Synaptic Actions of Amyotrophic-Lateral-Sclerosis-Associated G85R-SOD1 in the Squid Giant Synapse

ABSTRACT

Altered synaptic function is thought to play a role in many neurodegenerative diseases, but little is known about the underlying mechanisms for synaptic dysfunction. The squid giant synapse (SGS) is a classical model for studying synaptic electrophysiology and ultrastructure, as well as molecular mechanisms of neurotransmission. Here, we conduct a multidisciplinary study of synaptic actions of misfolded human G85R-SOD1 causing familial Amyotrophic Lateral Sclerosis (fALS). G85R-SOD1, but not WT-SOD1, inhibited synaptic transmission, altered presynaptic ultrastructure, and reduced both the size of the Readily Releasable Pool (RRP) of synaptic vesicles and mobility from the Reserved Pool (RP) to the RRP. Unexpectedly, intermittent high frequency stimulation (iHFS) blocked inhibitory effects of G85R-SOD1 on synaptic transmission, suggesting aberrant Ca²⁺ signaling may underlie G85R-SOD1 toxicity. Ratiometric Ca²⁺ imaging showed significantly increased presynaptic Ca²⁺ induced by G85R-SOD1 that preceded synaptic dysfunction. Chelating Ca²⁺ using EGTA prevented synaptic inhibition by G85R-SOD1, confirming the role of aberrant Ca²⁺ in mediating G85R-SOD1 toxicity. These results extended earlier findings in mammalian motor neurons and advanced our understanding by providing possible molecular mechanisms and therapeutic targets for synaptic dysfunctions in ALS as well as a unique model for further studies.

SIGNIFICANCE STATEMENT The squid giant synapse presents one of the few mature nervous systems in situ that mimics mammalian neuromuscular junctions, while allowing precise experimental manipulations and live measurement with superior spatial and temporal resolution. Applying these unique features to studying the molecular mechanisms of ALS, a devastating adult-onset neurodegenerative disease without cure, offers clues to understand the pathogenesis of the disease. Our results demonstrating synaptic dysfunction caused by ALS-associated mutant SOD1 protein and its underlying molecular pathways may suggest a novel approach to an effective therapeutic intervention as well as identify biomarkers for early diagnosis. Furthermore, the altered synaptic vesicle behavior and Ca²⁺ dynamics revealed through the perturbation of neurotransmission by ALS extends our understanding of fundamental synaptic physiology at both molecular and cellular levels.