Yuuta Imoto Lab

Decoding the Dynamin Code: Splice variants in synaptic function and disease

The fastest pathway for SV recycling, known as ultrafast endocytosis, occurs within 100ms after neurotransmitter release. This is 100-fold faster than the textbook mechanism, clathrin-mediated endocytosis. Despite these vastly different kinetics, both pathways share the same regulatory genes. It has remained unclear how the same genes can mediate such a wide range of speeds. 

Using time-resolved electron microscopy, we identified that ultrafast endocytosis is mediated by a brain-enriched splice variant of a membrane pinchase called dynamin (Dyn1xA). Super-resolution microscopy techniques, including gated-Stimulated Emission Depletion (gSTED) microscopy, revealed that Dyn1xA and other endocytic proteins form molecular condensates in presynapses to concentrate these proteins. This structure accelerates endocytic kinetics by 100-fold. 

As exemplified by Dyn1xA, alternative would be a key regulator for rapid synaptic function. Brain tissue expresses over 30 different combinations of dynamin splice variants in response to different neuronal activities, developmental stages and likely specific neuronal subtypes. Our lab focuses on deciphering the 'Dynamin code'—the puzzle pieces of splicing regions that allow neurons to adapt to a wide range of brain functions. 

Our findings have translational potential as some human diseases such as Down syndrome, epilepsy and autism are associated with abnormal SV recycling. In fact, specific Dynamin exons exhibit mutations that are associated with these neuronal disorders. Being able to pinpoint the proteins responsible for affecting these processes gives researchers targets to consider for therapeutic purposes. 

The high frequency of synaptic transmissions leads to an accumulation of old and/or damaged synaptic vesicle proteins. The overall health of the neuron depends on its ability to identify, isolate and deliver these aberrant proteins to the cell body for degradation. Our laboratory is interested in deciphering the molecular underpinnings driving this process. A key organelle for sorting damaged proteins is the late endosome. While traditional models suggest that late endosome maturation takes more than five minutes, we observe the appearance of late endosomes as early as 10 seconds after neurotransmitter release. To investigate molecular mechanisms of this rapid maturation in presynapses, we use time-resolved electron microscopy and gSTED microscopy, combined with genetic engineering. This approach allows us to directly observe membrane dynamics during endosomal maturation with millisecond precision and localize proteins on 100-300 nm diameter endosomes within presynapses. These insights help us understand how abnormal synaptic vesicle proteins are sorted in both healthy and pathological conditions.

Modern animals have well-established centralized nervous systems, enabling complex behaviors and cognitive functions, including learning and memory formation. In contrast, our common ancestors such as cnidarians exhibit simpler nervous system networks and low-frequency neurotransmission, but they already possess similar sets of genes related to synaptic formation and SV release as found in mammals. However, genes involved in rapid SV recycling, including dynamin splice variants, only emerged in the earliest species with centralized nervous systems. 

Our laboratory investigates how these early, rapid SV recycling genes contributed to the establishment of modern synaptic structures and functions. To address this question, we apply advanced microscopy techniques and genetic engineering in various bilaterian and non-bilaterian organisms.