Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones

Abstract

Key pointsr Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process.r We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle.r We separated two sequential phases of endocytosis taking place during the stimulation train:early and late endocytosis.r Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity.r Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin-phospholipid interaction.Abstract Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo-and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB.

Early auto‐immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis

“…We focused on photoreceptor ribbon synapses because these were particularly sensitive and the most affected synapses (Figs C and C, and ). To address this question at the cellular level, we generated a transgenic mouse line in which the SypHy protein, a fusion protein of synaptophysin with pH‐sensitive GFP (Dreosti & Lagnado, ; Linares‐Clemente et al , ), is expressed under the control of the mouse rod opsin promotor to report synaptic vesicle cycling in mouse rod photoreceptor ribbon synapses (Fig ).…”

“…A transgenic mouse was generated in which the pH‐sensitive SypHy (acronym for a fusion construct of pH‐sensitive GFP fused to the lumenal domain of the synaptic vesicle protein synaptophysin; Linares‐Clemente et al , ) was expressed under the control of the mouse rod opsin promotor (Geppert et al , ). The mouse opsin promotor was cloned into the HindIII/SalI site, the SypHy cDNA into the XhoI/BamHI site, and the human growth hormone polyadenylation signal into the BamHI/NotI site of a pEGFP‐N1‐based plasmid vector.…”

Synaptic Vesicle-Recycling Machinery Components as Potential Therapeutic Targets

“…2). Furthermore, recent studies have provided several examples in which synaptic vesicle-recycling process and subsequent dynamics of neurotransmitter release can be modulated by small molecules that target dynamin-dependent endocytosis or myosin light chain kinase-dependent vesicle transport (Chung et al, 2010;Maeno-Hikichi et al, 2011;Linares-Clemente et al, 2015). From a neurotherapeutics perspective, targeting the synaptic vesicle-recycling machinery may present a key advantage as it induces frequency-dependent changes in the efficacy of neurotransmission to counter or correct disease processes, in contrast to typical blockers of neurotransmission that trigger global suppression or augmentation of neurotransmitter release that may potentially yield broader side effects (Kavalali, 2006).…”

Monitoring activity-dependent bulk endocytosis with the genetically-encoded reporter VAMP4-pHluorin

“…Finally, the discovery of VAMP4-pHluorin as a selective reporter of ADBE opens up the possibility of incorporating the probe at the genomic level to facilitate in vivo studies of this endocytosis mode. This approach has yielded important information in previous studies in both synaptobrevin II-pHluorin-expressing invertebrate and mammalian systems ( Araki et al, 2005 , Li et al, 2005 , Dason et al, 2010 , Linares-Clemente et al, 2015 ). The potential power of this approach is still untapped, however it may offer the tantalising possibility of monitoring ADBE in intact neuronal circuits, in real time, in vivo .…”