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 (). AMPK and ACC phosphorylation was evident at 30 min and persisted for at least 8 h (). At 24 h, phosphorylation of AMPK returned to baseline but that of ACC remained increased (). 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 ().
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 (), whereas cells treated with AICAR exhibited widespread stellation (). 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 (). 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 () and 24 () h after treatment. At 4 h, cells 1 and 2 are stellate (). After 24 h, cell 2 remains stellate, whereas cell 1 has returned to its original polygonal morphology (). This time course of AICAR-induced stellation was similar to other stellating agents, including adenosine and phorbol myristate acetate [3
]. Stellation was significant at 2mM AICAR with maximal effect at 10mM ().
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 (), or via immunostaining against GFAP () or ezrin (), 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
]. 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).
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 () [3
]. In contrast, preincubation with XAC did not diminish AICAR-induced stellation (). Thus, AICAR-induced stellation does not require adenosine A1 receptor activation, supporting the hypothesis that AMPK activation induces astrocyte stellation.
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, ). Immunofluorescence with antibodies against vinculin revealed dispersal of focal adhesions after AICAR treatment ().
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
]. Merely inhibiting Rho and/or its downstream effectors is sufficient to induce stellation in astrocytes [1
]. 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 (). Of note, transient transfection of RhoV14 also abolished basal stellation induced by media change to HBSS (data not shown).
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 (). In cultured rat cortical astrocytes preincubated with 40 μM compound C (CC), AICAR-induced stellation was prevented (). These results suggest that AMPK activation is necessary for AICAR-induced stellation.
AICAR-induced stellation requires AMPK activity