Neuronal nitric oxide synthase (nNOS) mRNA expression and NADPH-diaphorase staining in the frontal cortex, visual cortex and hippocampus of control and Alzheimer's disease brains

Abstract

Neuronal nitric oxide synthase (nNOS) mRNA levels and NADPH diaphorase (NADPH-d) staining were compared in the frontal cortex, visual cortex and hippocampus (dentate gyrus and CA subfields of Ammon's horn) of five Alzheimer's disease (AD) and six control brains. The cellular abundance of nNOS mRNA was quantified by in-situ hybridisation using ³⁵S-labelled antisense oligonucleotides complementary to the human nNOS sequence. Although the mean level of nNOS expression was decreased in all three regions in AD cases as compared to controls, it did not reach significance. Neurones positively labelled for nNOS mRNA and neurones positive for NADPH-d histochemistry displayed similar distributions in control and AD cases. In AD brains the density of neurones having detectable levels of nNOS mRNA was significantly decreased in the white matter underlying the frontal cortex (P < 0.05) but not in the frontal cortex gray matter; no change was observed in the gray or white matter of the visual cortex in AD. The number of cells expressing detectable levels of nNOS mRNA in the hippocampus was also significantly decreased (P < 0.05) in AD. The density of NADPH-d-positive cells was not significantly decreased in the gray or white matter of the frontal or visual cortices in AD compared to controls; however, the number of NADPH-d-positive cells was significantly decreased in the hippocampus (P < 0.01). These data indicate that although the cellular abundance of nNOS mRNA is not significantly decreased in these three regions in AD, there is significant decrease in the number of cells expressing detectable levels of nNOS mRNA in the white matter underlying the frontal cortex and in the dentate gyrus and CA subfields of the hippocampus in AD. Futhermore, there was also a significant decrease in the number of NADPH-d-positive cells in the dentate gyrus and CA subfields of the hippocampus in AD as compared to controls. These results suggest specific populations of nNOS/NADPH-d cells in the white matter underlying the frontal cortex and in the hippocampus are vulnerable in AD. The implications of these findings are discussed.

Introduction

Alzheimers disease (AD) is characterised by the presence of neurofibrillary tangles, senile plaques and neuronal degeneration. The parts of the brain that are most vulnerable include the hippocampus, parahippocampal gyrus, entorhinal cortex and the amygdaloid nuclei. The parahippocampal gyrus receives connections originating in the association cortex of the temporal, parietal and frontal lobes and these areas of cortex are also severely affected in AD. The neuronal isoform of the enzyme nitric oxide synthase (nNOS) and NADPH-diaphorase (NADPH-d, a histochemical marker for NOS-containing cells) 24, 16, 33have been shown to co-localize with somatostatin (SOM) immunoreactivity and neuropeptide Y (NPY) immunoreactivity in several forebrain regions in the rat and human brain including the cerebral cortex, neostriatum and amygdala 45, 46, 28, 43, 19, 39. In the cerebral cortex nNOS/NADPH-d, NPY and SOM are reported to have greater than 90% co-localisation in non-pyramidal neurones [27]and they are estimated to constitute approximately 2% of all neurones in the cerebral cortex [44]. The activation of the enzyme nNOS is dependent on calcium concentration and a number of other co-factors [6]; once activated nNOS converts the substrate l-arginine to l-citrulline and nitric oxide (NO). NO has been implicated in excitotoxicity. NO released by nNOS-containing neurones has been shown to mediate the neurotoxicity induced by NMDA in cultured cerebral cortical neurones [17]. Although neurones containing nNOS/NADPH-d are reported to be relatively resistant to degeneration in Huntington's disease (HD) [20], in AD [25], ischaemia and neurotoxin-induced insults 3, 42, 12, NADPH-d-containing neurones have been shown to undergo dystrophic changes in the cerebral cortex and the hippocampal formation in patients with AD 27, 25, 43, 31. In a recent study on HD we found that the abundance of nNOS mRNA is decreased in the striatum, particularly in the dorsal regions of the striatum which usually show more advanced pathology [34].

The reported sparing of NADPH-d neurones in AD and the potential involvement of NO in the pathology of this disease led us to investigate whether nNOS gene expression is altered in the frontal cortex, and hippocampus (regions which are particularly vulnerable to pathological changes in AD) relative to an area that is less damaged, the visual cortex, in AD; we also examined the distribution of cells positive for nNOS gene expression in these regions in control and AD brains. For comparative purposes consecutive sections from each region have also been processed for NADPH-d histochemistry, a marker for NOS-containing cells.