Research reportUp-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer’s disease
Introduction
A major neuropathological feature of Alzheimer’s disease (AD) is the deposition of paired helical filaments (PHF) as neurofibrillary tangles (NFTs) in neuronal cell bodies, neuropil threads (NTs), and the dystrophic neurites surrounding β-amyloid cores in neuritic plaques [10]. A number of studies have shown that these neurofibrillary changes correlate with dementia in AD patients [1], [4], [14].
The principal protein subunit of PHFs is an abnormally hyperphosphorylated form of the microtubule-associated protein tau, PHF-tau [27], [28], [35], [37], which has been suggested to result from an altered balance between the activities of tau protein kinase and protein phosphatase [28], [36]. A number of proline-directed kinases have been implicated in the hyperphosphorylation of PHF-tau, including cyclin-dependent kinase 5 (cdk5), glycogen synthase kinase-3 (GSK-3) and the cell division cycle 2 (cdc2) kinase [29], [38], [41], [55]. The mitogen-activated protein (MAP) kinase family belongs to the proline-directed protein kinases that have received attention with respect to tau hyperphosphorylation. The MAP kinases include the extracellular signal-regulated protein kinases (ERKs), the stress-activated protein kinase/C-jun amino terminal kinase (SAPK/JNK), and p38 kinase. They can be distinguished from one other on the basis of the tripeptide dual phosphorylation motifs required for kinase activation. The ERKs include p44 ERK1 and p42 ERK2 [7], and PK40 ERK [49], all of which have been shown capable of phosphorylating recombinant tau at several of the same sites as PHF-tau [15], [23], [33], [41], [43], [53].
Despite in vitro data implicating MAP kinases in hyperphosphorylation of tau protein, studies with intact cells have provided conflicting results. Lu et al. [43] demonstrated that microinjection of rat hippocampal primary neurons with purified sea star p44 ERK1 resulted in PHF-like tau hyperphosphorylation associated with compromised microtubule assembly. In contrast, others have shown that expression of p44 ERK1 and p42 ERK2 in primary neurons and either COS cells or fibroblasts transfected with tau does not result in Alzheimer-like phosphorylation of tau [40], [42]. An induction of PHF-like tau following inhibition of protein phosphatase with okadaic acid has been shown to involve the activation, amongst other kinases, of p44 ERK1 and p42 ERK2 concomitant with reduced dephosphorylation of tau protein [22], [57], [59]. However, since okadaic acid-induced tau hyperphosphorylation in rat primary cortical neurons can occur even in the presence of a MAP kinase inhibitor [31], tau hyperphosphorylation can occur independently of MAP kinases [32]. Immunohistochemical studies have shown the presence of p42 ERK2 immunoreactivity in both normal and tangle-bearing neurons and senile plaque neurites of the hippocampus in AD cases [32], [46], [58]. Neurons containing the highest levels of MAP kinases, however, are not necessarily those most likely to develop NFTs, thereby indicating that the presence of these enzymes is not in itself sufficient to predispose neurons to NFT formation [32].
MAP kinase activation occurs in response to a variety of hormones and growth factors and requires phosphorylation at threonine and tyrosine residues by MAP kinase kinase (MEK) [2], [7], [24]. To study the status of the activation of MAP kinase in the AD brain, we employed phospho-specific antibodies that detect phosphorylated MEK1/2 at serine217 and serine221 (active MEK1/2, p-MEK1/2), and both phosphorylated p44 ERK1 and p42 ERK2 at threonine 202 and tyrosine 204 residues (active ERK1/2, p-ERK1/2) to immunostain a series of brains that had been staged for AD neurofibrillary changes as described by Braak and Braak [11]. According to this staging protocol, the formation of AD neurofibrillary changes is seen first in the projecting cells of the transentorhinal region. The process continues into the entorhinal cortex, the hippocampus, following a predictable, non-random pattern, and finally extends into the association cortices [11]. Using confocal microscopy, the activities of p-MEK1/2 and p-ERK1/2 in neurons were investigated in relationship to PHF-tau labeled by antibody AT8 to tau phosphorylated at Ser-202/Thr-205. The distribution of p-MEK1/2 and p-ERK1/2 was found to correspond to the distribution of neurofibrillary changes in Alzheimer’s disease.
Section snippets
Materials
Affinity-purified rabbit polyclonal phospho-specific MEK1/2 (Ser217/221) and phospho-specific p44/p42 MAP kinase (Thr202/Tyr204) antibodies that detect the active forms of MEK1/2 and ERK1/2 were purchased from New England Biolabs, Inc. (Beverly, MA). The specificities of phospho-specific antibodies to MEK1/2 and ERK1/2 have been confirmed in brain homogenates [50], [51]. The mouse monoclonal antibody (mAb) AT8 that recognizes tau phosphorylated Ser-202/Thr-205 was purchased from Innogenetics
Distribution of p-MEK1/2 and p-ERK1/2 in brains at NFT stages I–VI and amyloid deposits stages A–C
In control cases (Stage 0), which were devoid of NFTs and amyloid deposits, the transentorhinal, the hippocampal complex and the temporal isocortex were only weakly stained with antibodies to p-MEK1/2 (Fig. 1; Normal Control) or p-ERK1/2 (Fig. 2). At the transentorhinal stage (Stages I and II), a significant increase in immunostaining in the layer Pre-α of the entorhinal region but neither in the hippocampus nor in the temporal cortex was observed with antibodies to p-MEK1/2 (Fig. 1;
Discussion
The involvement of MAP kinases, p44 ERK1 and p42 ERK2 in the AD-like hyperphosphorylation of tau is controversial. Previous immunohistochemical studies carried out using antibodies that did not distinguish between active and inactive forms of MEK and ERK reported the association of these kinases with microtubules and with the abnormally hyperphosphorylated tau [3], [19], [46], [52], [54]. Subsequent studies found the association of the active MEK1/2, ERK1/2, JNK and P38 with early deposition of
Conclusion
Active forms of MEK1/2 and ERK1/2 were found to be co-distributed with the progressive accumulation of neurofibrillary changes in AD. In particular, accumulation of the active forms of MEK1/2 and ERK1/2 was found not only in pretangle-like neurons and neurons with classic tangles but also in neurons which did not show abnormally hyperphosphorylated tau. The neurofibrillary pathology positive for active forms of MEK1/2 and ERK1/2 was also found to occur prior to the deposition of β-amyloid in
Acknowledgements
This paper is dedicated to Professor Eva Braak. The Gamla Tjänarinnor Foundation, Erik och Edith Fernströms Stiftelse, Sigurd Och Elsa Goljes Minne, Åke Wibergs Stiftelse, Fredrik och Ingrid Thurings Stiftelse, Gun och Bertil Stohnes Stiftelse, Karolinska Institutets Stiftelser, Loo and Hans Ostermans Foundation, the Deutsche Forschungsgemeinschaft, and NIH grants NS18105, AG08076 and AG19158 provided financial support.
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