J. Neurochem. (2011) 117, 403–411.
Oxidative stress, associated with a variety of disorders including neurodegenerative diseases, results from accumulation of reactive oxygen species (ROS). Oxidative stress is not only responsible for neuron apoptosis, but can also provoke astroglial cell death. Numerous studies indicate that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuron survival, but nothing is known regarding the action of PACAP on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of PACAP on H2O2-induced astrocyte death. Pre-treatment of cultured rat astrocytes with nanomolar concentrations of PACAP prevented cell death provoked by H2O2 (300 μM), whereas vasoactive intestinal polypeptide was devoid of protective activity. The effect of PACAP on astroglial cell survival was abolished by the type 1 PACAP receptor antagonist, PACAP6-38. The protective action of PACAP was blocked by the protein kinase A inhibitor H89, the protein kinase C inhibitor chelerythrine and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. PACAP stimulated glutathione formation, and blocked H2O2-evoked ROS accumulation and glutathione content reduction. In addition, PACAP prevented the decrease of mitochondrial activity and caspase 3 activation induced by H2O2. Taken together, these data indicate for the first time that PACAP, acting through type 1 PACAP receptor, exerts a potent protective effect against oxidative stress-induced astrocyte death. The anti-apoptotic activity of PACAP on astrocytes is mediated through the protein kinase A, protein kinase C and MAPK transduction pathways, and can be accounted for by inhibition of ROS-induced mitochondrial dysfunctions and caspase 3 activation.
Dulbecco’s modified Eagle’s medium
foetal bovine serum
pituitary adenylate cyclase-activating polypeptide
protein kinase A
protein kinase C
reactive oxygen species
vasoactive intestinal polypeptide
- VPAC1-R and VPAC2-R
VIP/PACAP mutual receptors
Glial cells were traditionally thought to be detrimental in neurodegenerative disorders, but increasing evidence suggests that reactive astrocytes may actually protect neurons against oxidative stress in diverse neuropathological conditions such as stroke, Alzheimer’s disease and Parkinson’s disease (Emerit et al. 2004; Takuma et al. 2004; Reynolds et al. 2007). In fact, astrocytes contribute to the defense of surrounding neurons by providing trophic supports and antioxidant molecules such as the glial cell-line derived neurotrophic factor (Sandhu et al. 2009) and glutathione precursors (Bolanos et al. 1996; Dringen et al. 2000; Takuma et al. 2004). These data emphasize the prominent role of astrocytes in the protection of neurons against oxidative stress and cell death. Nevertheless, it has recently been reported that sustained oxidative stress could also induce astrocyte cell death (Lu et al. 2008). Among the reactive oxygen species (ROS) that are responsible for oxidative stress, H2O2 is regarded as a key substance involved in both neuronal (Vaudry et al. 2002) and astroglial cell death (Ferrero-Gutierrez et al. 2008). As, loss of glial cells may critically impair neuronal survival, protection of astrocytes from oxidative insult appears essential to maintain brain function.
It is well documented that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)-secretin-glucagon family (Miyata et al. 1989), exerts potent neuroprotective effects in models of neurodegenerative diseases, traumatic brain injury and stroke (Vaudry et al. 2009), but nothing is known regarding the protective role of PACAP in astroglial cells. PACAP and VIP act through three types of receptors, the PACAP-selective PAC1-R and the PACAP/VIP mutual receptors VPAC1-R and VPAC2-R (Vaudry et al. 2009). These three PACAP receptors are expressed in both resting (Joo et al. 2004) and reactive astrocytes (Van Landeghem et al. 2007), and their activation modulates proliferation, differentiation (Hashimoto et al. 2003; Nishimoto et al. 2007), plasticity (Perez et al. 2005) and metabolism of astroglial cells (Magistretti et al. 1998), as well as the release of neuroprotective factors (Dejda et al. 2005; Masmoudi-Kouki et al. 2007). In particular, it has been shown that PACAP induces the release of interleukin-6 (Ohtaki et al. 2006) and regulated on activation, normal T expressed and secreted (RANTES)/chemokines (Brenneman et al. 2002) from cultured astrocytes. Moreover, recent data indicate that PACAP protects astrocytes from β-amyloid peptide-induced cell death (Shieh et al. 2008), suggesting that endogenous PACAP may exert glioprotective effects and thereby neuroprotective activities. In agreement with this hypothesis, up-regulation of both PACAP and PACAP receptor expression has been observed in astrocytes after stroke (Shioda et al. 2004; Stumm et al. 2007).
Although there is clear evidence that PACAP receptors are expressed by glial cells, a possible effect of PACAP on astrocyte survival has never been investigated. The purpose of the present study was thus to examine the possible protective effect of PACAP against H2O2-induced astroglial cell death and to investigate some of the mechanisms involved.
Materials and methods
Wistar rats (Pasteur Institute, Tunis, Tunisia) were kept in a temperature-controlled room (21 ± 1°C) under an established photoperiod (lights on from 7:00 am to 7:00 pm) with free access to food and water. All experiments were performed according to the recommendations of the ethic committee of Tunis University for care and use of animals in conformity with NIH guidelines.
The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide, PACAP6-38 and VIP were synthesized by solid-phase methodology as previously described (Jolivel et al. 2009). Dulbecco’s modified Eagle’s medium (DMEM), F12 culture medium, d(+)-glucose, l-glutamine, HEPES buffer solution, foetal bovine serum (FBS), trypsin-EDTA, and the antibiotic-antimycotic solution were obtained from Gibco (Invitrogen, Grand Island, NY, USA). Cytosine-β-d-arabinofuranoside, sodium pyruvate, fluorescein diacetate-acetoxymethyl ester, chelerythrine, H89, U73122 and insulin were purchased from Sigma Aldrich (St. Louis, MO, USA). 5-6-Chloromethyl 2′-7′-dichlorodihydrofluorescein diacetate, monochlorobimane (mBCl) and the mitochondrial potential sensor JC-1 were obtained from Molecular Probes (Eugene, OR, USA). U0126 and Apo-ONE Homogeneous Caspase-3/7 assay kit were supplied by Promega (Charbonnières, France).
Secondary cultures of rat cortical astrocytes
Secondary cultures of rat cortical astrocytes were prepared from 1- or 2-day-old Wistar rats as previously described (Brown and Mohn 1999) with minor modifications. Briefly, cerebral hemispheres were collected in DMEM/F12 (2 : 1; v/v) culture medium supplemented with 2 mM glutamine, 1% insulin, 5 mM HEPES, 0.4% glucose and 1% of the antibiotic-antimycotic solution. Dissociated cells were resuspended in culture medium supplemented with 10% FBS, plated in 150-cm2 flasks (Greiner Bio-one GmbH, Frickenhausen, Germany) and incubated at 37°C in a 5% CO2/95% O2 atmosphere. When cultures were confluent, astrocytes were isolated by shaking overnight the flasks with an orbital agitator. Adhesive cells were detached by trypsination and pre-plated for 5 min to discard contaminating microglial cells. Then, non-adhering astrocytes were harvested and plated in 24-well plates at a density of 8 × 104 cells/mL. The cells were incubated at 37°C in a humid atmosphere (5% CO2). In these conditions, after 5 days, more than 99% of the cells were labeled with antibodies against glial fibrillary acidic protein (Castel et al. 2006). All experiments were performed on 5- to 7-day-old secondary cultures.
Primary culture of cerebellar granule neurons
Granule cell suspensions were prepared from cerebellums of 8-day-old rats, as described previously (Vaudry et al. 1999). The cells were cultured in a chemically defined medium consisting of 75% DMEM and 25% F12 supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 25 mM KCl, 1% of an antibiotic–antimicotic solution and 10% FBS. 10 μM cytosine-β-d-arabinofuranoside was added to the cultures 20 h after plating to prevent proliferation of non-neuronal cells. Granule neurons were incubated in the presence of 10% FBS, at 37°C in a humid atmosphere (5% CO2) for 5–7 days.
Measurement of cell survival
For cell survival experiments, astrocytes and granule neurons were maintained at 37°C with fresh serum-free astrocyte medium in the absence or presence of test substances. At the end of the treatment period, cells were incubated for 8 min with fluorescein diacetate (15 μg/mL), rinsed twice with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and lysed with a Tris/HCl solution containing 1% sodium dodecyl sulfate. Fluorescence intensity was measured (λ excitation = 485 nm and λ emission = 530 nm) with a FL800TBI fluorescence microplate reader (Bio-Tek Instruments, Winooski, VT, USA).
Measurement of intracellular ROS formation
Reactive oxygen species were detected by measuring the fluorescence of 2′,7′-dichlorofluorescein (DCF) resulting from the deacetylation and oxidation of the non-fluorescent compound DCFH2-DA. Cells seeded into 24-well plates were first incubated with 10 μM of cell-permeant DCFH2-DA in serum-free loading medium at 37°C for 30 min. At the end of the incubation, DCFH2-DA was removed and cells were washed twice with PBS, and then exposed to H2O2 in the absence or presence of PACAP. Fluorescence intensity was measured (λ excitation = 485 nm and λ emission = 530 nm) with a FL800TBI fluorescence microplate reader (Bio-Tek Instruments, Winooski, VT, USA).
Measurement of glutathione concentrations
Reduced glutathione, which represents the majority of intracellular free thiols in cells, was measured by using the thiol-reactive probe mBCl. At the end of the treatment period, cells seeded into 24-well plates were incubated with the mBCl probe (40 μM) for 30 min and then washed twice with PBS. Fluorescence intensity was measured (λ excitation = 360 nm and λ emission = 485 nm) with a FL800TBI fluorescence microplate reader (Bio-Tek Instruments, Winooski, VT, USA).
Measurement of mitochondrial activity
Mitochondrial membrane potential was quantified using the ratiometric probe JC-1. At the end of the treatment period, cells seeded into 24-well plates were incubated in the presence of the JC-1 probe (10 μg/mL) at 37°C for 15 min and then washed twice with PBS. In healthy astrocytes, the intact membrane potential allows the lipophilic dye JC-1 to enter into the mitochondria where it aggregates and produces an intense orange signal. In dead cells, the mitochondrial membrane potential collapses so that the monomeric JC-1 probe remains cytosolic and stains the cells in green. Fluorescence intensity was measured with a FL800TBI fluorescence microplate reader and expressed as a ratio of the emission at 590 nm (orange) over 530 nm (green) to evaluate mitochondrial integrity.
Measurement of caspase 3 activity
At the end of the incubation with PACAP and/or H2O2, cultured astrocytes were washed twice with PBS at 37°C, resuspended in DMEM (100 μL) mixed with 1× caspase assay buffer containing 25 μM caspase 3 substrate (Apo-ONE Homogeneous Caspase-3/7 kit, Promega). Caspase 3 activity was calculated from the slope of the fluorescence measured every 15 min for 3 h with excitation at 485 nm and emission at 530 nm.
All data are expressed as mean ± SEM from three to four independent experiments. For statistical evaluation of the results, anova followed by the Bonferroni’s test was performed. A p-value of 0.05 or less was considered as statistically significant.
Effect of PACAP on H2O2-induced astroglial cell death
Incubation of astrocytes with graded concentrations of H2O2 (100–400 μM) for 1 h provoked a dose-dependent decrease of the proportion of surviving cells. Addition of PACAP (10−9 M) to the culture medium markedly suppressed the toxic effect of moderate concentrations of H2O2 (100–300 μM) and partially reversed the deleterious effect of higher doses of H2O2 (350 and 400 μM) on astrocytes (Fig. 1a). The dose of 300 μM H2O2, which killed 50% of the cells, was used in all subsequent experiments. Examination of cultures by phase-contrast microscopy revealed that PACAP-treated cells displayed a flat polygonal morphology similar to that of untreated-astrocytes (Fig. 1b and d), while H2O2 (300 μM) induced cell shrinkage and the appearance of long thin processes (Fig. 1c). Pre-treatment of cells with PACAP (10−9 M) totally prevented the morphological changes induced by 300 μM H2O2 (Fig. 1e).
Co-incubation of astroglial cells with H2O2 and graded concentrations of PACAP (10−16 M to 10−6 M) resulted in a dose-dependent increase in the number of surviving cells (Fig. 2a). The half-maximum effect of PACAP was observed at a dose of 3 × 10−11 M and complete reversal of the toxic effect of H2O2 was obtained with 10−9 M PACAP. Incubation of astrocytes with VIP, even at concentrations up to 10−6 M, had no significant effect on H2O2-evoked cell death (Fig. 2a). Pre-incubation of astrocytes for 20 min with the PACAP receptor antagonist PACAP6-38 (10−6 M), which had no effect by itself on cell survival, totally abolished the protective effect of PACAP (Fig. 2b).
Signal transduction pathways involved in the glioprotective effect of PACAP on H2O2-induced astrocyte death
As, in cultured rat astrocytes, PACAP stimulates the adenylyl cyclase/protein kinase A (PKA), the phospholipase C (PLC)/protein kinase C (PKC) and the MAPK transduction pathways (Masmoudi-Kouki et al. 2007), we have investigated the signaling cascade involved in the protective action of PACAP. Incubation of astrocytes with the PKA inhibitor H89 (2 × 10−5 M), the PLC inhibitor U73122 (10−5 M), the PKC inhibitor chelerythrine (10−6 M) or the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126 (5 × 10−5 M) did not affect H2O2-induced cell death but totally abrogated the protective action of PACAP on astroglial cell survival (Fig. 3).
Effect of PACAP on H2O2-induced intracellular ROS accumulation and glutathione depletion
To examine whether PACAP could also block H2O2-induced intracellular ROS accumulation, astrocytes were labeled with 5-6-chloromethyl 2′-7′-dichlorodihydrofluorescein diacetate which forms the fluorescent DCF compound upon oxidation with ROS. Incubation of cultured astrocytes with 300 μM H2O2 for 1 h induced a 2.6-fold increase in DCF fluorescence intensity (Fig. 4a). Pre-incubation of cells with graded concentrations of PACAP (10−16 M to 10−6 M) reduced in a dose-dependent manner the effect of H2O2 on DCF formation. As glutathione is the most abundant thiol tripeptide for ROS scavenger in cells, we examined the effect of PACAP on the content of intracellular glutathione by using mBCl, a probe which forms a fluorescent compound when conjugated with glutathione. Graded concentrations of PACAP (10−16 M to 10−6 M) induced a dose-related increase in mBCl fluorescence intensity (Fig. 4b). In contrast, H2O2 (300 μM) reduced by 60% mBCl fluorescence intensity (Fig. 4c). Exposure of astroglial cells to graded concentrations of PACAP (10−16 M to 10−6 M) dose-dependently prevented the depletion of mBCl fluorescence induced by H2O2. The half-maximum effect of PACAP was observed at a concentration of 1.6 × 10−14 M and the maximum effect was obtained at a concentration of 10−10 M PACAP (Fig. 4c).
Effect of PACAP on H2O2-induced alteration of mitochondrial activity and activation of caspase 3
Considering the major effect of ROS in the permeabilization of the mitochondrial outer membrane, we examined the ability of PACAP to prevent the effect of H2O2 on mitochondria integrity by measuring the membrane potential with the fluorescent ratiometric probe JC-1. Treatment of astrocytes with 300 μM H2O2 for 1 h induced a significant reduction (−48%) of the 590/530 nm ratio, indicating that the mitochondrial integrity was severely altered by H2O2 (Fig. 5a). PACAP (10−9 M) had no effect on the 590/530 nm fluorescent ratio by itself, but suppressed the deleterious effects of H2O2 on mitochondrial membrane potential (Fig. 5a).
To further explore the mechanism involved in the protective action of PACAP, we monitored the activity of caspase 3. Incubation of cultured astrocytes with 300 μM H2O2 for 1 h provoked a 2.3-fold increase of caspase 3 activity (Fig. 5b). Pre-treatment of the cells with PACAP (10−9 M), which had no effect on capsase-3 activity by itself, totally blocked H2O2-induced caspase 3 activation (Fig. 5b).
Effect of supernatants from PACAP-treated astrocytes on H2O2-induced cerebellar granule neuron death
To examine whether PACAP can indirectly promote neuron survival through an astroglial-dependent mechanism, cerebellar granule neurons were treated with astrocyte-conditioned medium. Incubation of cultured granule cells with 50 μM H2O2 for 1 h provoked a decrease (−52%) in the proportion of surviving cells (Fig. 6). Treatment of cultured cerebellar granule cells for 1 h with supernatants from PACAP (10−9 M)-treated astrocytes provoked a significant reduction of neuron death induced by H2O2. Pre-treatment of cultured astrocytes with PACAP6-38 (10−6 M) attenuated the protective action of conditioned medium from PACAP-exposed astrocytes on granule cells (Fig. 6).
Pituitary adenylate cyclase-activating polypeptide receptors are not only present on neurons but are also expressed in astroglial cells (Vaudry et al. 2009), and several data indicate that PACAP plays a key role in the control of proliferation and differentiation of astrocytes (Hashimoto et al. 2003; Nishimoto et al. 2007). Here, we demonstrate for the first time that PACAP protects cultured rat astrocytes from apoptosis induced by H2O2, and we show that PACAP exerts its glioprotective effect through inhibition of ROS generation and caspase 3 activation.
In agreement with previous reports (Ferrero-Gutierrez et al. 2008; Lu et al. 2008), we observed that exposure of cultured astrocytes to H2O2 provoked cell death associated with modifications of cell morphology. Incubation of astrocytes with subnanomolar concentrations of PACAP dose-dependently prevented H2O2-induced cell death and suppressed H2O2-evoked morphological changes, indicating that PACAP is a potent glioprotective agent. While the beneficial effect of PACAP against neuronal cell death is well documented (Vaudry et al. 2000; Shioda et al. 2006; Allais et al. 2010; Deguil et al. 2010; May et al. 2010) this is the first report showing that PACAP exerts a neuroprotective action on glial cells.
Pituitary adenylate cyclase-activating polypeptide acts through three distinct receptors i.e. PAC1-R which specifically binds PACAP, and VPAC1 and VPAC2 receptors which exhibit similar affinities for PACAP and VIP (Vaudry et al. 2009). All three receptor subtypes are expressed in cultured cortical astrocytes from newborn rats, but PAC1 and VPAC2 receptors are the predominant forms (Jaworski 2000). Administration of VIP, even at high concentrations, was unable to reduce H2O2-evoked astrocyte death, and the PAC1-R antagonist PACAP6-38 totally suppressed the protective effect of PACAP. These data suggest that the glioprotective action of PACAP upon H2O2-evoked cell death is mediated through activation of PAC1-R. Consistent with this notion, numerous in vitro studies indicate that, in neurons, PACAP exerts its protective action through activation of PAC1-R (Somogyvari-Vigh and Reglodi 2004, for review).
We have previously demonstrated that, in cultured astrocytes, PAC1-Rs are positively coupled to both the adenylyl cyclase/PKA and the PLC/calcium/PKC transduction pathways (Masmoudi et al. 2003). We now show that treatment of astrocytes with the PKA inhibitor H89, the PLC inhibitor U73122 or the PKC inhibitor chelerythrine abrogated the effect of PACAP on cell survival. It is clearly established that the MAPK signaling cascade is involved in apoptotic death of astrocytes (Dong et al. 2009; Huang et al. 2009) and it has been shown that survival of astrocytes requires phosphorylation of extracellular signal-regulated kinase (ERK) (Takuma et al. 2000). There is also evidence that PACAP stimulates, in a cAMP-dependent manner, the phosphorylation of ERK1/2 in cultured rat astrocytes (Moroo et al. 1998; Hashimoto et al. 2003). Here, we show that the protective action of PACAP on astrocytes against H2O2 was abolished by the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. Collectively, these data suggest that the glioprotective activity of PACAP can be accounted for by activation of the PKA, PKC and MAPK transduction pathways. Consistent with these results, it has been reported that PACAP protects cerebellar neurons against H2O2- and C2 ceramide-induced apoptosis through PKA-, PKC- and ERK-dependent mechanisms (Vaudry et al. 2002; Falluel-Morel et al. 2004).
Generation of ROS has a fundamental role in the signalling of H2O2-induced cell toxicity (Fubini and Hubbard 2003). The fact that PACAP strongly attenuates ROS formation evoked by H2O2 in astrocytes suggests that PACAP may protect astrocytes from oxidative stress. The tripeptide glutathione, which acts as a free radical scavenger, is essential for the antioxidative defense system of the brain (Dringen et al. 2000). As astrocytes play a crucial role in cerebral glutathione metabolism by providing neighboring neurons with precursors for glutathione synthesis (Rana and Dringen 2007), we investigated the possible effect of PACAP on glutathione formation. Subnanomolar concentrations of PACAP increased in a concentration-dependent manner glutathione production in astrocytes, and markedly reduced the decrease of cellular content of glutathione induced by H2O2 treatment. These data strongly suggest that PACAP promotes astrocyte survival by increasing glutathione production, which in turn attenuates ROS formation. The involvement of glutathione in the protective effect of PACAP on astrocytes is supported by data showing that depletion of glutathione in cultured cerebellar and cortical astrocytes induces the production of free radicals and increases the vulnerability of cells to oxidative injuries (Kaur et al. 2007; Lu et al. 2008). Furthermore, pre-treatment of hippocampal slices with glutathione, protects astrocytes against H2O2 toxicity (Feeney et al. 2008).
The signalling mechanisms that trigger apoptosis in astrocytes are not completely elucidated. Nevertheless, it is widely accepted that ROS cause mitochondria damages yielding to the release of cytochrome c into the cytosol which in turn induces caspase activation. Measurement of mitochondrial activity, using the membrane potential-sensitive probe JC-1, revealed that PACAP prevented the deleterious effect of H2O2 on mitochondria. Concurrently, PACAP markedly suppressed caspase 3 activation induced by H2O2, indicating that the protective effect of PACAP on astrocytes is attributable to an inhibition of caspase 3 activity through a mitochondrial-dependent pathway. These results are consistent with previous data indicating the involvement of the apoptotic pathway in H2O2-induced cell death of both neurons and astrocytes (Vaudry et al. 2002; Takuma et al. 2004).
The glioprotective effect of PACAP might have a physiopathological significance in neurodegenerative diseases and stroke. It is established that astrocytes might relay, at least in part, the neuroprotective action of PACAP by releasing neuroprotective compounds (Dejda et al. 2005; Masmoudi-Kouki et al. 2007). In agreement with this notion, we found that supernatants from astrocytes treated with 10−9 M PACAP, a concentration which had no protective action by itself, prevented H2O2-induced death of cultured cerebellar granule cells. Concurrently, the expression of PAC1-R is increased in reactive astrocytes after cerebral injury (Suzuki et al. 2003; Shioda et al. 2004; Stumm et al. 2007) and in reactive astrocytes in culture (Nakamachi et al. 2010), highlighting the importance of astroglial cells in the neuroprotective activity of PACAP. Despite their high antioxidative activities, astrocytes cannot survive and protect neurons under massive oxidative stress (Feeney et al. 2008). Thus, up-regulation of PACAP receptors in astrocytes may increase protection of cells from oxidative insults, and PACAP, acting in part via astroglial cells, might have a therapeutic potential for treatment of cerebral injuries involving oxidative neurodegeneration.
In conclusion, the present study demonstrates that PACAP, acting likely through PAC1-R, exerts a potent glioprotective effect against oxidative stress. The anti-apoptotic activity of PACAP can be ascribed to stimulation of the MAPK transduction pathway, inhibition of ROS-induced mitochondrial dysfunctions and activation of caspase 3. An increase of astrocyte viability may thus contribute to the neuroprotective effect of PACAP.
The authors wish to thank Mr Samir Elbahi for skilful technical assistance. Y.H. and H.K. were recipients of fellowships from the University of Tunis El Manar and a France-Tunisia exchange program CMCU-Utique. S.B. and S.D. were recipients of fellowships from the University of Tunis El Manar and a France-Tunisia exchange program Inserm-DGRS. D.V. and H.V. are Affiliated Professors at the INRS – Institut Armand-Frappier. This study was supported by the Research Unit 00-UR-08-01, an Inserm-DGRS program (to M.A. and M.C.T.), an Inserm-FRSQ program (to A.F. and D.V.), Inserm (U982), the European Institute for Peptide Research (IFRMP23) and the Region Haute-Normandie.