Conclusion & Discussion

The studies above enabled us to characterize purified embryonic stem cell-derived motor neurons exposed to mutant astrocyte-conditioned medium as a powerful in vitro model to study ALS- linked non-cell autonomous motor neuron degeneration. In these studies, a survival timeline was demonstrated for the purified embryonic stem cell-derived motor neurons upon 7 days of exposure to mutant astrocyte-conditioned medium that is similar to the phenomenon observed in primary motor neurons (Nagai et al., 2007). Interestingly, the 50% motor neuronal loss observed after 7 days of exposure to mutant astrocyte-conditioned medium cannot be due to the loss of toxic activity of the medium, because even if this conditioned medium is changed half-way into the exposure duration, there is no significant further loss of motor neurons. Remarkably, a similar level of motor neuron loss has been reported by Raoul et al. (2002) in their study on Fas ligand toxicity in motor neurons (Raoul et al., 2002). Together these studies suggest that the observed 50% of the motor neurons that are spared symbolize a more resistant subpopulation of motor neurons.

Since the purpose of this project is to elucidate the molecular mechanism of non-cell autonomous motor neuron degeneration before neurons become committed to die, a point of no return was identified which will allow the capture of its molecular correlate by the RNA-Seq assay. Accordingly, by testing different exposure times, around 72 hours of exposure to mutant astrocyte-conditioned medium was noted as the time that motor neurons become irreversibly committed to die.

Moreover, compared to the other currently available in vitro ALS models, the model system characterized above has multiple advantages: (1) embryonic stem cell-derived motor

neurons combined both the characteristics of postmitotic motor neurons and the fact that they were readily expandable; (2) mutant astrocyte-mediated toxicity was specific for motor neurons; and, (3) this culture system could model disease processes both extrinsic and intrinsic to motor neurons. All of these advantages render embryonic stem cell-derived motor neurons a unique tool to unravel early molecular perturbations that take place in motor neurons in response to ALS-linked astrocyte toxicity.

Before the characterization of this in vitro model, there was a question as to whether the purification procedure might render the cells more resistant to the mutant astrocyte-induced toxicity. This was because the isolation method itself caused a physical stress on the cells and could cause or contribute to the demise of the sensitive cell subpopulation in the process. To avoid this scenario, purified embryonic stem cell-derived motor neurons were kept in fresh medium for two days prior to exposing them to mutant astrocyte-conditioned medium. This duration was enough time for the motor neurons to recover from the stress of the purification procedure and to grow processes in culture, suggesting healthy conditions of the cells (data not shown). Furthermore, purified embryonic stem cell-derived motor neurons showed about 50% decline in number after being in fresh medium for 2 days and being exposed to mutant astrocyte- conditioned medium for the following 7 days. This confirmed the stable existence of both the resistant and the sensitive subpopulations in this model system similar to what was observed in the mixed motor neuron cultures.

Following the above-mentioned findings, there are a number of interesting future studies that can be performed to study these subpopulations. For one, another batch of mutant astrocyte- conditioned medium can be added to purified embryonic stem cell-derived motor neurons after

168 hours of exposure to mutant medium to see if a similar molecular mechanism is activated in the resistant subpopulation as was in the sensitive subpopulation. Similarly, the transcriptional alterations that occur in purified embryonic stem cell-derived motor neurons after 168 hours of exposure to mutant astrocyte-conditioned medium, when only the resistant cells survive, can be compared to an earlier exposure time, when sensitive cells are present but are dying. In this manner, the molecular mechanism that is different between the sensitive and resistant subpopulations may be better understood. As of yet, no one has discerned if there are molecular differences between these two motor neuron subpopulations or that the resistant motor neurons merely need more time to become committed to death.

Another interesting future study would be to add the Bax inhibitor, V5, to purified embryonic stem cell-derived motor neurons after 72 hours of exposure to mutant astrocyte- conditioned medium, when motor neurons are already committed to cell death, and to see whether the full rescue of motor neurons can still be observed. If a rescue is not observed, this experiment would further validate that 72 hours of exposure to mutant astrocyte-conditioned medium is the point of no return for motor neurons and their death mechanism cannot be reversed.

Finally, all of the experiments performed on primary motor neurons displayed the same results as those performed on purified embryonic stem cell-derived motor neurons, suggesting strong similarities between these two types of motor neurons. However, even though embryonic stem cell-derived motor neurons are a powerful model system to elucidate the molecular mechanism underlying the ALS-linked astrocyte toxicity for motor neurons in vitro, it is still necessary to validate master regulators inferred from the RNA-Seq assay in primary motor

neurons and in transgenic animal models. In the case that a pathway would show pathogenic significance, both in vitro and in vivo, it would open new doors for therapeutic interventions aimed at targeting deleterious pathways before irreversible damage or commitment to death occurs during ALS-linked motor neuron degeneration.