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Brain Res. Author manuscript; available in PMC 2008 September 7.
Published in final edited form as:
PMCID: PMC2000700
NIHMSID: NIHMS30283

A pharmacological activator of AMP-activated protein kinase (AMPK) induces astrocyte stellation

Abstract

AMP-activated protein kinase (AMPK) represents a key energy-sensing molecule in many cell types. Because astrocytes are key mediators of metabolic signaling in the brain, we have initiated studies on the expression and activation of AMPK in these cells. Treatment of cultured rat cortical astrocytes with a pharmacological AMPK activator, AICA-riboside (AICAR) resulted in a time- and concentration-dependent increase in phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a direct substrate. AICAR treatment also induced a transition from epithelioid to stellate morphology in a time- and concentration-dependent manner. As stellation is indicative of actin cytoskeletal reorganization, the formation of stress fibers and focal adhesions in response to AICAR was assessed. AICAR-induced stellation correlated with F-actin disassembly and focal adhesion dispersal. Furthermore, transient transfection of an activated RhoA construct prevented AICAR-induced stellation, indicating a mechanism upstream of RhoA. Use of pharmacological inhibitor compound C prevented AICAR-induced stellation demonstrating necessity of AMPK activity for the response. Our findings suggest that AMPK mediates morphological alterations of astrocytes in response to energy depletion.

Keywords: AMPK, AICAR, astrocyte, stellation

INTRODUCTION

Astrocytes exhibit multiple branched processes in vivo. In pathological states, reactive astrocytes undergo characteristic morphological changes such as greater branching and thickening of processes and somatic hypertrophy [34]. In contrast, cultures of primary rodent cortical astrocytes exhibit a flat and polygonal shape. Knowledge about the mechanisms that govern astrocyte morphology changes comes from the study of agents that induce cultured astrocytes to exhibit a process bearing, or “stellate”, morphology. Agents that induce stellation include cyclic AMP [37,48]; neuromodulators, such as ATP and adenosine; growth factors, such as FGF2; hormones, such as vasopressin; PKC activators, such as phorbol esters; cytokines, such as TNF alpha [36]; and peptides, such as β amyloid [32]. Because these molecules are also released in brain injury or upregulated during gliosis [15], they could underlie morphological plasticity of astrocytes in vivo.

Recent studies revealed PI3K inhibition as the mechanism governing cyclic AMP-induced stellation [41]. Downstream of PI3K signaling, Akt phosphorylates and inhibits tuberous sclerosis complex 2, TSC2 [20,29]. AMP-activated protein kinase (AMPK) also phosphorylates TSC2, at sites distinct from Akt, resulting in its activation [30]. One function of TSC2 is to antagonize mTOR signaling [28]. There are two functionally distinct mTOR complexes, mTORC1, responsible for cell growth and proliferation, and mTORC2, which signals through Rho GTPases to stimulate actin stress fiber formation [31]. TSC2 can also inhibit Rho by binding to TSC1, and thus promote stress fiber disassembly and focal adhesion remodeling [23]. Therefore, AMPK modulates substrates that potentially control actin stress fiber dynamics. Indeed, stable overexpression SNARK, a member of the AMPK catalytic subunit family, caused HepG2 cells to exhibit a rounded morphology that was attributed to conversion of F-actin filaments to G-actin monomers [50]. SNARK overexpression also prevented focal adhesion kinase phosphorylation in response to glucose deprivation in these cells, implying energy levels modulate the actin cytoskeleton [50].

In vivo, severe hypoglycemia results in gliosis after neuronal necrosis [6]. Energy depletion by hypoglycemia, hypoxia, and ischemia activates AMPK [17,35] to restore ATP levels [26]. Interestingly, brain injury increases AMPK catalytic subunit expression in reactive astrocytes [53]. In the present study, we investigate the consequence of AMPK activation on morphology of cultured astrocytes using AICAR, a pharmacological AMPK activator.

RESULTS

To activate AMPK in cultured astrocytes, we used AICA-riboside (AICAR), a widely used pharmacological activator. To confirm that AMPK activation had occurred, we assessed the phosphorylation of AMPK and its direct substrate, acetyl-CoA carboxylase (ACC), indicative of AMPK pathway activation. Treatment with AICAR induced a time- and concentration-dependent increase in AMPK and ACC phosphorylation (Fig. 1). AMPK and ACC phosphorylation was evident at 30 min and persisted for at least 8 h (Fig. 1A). At 24 h, phosphorylation of AMPK returned to baseline but that of ACC remained increased (Fig. 1A). Possible explanations for the divergence of AMPK and ACC phosphorylation include differential dephosphorylation kinetics by the relevant phosphatases or activation of other pathways by AICAR resulting in ACC phosphorylation. Maximal AMPK and ACC phosphorylation was evident at a concentration of 0.5mM AICAR (Fig. 1B).

Figure 1
Treating cultured rat cortical astrocytes with a pharmacological AMPK activator, AICAR, induces phosphorylation of AMPK and its direct substrate, acetyl-CoA carboxylase (ACC)

Next, we examined the relationship between AMPK activation and astrocyte morphology. Because individual cell morphologies were difficult to assess in confluent cultures, cells were infected with GFP adenovirus to highlight a subset of cells for quantification. Stellate cells were classified as those exhibiting at least 3 discrete processes and excluded unbranched short filopodia less than one soma diameter. GFP adenovirus-infected astrocytes treated with vehicle (dH2O) exhibited rare stellate cells (Fig. 2A), whereas cells treated with AICAR exhibited widespread stellation (Fig. 2B). Stellation correlated with the time- and concentration-dependence of AMPK and ACC phosphorylation induced by AICAR. AICAR-induced stellation was evident at 1 h of incubation, with statistical significance at 2 and 4 h, and began to reverse by 6 h (Fig. 2C). These results suggest that AICAR-induced stellation was transient which was confirmed by photomicrographs of live cells taken on a marked coverslip in the same field at 4 (Fig. 2E) and 24 (Fig. 2F) h after treatment. At 4 h, cells 1 and 2 are stellate (Fig. 2E). After 24 h, cell 2 remains stellate, whereas cell 1 has returned to its original polygonal morphology (Fig. 2F). This time course of AICAR-induced stellation was similar to other stellating agents, including adenosine and phorbol myristate acetate [3,4]. Stellation was significant at 2mM AICAR with maximal effect at 10mM (Fig. 2D).

Figure 2
AICAR treatment induces stellate morphology in a time- and concentration-dependent manner in cultured rat cortical astrocytes

We compared the stellate morphologies of astrocytes treated with AICAR with that induced by conventional stellating agents, forskolin and AMP. Forskolin-induced stellation is more complete than that induced by AMP, involving complete retraction of the cell membrane [24]. Morphology was visualized using phase contrast microscopy (Fig. 3A-D), or via immunostaining against GFAP (Fig. 3E-H) or ezrin (Fig. 3I-L), an actin-binding protein revealing the fine peripheral processes of astrocytes [14]. AICAR-induced stellation was observed to be similar to that induced by AMP, with fewer cells exhibiting complete membrane retraction. In several cases using cultured astrocytes, stellate morphology correlates with increased GFAP expression [5,39,46]. Therefore, we investigated GFAP protein levels over the time course of AICAR-induced stellation. Western blots did not reveal any increase in GFAP during AICAR incubation up to 24 h (data not shown).

Figure 3
AICAR-induced stellation is similar to AMP-induced stellation, with fewer cells exhibiting complete membrane retraction as seen in forskolin-induced stellation

AICAR enters the cell through the adenosine transporter [21]. Intracellular phosphorylation by adenosine kinase results in its AMPK activating form, “ZMP” [45]. In rat brain slices, AICAR treatment induced depression of excitatory synaptic transmission in CA1 hippocampus [21]. This effect of AICAR was due to indirect activation of adenosine receptors because AICAR competes with adenosine for access to the transporter, increasing extracellular adenosine [21]. Treating astrocytes with adenosine, and its derivatives AMP, ADP, and ATP, induces stellation by P1 purinoceptor activation [3]. To rule out this potential alternate mechanism of AICAR-induced stellation, we examined the effect of an adenosine A1 receptor inhibitor on AICAR-induced stellation. As previously reported, preincubation with 10 μM A1 adenosine receptor inhibitor, xanthine amine congener (XAC), prevented astrocyte stellation induced by 100 μM extracellular AMP (Fig. 4A) [3]. In contrast, preincubation with XAC did not diminish AICAR-induced stellation (Fig. 4B). Thus, AICAR-induced stellation does not require adenosine A1 receptor activation, supporting the hypothesis that AMPK activation induces astrocyte stellation.

Figure 4
Adenosine A1 receptor inhibitor, xanthine amine congener (XAC), blocks AMP-induced stellation with no effect on AICAR-induced stellation in cultured rat cortical astrocytes

Actin stress fibers contact the extracellular matrix at focal adhesions in the plasma membrane [10]. Astrocyte stellation is usually the result of actin stress fiber disassembly [22]. To demonstrate the involvement of stress fibers and focal adhesions in AICAR-induced stellation, we stained cells with phalloidin, a probe that specifically binds to F-actin [1], and vinculin, an actin-binding protein enriched at focal adhesions [10]. AICAR-induced stellation resulted in marked loss of stress fibers as evidenced by phalloidin staining (arrows, Fig. 5A-C). Immunofluorescence with antibodies against vinculin revealed dispersal of focal adhesions after AICAR treatment (Fig. 5D-F).

Figure 5
AICAR-induced stellation is associated with disassembled actin stress fibers and dispersed focal adhesions

Modulation of the actin cytoskeleton and focal adhesions is directly linked to RhoA activity [25]. In addition, several modes of stellation, including adenosine and cAMP, require Rho inactivation [42,44]. Merely inhibiting Rho and/or its downstream effectors is sufficient to induce stellation in astrocytes [1,44]. To directly test whether AICAR-induced stellation was upstream of RhoA, we transiently transfected cultured rat cortical astrocytes with a plasmid expressing constitutively active RhoA (RhoV14) [40]. In cells transfected with RhoV14, AICAR-induced stellation was completely prevented, in contrast to cells transfected with GFP plasmid as a control (Fig. 6B,D). Of note, transient transfection of RhoV14 also abolished basal stellation induced by media change to HBSS (data not shown).

Figure 6
AICAR-induced stellation is prevented by constitutive RhoA activation

To assess whether the morphology changes induced by AICAR in cultured astrocytes required AMPK activation, we used a pharmacological AMPK inhibitor, compound C [55]. The use of compound C as an AMPK inhibitor has been previously described and characterized [55]. The mechanism of compound C action is to inhibit AMPK kinase activity, but not the activity of its upstream kinases. As expected, preincubation with compound C caused diminished AICAR-induced phosphorylation of the AMPK substrate ACC, but not of AMPK itself (Fig. 7B). In cultured rat cortical astrocytes preincubated with 40 μM compound C (CC), AICAR-induced stellation was prevented (Fig. 7A). These results suggest that AMPK activation is necessary for AICAR-induced stellation.

Figure 7
AICAR-induced stellation requires AMPK activity

DISCUSSION

In this report we have shown that treatment with a pharmacological AMPK activator, AICAR, induces stellation, a characteristic morphological change in cultured rat cortical astrocytes. AICAR-induced stellation is rapid, transient, concentration-dependent, and involves disassembly of F-actin stress fibers and dispersal of focal adhesions. Our initial investigation of the mechanism of AICAR-induced stellation revealed that AICAR-induced stellation depends on RhoA inactivation. Lastly, we present evidence to suggest that AMPK activity is necessary to induce stellation in astrocytes.

Studies investigating upstream and downstream components of the AMPK pathway provide evidence that AMPK could regulate cell morphology. LKB1, recently identified as an upstream activator of AMPK [27], is a polarizing stimulus in C. elegans embryo asymmetry formations [54]. In an in vitro scratch wound assay, astrocytes polarize, with their centrosome facing the wound, before migrating to close the wound [16]. When an LKB1 expression plasmid with a disrupted ATP-binding site, rendering it kinase dead and therefore unable to activate AMPK, was injected into the leading edge of astrocytes, this wound-induced reorientation was abolished [19]. In Saccharomyces cerevisiae, pharmacological mTOR inactivation by rapamycin treatment results in depolymerization of the actin cytoskeleton [52]. In mammalian cells, mTOR signaling is antagonized by AMPK activation of TSC2. The results of this study suggest that direct activation of AMPK can alter astrocyte morphology.

In this report, we present evidence to support the fact that AICAR-induced stellation is associated with disassembly of stress fibers at focal adhesions. Stress fiber assembly and focal adhesion formation depends on RhoA activation, as initially demonstrated in Swiss 3T3 fibroblasts [33,43]. In cultured astrocytes, RhoA activation is thought to maintain polygonal morphology [2,49]. Treating pituicytes with lysophosphatidic acid, a RhoA activator, prevented stellation in response to adenosine [44]. Astrocytes expressing constitutively activated RhoA fail to undergo cAMP induced stellation [42]. Manganese-induced stellation in cultured rat cortical astrocytes was recently demonstrated to be dependent on RhoA pathway inactivation [12]. In this study, astrocytes transiently transfected with a constitutively active RhoA construct, RhoV14, failed to undergo stellation when treated with AICAR. Thus, AICAR-induced stellation, like manganese and cAMP, acts upstream of the RhoA pathway in astrocytes.

Treating astrocytes with an AMPK inhibitor, compound C, prevented AICAR-induced ACC phosphorylation and stellation, supporting the necessity of AMPK activation for morphology changes. Interestingly, lower doses of AICAR resulting in AMPK phosphorylation were not sufficient to induce significant stellation. The higher doses of AICAR required to induce stellation suggest that another pathway may cooperate with AMPK. Possible candidates would be atypical PKC isoforms. Incubation of isolated rat extensor digitorum longus muscles with two different pharmacological activators of AMPK, AICAR and dinitrophenol, resulted in PKC zeta activation [13]. In epithelial cells, polarity is regulated by activation of Par4, a LKB1 homolog [7]. When LKB1 is activated it then leads to activation of a network of Par proteins, some of which recruit Rho GTPases and others recruit atypical PKCs, to modulate actin cytoskeleton dynamics. Similarly in astrocytes, activation of the Par6/PKC zeta complex is essential for polarization of migrating cells during in vitro wound healing [16].

AICAR-induced stellation of cultured astrocytes represents a useful model to study morphological changes that may occur in vivo in response to energy deprivation. AMPK-mediated regulation of the actin cytoskeleton could play an important role in coupling metabolic signals to morphological plasticity of astrocytes. The terminal processes of astrocytes that ensheath blood vessels, termed endfeet, regulate cerebral blood flow in response to increased neuronal activity [18,38,51]. Astrocytic release of vasoactive molecules is a response to regional energy depletion because neurons need a continuous supply of glucose to produce ATP and maintain excitability, but their effectiveness is determined by proximity of astrocyte processes [18]. Astrocytes also respond to pathological energy depletion, such as hypoglycemia, by releasing lactate [9]. Interestingly, several agents that induce stellation in cultured astrocytes also stimulate glycogenolysis [11,47]. Thus, changes in astrocyte morphology upon energy deprivation may be indicative of underlying neuroprotective responses. As energy depletion activates AMPK, this suggests a novel role to modulate the actin cytoskeleton in astrocytes.

EXPERIMENTAL PROCEDURE

Cell culture

Cortical astrocytes were prepared from the cerebral cortices of 1-day-old Sprague-Dawley rats and maintained as described previously [8]. The resultant cells were used for experiments 2-7 weeks after preparation. Cells were trypsinized, replated onto 18mm glass coverslips at an average density of 7 × 104 cells per coverslip, and grown in Dulbecco's Modified Eagle media (DMEM) containing antibiotic-antimycotic and 10% fetal bovine serum until confluent. Coverslips were not coated with poly-L-lysine because this substrate may induce morphological changes in astrocytes [24]. Where indicated, the culture medium was switched to serum-free DMEM (SF DMEM) 24 h before the start of experiments. For experiments, cells were incubated in SF DMEM or Hank's Balanced salt solution (HBSS). In typical cultures >98% of the cells were identified as astrocytes by immunostaining for glial fibrillary acidic protein (GFAP) and by their flattened, polygonal appearance. HBSS was purchased from Hyclone (Logan, UT, USA). The formulation for HBSS is as follows (in g/L): 8 NaCl, 0.05 HNa2PO4, 0.4 KCl, 0.06 KH2PO4, 0.098 MgSO4, 0.14 CaCl2, 1 D-glucose, 0 phenol red. DMEM, FBS, trypsin-EDTA, and antibiotic-antimycotic were purchased from GIBCO (Grand Island, NY, USA). Cell culture 12 well plastic plates, 60-mm plastic dishes, and glass coverslips were purchased from Fisher Scientific Company (Newark, DE, USA). CELLocate marked glass coverslips were purchased from Eppendorf (square size 175 μm; Hamburg, Germany).

Materials

AICA-Riboside (AICAR) and compound C (CC) were from Calbiochem (La Jolla, CA, USA). Xanthine amine congener (XAC) and adenosine monophosphate (AMP) were from Sigma (St Louis, MO, USA).

Transient transfections

Plasmid DNA expressing myc-tagged constitutively active RhoA (RhoV14), a gift from Dr. Alan Hall (Memorial Sloan-Kettering Cancer Center), was created as described previously [40]. The pmaxGFP control plasmid was from Amaxa (Gaithersburg, MD, USA). Plasmid expression was confirmed by immunofluorescence with a mouse monoclonal antibody against c-myc clone 9E10 (1:100, BD Biosciences, NY, USA) or a rabbit polyclonal antibody against GFP (1:2500, Abcam, Cambridge, MA, USA). Astrocytes were transfected, according to manufacturer's instructions, with Transfast reagent (Promega, Madison, WI, USA) at a DNA/liposome ratio of 1:3 and fixed 48 h after transfection.

Adenoviral infections

Control adenovirus expressing EGFP was purchased from Baylor College of Medicine Vector Core Facility. Astrocytes were incubated for 48 h with adenovirus at a concentration of 2.5 × 109 cfu/ml to infect 30–50% of cells.

Immunofluorescence

Cells were rinsed with phosphate-buffered saline (PBS) and fixed for 20 min at room temperature with 4% paraformaldehyde in PBS. Following two wash steps, each for 5 min in PBS, fixed cells were incubated for 1 h in blocking solution containing 0.1% Triton X-100, 2% goat serum, and 0.1% Na azide dissolved in PBS. Subsequently, the cells were incubated with primary antibody diluted in blocking solution for 1 h at room temp. Following three wash steps, Tetramethylrhodamine- or Oregon Green 488-labeled goat anti-rabbit or anti-mouse (1:200, Molecular Probes, Eugene, OR, USA) was applied in blocking solution for 1 h at room temp. DAPI (1μg/ml, Sigma, St. Louis, MO, USA) was included in the secondary antibody solution for nuclear staining. In the case of Oregon Green 514-labeled phalloidin (1:100, Molecular Probes, Eugene, OR, USA), the cells were blocked and then incubated in PBS containing 0.1% Triton X-100 for 30 min at room temperature. Cells stained with fluorescent-conjugated secondary antibody were washed again in PBS and mounted onto slides with GELMOUNT (Biomeda, Foster City, CA, USA). Micrographs were taken with an Olympus BX40 upright microscope equipped with a Scion color Firewire CCD camera (Scion, Frederick, MD). Immunofluorescence was performed using the following primary antibodies: rabbit polyclonal antibodies against GFP (1:2500, Abcam, Cambridge, MA, USA), GFAP (1:5000, Dako, Carpinteria, CA, USA) and mouse monoclonal antibodies against c-myc clone 9E10 (1:100, BD Biosciences, NY, USA), HA (monoclonal 1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), ezrin clone 3C12 (1:2000, a gift from Dr. Kivela, Helsinki University, Finland), and vinculin (1:200, Sigma, St Louis, MO, USA).

Western blots

After rinsing with PBS, cells were scraped in 5μl (12 well plates) or 15μl (60-mm dishes) of 2X Laemmli sample buffer. Prior to polyacrylamide gel electrophoresis, lysates were boiled for 10 min then centrifuged briefly at 200 × g. Lysates were loaded onto a 10% precast gel (Cambrex, Rockland, ME, USA), run at 125 volts for 90 min, and transferred to Hybond ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA) at 50 milliamps per gel. Nitrocellulose membranes were washed three times in Tris-buffered saline with 0.1% Tween-20 (TBST) and then incubated in TBST with 5% nonfat dry milk for 1 h at room temp. After three washes in TBST, membranes were incubated with primary antibody diluted in TBST with 5% BSA and 0.1% sodium azide for 1 h. After three washes in TBST, the membranes were incubated in TBST with 5% nonfat dry milk and goat secondary antibodies coupled to horseradish peroxidase (1:1000, Sigma, St Louis, MO, USA) for 1 h. After three washes in TBST, immunoreactive bands were visualized using enhanced chemiluminescence (West Dura reagent, PIERCE, Rockford, IL, USA). As a loading control, immunoblots were probed with a monoclonal antibody against alpha-tubulin clone DM1A (1:5000, Neomarkers, Fremont, CA, USA). Immunoblots were probed with the following primary antibodies: rabbit polyclonal antibodies against phosphothreonine 172 AMP-activated protein kinase alpha (1:1000, Upstate, Charlottesville, VA, USA) and phosphoserine 79 acetyl-CoA carboxylase (1:1000, Cell Signaling, Danvers, MA, USA).

Morphological analysis

An observer blinded to the treatment conditions analyzed stellation. Stellate cells were classified as those exhibiting at least 3 discrete processes and excluded unbranched short filopodia less than one soma diameter. For each coverslip GFP-positive cells at 10X magnification over five arbitrarily chosen areas were counted and the resulting average taken. Percent stellation was calculated as the number of stellate GFP positive cells divided by the total number of GFP positive cells.

Statistical analysis

Data points from individual assays represent mean values ± SEM. Statistically significant differences among groups were assessed with ANOVA followed by t-test using Bonferroni's correction for multiple comparisons, with values of p < 0.05 sufficient to reject the null hypothesis for all analyses. All experiments were designed with matched control conditions within each experiment to enable statistical comparison.

Abbreviations used in text

AMPK
AMP-activated protein kinase
AICAR
AICA-riboside
ACC
acetyl-CoA carboxylase
DMEM
Dulbecco's Modified Eagle media
HBSS
Hank's balanced salt solution
SF
serum-free
DMSO
dimethyl sulfoxide
GFAP
glial fibrillary acidic protein
GFP
green fluorescent protein
XAC
xanthine amine congener
PBS
phosphate buffered saline
TBST
Tris buffered saline with 0.1% Tween-20
PKC
protein kinase C
FGF2
fibroblast growth factor 2
TNF alpha
tumor necrosis factor alpha
TSC2
tuberous sclerosis complex 2
PI3K
phosphatidylinositol 3-kinase
Akt
cellular homolog of the viral oncogene v-akt
Rheb
ras homolog enriched in brain
PMA
phorbol myristate acetate
CC
compound C
LKB1
serine/threonine protein kinase 11
MAPK
mitogen activated protein kinase
ERK
extracellular signal regulated kinase
SNARK
sucrose-non-fermenting protein kinase/AMPK-related protein kinase

Footnotes

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Contributor Information

Carlita B Favero, Neuroscience Graduate Program, University of Virginia, Charlottesville, VA.

James W Mandell, Department of Pathology (Neuropathology), University of Virginia, Charlottesville, VA.

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