Reelin-mediated signaling in neuropsychiatric and neurodegenerative diseases

Reelin is a conserved extracellular glycoprotein crucial for neurodevelopment. In adulthood, Reelin is an important modulator of NMDA receptor-mediated neurotransmission, required for synaptic plasticity, learning and memory. Consequently, abnormal Reelin-mediated signaling has been associated with many human brain disorders involving directly or indirectly altered NMDA receptor function. For most neurological and neuropsychiatric disorders, abnormalities during brain development appear central in the disease etiology. However, a similar causative relationship for neurodegenerative diseases, like Alzheimer's disease (AD), has not been investigated yet.     

Postsynaptic density-93 deficiency protects cultured cortical neurons from N-methyl-d-aspartate receptor-triggered neurotoxicity

It has been reported that N-methyl-d-aspartate receptor (NMDAR)-triggered neurotoxicity is related to excessive Ca²⁺ loading and an increase in nitric oxide (NO) concentration. However, the molecular mechanisms that underlie these events are not completely understood. NMDARs and neuronal NO synthase each binds to the scaffolding protein postsynaptic density (PSD)-93 through its PDZ domains. In this study, we determined whether PSD-93 plays a critical role in NMDAR/Ca²⁺/NO-mediated neurotoxicity. We found that the targeted disruption of the PSD-93 gene attenuated the neurotoxicity triggered by NMDAR activation, but not by non-NMDAR activation, in cultured mouse cortical neurons. PSD-93 deficiency reduced the amount of NMDAR subunits NR2A and NR2B in synaptosomal fractions from the cortical neurons and significantly prevented NMDA-stimulated increases in cyclic guanosine 3′,5′-monophosphate and Ca²⁺ loading in the cortical neurons. These findings indicate that PSD-93 deficiency could block NMDAR-triggered neurotoxicity by disrupting the NMDAR-Ca²⁺-NO signaling pathway and reducing expression of synaptic NR2A and NR2B. Since NMDARs, Ca²⁺, and NO play a critical role during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders.     

Rapid regrowth of hippocampal mossy fibres and preceding maturation of NMDA receptor-mediated neurotransmission

Early in postnatal development, glutamatergic synapses contain primarily NMDA receptors and progressively acquire AMPA receptor function. To determine whether this transformation occurs in a process of regenerative synaptogenesis following axotomy, we investigated the recovery of AMPA and NMDA receptor-mediated neurotransmission after the transection of mossy fibres (MF) in organotypic hippocampal cultures. An NMDA component could already be elicited 1 day after the lesion and reached a saturated level after 3 days. Thereafter, an AMPA component appeared and slowly matured after 10 days. The preceding establishment of NMDA receptor function implies that immature MF synapses are functionally silent at least for the first several days of recovery. The appearance of AMPA receptor-mediated neurotransmission was unchanged in the presence of an NMDA-receptor antagonist or tetrodotoxin, which suggests that the AMPA receptor maturation is virtually independent of neuronal activity. Thus, the conversion of silent to functional synapses is not unique to synaptic plasticity or developmental processes but also occurs in recovery after brain damage, but its mechanism is likely to differ from NMDA receptor-dependent recruitment of AMPA receptors in synaptic plasticity.     

Cardiovascular and vocalization reactions elicited by N-methyl-D-aspartate in the pretentorial periaqueductal grey of cats

1. Cats were anaesthetized with urethane (1100-1200 mg/kg, i.p.), supplemented with halothane inhalation during surgery. Responses in terms of systemic arterial pressure, mean systemic arterial pressure(MSAP), heart rate, mean blood flow of the common carotid artery and femoral artery, amplitude and frequency of vocalizations, thoracic-abdominal contractions and limb movements were recorded. 2. Microinjection of N-methyl-D-aspartate (NMDA; 20 mmol/L, 200 nL) into the pretentorial periaqueductal grey (PAG) produced two classes of response: (i) cardiovascular responses and vocalization; and (ii) cardiovascular responses without vocalization. 3. For class 1 responses, five types of vocalization concomitant with pressor (VPR) or depressor (VDPR) responses were observed: (i) type 1 VDPR and VPR, elicited in the rostral and caudal part of the dorsal PAG, produced vocalization of slight hissing, with or without limb movement, moderately increased flow of the common carotid and slightly increased flow of the femoral arteries; (ii) types 2 and 3 VPR, elicited in the dorsolateral and intermediate-lateral PAG, produced hissing-howling and growling, increased respiratory movement, with or without repetitive burst limb movements or stretching of paws, slightly decreased common carotid artery flow and inconsistent changes (increased or decreased slightly) in femoral artery flow; (iii) type 4 VPR, elicited in the dorsomedial and intermediate-medial PAG, produced meowing-crying but without limb movements, common carotid artery flow was increased, but the femoral artery flow was slightly decreased or increased markedly; and (iv) type 5 VPR, elicited in the ventromedial PAG, produced meowing-screaming with or without limb movements, common carotid artery flow increased moderately and femoral artery flow increased markedly. Vocalization was loud and wild in type 4 and 5 responses. 4. For class 2 responses, two types of responses were observed: (i) a pressor response (PR) alone, elicited in the dorsolateral and intermediate-lateral PAG, produced inconsistent changes in common carotid and femoral artery flow, which increased, decreased or underwent no change; and (ii) a depressor response (DPR) elicited in the ventrolateral PAG produced moderate increases of common carotid and femoral artery flow. 5. The reduction of resistance in the femoral artery was more prominent (P < 0.05) in type 1 VDPR than in DPR. Similar changes occurred in the femoral artery among types 3 (P < 0.05) and 5 VPR (P < 0.005) and PR. In addition, the frequency of vocalization was significantly positively correlated with the increase in MSAP (P < 0.05) and mean common carotid and femoral artery flow (both P < 0.01) in all types of VPR. 6. These results suggest the presence of neurons involved in the expression of defence reactions in a longitudinal, complicated functional organization in the entire PAG column. In particular, NMDA stimulation of the medial, dorsal and dorsolateral PAG may elicit five different types of defence reactions, expressed by various forms of cardiovascular alterations concomitant with vocalization responses.     

Why do neuroprotective drugs that are so promising in animals fail in the clinic? An industry perspective

1. No neuroprotective drug has yet been shown to be effective in treating acute ischaemic stroke in the clinic, despite evidence of efficacy in animal models. 2. An academic/industry round-table group recently published guidelines to be met if a drug was to be progressed to clinical trial. 3. Major points included obtaining full dose-response evaluation and measurement of the therapeutic time window for efficacy, functional behavioural testing in addition to measurement of infarct volume, measurement of physiological parameters, use of appropriate models (transient and permanent focal ischaemia) and reproducibility of data by external laboratories. 4. The present paper examines both failed compounds and disodium 4-[(tert-butylimino) methyl] benzene-1, 3-disulphonate N-oxide (NXY-059), a nitrone radical-trapping agent currently in clinical development. It aims to determine whether these guidelines were met by compounds that have failed and, thus, determine whether following the guidelines, as is being done with NXY-059, will increase the chances of developing efficacious drugs for treating acute ischaemic stroke. 5. It is concluded that we will only achieve the goal of producing a clinically effective neuroprotective agent if the guidelines have been met by the novel compound under investigation.     

Neurotoxicity of glutamate in chick telencephalon neurons: reduction of toxicity by preincubation with carbachol, but not by the endogenous fatty acid amides anandamide and palmitoylethanolamide

Exposure of chick telencephalon neurons in serum-free primary culture to glutamate produced a concentration-dependent cell toxicity as seen by an increase in lactate dehydrogenase (LDH) release that was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine and was reduced by preincubation with the cholinergic agonist carbachol. Preincubation with a threshold concentration of NMDA did not prevent glutamate toxicity, suggesting that chick NMDA receptors do not desensitize in the manner reported for their rodent counterparts. Neither anandamide (arachidonyl ethanolamide, AEA) nor palmitoylethanolamide (PEA) was able to prevent the neurotoxicity produced by prolonged glutamate incubation, even under conditions in which the metabolism of the compounds by fatty acid amide hydrolase or AEA cellular uptake was blocked. It is concluded that treatments reported as granting neuroprotection towards glutamate toxicity in rodent primary neuronal cultures do not necessarily show the same properties in the chick. Received: 15 November 1999 / Accepted: 2 February 2000     

GABA mediated excitation in immature rat CA3 hippocampal neurons

Intracellular recordings from rat hippocampal neurons in vitro during the first postnatal week revealed the presence of spontaneous giant depolarizing potentials (GDPs). These were generated by the synchronous discharge of a population of neurons. GDPs reversed polarity at -27 and -51 mV when recorded with KCl or K-methylsulphate filled electrodes, respectively. GDPs were blocked by the GABAA receptor antagonist bicuculline (10 microM). Iontophoretic or bath applications of GABA (10-300 microM) in the presence of tetrodotoxin (1 microM), induced a membrane depolarization or in voltage clamp experiments an inward current which reversed polarity at the same potential as GDPs. The response to GABA was blocked in a non-competitive manner by bicuculline (10 microM) and did not desensitize. GABA mediated GDPs were presynaptically modulated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Their frequency was reduced or blocked by NMDA receptor antagonists and by the rather specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The frequency of GDPs was enhanced by glycine and D-serine (10-30 microM) in a strychnine insensitive manner. This effect was blocked by AP-5, suggesting that it was mediated by the allosteric modulatory site of the NMDA receptor. These observations suggest that most of the 'excitatory' drive in immature neurons is mediated by GABA acting on GABAA receptors; furthermore excitatory amino acids modulate the release of GABA by a presynaptic action on GABAergic interneurons.     

Activation of ionotropic glutamate receptors reduces the production of transforming growth factor-beta2 by developing neurons

Neuronal cultures derived from developing rat cerebral cortex were used to investigate the influence of glutamate receptors on the neuronal production of transforming growth factor-beta2 (TGFbeta2), a multifunctional cytokine that modulates neuronal and glial growth. Long-term exposure (48 h) of cortical neurons to selective antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors markedly increased TGFbeta2 levels in the culture medium. Conversely, treatment with NMDA or kainate reduced TGFbeta2 to levels below those in untreated cultures. The effect of kainate did not require NMDA receptor activity. Neuronal depolarization with K+ also reduced TGFbeta2 levels by opening voltage-gated L-type Ca2+ channels. Semi-quantitative RT-PCR measurements of neuronal TGFbeta2 mRNA showed that NMDA or AMPA/kainate receptor stimulation reduced TGFbeta2 mRNA levels. These results demonstrate that tonic activation of glutamate-gated cation channels downregulates neuronal expression of the TGFbeta2 gene and provide evidence for a novel mechanism whereby excitatory amino acids could influence the development of glial and neuronal lineages.     

The induction and modification of voltage-sensitive responses in cat neocortical nuerons by N-methyl-d-aspartate

The actions of the excitatory amino acid, N-methyl-D-aspartate (NMDA), on layer V neurons of cat sensorimotor cortex were examined in an in vitro slice preparation using current clamp, single electrode voltage clamp (SEVC), and ionic substitution techniques. Low doses of NMDA evoked a slow depolarization with a net decrease of input conductance. Larger doses additionally evoked repetitive firing, rhythmic depolarization shifts (DSs), low-threshold calcium spikes (in the presence of TEA+) and bistable membrane potential behavior. Ionic substitution experiments suggested that entry of both Ca2+ and Na+ ions contributed to the NMDA responses. Attention was focused on the NMDA response with Ca2+ entry blocked. Examination by SEVC revealed that, in both normal cells and in the presence of several blocking agents, NMDA induced a highly voltage-dependent inward ionic current which could result in a region of negative slope conductance on the cell's current-voltage relation. The development of this current seems capable of accounting for all aspects of the observed response, including the DSs and low-threshold Ca2+ spikes. Substitution of TEA+ for most external Na+ (with Ca2+ entry blocked) largely eliminated the NMDA responses and corresponding ionic current. Our results in neocortical neurons are compared to those recently obtained in cultured murine neurons.     

丙泊酚麻醉对孕中期大鼠子代学习记忆功能及HDAC2-CREB-NR2B信号通路的影响

目的 探讨孕中期大鼠经丙泊酚麻醉对子代大鼠学习与记忆功能的影响,以及对组蛋白去乙酰化酶2(HDAC2)-环磷腺苷效应元件结合蛋白(CREB)-天冬氨酸受体亚型(NR2B)信号通路的影响.方法 取36只妊娠14 d的SD大鼠,随机分为对照组和丙泊酚组,各18只,分别于颈静脉注射生理盐水(2 mL/kg)和1.5％丙泊酚(...