A novel artificially inducible cargo assay to probe intracellular transport defects in neurodevelopmental diseases

/abstract Microtubule-based intracellular transport is a fundamental process that is highly regulated for the growth, survival, and function of all cell types. The best example is found in neurons, which have long axons that extend from the cell body and connect with synaptic targets at their terminals. Long range transport of cargos is mediated by the plus-end motor kinesins walking along polarized microtubules which provide an active mechanism to deliver RNA, synaptic proteins and membrane made in the cell body to axonal terminals. This is a highly regulated process, highlighted by defects in transport contributing to many neurodevelopmental neurodegenerative diseases. Many approaches to studying motor function have relied on purified systems to identify molecular, structural, and biophysical properties. However, how to translate these in vitro findings to a cellular context remains a challenge. Other assays have successfully used the forced redistribution of endogenous organelles and vesicles as readout for motor behviour, but these systems can lead to negative changes within the neuron and further, many endogenous cargos are already bound by endogenous proteins and motors interfering with the interpretation of the results. What is needed is a new inducible system to assay motors and cargos with high degrees of spatial and temporal control to compliment existing methods. Here we propose a new optogenetic method to assay intracellular transport of expressed and endogenous motors. We have developed a method to induce artificial cargos which do not interact with endogenous proteins and do not affect normal neuronal function. Through an additional optogenetic system, we can control recruitment to artificial cargos binding specific motors, both expressed or endogenous. The inducibility of this system allows for a high degree of spatial and temporal control, while also providing a high throughout assay. This system can be used to not only identify fundamental in vivo properties of intracellular transport, but can also be used as an unbiased readout for motors with pathogenic mutations that contribute to neurodevelopmental disorders in humans. Further, we can utilize this approach to address how motors and cargos are regulated at precise regions throughout the neuron. Combined, this approach will novel way of assembling artificial protein cargos, which can be used to dissect the properties of molecular motors and mutations responsible for neurological diseases. Project Number: 1R15NS149117-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Stephen Tymanskyj | Institution: AUGUSTA UNIVERSITY, AUGUSTA, GA | Award Amount: $555,000 | Activity Code: R15 | Study Section: Neuronal Communications Study Section[NC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11360921