GDNF regulates the Aβ-induced endoplasmic reticulum stress response in rabbit hippocampus by inhibiting the activation of gadd 153 and the JNK and ERK kinases

www.elsevier.com/locate/ynbdi Neurobiology of Disease 16 (2004) 417 – 427

GDNF regulates the Ah-induced endoplasmic reticulum stress response in rabbit hippocampus by inhibiting the activation of gadd 153 and the JNK and ERK kinases Othman Ghribi, a,* Mary M. Herman, b Patcharin Pramoonjago, a Natalie K. Spaulding, a and John Savory a,c a

Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA IRP, NIMH, NIH, Bethesda, MD, USA c Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA b

Received 19 September 2003; revised 23 January 2004; accepted 1 April 2004 Available online 6 May 2004 Glial cell line-derived neurotrophic factor (GDNF) is a potent survival agent for neurons, however, its effect on AB-evoked neuronal death has not been examined. We show that the injection of AB into New Zealand white rabbit brain activates the endoplasmic reticulum (ER) chaperones, grp 78 and grp 94, and the transcription factor, gadd 153. These effects correlate with the activation of JNK and ERK as well as of microglia and with the phosphorylation of tau protein. Treatment with GDNF inhibits the activation of gadd 153, reduces the phosphorylation of JNK, abolishes the phosphorylation of ERK, prevents microglial activation, greatly reduces apoptotic cells, and does not affect the phosphorylation of tau. Our data suggest that the tau hyperphosphorylation and apoptosis triggered by AB are two independent events, and that the neuroprotective effect of GDNF against AB may result either directly by the inhibition of ER stress or indirectly through the inhibition of JNK and ERK activation. D 2004 Elsevier Inc. All rights reserved. Keywords: Ah; GDNF; Endoplasmic reticulum; grp 78; grp 94; gadd 153; JNK; ERK; Tau

Introduction Alzheimer’s disease (AD) is characterized by the deposition of amyloid h peptide (Ah), the accumulation of hyperphosphorylated tau in neurofibrillary tangles and progressive neurodegeneration. Although a key role for Ah is strongly suggested in mediating the neurodegenerative process, mechanisms that underlie Ah neurotoxic effects are still to be fully elucidated. Mitochondrial stress involving the release of cytochrome c into the cytoplasm (Keller et al., 1997; Parks et al., 2001; Rodrigues et al., 2000) and activation of the effector caspases (Allen et al., 2001) has been shown in cell cultures incubated with Ah. Mounting evidence now indicates that * Corresponding author. Department of Pathology, University of Virginia Health Sciences Center, Box 800214, Charlottesville, VA 22908. Fax: +1-434-924-2574. E-mail address: [email protected] (O. Ghribi). Available online on ScienceDirect (www.sciencedirect.com.) 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.04.002

the endoplasmic reticulum (ER) may also mediate Ah-induced neurodegenerative processes (Ghribi et al., 2001a, 2003a; Mattson et al., 2001; Nakagawa et al., 2000). Disturbance of the ER homeostasis activates stress-responsive genes, such as grp 78 and grp 94, to deal efficiently and appropriately with encountered stress (see for review Cudna and Dickson, 2003). However, prolonged ER stress leads to the activation of gadd 153 (also called CHOP), a transcription factor that can induce apoptosis (for review, see Cudna and Dickson, 2003) by mechanisms that still remain to be determined. One possible explanation is that gadd 153 activates the ERspecific caspase, caspase-12, and/or the MAP kinase signaling pathway, such activation plays a critical role in regulating apoptosis. Therefore, compounds that prevent the perturbation of ER homeostasis or the activation of MAP kinase may represent a potential neuroprotective approach in conditions where the ER homeostasis is compromised, such as in the presence of Ah peptide. GDNF is a neurotrophic factor that promotes the survival of many neuronal populations. Administration of exogenous recombinant GDNF has been shown to protect dopaminergic neurons in postnatal primary culture (Oo et al., 2003) in different models of cerebral ischemia (Abe et al., 1997; Hermann et al., 2001; Wang et al., 1997), after exposure to neurotoxins (Ugarte et al., 2003) or ethanol (McAlhany et al., 2000), and after spinal cord contusion (Cheng et al., 2002). The neuroprotective effect of GDNF in these conditions has been attributed to its anti-apoptotic action. The protective effect of GDNF involves a variety of intracellular signal transductions mediated by different pathways, including the MAP kinase signaling pathway (McAlhany et al., 2000; Neff et al., 2002; Nicole et al., 2001; Ugarte et al., 2003; Wiklund et al., 2002). On the other hand, we have previously shown that GDNF also protects the rabbit brain against a neurotoxic dose of aluminum by mechanisms that involve the ER stress response (Ghribi et al., 2001b). Despite the use of GDNF to protect against neurodegeneration in a wide range of model systems, the present study is the first to examine whether GDNF can oppose Ah-induced neurodegenerative effects, a condition that includes ER stress-mediated apoptosis (Ghribi et al., 2001a, 2003a; Mattson et al., 2001; Nakagawa et al., 2000) and the activation of MAP kinases (Pyo et al., 1998; Savage et al., 2002).

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Material and methods Antibodies The following antibodies were used for immunohistochemistry and Western blot analyses: mouse monoclonal antibodies (mAb) to

KDEL (detects both grp 94 and grp 78) (Stressgen, San Diego, CA); gadd 153, JNK, p-JNK, ERK, p-ERK and histone H1 (Santa Cruz Laboratories, Santa Cruz, CA); calnexin (Transduction Laboratories, Lexington, MD); caspase-12 (Sigma, Saint Louis, MO); AT8 (Innogenetics, Ghent, Belgium); PHF-1(gift from Dr. Peter Davis, Albert Einstein College of Medicine); tau 5 (from Dr. Lester

Fig. 1. Ah activates the endoplasmic reticulum chaperones. (a) Representative Western blot of grp 94 and grp 78, both detected by KDEL mAb in tissue fractions of hippocampus in control, Ah-treated and GDNF/Ah-treated rabbits. In the controls, grp 94 and grp 78 are detected in the endoplasmic reticulum (er) fraction. In the Ah-treated rabbits, grp 94 and grp 78 bands are detected in both the cytosolic (c) and nuclear (n) fractions. In the GDNF/Ah-treated rabbits, grp 94 is restricted to the nuclear fraction and grp 78 is localized in the cytosolic and endoplasmic reticulum fractions. Calnexin and Histone H1 are used, respectively, as markers for the endoplasmic reticulum and nuclear fractions. (b) Representative photomicrographs of sections immunostained with KDEL (green, detects both grp 78 and grp 94) and NeuN (red, a marker for neuronal nuclei) in the CA1 layer of the hippocampus (Box in A – C, low magnification from a control). In the controls, staining for grp 78 and grp 94 (panel D) and NeuN (panel E) do not colocalize (panel F). In the Ah-treated animal, staining of grp 78 and grp 94 (panel G) is more diffuse, and overlay with NeuN (panel H ) shows that grp 78 and grp 94 staining is either in or near the nuclei (panel I, arrowheads). In a GDNF/Ah-treated animal, the overlay of grp 78 and grp 94 staining (panel J) with NeuN staining (panel K) demonstrates that grp 78 and grp 94 immunostaining occurs mainly outside the nucleus (panel I). (Scale bar in C, 100 Am; I, 10 Am).

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Binder, Northwestern University, Chicago, IL); NeuN (Chemicon Inc., Temecula, CA); and to RCA-1 lectin (Vector Laboratories, Burlingame, CA) or Lectin BS-1 (Sigma). Animals and treatment Adult (2 – 3 years and 4 – 5 kg) female New Zealand white rabbits received either intracisternal injections of 100 Al normal saline (n = 6; controls); 100 Al of 2 mg/ml Ah(1-42) (n = 6; Ah-treated group); or 100 Al of 2 mg/ml Ah(1-42) plus 100 Al of 500 ng/ml GDNF in saline (n = 6; Ah/GDNF-treated group). The GDNF was obtained commercially (R&D systems, Inc., Minneapolis, MN). Aggregated Ah(1-42) (American Peptide Company, Sunnyvale, CA) was prepared by incubating freshly solubilized Ah(1-42) at a concentration of 2 mg/ml in saline at 37jC for 3 days. All rabbits were sacrificed

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7 days after the intracisternal administration, and at necropsy were perfused with Dulbecco’s phosphate-buffered saline (GIBCO, Grand Island, NY) at 37jC. All animal procedures were carried out in accordance with the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Western blot analysis Proteins from whole homogenates or from the nuclear, mitochondrial, cytosolic, and microsomal fractions of the entire hippocampus, extracted as we have described previously (Ghribi et al., 2002, 2003a), were used when indicated. In brief, freshly obtained hippocampal tissue was gently homogenized using a teflon homogenizer (Thomas Scientific, Philadelphia PA) in 7 volumes of cold suspension buffer (20 mM HEPES-KOH

Fig. 2. Ah activates ER stress-induced initiators of apoptosis and TUNEL positivity, effects that are reversed by GDNF. (a) Representative Western blot of gadd 153 in tissue fractions of hippocampus in controls, Ah-treated and GDNF/Ah-treated rabbits. In the control, gadd 153 is detected in the cytosolic fraction. Following Ah administration, gadd 153 is present in the cytosolic fraction but is also present in the nucleus; treatment with GDNF prevents the translocation of gadd 153 into the nucleus. Caspase-12 predominantly resides in the endoplasmic reticulum fraction (er) as procaspase-12 (60 kDa) in the controls. Ah induces cleavage of caspase-12 into 40, 30 and 25 kDa bands in the cytosol (c) and GDNF completely prevents the Ah-induced activation of caspase-12. Calnexin and histone H1, respectively, stain predominantly the endoplasmic reticulum (er) and the nuclear (n) fractions. (b) Representative photomicrographs of sections immunostained with TUNEL (green) in the CA1 layer of the hippocampus in a control, Ah-treated and GDNF/Ah-treated rabbit. (A) TUNEL labeling of DNA fragmentation from the control is negligible (B) TUNEL-positive cells increase dramatically following Ah administration. (C) Addition of GDNF reduces the number of neurons exhibiting DNA fragmentation (Scale bar in C, 10 Am). (D) Quantitative analysis showing that the number of TUNEL-positive neurons are markedly reduced following treatment with GDNF/Ah (n = 6) in comparison to animals treated only with Ah (n = 6).

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(pH 7.5), 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and protease inhibitor cocktail). The nuclear fractions were first isolated by centrifuging the homogenates at 750  g for 10 min at 4jC. The mitochondrial fractions were then isolated from the soluble (cytosolic) fraction at 8000  g for 20 min at 4jC. The 8000 g pellets were resuspended in cold buffer without sucrose and were used as the mitochondrial fraction. The supernatant was further centrifuged at 100,000  g for 60 min at 4jC to separate the cytosolic from the microsomal fractions. Protein concentrations were determined with the BCA protein assay reagent (Pierce, Rockford, IL). Proteins (5 Ag) were separated by SDS-PAGE (10% gel), followed by transfer to a polyvinylidene difluoride membrane (Millipore, Bedford, MD) and incubation with antibodies recognizing grp 94 and grp 78 at a 1:100 dilution; gadd 153, caspase-12, JNK and p-JNK, ERK and pERK at a 1:250 dilution; and AT8, PHF1 and tau 5 at a 1:1000 dilution. Calnexin mAb was applied as an ER marker at 1:500, histone H1 mAb as a nuclear marker at 1:500, and cytochrome c oxidase subunit IV mAb as a mitochondrial marker at a 1:1000 dilution. A mAb reacting with h actin (Sigma) was applied at a 1:250 dilution as a gel loading control. The blots were developed using an enhanced chemiluminescence detection kit (BioRad, Hercules, CA). Since the number of specimens from the various animals and subcellular fractionations did not allow all of the Western blot analyses to be carried out on a single gel, we evaluated the blots in a semiquantitative manner with three independent observers.

Immunohistochemistry Dual labeling. At the time of animal sacrifice, coronally sectioned tissues were rapidly frozen on a platform in liquid nitrogen fumes, placed into zipper closure plastic bags, buried in dry ice, and then stored at 76jC until used. For double labeling with KDEL and NeuN, gadd 153 and lectin-BS-1, AT8 and lectin BS-1, or AT8 and gadd 153, frozen coronal brain sections (14-Am thick) were obtained from the hippocampal level of control, Ah-treated and GDNF/Ah-treated animals were dried for 15 min at room temperature (RT) and fixed in cold acetone for 10 min, followed by a 30-min incubation in methanol/0.03% H2O2. Sections were washed three times in PBS for 5 min each, blocked with 2% goat serum, and incubated overnight at 4jC with mouse anti NeuN antibody (1:100), Lectin BS-1 or AT8. Sections were then washed three times in PBS for 5 min and incubated for 2 h at 37jC in a 1:500 dilution of a-mouse IgG FITC. They were washed in PBS buffer followed by distilled H2O, blocked with 2% goat serum and incubated for 2 h at 37jC in a 1:100 dilution of KDEL mouse mAb, gadd 153 (1:50), or AT8 (1:100), then incubated for 2 h at 37jC in a 1:250 dilution of Cy3-conjugated goat antimouse IgG. After 3  5 min washes in PBS and a 5-min wash in distilled H2O, sections were mounted with Vectashield (Vector Laboratories), coverslipped, and examined with a fluorescence Olympus BH2 microscope (Melville, NY) using an excitation/ emission wavelength of 365/490 nm, and utilizing Image Pro Plus 4.1 analysis software (Media Cybernetics, Baltimore, MD).

Fig. 3. ER stress response correlates with JNK and ERK activation. Representative Western blots of JNK, p-JNK, ERK, and p-ERK and the ratios of levels of p-JNK to JNK and of p-ERK to ERK measured by densitometric scanning from hippocampal tissue. (a) JNK is detected as a double band of 54 and 46 kDa, with similar levels in the control, Ah-treated and GDNF/Ah-treated animals. The active p-JNK is also detected as a double band and is increased by Ah and partially reduced by the addition of GDNF. (b) The ratio of p-JNK to non-phosphorylated JNK of both the 54 and 46 kDa band is increased by treatment with Ah and is reduced by the addition of GDNF. (c) ERK is detected as a double band at 44 kDa and a more intense band at 42 kDa. The active p-ERK is also detected as a double band, barely detectable in controls, but significantly elevated following Ah treatment; GDNF treatment dramatically reduces the Ahinduced increase in p-ERK. (d) The ratio of p-ERK to non-phosphorylated ERK of both the 44- and 42-kDa bands is increased by treatment with Ah and reduced by the addition of GDNF. **P < 0.01, ***P < 0.001 vs. control, +P < 0.05, ++P < 0.01 vs. Ah (ANOVA followed by the Student’s t test).

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TUNEL assay. Apoptosis detection was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) technique on frozen coronal brain sections (14 Am thick) from the hippocampal level of control, Ah-treated and GDNF/Ah-treated animals. Detection of DNA fragmentation was performed using the Apoptosis Detection System (Fluorescein, Promega, Madison, WI). Eight fields at a magnification of 400 were captured from the CA 1 region of the hippocampus from each animal, and results were compared between the Ahtreated and the GDNF/Ah-treated animals. Results from the Ahtreated rabbits were assigned with a value of 100%. The GDNF effect was then expressed as the percent reduction in the number of positive neurons in the GDNF/Ah group when compared to the Ah-treated rabbits. Phosphorylated tau and microglia. A 14-Am thick frozen sections from control, Ah(1-42)-treated, and GDNF/Ah(1-42)treated animals cut at the level of the hippocampus were airdried at room temperature, fixed in cold acetone for 10 min, treated with 1% hydrogen peroxide in PBS and incubated with a blocking solution of 2% normal serum (also in PBS). Subsequently, sections were reacted overnight at 4jC with AT8 (a mouse mAb against phosphorylated tau) at a 1:100 dilution, or with RCA-1 lectin (a marker for microglia) at a 1:1000 dilution. After washing with PBS and incubating with the biotinylated secondary antibody, sections were processed

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with a Vectastain Elite avidin – biotin complex technique kit (Vector Laboratories), and visualized by diaminobenzidine/hydrogen peroxide, and counterstained lightly with hematoxylin. All procedures were carried out at room temperature unless otherwise indicated.

Results and discussion Injection of aggregated Ah into the brain of experimental animals may represent a valuable tool for studying the neurotoxic effect of this peptide. Rabbits more closely resemble primates genetically than they do rodents (Graur et al., 1996) and therefore they may be a useful system to study the pathogenesis of AD to enhance information already obtained from transgenic rodents, aged primates and cell culture. Furthermore, the amino acid sequence for Ah in rabbit, unlike that of rodents, is identical to the human sequence (Johnstone et al., 1991). In addition, rabbits may develop eyeblink conditioning impairment (Woodruff-Pak and Trojanowski, 1996). ER stress response correlates with apoptosis, an effect induced by Ab and prevented by GDNF In controls, the ER-resident proteins, grp 94 and grp 78, are detected in the ER fraction (Fig. 1a). In Ah-treated rabbits, grp 94

Fig. 4. Ah-induced microglial activation. RCA-1 lectin staining, used as a marker for microglia, is examined in the pyramidal cell layer in CA1 of the hippocampus (panel A, box). In the control, only faint staining is observed with RCA-1 (panel B), while in a section from the Ah-treated animal the RCA immunoreactivity is intense and shows processes suggestive of active microglia (panel C, arrowheads). Addition of GDNF to the Ah treatment attenuates the RCA-1 immunoreactivity to a level comparable to that in the control (panel D). (Scale bar in D, 20 Am).

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and grp 78 are localized in the cytosol and nuclear fractions. In GDNF/Ah-treated rabbits, the grp 94 band is restricted to the nucleus and 2 bands for grp 78 are detected, a less intense band in the cytosolic fraction and a more intense band in the ER fraction (Fig. 1a). Calnexin mAb, a specific marker for the endoplasmic reticulum, and histone H1, a marker for nuclei, stain only their respective subcellular fractions (Fig. 1a). The immunofluorescence assay shows that in controls, grp 94, and grp 78 staining is located exclusively outside the nucleus (Fig. 1b, panel F). Following Ah, grp 94 and grp 78 are found in either a nuclear location (nuclei are stained with NeuN, red, arrows) or outside the nuclei (Fig. 1b, panel I). In the GDNF/Ah-treated animals, grp 94 and grp 78 staining are mainly extranuclear with only very few cells exhibiting nuclear staining (Fig. 1b, panel L). The protein gadd 153 is present in the cytosolic fractions in controls and is detected in both the cytosolic and the nuclear fractions in Ah-treated animals; GDNF treatment prevents the translocation of gadd 153 into the nucleus (Fig. 2a).

Procaspase-12 (approximately 60 kDa) is detected primarily in the endoplasmic reticulum from controls; in the Ah-treated animals, cleavage product bands (approximately 40, 30, and 25 kDa) corresponding to activated caspase-12 are localized in the cytosol, and treatment by GDNF fully prevents this cleavage (Fig. 2a). Sections from control animals demonstrate very few scattered TUNEL-positive neurons in the pyramidal layer (CA1) of the hippocampus (Fig. 2b, panel A). Ah administration induces widespread TUNEL-positive cells in this same region of the hippocampus (Fig. 2b, panel B), a process which is decreased following GDNF treatment (Fig. 2b, panel C). Quantitative analysis of TUNEL-positive cells, carried out at a magnification of 400, shows that the Ah-induced TUNEL staining is markedly reduced by GDNF treatment (Fig. 2b, graph D). Expressional patterns of ER chaperones in the brain of AD subjects has shown that grp 78 is increased only in surviving neurons, especially in the CA3 subfield of the hippocampus and in the deep layers of the entorhinal cortex, suggesting that this protein

Fig. 5. Ah-induced phosphorylation of tau is not reduced by GDNF. (a) Representative Western blots of mAbs AT8, PHF and tau 5 in tissue from hippocampus from control, Ah-treated and GDNF/Ah-treated animals. Phosphorylated tau, detected by AT8 and PHF-1, is highly increased in the Ah-treated animal in comparison to the control, and the addition of GDNF does not affect the Ah-induced increase in phosphorylated tau. Non-phosphorylated tau levels, detected by tau 5, do not differ significantly between control, Ah-treated or GDNF/Ah-treated animals. (b) Immunostaining with mAb AT8 in the pyramidal cell layer in CA1 of the hippocampus (box in panel A) from a control (panel B), Ah-treated (panel C) and GDNF/Ah-treated (panel D) animal. Ah administration induces the accumulation of phosphorylated tau in cell processes, as revealed by AT8; this AT8 immunoreactivity is not prevented by the addition of GDNF. (Scale bar in D, 50 Am; scale bar of box in D, 10 Am).

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may protect such cells from AD-specific damage (Hamos et al., 1991). Other studies have demonstrated that grp 78 immunostaining is diminished in AD choroid plexus (Anthony et al., 2003). It has also been found that administration of 2-deoxy-D-glucose to adult rats procures the resistance of synaptic terminals to the dysfunction and degeneration induced by Ah peptide, effects that correlate with increased levels of grp 78 in synaptosomes (Guo and Mattson, 2000). In human embryonic kidney 293 cells, grp 78 has been found to bind to amyloid precursor protein (APP) in the ER and to decrease the levels of Ah40 and Ah42 secretion, suggesting that grp 78 facilitates the correct folding of APP in the ER (Yang et al., 1998). While changes in grp 78 expression are relevant to the pathology of AD, little is known about grp 94 expression. Yoo et al. (2001) have found that the expression of grp 94, but not grp 78, is increased in the parietal cortex of AD patients, and have suggested that this increase may account for abnormalities in the intracellular translocation of protein kinases and the intracellular signal transduction in AD brain. Our results demonstrate that Ah neurotoxicity correlates with a redistribution of both grp 78 and grp 94 from the ER to the cytosol and nucleus. The neuroprotective effect of GDNF affects the Ah-induced subcellular distribution of grps differently, with a confinement of grp 94 exclusively in the nucleus while grp 78 localizes partially in the ER. These results suggest that grp 94 expression in the nucleus is required in order for GDNF to prevent the neuronal stress induced by Ah. Activation of grp 94 and grp 78 is considered to be a mechanism for cells to cope with the toxic buildup of misfolded

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proteins and with changes in Ca2+ levels. However, sustained or intense ER stress leads to activation of the transcription factor gadd 153, a mediator of apoptosis. We demonstrate in our study that gadd 153 exists in the cytoplasm in controls, while following Ah administration, it is activated and translocates to the nucleus. The mechanism by which gadd 153 translocates involves the activation of multiple signaling pathways that include the three sensors of the UPR, PERK (PKR-like ER kinase), Ire1 (high inositol-requiring) a and h, and ATF-6 (activating transcription factor) a and h (Forman and Trojanowski, 2003). Activation of one or more of these sensors leads to the activation of gadd 153. Activated gadd 153 has been implicated in the induction of apoptosis (for review, see Cudna and Dickson, 2003) by mechanisms that are still undetermined. Activation of the ER-specific caspase, caspase-12 (Nakagawa et al., 2000) as well as activation of the MAP kinases (Urano et al., 2000) may be targets for the gadd 153 activation which is induced by ER stress. GDNF inhibits Ab(1-42)-induced JNK and ERK phosphorylation The mAb used in these studies reacts with both JNK1 and 2 and detects a 54- and 46-kDa band in control, Ah-treated and in GDNF/Ah-treated rabbits (Fig. 3a). The mAb recognizing phosphorylated JNK (p-JNK) is immunoreactive with both p-JNK1 and p-JNK2 (54 and 46 bands). In controls, p-JNK levels are low and administration of Ah peptide significantly raises the levels of pJNK; treatment with GDNF significantly reduces the Ah-induced elevation of p-JNK (Fig. 3a).

Fig. 6. Representative immunostaining in the pyramidal cell layer in CA1 of the hippocampus of gadd 153 (green) and AT8 (red) in control (panels A – C), Ah-treated (panels D – F) and GDNF/Ah-treated (panels G – I) animals. Overlay of Gadd 153 and AT8 staining shows that gadd 153 and p-tau do not colocalize in either the Ah-treated (panel F) or GDNF/Ah-treated animals (panel I). Scale bar in I, 10 Am.

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Both 42 and 44 kDa ERK are detected by the antibody to ERK in tissue from hippocampus in the control, Ah-treated, and GDNF/Ah-treated animals (Fig. 3c). The 44-kDa band is less intense than the 42-kDa band in all groups. Activated ERK (p-ERK) is barely detected in controls as a single 42-kDa band, and administration of Ah induces an increase in the level of p-ERK; treatment by GDNF dramatically inhibits this Ah induction of p-ERK (Fig. 3c). The MAP kinases play an important role in regulating apoptosis, and caspase12 activation triggers apoptosis by activating the effector caspases. Our results demonstrate that the Ah-induced activation of gadd 153 correlates with the cleavage of caspase-12 as well as the activation of JNK and ERK kinase and of TUNELpositive neurons. Whether caspase-12 or the JNK and ERK kinase pathway is the determinant for the induction of apoptosis in our animal model system is still to be elucidated. Furthermore, whether caspase-12 activation, following Ah, results from the activation of JNK and ERK or whether JNK and ERK are activators of caspase-12 is also to be determined. Intracisternal perfusion of GDNF suppresses Ah-induced apoptosis by preventing the nuclear translocation of gadd 153 and the cleavage of caspase-12 and by markedly reducing the activation of ERK and, to a lesser extent, JNK. Whether the reduction of phosphorylation of JNK, ERK, or both is required for the anti-apoptotic effect of GDNF remains to be elucidated. JNK and ERK kinases mediate some of the intracellular signal transduction pathways of GDNF.

GDNF has been reported to stimulate (Mograbi et al., 2001; Nicole et al., 2001; Wiklund et al., 2002) or inhibit (McAlhany et al., 2000) the MAP kinase signaling pathway or to be independent of the pathway (Hirata and Kiuchi, 2003). GDNF prevents the Ab-induced activation of microglia RCA-1 lectin staining has been examined in the hippocampus using a region of the CA1 pyramidal cell layer (Fig. 4, box in panel A) for comparison between animals. In the hippocampus of the control, RCA-1 lectin faintly stains microglia (Fig. 4, panel B), whereas following Ah treatment a more intense staining is observed (Fig. 4, panel C). In the GDNF/Ah-treated animal, RCA-1 staining is similar to that observed in the control (Fig. 4, panel D). Vulnerable brain regions in AD exhibit activated microglia (see, for review, Mattson and Chan, 2003) and administration of Ah(142) into rat brain results in a marked increase in microglia (Jantaratnotai et al., 2003). However, whether the response of microglia to Ah represents a defensive mechanism or is a contributor to the neurodegenerative process is not yet clear. Ab-induced tau phosphorylation is not prevented by GDNF Ah treatment results in phosphorylation of the microtubuleassociated protein tau as demonstrated by Western blotting using AT8 or PHF-1 mAbs; this phosphorylation is not affected by

Fig. 7. Representative immunostaining in the pyramidal cell layer in CA1 of the hippocampus with gadd 153 (green) and lectin BS-1 (red) in control (panels A – C), Ah-treated (panels D – F) and GDNF/Ah-treated (panels G – I) animals. All gadd 153-positive cells are distinct from microglia in the control (panel C), Ah-treated (panel F) and in GDNF/Ah-treated (panel I) animals. Note the minimal lectin in the GDNF/Ah-treated animal (panel H) in comparison to the Ahtreated animal (panel E). Scale bar in I, 10 Am.

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Fig. 8. Representative immunostaining in the pyramidal cell layer in CA1 of the hippocampus of AT8 (green) and lectin BS-1 (red) in control (panels A – C), Ah-treated (panels D – F) and GDNF/Ah-treated (panels G – I) animals. The overlay of AT8 and lectin staining shows that some lectin BS1-positive microglia express p-tau, as revealed by AT8 mAb in Ah-treated (panel F, arrowheads) and GDNF/Ah-treated (panel I, arrowheads) animals. Scale bars in I, 10 Am.

GDNF treatment (Fig. 5a). The level of total nonphosphorylated tau at epitope tau 5 does not differ from one group to another. Immunohistochemistry, using AT8, also shows that following Ah

treatment, phosphorylation of tau occurs in the pyramidal cell layer of the hippocampus and GDNF treatment does not prevent the immunoreactivity to AT8 (Fig. 5b). GDNF does not appear to

Fig. 9. Ah induces stress in the endoplasmic reticulum (ER) and hyperphosphorylation of tau (p-tau) by two distinctive mechanisms. Ah perturbs ER functions, either by perturbing Ca2+ homeostasis and/or by activating the unfolded protein response (UPR). Ah-induced perturbation of Ca2+ homeostasis leads to activation of the ER-specific apoptosis pathway involving the activation of caspase-12. In response to the UPR, the ER-resident chaperones are activated to help cells resist stress. However, sustained or intense stress activates the transcription factor gadd 153, which recruits JNK and/or ERK kinases, leading to the activation of apoptosis. GDNF protects against Ah-induced apoptosis and oxidative stress by preventing the perturbation of Ca2+ homeostasis, misfolding of proteins or activation of NK and/or ERK kinases; the p-tau pathway is not affected.

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interfere with the phosphorylation of tau protein induced by Ah in our rabbit system. Gadd 153 does not colocalize with p-tau The ability of GDNF to prevent ER-mediated apoptosis, but not the production of p-tau, suggests that either the apoptosis pathway is more sensitive to GDNF treatment than is p-tau or that apoptosis and p-tau occur through two independent pathways. Immunostaining of gadd 153 (green) is distinct from that of p-tau (red), as assessed by mAb AT8 in both the Ah-treated (Fig. 6, panel F) and GDNF/Ah-treated (Fig. 6, panel I) rabbits. These results indicate that the activation of gadd 153 occurs in cells that do not exhibit ptau. Further dual immunostaining of gadd 153 (Fig. 7, green) and microglia (Fig. 7, red), using lectin BS-1, demonstrates that gadd 153 does not reside in microglia either in control (Fig. 7, panel C), Ah-treated (Fig. 7, panel F) or in GDNF/Ah-treated (Fig. 7, panel I) rabbits. Rather the opposite is observed, as illustrated in Fig. 8: p-tau, stained green with AT8, localizes in both microglial (red) and non-microglial cells in Ah-treated (Fig. 8, panel F) and in GDNF/Ah-treated (Fig. 8, panel I) rabbits. Our results demonstrate that following Ah administration, the ER stress response-induced activation of gadd 153 does not occur in the same cells in which hyperphosphorylation of tau protein is found. Furthermore, while tau phosphorylation is present in both microglial and neuronal cells, gadd 153 staining is not seen in microglial cells. Target cells of GDNF have been considered to be neurons; however, the expression of the GDNF receptors, ret and GFR alpha-1, have also been demonstrated in primary cultures of rat microglia (Honda et al., 1999). Thus, it is unlikely that the inability of GDNF to prevent p-tau is related to whether p-tau is derived from a neuronal or a glial pool. Rather, gadd 153 activation and p-tau might involve two different underlying pathways (Fig. 9), suggesting that glial cells and neurons that exhibit p-tau are more resistant to the Ahinduced ER stress-mediated apoptosis. Although it will be necessary to investigate whether p-tau-positive cells are resistant to apoptosis after a long-term (>7 days) exposure to Ah, it is possible that phosphorylation of tau and apoptosis may be two distinct pathological processes. We have recently shown similar data where treatment with lithium prevents Ah-induced apoptosis but does not prevent the accumulation of p-tau (Ghribi et al., 2003a,b). GDNF mRNA-containing neurons are found in adult human hippocampus in the proximal CA1 to CA3 pyramidal layer, granular layer and hilus, and are sparse in the oriens and molecular layers (Serra et al., 2002). This suggests that GDNF may play a role in the development of intrahippocampal circuitry and in neuronal function and maintenance throughout life (Serra et al., 2002). It may then be possible that a decrease in GDNF activity participates in the degeneration of brain cells in the course of Alzheimer’s disease. Treatment with GDNF has been demonstrated to protect against a variety of degenerative insults, and other growth factors, such as bFGF, have been shown to reduce Ah toxicity in primary hippocampal cultures by mechanisms involving the suppression of oxidative stress (Mattson et al., 1993). Insulinlike growth factor I also protects and rescues hippocampal neurons against Ah-induced toxicity (Dore et al., 1997). It is then possible that the GDNF neuroprotective effect involves the inhibition of oxidative stress, stabilization of Ca2+ homeostasis, and the maintenance of ER functions. In summary, we have demonstrated that Ah administration into rabbit brain induces a cascade of apoptosis, an activation of

microglial cells and tau phosphorylation. The Ah-induced apoptosis involves the activation of the ER stress response which recruits the transcription factor, gadd 153, and the kinases, JNK and ERK. Activation of gadd 153 and the induction of the apoptosis cascade are downstream events to the initial activation of grp 94 and grp 78, which follows Ah administration. Interestingly, GDNF favors a nuclear restriction of grp 94, suggesting that translocation of grp 94 into the nucleus may represent one early defensive mechanism by which GDNF accomplishes its anti-apoptotic action against the effects of Ah. Our data show for the first time that GDNF promotes cell survival in the presence of Ah peptide by preventing the ER stress response-induced activation of gadd 153, JNK and ERK. GDNF also prevents the activation of microglial cells. However, GDNF does not prevent the hyperphosphorylation of tau, another consequence of Ah administration. These results indicate that GDNF can confer protection to cells against the toxic effects of Ah peptide, although this protection is incomplete since the hyperphosphorylation of tau is not prevented by GDNF. Pharmacological agents that specifically prevent the hyperphosphorylation of tau, in addition to GDNF, may thus represent an effective approach to efficiently reverse the toxic effects induced by Ah.

Acknowledgments Supported by the Virginia Center on Aging. We thank Dr. Peter Davies for his gift of antibody to PHF1.

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