Grebenik, E. | bioRxiv Science

bioRxiv2025

BASP1 Couples Ca2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

Actin-mediated mitochondrial fission is essential for cellular homeostasis, yet the mechanisms by which actin is recruited to mitochondria and how it couples the outer and inner mitochondrial membranes (IMM) remain poorly understood. Using a phosphoproteomic screen in a rat model of -synucleinopathy, we identified BASP1 as a calcineurin-dependent substrate that is constitutively dephosphorylated under pathological Ca2+ elevations and phosphorylated under neuroprotective calcineurin inhibition. Immunoprecipitation and mass spectrometry of phosphomutant BASP1 expressed in neurons revealed that dephosphorylation promotes interactions with actin and IMM proteins. Dephosphorylated BASP1 recruits actin to mitochondria, while subsequent phosphorylation enables actin-mediated mitochondrial fission and neurite elongation. Constitutive dephosphorylation, as it occurs in -synucleinopathy, impairs mitochondrial fission, inhibits neurite growth and promotes -synuclein aggregation. Our findings position BASP1 as a Ca2+-CaN-regulated hub that coordinates actin remodeling and couples mitochondrial membranes to drive fission, revealing a mechanistic axis linking mitochondrial dysfunction to neuronal morphogenesis and -synuclein pathobiology.

cell biology↗

bioRxiv2025

Calcineurin-mediated regulation of GAP-43 is essential for neurite and synapse formation and protects against α-synuclein-induced degeneration

IntroductionElevated calcium (Ca2+) levels and hyperactivation of the Ca2+-dependent phosphatase calcineurin are key factors in -synuclein (-syn) pathobiology in Dementia with Lewy Bodies and Parkinsons Disease (PD). Calcineurin activity can be inhibited by FK506, an FDA-approved compound. Our previous work demonstrated that sub-saturating doses of FK506 provide neuroprotection against -syn pathology in a rat model of -syn neurodegeneration, an effect associated with the phosphorylation of growth-associated protein 43 (GAP-43). MethodsTo investigate the role of GAP-43 phosphorylation, we generated phosphomutants at the calcineurin-sensitive sites and expressed them in PC12 cells and primary rat cortical neuronal cultures to assess their effects on neurite morphology and synapse formation. Additionally, we performed immunoprecipitation mass spectrometry in HeLa cells to identify binding partners of these phosphorylation sites. Finally, we evaluated the ability of these phosphomutants to modulate -syn toxicity. ResultsIn this study, we demonstrate that calcineurin-regulated phosphorylation at S86 and T172 of GAP-43 is a crucial determinant of neurite branching and synapse formation. A phosphomimetic GAP-43 mutant at these sites enhances both processes and provides protection against -syn-induced neurodegeneration. Conversely, the phosphoablative mutant prevents neurite branching and synapse formation while exhibiting increased interactions with ribosomal proteins. DiscussionOur findings reveal a novel mechanism by which GAP-43 activity is regulated through phosphorylation at calcineurin-sensitive sites. These findings suggest that FK506s neuroprotective effects may be partially mediated through GAP-43 phosphorylation, providing a potential target for therapeutic intervention in synucleinopathies.

neuroscience↗