Adam Doble

The Role of Excitotoxicity in Neurodegenerative Disease: Implications for Therapy

Glutamic acid is the principal excitatory neurotransmitter in the mammalian central nervous system. Glutamic acid binds to a variety of excitatory amino acid receptors, which are ligand-gated ion channels. It is activation of these receptors that leads to depolarisation and neuronal excitation. In normal synaptic functioning, activation of excitatory amino acid receptors is transitory. However, if, for any reason, receptor activation becomes excessive or prolonged, the target neurones become damaged and eventually die. This process of neuronal death is called excitotoxicity and appears to involve sustained elevations of intracellular calcium levels. Impairment of neuronal energy metabolism may sensitise neurones to excitotoxic cell death. The principle of excitotoxicity has been well-established experimentally, both in in vitro systems and in vivo, following administration of excitatory amino acids into the nervous system. A role for excitotoxicity in the aetiology or progression of several human neurodegenerative diseases has been proposed, which has stimulated much research recently. This has led to the hope that compounds that interfere with glutamatergic neurotransmission may be of clinical benefit in treating such diseases. However, except in the case of a few very rare conditions, direct evidence for a pathogenic role for excitotoxicity in neurological disease is missing. Much attention has been directed at obtaining evidence for a role for excitotoxicity in the neurological sequelae of stroke, and there now seems to be little doubt that such a process is indeed a determining factor in the extent of the lesions observed. Several clinical trials have evaluated the potential of antiglutamate drugs to improve outcome following acute ischaemic stroke, but to date, the results of these have been disappointing. In amyotrophic lateral sclerosis, neurolathyrism, and human immunodeficiency virus dementia complex, several lines of circumstantial evidence suggest that excitotoxicity may contribute to the pathogenic process. An antiglutamate drug, riluzole, recently has been shown to provide some therapeutic benefit in the treatment of amyotrophic lateral sclerosis. Parkinsons disease and Huntingtons disease are examples of neurodegenerative diseases where mitochondrial dysfunction may sensitise specific populations of neurones to excitotoxicity from synaptic glutamic acid. The first clinical trials aimed at providing neuroprotection with antiglutamate drugs are currently in progress for these two diseases.

Possible involvement of nitric oxide in long-term potentiation

Long-term potentiation (LTP) in the hippocampus is known to involve NMDA (N-methyl-D-aspartate) receptors. Since activation of NMDA receptors in the cerebellum results in the formation of nitric oxide (NO), we studied the possible involvement of this messenger in hippocampal synaptic plasticity. We report here that the NO-synthase inhibitor, L-N omega-nitro-arginine, blocks LTP and that sodium nitroprusside, which releases NO, produces a long-lasting enhancement in synaptic efficacy which is not additive with tetanus-induced LTP.

Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals.

The action of gamma-aminobutyric acid (GABA) and related compounds on rat isolated atria and mouse and guinea pig isolated vas deferens has been studied. GABA depressed the evoked but not basal release of [3H]noradrenaline from atria (IC50 4 micro M) and reduced the twitch responses of the vas deferens (IC50 3 micro M) in a dose-dependent manner. These depressant effects were not prevented by recognized GABA antagonists such as bicuculline and picrotoxin. Numerous GABA analogues, in particular 3-aminopropanesulphonic acid, failed to mimic the action of GABA. However, beta-p-chlorophenyl GABA (baclofen) was stereospecifically active. Other related beta-substituted derivatives were also active but to a lesser degree than GABA. Pretreatment of the vas deferens with the neuronal GABA uptake inhibitors 2,4-diaminobutyric acid or cis-3-aminocyclohexanecarboxylic acid potentiated the action of GABA. These data suggest the presence of a bicuculline-insensitive GABA receptor on autonomic nerve terminals. Preliminary observations indicate a lack of chloride ion dependence in the action of GABA at this site.

Multiple benzodiazepine receptors: no reason for anxiety

Since the introduction of the benzodiazepines into clinical practice in 1960, these drugs have been widely employed as anxiolytics, sedative/hypnotics and anticonvulsants. In recent years, concern has been expressed about their side-effects, and their use has declined. During this latter period many advances have been made in understanding the molecular mechanisms by which these drugs produce their effects. Adam Doble and Ian Martin review this progress and highlight the possibilities that these advances may hold for the development of more efficacious and specific medicines.

Inhibition by riluzole of electrophysiological responses mediated by rat kainate and NMDA receptors expressed in Xenopus oocytes

The effects of riluzole, an anticonvulsant and neuroprotective compound, on excitatory amino acid-evoked currents were studied in Xenopus laevis oocytes injected with mRNA from rat whole brain or cortex. Responses to kainic acid were blocked by riluzole (IC50 = 167 microM) as well as by the quinoxalinedione antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX: IC50 = 0.21 microM) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX: IC50 = 0.043 microM). Riluzole was somewhat more potent at blocking responses to N-methyl-D-aspartic acid (NMDA: IC50 = 18.2 microM); the competitive NMDA receptor antagonist 2-amino-phosphonovaleric acid (2-APV) yielded an IC50 of 6.1 microM in this system. The inhibition by both riluzole and 2-APV was reversible and did not appear to be use dependent, unlike that of the channel blocker MK-801 ([+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate). It was impossible to demonstrate an interaction of riluzole with any of the known ligand recognition sites on either the kainate or the NMDA receptor in radioligand binding studies. These results suggest a direct but non-competitive action of riluzole on ionotropic glutamate receptors.

Riluzole in Huntington's disease: a 3‐year, randomized controlled study

We conducted a randomized double‐blind trial of riluzole in Huntingtons disease to investigate the efficacy of this antiexcitotoxic drug in slowing disease progression.

A Role for Nitric Oxide in Long‐term Potentiation

Nitric oxide production in the cerebellum and induction of long‐term potentiation (LTP) in the hippocampus have some characteristics in common: both phenomena are induced by activation of N‐methyl‐D‐aspartate receptors and both are highly dependent on calcium‐mediated processes. Here we provide evidence that endogenous nitric oxide production is necessary for synaptic plasticity in the CA1 hippocampus of the rat. LTP recorded in slices was blocked in a concentration‐dependent manner by the nitric oxide synthase inhibitors L‐NG‐nitroarginine and L‐NG‐nitroarginine methyl ester, but L‐NG‐monomethylarginine was only marginally active. Bathing the slices with haemoglobin, a protein that scavenges nitric oxide, also resulted in a concentration‐dependent blockade of LTP. Nitric oxide released locally from hydroxylamine produced a stable potentiation of synaptic transmission that was not additive with LTP induced by high‐frequency stimulation. These results are fully consistent with the presumed retrograde messenger role of nitric oxide in LTP.

Electrophysiological and pharmacological evidence that peripheral type benzodiazepine receptors are coupled to calcium channels in the heart.

PK 11195, an antagonist of the peripheral type benzodiazepine receptor, does not affect either the duration of the action potential or the tension of the guinea pig papillary muscle. However, it antagonized the effects of the calcium channel blockers, nitrendipine, verapamil, diltiazem, and of BAY K8644, a calcium channel agonist in this heart preparation. On the other hand, PK 11195 does not change the increase in the action potential duration provoked by the potassium channel blocker tetraethylammonium. RO5-4864, an agonist of the peripheral type benzodiazepine receptor, decreased the tension of the guinea pig papillary muscle. The effect was reversed by increasing extracellular Ca2+ concentrations up to 4 mM. These results suggest that in the heart the peripheral type benzodiazepine receptors are coupled to calcium channels.

Antagonism by riluzole of entry of calcium evoked by NMDA and veratridine in rat cultured granule cells: evidence for a dual mechanism of action

1 Intracellular calcium levels were measured in cultured cerebellar granule cells of the rat by use of the fluorescent dye, indo‐1/AM. 2 Intracellular calcium levels were increased by depolarizing stimuli such as N‐methyl‐d‐aspartate (NMDA) (100 μm), glutamic acid (20 μm), and veratridine (10 μm). This increase was essentially due to entry of external calcium. 3 Riluzole (10 μm) blocked responses to all the depolarizing agents. 4 Riluzole could still block the increase in intracellular calcium evoked by NMDA or glutamic acid when sodium channels were blocked by tetrodotoxin, suggesting that this effect is not mediated by a direct action of riluzole on the voltage‐dependent sodium channel. 5 Pretreatment of the cells with pertussis toxin (0.1 μg ml−1) did not modify the increases in intracellular calcium evoked by NMDA, glutamic acid or veratridine. 6 In pertussis toxin‐treated cells, riluzole could no longer block responses to excitatory amino acids, but still blocked responses to veratridine. 7 It is concluded that riluzole has a dual action on cerebellar granule cells, both blocking voltage‐dependent sodium channels and interfering with NMDA receptor‐mediated responses via a pertussis toxin‐sensitive mechanism. Furthermore, these two processes have been shown to be independent.

Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PK 11195: Anatomical and subcellular distribution

The peripheral-type benzodiazepine binding site, erstwhile characterized in the rodent and feline brain, has now been characterized in post-mortem human brain using [3H]PK 11195. The kinetics and pharmacological properties of the binding of this ligand are similar to peripheral-type benzodiazepine binding sites elsewhere. The potency of RO5-4864 for this site in human brain is close to that seen in ruminant and carnivore tissues but considerably lower than in rodent tissues. The regional distribution of these binding sites would suggest a neuronal rather than a glial localization. [3H]PK 11195 bound in a similar fashion to slide-mounted sections of human brain, thus allowing quantitative studies of the regional distribution of peripheral-type benzodiazepine binding sites to be made. The binding sites were distributed heterogeneously, but were restricted to the grey matter. Highest densities of binding sites were found in forebrain structures. The localization was not limited to any functional system, nor did it resemble any previously described transmitter system. The similarities between peripheral-type benzodiazepine binding sites in human and in feline brain in terms of their pharmacological characteristics and their regional and subcellular distribution suggest that the cat, rather than the rat, may be the better model for studying a possible role for this site in human cerebral function.