Lemaitre, F. | bioRxiv Science

bioRxiv2020

ALG-2 interacting protein-X (Alix) is required for activity-dependent bulk endocytosis at brain synapses

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 which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with, and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ABDE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix alters synaptic recovery during physiological or pathological repeated stimulations.

neuroscience↗

bioRxiv2020

Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

Anti-CD20 monoclonal antibody (mAb) represents an effective strategy for the treatment of B cell malignancies that may involve complement activity, antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP). While ADP mediated by Kupffer cells is essential to deplete circulating tumors, the relative contribution of each mechanism to the elimination of non-circulating targets has yet to be clarified. Using intravital imaging in a model of MYC-driven B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To quantify the contribution of different cytotoxic mechanisms in situ, we designed a dual fluorescent reporter to track phagocytosis and apoptosis in real-time. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting only in partial depletion. Moreover, macrophage density was strongly reduced in tumor-rich regions. Given their sessile phenotype, macrophages primarily targeted neighboring tumors, resulting in a substantial spatial constraint. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy represents a critical path towards the design of optimized therapies. Key pointsO_LIFunctional intravital imaging establishes antibody-dependent phagocytosis as the major mechanism acting at the tumor site during anti-CD20 therapy. C_LIO_LIA transient wave of phagocytosis and a limited macrophage density restrict the efficiency of anti-CD20 anti-tumor activity. C_LI

immunology↗