Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death

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Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death Isaac G. Onyango,a,b,c,* Jeremy B. Tuttle,a,c and James P. Bennett Jr.b,c,* a

Department of Neuroscience, University of Virginia School of Medicine, PO Box 801392, Charlottesville, VA 22908, USA Department of Neurology, University of Virginia School of Medicine, PO Box 801392, Charlottesville, VA 22908, USA c Center for the Study of Neurodegenerative Diseases, University of Virginia School of Medicine, PO Box 801392, Charlottesville, VA 22908, USA b

Received 20 September 2004; revised 21 February 2005; accepted 23 February 2005 Available online 12 April 2005

The cause of idiopathic PD is obscure, and most cases are sporadic. Oxidative stress and deficiency of various neurotrophic factors (NTFs) could be factors triggering neurodegeneration in the substantia nigra (SN). Cytoplasmic hybrid cells (cybrids) made from mitochondrial DNA of idiopathic PD subjects have reduced glutathione (GSH) levels and increased vulnerability to H2O2. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) rescue PD cybrids from H2O2-induced cell death. GDNF mediated effects require Src kinase and phosphatidylinositol 3-kinase (PI3K)/ Akt activation. Inhibiting either PI3K/Akt or ERK pathways blocks the effects of BDNF. Inhibiting p38MAPK and c-Jun N-terminal kinase (JNK) pathways enhances the neuroprotective effects of both NTFs. These results demonstrate that expression of PD mitochondrial genes in cybrids increases vulnerability to oxidative stress that is ameliorated by both BDNF and GDNF, which utilize distinct signaling cascades to increase intracellular GSH and enhance survival-promoting cell signaling. D 2005 Elsevier Inc. All rights reserved. Keywords: Parkinson’s disease; Cybrids; Neurotrophins; Oxidative stress; Apoptosis; Mitochondria

Introduction The degeneration of dopaminergic neurons of the substantia nigra (SN) is responsible for primary motor symptoms observed in

* Corresponding authors. I.G. Onyango is to be contacted at Department of Neuroscience, University of Virginia School of Medicine, PO Box 801392, Charlottesville, VA 22908, USA. Fax: +1 434 982 4380. J.P. Bennett Jr., Department of Neurology, University of Virginia School of Medicine, PO Box 801392, Charlottesville, VA 22908, USA. E-mail address: [email protected] (I.G. Onyango). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.02.009

Parkinson’s disease (PD). Although the etiology of nigral neuron degeneration remains unknown, post mortem studies show that dying cells bear the signs of apoptosis, in particular chromatin condensation, DNA fragmentation, oxidative damage, mitochondrial dysfunction, and caspase activation (Hajimohamadreza and Treherne, 1997; Mochizuki et al., 1996; Tatton et al., 1998). One of the features in the PD nigrostriatal region is a severe deficiency of various neurotrophic factors (NTFs) (Mogi et al., 1999; Nagatsu, 2002) which could further enhance dopaminergic neuron degeneration and disease symptoms. Although current therapies for PD focus on symptomatic treatment, attention has begun to turn to the possibility of neuroprotective therapies that will slow down disease progression. NTFs specific to dopaminergic neurons could be of significant clinical importance, and gaining insight into the cellular mechanisms underlying the survival signals of NTFs may reveal cellular targets for treating PD. However, individual NTFs may have different effects on the same population of cells through differential activation of signaling mechanisms. Brain-derived growth factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) belong to two different families of NTFs and regulate diverse neuronal functions (Lapchak et al., 1996; Lewin and Barde, 1996; Shen et al., 1997), and both have been shown to play critical roles in the development and function of dopaminergic neurons. BDNF mediates its effects via two classes of receptors: a member of the tropomyosin receptor kinase (Trk) family of receptors, TrkB, and a member of the tumor necrosis factor family of receptors, the p75 neurotrophin receptor (p75NTR) (Friedman and Greene, 1999). Upon ligand binding Trk autophosphorylates on tyrosine residues and stimulates intracellular signaling pathways by activating a variety of enzymes and effectors, including phospholipase C-g, phosphatidylinositol 3-kinase (PI3K), Shc, and mitogen-activated protein kinase (MAPK) (Gunn-Moore et al., 1997; Kaplan and Miller, 2000; Marsh and Palfrey, 1996; Segal and Greenberg, 1996).

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GDNF acts by binding to its receptor complex, which includes the GDNF family receptor a (GFRa) and a membrane Ret protein tyrosine kinase (Ret) (Jing et al., 1996; Saarma and Sariola, 1999; Trupp et al., 1996). Ret activates several intracellular signaling cascades which include the extracellular signal-regulated kinase (ERK), PI3K, p38MAPK (p38), and the c-Jun N-terminal kinase (JNK) pathways (Chiariello et al., 1998; Murakami et al., 1999; Worby et al., 1996). To achieve efficient downstream signaling and maximum levels of GDNF-mediated bioactivity, Ret must be recruited to lipid rafts by GFRa1. Activated Ret interacts with Src family of non-receptor tyrosine kinases (SFKs) only when they are recruited to lipid rafts, although the Src-SH2 docking site on Ret is generated after GDNF stimulation irrespective of the localization of the receptor (Airaksinen and Saarma, 2002; Encinas et al., 2001; Tansey et al., 2000). PI3K and members of the MAPK superfamily, i.e. ERK, JNK, and p38, play central roles in diverse cellular responses, including cell growth, differentiation, survival, and death. Each MAPK is activated by a specific phosphorylation cascade. The balance between these pathways may determine the cell fate (Xia et al., 1995). PLCg is rapidly activated in response to growth factor stimulation and plays an important role in regulating cell proliferation and differentiation and may have a protective function during cellular response to oxidative stress (Wang et al., 2001). Receptor tyrosine kinases interact with adaptor proteins such as Shc (which exists in three isoforms: p66, p52, and p46) to recruit second messengers (Bonfini et al., 1996). Cytoplasmic hybrid cells (cybrids) from patients with idiopathic PD have revealed that expression of PD mitochondrial genes generates multiple detrimental consequences for cell survival that correlate with changes in PD brain. These consequences include decreased complex I activity (Dawson and Dawson, 2003; Schapira et al., 1998; Swerdlow et al., 1996), increased production of reactive oxygen species, increased numbers of morphologically abnormal mitochondria (Trimmer et al., 2000), as well as spontaneous production of true Lewy body inclusions (Trimmer et al., 2004). We have previously shown that, under basal replicating conditions, PD cybrids have decreased viability compared to control cybrids (CNT) that have mitochondria and mitochondrial genes from age-matched, disease free control subjects (Onyango et al., 2005). These multiple changes demonstrate the pervasive cellular consequences of expressing PD mitochondrial genes and provide compelling circumstantial evidence for the involvement of mitochondrial genes in PD pathogenesis. We have previously shown that expression of PD mitochondrial genes in PD cybrids results in the activation of the p38 and JNK signaling pathways leading to apoptotic death. Treatment with NAC reduced or eliminated these death-promoting activities by activating NF-KB downstream of the PI3K pathway suggesting that increased oxidative stress was responsible for the shift towards apoptosis in PD cybrids (Onyango et al., 2005). Protein phosphorylation appears to be an important molecular mechanism for transducing biochemical signals initiated by ROS (Kyriakis and Avruch, 1996). ROS can activate ERK, JNK, p38, and PI-3K/Akt pathway leading to increased gene transcription (Adler et al., 1999; Guyton et al., 1996). In the present study, we found that PD cybrids showed increased sensitivity to death induced by exogenous oxidative stress (H2O2). We then examined the effects of saturating concentrations of BDNF and GDNF on the viability of PD cybrids

exposed to H2O2 and used specific pharmacological inhibitors to systematically characterize the signaling mechanisms that might be involved in protective effects of these NTFs. H2O2-induced apoptosis in PD cybrids was evidenced by increased DNA condensation and poly-ADP ribose polymerase (PARP) cleavage as well as decreased MTT reduction. This loss of viability was associated with a decline in the levels of reduced glutathione (GSH). We found that addition of BDNF or GDNF rescued cybrids from H2O2-induced cell death, and the survivalpromoting effects of both these NTFs were mediated by the activation of the PI3K/Akt and ERK signaling pathways with concomitant reduction in the activation of the p38 and JNK signaling pathways. These findings demonstrate that BDNF and GDNF have an important role in preventing apoptosis in this model and activation of PI3K and ERK dependent pathways plays a pivotal role in mediating these protective effects.

Materials and methods Subject characteristics Patient (PD) and control (CNT) volunteers were recruited after Institutional Review Board approval. Volunteers provided informed consent by signing an approved informed consent document in the presence of witnesses. PD volunteers were enrolled in PD drug trials at the University of Virginia, examined, diagnosed, and staged according to accepted criteria (Hoehn and Yahr, 2001) by sub-specialists experienced with PD diagnosis. Only stages I – IV were eligible but most were stage III. None of the patients was believed to have alternative diagnoses, drug-induced Parkinsonism, another neurodegenerative disease, or any other serious medical illness. The mean age was 75.66 T 3.05 SD for the PD group and 67.00 T 6.24 SD for the CNT group. Both sexes were represented in the PD and CNT group. Generation of cybrids To create cybrids for this study, Rho0 SH-SY5Y cells lacking mtDNA were repopulated with mitochondria containing platelet mtDNA from volunteer patients or age-matched CNT as previously described (Swerdlow et al., 1996). Platelets were isolated from aseptically drawn blood samples collected in acidcitrate-dextrose. Suspensions of Rho0 cells and platelets were mixed and incubated with a polyethylene glycol solution (PEG 1000, W.T. Baker, Phillipsburg, NJ). Next, the cell/PEG solution was diluted with culture medium (10 min) and then transferred to a culture flask. The culture medium on new cybrids was changed every 2 – 3 days. To eliminate cells that were not repopulated with platelet mtDNA, the culture medium was replaced with selection medium that consisted of pyruvate and uridine free Dulbecco’s modified Eagle’s medium with high glucose plus 10% dialyzed fetal calf serum (Hyclone, Logan, UT), penicillin, and streptomycin (Swerdlow et al., 1996). Rho0 cells lacking sufficient donor mitochondria repopulation ultimately died during the 5 – 6 weeks in selection medium. Cybrids that tested negative for mycoplasma were aliquoted and frozen in liquid nitrogen. All experiments were performed on cells at passage 21 – 23 after thawing.

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Cell culture Cybrid cell lines were maintained in 75 cm2 culture flasks in culture medium that consisted of Dulbecco’s modified Eagle’s medium high glucose, 10% fetal bovine serum (Hyclone, Logan, UT), penicillin, streptomycin, uridine, and pyruvate as previously described (Trimmer et al., 2000). The media was changed every 2 – 3 days and the cells were passed every 4 – 5 days. Analysis of cybrid viability Cells were seeded in 96-well plates at a density of 5  103 cells/ well and cultured for 6 – 8 h before treatment with or without either BDNF or GDNF (Sigma, St. Louis, MO), kinase inhibitors (PI-3K inhibitor LY294002, the p38MAPK-specific inhibitor SB203580, MEK specific inhibitor PD98059, and JNK inhibitor I were from Calbiochem, La Jolla, CA). After treatment, the cells were cultured in medium containing 10% fetal bovine serum for 24 h. The cells were treated with 500 Ag/ml 3-(4,5-dimethyl-thiazol-2-yl) 2,5diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO) and cultured for 4 h in a CO2 incubator. Viable cells, i.e. those having functional mitochondrial succinate dehydrogenase, can convert MTT to formazan that generates a blue color when dissolved in dimethyl sulfoxide (Mosmann, 1983). The intensity was measured spectrophotometrically at an absorption wavelength of 565 nm. The results are expressed as percentage MTT reduction relative to CNT cultures. Quantification of apoptotic nuclei PD and CNT cybrids were cultured on LabTek 4 well chamber slides (Nalge Nunc International, Naperville, IL). In these experiments, the effects of the anti-oxidant N-Acetylcysteine (NAC) (1 mM), 20 AM buthionine sulfoximine (BSO), which depletes GSH in cells, 50 ng/ml BDNF, 10 ng/ml GDNF, or various concentrations of H2O2 were tested. Hoechst staining was performed 24 h after treatment. Briefly, the slides were washed with PBS and then fixed with ice-cold 4% paraformaldehyde for 30 min. After washing with PBS, the cells were stained with Hoechst 33258 (0.01 mg/ml in PBS) for 20 min at room temperature under dim light. The slides were then washed and mounted with coverslips in glycerol – methanol (1:1 v/v) and stored at 4-C until quantification by epifluorescence microscopy using a UV 2A DM 400 filter (330 – 380 excitation wavelength). Three slides were used per experimental group, with at least 200 cells in four random fields being counted on each slide. GSH assay GSH levels were measured using a fluorometric assay method with monochlorobimane (mBCl) as previously described (Sebastia et al., 2003). Briefly, PD and CNT cybrids were seeded at 3 105/ ml in 96-well plates (Corning, Corning, NY) and used after 24 h. mBCl was dissolved in dimethyl sulfoxide. Working solutions in PBS at 1 mM were prepared immediately before use and 10 Al added directly to the each well 96-well plate of cybrid cultures in 190 Al of media. Fluorescence was recorded before (blank) and 40 min after adding the probe in a Fluoroskan Ascent FL plate reader (MTX Labsystems Inc., Vienna, VA) at the following excitation/ emission wavelengths T bandpass: 365 nm/460 nm. Results were obtained by subtracting blank values and presented as fluorescence

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arbitrary units or as a percentage of CNT. Parallel plates were grown to normalize sample values to protein values. Immunoprecipitation and Western blot analysis Cells at ¨80% were stressed with a 15 min pulse of 50 AM H2O2 and then switched to control media or media supplemented with either 50 ng/ml BDNF or 10 ng/ml GDNF. For the pharmacological inhibitor studies, cybrids were treated with or without either PP2, PD98059, LY29402, SB203580, or JNK inhibitor I 30 min before the 15 min H2O2 pulse. The control and neurotrophic media were supplemented with the respective kinase inhibitor. The cultures were washed in ice-cold PBS and immediately lysed in modified radioimmunoprecipitation (RIPA) buffer. After 30 min incubation on ice, cells were disrupted by a 10 s sonication. Lysates were then centrifuged at 14,000  g for 10 min. The precipitates were discarded and the protein concentrations of the supernatants determined by the BCA protein assay kit (BioRad, Hercules, CA) using bovine serum albumin as a standard and then stored at 80-C. For immunoprecipitation, 800 Ag total cellular protein was precleared by the addition of 1 Ag of the appropriate control IgG together with 20 Al of volume of suspended (25% v/v) protein A/G agarose conjugate and incubated at 4-C for 30 min. After a 30 s centrifugation at 500  g, the supernatant was transferred into a microcentrifuge tube and 1 Ag of the appropriate primary antibody added. This mixture was mixed by end over end rotation at 4-C for 2 h. 20 Al of protein A/G agarose suspension was added to the mixture and incubation continued with end over end rotation at 4-C for 1 h. The immunoprecipitates were collected by centrifugation at 500  g for 30 s and washed gently 4 times with PBS each time repeating the centrifugation step. After the final wash, the supernatant was carefully aspirated and discarded and the pellet resuspended in 40 Al of 2  electrophoresis sample buffer, boiled for 3 min, and analyzed by SDS-PAGE. For Western blot analyses, 50 Ag of protein was resolved by SAD-PAGE and transferred onto nitrocellulose membrane by the standard Western blotting technique. The primary antibodies were Trk, Ret, GFRa1, ERK, pERK, Akt, pAkt, p38MAPK, p p38MAPK, PLCg1, Shc, a-actin, phosphotyrosine, PARP, and secondary HRP conjugated IgGs were from Santa Cruz Biotech (Santa Cruz, CA) and pJNK from Upstate (Charlottesville, VA). The antigen – antibody complexes on the membranes were detected with HRP-conjugated secondary antibody and visualized using the ECL system (Amersham). The membranes were stripped with Restorei Western blot stripping buffer from Pierce (Rockford, IL) and re-probed for total protein or a-actin. Horizontal scanning densitometry was performed on Western blots using Kodak 1Di image analysis system. Statistics Data were analyzed for statistical significance by a one-way analysis of variance using GraphPad Prism software (GraphPad Software Inc, San Diego, CA). If a significant F value was obtained (P < 0.05), the Dunnett’s multiple comparison test was used for post-hoc significance testing. The level of significance for all comparisons was set at P < 0.05. In all experiments, the different experimental protocols were tested at least three times on the 3 different PD and 3 different CNT cybrids cell lines and representative data shown.

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Results Table 1 lists the age and gender of the subjects from whom platelets were obtained to create cybrid cell lines. In the PD group, there are more men than women, which is well recognized as part of the disease demographic characteristics.

(Fig. 3). Fluorescent microscopy also revealed more cells with condensed nuclei apoptotic nuclei (72% and 44%) compared to CNT cybrids (not shown). Based on these results, all subsequent experiments utilized a dose of 50 AM H2O2. BDNF and GDNF protect PD cybrids against H2O2-induced cell death

Ret, TrkB, and GFRa1 receptors are expressed in PD cybrids Immunocytochemistry with antibodies specific for TrkB, Ret, and GFRa1 was used to determine whether receptors for BDNF and GDNF are expressed in PD cybrids. We found that TrkB, Ret, and GFRa1 are expressed in PD and CNT cybrids (Figs. 1B – D). There was no significant difference in the expression levels of these receptors in PD and CNT cybrids. Furthermore, we were able to confirm/detect expression of these receptors by Western blot analysis (Fig. 1E). This raised the possibility that cybrids may be capable of responding to both BDNF and GDNF. BDNF and GDNF increase PD cybrid viability The effects of NTFs were initially assessed by treating the cultures over a 24 h period with either 50 ng/ml BDNF or 10 ng/ml GDNF and examining their impact on viability. Both NTFs significantly enhanced PD cybrid viability as measured by the MTT reduction assay (Fig. 2A) and also by counting the numbers of Hoechst stained condensed nuclei (Fig. 2B). Both GDNF and BDNF suppress PD cybrid PARP cleavage BDNF and GDNF-induced inhibition of caspase 3 activity was provided by experiments on the stability of poly-ADP ribose polymerase (PARP), a DNA repair enzyme and important endogenous nuclear substrate of caspase 3 and caspase 7 (Decker and Muller, 2002). PD cybrids have enhanced cleavage of the native 116 kDa PARP that results in the appearance of a 85 kDa degradation product. GDNF as well as BDNF blocked PARP degradation while CNT cybrids do not exhibit detectable PARP cleavage (Fig. 2C). Effects of H2O2 on PD cybrid viability Because oxidative stress may contribute to neurodegeneration, we evaluated PD cybrid viability after exposure to H2O2. We induced oxidative stress with a 15 min pulse of H2O2 (50, 100, or 500 AM) and monitored MTT reduction and nuclear condensation 24 h later. 500 AM H2O2 killed virtually all PD cybrids after 24 h (not shown). 100 AM H2O2 and 50 AM H2O2 reduced PD cybrid viability to ca. 16% and 39% of untreated CNT respectively

Table 1 Characteristics of subjects Cybrid line number

Age (years)

Gender

CNT 56 CNT 62 CNT 68 PD 60 PD 63 PD 66

72 60 69 79 73 75

M F M F M M

We evaluated the effects of BDNF and GDNF treatment on the viability of PD cybrids treated with 50 AM H2O2. Cybrids at ca. 80% confluence were stressed with a 15-min pulse of 50 AM H2O2. The cells were then switched to control media or media supplemented with the either 50 ng/ml BDNF or 10 ng/ml GDNF. Both BDNF and GDNF protected PD cybrids from H2O2-induced cell death (Fig. 4). Effects of BDNF and GDNF on PD cybrid GSH levels GSH deficiency may be one of the primary events in the cascade of cell death (Ebadi and Hiramatsu, 2000). GSH provides an important non-enzymatic cellular defense against free radicals. The levels of GSH are selectively reduced in substantia nigra of PD patients and this phenomenon does not occur in other neurodegenerative disorders (Sian et al., 1994). In order to test whether GSH depletion is critical for loss of PD cybrid viability, we examined the levels of GSH in PD cybrids under basal conditions and after treatment with either BDNF, GDNF, or NAC, a precursor for GSH synthesis. PD cybrids under basal conditions have decreased GSH levels compared to CNT cybrids, and PD cybrid GSH levels were reduced further by H2O2 and to a greater degree than were CNT cybrid GSH levels (Fig. 5A). While BDNF and GDNF did not significantly alter CNT cybrid GSH levels, they both significantly increased PD cybrid GSH levels. NAC significantly enhanced GSH levels while H2O2 dramatically reduced GSH levels in both PD and CNT cybrids (Fig. 5A). To further assess the role of GSH in NTF mediated neuroprotection, we examined the effect of GSH depletion in BDNF and GDNF treated cybrids. Cybrids were pretreated with BSO, an inhibitor of g-glutamylcysteine synthetase (Griffith and Meister, 1979), 24 h before exposure to either BDNF or GDNF. BSO reduced GSH levels in both CNT and PD cybrids (Fig. 5B). This decline in GSH levels reduced the protective effects of both BDNF and GDNF (Fig. 5C). This suggests that the H2O2-induced death in PD cybrids may be affected by decreased levels of GSH and that the protective effects of BDNF and GDNF may derive from increased GSH levels. Involvement of PI3K in BDNF and GDNF stimulated survival promotion The H2O2-induced neuronal death of PD cybrids is prevented by the addition of BDNF or GDNF. To examine the reason why the strengths of the survival-promoting effects of BDNF and GDNF were dissimilar in PD cybrids, we compared the signaling pathways that mediate the effects. BDNF and GDNF are known to exert their survival-promoting effects by activating the PI3K pathway via Trk and Ret respectively. We first examined the effects of 10 AM LY294002, a widely used specific inhibitor of PI3K, on the viability of cybrids treated with H2O2 in the presence or absence of either BDNF or GDNF. LY294002 abrogated the

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Fig. 1. Morphological appearance of PD and CNT cybrids after 24 h in culture (A). Phase contrast micrograph of cybrids. (B). Cybrids labeled with a polyclonal antibody against the BDNF receptor TrkB (C). Cybrids labeled with a polyclonal antibody against the GDNF receptor Ret (D). Cybrids labeled with a polyclonal antibody against the GDNF receptor GFRa-1 (magnification: 40). (E) Western blot analyses confirming expression of Trk, Ret, and GFRa-1 in PD and CNT cybrids. a-actin was used as the loading control.

survival-promoting effects of both BDNF (Fig. 6A) and GDNF (Fig. 6B). This suggests that Akt is essential for the survivalpromoting effects of these NTFs in PD cybrids. We also examined the phosphorylation of Akt. Lysates from cybrids treated with

H2O2 in the presence or absence of either BDNF or GDNF were immunoblotted with anti-phospho-Akt antibody, which reacts only with phosphorylated and activated Akt. Akt is phosphorylated in response to both BDNF and GDNF (Fig. 6C).

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Fig. 3. Effects of exogenous H2O2 on PD cybrid viability. Exogenous stress was induced in cybrids by a 15 min pulse of (50, 100, or 500 AM) H2O2. The cybrids were then transferred to control media and survival measured 24 h later by the MTT assay. Data represent mean T SEM of three separate determinations with the 3 different PD and 3 different CNT lines.

ERK activation is not required in GDNF stimulated survival promotion, but it is important for BDNF mediated protection of H2O2 treated PD cybrids An increasing body of evidence demonstrates that signaling through Ras contributes to the regulation of differentiation and survival of neurons and these effects normally involve activation of the ERK pathway (Bonni et al., 1999; Nobes and Tolkovsky, 1995). To further characterize the difference in survival-promoting effects of BDNF and GDNF in PD cybrids, we compared their sensitivity to PD98059, after treatment with H2O2. PD98059 is a specific inhibitor for MAP/ERK kinase (MEK-1), the enzyme that phosphorylates and activates ERK. While PD98059 did not have a significant effect on the survival-promoting effects of GDNF (Fig. 6B), it blocked the survival-promoting effects of BDNF (Fig. 6A). We assayed lysates from cybrids treated with H2O2 and stimulated with either BDNF or GDNF in the presence of PD98059 by Western blot analysis for ERK activation using pERK specific antibodies. PD98059 inhibited both BDNF and GDNF stimulated ERK phosphorylation (Fig. 6C).

Fig. 4. BDNF and GDNF protect PD cybrids from H2O2 injury induced cell death. Exogenous stress was induced by a 15 min pulse of 50 AM H2O2 in the presence or absence of NTFs. Survival was measured 24 h later by the MTT assay. BDNF and GDNF treated cybrids had significantly higher viability compared to their untreated counterparts after H2O2 injury. Data represent mean T SEM of three separate determinations with the 3 different PD and 3 different CNT lines. aP < 0.05 relative to CNT cultures; bP < 0.05 relative to PD cultures; and cP < 0.05 relative to 50 AM treated PD cultures. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test for post-hoc significance testing.

neurotrophic mediated PI3K and ERK activation and cybrid survival. To test the functional relevance of SFKs in BDNF and GDNF mediated PD cybrid viability, we used PP2, a selective inhibitor of SFKs (Salazar and Rozengurt, 1999). Cybrids at 80% confluence were switched to a culture medium supplemented with either BDNF or GDNF in the presence or absence of 1 AM PP2. After 24 h, evaluation of PD survival showed that PP2 blocked the survival-promoting properties of GDNF (Fig. 6B). The inhibition of SFK activity had no significant effect on BDNF mediated survival (Fig. 6A). We did not identify the member(s) of the SFKs that mediate GDNF survival in PD cybrids.

SFK activity is selective for GDNF mediated PD cybrid survival

BDNF induces a stronger phosphorylation of PD cybrid Shc proteins

SFKs may present proximal signaling elements necessary for maximal downstream signaling or the optimal biological effects of NTFs. We examined if SFK activity was necessary for optimal

To study the respective effects of BDNF and GDNF upstream of PI3K and ERK signaling, we immunoprecipitated Shc from lysates of cells treated with either BDNF or GDNF. The

Fig. 2. BDNF and GDNF decrease apoptotic cell death in PD cybrids. Cybrids were plated in 96-well plates or LabTek 4 well chamber slides for 6 – 8 h before treatment with or without either BDNF or GDNF for 24 h. Cell viability was assessed by the MTT assay (A) or Hoechst staining (B). Under basal conditions, PD cybrids are less viable than CNT cybrids. Both BDNF and GDNF significantly enhance PD cybrid viability. For Hoechst staining, three slides were used per experimental group, with at least 200 cells in four random fields being counted on each slide. Data represent mean T SEM of three separate determinations with the 3 different PD and 3 different CNT lines. aP < 0.05 relative to CNT cultures; bP < 0.05 relative to PD cultures. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test for post-hoc significance testing. (C) CNT cybrids and PD cybrids treated with either BDNF or GDNF were rapidly lysed and aliquots used to determine PARP processing by Western blot analysis. Analysis was performed for the full-length PARP (116 kDa) and its cleaved fragment (85 kDa). As a loading control, a-actin was used.

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Fig. 5. The effects of BDNF and GDNF on cybrid GSH levels. GSH levels were determined using a fluorometric assay method of the dye monochlorobimane (mBCl) in cybrids under basal conditions and after (A). Treatment with either BDNF, GDNF, H2O2, or NAC. (B) Treatment with 20 AM BSO before exposure to either BDNF or GDNF (C). Data represent mean T SEM of three separate determinations with the 3 different PD and 3 different CNT lines. (C) Exogenous stress was induced by a 15 min pulse of 50 AM H2O2 in the presence or absence of NTFs in 20 AM BSO pre-treated cybrids. Viability was measured 24 h later by the MTT assay. aP < 0.05 relative to CNT cultures; bP < 0.05 relative to PD cultures; bP < 0.05 relative to PD cultures treated with BSO; and dP < 0.05 relative to BSO treated CNT cultures. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test for post-hoc significance testing.

immunoprecipitates were then immunoblotted with anti-phosphotyrosine antibody. Both NTFs also stimulated phosphorylation of the Shc isoforms p66 and p46 although GDNF stimulated Shc phosphorylation to a lesser extent that BDNF (Fig. 7). Since Shc proteins are the predominant activators of the Ras-ERK pathway (Bruning et al., 1997; Ricketts et al., 1996; Sasaoka et al., 1994, 1996), this may explain why inhibition of the ERK pathway blocked the survival-promoting effects of BDNF.

significantly enhanced PD cybrid viability in the face of H2O2induced stress. The addition of either BDNF (Fig. 8A) or GDNF (Fig. 8B) enhances PD cybrid survival to basal CNT levels. To ascertain whether SB203580 and JNK inhibitor I could actually block p38MAPK and JNK activation respectively, we examined the effects of these inhibitors on p38MAPK and JNK phosphorylation. As shown in Fig 8C, pretreatment of the cells with SB203580 inhibited p38MAPK phosphorylation and JNK inhibitor I inhibited JNK phosphorylation.

BDNF and GDNF activate PLCc to the same extent BDNF and GDNF are also capable of activating PLCg. To examine whether these NTFs show differences in intracellular signaling via PLCg, we examined their effects on PD cybrid PLCg phosphorylation. We immunoprecipitated PLCg from lysates of cybrids treated with either BDNF or GDNF and immunoblotted the immunoprecipitates with anti-phosphotyrosine antibody. We found no difference in the tyrosine phosphorylation signaling of PLCg induced by BDNF and GDNF (not shown). Inhibition of p38MAPK and JNK pathways increases survival in H2O2 treated PD cybrids while NTFs enhance PD cybrid survival to CNT levels without affecting the phosphorylation activation of either p38MAPK or JNK. JNK and p38MAPK are often implicated in cell death (Chang and Karin, 2001) and stress signals are known to upregulate p38MAPK in neurons (Kawasaki et al., 1997). We have previously demonstrated that the decrease in PD cybrid viability is mediated by the stress activated protein kinases (SAPK) (Onyango et al., 2005). To address the potential involvement of p38MAPK and JNK pathways in H2O2-induced neurotoxicity, we pretreated cybrids with inhibitors specific for p38MAPK (SB203580) and JNK (JNK inhibitor I) pathways prior to H2O2 exposure in the presence or absence of either BDNF or GDNF. Both inhibitors

Discussion There are four major new findings of the present study. First, PD cybrids are more susceptible to H2O2-induced stress than CNT cybrids. This loss of viability is associated with a decline in the levels of GSH. Second, PD cybrids express the NTF receptors TrkB, Ret, and GFRa1 at about the same level as CNT cybrids, and the addition of either BDNF or GDNF rescues these cells from H2O2-induced neuronal death. Third, comparison of the intracellular signaling stimulated by each NTF revealed that BDNF and GDNF utilize distinct signaling mechanisms to exert these effects. While the PI3K/Akt pathway and SFK activity are primarily important for the survival-promoting effects of GDNF, BDNF utilizes both the PI3K/Akt and ERK pathways to protect PD cybrids against H2O2-induced apoptotic death. BDNF also stimulates phosphorylation of Shc 66 and 46. Finally, the survival-promoting effects of both NTFs are associated with decreased activation of the p38MAPK and JNK pathways. We have previously shown that, under basal conditions, PD cybrids have a significant activation of pro-apoptotic cell signaling and biochemistry that appears to derive from oxidative stress (Onyango et al., 2005). Indeed, neurons undergoing oxidative

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Fig. 6. Effects of SFK, PI3K, and ERK1/2 inhibitors on the protective effect of BDNF and GDNF on H2O2 treated PD cybrids. Cybrids were treated for 30 min with either 1 AM PP2, 25 AM LY294002, or 25 AM PD98059 before being exposed to a 15 min H2O2 pulse in the presence of NTFs. Cybrid survival was evaluated after 24 h in culture with the respective NTF: (6A = BDNF; 6B = GDNF). Extracts of the cybrids were subjected to SDS-PAGE with subsequent Western blot analysis with antibodies against phosphospecific Akt or ERK1/2 antibodies. Data represent mean T SEM of three separate determinations with the 3 different PD and 3 different CNT lines. aP < 0.05 relative to CNT cultures; bP < 0.05 relative to PD cultures; and cP < 0.05 relative to 50 AM treated PD cultures. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test for post-hoc significance testing.

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Fig. 7. (A) BDNF induces robust tyrosine phosphorylation of Shc proteins. Shc proteins were immunoprecipitated from lysates of cybrids treated with either BDNF or GDNF and probed with an anti-phosphotyrosine antibody. The lower panel shows reprobing of the filter with anti-Shc antibodies. (B) Quantitative analysis of phospho Shc values (means T SEM, n = 3), *P < 0.05.

stress and apoptosis have been observed in the SN of post mortem brains with PD (Mochizuki et al., 1996; Sian et al., 1994). Because PD cybrids show evidence of increased production of reactive oxygen species (Swerdlow et al., 1996), in spite of increased expression of anti-oxidative enzymes (Cassarino and Bennett, 1999), the question arises as to how this oxidative stress is generated. In the present study, we found that depletion of GSH may explain both our previously reported oxidative stress and our recent findings of increased neuronal death (Onyango et al., 2005) in PD cybrids. GSH functions as an essential antioxidant neutralizing oxidative stress caused by free radicals. In this study, we found that GSH levels in PD cybrids are significantly reduced compared those in CNT cybrids. Multiple studies confirm that the brain’s SN region is abnormally depleted of GSH in PD patients (Jenner, 1998; Kidd, 2000; Perry et al., 1982; Riederer et al., 1989). In animal and cell-level experiments, depletion of nigrostriatal GSH is associated with enhanced sensitivity to oxidants and to complex I impairment. In cell culture of immature cortical neurons and neuronal cell lines, GSH depletion induces cell death (Heales et al., 1995; Li et al., 1997) and GSH deficiency leads to mitochondrial damage in the brain (Jain et al., 1991). GSH depletion seemingly occurs early in the disease pathogenesis, so it may be a central factor in the process (Foley and Riederer, 2000; Youdim et al., 1989) rendering the cells vulnerable to toxin induced degeneration (Jenner and Olanow, 1998). The decreased

levels of GSH may partly explain why PD cybrids are more vulnerable to exogenous stress compared to CNT cybrids. Neurotrophic factors have been shown to protect neurons against oxidative stress by inducing an increase in the activities of antioxidant enzymes and modulate expression of apoptosis-related proteins in a pro-survival manner. It is speculated that the differences in relative abilities of different NTF’s to suppress ROS are likely due to differences in their specific signal transduction mechanisms and gene products affected (Heaton et al., 2003a,b; Mattson et al., 1995). GDNF has been shown to prevent degeneration of nigral dopaminergic (DAergic) neurons following 6-hydroxydopamine (6-OHDA) injection (Lin et al., 1993; Sauer et al., 1995) and BDNF has been demonstrated as a trophic factor for cultured DAergic neurons (Hyman et al., 1991), as well as a protective agent against 6-OHDA neurotoxicity (Spina et al., 1992). We found that BDNF and GDNF promoted viability of PD cybrids and offered protection against H2O2-induced cell death. We found no significant difference in the protein expression levels of TrkB, Ret, and GFRa1 in PD cybrids compared to CNT cybrids. Although the neuronal content for TrkB in PD has been shown to be unchanged relative to age-matched controls (Benisty et al., 1998), the levels of the Ret component of the GDNF receptor have been found to be slightly reduced in PD compared to age-matched control brains (Walker et al., 1998). Ret must be recruited to lipid rafts by GFRa1 to achieve efficient downstream signaling and maximal GDNF mediated bioactivity (Tansey et al., 2000). These microdomains may function as specialized signaling organelles since they are rich in signaling proteins such as SFKs (Anderson, 1998). SFKs may therefore present proximal signaling elements necessary for maximal downstream signaling of GDNF. We found that SFK activity was necessary for optimal GDNF mediated PD cybrid survival after exposure to H2O2. Treating cybrids with PP2 selectively blocked the ability of GDNF to protect PD cybrids against H2O2-induced death. Src has been shown to activate PI3K/ Akt to ultimately promote cell survival (Schlessinger, 2000). Consistent with this, our results show that SFK inhibition attenuated the survival-promoting effects induced by GDNF. We then focused on elucidating the potential role of PI3K and MAPKs in the NTF mediated neuroprotection against H2O2induced apoptosis in PD cybrids. We found that PI3K/Akt signal transduction system is activated by both BDNF and GDNF in H2O2 treated PD cybrids. Inhibition of the PI3K/Akt pathway was very effective in reversing the protective effects of both BDNF and GDNF suggesting that this pathway is important for the NTFs protection against H2O2-induced cybrid death. This is consistent with its functions in many different cells, where PI3K pathway may be activated directly by various growth factors and indirectly through activated Ras to regulate the localization and the activity of a key enzymes in the cell survival and anti-apoptotic pathway such as PDK1 and Akt/PKB (Cantley, 2002; Franke et al., 1997; Fruman et al., 1998; Vanhaesebroeck and Alessi, 2000; Wymann et al., 2003). The effects of GDNF in PD cybrids are possibly mediated through the PI3K/Akt pathway downstream of SFK. BDNF and GDNF activated the ERK signal transduction systems in H2O2 treated PD cybrids. Blocking the ERK pathway using PD98059 inhibited protective effects of BDNF but did not significantly alter the survival-promoting effects of GDNF. This supports the observation that activation of ERK by some conditions of stress, particularly oxidant injury, or by growth

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factors has been shown to confer a survival advantage to cells (Guyton et al., 1996; Wang et al., 1998; Xia et al., 1995). Shc is involved in signal transduction between the tyrosine kinases and

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the Ras proteins which stimulates the ERK signaling cascade (Marshall, 1996). While the tyrosine phosphorylation levels of PLCg1 remained unaltered, BDNF elicited a stronger phosphor-

Fig. 8. Effects of SAPKs inhibitors on the protective effect of BDNF and GDNF on H2O2 treated PD cybrids. Cybrids were treated for 30 min with 5 AM SB203580 or 1 AM JNK inhibitor I before being exposed to a 15 min H2O2 pulse in the presence of NTFs. Cybrid survival was evaluated after 24 h in culture with the respective NTF. Extracts of the cybrids were subject to SDS-PAGE with subsequent Western blot analysis with antibodies against phosphospecific p38MAPK or JNK antibodies. Data represent mean T SEM of three separate determinations with the 3 different PD and 3 different CNT lines. *P < 0.05 relative to CNT cultures; data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test for post-hoc significance testing.

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ylation of Shc proteins than GDNF in H2O2 treated PD cybrids. This may partly explain why inhibition of the ERK pathways blocked the survival-promoting effects of BDNF and not GDNF. These data suggest that, while the PI3K/Akt pathway is primarily important for protective effects of GDNF, BDNF utilizes both the PI3K/Akt and ERK pathways to protect PD cybrids against H2O2-induced neuronal death. The survival-promoting effects of both NTFs in PD cybrids seem independent of p38MAPK and JNK activity suggesting that the NTF effect may be different from blockade of p38MAPK or JNK. In summary, in this study, we have shown that PD cybrids are more vulnerable than CNT to H2O2 injury. The cell death outcome after acute oxidative injury in PD cybrids is dependent upon the suppression of PI3K/Akt pathway and activation of the p38MAPK and JNK pathways. Revealing the mechanism of this phenomenon will require additional studies. Exogenous addition of either BDNF or GDNF significantly blocked neuronal death by distinct usages of the PI3K/Akt and ERK pathways and suppression of the activities of the p38MAPK and JNK pathways in this cellular model. These findings in a biologically faithful, mitochondrial gene-derived cybrid cell model of sporadic PD replicate the GSH deficiency found in PD brain, demonstrate how PD neurons may have increased vulnerability to oxidative stress, and demonstrate the potential therapeutic value of both NTFs and suppression of pro-apoptotic JNK and p38MAPK signaling.

Acknowledgments Supported by the generosity of American taxpayers through NIH grants AG14373, NS39005, and NS39788.

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