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. 2018 Aug 1:384:54-63.
doi: 10.1016/j.neuroscience.2018.05.019. Epub 2018 May 23.

A-Kinase-Anchoring Protein (AKAP150) is expressed in Astrocytes and Upregulated in Response to Ischemia

Aixa F Rivera-Pagán  1 , Miguel P Méndez-González  2 , David E Rivera-Aponte  1 , Christian J Malpica-Nieves  1 , Katya V Melnik-Martínez  3 , Astrid Zayas-Santiago  4 , Gerónimo Maldonado-Martínez  5 , Yaroslav M Shuba  6 , Serguei N Skatchkov  7 , Misty J Eaton  8
Affiliations

A-Kinase-Anchoring Protein (AKAP150) is expressed in Astrocytes and Upregulated in Response to Ischemia

Aixa F Rivera-Pagán et al. Neuroscience. .

Abstract

A-kinase-anchoring proteins, AKAPs, are scaffolding proteins that associate with kinases and phosphatases, and direct them to a specific submembrane site to coordinate signaling events. AKAP150, a rodent ortholog of human AKAP79, has been extensively studied in neurons, but very little is known about the localization and function of AKAP150 in astrocytes, the major cell type in brain. Thus, in this study, we assessed the localization of AKAP150 in astrocytes and elucidated its role during physiological and ischemic conditions. Herein, we demonstrate that AKAP150 is localized in astrocytes and is up-regulated during ischemia both in vitro and in vivo. Knock-down of AKAP150 by RNAi depolarizes the astrocytic membrane potential and substantially reduces by 80% the ability of astrocytes to take up extracellular potassium during ischemic conditions. Therefore, upregulation of AKAP150 during ischemia preserves potassium conductance and the associated hyperpolarized membrane potential of astrocytes; properties of astrocytes needed to maintain extracellular brain homeostasis. Taken together, these data suggest that AKAP150 may play a pivotal role in the neuroprotective mechanism of astrocytes during pathological conditions.

Keywords: cortical astrocytes; membrane potential; middle cerebral artery occlusion; potassium uptake; siRNA.

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Figures

Fig. 1.
Fig. 1.. AKAP150 expression in cultured astrocytes during control and ischemia-like conditions.
By using Western Blot, levels of AKAP150 protein were determined in control astrocytes and astrocytes exposed to hypoxia/hypoglycemia for 24 hours (Left, n= 4). Quantification of the effect of AKAP150 siRNA on protein levels in astrocytes 4 days after transfection with 20 nM siRNA during normal (Middle, n=3) and hypoxia/ hypoglycemia for 24 hrs (Right, n=3). AKAP150 was detected as a band of around 150KDa which is consistent with the predicted molecular weight. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM). The asterisks indicate a significant difference from control (t-test; p<0.05). Representative Western blots are shown above the graph. Data are expressed relative to control.
Fig. 2.
Fig. 2.. Localization of AKAP150 protein in vivo in astrocytes after tMCAO.
Immunostaining for AKAP150 (green labeling) and GFAP (red labeling) in hippocampus and cortex after tMCAO. Representative images show a qualitative increase of AKAP150 levels in hippocampus (A) and cortex (B) on the ipsilateral (lesion) side of the brain. White arrows point to astrocytic cell bodies and processes. Insets show higher magnification of the merged image to highlight colocalization between GFAP and AKAP150 in astrocytes. Note: There is an appreciable increase of AKAP150 immunoreactivity in astrocytes on the ipsilateral (lesion) side of the brain.
Fig. 3.
Fig. 3.. Distribution of the Membrane Potentials of Control and AKAP150 siRNA transfected astrocytes with and without exposure to hypoxia/hypoglycemia.
A. Control (mock-transfected) astrocytes in normoxic conditions. B. Astrocytes transfected with AKAP150 siRNA (20 nM) in normoxic conditions. C. Control (mock-transfected) astrocytes exposed to hypoxia/hypoglycemia for 24 hours. D. Astrocytes transfected with AKAP150 siRNA (20 nM) exposed to hypoxia/hypoglycemia for 24 hours. In all cases, astrocytes were transfected 4 days prior to electrophysiology studies. Knock-down of AKAP150 in astrocytes exposed to hypoxia/hypoglycemia for 24 hours alters the distribution of the resting membrane potentials towards depolarization.
Fig.4.
Fig.4.. Suppression of AKAP150 modulates ability of astrocytes to buffer K+.
Mock-transfected astrocytes and astrocytes transfected with AKAP150siRNA were subjected to control (normoxic) or hypoxic/hypoglycemic conditions for 24 hours and subsequently utilized for electrophysiology. Cells were bathed in a solution containing 3 mM [K+]o and this solution was changed to either 1 or 10 mM [K+]o. The currents induced by altering the external concentrations of K+ were measured using the whole-cell voltage-clamp technique. (Control/Mock n=14, Control/siRNA n=14, Ischemia/Mock n= 7 and Ischemia/siRNA n=8). Results are expressed as mean current (pA) ± standard error of the mean (SEM). The * indicates a significant difference between Ischemia Control and Ischemia siRNA and ** indicates a significant difference between Control Mock and ischemia Mock (ANOVA followed by Tukey’s; p<0.05).
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