Intermediate filaments attenuate stimulation‐dependent mobility of endosomes/lysosomes in astrocytes

Abstract

Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis-mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild-type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1-positive vesicles and reduced the mobility of ANP-positive vesicles in WT astrocytes. In GFAP(-/-)Vim(-/-) astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1-positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation-dependent attenuation of ANP-positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP(-/-)Vim(-/-) astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation-dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking.

Single-vesicle architecture of synaptobrevin2 in astrocytes

“…We next assessed vesicular diameter using an approach involving filling the vesicle lumen in live astrocytes using LysoTracker Red DND-99, which stains acidic organelles within a cell 25 . The specificity of monitoring secretory vesicles was obtained by transfecting astrocytes to express YSpH and hence label Sb2-positive vesicles, before their exposure to LysoTracker.…”

“…For our present measurements, only a subset of vesicles was interrogated due to the fact that a vesicle expressing a visible YSpH requires optimized pH in the vesicle lumen along with co-labelling with LysoTracker. Namely, LysoTracker stains mostly acidic compartments of astrocytes 25 , whereas YSpH becomes fluorescent once the fusion pore is open or by other mechanisms that lead to alkalinization.…”

Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system

“…When the GFAP À/À Vim À/À mice were subjected to focal brain ischemia, they developed larger infarction compared to wild-type mice [73 ]. We are still seeking the mechanistic understanding of some of these phenomena, but a number of studies linked the astrocyte IF system to cell migration [54], viscoelastic properties [74], vesicle trafficking [75][76][77], activation of Erk and c-fos [78], control of glutamate transport and astrocyte gap junctional communication [73 ], reaction to hypoosmotic and oxidative stresses [49,79], reconstruction of blood-brain barrier [80], and interaction with microglia [81,82].…”

Astrocyte Reactivity and Reactive Astrogliosis: Costs and Benefits

“…The same conclusion was made by exposing the GFAP Ϫ/Ϫ Vim Ϫ/Ϫ mice to focal brain ischemia (induced by the transection of the middle cerebral artery), a mouse model of ischemic stroke, which resulted in larger infarction compared with wild-type mice (132). The underlying cellular and molecular mechanisms remain incompletely understood, although the astrocyte IF system has been linked to astrocyte motility (131); viscoelastic properties, which might affect cell migration (139); vesicle trafficking (184,185,241); activation of Erk and c-fos (156); the efficiency of glutamate transport and astrocyte gap junctional communication (132) (135); response to hypoosmotic and oxidative stress and neuroprotective properties (55,58); reconstruction of blood-brain barrier (177); MHC class II molecule presentation (241); and interaction with microglia (125,143). The functions of astrocytes in healthy CNS and in acute and late stages of neurological diseases are exemplified in FIGURE 8C.…”