Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer’s disease

Abstract

The abnormal hyperphosphorylation of tau in Alzheimer’s disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braak’s protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I–III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation of tau and the presence of this lesion before deposition of β-amyloid in AD.

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.