Secreted β-amyloid precursor protein activates microglia via JNK and p38-MAPK

Neurobiology of Aging 26 (2005) 9–16

Secreted ␤-amyloid precursor protein activates microglia via JNK and p38-MAPK Angela M. Bodles a , Steven W. Barger a,b,c,∗ b

a Donald W. Reynolds Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Department of Neurobiology and Developmental Science, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA c Geriatric Research Education Clinical Center, Central Arkansas Veterans Healthcare System, Little Rock, AR 72205, USA

Received 16 May 2003; received in revised form 18 December 2003; accepted 18 February 2004

Abstract Reactive microglia are thought to play a role in the pathogenesis of Alzheimer’s disease (AD) and are localized to the senile plaques that are associated with cognitive decline. The ␤-amyloid precursor protein (␤APP) is over-expressed in the dystrophic neurites near such plaques, and secreted forms of ␤APP (sAPP␣) activate inflammatory responses in microglia. To characterize the mechanisms by which sAPP␣ activates microglia, we assayed its effects on MAP kinases, including c-Jun N-terminal kinases (JNK), extracellular signal-regulated protein kinases (ERK), and p38-MAPK. sAPP␣ was found to rapidly activate JNKs, ERKs and p38-MAPK in a dose-dependent manner. The JNK inhibitor SP600125 and the p38 inhibitor SB203580 independently reduced both nitrite accumulation and induction of inflammatory nitric oxide synthase (iNOS). By contrast, inhibition of the ERK pathway with U0126 did not appreciably affect either outcome measure. These findings suggest that sAPP activates the ERK, JNK and p38 classes of MAP kinases but that only JNK and p38-MAPK are critical for activation of microglia by sAPP␣, a process that compromises neuronal function and survival. © 2004 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; APP; Inflammation; MAP kinase; Microglia; Nitric oxide synthase; Stress-activated kinase

1. Introduction Alzheimer’s disease is the most common degenerative brain disorder affecting today’s elderly population and is characterized by dementia and a specific constellation of neuropathological findings. Among the latter are deposits of the amyloid ␤-peptide (A␤) associated with glial cells expressing inflammatory phenotypes [19] and dystrophic neurites expressing high levels of the A␤ precursor protein (␤APP) [19]. ␤APP is a 695- to 770-amino acid, membrane-spanning glycoprotein (reviewed in [8]). Proteolysis by ␤- and ␥-secretases produces A␤; it also liberates sAPP␤, a polypeptide composed of the residues amino-terminal to the ␤-cleavage site (e.g., the amino-terminal 596 amino acids of ␤APP695 ). An alternative pathway involves ␣-secretase cleavage within the A␤ sequence (after position 16 in the A␤ sequence), leading to the release of sAPP␣ (e.g., the amino-terminal 612 amino acids of ␤APP695 ). ∗

Corresponding author. Tel.: +1-501-526-5811; fax: +1-501-526-5830. E-mail address: [email protected] (S.W. Barger).

0197-4580/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2004.02.022

Neuroprotective and neuromodulatory activities have been attributed to sAPP␣ [5]. However, we have previously shown that both sAPP␣ and sAPP␤ elevate markers of inflammation in microglia, including their elaboration of neurotoxicity [3,4,25,26]. This effect involves release of the excitotoxin glutamate from microglia [4], mediates a proinflammatory stimulus from injured neurons [26], and is modulated by apolipoprotein E [3]. Neuroinflammation has become increasingly suspected of playing a crucial role in diseases of the central nervous system, including Alzheimer’s disease (reviewed in [1]). The MAP kinase family is comprised of four sub-groups: ERKs, JNKs, BMK1, and p38-MAPK. All are involved in mediating cellular responses to extracellular stimuli. ERK 1 and ERK 2 are mostly thought of as regulators of cell growth and differentiation, but in the brain they also respond to stress stimuli such as oxidative stress, glutamate and changes in calcium levels. JNK is activated by similarly stressful stimuli as well as by a deficit of trophic factors and through the activation of death domain receptors by tumor necrosis factor-␣. The expression of ERKs, JNKs, and p38-MAPK has been investigated in mild to severe cases of AD (assessed by Braak staging) and related to

10

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

the progression of the disease [30,44]. The p38-MAPK has been implicated in the hyperphosphorylation of tau [17,35], including that observed after activation of nearby microglia [25]. In neurons, sAPP␣ stimulates phosphorylation of ERK1 and ERK2 via a phosphatidylinositol-3-kinase signaling pathway; neurotrophic effects of sAPP␣ in neurons are eliminated or decreased when either PI3 K or ERK1/ERK2 are selectively blocked [10]. NF-␬B is activated by sAPP␣ [3], which provides further evidence for a neurotrophic effect of sAPP␣ in neurons [5,10] yet simultaneous connections to inflammation. Analysis of non-demented and AD cases revealed ERK activation, in conjunction with JNK, as one of the earliest events in the disease pathogenesis [44]. Here, we propose that similar events mediate a component of sAPP␣’s bioactivity towards microglia. We found that sAPP␣ activated ERK1 and ERK2, as well as JNK and p38, in a dose-dependent manner. Inhibition of the ERK pathway by the MEK inhibitor U0126 attenuated responses to sAPP␣ only at doses that were somewhat toxic, suggesting that ERKs are not critical in transducing the sAPP␣ stimulus. However, a JNK inhibitor and p38 inhibitor independently blocked sAPP␣’s elevation of inducible nitric oxide synthase (iNOS) expression, suggesting that the JNK and p38-MAPK signaling pathways contribute to neurodegenerative microglial activation by sAPP␣.

lution was then mixed with the LAL/sample mixture for an additional 6 min before stopping the reaction. The absorbance was determined spectrophotometrically at 405 nm, and the concentration of endotoxin present was calculated from a standard curve. Fractions with endotoxin levels below 0.1 EU/mL at 30 nM were used for treatments of microglia.

2. Experimental procedures

2.3. Nitrite measurement

2.1. sAPPα production

Primary microglia plated in 96-well plates were changed to serum-free MEM 1 day after plating, and treatments were applied. When present, U0126 (Calbiochem), SP600125 (Tocris) or SB203580 (Calbiochem) were added for 60 min before sAPP or LPS. Conditioned medium (100 ␮L) was incubated with Griess reagent (100 ␮L) (0.5% sulfanilamide and 0.05% N-(1-naphthyl)-ethylenediamine dihydrochloride in 0.25% phosphoric acid) in a new plate. After 10 min the absorbance of the samples were read spectrophotometrically at 540 nm. Absolute concentrations of nitrite were calculated by interpolation in a standard curve generated with known concentrations of sodium nitrite. Values represent the mean ± S.E.M. of quadruplicate determinations. After removal of the medium for nitrite measurements, a methyltetrazolium (MTT) assay was performed to ascertain viability. The cultures were replenished with fresh medium containing 125 ␮g/mL MTT and incubated an additional hour. The formazan reaction product was solubilized in DMSO, and absorption was read at 570 nm on a spectrophotometric plate reader.

TOP10TM E. coli expressing His-tagged sAPP␣ from the pTrcHis vector (Invitrogen) were grown overnight in Luria broth (DIFCO). Isopropyl-␤-d-thiogalactoside (1 mM) was added at an optical density of 0.6. The cultures were collected by centrifugation, then lysates were applied to a nickel-affinity resin column. The column was washed successively with buffers at pH 6.0 and 5.3 before being eluted at pH 4.0 in 0.5 mL fractions. After analysis by SDS-PAGE (7.5%) fractions enriched in sAPP␣ were pooled and dialyzed against 0.1% Triton X-100, 20 mM triethanolamine (pH 7.4). Dialysate was further resolved by low-pressure, preparative chromatography on a MonoQ (Pharmacia) column. After loading, the column was washed with 2 mL Buffer A (100 mM NaCl, 20 mM triethanolamine, pH 7.4), then eluted at 1.0 mL/min under the following gradient: 0–45% Buffer B (1 M NaCl, 20 mM triethanolamine, pH 7.4), 2 min; 45–85% Buffer B, 20 min. Fractions were collected as 0.5 mL and screened by SDS-PAGE and endotoxin assay. Endotoxin levels were determined for each chromatography fraction using the chromogenic Limulus amoebocyte lysate (LAL) test (BioWhittaker). Briefly, the samples were mixed with LAL and incubated at 37 ◦ C for 10 min. A substrate so-

2.2. Primary microglia culture Rats were euthanized at 1–3 days for the removal of cerebral cortices. The meninges were removed, and the cortical tissue was disrupted by mincing, trypsinization and trituration before plating the cells in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 10 ␮g/mL gentamycin. After approximately 10–14 days the astrocytes had reached confluency and the microglia were sufficiently numerous. Microglia were harvested by vigorous lavage with a 10 mL pipette, collected by centrifugation, and counted on a hemacytometer. Viable cells were plated at 6 × 105 /plate in 35 mm culture plates for western analysis or 1 × 105 /well in 96-well plates for nitrite assay. After 30 min, the plates were washed vigorously to remove astrocytes and oligodendrocytes; resulting cultures were typically comprised of >95% microglia, as determined by staining with Griffonia isolectin B4. Treatments were administered 18–24 h after replating.

2.4. Western blot analysis Microglia cells were harvested in lysis buffer (2% sodium dodecyl sulfate, 62 mM Tris–HCl, pH 6.8). Lysates were

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

11

Fig. 1. Effect of sAPP␣ on JNK activation in microglia. Primary rat microglia were treated with (A) sAPP␣ at 30 nM for the times indicated and (B) sAPP␣ for 30 min at concentrations ranging 1–100 nM. Equal amounts of protein were subjected to Western blot with an anti-phospho JNK antibody (upper) and an anti-phospho-independent JNK antibody (lower).

briefly sonicated and quantified for protein by bicinchoninic acid (BCA; Pierce). Equivalent quantities of protein were resolved by SDS-PAGE (12%) and transferred onto a nitrocellulose membrane at 100 mV for 1 h at ∼4 ◦ C. Membranes were blocked for 1 h at room temperature with 3% bovine serum albumin (BSA) in Tris-buffered saline (10 mM Tris–HCl, 100 mM NaCl, pH 7.4) containing 0.1% Tween-20 (TTBS). Anti-phospho-ERK (1:2000) (BioLabs), anti-phospho-JNK (1:500) (Santa Cruz), anti-phospho-p38 (1:1000) (Biolabs) or anti-iNOS (1:2000) (Becton-Dickinson/Transduction Laboratories) was diluted in TTBS containing 3% BSA and applied at room temperature for 2 h. After washing, the blot was incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse secondary antibody diluted 1:400 in TTBS containing 3% BSA. Immunoblots were developed using a BCIP/NBT kit (Vector Laboratories). The anti-phospho-ERK antibody detects phosphorylated forms of both ERK1 and ERK2; the anti-phospho-JNK antibody is similarly cross-reactive with the JNK family. As a control for changes in the total amount of the kinases, blots were also probed with antibodies specific for each class of MAPK irrespective of phosphorylation state. ERK, JNK or p38-MAPK antibody (1:1000) (Biolabs) was applied to the blots as above, but immunodetection was provided through chemiluminescence with the Western-LightTM system (Applied Biosystems, Bedford, MA). Through this strategy, the total amount of a given kinase (chemiluminescence) and its phosphorylated form (colorimetric development) could be measured on each blot.

3. Results To test the possible involvement of JNK in the activation of microglia by sAPP␣, Western blot analysis was performed on sAPP-treated cultures with a phospho-specific JNK antibody. Rat primary microglia were treated with sAPP␣ at 1–100 nM for 10–120 min. JNK phosphorylation was stimulated by sAPP␣, peaking at 30 min (Fig. 1A) in a dose-dependent manner (Fig. 1B). Activation of p38-MAP kinase was detected under similar conditions (e.g., Fig. 2) also peaking at 30 min. Similar assays for ERKs indicated that sAPP␣ also evoked activation of this class of MAP kinase. Dosimetrically, the response was maximal at 30 nM and was saturated beyond this concentration (Fig. 3).

Fig. 2. Effect of sAPP␣ on p38-MAPK in microglial cells. Primary rat microglia were treated with sAPP␣ at the times indicated. Equal amounts of protein were subjected to Western blot with an anti-phospho p38-MAPK antibody (upper) and an anti-phospho-independent p38-MAPK antibody (lower).

12

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

Fig. 3. Effect of sAPP␣ on ERK1 and ERK2 activation in microglial cells. Primary rat microglia were treated with sAPP␣ for 20 min, at concentrations ranging from 1 to 100 nm as indicated, or vehicle (control). Equal amounts of protein were subjected to Western blot with an anti-phospho ERK antibody (upper) and an anti-phospho-independent ERK antibody (lower).

Activation of the ERKs by sAPP␣ peaked at 20 min and declined thereafter. The same blots were also assayed using phosphorylation-independent antibodies to confirm that results with phosphorylation-dependent antibodies did not involve changes in expression of the kinases. None of these kinases showed an increase in total-protein levels (Figs. 1–3). To test the role of these kinases in transducing the proinflammatory signal of sAPP, biological responses to sAPP␣ were examined in microglia pretreated with inhibitors that are relatively selective for each class. Nitrite production from microglia after treatment with sAPP␣ was measured as one indicator of proinflammatory activation. As previously reported, microglia treated with sAPP␣ showed a large, dose-dependent accumulation of nitrite in their culture medium. Application of the JNK inhibitor SP600125 suppressed this nitrite response at concentrations ranging from 1 to 30 ␮M (Fig. 4). In a similar manner, the nitrite response was suppressed upon application of the

p38-MAPK inhibitor SB203580 in a dose-dependent manner (0.01–10 ␮M) (Fig. 4). As these results implied a role for both JNK and p38-MAPK, a combination of SP600125 with SB203580 was tested in a nitrite assay. At higher concentrations, these compounds can cross-react with unintended kinases. Therefore, we tested the effects of combination within the middle concentrations of the inhibitory dose range for each compound (Table 1). A combination of 0.1 ␮M SB203580 with 3 ␮M SP600125 resulted in suppression of the sAPP␣ response that was statistically lower than that achieved with either compound alone. Inducible NOS generates much of the nitric oxide responsible for measured nitrite accumulation in microglial cultures, and the expression of iNOS is elevated by sAPP␣ [3]. We assayed iNOS levels by Western blot analysis in microglia that had been exposed to sAPP␣ in the absence or presence of SP600125. A dose-dependent suppression of the iNOS induction by SP600125 was apparent (Fig. 5). Similarly, we assayed iNOS levels by Western blot analysis in microglia that had been exposed to sAPP␣ in the absence and presence of SB203580. A dose-dependent suppression of the iNOS induction by SB203580 was also detected (Fig. 6). To test of the role of ERKs in the sAPP␣ effects on microglia we utilized U0126, an effective inhibitor of MEK. As an upstream activator of the ERKs, MEK activity is necessary for ERK-mediated signal transduction. However, U0126 had no appreciable effect on sAPP␣-stimulated nitrite levels (Fig. 4) (though MTT assays showed that U0126

Table 1 Combination of kinase inhibitors

dosea

Fig. 4. Effect of JNK inhibitor (SP600125), p38 inhibitor (SB203580), and MEK inhibitor (U0126), on sAPP␣ induced nitrite production in microglia. Primary rat microglial cells were treated with SP600125, SB203580 and U0126, independently, at the concentrations indicated for approximately 1 h before the addition of sAPP␣ at 30 nM for 18 h. Results shown are the mean ± S.E.M. of four independent experiments (∗ P < 0.001).

Low High doseb

SP600125

SB203580

Both

58.40 ± 4.521 39.86 ± 7.788

70.96 ± 6.787 45.79 ± 2.750

37.65 ± 4.649∗ 14.90 ± 7.212

Values represent nitrite (␮M) produced by primary microglia treated with 30 nM sAPP; values reflect percentage of response to sAPP alone ± S.E.M. a SP600125 = 3 ␮M, SB203580 = 0.1 ␮M. b SP600125 = 10 ␮M, SB203580 = 1 ␮M. ∗ P < 0.05 versus either drug alone.

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

13

Fig. 5. Effect of JNK inhibitor, SP600125, on sAPP␣ induced iNOS production in microglia. Primary rat microglial cells were pretreated with SP600125 at concentrations indicated for 1 h before treatment with sAPP␣ at 30 nM for 18 h. Equal amounts of protein were subjected to Western blot with an anti-iNOS antibody.

Fig. 6. Effect of p38-MAPK inhibitor, SB203580, on sAPP␣ induced iNOS production in microglia. Primary rat microglial cells were pretreated with SB203580 at concentrations indicated for 1 h before treatment with sAPP␣ at 30 nM for 18 h. Equal amounts of protein were subjected to Western blot with an anti-iNOS antibody.

Fig. 7. Effect of MEK inhibitor, U0126, on sAPP␣ induced iNOS production in microglia. Primary rat microglial cells were pretreated with U0126 at concentrations indicated for 1 h prior to treatment with sAPP␣ at 30 nM for 24 h. Equal amounts of protein were subjected to Western blot with an anti-iNOS antibody.

Fig. 8. Inhibition of ERK1 and ERK2 activation by sAPP␣ with the MEK inhibitor U0126. Primary rat microglia were pretreated with U0126 for 30 min at concentrations ranging from 0.3 to 30 ␮M before treatment with sAPP␣ for 20 min at 30 nm. Equal amounts of protein were subjected to Western blot with an anti-phospho ERK antibody.

was marginally toxic at 30 nM). The ineffectiveness of U0126 on sAPP␣-stimulated nitrite accumulation was consistent with the fact that this drug did not affect the ability of sAPP␣ to elevate the expression of iNOS (Fig. 7). Negative results with U0126 could possibly result from an inactive drug preparation. To verify that the drug inhibited ERK activation under the experimental conditions applied, U0126-treated cultures were subjected to Western blot analysis with an anti-phospho-ERK antibody (Fig. 8). U0126 inhibited ERK1 and ERK2 activation by sAPP␣ in a dose-dependent manner; ERK activation was only partially inhibited at 1 ␮M but completely inhibited at 3, 10, and 30 ␮M.

4. Discussion Neuroinflammation, resulting from the activation of microglia, has been linked to many neurodegenerative conditions, including Alzheimer’s disease. The extent to which inflammatory events initiate such neurodegeneration remains to be determined. However, it is clear from paradigms that either stimulate or suppress microglial activation that proinflammatory mechanisms can contribute substantially to structural and functional deficits in the CNS [7]. As the resident phagocytes of the CNS, microglia have primary roles in elimination of infectious agents and clearance of damaged tissue. However, microglia also secrete numerous

14

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

factors that are proinflammatory and/or neurotoxic; chief among these may be excitotoxins [4,16,31]. Postmortem analysis of AD brain reveals the presence of microglia colocalized with neuritic plaques in the cerebral cortex [19]. Furthermore, analysis of non-demented, age-matched controls reveals that microglia are associated with the relatively rare senile plaques in these individuals as well [15], suggesting that the presence of the microglia may play a role in the development and maturation of plaques. Overall, it appears that activated microglia are present at an early stage of the disease process and remain associated with plaques as they develop. This phenomenon corresponds to the elevation of ␤APP in dystrophic neurites in such plaques [19]. Together with coincident elevations of ␤APP in several neurodegenerative conditions (i.e., head injury, epilepsy, stroke, and Down syndrome), all of which predispose to microglial activation and AD-like pathology [18], these data suggest that overproduction of sAPP could contribute to AD genesis or progression. This possibility seems quite likely considering the connections of ␤APP and/or sAPP to neurotoxic phenotypes in microglia [3,4,13,23,25,26]. Here we have shown that the proinflammatory activity of sAPP␣ is correlated with activation of the JNK, p38-MAPK and ERK signaling cascades in microglia. Treatment of primary rat microglia with sAPP␣ and Western blot analysis using phospo-specific antibodies revealed a time- and concentration-dependent increase in JNK, p38-MAPK, and ERK phosphorylation events associated with the activation of these kinases. However, blockade of ERK activation with the MEK inhibitor U0126 had no effect on sAPP␣-triggered proinflammatory activation of microglia. By contrast, inhibition of JNK or p38-MAPK partially blocked the induction of iNOS expression and the resultant accumulation of nitrite in microglial cultures in a concentration-dependent manner. Application of a combination of inhibitors, SP600125 and SB203580, increased the potential to reduce nitrite production significantly. SB203580 is relatively selective for p38-MAPK, but it can influence JNK at approximately 10-fold higher concentrations [21,40]. Hence, it would be difficult to interpret the results of combination of the two inhibitors at the higher ends of their effective concentration ranges. It is also possible that SP600125 may have owed some of its effectiveness at higher concentrations to inhibition of p38-MAPK, and vice versa. However, it is reassuring that the dose-response curves differed by about the same order of magnitude as the Ki ’s of these drug for their respective targets; if SB203580 worked by crossover inhibition of JNK, for instance, it should have done so at doses roughly equivalent to those by which SP600125 inhibited JNK. The requirement for both kinases in the response to sAPP␣ suggests that the two combine their efforts in a mechanistic way, e.g., in a sequential relationship rather than a redundantly parallel manner. It is likely that various MAP kinases participate differentially in proinflammatory activation by distinct stimuli or in different cell types. Pyo et al. [32] found that all three of the

MAP kinase classes investigated here were activated by LPS or A␤25–35 in primary rat microglia, and LPS-induced NO was mutually dependent on ERK and p38-MAPK pathways; similar results with LPS were reported by Bhat et al. [6]. McDonald et al. [28], by contrast, found that A␤25–35 could activate ERKs and p38-MAPK in THP-1 monocytes, but activation of JNKs was not observed. Using the same system, Combs et al. [12] demonstrated a requisite role for the ERK pathway in the ability of an A␤ peptide to elicit neurotoxicity from THP-1 cells. However, the ERKs are not essential for elevations of NO, IL-1␤, or NF-␬B activity upon LPS stimulation in the BV-2 microglia cell line, where p38-MAPK was identified as a key component of activation [20,39]. A similar study in a macrophage line revealed that LPS activates both ERK and p38-MAPK, but only the latter was involved in the expression of iNOS and NO release [9]. Valledor et al. [38] concluded that the time frame of ERK activation is a critical determinant of its contribution to macrophage activation, analogous to the time-dependent switch between ERK contributions to neuronal differentiation [33]. Given these varied results, the relative roles of ERK and other MAP kinases in AD are far from clear. To some extent, the answer may depend upon the degree to which A␤ or other stimuli are primarily responsible for microglial activation in this condition. Notably, there is no evidence of ERK activation in combination with apoptosis or cell death in AD or a range of other neurodegenerative diseases [14]. Indeed, ERK activation may suppress partially the production of toxic mediators [39]. ERK activation is associated with anti-apoptotic or otherwise survival-promoting signals in many systems; more specifically, macrophages depend on the ERK pathway for trophic support by TGF␤1 [11]. These findings suggest that if ERKs play any role in neuroinflammation, they may serve only to promote survival of the microglia in the face of harsh conditions they create for themselves [27]. Even the epidemiological suggestions that non-steroidal anti-inflammatory drugs (NSAIDs) prevent AD [42] may speak to a non-pathogenic role for ERKs, as NSAIDs activate the ERK signaling cascade [2,24]. Other components of the JNK pathway, provide mechanistic connections to AD and neuroinflammation. The upstream cascade for JNK activation has recently been elucidated, indicating that JNK kinase 1 (JKK1) plays an important role in the pathway [43]. Brain tissue from AD and AMC cases were examined for JKK1, and immunodetectable levels were localized to neurofibrillary tangles, neuritic senile plaques, granulovacuolar degeneration, and neurophil threads in the hippocampus of the AD cases; little staining was observed in the AMC cases. Overall levels of phospho-JKK1 were elevated in AD versus AMC. As Jun is a critical component of AP-1 and other transcription factor complexes, JNK activation logically predicts induction of proinflammatory genes controlled by Jun such as iNOS [34], cyclooxygenase 2 [41], and various cytokines [29]. Indeed, a role for JNKs in activation of iNOS has been demonstrated for several stimuli [34].

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

Associations have also been drawn between p38-MAPK and AD, but mostly in relation to activity within neurons. Neurofibrillary tangles (NFTs) comprised of the microtubule-associated protein tau are one of the disease’s sine qua non, and p38-MAPK can phosphorylate tau on some of the amino acids hyperphosphorylated in NFTs [36]. The active form of p38-MAPK appears to be elevated in AD, with immunohistochemical localization to tangle-bearing neurons [22,37,44]. Interestingly, chronic elevation of IL-1␤ in the rat brain can convert tau to an NFT-like immunoreactivity [37], providing one link (among many) between microglial activation and characteristic AD neuropathology. We previously reported that activation of microglia by sAPP␣ results in their ability to activate p38-MAPK in neurons, an effect probably mediated by IL-1␤ [25]. The present data suggest that this kinase may participate in an array of different cell types and mechanisms in AD. In conclusion, our results demonstrate that sAPP␣ activates microglia largely through the JNK and p38-MAPK pathways and that such activation has the potential to produce factors that are proinflammatory and toxic to neurons. Therapeutic strategies aimed at increasing production of sAPP␣ may cause unintentional neurodegeneration through the activation of microglia. However, greater understanding of the mechanisms involved may help to circumvent this complication. Arresting the JNK or p38-MAPK pathways (perhaps, combined with a stimulation of the ERK cascade) may prove beneficial in the quest for pharmacotherapy targets for neurodegenerative diseases. 5. Conflict of interest statement Dr. Barger holds a U.S. patent (6,440,678,B1) entitled “Materials and methods related to the inflammatory effects of secreted amyloid precursor proteins.” The application is limited to screening molecules for the ability to inhibit such effects through direct binding to amyloid precursor proteins. Dr. Bodles has no conflict of interest to declare.

Acknowledgments This work was supported by NIH funds AG12411 and AG17498. References [1] Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, et al. Inflammation and Alzheimer’s disease. Neurobiol Aging 2000; 21(3):383–421. [2] Avramovich Y, Amit T, Youdim MB. Non-steroidal anti-inflammatory drugs stimulate secretion of non-amyloidogenic precursor protein. J Biol Chem 2002;277(35):31466–73. [3] Barger SW, Harmon AD. Microglial activation by secreted Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature 1997;388(6645):878–81.

15

[4] Barger SW, Basile AS. Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function. J Neurochem 2001;76(3 Part 3):846–54. [5] Barger SW, Mao X, Moerman AM, Ranganathan A. Mechanistic and metaphorical connections between NF-␬B and the secreted Alzheimer’s ␤-amyloid precursor protein. In: Patterson P, Kordon C, Christen YS, editors. Neuro-immune interactions in neurologic and psychiatric disorders. Berlin: Springer, 2000. p. 57–72. [6] Bhat NR, Zhang P, Lee JC, Hogan EL. Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-␣ gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 1998;18(5):1633–41. [7] Bruce-Keller AJ. Microglial-neuronal interactions in synaptic damage and recovery. J Neurosci Res 1999;58(1):191–201. [8] Chan SL, Furukawa K, Mattson MP. Presenilins and APP in neuritic and synaptic plasticity: implications for the pathogenesis of Alzheimer’s disease. Neuromol Med 2002;2(2):167–96. [9] Chen CC, Wang JK. p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages. Mol Pharmacol 1999;55(3):481–8. [10] Cheng G, Yu Z, Zhou D, Mattson MP. Phosphatidylinositol-3-kinaseAkt kinase and p42/p44 mitogen-activated protein kinases mediate neurotrophic and excitoprotective actions of a secreted form of amyloid precursor protein. Exp Neurol 2002;175(2):407–14. [11] Chin BY, Petrache I, Choi AM, Choi ME. Transforming growth factor beta1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages. J Biol Chem 1999;274(16):11362–8. [12] Combs CK, Johnson DE, Cannady SB, Lehman TM, Landreth GE. Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins. J Neurosci 1999;19(3):928–39. [13] DeGiorgio LA, Shimizu Y, Chun HS, Cho BP, Sugama S, Joh TH, et al. APP knockout attenuates microglial activation and enhances neuron survival in substantia nigra compacta after axotomy. Glia 2002;38(2):174–8. [14] Ferrer I, Blanco R, Carmona M, Ribera R, Goutan E, Puig B, et al. Phosphorylated map kinase (ERK1, ERK2) expression is associated with early tau deposition in neurons and glial cells, but not with increased nuclear DNA vulnerability and cell death, in Alzheimer disease, Pick’s disease, progressive supranuclear palsy and corticobasal degeneration. Brain Pathol 2001;11(2):144–58. [15] Fukumoto H, Asami-Odaka A, Suzuki N, Iwatsubo T. Association of A beta 40-positive senile plaques with microglial cells in the brains of patients with Alzheimer’s disease and in non-demented aged individuals. Neurodegeneration 1996;5(1):13–7. [16] Giulian D, Yu J, Li X, Tom D, Li J, Wendt E, et al. Study of receptormediated neurotoxins released by HIV-1-infected mononuclear phagocytes found in human brain. J Neurosci 1996;16(10):3139–53. [17] Goedert M, Hasegawa M, Jakes R, Lawler S, Cuenda A, Cohen P. Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases. FEBS Lett 1997;409(1):57–62. [18] Griffin WS, Mrak RE. Interleukin-1 in the genesis and progression of and risk for development of neuronal degeneration in Alzheimer’s disease. J Leukocyte Biol 2002;72(2):233–8. [19] Griffin WS, Sheng JG, Roberts GW, Mrak RE. Interleukin-1 expression in different plaque types in Alzheimer’s disease: significance in plaque evolution. J Neuropathol Exp Neurol 1995; 54(2):276–81. [20] Han IO, Kim KW, Ryu JH, Kim WK. p38 mitogen-activated protein kinase mediates lipopolysaccharide, not interferon-gamma, -induced inducible nitric oxide synthase expression in mouse BV2 microglial cells. Neurosci Lett 2002;325(1):9–12. [21] Harada J, Sugimoto M. An inhibitor of p38 and JNK MAP kinases prevents activation of caspase and apoptosis of cultured cerebellar granule neurons. Jpn J Pharmacol 1999;79(3):369–78.

16

A.M. Bodles, S.W. Barger / Neurobiology of Aging 26 (2005) 9–16

[22] Hensley K, Floyd RA, Zheng NY, Nael R, Robinson KA, Nguyen X, et al. p38 kinase is activated in the Alzheimer’s disease brain. J Neurochem 1999;72(5):2053–8. [23] Ikezu T, Luo X, Weber GA, Zhao J, McCabe L, Buescher JL, et al. Amyloid precursor protein-processing products affect mononuclear phagocyte activation: pathways for sAPP- and Abeta-mediated neurotoxicity. J Neurochem 2003;85(4):925–34. [24] Lennon AM, Ramauge M, Pierre M. Role of redox status on the activation of mitogen-activated protein kinase cascades by NSAIDs. Biochem Pharmacol 2002;63(2):163–70. [25] Li Y, Liu L, Barger SW, Griffin WS. Interleukin-1 mediates pathological effects of microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway. J Neurosci 2003;23(5):1605–11. [26] Li Y, Liu L, Kang J, Sheng JG, Barger SW, Mrak RE, et al. Neuronalglial interactions mediated by interleukin-1 enhance neuronal acetylcholinesterase activity and mRNA expression. J Neurosci 2000;20(1):149–55. [27] Liu B, Wang K, Gao HM, Mandavilli B, Wang JY, Hong JS. Molecular consequences of activated microglia in the brain: overactivation induces apoptosis. J Neurochem 2001;77(1):182–9. [28] McDonald DR, Bamberger ME, Combs CK, Landreth GE. Beta-amyloid fibrils activate parallel mitogen-activated protein kinase pathways in microglia and THP1 monocytes. J Neurosci 1998;18(12):4451–60. [29] Nicholson WJ, Slight J, Donaldson K. Inhibition of the transcription factors NF-kappa B and AP-1 underlies loss of cytokine gene expression in rat alveolar macrophages treated with a diffusible product from the spores of Aspergillus fumigatus. Am J Respir Cell Mol Biol 1996;15(1):88–96. [30] Pei JJ, Braak H, An WL, Winblad B, Cowburn RF, Iqbal K, et al. Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer’s disease. Brain Res Mol Brain Res 2002;109(1–2):45–55. [31] Piani D, Spranger M, Frei K, Schaffner A, Fontana A. Macrophageinduced cytotoxicity of N-methyl-d-aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur J Immunol 1992;22(9):2429–36. [32] Pyo H, Jou I, Jung S, Hong S, Joe EH. Mitogen-activated protein kinases activated by lipopolysaccharide and beta-amyloid in cultured rat microglia. Neuroreport 1998;9(5):871–4.

[33] Qui MS, Green SH. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron 1992;9(4):705–17. [34] Rao KM. Molecular mechanisms regulating iNOS expression in various cell types. J Toxicol Environ Health B: Crit Rev 2000;3(1):27–58. [35] Reynolds CH, Utton MA, Gibb GM, Yates A, Anderton BH. Stressactivated protein kinase/c-jun N-terminal kinase phosphorylates tau protein. J Neurochem 1997;68(4):1736–44. [36] Reynolds CH, Nebreda AR, Gibb GM, Utton MA, Anderton BH. Reactivating kinase/p38 phosphorylates tau protein in vitro. J Neurochem 1997;69(1):191–8. [37] Sheng JG, Jones RA, Zhou XQ, McGinness JM, Van Eldik LJ, Mrak RE, et al. Interleukin-1 promotion of MAPK-p38 overexpression in experimental animals and in Alzheimer’s disease: potential significance for tau protein phosphorylation. Neurochem Int 2001;39(5–6):341–8. [38] Valledor AF, Comalada M, Xaus J, Celada A. The differential time-course of extracellular-regulated kinase activity correlates with the macrophage response toward proliferation or activation. J Biol Chem 2000;275(10):7403–9. [39] Watters JJ, Sommer JA, Pfeiffer ZA, Prabhu U, Guerra AN, Bertics PJ. A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production. J Biol Chem 2002;277(11):9077–87. [40] Whitmarsh AJ, Yang SH, Su MS, Sharrocks AD, Davis RJ. Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol 1997;17:2360–71. [41] Xie W, Herschman HR. v-src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor. J Biol Chem 1995;270(46):27622–8. [42] Zandi PP, Anthony JC, Hayden KM, Mehta K, Mayer L, Breitner JC. Reduced incidence of AD with NSAID but not H2 receptor antagonists: the Cache County Study. Neurology 2002;59(6):880–6. [43] Zhu X, Ogawa O, Wang Y, Perry G, Smith MA. JKK1, an upstream activator of JNK/SAPK, is activated in Alzheimer’s disease. J Neurochem 2003;85(1):87–93. [44] Zhu X, Castellani RJ, Takeda A, Nunomura A, Atwood CS, Perry G, et al. Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer disease: the two hit hypothesis. Mech Ageing Develop 2001;123(1):39–46.