Differential susceptibility to neurotoxicity mediated by neurotrophins and neuronal nitric oxide synthase

Abstract

NMDA neurotoxicity, which is mediated, in part, by formation of nitric oxide (NO) via activation of neuronal NO synthase (nNOS), is modulated by neurotrophins. nNOS expression in rat and mouse primary neuronal cultures grown on a glial feeder layer is significantly less than that of neurons grown on a polyornithine (Poly-O) matrix. Neurotrophins markedly increase the number of nNOS neurons, nNOS protein, and NOS catalytic activity and enhance NMDA neurotoxicity via NO-dependent mechanisms when neurons are grown on glial feeder layers. In contrast, when rat or mouse primary cortical neurons are grown on a Poly-O matrix, neurotrophins have no influence on nNOS neuronal number or NOS catalytic activity and reduce NMDA neurotoxicity. Primary neuronal cultures from mice lacking nNOS grown on a glial feeder layer fail to respond to neurotrophin-mediated enhancement of neurotoxicity. Together, these results indicate that nNOS expression and NMDA NO-mediated neurotoxicity are dependent, in part, on the culture paradigm, and neurotrophins regulate the susceptibility to NMDA neurotoxicity via modulation of nNOS. Furthermore, these results support the idea that NMDA neurotoxicity in culture is critically dependent on the developmental state of the neurons being assessed and suggest that, when cortical neurons are cultured on a glial feeder layer, they do not reach nearly as mature a phenotype as when grown on a Poly-O matrix.

Fig. 1.

Culture-dependent regulation of nNOS expression. Shown are Hoffman modulation photomicrographs of cortical neurons stained with NADPH–diaphorase depicting neuronal nitric oxide synthase (nNOS) neurons 24 hr after treatment with brain-derived neurotrophic factor (BDNF; 100 ng/ml). A,B, Control cultures grown either on a glial layer or a polyornithine (Poly-O Matrix) matrix, respectively.C, D, Cultures that were treated for 24 hr with 100 ng/ml BDNF. Neurons cultured on a glial layer express negligible levels of nNOS neurons, the expression of which is increased markedly with BDNF treatment (A, C). Neurons cultured on a Poly-O matrix have a relatively higher expression of nNOS neurons (arrowheads), which is unchanged with BDNF treatment (B, D).

Fig. 2.

Neurotrophins enhance nNOS expression in cortical neurons grown on a glial layer. A, Pretreatment (24 hr) with neurotrophins (100 ng/ml) markedly enhances the number of nNOS neurons, as indicated by NADPH–diaphorase staining of cortical neurons. Each data point represents the mean ± SEM (n = 8–12) of at least two separate experiments. *p < 0.0001 for NT-3, NT-4, GDNF, and BDNF, as compared with control cultures.NGF is not significantly different from control.B, Neurotrophins increase the amount ofnNOS protein, as measured by Western blot analysis.C, A parallel increase in NOS catalytic activity is observed. ^†p < 0.02 for NT-3, NT-4, GDNF, and BDNF, as compared with control cultures. Each data point represents the mean ± SEM of at least two separate experiments performed in duplicate.

Fig. 3.

Neurotrophins do not enhance expression of nNOS in cortical neurons grown on a polyornithine (Poly-O) matrix.A, Cortical neurons grown on a Poly-O matrix express a significantly higher quantity of nNOS neurons, as indicated by NADPH–diaphorase staining, than cortical neurons grown on a glial layer. Pretreatment (24 hr) with BDNF does not influence the total number of nNOS neurons. Each data point represents the mean ± SEM (n = 8–12) of at least two separate experiments. B, BDNF increases the amount of nNOS protein, as measured by Western blot analysis; however, (C) nNOS catalytic activity remains unchanged. Each data point represents the mean ± SEM of at least two separate experiments performed in duplicate. The number of NADPH–diaphorase neurons and NOS catalytic activity in BDNF-treated cultures is not significantly different from controls.

Fig. 4.

Increases in nNOS levels mediate potentiation of NMDA neurotoxicity by neurotrophins. A, Rat cortical neurons grown on a glial layer exhibit NMDA-mediated (NMDA; 500 μm) neurotoxicity, which is not influenced by the competitive nNOS inhibitor nitroarginine–methyl ester (LNAME; 500 μm) or by excessl-arginine (LARG; 5 mm). Pretreatment (24 hr) with BDNF (100 ng/ml) markedly enhances NMDA (500 μm) neurotoxicity (^†p < 0.0001), which is prevented with LNAME (500 μm;^§p < 0.0001), and LARG(5 mm; ^‡p < 0.0001) reverses this neuroprotection. B, Cortical neurons grown on a Poly-O matrix demonstrate an equivalent amount ofNMDA neurotoxicity to that observed in cortical neurons grown on a glial layer and pretreated with BDNF (100 ng/ml). This neurotoxicity is also NO-dependent (*p< 0.0001). BDNF treatment of neurons grown on a Poly-O matrix attenuates the neurotoxic response to NMDA (500 μm) by ∼50% (^†p < 0.0001).LNAME (500 μm;^§p < 0.05) provides further protection, and LARG (5 mm;^‡p < 0.0001) reverses this protection. Each data point represents the mean ± SEM (n= 6–12) of at least two separate experiments; a minimum of 4000–10,000 neurons was counted.

Fig. 5.

Culture-independent sensitivity to NO neurotoxicity. Cultures were exposed to increasing concentrations of the NO donor sodium nitroprusside (SNP Concentration). Both neuronal culture systems are equally sensitive to the toxic effects of SNP. SNP depleted of NO by preincubating in culture media for 24 hr has no intrinsic toxicity. Pretreatment (24 hr) withBDNF (100 ng/ml) does not influence SNP toxicity in cortical neurons grown on a glial layer; however, cortical neurons grown on a Poly-O matrix are less susceptible to the toxic effect of 500 μm SNP (*p < 0.001). Each data point represents the mean ± SEM (n = 4–6) of at least two separate experiments; a minimum of 4000–8000 neurons was counted.

Fig. 6.

Increases in nNOS levels mediate potentiation of NMDA neurotoxicity by neurotrophins. A, Murine cortical neurons grown on a glial layer exhibit NMDA-mediated (NMDA; 500 μm) neurotoxicity, which is not influenced by the competitive nNOS inhibitor nitroarginine–methyl ester (LNAME; 500 μm) or by excessl-arginine (LARG; 5 mm). Pretreatment (24 hr) with BDNF (100 ng/ml) markedly enhances NMDA (500 μm) neurotoxicity (^†p < 0.0001), which is prevented with LNAME (500 μm; ^§p < 0.0001), and LARG(5 mm; ^‡p < 0.0001) reverses this neuroprotection. B, Cortical neurons grown on a Poly-O matrix demonstrate an equivalent amount ofNMDA (500 μm) NO-dependent neurotoxicity (*p < 0.0001) to that observed in cortical neurons grown on a glial layer pretreated with BDNF (100 ng/ml).BDNF treatment of neurons grown on a Poly-O matrix attenuates the neurotoxic response to NMDA (500 μm) by ∼50% (^†p < 0.0001),LNAME (500 μm;^§p < 0.01) provides further protection, and LARG (5 mm;^‡p < 0.0001) reverses this protection. Each data point represents the mean ± SEM (n= 6–12) of at least two separate experiments; a minimum of 4000–10,000 neurons was counted.

Fig. 7.

Neurotrophin-mediated enhancement of NMDA neurotoxicity is prevented in nNOS⁻ mice.A, nNOS⁻ cultures are markedly resistant to NMDA neurotoxicity, and BDNF(100 ng/ml) fails to enhance NMDA neurotoxicity in cortical cultures grown on a glial layer. B,BDNF fails to provide neuroprotection to cortical neurons grown on a Poly-O matrix. Each data point represents the mean ± SEM (n = 6–12) of at least two separate experiments; a minimum of 4000–10,000 neurons was counted. None of the determinations is statistically different from another.