Morphological Plasticity Of Supraoptic Astrocyte And The Interactions Between Its Functional Proteins

Background and PurposeAstrocytes play an important regulatory role in neural functions.Glial fibrillary acidic protein(GFAP)is not only the basic cytoskeletal elements of astrocytes,but also closely related to the functional activities of astrocytes,thereby affecting the interaction between astrocytes and neurons.In GFAP-associated changes in astrocytic structures and functions or astrocytic plasticity,expressions and distributions of many astrocytic functional proteins also change,such as aquaporin 4(AQP4),vesicle-associated membrane protein(VAMP),Ezrin and filamentous actin(F-actin).Among them,AQP4,mainly localized in the membrane of astrocyte processes,has close association with many ion channels and transporters and they together maintain the stability of osmotic pressure and cell volume.VAMP takes a part in transport of cell membrane proteins and exocytotic substances,and plays a vital role in astrocyte membrane functions.Ezrin is a linking protein in cytoskeletal network,and its binding with cell membrane and cytoskeletal elements is a necessity for the expansion and retraction of astrocyte processes.F-actin is a major protein responsible for cell transport and shape alteration,and its reorganization is responsible for the relative movement of astrocyte functional proteins.Our previous work found that GFAP in the supraoptic nucleus(SON)of the hypothalamus has extensive molecular associations with other functional proteins in astrocytes,such as AQP4.Under different functional conditions,the interactions between GFAP and other functional proteins as well as their spatial distribution are significantly different.After changing F-actin reorganization,changes in astrocyte morphological plasticity and its associated oxytocin(OT)neuronal activity occur,indicating that F-actin reorganization may influence the interactions between GFAP and other membrane functional proteins.Based on these findings,we propose that F-actin reorganization plays motor roles in the interaction between GFAP and its associated membrane functional proteins.MethodsIn this study,the SON of SD lactating female rats and male rats were selected as experimental models.In the in vivo studies,three different stages of observations were selected,i.e.non-suckling,suckling stimulation and milk-ejection reflex(MER)on the 8th postpartum day.Lactating rats were decapitated to obtain blood and to dissect the SON.In the in vitro studies,hypothalamic slices were prepared from male rats after decapitation.Then the slices were incubated in vitro with different drugs including OT,potassium(K~+)and cytochalasin D(CD).These drugs were used to simulate SON neurochemical environment at the three different lactating stages in vivo and block F-actin polymerization.The treated SON was punched out and used for Western blot(WB),immunohistochemistry(IHC),and co-immunoprecipitation(CO-IP)that were used to analyze the protein expressions of GFAP,AQP4,VAMP and Ezrin,and molecular association of GFAP with other proteins,and to ascertain role of F-actin in them.ResultsIn the in vivo studies,(1)the expression of GFAP monomer of SON during suckling increased compared to that during non-suckling,and decreased after the MER.(2)Correspondingly,the expression of VAMP and AQP4tended to increase during suckling but decreased after the MER;the expression of AQP4 and VAMP were consistent with the expression of GFAP(i.e.increase and then decrease),and was moderately correlated with the expression of GFAP;however,Ezrin continued to decrease.(3)The molecular association of GFAP with VMAP and AQP4 decreased during suckling and increased after the MER.However,the molecular association of GFAP with Ezrin continued to increase from suckling to the MER.(4)Immunohistochemical analysis showed that the colocalization of GFAP filaments with AQP4 decreased but that with F-actin increased during suckling.After the MER,the colocalization of GFAP filaments with AQP4,F-actin changed little compared to that in non-suckling.In the in vitro studies,(1)after blocking F-actin accumulation with CD,the increases of GFAP,VAMP and Ezrin evoked by OT that simulates suckling were not affected significantly and the reverse decrease of GFAP.The continuous increase of Ezrin and AQP4 caused by the OT plus K~+was not affected significantly.(2)The presence of CD also reversed OT-evoked decreases in the molecular association between GFAP,VAMP and AQP4,but it did not change the molecular association between GFAP and Ezrin.(3)In the presence of CD,OT-K~+-evoked decreases in the molecular associations between GFAP and Ezrin,and between GFAP and VAMP were changed whereas,OT-K~+-elicited decrease in the molecular association between GFAP and AQP4 did not change.ConclusionThe extension of GFAP filaments during astrocyte process expansion can guide astrocyte secretory protein transport and membrane assembly,and the retraction of astrocyte processes is related to increased interactions between F-actin and GFAP.The polymerization of F-actin can promote the depolymerization of GFAP filaments and reduce the expression of secretory and membrane functional proteins while promoting their transport and membrane assembly.