Research reportAberrant expression of nNOS in pyramidal neurons in Alzheimer's disease is highly co-localized with p21ras and p16INK4a
Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disorder neuropathologically characterized by extracellular depositions of Aβ and intracellular formations of paired helical filaments. Although major molecular components of paired helical filaments are well characterized [43], mechanisms leading to neurofibrillary degeneration are still not understood very well. The recent finding that a high degree of structural plasticity predisposes neurons to neurofibrillary degeneration [8] corroborates previous observations on the link between aberrancies of growth and proliferation-regulating mechanisms and neurodegeneration and supports the assumption that mechanisms mediating neuronal plasticity, proliferation and differentiation might be critically involved in these processes 1, 2, 5, 7, 19, 48. Levels of a variety of molecules with potential neurotrophic and mitogenic activity and their binding sites such as NGF 21, 23, 29, bFGF 44, 58, 94, EGF 12, 95, IL-1 [20], IL-2 [53], IL-6 [30], IGF-1 [22], IGF-2 [97], PDGF [73] and HGF/SF [32] are elevated in AD. Activation of cell surface receptors of most of these trophic factors is linked to the downstream MAPK cascade by the small G-protein p21ras that is localized to the inner aspect of the plasma membrane [47]. Expression of p21ras 40, 42, of MEK, ERK1/2 [3] and p14-3-3 10, 41, 67, major elements of the MAP kinase, as well as of further downstream molecules involved in regulation of proliferation and differentiation such as cyclins, cyclin-dependent kinases and their inhibitors as, for example, those of the p16INK4a family, are all elevated early during the course of AD 6, 9, 10, 81, 82, 83 indicating some aberrancy of proliferation- and differentiation-regulating mechanisms.
The small G-protein p21ras, a critical molecular switch for the induction of these processes, is activated through conversion from its inactive, GDP-bound, to the active, GTP-bound, state [51]. This activation of p21ras can also directly be triggered by nitric oxide (NO), one of the smallest biological active messenger molecules 64, 111. In the nervous system, among other processes, NO is involved in the regulation of synaptogenesis, synaptic plasticity and remodelling of neurites and synapses 15, 16, 17, 18, 24, 35, 38, 39, 57, 6970, 71, 76, 92, 101, 106, 113, 114. NO, however, apparently plays also a pathophysiological role in neurodegenerative diseases, stroke and other psychiatric diseases 54, 62, 84, 91, 96, 106.
Based on observations on alterations of the different isoforms of the NO-generating enzyme, nitric oxide synthase (NOS), in brains of AD patients, NO was suggested to be involved in neurodegeneration in AD 11, 26, 61, 72, 78, 84, 89, 98, 99, 102[105]. Tangle-bearing neurons in the cerebral cortex, for example, start to express neuronal NOS (isoform nNOS; NOS I), an enzyme that is not expressed in healthy pyramidal neurons. Another isoform of NOS (isoform iNOS; NOS II) is preferentially found in astrocytes associated with plaques 72, 98, 105, 109. Other authors described an aberrant localization of iNOS in tangle-bearing neurons [102] or of endothelial NOS (eNOS, NOS III) in neuritic processes and astrocytes [78], findings that have not been confirmed by others. Thus, the involvement of NO/NOS in the pathogenesis of AD is still somewhat conflicting and poorly understood 55, 72, 84, 109.
To study the potential link of nNOS and critical regulators of cellular proliferation and differentiation in the process of neurofibrillary degeneration, we analyzed the expression pattern of NOS-isoforms, p21ras and p16INK4a in comparison to neurofibrillary degeneration in AD.
Section snippets
Cases
Brains used in the present study were obtained from seven controls (four females, three males) and from 14 patients with AD (eight females, six males). Cases were matched with respect to age (mean age±S.D.: controls, 92.4±3.8 years; AD, 89.6±5.3 years; p>0.20, Student's t-test), postmortem interval (controls: 30.7±9.4 h; AD: 30.5±10.0 h; p>0.20) and the “Premortem Severity Index” (PMSI) by Monford et al. [77] (p>0.20) to minimize the likelihood of an artificial influence by premortem hypoxia
Specificity of NOS antibodies
Isoform-specificity of NOS antibodies was verified by Western blotting using recombinant NOS proteins and cytosolic protein extract obtained from the temporal cortex of a patient with AD. The rabbit polyclonal anti-iNOS-antibody labeled exclusively the 130-kDa iNOS recombinant protein and showed very weak crossreactivity with the 150-kDa nNOS from brain extract. Both the mouse monoclonal anti-nNOS and rabbit polyclonal anti-nNOS antibodies reacted preferentially with the 150-kDa nNOS of the
Localization of NOS isoforms in the cortex of normal human brain and AD
In the present study, we have analyzed the distribution of NOS isoforms in the cortex of normal human brain and in AD. Expression of nNOS in the isocortex of normal brain is restricted to a subset of interneurons and axonal plexus. The number of these neurons is relatively small and has been estimated to constitute approximately 2% of all cortical neurons in rodents [101] but seems much higher in human cortex [109]. In addition, multipolar neurons of entorhinal cortical layer Pre-α (lamina
Acknowledgements
This study was supported by the Bundesministerium für Bildung, Forschung and Technologie (BMBF), Interdisciplinary Centre for Clinical Research at the University of Leipzig (01KS9504, Project C1). We thank B. Bär and H. Gruschka for her skillfull assistance and J. Grosche for technical help with the laser scanning microscope.
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