Upstream eventBDNF, Reduced
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment||non-adjacent||Moderate||Low|
Life Stage Applicability
|During brain development||High|
Key Event Relationship Description
Disruption of BDNF signaling (and other factors, such as NGF or Reelin, etc.) during brain development was shown to interfere with synaptogenesis in the hippocampus (Sanchez-Martin et al., 2013; Neal et al., 2010; Stansfiled et al., 2012). In the adult brain, BDNF is involved in synaptic plasticity (Lu et al., 2013; Leal et al., 2014), which is a fundamental process linked with learning and memory. Synaptic dysfunction is a key pathophysiological hallmark in neurodegenerative disorders, including Alzheimer's disease, and synaptic repair therapies based on the use of trophic factors, such as BDNF, are currently under consideration (Lu et al., 2013).
BDNF is released by the BDNF-producing neurons of the CNS and binds to Trk-B of the PV-interneurons, an interaction necessary for the subsequent developmental effects of this neurotrophin (Polleux et al., 2002; Jin et al., 2003; Rico et al., 2002; Aguado et al., 2003). BDNF promotes the morphological and neurochemical maturation of hippocampal and neocortical interneurons and promotes GABAergic synaptogenesis (Danglot et al., 2006; Hu and Russek, 2008).
BDNF plays an important role in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development (Chapleau et al., 2009). Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, as well as in mouse models of these disorders (Chapleau et al., 2009).
Evidence Supporting this KER
BDNF, in addition to its pro-survival effects, has powerful synaptic effects, promoting synaptic transmission, synaptic plasticity and synaptogenesis (Lu et al., 2013; Sanchez-Martin et al., 2013; Neal et al., 2010; Stansfiled et al., 2012; Danglot et al., 2006; Hu and Russek, 2008). NMDAR activity has been linked to the signaling of the trans-synaptic neurotorophin BDNF (Neal et al., 2010).
Use of selective agonist or antagonist of BDNF receptor TrkB demonstrates the contribution of BDNF in synaptogenesis in adult-generated neurons in the rat dentate gyrus (Ambrogini et al., 2013). In this regard, exogenous application of BDNF significantly increased the number of functional synapses in culture (Vicario-Abejon et al., 1998; Marty et al., 2000), while blocking of BDNF with antibodies greatly reduced the formation of inhibitory synapses (Seil and Drake-Baumann, 2000). Similar results were described also in an in vivo study on mutant mice characterized by deletion of the trkB gene in cerebellar precursors (obtained by Wnt1-driven Cre--mediated recombination). TrkB mutant mice showed reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations, whilst granule and Purkinje cell dendrites appeared normal and the former presented typical numbers of excitatory synapses. This study demonstrated that TrkB is essential to the development of GABAergic neurons and the regulation of synapse formation (Rico et al., 2002). BDNF is also a potent regulator of spontaneous neuronal activity in GABAergic neurons and interneurons, as shown in in embryonic (E18) hippocampal slices (Aguado et al., 2003), and plays a critical role in controlling the emergence, complexity and networking properties of spontaneous networks.
TH deficiency during the foetal and/or the neonatal period, apart from reducing synaptogenesis, can produce several other deleterious effects for neural growth and development (e.g., such as reduced synaptic connectivity, delayed myelination, disturbed neuronal migration, deranged axonal projections, and alterations in neurotransmitters' levels), possibly through decreased BDNF levels (Koromilas et al., 2010; Shafiee et al., 2016).
Several studies (in vitro, ex vivo, and in vivo) have shown correlations between downregulation of BDNF signaling (e.g., in trasgenic animals, or upon treatment with K252a (a BDNF receptor inhibitor) or with an antibody anti-BDNF) and synaptogenesis (and synapses) decrease:
- Westerholz et al., 2013 In recent in vitro studies with rat T3-deficient cultures of cortical GABAergic PV+ interneurons, which are subject to BDNF regulation, it was shown that the number of synaptic boutons (i.e., presynaptic terminals containing the presynaptic marker synaptophysin) was reduced, an effect that was abolished after exogenous BDNF application. Additionally, inhibition of BDNF TrkB receptors by K252a in cultures containing T3 resulted also in decreased number of synaptic boutons, as in the T3-deprived cultures. These results indicate that BDNF signaling promotes the formation of synaptic boutons and that this function is mediated by THs (T3 and T4). Additionally, T3-related increase of spontaneous network activity was remarkably reduced after addition of K252a, and also upon inhibition of mTOR pathway (with rapamycin), a pathway known to control synaptogenesis (Buckmaster et al., 2009).
- Sato et al., 2007 This study on rat cultured hippocampal slices showed that beta-estradiol (E2) induced synaptogenesis between mossy fibers (one of the major inputs to cerebellum) and hippocampal CA3 neurons by enhancing BDNF release from dentate gyrus (DG) granule cells, by increasing the expression of PSD95, a postsynaptic marker. E2 effects on in hippocampal slice cultures and subregional neuron cultures were completely inhibited by blocking the BDNF receptor (TrkB) with K252a (200 nM) or by using a function-blocking antibody to BDNF (10 μg/ml), which inhibited the expression of PSD95 induced by E2. Both K252a and the antibody anti-BDNF elicited ~ 60-70% decrease of spine density and ~ 55% decrease of presynaptic sites in dentate gyrus granule cells (measured as number of puncta/neuron).
- Schjetnan and Escobar, 2012 In this study, intrahippocampal microinfusion of BDNF (3 µg/3 µl; 0.2 µl/min,) in adult rats modified the ability of the hippocampal mossy fiber pathway to present long-term potentiation (LTP, i.e., a persistent strengthening of synapses based on recent patterns of activity) by high frequency stimulation (HFS). This indicates that BDNF initiates the metaplastic mechanisms that allow the modiﬁcations of the ability of the mossy ﬁber pathway to present LTP induced by subsequent HFS. On the contrary, microinfusion of K252a (administered in combination with BDNF: 3 μg of BDNF/3 μl of K252a 20 μM; 0.2 μl/min) blocked the functional and morphological effects produced by BDNF (shown by densitometric analysis on synaptic reorganization: ~ 30% reduction of the relative area of the dorsal hippocampus in the contralateral side of HFS, and ~ 70% reduction in the ipsilateral side of HFS, compared to BDNF administered alone), supporting the role of BDNF in the regulation of synaptic plasticity.
- Schildt et al., 2013 Using field potential recordings in CA3 of adult heterozygous BDNF knockout (BDNF+/-) mice, an impairment of NMDAR-independent mossy fiber (MF)-LTP (~ 50% decrease) was observed. Additionally, inhibition of TrkB/BDNF with K252a (slices preincubated for 3 hr with 100 nM), or with the selective BDNF scavenger TrkB-Fc (slices preincubated for 3 hr with 5 μg/ml), both inhibited MF-LTP to the same extent as observed in BDNF+/- mice (K252a: ~ 60% decrease vs control slices; TrkB-Fc: ~ 50% decrease vs control slices).
- Cortés et al., 2012 Adult male Sprague-Dawley rats were treated with 6-propyl-2-thiouracil (PTU, a TPO inhibitor) (0.05% in drinking water) for 20 days to induce hypothyroidism. PTU-treated rats showed decrease serum fT4 (~ 70% decrease vs control) and tT3 (~ 45% decrease vs control) levels, and increased TSH levels (~ 9.5-fold increase over control). The hippocampus of hypothyroid adult rats displayed increased apoptosis levels in neurons and astrocyte and reactive gliosis compared with controls. The glutamatergic synapses from the stratum radiatum of CA3 from hypothyroid rats, contained lower postsynaptic density (PSD) than control rats (~ 25% lower PSD than control). This observation was in agreement with a reduced content of NMDAR subunits (NR1 and NR2A/B subunits, both subunits: ~ 25% decrease vs control) at the PSD in hypothyroid animals. Additionally, the hippocampal amount of BDNF mRNA (assessed by in situ hybridization) was higher (~ 4.8-fold increase over control) of hypothyroid rats, while the content of TrkB protein (BDNF receptor) was reduced (~ 30% decrease vs control) at the PSD of the CA3 region of hypothyroid rats, compared with controls. Even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.
- Koibuchi et al., 2001 Here newborn mice were rendered hypothyroid by administering MMI (TPO inhibitor) and perchlorate (NIS inhibitor) in drinking water to their mothers. Neurotrophin-3 (NT-3) and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (ROR-alpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased in hypothyroid rats. Dendritic arborization is used as readout for synapse formation, as post-synaptic side (synaptogenesis) is mainly located on dendrites.
- Aguado et al., 2003 BDNF overexpression in transgenic embryos raised the spontaneous activity of E18 hippocampal neurons, as shown by increased number of synapses (63% more synapses in the hippocampus of BDNF transgenic embryos than in controls), and increased spontaneous neuronal activity (2.3 times more active neurons than wild type embryos, and 36.3% greater rates of activation). Moreover, BDNF transgenic embryos had higher number of GABAergic interneuron synapses, as shown by higher GAD67 mRNA (by 3-fold) and K(+)/Cl(-) KCC2 mRNA expression (by 4.3-fold) (responsible for the conversion of GABA responses from depolarizing to inhibitory), without altering the expression of GABA and glutamate ionotropic receptors. These data indicate that BDNF controls both GABAergic pre- and postsynaptic sites.
KEs proceeding the AO (decreased cognition), such as "Reduced BDNF Release" and "Decreased synaptogenesis" are also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure, as a reference chemical, are reported. These studies do not refer to TH disruption; however, they provide empirical support for this KER (Reduced release of BDNF leads to decreased synaptogenesis).
Synaptic structural plasticity was shown to be modified by Pb treatment during early (pre-weaning) or late (post-weaning) brain development in rats exposed to 2 mM Pb in drinking water for 3 weeks (Xiao et al., 2014). An iron chelator (clioquinol) can rescue the Pb-induced impairment of synaptic plasticity in hippocampus (Chen et al., 2007), showing that Pb can affect synaptogenesis and synaptic plasticity. Primary hippocampal neurons obtained from ED18 rat pups and treated with Pb (1, 2 microM) for 5 days exhibited pre-synaptic deficits due to disruption of NMDAR-dependent BDNF signaling (Neal et al., 2010; Stansfield et al., 2012). A decrease in bdnf expression was observed in mouse embryonic stem cells differentiated into neurons, if they were exposed to Pb 0.1 microM throughout the whole differentiation process (Sanchez-Martin et al., 2013). Similar alterations in gene expression patterns of neural markers (synapsin 1), neurotrophins (bdnf), transcription factors and glutamate-related genes were found in mice, when their mothers were exposed to 0-3 ppm of Pb in drinking water from 8 weeks prior to mating, through gestation and until postnatal day 10 (Sanchez-Martin et al., 2013).
Uncertainties and Inconsistencies
Alterations of BDNF signaling is probably not the only mechanism leading to impaired synaptogenesis and synaptic plasticity. Indeed NMDAR activity can also modulate nitric oxide (NO) signaling. Exogenous NO addition during Pb exposure results in complete recovery of whole-cell synaptophysin levels and partial recovery of synaptophysin and synaptobrevin in synapses in Pb-exposed neurons (Neal et al., 2012). In addition, in Wistar rats, the anti-oxidant and radical scavenger quercetin was able to relieve the impairment of synaptic plasticity induced by chronic Pb exposure (from parturition through adulthood (PND 60); 0.2% Pb in drinking water of mothers and post-weaning pups) (Hu et al., 2008), suggesting that oxidative stress can also interfere with synapse formation.
Additionally, while PTU (a TPO inhibitor) has been shown to decrease brain BDNF levels and expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in the study from Cortés and colleagues (Cortés et al., 2012), treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB protein, the BDNF receptor, resulted reduced at the PSD of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD. These indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain. However, even though BDNF levels were increased, the decrease of BDNF receptor (TrkB) compromises the signalling pathway under BDNF control.
Results variability from study to study is due to different experimental study designs, accounting for differences in brain development stages (PND vs adult), times of exposures to chemicals, and regional brain differences.
Quantitative Understanding of the Linkage
There is a lack of studies directly linking BDNF levels (gene and/or protein) with the quantitative analysis of synaptogenesis induced by decreased TH levels, and therefore no robust quantitative information can be provided. However, in the AOP 13 ("Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" https://aopwiki.org/aops/13) direct associations between Pb-induced decrease of BDNF (protein and/or mRNA) and decrease of pre- and post-synaptic proteins are discussed, supported also by quantitative analyses of spines and dendrite morphology.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Empirical evidence comes from work with laboratory rodent-derived cells and brain slices, and rodent in vivo studies.
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Neal AP, Stansfield KH, Guilarte TR. (2012). Enhanced nitric oxide production during lead (Pb(2)(+)) exposure recovers protein expression but not presynaptic localization of synaptic proteins in developing hippocampal neurons. Brain Res 1439: 88-95.
Polleux F, Whitford KL, Dijkhuizen PA, Vitalis T, Ghosh A. (2002). Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling. Development 129:3147–60.
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Sato K, Akaishi T, Matsuki N, Ohno Y, Nakazawa K. (2007). beta-Estradiol induces synaptogenesis in the hippocampus by enhancing brain-derived neurotrophic factor release from dentate gyrus granule cells. Brain Res. May 30;1150:108-20.
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