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Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP+-induced neuronal toxicity

  • Translational Neurosciences - Original Article
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Abstract

Nitrative stress is a key component of the pathogenic process in Parkinson’s disease (PD), but the relative roles of constitutive neuronal nitric oxide synthase (n-NOS) and inducible nitric oxide synthase (i-NOS) in glial cells remain unresolved. We have investigated the effects of a range of concentrations of the selective n-NOS inhibitor ARR17477, and the selective i-NOS inhibitor 1400W, on MPP+-induced cell death in foetal ventral mesencephalic (VM) dopaminergic cultures. MPP+ induced a loss of TH-positive neurones accompanied by an increase in immunoreactivity for GFAP and OX-6 as markers of astrocytes and activated microglia, respectively, and induced i-NOS immunoreactivity. Unexpectedly, MPP+ treatment did not induce 3-NT immunoreactivity in the cultures. ARR17477 and 1400W alone had no effect on the number of TH-positive cells or on the number of GFAP or OX-6 positive cells. ARR17477 did not prevent the MPP+-induced decrease in TH-positive neurones and had no effect on the increased number of GFAP- and OX-6-positive cells. By contrast, 1400W caused a concentration-dependent preservation of TH-positive neurones in the presence of MPP+. It also significantly reduced the number of OX-6-immunoreactive cells and there was a small reduction in GFAP immunoreactivity. The results suggest a major role for i-NOS-mediated nitrative stress in microglia in MPP+-induced dopaminergic cell death and this may have important implications for developing neuroprotective strategies for PD.

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Abbreviations

3-NT:

3-Nitrotyrosine

BSA:

Bovine serum albumin

DAB:

3,3′-Diaminobenzidine tetrahydrochloride

DI:

Deionised

DIV:

Days in vitro

D-MEM:

Dulbecco’s modified Eagle medium

DPX:

Di-n-butylphthalate in xylene

DS:

Donkey serum

GFAP:

Glial fibrillary acidic protein

i-NOS:

Inducible nitric oxide synthase

MPP+ :

4-Methylpyridinium

n-NOS:

Neuronal nitric oxide synthase

OX-6:

Major histocompatibility complex class II

PBS:

Phosphate buffered saline

PD:

Parkinson’s disease

PSN:

Penicillin, streptomycin and neomycin solution

TH:

Tyrosine hydroxylase

VM:

Ventral mesencephalic

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Acknowledgments

This research was funded by Proximagen Neurosciences Ltd., London, UK. There is no actual or potential conflict of interest in relation to this article.

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Correspondence to Peter Jenner.

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Brzozowski, M.J., Jenner, P. & Rose, S. Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP+-induced neuronal toxicity. J Neural Transm 122, 779–788 (2015). https://doi.org/10.1007/s00702-014-1334-8

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  • DOI: https://doi.org/10.1007/s00702-014-1334-8

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