Carlos B. Duarte

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways.

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.

Role of the brain‐derived neurotrophic factor at glutamatergic synapses

The neurotrophin brain‐derived neurotrophic factor (BDNF) plays an important role in the activity‐dependent regulation of synaptic structure and function, particularly of the glutamatergic synapses. BDNF may be released in the mature form, which activates preferentially TrkB receptors, or as proBDNF, which is coupled to the stimulation of the p75NTR. In the mature form BDNF induces rapid effects on glutamate release, and may induce short‐ and long‐term effects on the postsynaptic response to the neurotransmitter. BDNF may affect glutamate receptor activity by inducing the phosphorylation of the receptor subunits, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. Stimulation of the local protein synthesis and transcription activity account for the delayed effects of BDNF on glutamatergic synaptic strength. Several evidences show impaired synaptic plasticity of glutamatergic synapses in diseases where compromised BDNF function has been observed, such as Huntingtons disease, depression, anxiety, and the BDNF polymorphism Val66Met, suggesting that upregulating BDNF‐activated pathways may be therapeutically relevant. This review focuses on recent advances in the understanding of the regulation of the glutamatergic synapse by BDNF, and its implications in synaptic plasticity.

Bdnf regulates the expression and traffic of NMDA receptors in cultured hippocampal neurons

The neurotrophin BDNF regulates the activity-dependent modifications of synaptic strength in the CNS. Physiological and biochemical evidences implicate the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present study, we investigated the effect of BDNF on the expression and plasma membrane abundance of NMDA receptor subunits in cultured hippocampal neurons. Acute stimulation of hippocampal neurons with BDNF differentially upregulated the protein levels of the NR1, NR2A and NR2B NMDA receptor subunits, by a mechanism sensitive to transcription and translation inhibitors. Accordingly, BDNF also increased the mRNA levels for NR1, NR2A and NR2B subunits. The neurotrophin NT3 also upregulated the protein levels of NR2A and NR2B subunits, but was without effect on the NR1 subunit. The amount of NR1, NR2A and NR2B proteins associated with the plasma membrane of hippocampal neurons was differentially increased by BDNF stimulation for 30 min or 24 h. The rapid upregulation of plasma membrane-associated NMDA receptor subunits was correlated with an increase in NMDA receptor activity. The results indicate that BDNF increases the abundance of NMDA receptors and their delivery to the plasma membrane, thereby upregulating receptor activity in cultured hippocampal neurons.

Regulation of hippocampal synaptic plasticity by BDNF.

The neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a major regulator of activity-dependent plasticity at excitatory synapses in the mammalian central nervous system. In particular, much attention has been given to the role of the neurotrophin in the regulation of hippocampal long-term potentiation (LTP), a sustained enhancement of excitatory synaptic strength believed to underlie learning and memory processes. In this review we summarize the evidence pointing to a role for BDNF in generating functional and structural changes at synapses required for both early- and late phases of LTP in the hippocampus. The available information regarding the pre- and/or postsynaptic release of BDNF and action of the neurotrophin during LTP will be also reviewed. Finally, we discuss the effects of BDNF on the synaptic proteome, either by acting on the protein synthesis machinery and/or by regulating protein degradation by calpains and possibly by the ubiquitin-proteasome system (UPS). This fine-tuned control of the synaptic proteome rather than a simple upregulation of the protein synthesis may play a key role in BDNF-mediated synaptic potentiation. This article is part of a Special Issue entitled SI: Brain and Memory.

Regulation of AMPA receptors and synaptic plasticity.

Neuronal activity controls the strength of excitatory synapses by mechanisms that include changes in the postsynaptic responses mediated by AMPA receptors. These receptors account for most fast responses at excitatory synapses of the CNS, and their activity is regulated by various signaling pathways which control the electrophysiological properties of AMPA receptors and their interaction with numerous intracellular regulatory proteins. AMPA receptor phosphorylation/dephosphorylation and interaction with other proteins control their recycling and localization to defined postsynaptic sites, thereby regulating the strength of the synapse. This review focuses on recent advances in the understanding of the molecular mechanisms of regulation of AMPA receptors, and the implications in synaptic plasticity.

Regulation of local translation at the synapse by BDNF

The neurotrophin brain-derived neurotrophic factor (BDNF) plays a key role in synaptic plasticity, in part due to changes in local protein synthesis. Activation of TrkB (tropomyosin-related kinase B) receptors for BDNF triggers several parallel signaling pathways, including the Ras/ERK, the phosphatidylinositol 3-kinase (PI3-K) and the phospholipase C-γ pathways. Recent studies have elucidated some of the signaling mechanisms that contribute to the regulation of translation activity by BDNF, through modulation of initiation and elongation phases, but the resulting changes in the proteome are not yet fully characterized. The proteins synthesized in response to activation of TrkB receptors by BDNF depend on the mRNAs that are available locally, after delivery and transport along dendrites. Recent studies have shown that BDNF may also play a regulatory role at this level. Furthermore, BDNF regulates transcription activity, thereby affecting the array of mRNAs available to be transported along dendrites. This review highlights the recent advances in the understanding of the diversity of mechanisms that contribute to the regulation of the synaptic proteome by BDNF, which may account for its role in synaptic plasticity.

Regulation of AMPA receptors by phosphorylation.

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+ - and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity.

Ca2+ influx through glutamate receptor-associated channels in retina cells correlates with neuronal cell death

We studied the effect of glutamate, N-methyl-D-aspartate (NMDA), kainate or K+ depolarization, on neurotoxicity in cultured chick retinal cells, under conditions in which we could discriminate between Ca2+ entering through ionotropic glutamate receptors and voltage-sensitive Ca2+ channels (VSCCs). When neurons were challenged with NMDA, kainate or glutamate, in Na(+)-containing medium, a decrease in cell survival was observed, whereas K+ depolarization did not affect the viability of the cells. The Mg2+ ion completely prevented the toxic effect mediated by the NMDA receptor, and had a small but significant protective effect at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) receptor-induced cell death. We observed that, in a Na(+)-free N-methyl-D-glucamine (NMG) medium, to avoid the activation of VSCCs indirectly by the glutamate receptor agonists, stimulation of the glutamate receptors causes Ca2+ influx only through NMDA and AMPA/kainate receptor-associated channels, and that Ca2+ entry correlates well with subsequent cell death. These results show that the activation of NMDA or AMPA/kainate receptors can cause excitotoxicity in retinal neurons by mechanisms not involving Na+ influx, but rather depending on the permeation of Ca2+ through glutamate receptor-associated channels. For small Ca2+ loads the entry of Ca2+ through the NMDA receptor-associated channel was more efficient in triggering cell death than the influx of Ca2+ through the AMPA/kainate receptor.

Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons.

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca(2+) dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca(2+) influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca(2+) channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A(1) receptor antagonist, showing that the release is tonically inhibited by the adenosine A(1) receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation.

Impairment of excitatory amino acid transporter activity by oxidative stress conditions in retinal cells: effect of antioxidants.

In the present study we analyzed how oxidative stress conditions induced by ascorbate/ Fe2+ affect the excitatory amino acid (EAA) transport systems in cultured chick retina cells. The uptake of D‐[3H]aspartate, which is transported by the same carrier as glutamate, was determined in control cells and in cells subjected to ascorbate/Fe2+. The uptake of this EAA was Na+ dependent and was inhibited by about 40% under oxidative stress conditions. To clarify the molecular mechanisms involved in the inhibition of D‐[3H]aspartate uptake by ascorbate/Fe2+, we investigated the effect of vitamin E (Vit E), melatonin, reduced glutathione (GSH), and dithiothreitol (DTT) on the uptake of D‐[3H]aspartate and on the extent of lipid peroxidation in control and in peroxidized cells. Preincubation with Vit E (100 μM) abolished lipid peroxidation, but had no significant effect on the inhibition of D‐[3H]aspartate uptake evoked by ascorbate/Fe2+. Melatonin was more effective in reducing the formation of TBARS and conjugated dienes than in preventing the D‐[3H]aspartate uptake inhibition evoked by the oxidant pair. Conversely, GSH (4 mM) and DTT (4 mM) completely prevented the inhibition of D‐[3H]aspartate uptake in cells subjected to oxidative stress, but were without effect on the extent of peroxidation. Free fatty acids, such as ar‐achidonic acid, seem not to be involved in reducing the activity of the D‐[3H]aspartate uptake system, whereas the reduction of the Na+ electrochemical gradient that occurs under oxidative stress was in part involved in the reduction of D‐[3H]aspartate uptake by the cells. The inhibition of D‐[3H]aspartate uptake by ascorbate/Fe2+ persisted for at least 1 h, but could be partially reverted by disulfide reducing agents. It is concluded that oxidative stress causes long‐lasting modifications of the glutamate/D‐[3H]aspartate transport system (or systems), such as oxidation of protein sulfhydryl (SH) groups, which can be recovered by some antioxidants.—Agos‐tinho, P., Duarte, C. B., Oliveira, C. R. Impairment of excitatory amino acid transporter activity by oxidative stress conditions in retinal cells: effect of antioxidants. FASEB J. 11, 154‐163 (1997)