Molecular characterization of the AMPA-receptor potentiator S70340 in rat primary cortical culture: whole-genome expression profiling.

To improve our understanding of the molecular events underlying the effects of positive allosteric modulators of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (S)-AMPA-type glutamate receptors, gene expression profiles of primary cortical culture were measured by Agilent-Microarray technique under (S)-AMPA (1µM) stimulation for 0.5, 6, 24 and 48h in the presence or absence of S70340 (30µM), an allosteric potentiator of AMPA receptors. (S)-AMPA and S70340 treatment alone have little effect on gene expression whereas as early as 6h, their combination induced a large number of genes known to decrease apoptosis and mediate cell survival. Pathway analyses of (S)-AMPA+S70340 treatment-mediated gene expression from 6 to 48h further suggested the activation of cellular functions including neuron differentiation and neurite outgrowth. A proportion of genes implicated in these functions encode proteins involved in environmental cues and are expressed in growth cones, such as extracellular matrix component proteins and filopodia microfilament-associated proteins. Time course analysis of mRNA expression combined with in silico promoter analysis revealed an enrichment in the cAMP response element (CRE) among co-regulated genes. This study demonstrated that S70340-mediated AMPA potentialisation activated genes and functional processes involved in neuroprotective and cognitive effects and describes putative new functional biomarkers.

S18986: a positive modulator of AMPA-receptors enhances (S)-AMPA-mediated BDNF mRNA and protein expression in rat primary cortical neuronal cultures.

The present study describes the effect of (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on (S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. (S)-AMPA (0.01-300 microM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC(50)=7 microM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (< or =300 microM) failed to increase basal BDNF expression. However, S18986 (300 microM) in the presence of increasing concentrations of (S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3-5-fold). S18986 (100-300 microM) potentiated BDNF mRNA induced by 3 microM (S)-AMPA (2-3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of (S)-AMPA-mediated BDNF expression (40-fold; EC(50)=18 microM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 microM) in the presence of 3 microM (S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 microM (S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl-D-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of (S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca(2+)-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin-Kinase II/IV inhibitor (KN-62, 25 microM) significantly inhibited (-85%, P<0.001) AMPA+S18986 stimulated expression of BDNF mRNA. The present study supports the observations that AMPA receptor allosteric modulators can enhance the expression of BDNF mRNA and protein expression via the AMPA receptor in cultured primary neurones. Consequently, the long-term elevation of endogenous BDNF expression by pharmacological intervention with this class of compounds represents a potentially promising therapeutic approach for behavioural disorders implicating cognitive deficits.

Pleiotrophin mediates the neurotrophic effect of cyclic AMP on dopaminergic neurons: analysis of suppression-subtracted cDNA libraries and confirmation in vitro.

To better understand the particular vulnerability of mesencephalic dopaminergic neurons to toxins or gene mutations causing parkinsonism, we have taken advantage of a primary cell culture system in which these neurons die selectively. Antimitotic agents, such as cytosine arabinoside or cAMP, prevent the death of the neurons by arresting astrocyte proliferation. To identify factors implicated in either the death of the dopaminergic neurons or in the neuroprotective effect of cAMP, we constructed cDNA libraries enriched by subtractive hybridization and suppressive PCR in transcripts that are preferentially expressed in either control or cAMP-treated cultures. Differentially expressed transcripts were identified by hybridization of the enriched cDNAs with a commercially available cDNA expression array. The proteoglycan receptors syndecan-3 and the receptor protein tyrosine phosphatase zeta/beta were found among the transcripts preferentially expressed under control conditions, and their ligand, the cytokine pleiotrophin, was highly represented in the cDNA libraries for both conditions. Since pleiotrophin is expressed during embryonic and perinatal neural development and following lesions in the adult brain, we investigated its role in our cell culture model. Pleiotrophin was not responsible for the death of dopaminergic neurons under control conditions, or for their survival in cAMP-treated cultures. It was, however, implicated in the initial and cAMP-dependent enhancement of the differentiation of the dopaminergic neurons in our cultures. In addition, our experiments have provided evidence for a cAMP-dependent regulatory pathway leading to protease activation, and the identification of pleiotrophin as a target of this pathway.

Differential gene expression induced by chronic levodopa treatment in the striatum of rats with lesions of the nigrostriatal system.

Levodopa, the major treatment for patients with Parkinson's disease, has been shown to induce a variety of compensatory effects, including facilitation of sprouting by dopaminergic neurons, in experimental animals with lesions leading to denervation of the striatum. To better understand the cellular and molecular environment where most of these compensatory changes take place, in particular elements that might contribute to the recovery of dopaminergic innervation, we have constructed a differential expression library enriched in transcripts from the striata of rats with lesions of the medial forebrain bundle treated with levodopa for 6 months. We have used this library to screen an expression array of rat genes representing the major cell functions, and have identified several that are involved in neurotrophic mechanisms and plasticity. We have confirmed the differential expression of selected transcripts by non-radioactive in situ hybridization, and report that the growth factor pleiotrophin, myelin basic protein and calmodulin are overexpressed in the denervated striatum of levodopa-treated rats.

Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/glial mesencephalic cultures requires the repression of presumptive astrocytes.

Cyclic AMP-elevating agents are highly effective in preventing the loss of dopaminergic neurons that occurs spontaneously in neuronal-glial mesencephalic cultures. We demonstrate here that cAMP causes a concomitant decline in the number of dividing non-neuronal cells, suggesting that inhibition of proliferation contributes to neuroprotection. Consistent with this hypothesis, a transient treatment with the antimitotic cytosine arabinoside, at concentrations that induce long-term repression of glial cell proliferation, mimicked the neuroprotective action of cAMP and also obviated the need for the cyclic nucleotide. Treatment with cAMP-elevating agents reduced the population of OX-42-positive microglial cells and the number of immature astrocytes expressing vimentin and low levels of the astrocytic marker glial fibrillary acidic protein. The effect on the immature astrocytes, however, seemed essential for neuroprotection. Ciliary neurotrophic factor and leukemia inhibitory factor, which stimulate astrocyte differentiation without reducing cell proliferation, failed to reproduce the protective effects of the cyclic nucleotide. Cyclic AMP did not operate by counteracting the action of the astrocyte mitogen epidermal growth factor or by reducing activation of the mitogen-activated protein kinase signaling pathway. The neuroprotective and antiproliferative actions of cAMP, however, were closely mimicked by olomoucine and roscovitine, potent inhibitors of the cyclin-dependent kinase CDK1 that are structurally related to cAMP. Measurement of CDK1 activity confirmed that neuroprotection was closely correlated with inhibition of this kinase by cAMP. In summary, neuroprotection of mesencephalic dopaminergic neurons by cAMP most probably requires the repression of presumptive astrocytes through inhibition of CDK1.

Activation of the mitogen-activated protein kinase (ERK(1/2)) signaling pathway by cyclic AMP potentiates the neuroprotective effect of the neurotransmitter noradrenaline on dopaminergic neurons.

We have shown previously that low concentrations of noradrenaline (NA) confer long-term but partial protection to tyrosine hydroxylase (TH(+)) dopaminergic neurons by reducing spontaneously occurring oxidative stress. We demonstrate here that the effect of NA is strongly enhanced by cAMP-elevating agents, in particular forskolin (FK), through a mechanism that does not involve activation of adrenoceptors. FK also enhanced the neuroprotective action of antioxidants that mimic the trophic effects of NA, such as trolox and pyrocatechol, but was totally ineffective by itself, suggesting that inhibition of oxidative stress was a required step to reveal the cAMP-dependent mechanism. Neuroprotection afforded by FK was rapidly reversible, optimal when the treatment was initiated in the early phase of the culture and exquisitely specific to dopaminergic neurons. FK stimulated the phosphorylation of extracellular signal-activated kinases (ERK)(1/2) in a subpopulation of dopaminergic neurons, suggesting that the mitogen-activated protein kinase (MAPK) pathway was involved in the effects of cAMP-elevating agents. Accordingly, inhibition of the upstream kinases of ERK(1/2) by 2'-amino-3'-methoxyflavone (PD98059) not only suppressed MAPK activation caused by FK but also abolished the survival promoting activity that this compound exerts on TH(+) neurons. PD98059 did not reduce, however, the trophic effects provided by NA alone. Surprisingly, the archetypal cAMP-dependent protein kinase was apparently not responsible for ERK(1/2) activation. The data suggest that the MAPK signaling pathway plays a key role in the trophic effects that cAMP elevating agents and NA cooperatively exert on TH(+) neurons.