<0.05, ** p<0.01, *** p<0.001). Graphs showing (C) 783 normalized head area, (D) head/brain area ratio, (E) brain area, (F) width of fore-and midbrain junction 784 and (G) width of midbrain of NF45 tadpoles exposed to 10 -1 g/mL 4'-OH-PPF for 72 h. Values 785 normalized to the control group (Pool of 3 independent experiments