Isolated Axoplasm from Squid and Pathogenic Mechanisms in Amyotrophic Lateral Sclerosis.

Abstract

The squid giant axon is an experimental model that has been instrumental in providing answers to many fundamental questions in cell biology, including the discovery of the motor protein kinesin and probing molecular mechanisms of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The giant axon offers scale and ease of access that is not possible in mammalian models. The power of this model stems from the fact that one can introduce reagents and proteins that would not cross a plasma membrane at defined concentrations in a buffer environment that is closely modeled on normal components of the axoplasm. By leveraging the unique biology of the squid axon, early studies were able to define the channels and ion specificities of the action potential. Subsequent studies using video-enhanced contrast- differential Interference contrast microscopy (VEC-DIC) and the isolated axoplasm led to the identification of kinesins, a class of motor proteins responsible for intracellular vesicle trafficking toward the plus end of microtubules. This combination of isolated axoplasm and advanced imaging continues to provide insights into pathogenic mechanisms for adult-onset neurodegeneration, including ALS, and regulation of fast axonal transport, a process disrupted in motor neuron disease. Here, procedures for dissecting the giant axon, isolating axoplasm, and perfusing defined amounts of proteins and/or reagents into the axoplasm are detailed, as well as methods for visualizing and interactively analyzing vesicle traffic in real time. This model represents a singular avenue to understand the critical function of neurons, shedding light on the mechanisms driving ALS pathology and other neurodegenerative diseases. This system also offers a tractable platform for drug testing, where candidate therapeutic compounds can be introduced directly into the axoplasm, and their effects on vesicle trafficking can be assessed in real time, providing a rapid and physiologically relevant means of identifying potential ALS therapeutic targets.