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Brain-derived neurotrophic factor and epilepsy: a systematic review
Accepted Manuscript Brain-derived neurotrophic factor and epilepsy: a systematic review
Lorenzo Iughetti, Laura Lucaccioni, Francesco Fuggetto, Barbara Predieri, Alberto Berardi, Fabrizio Ferrari PII: DOI: Reference:
S0143-4179(18)30065-9 doi:10.1016/j.npep.2018.09.005 YNPEP 1891
To appear in:
Neuropeptides
Received date: Revised date: Accepted date:
17 April 2018 13 August 2018 19 September 2018
Please cite this article as: Lorenzo Iughetti, Laura Lucaccioni, Francesco Fuggetto, Barbara Predieri, Alberto Berardi, Fabrizio Ferrari , Brain-derived neurotrophic factor and epilepsy: a systematic review. Ynpep (2018), doi:10.1016/j.npep.2018.09.005
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ACCEPTED MANUSCRIPT Brain-derived neurotrophic factor and epilepsy: a systematic review. Lorenzo Iughetti Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy
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Laura Lucaccioni
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Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of
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Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy
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Francesco Fuggetto
Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of
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Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy Barbara Predieri
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Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of
Alberto Berardi
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Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy
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Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy Fabrizio Ferrari
Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy Correspondig Author: Prof. Lorenzo Iughetti,
ACCEPTED MANUSCRIPT Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy. Tel. +39 059 4225382. Email: [email protected] Financial and competing interest disclosure
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The authors have no relevant financial and competing interests to declare.
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Abstract
Several in vitro, ex vivo and in vivo studies imply brain-derived neurotrophic factor (BDNF) in the
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pathophysiology of epilepsy. Aim of our work is to report the most important findings regarding
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BDNF and its potential role in epilepsy. We targeted those publications addressing both in vitro and in vivo evidences of relationship between BDNF and epilepsy. Basic researches, randomized
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trials, cohort studies, and reviews were contemplated to give a breadth of clinical data. Medline, CENTRAL, and Science Direct were searched till August 2017 using keywords agreed by the
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authors. Together with a defined role in developmental and mature brain, BDNF has excitatory effects in neuronal cultures and animal brain slices. Furthermore, both BDNF and its conjugated
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receptor (i.e. Tropomyosin receptor kinase B or TrkB) are increased in animal models and humans with epilepsy, particularly in the temporal and hippocampal areas. Acute injection of BDNF in brain
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of mice induces seizures, which are almost or totally abolished blocking its transcription and pathway. Chronic infusion of BDNF is conversely associated with a decreased neuronal excitability, probably via several mechanism including an increase in central levels of neuropeptide Y (NPY), altered conductance of chloride, and downregulation of TrkB. While genetic studies are inconclusive, serum BDNF is more frequently higher in patients with epilepsy and appears to be correlated to severity of disease. Current evidences suggest that inhibiting BDNF-TrkB signaling and reinforcing the NPY system could represent a potential therapeutic strategy for epilepsy, especially for temporal lobe epilepsy.
ACCEPTED MANUSCRIPT Keywords: Neurotrophins, brain-derived neurotrophic factor, epilepsy, epileptogenesis
Introduction Epilepsy is a prevalent neurological disorder characterized by an enduring predisposition to generate epileptic seizures. Such condition is also associated with a relatively high mortality and
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morbidity. Although new anti-epileptic drugs (AEDs) have been introduced in the last decades,
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seizures control cannot be achieved for all cases.
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The discovery that limbic seizures increase nerve growth factor (NGF) and mRNA levels (Gall and Isackson, 1989) developed the idea that seizure-induced expression of neurotrophic factors may
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contribute to the lasting structural and functional changes underlying epileptogenesis. From then, a number of studies have demonstrated that brain-derived neurotrophic factor (BDNF), one of such
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NGF, has plastic and pro-excitatory effects on the neurons and is both localized and upregulated in areas implicated in epileptogenesis and has clear pro-epileptogenic properties.
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In clinical practice, novel therapeutic approaches for the management of epilepsy are required.
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Although there are new anti-epileptic strategies through the use of neuropeptides (Kovac et al, 2013), elucidating the cellular and molecular mechanisms of epileptogenesis could provide the answer to this need. Aim of the present review is to collect and present clearly the most important
Methods
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findings regarding BDNF and its potential role in epilepsy.
This review was conducted and is reported in accordance with the PRISMA guidelines (Moher et al, 2009, 2015; Iqbal et al, 2017).
ACCEPTED MANUSCRIPT Eligibility criteria We included studies conducted about BDNF in epilepsy and epileptogenesis with the following characteristics: Basic researches: we included in vitro and ex vivo studies assessing cellular models of neuronal hyper-excitability and the expressed levels of BDNF. We also included in vivo
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studies on mutant mice and rat models assessing the influence of BDNF on epileptogenesis.
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Cohort studies and case-control studies: we included studies assessing the concentration of
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BDNF in different body fluids (plasma, serum, human milk, cerebrospinal fluid) and its link
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with epilepsy in humans.
Reviews: We included high-quality reviews to give a breadth of pre-clinical data. We also
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missed by the research strategy.
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performed a manual check of reviews’ references list, in view of identifying any relevant study
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Research strategy for identification of relevant studies: Four members of the research team (F.F., A.B., B.P., L.I.) performed a comprehensive literature research in Medline, CENTRAL, and Science Direct using terms identified and agreed by the
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authors: “BDNF”; “BDNF AND epilepsy”; “BDNF AND epileptogenesis”. Only publications in English were considered. The reference lists of retrieved studies have also been reviewed to identify studies that may have not been spotted by the search strategy. Those studies identified as potentially able to answer the study question but contained missing data, authors were contacted in attempt to fill the gap.
Data extraction, study quality and bias assessment Two authors (F.F. and L.L.) independently assessed both titles and abstracts of 19 957 Medline +
ACCEPTED MANUSCRIPT 427 CENTRAL + 39 564 Science Direct potentially eligible studies. Disagreements were resolved with discussion between all the authors. Studies with ambiguous or unclear result were retrieved in full and further assessed by all authors independently and included if pertinent. We excluded all those studies with insufficient statistical analysis, possible biases and contradictions, not clear end-points, and inconsistent or arbitrary conclusions.
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We assessed the study quality of each basic research publication using the 10-item
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collaborative approach to meta-analysis and review of animal data in experimental studies
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(CAMARADES) study quality checklist (Macleod et al, 2004). For human studies we used the NIH Quality Assessment Tool for Observational Cohort and Cross -Sectional Studies
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(National Heart, Lung, and Blood Institute, 2018). Two reviewers (F.F., L.L.) independently assessed the risk of bias using SYRCLE's risk of bias tool for animal studies (Hooijmans et al,
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2014) and the ROBINS’I tool for assessing risk of bias in non-randomized studies (Sterne et
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al, 2016).
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Results
Study selection
Of the 59 948 records found, 1 113 matched our inclusion criteria (Figure 1). After reading the fulltext articles, we decided to exclude 1 073 articles because of one or more of the following: (1) no innovative or important content, (2) no multivariable analysis, (3) insufficient data, (4) no clear potential biases or strategies to solve them, (5) no clear end points, (6) inconsistent or arbitrary conclusions. The final set included 40 articles.
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Fig. 1. Flow diagram of study inclusion.
Results of individual studies: BDNF and epilepsy In vitro and ex vivo studies
ACCEPTED MANUSCRIPT BDNF mRNA and mature protein are markedly higher in the hippocampus of animal models following a seizure (Ernfors et al, 1991; Isackson et al, 1991; Nibuya et al, 1995), and high levels of BDNF and Trk mRNA are found in the areas classically known to be prone to seizures such as hippocampus and enthorhinal cortex (Lindvall et al, 1994). Immunoreactivity-based studies also depose for constitutively high concentrations of BDNF mature protein in the hippocampus (Conner
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et al, 1997), and allowed to find that phosphorylated (activated) Trk were in greater amounts in the
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hippocampus of mice, following partial kindling or kainate acid (KA)-induced seizures when
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compared with the controls (Binder et al, 1999).
Post-traumatic epilepsy is a useful model for the elucidation of molecular mechanisms underlying
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neuronal hyper-excitability. Post-traumatic epilepsy undoubtedly involves several pathogenic factors, probably acting in parallel. However, two events represent a potential base for this
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condition: 1) disinhibition, and 2) formation of new excitatory neuronal connections (Prince et al, 2009). In a model of trauma-induced epileptogenesis, levels of BDNF and TrkB protein increased
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two- to three-fold in area CA (cornus ammonis) 3 by 24-48 h after Schaffer collateral transection, which led to axonal sprouting and hyperexcitability in area CA3 of hippocampal slice cultures, thus
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representing a model of post-traumatic neuronal hyperexcitability (Dinocourt et al, 2006). Injuryinduced axonal sprouting of axons was impaired in hippocampal slice cultures from mice,
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expressing low levels of TrkB receptors, thus pointing to the important role of BDNF/TrkB signaling in post-traumatic plasticity (Dinocourt et al, 2006). As demonstrated by basic science researches, excitatory GABA actions, induced by altered expression of chloride transporters as potassium-chloride transporter member 5 (KCC2) and Na-KCl cotransporter 1 (NKCC1), can contribute to seizure generation in temporal lobe epilepsy (TLE). Recently, Eftekhari et al. (2014) reported a marked upregulation of NKCC1 protein expression in mice with pilocarpine-induced status epilepticus, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Following a single high dose or multiple injections of BDNF during the latent phase of TLE, KCC2 levels were found to be raised
ACCEPTED MANUSCRIPT in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyper-excitability behaviors were ameliorated for a short period of time. Brain neuropeptide Y (NPY) was found to be increased following spontaneous convulsions in rat
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epilepsy models (Vezzani et al, 1999), as well as at the end of a seven-day long infusion of BDNF
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(12 microg/day) in the hippocampal region of mice (Reibel et al, 2000). Infusion of NPY into the
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rat hippocampus has shown to retard the development of hippocampal kindling, while anti-NPY antibodies could reverse this effect (Reibel et al, 2003). Furthermore, NPY-deficient mice develop
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spontaneous seizures and show a higher susceptibility to seizures induced by GABA receptor antagonists (Erickson et al, 1996). NPY appears to selectively inhibit sprouted MF-mediated field
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excitatory postsynaptic potentials mainly via the activation of presynaptic Y2 receptors (Koyama
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and Ikegaya, 2005).
In vivo studies
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Mice lacking both BDNF alleles in the germline die in the neonatal period; thus experimental studies have been conducted using mice of BDNF heterozygous (+/-) type. In these models, a
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striking reduction of development of kindling can be appreciated (Kokaia et al, 1995). Moreover, the conditional forebrain knockout of TrkB was followed by the complete abolishment of kindling (Scharfman, 2005). Other mutant mice models supported the previous observations: in truncated TrkB-overexpressing animals (which also work as decoy receptors), Carim-Todd and Lähteinen reported a decrease of spontaneous seizures, seizure severity and seizure onset delay in mice with KA-induced status epilepticus (Carim-Todd et al, 2009; Lähteinen et al, 2002). On the contrary, overexpression of BDNF in transgenic mice led to spontaneous seizures and more severe seizures in response of KA (Croll et al, 1999). Intra-hippocampal infusion of BDNF is enough to induce seizure activity in vivo models of
ACCEPTED MANUSCRIPT spontaneous limbic seizures (Scharfman et al, 2002). On the other side, in a study involving male Wistar rats, amplitude and frequency of epileptiform burst discharges were significantly decreased in epileptic animals treated with single high dose (10 μL on the 13th day) or multiple low doses (1μg/ μL on days 10, 11, 12, and 13) of BDNF injections after the induction of status epilepticus within the dorsal hippocampus, compared to the group that received pilocarpine hydrochloride
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(350 mg/kg) intraperitoneally, the latter procedure being an experimental model of TLE (Eftekhari
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et al, 2016). Other studies demonstrated that a chronic intrahippocampal infusion of BDNF inhibits
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the local kindling development (Binder et al, 2001).
Another approach consisted in the intracerebroventricular infusion of TrkB-Fc, i.e. antibodies
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against TrkB receptor, to inhibit the development of kindling. However, the same results could not be appreciated when TrkA-Fc or TrkC-Fc were administrated. Importantly, the magnitude of
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inhibition of kindling development correlated with the degree of penetration of TrkB-Fc into hippocampus, as seen via immune- histochemical reactions (Binder et al, 1999b). Kang and
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colleagues also showed that acute TrkB inhibition resolve phenobarbital-resistant seizures in a mouse model of neonatal ischemia (Kang et al, 2015).
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The relationship between central and peripheral levels of BDNF has been studied mostly in rodent models. Two series suggest that there is a significant positive correlation between brain tissue levels
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and serum or whole blood levels (the latter being influenced by BDNF storage in platelets) (Sartorius et al, 2009; Klein et al, 2011). However, in a study on adult rats with experimental epilepsy, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable, despite robust elevation in both mRNA and protein in multiple brain regions (Lanz et al, 2012).
Evidences for BDNF in humans Several experimental techniques have been adopted in order to study BDNF-related effects in humans with epilepsy.
ACCEPTED MANUSCRIPT Only four studies (Hong et al, 2014; Ismail et al, 2015; LaFrance et al, 2010; Chen et al, 2016) investigated the relationship between BDNF serum levels and epilepsy in both children and adults (Table 1). Hong et al. (2014) were the only who used Luminex Human BDNF Antibody Bead Kit (Invitrogen, Camarillo, CA, USA) and Invitrogen’s Growth Factor Buffer Reagent Kit for serum BDNF measurement; all the others used the enzyme- linked immunosorbent assay (ELISA)
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technique. No series evaluated BDNF levels in the CSF of subjects with epilepsy.
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Gender, but not age, was found to be a significant factor related to serum BDNF levels in controls and people with epilepsy Serum BDNF levels in people with epilepsy were not different from those of controls Seizure frequency and epilepsy duration negatively correlate with serum BDNF levels regardless other factors When BDNF cut-off values of 6260 pg/ml were used, the sensitivity to identify people with daily or more frequent seizures from those with fewer seizures was 80% and specificity was 90% Serum BDNF levels of epileptic infants and milk BDNF levels of their mothers were significantly higher than values for controls Serum BDNF levels of epileptic infants and milk BDNF levels of their mothers correlated with age, weight,
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135 people with epilepsy mean age: 27.2 y range: 11–65 y 34 controls mean age 31.8 y range: 22-68 y
RESULTS
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DEMOGRAPHICS
COMMENTS AND CONCLUSIONS Concentration of BDNF in serum is associated with disease severity in people with epilepsy and may be a helpful marker for severity
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AUTHOR AND REFERENCE Hong, 2014
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Table 1. Studies evaluating serum BDNF levels in patients with epilepsy
Ismail, 2015
30 breastfed infants (<2 years old) with idiopathic epilepsy 15 control healthy breastfed infants
Serum and milk BDNF levels are higher in epileptic infants than in controls and may be used as marker of disease severity
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Chen, 2016
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LaFrance, 2010.
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length, and head circumference of epileptic children Serum and milk BDNF levels were significantly increased with increased duration of illness and frequency of seizures There was a significant positive correlation between serum and breastmilk levels of BDNF and significantly higher levels in severe cases of epilepsy 15 patients with ES Healthy controls showed 12 patients with higher BDNF levels compared PNES to patients with PNES age range: 19-76 y Healthy controls showed 17 healthy volunteers higher levels of BDNF compared to patients with ES without comorbid major depressive disorder
34 patients with TLE, of which 23 unilateral TLE 11 bilateral TLE 22 healthy controls
Significantly lower serum BDNF levels in patients with TLE compared with the controls, with significant contribution mainly from the subgroup with bilateral TLE, which also had more frequent seizures The BDNF levels correlated with epilepsy duration (σ=0.355; p=0.040) and fractional
Unlike children, adults with epilepsy appear to have lower levels of serum BDNF, which can be used to distinguish adult patients with ES or PNES from healthy controls. Further human studies are needed to better understand the pathophysiology explaining the decreased serum BDNF levels found in epilepsy and in PNES Serum BDNF levels reflected longer epilepsy duration, impaired white matter integrity, and poor cognitive function in patients with chronic TLE
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anisotropy (FA) in the left temporal lobe, left thalamus, and right hippocampus. Using a regression model, BDNF level predicted verbal memory score. Furthermore, design fluency scores were predicted by serum BDNF level via the interactions with left temporal FA BDNF = brain-derived neurotrophic factor; PNES= psychogenic nonepileptic seizures; TLE = temporal lobe epilepsy
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Wang and colleagues measured BDNF total mRNA and its six transcripts in the hippocampal tissue
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of TLE patients with or without hippocampal sclerosis (HS) by real-time fluorescence quantitative polymerase chain reaction and compared with those from healthy controls. Furthermore, they also
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assessed excitatory actions induced by BDNF on hippocampal cells by whole-cell patch-clamp recordings (Wang et al, 2011). The Authors found a statistically significant increase of three human
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BDNF mRNA transcripts in TLE patients with HS, compared with those without HS (transcripts 2, 3 and 5 exhibited 2.1-, 2.3- and 4.1-fold increases, respectively). They failed to find any difference
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of the other transcripts. Furthermore, BDNF appeared to directly induce N-methyl-D-aspartate (NMDA) currents in dentate granule cells of the sole patients with HS-related TLE. Similarly,
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Murray and colleagues (Murray et al, 2000) found lower levels of alpha subunit of calcium/calmodulin-dependent protein kinase II’s mRNA and higher levels brain-derived neurotrophic factor mRNA throughout the granule cells of the hippocampus of patients with intractable TLE when compared with controls; furthermore, there also was a significant negative correlation between the duration of epilepsy and the expression of mRNA for brain-derived neurotrophic factor. Moreover, Takahashi et al. (1999) found that patients with intractable TLE are characterized by a marked increase in BDNF levels (2.6-fold, p<0.01) but no other neurotrophins. In a study involving 40 patients suffering from pharmaco-resistant TLE, it has been demonstrated (Hou et al, 2010) a marked increase in BDNF/TrkB expression in the dentate gyrus and CA3
ACCEPTED MANUSCRIPT regions of HS and non-HS groups. Treatment with Valproic acid resulted in a significant downregulation of BDNF/TrkB protein expression in sclerotic and non-sclerotic hippocampus. Recently, Martínez-Levy et al. investigated BDNF expression of transcripts with exons I (BDNFI), II (BDNFII), IV (BDNFIV) and VI (BDNFVI) and methylation levels of promoters 4 and 6 in the hippocampi of 24 patients with pharmaco-resistant TLE (Martínez- Levy et al, 2016). They found:
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1) a statistical significant increase for BDNFVI in patients compared to the control group (n=8), and
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2) a decreased BDNFVI expression (p<0.05) in patients treated with Topiramate (n=3) when
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compared to the remaining group of patients.
Western blot analysis of proteins contained in postsynaptic density from seven human epileptic
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neocortex testified that TrkB were upregulated to 2.6 ± 0.26-fold compared with control patients (n=3) (Wyneken et al, 2003).
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Past genetic studies evaluating differences in BDNF (and in particular BDNF Val66Met polymorphism) and other related genomic sequences between patients with epilepsy and normal
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controls have been inconclusive (Bragatti et al, 2010; Lohoff et al, 2005; Chou et al, 2004).
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In 2015 Shen et al investigated the association between BDNF Val66Met polymorphis m and the occurrence of temporal lobe epilepsy (TLE) and its clinical phenotypes. A Case -control study was employed and results showed that the frequency of Met allele was found to be lower
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in the TLE patients compared with the control subjects (43.9% vs. 48.6%, p:0.012, OR:1.21, 95% CI:1.04–1.41), and the frequency of Met66 allele carriers in the TLE with hippocampal sclerosis was significantly lower than those non carriers (20.5% vs. 29.1%, p:0.040). Authors suggested that BDNF Val66Met polymorphism might be correlated with epileptogenesis, and Met66 allele might play a protective role against the occurrence of TLE (Shen et al, 2015).
Discussion Research Effects
ACCEPTED MANUSCRIPT Epilepsy is a disease characterized by an enduring predisposition to generate epileptic seizures and by neurobiological, cognitive, psychological, and social consequences: it is a prevalent neurological disorder, associated with a relatively high mortality and morbidity. Despite new AEDs, seizures control cannot be achieved in all cases, in particular with complex partial epilepsy. In a French study (Picot et al, 2008) conducted on a population over 15 years of age, the proportion
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of non-controlled epilepsy (i.e. seizure-frequency at least one per month for 18 months) on the total
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number of the epilepsy cases was 15.6%, and up to 19% in children (Ramos-Lizana et al, 2012).
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In 1951, Levi-Montalcini and Hamburger discovered the first nerve growth factor, a peptide with trophic actions on sensory and sympathetic neurons (Levi-Montalcini and Hamburger, 1951).
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Thirty-one years later, Barde et al. described BDNF, which was shown to promote survival of a population of dorsal root ganglion neurons (Barde et al, 1982), and entered the literature as the
neurotrophin family were discovered.
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second member of the family of neurotrophic factors. Since then, other members of the so-called
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With the discovery that limbic seizures increase NGF mRNA levels (Gall and Isackson, 1989), the
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idea that seizure-induced expression of neurotrophic factors may contribute to the lasting structural and functional changes underlying epileptogenesis started to motivate further researches. BDNF is diffusely identified through all the CNS (Conner et al, 1997; Merlio et al, 1991), but
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constitutively high BDNF expression is found in the hippocampus, mostly in areas corresponding to MF axons of dentate granule cells (Conner et al, 1997). Moreover, BDNF is not only expressed by neurons, but is also found in astrocytes and microglia (Parpura and Zorec, 2010), both playing an important role in neuronal excitability in physiologic and pathologic conditions, including epilepsy. BDNF is important in development and maintenance of neuronal populations within the central nervous system or cells directly associated with it (Chen et al, 2013). From the first observation, that showed that BDNF, but not NGF, increased the frequency of miniature excitatory postsynaptic currents in Xenopus culture, a number of studies focused on the effects of BDNF on neuronal excitability and its potential roles in epileptogenesis.
ACCEPTED MANUSCRIPT While it is still unclear whether BDNF-induced synaptic potentiation occurs primarily by a presynaptic (e.g. trough enhancement of glutamate release) or post-synaptic (e.g. via phosphorylation of neurotransmitter receptors) mechanism, both pre- and post-synaptic TrkB receptors are important, at least in the hippocampus. Overall, BDNF appears to reduce the inhibitory
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(GABAergic) currencies and strengthen the excitatory (glutamatergic) synapses.
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Although BDNF upregulation seen in response of seizures might be aimed to neuroprotective and
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morphological purposes, little direct evidence exists to support such actions (Binder et al, 2001; Soysal et al, 2016). By contrast, a number of recent works described the striking role of BDNF on
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the neuronal excitability and epileptogenesis in animal models and patients with epilepsy. First, following an induced seizure endogenous BDNF and TrkB proteins appear to be upregulated
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in the areas of CNS which are well known to represent the anatomical substrate of epilepsy (namely: hippocampus and entorhinal cortex). Furthermore, BDNF/TrkB signaling promotes MF
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sprouting in dentate gyrus of hippocampal slice preparations obtained from the epileptic animals,
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and regulate the progression of hippocampal kindling in a dose dependent, long lasting, not secondary to neuronal toxicity and location-specific (hippocampus) manner. Moreover, although high levels of exogenous BDNF do not seem to affect severity of seizure, they exacerbate the injury
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caused by KA, specifically to CA3 pyramidal neurons. Increased levels of BDNF and TrkB protein in hippocampal slice cultures are also appreciated in models of post-traumatic neuronal hyperexcitability, thus pointing to the important role of BDNF/TrkB signaling in post-traumatic hyperexcitability and plasticity. It is important to underline that BDNF mRNA upregulation by seizure or perhaps by other stimuli, such as traumatic brain injury or hypoxia, leads to increased BDNF production by the dentate granule cells and increased anterograde transport and release of BDNF from MF axons resulting in activation of TrkB receptors in the hilus and CA3 stratum lucidum. This suggests that strengthening of the excitatory MF input onto CA3 pyramidal cells might be a primary mechanism by which BDNF promotes epileptogenesis (Binder et al, 2001).
ACCEPTED MANUSCRIPT Confirmations about the pro-epileptogenic potential of BDNF also come from studies in live mice. Transgenic mice models proved that a partial (in BDNF heterozygous (+/-) animals) or complete (in mice with conditional forebrain knockout of TrkB) abolition of development of kindling can be reached. The reason for a not fully-suppressed epileptogenesis in mice lacking almost the entire synthesis of BDNF has to be searched in the activation of TrkB by the other upregulated NTs. The
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excitatory effects of BDNF were demonstrated with the registration of seizure activity and
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increased MF sprouting in mice following the intrahippocampal infusion of BDNF. A recent
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approach involved the usage of Trk receptor-specific antibodies during kindling development in vivo. These compounds consist in divalent homodimers that contain the ligand-binding domain of a
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specific trk receptor, thus acting as decoy receptors for the endogenous NTs. Antibody-based studies firmly point to TrkB as the receptor involved in pro-epileptogenic effects of BDNF.
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In humans, BDNF can be measured in CSF as well as in plasma or serum. BDNF is detectable in blood because it is also abundantly expressed and secreted in other non-neuronal tissues
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(endocrine and salivary glands, respiratory system, urinary tract, female gonads, macrophages, lymphocytes, vascular endothelial and smooth muscle cells). Platelets (PLTs) represent a major
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storage site of BDNF in peripheral blood, and consequently its serum levels are higher than plasma ones. Recently, Iughetti et al. (Iughetti et al, 2011) found that in children: (1) plasma BDNF levels
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seem to be influenced by hormonal status, (2) BDNF was positively correlated with platelet count and negatively associated with both BMI and age, (3) BDNF levels in pubertal males were significantly lower than prepubertal males and both prepubertal and pubertal females, and (4) platelets remain the most important predictor of their concentration. Human evidences come mainly from four different approaches. First, serum BDNF levels were analyzed in children and adults with epilepsy and then compared with those appreciated in healthy controls. While theoretically weak because of possible influences due to BDNF production from peripheral sources, the great bulk of the evidences suggests a significant positive correlation between brain tissue levels and serum or whole blood levels (the latter being influenced by BDNF
ACCEPTED MANUSCRIPT storage in platelets) and thus it appears rational to assess serum or plasma BDNF levels to appreciate differences in both models and humans with epilepsy and controls. Three studies found that higher BDNF levels in serum (and milk) are associated with disease severity in people with temporal lobe and other forms of primary epilepsy, thus representing a potentially helpful marker for disease severity. However, one study stated that adults with epilepsy have decreased levels of
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serum BDNF (LaFrance et al, 2010). A second approach is based on the assessment of BDNF
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mRNA and protein expression in hippocampal tissue of TLE patients. All the evidences support the
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view that BDNF levels are higher in patients with TLE, particularly in those with hippocampal sclerosis. Interestingly, while a marked increase in BDNF/TrkB expression in the dentate gyrus and
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CA3 regions of patients with pharmaco-resistant was appreciated by Hou and colleagues, the treatment with Valproic acid resulted in a significant downregulation of BDNF/TrkB protein of
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expression (Hou et al, 2010). These results suggest that BDNF transcript is specifically upregulated in patients with TLE, an effect that seems to be influenced by the use of specific antiepileptic drugs.
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A third approach consists in whole-cell patch-clamp recordings dentate granule cells in transverse hippocampal slices obtained hippocampal specimens from patients with TLE during surgical
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removal of the anterior temporal lobe. Such technique confirmed the post-synaptic pro-excitatory effects of BDNF and the key role of its receptor TrkB. The fourth and the last strategy were based
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on the study of a potential relationship between specific BDNF gene polymorphisms and the epileptogenic phenotype. Despite the enthusiasm on Val66Met polymorphism, which appeared to be less present in patients with TLE compared to the healthy population, it cannot be found any convincing and definitive evidence of such link. Similar inferences can be done for all the other polymorphisms studied. While all the evidences coming from in vitro studies point to a pro-epileptogenic role of BDNF, other experimental evidences attribute to the neurotrophin anti-convulsant properties and could at least in part justify the lower levels of BDNF seen in patients with epilepsy in the reported series. In particular, chronic seven-day infusion of BDNF into the adult rat hippocampus significantly delays
ACCEPTED MANUSCRIPT the development of kindling induced by repeated electrical stimuli, a model of progressive TLE. Despite the possibility that such prolonged BDNF infusion downregulates or desensitizes the TrkB signaling pathway, the antiepileptic effect of BDNF is thought to be attributable to NPY, which has shown to be genetically induced by the neurotrophin and acts as a neuronal- membrane stabilized through its receptor Y2 . Finally, BDNF-induced expression of KCC2 channels and NKCC1
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downregulation in epileptic hippocampi are associated with a delayed development of spontaneous
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recurrent as well as better hyper excitability behaviors in treated mice. These evidences suggest
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possible therapeutic effects of BDNF via altering chloride transporters expression, pointing to chloride transporters as major culprits in temporal lobe epileptogenesis. In fact, it can be speculated
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a self-limited potential of BDNF-induced neuronal excitability, as represented in Figure 2. Figure 2. Schematic view of speculated role of BDNF in epilepsy. Epileptogenesis involves multiple steps,
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including the expression of BDNF and NPY. While BDNF exerts itself a pro-epileptogenic effect (green arrow), the induced NPY along with a TrkB down-regulation/desensitization and variation in neuronal Cl-
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conductances reduce CNS excitability (red arrow).
The major limitation of the present review lays in the type of studies analyzed, i.e. mainly observational studies. In fact, no randomized-control trials were available for the subject of our
ACCEPTED MANUSCRIPT review, seen that the role of BDNF in epileptogenesis and the consequent possible therapeutic application as biomarker for epilepsy severity is still uncertain. Furthermore, human evidences come from low-quality and non-homogeneous series. However, potential experimental and methodological confounders (such as BDNF doses employed and techniques of measures of this
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neurotrophin) have been minimized.
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Conclusions
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Taken together, the presented evidences suggest that BDNF has important effects on neuronal
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activity, particularly in the hippocampus, with a rapid modulation of excitatory and inhibitory synaptic transmission. Inhibiting BDNF-TrkB signaling and reinforcing NPY system could
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represent a potential therapeutic strategy for epilepsy, particularly for TLE. Current studies involving humans are confirming the animal results, although the low number and the
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methodologically weak nature of these leave unsolved the questions. Furthermore, reliable inferences are difficult to be obtained when basic and animal studies are considered. In particular,
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as epilepsy is a chronic, enduring disease, the short life span and the speed of progress of epilepsy in animals could be very different from those actually seen in humans. Finally, researchers should
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pay attention to the individual differences in sensitivity to BDNF and its pro-epileptogenic effects, with BDNF gene in human being a potential cause of such diversity in patients. It is thus explicit that further studies are needed in order to link, unequivocally, BDNF and epileptogenesis.
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ACCEPTED MANUSCRIPT BDNF as member of the family of neurotrophic factors is important in development and maintenance of neuronal populations within the central nervous system. BDNF is not only expressed by neurons but is also found in astrocytes and microglia and constitutively high BDNF expression is found in the hippocampus, BDNF can potentiate synaptic transmission and influence both structure and function of inhibitory cells, especially GABA-ergic neurons.
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BDNF reduction in specific brain areas is are associated with neurodegenerative diseases, neuropsychiatric disorders, and obesity
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Inhibiting BDNF pathway could represent a potential therapeutic strategy for epilepsy.
Lorenzo Iughetti, Laura Lucaccioni, Francesco Fuggetto, Barbara Predieri, Alberto Berardi, Fabrizio Ferrari PII: DOI: Reference:
S0143-4179(18)30065-9 doi:10.1016/j.npep.2018.09.005 YNPEP 1891
To appear in:
Neuropeptides
Received date: Revised date: Accepted date:
17 April 2018 13 August 2018 19 September 2018
Please cite this article as: Lorenzo Iughetti, Laura Lucaccioni, Francesco Fuggetto, Barbara Predieri, Alberto Berardi, Fabrizio Ferrari , Brain-derived neurotrophic factor and epilepsy: a systematic review. Ynpep (2018), doi:10.1016/j.npep.2018.09.005
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ACCEPTED MANUSCRIPT Brain-derived neurotrophic factor and epilepsy: a systematic review. Lorenzo Iughetti Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy
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Laura Lucaccioni
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Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of
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Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy
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Francesco Fuggetto
Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of
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Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy Barbara Predieri
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Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of
Alberto Berardi
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Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy
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Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy Fabrizio Ferrari
Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy Correspondig Author: Prof. Lorenzo Iughetti,
ACCEPTED MANUSCRIPT Department of Medical and Surgical Sciences of the Mother, Children and Adults. University of Modena and Reggio Emilia, Via del Pozzo n. 71, 41124, Modena, Italy. Tel. +39 059 4225382. Email: [email protected] Financial and competing interest disclosure
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The authors have no relevant financial and competing interests to declare.
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Abstract
Several in vitro, ex vivo and in vivo studies imply brain-derived neurotrophic factor (BDNF) in the
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pathophysiology of epilepsy. Aim of our work is to report the most important findings regarding
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BDNF and its potential role in epilepsy. We targeted those publications addressing both in vitro and in vivo evidences of relationship between BDNF and epilepsy. Basic researches, randomized
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trials, cohort studies, and reviews were contemplated to give a breadth of clinical data. Medline, CENTRAL, and Science Direct were searched till August 2017 using keywords agreed by the
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authors. Together with a defined role in developmental and mature brain, BDNF has excitatory effects in neuronal cultures and animal brain slices. Furthermore, both BDNF and its conjugated
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receptor (i.e. Tropomyosin receptor kinase B or TrkB) are increased in animal models and humans with epilepsy, particularly in the temporal and hippocampal areas. Acute injection of BDNF in brain
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of mice induces seizures, which are almost or totally abolished blocking its transcription and pathway. Chronic infusion of BDNF is conversely associated with a decreased neuronal excitability, probably via several mechanism including an increase in central levels of neuropeptide Y (NPY), altered conductance of chloride, and downregulation of TrkB. While genetic studies are inconclusive, serum BDNF is more frequently higher in patients with epilepsy and appears to be correlated to severity of disease. Current evidences suggest that inhibiting BDNF-TrkB signaling and reinforcing the NPY system could represent a potential therapeutic strategy for epilepsy, especially for temporal lobe epilepsy.
ACCEPTED MANUSCRIPT Keywords: Neurotrophins, brain-derived neurotrophic factor, epilepsy, epileptogenesis
Introduction Epilepsy is a prevalent neurological disorder characterized by an enduring predisposition to generate epileptic seizures. Such condition is also associated with a relatively high mortality and
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morbidity. Although new anti-epileptic drugs (AEDs) have been introduced in the last decades,
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seizures control cannot be achieved for all cases.
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The discovery that limbic seizures increase nerve growth factor (NGF) and mRNA levels (Gall and Isackson, 1989) developed the idea that seizure-induced expression of neurotrophic factors may
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contribute to the lasting structural and functional changes underlying epileptogenesis. From then, a number of studies have demonstrated that brain-derived neurotrophic factor (BDNF), one of such
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NGF, has plastic and pro-excitatory effects on the neurons and is both localized and upregulated in areas implicated in epileptogenesis and has clear pro-epileptogenic properties.
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In clinical practice, novel therapeutic approaches for the management of epilepsy are required.
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Although there are new anti-epileptic strategies through the use of neuropeptides (Kovac et al, 2013), elucidating the cellular and molecular mechanisms of epileptogenesis could provide the answer to this need. Aim of the present review is to collect and present clearly the most important
Methods
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findings regarding BDNF and its potential role in epilepsy.
This review was conducted and is reported in accordance with the PRISMA guidelines (Moher et al, 2009, 2015; Iqbal et al, 2017).
ACCEPTED MANUSCRIPT Eligibility criteria We included studies conducted about BDNF in epilepsy and epileptogenesis with the following characteristics: Basic researches: we included in vitro and ex vivo studies assessing cellular models of neuronal hyper-excitability and the expressed levels of BDNF. We also included in vivo
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studies on mutant mice and rat models assessing the influence of BDNF on epileptogenesis.
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Cohort studies and case-control studies: we included studies assessing the concentration of
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BDNF in different body fluids (plasma, serum, human milk, cerebrospinal fluid) and its link
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with epilepsy in humans.
Reviews: We included high-quality reviews to give a breadth of pre-clinical data. We also
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missed by the research strategy.
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performed a manual check of reviews’ references list, in view of identifying any relevant study
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Research strategy for identification of relevant studies: Four members of the research team (F.F., A.B., B.P., L.I.) performed a comprehensive literature research in Medline, CENTRAL, and Science Direct using terms identified and agreed by the
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authors: “BDNF”; “BDNF AND epilepsy”; “BDNF AND epileptogenesis”. Only publications in English were considered. The reference lists of retrieved studies have also been reviewed to identify studies that may have not been spotted by the search strategy. Those studies identified as potentially able to answer the study question but contained missing data, authors were contacted in attempt to fill the gap.
Data extraction, study quality and bias assessment Two authors (F.F. and L.L.) independently assessed both titles and abstracts of 19 957 Medline +
ACCEPTED MANUSCRIPT 427 CENTRAL + 39 564 Science Direct potentially eligible studies. Disagreements were resolved with discussion between all the authors. Studies with ambiguous or unclear result were retrieved in full and further assessed by all authors independently and included if pertinent. We excluded all those studies with insufficient statistical analysis, possible biases and contradictions, not clear end-points, and inconsistent or arbitrary conclusions.
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We assessed the study quality of each basic research publication using the 10-item
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collaborative approach to meta-analysis and review of animal data in experimental studies
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(CAMARADES) study quality checklist (Macleod et al, 2004). For human studies we used the NIH Quality Assessment Tool for Observational Cohort and Cross -Sectional Studies
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(National Heart, Lung, and Blood Institute, 2018). Two reviewers (F.F., L.L.) independently assessed the risk of bias using SYRCLE's risk of bias tool for animal studies (Hooijmans et al,
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2014) and the ROBINS’I tool for assessing risk of bias in non-randomized studies (Sterne et
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al, 2016).
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Results
Study selection
Of the 59 948 records found, 1 113 matched our inclusion criteria (Figure 1). After reading the fulltext articles, we decided to exclude 1 073 articles because of one or more of the following: (1) no innovative or important content, (2) no multivariable analysis, (3) insufficient data, (4) no clear potential biases or strategies to solve them, (5) no clear end points, (6) inconsistent or arbitrary conclusions. The final set included 40 articles.
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Fig. 1. Flow diagram of study inclusion.
Results of individual studies: BDNF and epilepsy In vitro and ex vivo studies
ACCEPTED MANUSCRIPT BDNF mRNA and mature protein are markedly higher in the hippocampus of animal models following a seizure (Ernfors et al, 1991; Isackson et al, 1991; Nibuya et al, 1995), and high levels of BDNF and Trk mRNA are found in the areas classically known to be prone to seizures such as hippocampus and enthorhinal cortex (Lindvall et al, 1994). Immunoreactivity-based studies also depose for constitutively high concentrations of BDNF mature protein in the hippocampus (Conner
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et al, 1997), and allowed to find that phosphorylated (activated) Trk were in greater amounts in the
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hippocampus of mice, following partial kindling or kainate acid (KA)-induced seizures when
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compared with the controls (Binder et al, 1999).
Post-traumatic epilepsy is a useful model for the elucidation of molecular mechanisms underlying
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neuronal hyper-excitability. Post-traumatic epilepsy undoubtedly involves several pathogenic factors, probably acting in parallel. However, two events represent a potential base for this
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condition: 1) disinhibition, and 2) formation of new excitatory neuronal connections (Prince et al, 2009). In a model of trauma-induced epileptogenesis, levels of BDNF and TrkB protein increased
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two- to three-fold in area CA (cornus ammonis) 3 by 24-48 h after Schaffer collateral transection, which led to axonal sprouting and hyperexcitability in area CA3 of hippocampal slice cultures, thus
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representing a model of post-traumatic neuronal hyperexcitability (Dinocourt et al, 2006). Injuryinduced axonal sprouting of axons was impaired in hippocampal slice cultures from mice,
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expressing low levels of TrkB receptors, thus pointing to the important role of BDNF/TrkB signaling in post-traumatic plasticity (Dinocourt et al, 2006). As demonstrated by basic science researches, excitatory GABA actions, induced by altered expression of chloride transporters as potassium-chloride transporter member 5 (KCC2) and Na-KCl cotransporter 1 (NKCC1), can contribute to seizure generation in temporal lobe epilepsy (TLE). Recently, Eftekhari et al. (2014) reported a marked upregulation of NKCC1 protein expression in mice with pilocarpine-induced status epilepticus, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Following a single high dose or multiple injections of BDNF during the latent phase of TLE, KCC2 levels were found to be raised
ACCEPTED MANUSCRIPT in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyper-excitability behaviors were ameliorated for a short period of time. Brain neuropeptide Y (NPY) was found to be increased following spontaneous convulsions in rat
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epilepsy models (Vezzani et al, 1999), as well as at the end of a seven-day long infusion of BDNF
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(12 microg/day) in the hippocampal region of mice (Reibel et al, 2000). Infusion of NPY into the
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rat hippocampus has shown to retard the development of hippocampal kindling, while anti-NPY antibodies could reverse this effect (Reibel et al, 2003). Furthermore, NPY-deficient mice develop
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spontaneous seizures and show a higher susceptibility to seizures induced by GABA receptor antagonists (Erickson et al, 1996). NPY appears to selectively inhibit sprouted MF-mediated field
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excitatory postsynaptic potentials mainly via the activation of presynaptic Y2 receptors (Koyama
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and Ikegaya, 2005).
In vivo studies
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Mice lacking both BDNF alleles in the germline die in the neonatal period; thus experimental studies have been conducted using mice of BDNF heterozygous (+/-) type. In these models, a
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striking reduction of development of kindling can be appreciated (Kokaia et al, 1995). Moreover, the conditional forebrain knockout of TrkB was followed by the complete abolishment of kindling (Scharfman, 2005). Other mutant mice models supported the previous observations: in truncated TrkB-overexpressing animals (which also work as decoy receptors), Carim-Todd and Lähteinen reported a decrease of spontaneous seizures, seizure severity and seizure onset delay in mice with KA-induced status epilepticus (Carim-Todd et al, 2009; Lähteinen et al, 2002). On the contrary, overexpression of BDNF in transgenic mice led to spontaneous seizures and more severe seizures in response of KA (Croll et al, 1999). Intra-hippocampal infusion of BDNF is enough to induce seizure activity in vivo models of
ACCEPTED MANUSCRIPT spontaneous limbic seizures (Scharfman et al, 2002). On the other side, in a study involving male Wistar rats, amplitude and frequency of epileptiform burst discharges were significantly decreased in epileptic animals treated with single high dose (10 μL on the 13th day) or multiple low doses (1μg/ μL on days 10, 11, 12, and 13) of BDNF injections after the induction of status epilepticus within the dorsal hippocampus, compared to the group that received pilocarpine hydrochloride
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(350 mg/kg) intraperitoneally, the latter procedure being an experimental model of TLE (Eftekhari
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et al, 2016). Other studies demonstrated that a chronic intrahippocampal infusion of BDNF inhibits
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the local kindling development (Binder et al, 2001).
Another approach consisted in the intracerebroventricular infusion of TrkB-Fc, i.e. antibodies
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against TrkB receptor, to inhibit the development of kindling. However, the same results could not be appreciated when TrkA-Fc or TrkC-Fc were administrated. Importantly, the magnitude of
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inhibition of kindling development correlated with the degree of penetration of TrkB-Fc into hippocampus, as seen via immune- histochemical reactions (Binder et al, 1999b). Kang and
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colleagues also showed that acute TrkB inhibition resolve phenobarbital-resistant seizures in a mouse model of neonatal ischemia (Kang et al, 2015).
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The relationship between central and peripheral levels of BDNF has been studied mostly in rodent models. Two series suggest that there is a significant positive correlation between brain tissue levels
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and serum or whole blood levels (the latter being influenced by BDNF storage in platelets) (Sartorius et al, 2009; Klein et al, 2011). However, in a study on adult rats with experimental epilepsy, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable, despite robust elevation in both mRNA and protein in multiple brain regions (Lanz et al, 2012).
Evidences for BDNF in humans Several experimental techniques have been adopted in order to study BDNF-related effects in humans with epilepsy.
ACCEPTED MANUSCRIPT Only four studies (Hong et al, 2014; Ismail et al, 2015; LaFrance et al, 2010; Chen et al, 2016) investigated the relationship between BDNF serum levels and epilepsy in both children and adults (Table 1). Hong et al. (2014) were the only who used Luminex Human BDNF Antibody Bead Kit (Invitrogen, Camarillo, CA, USA) and Invitrogen’s Growth Factor Buffer Reagent Kit for serum BDNF measurement; all the others used the enzyme- linked immunosorbent assay (ELISA)
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technique. No series evaluated BDNF levels in the CSF of subjects with epilepsy.
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Gender, but not age, was found to be a significant factor related to serum BDNF levels in controls and people with epilepsy Serum BDNF levels in people with epilepsy were not different from those of controls Seizure frequency and epilepsy duration negatively correlate with serum BDNF levels regardless other factors When BDNF cut-off values of 6260 pg/ml were used, the sensitivity to identify people with daily or more frequent seizures from those with fewer seizures was 80% and specificity was 90% Serum BDNF levels of epileptic infants and milk BDNF levels of their mothers were significantly higher than values for controls Serum BDNF levels of epileptic infants and milk BDNF levels of their mothers correlated with age, weight,
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135 people with epilepsy mean age: 27.2 y range: 11–65 y 34 controls mean age 31.8 y range: 22-68 y
RESULTS
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DEMOGRAPHICS
COMMENTS AND CONCLUSIONS Concentration of BDNF in serum is associated with disease severity in people with epilepsy and may be a helpful marker for severity
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AUTHOR AND REFERENCE Hong, 2014
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Table 1. Studies evaluating serum BDNF levels in patients with epilepsy
Ismail, 2015
30 breastfed infants (<2 years old) with idiopathic epilepsy 15 control healthy breastfed infants
Serum and milk BDNF levels are higher in epileptic infants than in controls and may be used as marker of disease severity
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Chen, 2016
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LaFrance, 2010.
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length, and head circumference of epileptic children Serum and milk BDNF levels were significantly increased with increased duration of illness and frequency of seizures There was a significant positive correlation between serum and breastmilk levels of BDNF and significantly higher levels in severe cases of epilepsy 15 patients with ES Healthy controls showed 12 patients with higher BDNF levels compared PNES to patients with PNES age range: 19-76 y Healthy controls showed 17 healthy volunteers higher levels of BDNF compared to patients with ES without comorbid major depressive disorder
34 patients with TLE, of which 23 unilateral TLE 11 bilateral TLE 22 healthy controls
Significantly lower serum BDNF levels in patients with TLE compared with the controls, with significant contribution mainly from the subgroup with bilateral TLE, which also had more frequent seizures The BDNF levels correlated with epilepsy duration (σ=0.355; p=0.040) and fractional
Unlike children, adults with epilepsy appear to have lower levels of serum BDNF, which can be used to distinguish adult patients with ES or PNES from healthy controls. Further human studies are needed to better understand the pathophysiology explaining the decreased serum BDNF levels found in epilepsy and in PNES Serum BDNF levels reflected longer epilepsy duration, impaired white matter integrity, and poor cognitive function in patients with chronic TLE
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anisotropy (FA) in the left temporal lobe, left thalamus, and right hippocampus. Using a regression model, BDNF level predicted verbal memory score. Furthermore, design fluency scores were predicted by serum BDNF level via the interactions with left temporal FA BDNF = brain-derived neurotrophic factor; PNES= psychogenic nonepileptic seizures; TLE = temporal lobe epilepsy
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Wang and colleagues measured BDNF total mRNA and its six transcripts in the hippocampal tissue
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of TLE patients with or without hippocampal sclerosis (HS) by real-time fluorescence quantitative polymerase chain reaction and compared with those from healthy controls. Furthermore, they also
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assessed excitatory actions induced by BDNF on hippocampal cells by whole-cell patch-clamp recordings (Wang et al, 2011). The Authors found a statistically significant increase of three human
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BDNF mRNA transcripts in TLE patients with HS, compared with those without HS (transcripts 2, 3 and 5 exhibited 2.1-, 2.3- and 4.1-fold increases, respectively). They failed to find any difference
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of the other transcripts. Furthermore, BDNF appeared to directly induce N-methyl-D-aspartate (NMDA) currents in dentate granule cells of the sole patients with HS-related TLE. Similarly,
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Murray and colleagues (Murray et al, 2000) found lower levels of alpha subunit of calcium/calmodulin-dependent protein kinase II’s mRNA and higher levels brain-derived neurotrophic factor mRNA throughout the granule cells of the hippocampus of patients with intractable TLE when compared with controls; furthermore, there also was a significant negative correlation between the duration of epilepsy and the expression of mRNA for brain-derived neurotrophic factor. Moreover, Takahashi et al. (1999) found that patients with intractable TLE are characterized by a marked increase in BDNF levels (2.6-fold, p<0.01) but no other neurotrophins. In a study involving 40 patients suffering from pharmaco-resistant TLE, it has been demonstrated (Hou et al, 2010) a marked increase in BDNF/TrkB expression in the dentate gyrus and CA3
ACCEPTED MANUSCRIPT regions of HS and non-HS groups. Treatment with Valproic acid resulted in a significant downregulation of BDNF/TrkB protein expression in sclerotic and non-sclerotic hippocampus. Recently, Martínez-Levy et al. investigated BDNF expression of transcripts with exons I (BDNFI), II (BDNFII), IV (BDNFIV) and VI (BDNFVI) and methylation levels of promoters 4 and 6 in the hippocampi of 24 patients with pharmaco-resistant TLE (Martínez- Levy et al, 2016). They found:
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1) a statistical significant increase for BDNFVI in patients compared to the control group (n=8), and
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2) a decreased BDNFVI expression (p<0.05) in patients treated with Topiramate (n=3) when
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compared to the remaining group of patients.
Western blot analysis of proteins contained in postsynaptic density from seven human epileptic
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neocortex testified that TrkB were upregulated to 2.6 ± 0.26-fold compared with control patients (n=3) (Wyneken et al, 2003).
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Past genetic studies evaluating differences in BDNF (and in particular BDNF Val66Met polymorphism) and other related genomic sequences between patients with epilepsy and normal
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controls have been inconclusive (Bragatti et al, 2010; Lohoff et al, 2005; Chou et al, 2004).
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In 2015 Shen et al investigated the association between BDNF Val66Met polymorphis m and the occurrence of temporal lobe epilepsy (TLE) and its clinical phenotypes. A Case -control study was employed and results showed that the frequency of Met allele was found to be lower
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in the TLE patients compared with the control subjects (43.9% vs. 48.6%, p:0.012, OR:1.21, 95% CI:1.04–1.41), and the frequency of Met66 allele carriers in the TLE with hippocampal sclerosis was significantly lower than those non carriers (20.5% vs. 29.1%, p:0.040). Authors suggested that BDNF Val66Met polymorphism might be correlated with epileptogenesis, and Met66 allele might play a protective role against the occurrence of TLE (Shen et al, 2015).
Discussion Research Effects
ACCEPTED MANUSCRIPT Epilepsy is a disease characterized by an enduring predisposition to generate epileptic seizures and by neurobiological, cognitive, psychological, and social consequences: it is a prevalent neurological disorder, associated with a relatively high mortality and morbidity. Despite new AEDs, seizures control cannot be achieved in all cases, in particular with complex partial epilepsy. In a French study (Picot et al, 2008) conducted on a population over 15 years of age, the proportion
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of non-controlled epilepsy (i.e. seizure-frequency at least one per month for 18 months) on the total
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number of the epilepsy cases was 15.6%, and up to 19% in children (Ramos-Lizana et al, 2012).
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In 1951, Levi-Montalcini and Hamburger discovered the first nerve growth factor, a peptide with trophic actions on sensory and sympathetic neurons (Levi-Montalcini and Hamburger, 1951).
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Thirty-one years later, Barde et al. described BDNF, which was shown to promote survival of a population of dorsal root ganglion neurons (Barde et al, 1982), and entered the literature as the
neurotrophin family were discovered.
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second member of the family of neurotrophic factors. Since then, other members of the so-called
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With the discovery that limbic seizures increase NGF mRNA levels (Gall and Isackson, 1989), the
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idea that seizure-induced expression of neurotrophic factors may contribute to the lasting structural and functional changes underlying epileptogenesis started to motivate further researches. BDNF is diffusely identified through all the CNS (Conner et al, 1997; Merlio et al, 1991), but
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constitutively high BDNF expression is found in the hippocampus, mostly in areas corresponding to MF axons of dentate granule cells (Conner et al, 1997). Moreover, BDNF is not only expressed by neurons, but is also found in astrocytes and microglia (Parpura and Zorec, 2010), both playing an important role in neuronal excitability in physiologic and pathologic conditions, including epilepsy. BDNF is important in development and maintenance of neuronal populations within the central nervous system or cells directly associated with it (Chen et al, 2013). From the first observation, that showed that BDNF, but not NGF, increased the frequency of miniature excitatory postsynaptic currents in Xenopus culture, a number of studies focused on the effects of BDNF on neuronal excitability and its potential roles in epileptogenesis.
ACCEPTED MANUSCRIPT While it is still unclear whether BDNF-induced synaptic potentiation occurs primarily by a presynaptic (e.g. trough enhancement of glutamate release) or post-synaptic (e.g. via phosphorylation of neurotransmitter receptors) mechanism, both pre- and post-synaptic TrkB receptors are important, at least in the hippocampus. Overall, BDNF appears to reduce the inhibitory
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(GABAergic) currencies and strengthen the excitatory (glutamatergic) synapses.
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Although BDNF upregulation seen in response of seizures might be aimed to neuroprotective and
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morphological purposes, little direct evidence exists to support such actions (Binder et al, 2001; Soysal et al, 2016). By contrast, a number of recent works described the striking role of BDNF on
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the neuronal excitability and epileptogenesis in animal models and patients with epilepsy. First, following an induced seizure endogenous BDNF and TrkB proteins appear to be upregulated
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in the areas of CNS which are well known to represent the anatomical substrate of epilepsy (namely: hippocampus and entorhinal cortex). Furthermore, BDNF/TrkB signaling promotes MF
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sprouting in dentate gyrus of hippocampal slice preparations obtained from the epileptic animals,
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and regulate the progression of hippocampal kindling in a dose dependent, long lasting, not secondary to neuronal toxicity and location-specific (hippocampus) manner. Moreover, although high levels of exogenous BDNF do not seem to affect severity of seizure, they exacerbate the injury
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caused by KA, specifically to CA3 pyramidal neurons. Increased levels of BDNF and TrkB protein in hippocampal slice cultures are also appreciated in models of post-traumatic neuronal hyperexcitability, thus pointing to the important role of BDNF/TrkB signaling in post-traumatic hyperexcitability and plasticity. It is important to underline that BDNF mRNA upregulation by seizure or perhaps by other stimuli, such as traumatic brain injury or hypoxia, leads to increased BDNF production by the dentate granule cells and increased anterograde transport and release of BDNF from MF axons resulting in activation of TrkB receptors in the hilus and CA3 stratum lucidum. This suggests that strengthening of the excitatory MF input onto CA3 pyramidal cells might be a primary mechanism by which BDNF promotes epileptogenesis (Binder et al, 2001).
ACCEPTED MANUSCRIPT Confirmations about the pro-epileptogenic potential of BDNF also come from studies in live mice. Transgenic mice models proved that a partial (in BDNF heterozygous (+/-) animals) or complete (in mice with conditional forebrain knockout of TrkB) abolition of development of kindling can be reached. The reason for a not fully-suppressed epileptogenesis in mice lacking almost the entire synthesis of BDNF has to be searched in the activation of TrkB by the other upregulated NTs. The
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excitatory effects of BDNF were demonstrated with the registration of seizure activity and
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increased MF sprouting in mice following the intrahippocampal infusion of BDNF. A recent
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approach involved the usage of Trk receptor-specific antibodies during kindling development in vivo. These compounds consist in divalent homodimers that contain the ligand-binding domain of a
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specific trk receptor, thus acting as decoy receptors for the endogenous NTs. Antibody-based studies firmly point to TrkB as the receptor involved in pro-epileptogenic effects of BDNF.
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In humans, BDNF can be measured in CSF as well as in plasma or serum. BDNF is detectable in blood because it is also abundantly expressed and secreted in other non-neuronal tissues
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(endocrine and salivary glands, respiratory system, urinary tract, female gonads, macrophages, lymphocytes, vascular endothelial and smooth muscle cells). Platelets (PLTs) represent a major
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storage site of BDNF in peripheral blood, and consequently its serum levels are higher than plasma ones. Recently, Iughetti et al. (Iughetti et al, 2011) found that in children: (1) plasma BDNF levels
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seem to be influenced by hormonal status, (2) BDNF was positively correlated with platelet count and negatively associated with both BMI and age, (3) BDNF levels in pubertal males were significantly lower than prepubertal males and both prepubertal and pubertal females, and (4) platelets remain the most important predictor of their concentration. Human evidences come mainly from four different approaches. First, serum BDNF levels were analyzed in children and adults with epilepsy and then compared with those appreciated in healthy controls. While theoretically weak because of possible influences due to BDNF production from peripheral sources, the great bulk of the evidences suggests a significant positive correlation between brain tissue levels and serum or whole blood levels (the latter being influenced by BDNF
ACCEPTED MANUSCRIPT storage in platelets) and thus it appears rational to assess serum or plasma BDNF levels to appreciate differences in both models and humans with epilepsy and controls. Three studies found that higher BDNF levels in serum (and milk) are associated with disease severity in people with temporal lobe and other forms of primary epilepsy, thus representing a potentially helpful marker for disease severity. However, one study stated that adults with epilepsy have decreased levels of
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serum BDNF (LaFrance et al, 2010). A second approach is based on the assessment of BDNF
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mRNA and protein expression in hippocampal tissue of TLE patients. All the evidences support the
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view that BDNF levels are higher in patients with TLE, particularly in those with hippocampal sclerosis. Interestingly, while a marked increase in BDNF/TrkB expression in the dentate gyrus and
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CA3 regions of patients with pharmaco-resistant was appreciated by Hou and colleagues, the treatment with Valproic acid resulted in a significant downregulation of BDNF/TrkB protein of
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expression (Hou et al, 2010). These results suggest that BDNF transcript is specifically upregulated in patients with TLE, an effect that seems to be influenced by the use of specific antiepileptic drugs.
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A third approach consists in whole-cell patch-clamp recordings dentate granule cells in transverse hippocampal slices obtained hippocampal specimens from patients with TLE during surgical
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removal of the anterior temporal lobe. Such technique confirmed the post-synaptic pro-excitatory effects of BDNF and the key role of its receptor TrkB. The fourth and the last strategy were based
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on the study of a potential relationship between specific BDNF gene polymorphisms and the epileptogenic phenotype. Despite the enthusiasm on Val66Met polymorphism, which appeared to be less present in patients with TLE compared to the healthy population, it cannot be found any convincing and definitive evidence of such link. Similar inferences can be done for all the other polymorphisms studied. While all the evidences coming from in vitro studies point to a pro-epileptogenic role of BDNF, other experimental evidences attribute to the neurotrophin anti-convulsant properties and could at least in part justify the lower levels of BDNF seen in patients with epilepsy in the reported series. In particular, chronic seven-day infusion of BDNF into the adult rat hippocampus significantly delays
ACCEPTED MANUSCRIPT the development of kindling induced by repeated electrical stimuli, a model of progressive TLE. Despite the possibility that such prolonged BDNF infusion downregulates or desensitizes the TrkB signaling pathway, the antiepileptic effect of BDNF is thought to be attributable to NPY, which has shown to be genetically induced by the neurotrophin and acts as a neuronal- membrane stabilized through its receptor Y2 . Finally, BDNF-induced expression of KCC2 channels and NKCC1
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downregulation in epileptic hippocampi are associated with a delayed development of spontaneous
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recurrent as well as better hyper excitability behaviors in treated mice. These evidences suggest
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possible therapeutic effects of BDNF via altering chloride transporters expression, pointing to chloride transporters as major culprits in temporal lobe epileptogenesis. In fact, it can be speculated
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a self-limited potential of BDNF-induced neuronal excitability, as represented in Figure 2. Figure 2. Schematic view of speculated role of BDNF in epilepsy. Epileptogenesis involves multiple steps,
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including the expression of BDNF and NPY. While BDNF exerts itself a pro-epileptogenic effect (green arrow), the induced NPY along with a TrkB down-regulation/desensitization and variation in neuronal Cl-
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conductances reduce CNS excitability (red arrow).
The major limitation of the present review lays in the type of studies analyzed, i.e. mainly observational studies. In fact, no randomized-control trials were available for the subject of our
ACCEPTED MANUSCRIPT review, seen that the role of BDNF in epileptogenesis and the consequent possible therapeutic application as biomarker for epilepsy severity is still uncertain. Furthermore, human evidences come from low-quality and non-homogeneous series. However, potential experimental and methodological confounders (such as BDNF doses employed and techniques of measures of this
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neurotrophin) have been minimized.
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Conclusions
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Taken together, the presented evidences suggest that BDNF has important effects on neuronal
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activity, particularly in the hippocampus, with a rapid modulation of excitatory and inhibitory synaptic transmission. Inhibiting BDNF-TrkB signaling and reinforcing NPY system could
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represent a potential therapeutic strategy for epilepsy, particularly for TLE. Current studies involving humans are confirming the animal results, although the low number and the
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methodologically weak nature of these leave unsolved the questions. Furthermore, reliable inferences are difficult to be obtained when basic and animal studies are considered. In particular,
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as epilepsy is a chronic, enduring disease, the short life span and the speed of progress of epilepsy in animals could be very different from those actually seen in humans. Finally, researchers should
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pay attention to the individual differences in sensitivity to BDNF and its pro-epileptogenic effects, with BDNF gene in human being a potential cause of such diversity in patients. It is thus explicit that further studies are needed in order to link, unequivocally, BDNF and epileptogenesis.
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P., 2009. Correlations and discrepancies between serum and brain tissue levels of neurotrophins after electroconvulsive treatment in rats. Pharmacopsychiatry 42:270-6.
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Scharfman HE., 2005. Brain-derived Neurotrophic Factor and Epilepsy—A Missing Link?
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on serum BDNF levels in offsprings of epileptic rats. Neuropeptides. 56:1-8. Sterne JA, Hernán MA, Reeves BC, Savović J, Berkman ND, Viswanathan M, Henry , Altman DG, Ansari M, Boutron I, Carpenter JR, Chan AW, Churchill R, Deeks JJ, Hróbjartsson A, Kirkham J, Jüni P, Loke YK, Pigott TD, Ramsay CR, Regidor D, Rothstein
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ACCEPTED MANUSCRIPT brain-derived neurotrophic factor protein and its correlation with neuropeptide Y. Brain Res. 818:579-82. Vezzani A, Ravizza T, Moneta D, Conti M, Borroni A, Rizzi M, Samanin R, Maj R., 1999. Brain-derived neurotrophic factor immunoreactivity in the limbic system of rats after acute
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ACCEPTED MANUSCRIPT BDNF as member of the family of neurotrophic factors is important in development and maintenance of neuronal populations within the central nervous system. BDNF is not only expressed by neurons but is also found in astrocytes and microglia and constitutively high BDNF expression is found in the hippocampus, BDNF can potentiate synaptic transmission and influence both structure and function of inhibitory cells, especially GABA-ergic neurons.
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BDNF reduction in specific brain areas is are associated with neurodegenerative diseases, neuropsychiatric disorders, and obesity
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Inhibiting BDNF pathway could represent a potential therapeutic strategy for epilepsy.