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Transient Maternal IL-6 boosts glutamatergic synapses and disrupts hippocampal connectivity in the offspring

Filippo Mirabella, Genni Desiato, Sara Mancinelli, Giuliana Fossati, Marco Rasile, Raffaella Morini, Marija Markicevic, Christina Grimm, Clara Amegandjin, Alberto Termanini, Clelia Peano, Paolo Kunderfranco, Graziella di Cristo, Valerio Zerbi, Simona Lodato, Elisabetta Menna, Michela Matteoli, Davide Pozzi

Preprint posted on November 02, 2020 https://www.biorxiv.org/content/10.1101/2020.11.02.364356v1

How maternal inflammation/IL-6 messes with fetal brain wiring: STAT3/RGS4 signaling boosts excitatory network connections.

Selected by Theresa Pohlkamp

Background:

Autism and schizophrenia are complex neurodevelopmental disorders that can be triggered by genetic and environmental factors. Filippo Mirabella and colleagues study the effect of maternal inflammation as an environmental factor on mouse brain development.

Neuropsychiatric behaviors are the result of abnormal neural connectivity within and across brain regions. To encode and transmit information, neurons use positive and negative electrical currents that, with the additional parameters of time and space, provide the computational power of our brain. Specific types of inhibitory and excitatory neurons transmit either positive or negative charges, respectively. Computational precision is highly dependent on the balance between inhibitory and excitatory neurons and their numerous synaptic connections. Neural networks develop step-wise, starting with the specification of neuronal identity, their migration to defined destinations, growth of processes within and across regions, formation of synaptic connections and their plasticity to adapt behavior. While neurons mature, they observe their environment and detect signals that shape them. These signaling molecules can be of local origin, for example coming from neighboring cells, but also of global source, such as cerebrospinal fluid that contains molecules derived from peripheral blood.

In a living organism, as part of a defense mechanism, inflammatory events evoke the release of cytokines by innate immune cells into the blood stream. Specific cytokines, for example interleukin-6 (IL-6), have the capacity to cross the placenta and the blood brain barrier. Thus, during pregnancy, maternal cytokines can reach the developing fetal brain and influence the maturation process of neurons and can be responsible for neuropsychiatric diseases of the offspring (Knuesel et al., 2014; Wu et al., 2017). The work published in this preprint demonstrates the impact of maternal inflammation and IL-6 on neural network formation of the developing fetus. They discovered that IL-6 boosts the formation of excitatory synapses and affects brain wiring. Moreover, they found that signal transducer and activator of transcription-3 (STAT3) and its target gene encoding regulator of G protein signaling 4 (RGS4) play key roles in the molecular pathway driving these changes and therefore provide potential drug-targets for early interventional strategies.

 

What was done?

Pregnant mice were infected with IL-6 at gestation day 15 (GD15), when fetal brain regions have formed and neurogenesis is peaking, but before neurons begin to establish connections. At postnatal day 15 (P15) and P30 the authors found an increased number of excitatory synapses in the hippocampus, while inhibitory synapses did not change. Accordingly, excitatory neurons fired more frequently with unaltered current amplitude, while inhibitory currents were unaffected in both parameters. Importantly, magnetic resonance imaging of 14 weeks old mice revealed hyperconnectivity across multiple different brain regions, mostly from hippocampus to midbrain or cortical regions (see figure). Changes in the overall architecture of the brain were not detected, and signs for astrogliosis and inflammation were absent, indicating that prenatal exposure to IL-6 does not trigger brain-intrinsic inflammation in the adult brain.

To define the underlying molecular mechanism how IL-6 causes long-lasting changes in neuronal connectivity, the authors looked at the expression of the transcription factor STAT3, a known IL-6 target, and found it elevated in the hippocampus. In primary neurons, STAT3 phosphorylation at Tyrosine705 was increased in a timely sensitive manner, peaking 30 minutes after IL-6 exposure. Next, using a single-cell RNAseq approach in cultured pre-mature neurons exposed to IL-6 the authors identified transcriptional alterations in STAT3 that were specific to excitatory neuron populations, and absent in interneurons or astrocytes. Strikingly, they found RGS4 as a downstream target of STAT3, as it was concomitantly increased. Using primary neurons and a neuroblastoma cell line, they convincingly demonstrated the relevance of the STAT3/RGS4 pathway by complementing their experiments in the presence of specific drugs.

Figure: Circos-plot showing the anatomical location of hyper-connected edges (n=242) in the offspring of IL-6-treated mice compared to vehicle-treated mice. Reproduced with permission from Figure 2C of the preprint.

 

Why I chose this preprint.

I came across this preprint right after a seminar at UT Southwestern by Maria Lehtinen covering the effect of maternal immune activation on the inflammation of the embryonic choroid plexus (Cui et al., 2020). I was fascinated by the extensive study by F. Mirabella and colleagues, not only providing a precise image of the impact that maternal IL-6 exposes on fetal brain development, but also nailing down a key pathway involved.

In the ongoing pandemic and as a neuroscientist, I am at the same time excited and alarmed by discoveries how Covid-19 messes with the brain. IL-6 is an important component of the Covid-19 cytokine storm (Liu et al., 2020). During pregnancy, IL-6 and IL-17a, another Covid-19 relevant cytokine, can cause inflammatory damage to the fetal brain, potentially leading to neonatal brain development abnormalities (Choi et al., 2016; Rudolph et al., 2018), which is supported by the finding of this preprint. Thus, special attention needs to be payed to pregnant women infected by SARS-CoV2, to reduce severity of Covid-19 and their chance to experience a prolonged cytokine storm that could potentially affect their fetus’ brain development.

 

Questions to the authors:

  1. While locomotor activity, anxiety, and episodic memory were not compromised, spatial memory was affected in mice prenatally exposed to IL-6. Did you also examine social behavior? (e.g. nest forming, social novelty preference, vocalization)
  2. To narrow down the time-window of vulnerability, primary mouse neurons (E18) were cultured and treated with IL-6 on various days and electrophysiologically examined after a total of 14 days in vitro (DIV14). Excitatory frequency was increased when IL-6 was given at DIV4 or earlier, but not if applied as late as DIV13. How can you exclude that it was not too early to examine the phenotype after only 24 hours in case of the DIV13 treatment, when you were waiting 10-13 days in case of the DIV1-4 treatments?
  3. The peaking time for STAT3 Tyr705 phosphorylation in primary neurons is 30 minutes after exposure to IL-6. Did you test how long the change in RGS4 expression persists?
  4. How fast do you think therapeutic action is required to counteract the changes in the STAT3/RGS4 pathway and prevent long-lasting network changes?
  5. You show that both, astrocyte depleted and astrocyte co-cultured neurons have increased excitatory frequency and STAT3 expression upon IL-6 induction. In addition, you cannot find astrogliosis in IL-6 exposed brains. However, in Figure 7H a significant increase of RGS4 expression in the astrocyte population is shown. How do you think this could contribute to the situation in vivo, particularly in long term?
  6. Are you planning to expand your studies to include Covid-19 research? What would you look at, first?

 

References

Choi, G.B., Yim, Y.S., Wong, H., Kim, S., Kim, H., Kim, S.V., Hoeffer, C.A., Littman, D.R., Huh, J.R., 2016. The maternal interleukin-17a pathway in mice promotes autismlike phenotypes in offspring. Science 351, 933–939. https://doi.org/10.1126/science.aad0314

Cui, J., Shipley, F.B., Shannon, M.L., Alturkistani, O., Dani, N., Webb, M.D., Sugden, A.U., Andermann, M.L., Lehtinen, M.K., 2020. Inflammation of the Embryonic Choroid Plexus Barrier following Maternal Immune Activation. Dev Cell. https://doi.org/10.1016/j.devcel.2020.09.020

Knuesel, I., Chicha, L., Britschgi, M., Schobel, S.A., Bodmer, M., Hellings, J.A., Toovey, S., Prinssen, E.P., 2014. Maternal immune activation and abnormal brain development across CNS disorders. Nat Rev Neurol 10, 643–660. https://doi.org/10.1038/nrneurol.2014.187

Liu, F., Li, L., Xu, M., Wu, J., Luo, D., Zhu, Y., Li, B., Song, X., Zhou, X., 2020. Prognostic value of interleukin-6, C-reactive protein, and procalcitonin in patients with COVID-19. J Clin Virol 127, 104370. https://doi.org/10.1016/j.jcv.2020.104370

Rudolph, M., Graham, A., Feczko, E., Miranda-Dominguez, O., Rasmussen, J., Nardos, R., Entringer, S., Wadhwa, P., Buss, C., Fair, D., 2018. Maternal IL-6 during pregnancy can be estimated from newborn brain connectivity and predicts future working memory in offspring. Nat Neurosci 21, 765–772. https://doi.org/10.1038/s41593-018-0128-y

Wu, W.-L., Hsiao, E.Y., Yan, Z., Mazmanian, S.K., Patterson, P.H., 2017. The Placental Interleukin-6 Signaling Controls Fetal Brain Development and Behavior. Brain Behav Immun 62, 11–23. https://doi.org/10.1016/j.bbi.2016.11.007

Tags: coronavirus, glutamatergic, long covid, vgat, vglut

Posted on: 24th November 2020

doi: https://doi.org/10.1242/prelights.25714

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Author's response

Davide Pozzi and Michela Matteoli shared

  1. While locomotor activity, anxiety, and episodic memory were not compromised, spatial memory was affected in mice prenatally exposed to IL-6. Did you also examine social behavior? (e.g. nest forming, social novelty preference, vocalization)

Answer: Previous studies based on maternal exposure of IL-6 or Poly:IC have already described autistic-like behavior in the offspring, including the occurrence of sociability defects. Autistic-like behaviors in mice prenatally exposed to IL-6 are therefore expected. For this reason, we focused our attention on cognitive behavior, as intellectual disabilities represent one of the most common comorbidity associated with neurodevelopmental disorders. The fact that an early immunological insult provided by IL-6 was sufficient to produce long-lasting consequences on hippocampal-dependent cognition represents the proof of concept that besides inducing autistic-like behavior these immune molecules may be responsible for additional phenotypic defects.

  1. To narrow down the time-window of vulnerability, primary mouse neurons (E18) were cultured and treated with IL-6 on various days and electrophysiologically examined after a total of 14 days in vitro (DIV14). Excitatory frequency was increased when IL-6 was given at DIV4 or earlier, but not if applied as late as DIV13. How can you exclude that it was not too early to examine the phenotype after only 24 hours in case of the DIV13 treatment, when you were waiting 10-13 days in case of the DIV1-4 treatments?

Answer: The process of synaptogenesis in cultured hippocampal neurons is considered to be almost concluded at 14 DIV, when synaptic transmission is clearly detectable. However, many studies have reported that, even in mature neurons, a pro-synaptogenic stimulus can modify synapse number within 24 hours. Given IL-6 is known to affect specific synaptic properties acutely in mature neurons (acting on NMDA receptors and calcium transients), we asked whether the prosynaptogenic effect of IL-6 could be observed even in mature neurons. For that reason, a 24 hours treatment was considered to be a sufficient period for such an effect. However, a longer treatment in mature neurons could be worth to be attempted.

  1. The peaking time for STAT3 Tyr705 phosphorylation in primary neurons is 30 minutes after exposure to IL-6. Did you test how long the change in RGS4 expression persists?

Answer: We did not test the persistence of RGS4 upregulation upon IL-6 stimulation, but we checked STAT3.  In particular, we found that the upregulation of STAT3 induced by IL-6 at 6 DIV is transient, going back to basal levels at 14 DIV. Based on these data, one could speculate that RGS4 follows the same trend. However, a clear evidence is still lacking.

  1. How fast do you think therapeutic action is required to counteract the changes in the STAT3/RGS4 pathway and prevent long-lasting network changes?

Answer: In a previous paper, we demonstrated that an early blockade of the cytokine storm induced by maternal immune activation (MIA) through a magnesium-enriched diet in the mother (magnesium is well-known as anti-inflammatory agent) prevented the detrimental effects of MIA in the offspring. Given the last-lasting effects produced by IL-6 in terms of synapses and brain connectivity, an early intervention could be required for this model as well. We are performing further analysis to clarify this point.

  1. You show that both, astrocyte depleted and astrocyte co-cultured neurons have increased excitatory frequency and STAT3 expression upon IL-6 induction. In addition, you cannot find astrogliosis in IL-6 exposed brains. However, in Figure 7H a significant increase of RGS4 expression in the astrocyte population is shown. How do you think this could contribute to the situation in vivo, particularly in long term?

Answer: We do not have a clear idea about this point and several explanations could be formulated. RGS4 is a modulator of G-coupled receptor-mediated signaling, which is an essential pathway in many different cells. Hence, the IL6-mediated upregulation of RGS4 in non-neuronal cells might lead to additional effects which may be related or not to neuronal connectivity.

  1. Are you planning to expand your studies to include Covid-19 research? What would you look at, first?

Answer: This is a very interesting point. One of the key features of Covid-19 infection is the cytokine storm produced by this virus, which is thought to be the main cause for severe clinical outcomes. Among proinflammatory cytokines, elevated serum IL-6 is one of the main biomarkers which correlates with respiratory failures and multi-organ damage. In pregnant women affected by Covid-19 a vertical transmission of the virus has been rarely described and the clinical features of babies born from infected mothers are not severe. However, elevated level of IL-6 (as well as other pro-inflammatory cytokines) might impinge fetal brain development with long-lasting consequences in the adulthood. We are now started investigating the possible correlation between IL-6 levels in the mother affected by Covid-19 with neurodevelopmental clinical outcomes of their babies. In parallel, mouse model of Covid-19 could be an alternative and interesting tool to be used to test the impact of maternal IL-6 on brain connectivity.

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