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  • All iPSC derived neurons demonstrated expression of function

    2018-10-24

    All iPSC-derived neurons demonstrated expression of functional neurotransmitter receptors, responding to glutamate/glycine, GABA and to a lower extend to acetylcholine. Interestingly, the neuronal cultures also developed synchronized spontaneous calcium oscillations, which were likely to reflect Sunitinib firing (Robinson et al., 1993). This was confirmed as the inhibitor of voltage-gated sodium channels, TTX, blocked the synchronization of the oscillations. In vivo, spontaneous calcium oscillations occur in individual neural precursors and immature neurons in early development where they regulate several processes (Blankenship and Feller, 2009). Both during late embryonic and early postnatal development, neurons from many different areas of the CNS have been shown to form synchronized oscillatory networks consisting of bursts of action potentials separated by periods without firing. Such networks are believed to be mediated by gap junctions during embryonic development, while chemical synaptic transmission tends to mediate the majority of the network activity later in development and in mature neurons (Blankenship and Feller, 2009; Sutor and Hagerty, 2005). The occurrence of spontaneous synchronized calcium oscillations as well as the expression of the presynaptic protein Synapsin 1 in our neuronal cultures therefore implies a certain level of neuronal maturity. The mediation by chemical synapses could be confirmed by applying inhibitors or knock down of neurotransmitter receptors or gab junctions (Blankenship and Feller, 2009). In vitro, synchronized network activity mediated by chemical synapses has been observed in primary (Dravid and Murray, 2004) and ESC derived neurons (Heikkilä et al., 2009). Only recently, such network activity was reported in one study of iPSC derived neurons. However, these neurons had to be cultured for 70–140days and form 3D structures before regular spontaneous oscillations dependent on chemical synaptic activity were observed (Kirwan et al., 2015). In our neuronal cultures, synchronized spontaneous activity occurred robustly in all cell lines already after 28days of maturation, which indicated that they have reached a relatively advanced state of maturity compared to the majority of other iPSC derived neurons. A key element of SCA3 is the presence of SDS-insoluble ataxin-3 aggregates. Here we strived to induce aggregate formation with glutamate stimulation as described previously (Koch et al., 2011), yet, without success. As we were unable to obtain a positive control such as transgenic mouse brain with ataxin-3 aggregates, it was not possible to evaluate the protocols for detection of ataxin-3 aggregates. In addition, several biological traits of the investigated neurons could have inhibited the formation and accumulation of aggregates. Firstly, the neuronal differentiation protocol applied by Koch et al. was significantly different than ours, and likely to have Sunitinib generated a different neuronal population. In support of this, their neurons expressed the GABAergic marker GABA, whereas our cells only expressed GABAergic marker mRNA but not protein. Secondly, it is possible that our neurons are better at clearing cleaved and aggregated ataxin-3. For example, db-cAMP, has been shown to increase proteasomal activity (Myeku et al., 2012) and is used in a 800× lower concentration in the maturation protocol applied by Koch et al. compared to our protocol. However, withdrawal of db-cAMP for the last week of maturation did not induce SDS-insoluble aggregation in our experiments. Finally, our neurons might express less calpain or the calpain-activity might be decreased, reducing the cleavage of exp.-ataxin-3 and, thus, the formation of aggregates. For example, the spontaneous calcium oscillations caused by action potential firing in the neurons could induce prolonged calpain activation, which is known to upregulate the expression of the endogenous calpain inhibitor calpastatin (Averna et al., 2003). Supporting this theory, we were unable to induce high levels of calpain mediated cleavage of ataxin-3 and the calpain substrate α-fodrin using several ways to increase intracellular calcium levels in order to activate calpain, including increased glutamate response, treatment with a calpain activator (dibucaine) and a purinoreceptor agonist (ATP). The ionophore ionomycin, however, increased ataxin-3 and α-fodrin cleavage, but it was not possible to find an ionomycin concentration, which induced ataxin-3 cleavage without being toxic to the neurons. In further experiments, it will be relevant to explore other methods, such as induction of oxidative stress, proteasomal inhibition or inhibition of autophagy to either induce aggregation or cause accumulation of aggregates by inhibiting their degradation.