The Rho kinase inhibitor Fasudil up‐regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

Abstract

BACKGROUND AND PURPOSE Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.EXPERIMENTAL APPROACH The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA‐1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F‐/G‐actin, and localizations of EAAT1/2.RESULTS Fasudil and Y27632 increased [3H]‐d‐aspartate (d‐Asp) uptake into astrocytes, and the action of Fasudil was time‐dependent and concentration‐related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F‐actin and increased Vmax for [3H]‐d‐Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2.CONCLUSION AND IMPLICATIONS These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function – this activity profile may contribute to their beneficial actions in neuropathologies.

Fasudil Hydrochloride Effects on Eaat2 and Rock2 Protein Expressions in Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy.

“…This increase of EAAT2 expression was more significant after 2 days of administration of fasudil. These findings are consistent with those observed by several studies that moreover, have given further details [10,12].…”

“…As well as reducing the potentially toxic build-up of extracellular Glutamate, it is likely that Glutamate uptake by glial EAATs also signals the energy needs of nearby neurons via activation of the Na+/K+ ATPase and glucose transporters, and changes in the levels of ATP and lactate [15]. Given that activity of EAAT contributes to the determinant role of astrocytes in synaptic function regulation and the increasing evidence for an inter-dependency between astrocytic phenotype and EAAT function, Lau CL et al [10] found a role not yet determined for the ROCK/Rho system in this relationship. Their findings were consistent with the observations of Abe K et al of stellation of cultured astrocytes after inhibition of Rho kinase by Fasudil and Y-27632 [16].…”

“…However, recent studies found that it can promote survival of neuronal stem cells, axonal regeneration and differentiation of bone marrow mesenchymal cell into neurons [9,10].…”

Astrocytes Grown in Alvetex® Three Dimensional Scaffolds Retain a Non-reactive Phenotype

“…We note recent evidence that the rho-kinase inhibitor Fasudil regulates astrocyte morphology through the re-distribution of actin, potentiating a neuroprotective astrocyte phenotype [56,57]. Other work has shown that inhibition of the Rho-ROCK axis inhibits CSPG induced myosin phosphatase phosphorylation and reverses coffilin phosphorylation [58,59] showing that CSPG and ROCK interaction regulate astrocyte morphology.…”

Fine Astrocyte Processes Contain Very Small Mitochondria: Glial Oxidative Capability May Fuel Transmitter Metabolism

“…Interestingly glutamate uptake and oxidative metabolism may be spatially organized when required. Mitochondria are not associated with glial membrane patches containing the high affinity glutamate transporter GLT-1 in pure astrocyte cultures, and this does not change when mitochondria redistribute during astrocyte stellation induced by ROCK inhibition (by Y27632 [63,64]). When astrocytes are however co-cultured with cortical mouse neurons, glial mitochondria relocate to processes and (modestly) associate with GLT-1 [64].…”