F. Nicoletti

Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders?

Metabotropic glutamate (mGlu) receptors are a large, heterogeneous family of G-protein coupled receptors, which modulate excitatory synaptic transmission through various transduction pathways. Evidence is now accumulating that individual mGlu-receptor subtypes mediate distinct, facilitatory (group I subtypes) or inhibitory (group II and group III subtypes), actions on neurodegenerative processes. Drugs interacting with mGlu receptors are expected to influence both the induction and progression of neuronal degeneration without hampering the efficiency of fast excitatory synaptic transmission. For these reasons, mGlu receptors can be considered as promising drug targets in the experimental therapy of acute or chronic neurodegenerative diseases.

Mitotic signaling by β-amyloid causes neuronal death

Aggregates of β‐amyloid peptide (βAP), the main constituent of amyloid plaques in Alzheimers brain, kill neurons by a not yet defined mechanism, leading to apoptotic death. Here, we report that both full‐length βAP(j_40) or (1–42) and its active fragment βAP(25_35) act as proliferative signals for differentiated cortical neurons, driving them into the cell cycle. The cycle followed some of the steps observed in proliferating cells, including induction of cyclin Di, phosphorylation of retinoblastoma, and induction of cyclin E and A, but did not progress beyond S phase. Inactivation of cyclin‐dependent protein kinase‐4 or ‐2 prevented both the entry into S phase and the development of apoptosis in βAP(25_35)‐treated neurons. We conclude that neurons must cross the G1/S transition before succumbing to βAP signaling, and therefore multiple steps within this pathway may be targets for neuroprotective agents.—Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Giuffrida Stella, A. M., Canonico, P. L., Nicoletti, F., Sortino, M. A. Mitotic signaling by β ‐amyloid causes neuronal death. FASEB J. 13, 2225–2234 (1999)

Activation of A1 adenosine or mGlu3 metabotropic glutamate receptors enhances the release of nerve growth factor and S-100β protein from cultured astrocytes

Pharmacological activation of A1 adenosine receptor with 2‐chloro‐N6‐cyclopentyladenosine (CCPA) or mGlu3 metabotropic glutamate receptors with (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV) or aminopyrrolidine‐2R,4R‐dicarboxylate (2R,4R‐APDC) enhanced the release of nerve growth factor (NGF) or S‐100β protein from rat cultured astrocytes. Stimulation of release by CCPA and DCG‐IV or 2R,4R‐APDC was inhibited by the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine and by the mGlu2/3 receptor antagonist (2S,1′S,2′S,3′R)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine (PCCG‐4), respectively. Time‐course studies revealed a profound difference between the release of S‐100β protein and the release of NGF in response to extracellular signals. Stimulation of S‐100β protein exhibited rapid kinetics, peaking after 1 h of drug treatment, whereas the enhancement of NGF release was much slower, requiring at least 6 h of A1 adenosine or mGlu3 receptor activation. In addition, stimulation of NGF but not S‐100β release was substantially reduced in cultures treated with the protein synthesis inhibitor cycloheximide. In addition, a 6–8 h treatment of cultured astrocytes with A1 or mGlu3 receptor agonists increased the levels of both NGF mRNA and NGF‐like immunoreactive proteins, including NGF prohormone. We conclude that activation of A1 adenosine or mGlu3 receptors produces pleiotropic effects in astrocytes, stimulating the synthesis and/or the release of protein factors. Astrocytes may therefore become targets for drugs that stimulate the local production of neurotrophic factors in the CNS, and this may provide the basis for a novel therapeutic strategy in chronic neurodegenerative disorders. GLIA 27:275–281, 1999.

Neuroprotective activity of the potent and selective mGlu1a metabotropic glutamate receptor antagonist, (+)-2-methyl-4 carboxyphenylglycine (LY367385) : comparison with LY357366, a broader spectrum antagonist with equal affinity for mGlu1a and mGlu5 receptors

(+)-2-Methyl-4-carboxyphenylglycine (LY367385), a potent and selective antagonist of mGlu1a metabotropic glutamate receptors, was neuroprotective in the following in vitro and in vivo models of excitotoxic death: (i) mixed cultures of murine cortical cells transiently exposed to N-methyl-D-aspartate (NMDA); (ii) rats monolaterally infused with NMDA into the caudate nucleus; and (iii) gerbils subjected to transient global ischemia. We have compared the activity of LY367385 with that of the novel compound (+/-)-alpha-thioxantylmethyl-4-carboxyphenylglycine (LY367366), which antagonizes both mGlu1a and -5 receptors at low micromolar concentrations, but also recruits other subtypes at higher concentrations. Although LY367366 was neuroprotective, it was in general less efficacious than LY357385, suggesting that inhibition of mGlu1 receptors is sufficient to confer significant neuroprotection. We conclude that endogenous activation of mGlu1a receptors (or perhaps other mGlu1 receptor splice variants) contributes to the development of neuronal degeneration of excitotoxic origin.

Ipsilateral Motor Responses to Focal Transcranial Magnetic Stimulation in Healthy Subjects and Acute-Stroke Patients

Background and Purpose— Prevalence and characteristics of ipsilateral upper limb motor-evoked potentials (MEPs) elicited by focal transcranial magnetic stimulation (TMS) were compared in healthy subjects and patients with acute stroke. Methods— Sixteen healthy subjects and 25 patients with acute stroke underwent focal TMS at maximum stimulator output over motor and premotor cortices. If present, MEPs evoked in muscles ipsilateral to TMS were analyzed for latency, amplitude, shape, and center of gravity (ie, preferential coil location to elicit them). In stroke patients, possible relationships between early ipsilateral responses and functional outcome at 6 months were sought. Results— With relaxed or slightly contracting target muscle, maximal TMS over the motor cortex failed to elicit ipsilateral MEPs in the first dorsal interosseous (FDI) or biceps of any of 16 normal subjects. In 5 of 8 healthy subjects tested, ipsilateral MEPs with latencies longer than contralateral MEPs were evoked in FDI muscle (in biceps, 6 of 8 subjects) during strong (>50% maximum) contraction of the target muscle. In 15 of 25 stroke patients, ipsilateral MEPs in the unaffected relaxed FDI (in biceps, 6 of 25 stroke patients) were evoked by stimulation of premotor areas of the affected hemisphere. Their latencies were shorter than those that MEPs evoked in the same muscle by stimulation of the motor cortex of the contralateral unaffected hemisphere. Such responses were never obtained in normal subjects and were mostly observed in patients with subcortical infarcts. Patients harboring these responses had slightly better bimanual dexterity after 6 months. Conclusions— Ipsilateral MEPs obtained in healthy individuals and stroke patients have different characteristics and probably different origins. In the former, they are probably conveyed via corticoreticulospinal or corticopropriospinal pathways, whereas in the latter, early ipsilateral MEPs could originate in hyperexcitable premotor areas.

Desensitization of metabotropic glutamate receptors in neuronal cultures

Abstract: Preexposure of cultured cerebellar neurons to glutamate reduced the stimulation of polyphosphoinositide (PPI) hydrolysis induced by subsequent addition of glutamate without affecting the response to the muscarinic receptor agonist carbamylcholine. Desensitization of glutamate‐stimulated PPI hydrolysis developed rapidly and persisted up to 48 h after removal of glutamate from the incubation medium. Stimulation of PPI hydrolysis by quisqualate was abolished in cultures pretreated with quisqualate or glutamate, but not with N‐methyl‐D‐aspartate (NMDA). In contrast, pretreatment with NMDA reduced the stimulation of PPI hydrolysis induced by a subsequent addition of NMDA, leaving the action of quisqualate intact. The lack of cross‐desensitization between NMDA and quisqualate supports the existence of two distinct subtypes of glutamate receptors coupled to PPI hydrolysis. Desensitization induced by a 30‐min (but not by a 6‐h) exposure to glutamate was attenuated or prevented by putative protein kinase C inhibitors, including mono‐ and trisialogangliosides, sphingosine, and polymyxin B, but not by inhibitors of arachidonic acid metabolism, nor by the nonselective calpain inhibitor leupeptin, nor by the lectin concanavalin A. These results suggest that desensitization of metabotropic glutamate receptors involves, at least in its rapid component, activation of protein kinase C.

Excitatory Amino Acids Stimulate Inositol Phospholipid Hydrolysis and Reduce Proliferation in Cultured Astrocytes

Abstract: Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate ([3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12‐h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl‐3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12‐tetradecanoate 13‐acetate. Glutamate and ibotenate reduced [methyl‐3H]‐thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2‐amino‐4‐phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective. Inhibition of [methyl‐3H]thymidine incorporation by quisqualate required a lag time of about 4 h and, in cells synchronized to proliferate, occurred when the drug was added during the transition between G0 and G1, but not during the S phase of the mitotic cycle. This suggests that an inducible factor may be involved in the antiproliferative effect of excitatory amino acids. Accordingly, activation of quisqualate receptors led to a rapid and transient increase in mRNA levels of the early inducible gene, c‐fos. These results suggest that activation of a specific class of “quisqualate‐preferring”excitatory amino acid receptors reduces proliferation of astrocytes in primary cultures.

Nootropic Drugs Positively Modulate α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-Sensitive Glutamate Receptors in Neuronal Cultures

Abstract: Micromolar concentrations of piracetam, aniracetam, and oxiracetam enhanced α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐stimulated 45Ca2+ influx in primary cultures of cerebellar granule cells. Nootropic drugs increased the efficacy but not the potency of AMPA and their action persisted in the presence of the voltage‐sensitive calcium channel blocker nifedipine. Potentiation by oxiracetam was specific for AMPA receptor‐mediated signal transduction, as the drug changed neither the stimulation of 45Ca2+ influx by kainate or N‐methyl‐d‐aspartate nor the activation of inositol phospholipid hydrolysis elicited by quisqualate or (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid. Piracetam, aniracetam, and oxiracetam increased the maximal density of the specific binding sites for [3H]AMPA in synaptic membranes from rat cerebral cortex. Taken collectively, these results support the view that nootropic drugs act as positive modulators of AMPA‐sensitive glutamate receptors in neurons.

Stimulation of inositol phospholipid hydrolysis by excitatory amino acids is enhanced in brain slices from vulnerable regions after transient global ischemia

Abstract: Stimulation of inositol phospholipid hydrolysis by transmitter receptor agonists was measured in slices from hippocampus, cerebral cortex, and corpus striatum at various intervals after transient global ischemia in rats. Ischemia was induced through the four‐vessel occlusion model. Stimulation of [3H]inositol monophosphate formation by excitatory amino acids was greatly enhanced in hippocampal slices prepared from ischemic rats at 24 h or 7 days after reperfusion. This potentiation was more evident using ibotenic acid and was also observed in cerebral cortex, but not in corpus striatum. This regional profile correlated with the pattern of ischemia‐induced neuronal damage observed under our experimental conditions. The enhanced responsiveness to excitatory amino acids was always accompanied by an increase in both basal and norepinephrine‐stimulated [3H]inositol monophosphate formation. In contrast, stimulation of [3H]inositol monophosphate formation by carbamylcholine was not modified in hippocampal or cortical slices from ischemic animals

The SSRI, citalopram, improves bradykinesia in patients with Parkinson's disease treated with L-dopa.

Idiopathic Parkinsons disease (IPD) is characterized by motor signs such as akinesia, rigidity, and often tremor at rest. In addition to these symptoms, depression is a common finding affecting 40% of patients with IPD. This study evaluates the effect of the selective serotonin reuptake inhibitor, citalopram, on motor and nonmotor symptoms of depressed and nondepressed patients with IPD. Forty-six nondemented patients with IPD (24 men, 22 women; mean age 64 ± 5.3 years; mean ± SD disease duration, 6.4 ± 3.2 years; mean ± SD Hoehn-Yahr stage, 2.8 ± 1.2) were included in the study. Patients were divided in two subgroups: depressed (n = 18) and nondepressed (n = 28). Citalopram was added in an unblinded manner, starting with 10 mg/d, and, after a week, increased up to 20 mg/d in the depressed subgroup (n = 18) and in half of the nondepressed subgroup (n = 14). Parkinsonian and depressive symptoms were evaluated before and after 1 and 4 months of treatment. Statistical evaluation was made by analysis of variance for repeated measures. Citalopram did not worsen motor performance in IPD, but improved bradykinesia and finger taps after 1 month and 4 months of treatment both in patients with and without depression (p < 0.05 versus baseline). A clear improvement in mood was also observed in 15 of 16 patients with depression. Although case reports indicate that citalopram can potentially worsen the motor symptoms in patients with PD, to date this effect has not been confirmed. Many of the symptoms, typically associated with depression, can be observed in nondepressed patients with IPD, because signs thought to represent depression can be produced by Parkinsons disease. In this study, we observed that when combined with levodopa, citalopram induces an improvement of motor performance, in particular of subscores 23 and 31 of Unified Parkinsons Disease Rating Scale both in depressed and in nondepressed patients with IPD.