Elsevier

Neurobiology of Disease

Volume 78, June 2015, Pages 115-125
Neurobiology of Disease

Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood–brain barrier dysfunction

https://doi.org/10.1016/j.nbd.2015.02.029Get rights and content

Highlights

  • Injection of albumin into the ventricles of mice results in gliosis and seizures.

  • TGF-β1 and albumin have similar epileptogenic effects.

  • Albumin and TGF-β1 induce similar excitatory synaptogenesis in vitro and in vivo.

  • The synaptogenic effect is astrocyte dependent.

  • Inhibition of TGF-β/ALK5 pathway prevents excitatory synaptogenesis and epilepsy.

Abstract

Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood–brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

Introduction

Traumatic, ischemic, and infectious brain injuries often initiate a cascade of epileptogenic events, ultimately leading to post-injury epilepsy (PIE) after a latent period of months to years. Accumulating evidence suggests a key role of post-injury dysfunction of the blood–brain barrier (BBB) in the development of PIE: BBB dysfunction is a well-documented finding in patients following brain trauma (occurring within hours of the event and often persisting for days to weeks; for a review, see Abbott and Friedman, 2012, Cunningham et al., 2005, Rosenberg, 2012, Shlosberg et al., 2010) and is more frequent in post-traumatic patients with epilepsy (Raabe et al., 2012, Schmitz et al., 2013, Tomkins et al., 2008). In experimental animals, BBB dysfunction was associated with increased propensity for symptomatic seizures and the development of epilepsy (Friedman et al., 2009, Frigerio et al., 2012, Marchi et al., 2007, Seiffert et al., 2004, Van Vliet et al., 2007).

Previous studies have linked the brain's immune response with seizures and epilepsy, as activation of the pro-inflammatory IL-1 receptor/Toll-like receptor (IL1R/TLR) system was shown to promote seizure onset and recurrence in mice and rats (for a review, see Devinsky et al., 2013, Marchi et al., 2013, Vezzani et al., 2011a, Vezzani et al., 2011b). Recent findings have highlighted the involvement of a specific inflammatory pathway in PIE, identifying the epileptogenic role of transforming growth factor beta (TGF-β) signaling in animal models of BBB dysfunction. Serum albumin was shown to enter BBB-disrupted brain tissue and bind to TGF-β receptors in astrocytes (Ivens et al., 2007), inducing inflammatory signaling (Cacheaux et al., 2009) and SMAD-2/3 phosphorylation (Bar-Klein et al., 2014), thereby modifying astrocytic function (Braganza et al., 2012, David et al., 2009, Seiffert et al., 2004). TGF-β signaling was also associated with immediate changes in extracellular potassium and glutamate, and a lower threshold for neuronal activation in slices (David et al., 2009, Lapilover et al., 2012). However, the immediate and short-lived nature of these changes (Ivens et al., 2007, Lapilover et al., 2012) suggests the involvement of additional modifications, underlying permanent network changes that sustain chronic recurrent seizures. While axonal sprouting and synaptogenesis were shown to contribute to seizures and persistent network alterations (Babb et al., 1991, Bragin et al., 2000, Marco and DeFelipe, 1997), the detailed changes and mechanisms underlying network reorganization in PIE are not well understood.

Glial cells are known to play a key role in the formation and elimination of both excitatory and inhibitory synapses in developing and mature brain (Chung and Barres, 2012, Clarke and Barres, 2013, Elmariah et al., 2005, Eroglu and Barres, 2010). Specifically, astrocytes were shown to regulate synaptogenesis through secretion of thrombospondins (TSPs) (Christopherson et al., 2005, Crawford et al., 2012, Eroglu et al., 2009, Liauw et al., 2008), Hevin (Kucukdereli et al., 2011) and glypicans 4 and 6 (Allen et al., 2012). Moreover, synapse formation was also associated with activation of TGF-β signaling in Schwann cells (Feng and Ko, 2008, Packard et al., 2003), and more recently in astrocytes via induction of Smad3 (Yu et al., 2014), secretion of d-serine (Diniz et al., 2012), and CaM kinase II signaling (Diniz et al., 2014). Since the dysfunction of astroglia has been shown following brain injury, in models of acquired epilepsy (Crunelli et al., 2014, Devinsky et al., 2013, Friedman et al., 1996, Marchi et al., 2013) and in the BBB-compromised brain, here we set out to study the role of albumin-induced TGF-β pathway activation in synaptogenesis and chronic seizures. We further challenged the hypothesis that the inhibition of the TGF-β ALK5 receptor is sufficient to prevent the development of epilepsy. Through a combination of in vitro and in vivo models of inflammation post-BBB dysfunction, our study sheds light on the epileptogenic cascade following brain injury and highlights novel targets for PIE prevention in at-risk patients.

Section snippets

Materials and methods

All animal procedures were approved by the UC Berkeley Animal Care and Use Committee or the animal care and use ethical committees at the Ben-Gurion University of the Negev, Beer-Sheva.

ICV albumin induces epileptogenesis in vivo

To simulate BBB dysfunction in naïve animals and to study the role of serum albumin in epileptogenesis, mice were infused with albumin (0.4 mM in ACSF, 1 μL/h) through an ICV mini-osmotic pump for 7 days. ECoG data were acquired for up to 32 days (32 days: n = 4; 14 days: n = 7; 10 days: n = 2) and analyzed using an automated seizure detection algorithm (see Materials and Methods and Bar-Klein et al., 2014). Albumin treatment did not induce status epilepticus (SE) nor seizures during the first 72 h after

Discussion

Here we demonstrate an albumin-induced and TGF-β-mediated synaptogenic mechanism critical in the epileptogenic process. We show both in vitro and in vivo that albumin induces excitatory synapse formation, and that this effect is astrocyte and ALK5 dependent.

TGF-β signaling in astrocytes following exposure to albumin was previously shown to promote several functional changes that may be associated with increased neuronal excitability, including induction of pro-ictogenic inflammatory cytokines (

Competing interests

The authors declare no competing interests.

Acknowledgments

The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) under grant agreement no.602102 (EPITARGET, AF), the Israel Science Foundation (713/11, AF), the National Institute of Health (RO1/NINDS NS066005, DK, AF), and the German Science Foundation (DFG-SFB TR3, AF).

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    1

    I.W & L.W contributed equally.

    2

    L.K & O.V contributed equally.

    3

    A.F & D.K contributed equally.

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