Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells

Abstract

The recycling of synaptic vesicle (SV) proteins and transmitter release occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the adaptor protein 1 and 3 complex (AP-1/AP-3). As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 min after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. Cover Image for this issue: doi. 10.1111/jnc.13801.

Photoconversion of FM1-43 Reveals Differences in Synaptic Vesicle Recycling and Sensitivity to Pharmacological Disruption of Actin Dynamics in Individual Synapses

“…We observed an increase in the proportion of PC + SVs when the fixative was added 10 min after stimulation relative to those found when it was added immediately after stimulation. These results, together with the reduction in endosome size, suggest the processing of endosomes and the formation of new SVs from these structures, in close agreement with previous observations. ,− However, endosomes are not fully transformed into SVs and the remains of these organelles (smaller endosomes) are still observed 10 min after the end of the stimulus, helping understand the differences in recycling efficiency observed in the FM1-43 experiments shown here and previously . It is conceivable that boutons that retain more endosomes also retain more fluorescence and thus, they will be considered as low recycling efficiency boutons .…”

“…The profiles of dye loss from 20 randomly selected synaptic boutons stimulated chemically (Figure B) or electrically (Figure C) reflected the wide heterogeneity of the responses in both cases. More synaptic boutons responded to chemical stimulation and as shown previously, the responses to this stimulus were faster and produced stronger dye release. Hence, and in contrast to hippocampal neurons, the heterogeneity of responses in cerebellar granule cells persists even when they are stimulated at high frequency or by strong depolarizing conditions.…”

“…Different mechanisms of endocytosis contribute to membrane retrieval at cerebellar granule cell boutons, CME and ADBE. The contribution of ADBE to membrane retrieval after strong stimulation of cerebellar granule cells has been demonstrated extensively elsewhere − and in our previous studies using horseradish peroxidase (HRP) to label endosomes . Using FM1-43 FX photoconversion to label endocytic organelles, here we found that most of the fluorescence is incorporated into SVs, with endosomes apparently distributed nonuniformly in all the boutons.…”

Altered Synaptic Membrane Retrieval after Strong Stimulation of Cerebellar Granule Neurons in Cyclic GMP-Dependent Protein Kinase II (cGKII) Knockout Mice

“…Thus, the extent by which FM1-43 staining is unloaded can be considered as a parameter of synaptic vesicle recycling at nerve terminals. Chemical stimulation was employed to mobilize the recycling pool and to incorporate the fluorescent styryl dye FM1-43 into SVs in cerebellar granule cells because it is necessary a strong stimulus [ 31 ]. This phenomenon is also observed in vivo as the synapses between mossy fibers (MFs) and granule cells are characterized by high rates of sustained vesicular release [ 32 ].…”

Nitric Oxide and Synaptic Transmission in the Cerebellum

“…This type of endocytosis is not involved in the immediate re-availability of vesicles but rather in preventing a deleterious increase of the presynaptic terminal's membrane (Clayton and Cousin 2009). The final destination of the endosomes formed seems to be their future budding to yield new vesicles (Clayton and Cousin 2009;Clayton et al 2008;Ramperez et al 2017;Teng et al 2007). The models used to explain this type of endocytosis assume a saturation of clathrin-mediated endocytosis (Lou et al 2008), and once this capacity is exceeded, bulk endocytosis will be triggered (Clayton and Cousin 2009).…”