Antonello Novelli

Nuclear factor κB is a critical determinant in N‐methyl‐d‐aspartate receptor‐mediated neuroprotection

The role of a nuclear factor κB (NF‐κB) in NMDA receptor‐mediated neuroprotection is not known. A candidate sequence from the 5′ flanking region of exon 3 of the rat brain‐derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 μm NMDA activated a specific DNA binding activity that was blocked by the NMDA receptor antagonist MK‐801. Anti‐p65 antibody or anti‐p50 antibody ‘supershifted’ the DNA binding activity, suggesting that the DNA–protein complex was composed of p65 and p50 subunits. NMDA receptor‐mediated neuroprotection was blocked when cerebellar neurons were transfected with a double‐stranded oligonucleotide containing the BDNF gene NF‐κB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double‐stranded NF‐κB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF‐κB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quantified the level of exon 3‐specific BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3‐specific BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF‐κB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF‐κB DNA binding activity. Taken together, these data support a mechanism where NF‐κB plays a critical role in NMDA‐mediated neuroprotection.

Aluminum-induced degeneration of astrocytes occurs via apoptosis and results in neuronal death.

The mechanisms by which aluminum interacts with the nervous system are only partly understood. In this study, we used cultured astrocytes and neurons to investigate the effects of long exposures to aluminum (1 mM). We found that aluminum accumulated both in neurons and astrocytes. After 8-12 days exposure, aluminum caused strong changes in the morphology of astrocytes including shrinkage of cell bodies and retraction of processes. Exposures over 15-18 days reduced astrocytes viability by 50%. Aluminum-induced degeneration of astrocytes involved the DNA fragmentation characteristic of apoptosis, and staining of aluminum-treated astrocytes with the DNA-binding fluorochrome Hoeschst 33258 revealed the typical apoptotic condensation and fragmentation of chromatin. Aluminum was also found to be neurotoxic, causing first (4-6 days) abnormal clustering and aggregation, and later (8-12 days) neuronal death. Interestingly, aluminum neurotoxicity occurred in neuroglial cultures containing approximately 10% astrocytes but not in near-pure neuronal cultures containing only 1% astrocytes. Staining of co-cultured cells with Hoeschst 33258 showed apoptotic condensation and fragmentation of chromatin in aluminum-treated astrocytes but not in co-cultured neurons. Our study demonstrates that aluminum can induce the apoptotic degeneration of astrocytes, and that this toxicity is critical in determining neuronal degeneration and death. Aluminum-mediated apoptosis of cultured astrocytes may be also a valuable model system to study the mechanisms underlying apoptosis in glial cells.

Basic fibroblast growth factor protects cerebellar neurons in primary culture from NMDA and non-NMDA receptor mediated neurotoxicity

We have investigated the ability of bFGF to protect cerebellar neurons from neurotoxicity by excitatory amino acids. We have found that preincubation with 1–2.5 nM bFGF for 1–6 days significantly protected neurons from excitotoxic damage via NMDA receptors as well as ionotropic non‐NMDA receptors. bFGF neuroprotection appeared not to be dependent upon neuronal differentiation and was not mimicked by other neurotrophins including BDNF, NT‐3 and NGF. A greater rise in extracellular calcium‐dependent cGMP formation, following either depolarization or excitatory amino acid receptor activation was observed in bFGF‐pretreated neurons. We suggest that neuroprotection from excitotoxicity following bFGF treatment may be associated to the modulation of neurochemical pathways dependent upon extracellular calcium influx.

Preconditioning and neurotrophins: a model for brain adaptation to seizures, ischemia and other stressful stimuli.

Summary.The amino acid glutamate, the major excitatory neurotransmitter in the central nervous system, activates receptors coupled to calcium influx. Excessive activation of glutamate receptors in conditions such as severe epileptic seizures or stroke can kill neurons in a process called excitotoxicity. However, subtoxic levels of activation of the N-methyl-D-aspartate (NMDA) type of glutamate receptor elicit adaptive responses in neurons that enhance their ability to withstand more severe stress. A variety of stimuli induce adaptive responses to protect neurons. For example, sublethal ischemic episodes or a mild epileptic insult can protect neurons in a process referred to as tolerance. The molecular mechanisms that protect neurons by these different stressful stimuli are largely unknown but they share common features such as the transcription factor, nuclear factor kappa B (NF-κB), which is activated by ischemic and epileptic preconditioning as well as exposure to subtoxic NMDA concentrations. In this article, we describe stress-induced neuroprotective mechanisms highlighting the role of brain-derived neurotrophic factor (BDNF), a protein that plays a crucial role in neuronal survival and maintenance, neurogenesis and learning and memory.

N‐methyl‐D‐aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal‐regulated kinase pathway

N‐Methyl‐D‐aspartate (NMDA) at a subtoxic concentration (100 μM) promotes neuronal survival against glutamate‐mediated excitotoxicity via a brain‐derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐3 kinase (PI3‐K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3‐K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration‐ and time‐dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK‐801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 μM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3‐K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl‐2, bcl‐XL, and bax mRNA levels,. NMDA increases Bcl‐2 and Bcl‐XL protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal‐regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate‐mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl‐2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.

Inhibition of protein phosphatases induces IGF-1-blocked neurotrophin-insensitive neuronal apoptosis.

We have previously described the marine toxin okadaic acid (OKA) to be a potent neurotoxin for cultured rat cerebellar neurons. Here we show that OKA‐induced neurodegeneration involves the DNA fragmentation characteristic of apoptosis and is protein synthesis‐dependent. DNA fragmentation and neurotoxicity correlated with inhibition of protein phosphatase (PP) 2A rather than PP1 activity. Neurotrophins NT‐3 and BDNF failed to protect from OKA‐induced apoptotic neurotoxicity that was, however, totally prevented by insulin‐like growth factor‐1. Neuronal death by OKA was significantly reduced by protein kinase C inhibitors and by the L‐type calcium channel agonist Bay K8644, while it was potentiated by the reduction of free extracellular calcium concentrations.

The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons

The tumor promoter okadaic acid (OKA), is a marine toxin of algal origin, identified as a potent inhibitor of protein phosphatases 1 and 2A, and possibly enhancing calcium influx through voltage dependent calcium channels (VSSC). We now report that OKA at concentrations as low as 0.5 nM produced neurotoxicity, characterized first by the desintegration of the neurites and swelling of cell bodies, and later by cellular death. Non-neuronal cells viability and morphology were unaffected up to at least 5 nM OKA. Neurons sensitivity to the toxin changed with age in culture. Maximum neurotoxicity was observed in neurons at 9 DIC, when the OKA concentration producing half of the maximum reduction in neuronal survival (EC50) was approximately 0.65 nM. At 5 DIC or 19 DIC (EC50 approximately 2.5 nM and approximately 4.5 nM respectively), neurons appeared to be less sensitive to OKA. Neurotoxicity by OKA was not reduced by VSCC antagonists such as nifedipine and verapamil, nor by antagonists of excitatory aminoacid (EAA) receptors including APV, MK801 or CNQX. VSCC antagonists and EAA receptors antagonists fully protected from neurotoxicity induced by depolarization with KCl. These results suggest that OKA mechanism of neurotoxicity may not directly involve VSCC, endogenous EAA release and EAA receptors, but may depend upon other neurochemical events.

Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels

Summary.Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways. We found that nefopam prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. Nefopam protection was concentration-dependent. 47 μM nefopam provided 50% protection while full neuroprotection was achieved at 100 μM nefopam. Neuroprotection was associated with a 73% reduction in the BayK8644-induced increase in intracellular calcium concentration. Nefopam also inhibited intracellular cGMP formation following BayK8644 in a concentration-dependent manner, 100 μM nefopam providing full inhibition of cGMP synthesis and 58 μM allowing 50% cGMP formation. Nefopam reduced NMDA receptor-mediated cGMP formation resulting from the release of glutamate following activation of channels by BayK8644. Finally, we also showed that nefopam effectively reduced cGMP formation following stimulation of cultures with domoic acid, while not providing neuroprotection against domoic acid. Thus, the novel action of nefopam we report here may be important both for its central analgesic effects and for its potential therapeutic use in neurological and neuropsychiatric disorders involving an excessive glutamate release.

Neuronal Sensitization and Its Behavioral Correlates in a Rat Model of Neuropathy Are Prevented by a Cyclic Analog of Orphenadrine

N-methyl-D-aspartic acid (NMDA) is an agonist at the homonymous receptor implicated in the development of neuronal sensitization and its behavioral correlates. An effective modulation of the NMDA effects, achieved also by uncompetitive antagonists, could contribute to controlling pain symptoms in several neuropathic syndromes. Because nefopam is a known analgesic derivative of orphenadrine and of its congener diphenhydramine, both uncompetitive NMDA receptor antagonists, we tested the effect of nefopam on the developing pain and neuronal anomalies in an animal model of chronic pain with NMDA receptor involvement. A single intraperitoneal injection of nefopam was administered twenty minutes prior to the chronic constriction injury of the sciatic nerve (CCI rats). In the first 10 days, nefopam (30 mg/kg) significantly decreased behavioral signs of neuropathic pain and the stimulus-evoked electrophysiological anomalies in recordings at 14 days, with only slight manifestation afterwards. The dose of 20 mg/kg was ineffective. Nefopam injected after constriction was ineffective. In normal non-operated rats, Nefopam had no effect on the electrophysiological and behavioral parameters. Iontophoretic nefopam (1 mM, 50-80 nA, positive current) in normal rats did not change the spontaneous neuronal activity, but reduced the mean response to noxious stimuli and the concurrent iontophoretic NMDA evoked activity. In CCI rats, iontophoretic nefopam did not significantly modify the spontaneous hyperactivity but reduced significantly both the frequency of the responses to noxious stimuli, and the duration of the afterdischarge. We propose that nefopam exerts a preventive analgesic effect, with a possible role in modulating NMDA receptor-mediated effects in central sensitization.

Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 microM) or glutamate (15 microM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 microM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25 microM), oxatomide (20 microM), and triprolidine (50 microM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (+)-alpha-tocopherol (200 microM) or the endogenous antioxidant melatonin (200 microM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.