Cell targeted phenotyping: Understanding molecular genetic mechanisms of C9ORF72 ALS in motor neurons

Amyotrophic lateral sclerosis (ALS) presents in adulthood with the loss of both upper and lower motor neurons. In 2011, a large hexanucleotide repeat expansion in the C9ORF72 gene was implicated in 7% of sporadic and 40% of familial cases, making this mutation the most frequent cause of ALS known to date.
The differentiation of lower motor neurons from induced pluripotent stem cells derived from patients with the C9ORF72 expansion provides a model allowing exploration of the downstream effects of the expansion whilst maintaining the patient’s genetic background. We have successfully generated high percentage motor neuron cultures from healthy individuals and patients in vitro. These cells are electrophysiologically active and express mature motor neuron markers including choline acetyltransferase and SMI-32. We compared methods and supplements to further increase the electrophysiological maturity of the cells allowing them to resemble physiological motor neurons as closely as possible. We saw a decrease in resting membrane potential when conditions were modified.
Our electrophysiological analysis focuses on the intrinsic properties of the cells as they mature in culture including: voltage gated sodium and potassium channel current, synaptic vesicle release and action potential characteristics. Further studies will look more specifically at channel subtypes present within the patient vs control motor neurons. A HB9:cre/lox lentiviral system to drive the expression of channelrhodopsin allows us to specifically track and stimulate the motor neurons non-invasively.
Overall this project aims to further elucidate C9ORF72 disease pathophysiology to allow future development of therapeutic approaches and targets.