Lysosomes Are the Major Vesicular Compartment Undergoing Ca²⁺-Regulated Exocytosis from Cortical Astrocytes

Abstract

Although Ca 2ϩ-dependent exocytosis is considered to be a pathway for gliotransmitter release from astrocytes, the structural and functional bases of this process remain controversial. We studied the relationship between near-membrane Ca 2ϩ elevations and the dynamics of single astroglial vesicles with styryl (FM) dyes. We show that cultured astrocytes, unlike neurons, spontaneously internalize FM dyes, resulting in the labeling of the entire acidic vesicle population within minutes. Interestingly, metabotropic glutamate receptor activation did not affect the FM labeling. Most FM-stained vesicles expressed sialin, CD63/LAMP3, and VAMP7, three markers for lysosomes and late endosomes. A subset of lysosomes underwent asynchronous exocytosis that required both Ca 2ϩ mobilization from intracellular stores and Ca 2ϩ influx across the plasma membrane. Lysosomal fusion occurred within seconds and was complete with no evidence for kiss and run. Our experiments suggest that astroglial Ca 2ϩ -regulated exocytosis is carried by lysosomes and operates on a timescale orders of magnitude slower than synaptic transmission.

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

“…11,12,20,44 We show that astrocyte cell membrane injury causes a greater [Ca 2+ ] c increase, triggering more robust lysosome exocytosis. Receptor stimulation causes a sustained and low rate of exocytosis where lysosomes release a part of their luminal content and none of their membrane proteins (Figures 2 and 6).…”

“…29,34 However, the existence of partial exocytosis of astrocyte lysosomes has been contested. 11,20 Using FITC dextran to label the lysosome lumen, we found that astrocyte lysosomes can exocytose their contents partially (Figure 2c) or fully (Figure 2d). In fibroblasts, lysosomes that partially release their contents have a restricted fusion pore that allows the release of smaller (10 kDa), but not larger (500 kDa) sized dextran.…”

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

“…42,43 In addition, the core secretory machinery for vesicle exocytosis consists of SNAREs (N-ethylmaleimide-sensitive factor attachment protein receptors) and sec1/STXBP/Munc18-like proteins. 44 The marker protein of secretory lysosomes as the major vesicular compartment for Ca 2C -regulated exocytosis from astrocytes 45 is VAMP7/TI-VAMP (vesicle associated membrane protein 7). 46 Verderio et al (2012) 47 report that VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes.…”

FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes

“…Short exposure of mouse cortical astrocytes in culture to micromolar concentrations of FM4-64 (6.7 M, 2 min) at room temperature (RT) followed by a 10-min wash showed the expected FM4-64 puncta (14,18,19) associated with a diffuse labeling. Rupturing the PM in zero extracellular Ca 2ϩ concentration ([Ca 2ϩ ] o ) reduced the diffuse labeling without affecting the puncta (Fig.…”

“…Cultured astrocytes internalize FM dyes (3,(12)(13)(14)(15)(16)(17)(18). However, this uptake is not modified by the activation of metabotropic glutamate receptors (19), and its mechanism remains elusive.…”