Abstract
In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity dependent bulk endocytosis (ADBE). Alix (ALG-2 interacting protein X)/ PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins and thereby driving deformation and fission of endosomal and cell surface membranes. In fibroblasts, Alix is required for clathrin-independent endocytosis. Here, using electron microscopy, we show that synapses from mice lacking Alix have subtle defects in presynaptic compartments, translating into flawed synaptic plasticity. Using cultured neurons, we demonstrate that Alix is required for ADBE. We further demonstrate that in order to perform ADBE, Alix must be recruited to synapses by the calcium-binding protein ALG-2 and interact with endophilin-A. Finally, we show that mutant mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus. Furthermore, propagation of the epileptiform activity to the contralateral side of kainate injection is reduced. These results thus highlight Alix ko mice as an invaluable model to study the exact role of ADBE at synapses undergoing physiological or pathological stimulations.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
List of abbreviations
- 4AP
- 4-Aminopyridine
- ADBE
- Activity-Dependent Bulk Endocytosis
- ALG-2
- Apoptosis Linked Gene 2
- Alix
- ALG-2 Interacting protein X
- BAPTA
- 1,2-Bis(o-AminoPhenoxy)ethane-N,N,N’,N’-Tetraacetic Acid
- BE
- Bulk Endosome
- CA1
- Cornu Ammonis 1
- CA3
- Cornu Ammonis 3
- CHMP4B
- Charged Multivesicular body Protein 4B
- CGN
- Cerebellar Granule Neuron
- CIE
- Clathrin-Independent Endocytosis
- CME
- Clathrin-Mediated Endocytosis
- CTxB
- Cholera Toxin chain B
- DIV
- Day In Vitro
- EGTA
- Ethylene Glycol-bis(β-aminoethyl ether)-N,N,N’,N’-Tetraacetic Acid
- EM
- Electron Microscope (Microscopic/ Microscopy)
- ESCRT
- Endosomal Sorting Complexes Required for Transport
- GABAA
- γ-AminoButyric Acid type-A
- GFP
- Green Fluorescent Protein
- HRP
- Horse Radish Peroxidase
- kDa
- kiloDalton
- KO
- KnockOut
- LTP
- Long-Term Potentiation
- N-BAR
- N-terminal amphipathic helix-Bin-Amphiphysin-Rvs
- PRD
- Proline-Rich Domain
- PSD95
- Post-Synaptic Density Protein 95
- ROI
- Region of interest
- SH3
- Src Homology 3
- SV
- Synaptic Vesicle
- Syp-pH
- Synaptophysin-pHluorin
- TSG-101
- Tumour Susceptibility Gene-101
- WT
- Wild Type
- YFP
- Yellow Fluorescent Protein