Abstract Body: Introduction: Clearing necrotic tissue from lesions by microglia/macrophage is crucial for recovery after acute ischemic stroke (AIS). Lysosomal activity is key for efferocytosis. In the chronic phase, reduced lysosomal activity impairs clearance, causing residual debris and worsening cognitive impairment. Migrasomes, formed during cellular migration, help maintain other organelles like mitochondria.
Hypothesis: This study explored how migrasomes in microglia/macrophage help load damaged lysosomes for expulsion, preserving lysosomal quality and efferocytosis post-stroke.
Methods: Male C57BL/6 mice including wild-type (WT) and Tspan14 conditional knockout (Tspan14^fl/flLyz2^Cre, TSPAN14 CKO) underwent transient middle cerebral artery occlusion (tMCAO). Migrasome production and TSPAN14 expression were evaluated through staining and nano flow cytometry. Neurobehavioral performance was measured by rotarod, foot fault, novel object recognition, and water maze tests up to 14 days post-stroke. In vitro, bone marrow-derived macrophages (BMDM) were cultured with blebbistatin to inhibit migrasome production. BMDM efferocytosis of dead neurons was assessed by staining and flow cytometry.
Results: In cultures, 76.9% of migrasomes from BMDM engulfing dead neurons contained damaged lysosomes. Inhibiting migrasome production in BMDM impaired efferocytosis and increased damaged lysosome accumulation. TSPAN14 on the lysosomal membrane mediated lysosome sorting into migrasomes. Migrasomes from TSPAN14 CKO BMDM engulfing dead neurons had 62.3% less lysosomal content than those from WT BMDM, though their numbers were similar (P<0.05). BMDM from TSPAN14 CKO mice had 212.4% more damaged lysosomes and 54.7% less efferocytosis compared to WT BMDM (P<0.05). In tMCAO model, migrasome production and TSPAN14 expression increased in Iba1⁺ microglia/macrophage after stroke, peaking on day 3 and stabilizing by day 7. By day 14, both decreased. TSPAN14 CKO mice had worse sensorimotor and spatial learning deficits than WT mice 14 days after tMCAO (P<0.05). Microglia/macrophage in TSPAN14 CKO lesions had more damaged lysosomes and reduced efferocytosis (P<0.01).
Conclusions: TSPAN14 regulates lysosomal quality through migrasome-dependent mechanisms. In the chronic stroke phase, reduced TSPAN14 impairs lysosomal control in microglia/macrophage, leading to incomplete clearance and worsened cognitive impairment. Thus, TSPAN14 is a promising target to enhance clearance and improve stroke outcomes.