Aberrant expression of nNOS in pyramidal neurons in Alzheimer's disease is highly co-localized with p21^ras and p16^INK4a

Abstract

Aberrancies of growth and proliferation-regulating mechanisms might be critically involved in the processes of neurodegeneration in Alzheimer's disease (AD). Expression of p21ras and further downstream signalling elements involved in regulation of proliferation and differentiation as, for example, MEK, ERK1/2, cyclins, cyclin-dependent kinases and their inhibitors such as those of the p16^INK4a family, are elevated early during the course of neurodegeneration. Activation of p21ras can also directly be triggered by nitric oxide (NO), synthesized in the brain by various isoforms of nitric oxide synthase (NOS) that might be differentially involved into the pathomechanism of AD. To study the potential link of NO and critical regulators of cellular proliferation and differentiation in the process of neurofibrillary degeneration, we analyzed the expression pattern of NOS-isoforms, p21ras and p16^INK4a compared to neurofibrillary degeneration in AD. Additionally to its expression in a subtype of cortical interneurons that contain the nNOS-isoform also in normal brain, nNOS was detected in pyramidal neurons containing neurofibrillary tangles or were even unaffected by neurofibrillary degeneration. Expression of nNOS in these neurons was highly co-localized with p21ras and p16^INK4a. Because endogenous NO can activate p21ras in the same cell which in turn leads to cellular activation and stimulation of NOS expression [H.M. Lander, J.S. Ogiste, S.F.A. Pearce, R. Levi, A. Novogrodsky, Nitric oxide-stimulated guanine nucleotide exchange on p21 ras, J. Biol. Chem. 270 (1995) 7017–7020], the high level of co-expression of NOS and p21ras in neurons vulnerable to neurofibrillary degeneration early in the course of AD thus provides the basis for an autocrine feedback mechanism that might exacerbate the progression of neurodegeneration in a self-propagating manner.

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 p16^INK4a 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 p16^INK4a in comparison to neurofibrillary degeneration in AD.