Although the CAG expansion in ATXN is known

Although the CAG-expansion in ATXN3 is known to cause SCA3, the underlying cellular mechanisms leading to neurodegeneration are not fully elucidated. It has been proposed that the polyQ expansion alters normal ataxin-3 functions in for example endoplasmic reticulum associated degradation (ERAD) and transcriptional regulation (Matos et al., 2011). The polyQ repeat expansion also leads to the formation of SDS-insoluble exp.-ataxin-3 aggregated oligomers and large neuronal intranuclear inclusions (NIIs) (Ellisdon et al., 2006), of which especially the oligomers are thought to be toxic (Pellistri et al., 2013). Aggregates can sequester proteins from diverse cellular processes (Chai et al., 2002; Matos et al., 2011; Mori et al., 2012) and may also disturb the cellular quality control systems, such as the ubiquitin proteasome system and autophagy by overloading (Matos et al., 2011). A prevailing hypothesis is that cleavage of exp.-ataxin-3 is important for its aggregation, as C-terminal polyQ containing fragments of exp.-ataxin-3 seed ataxin-3 aggregation and induce cell death to a higher extend than full length exp.-ataxin-3 (Goti, 2004). Additionally, fragments of ataxin-3 have been detected in affected brain areas of SCA3 patients but not in unaffected areas or in brains of healthy controls (Goti, 2004). In particular the role of calpain-mediated ataxin-3 cleavage in SCA3 pathogenesis is supported by convincing evidence (Hübener et al., 2013; Koch et al., 2011). Functional studies investigating molecular mechanisms of neurodegenerative diseases are generally limited by the lack of reliable in vitro models of human neurons. Induced pluripotent stem cell (iPSC)-derived neurons are a potential solution to this problem and several disease models have already been generated and published (Reviewed by Corti et al., 2015). The first iPSC lines derived from SCA3 patients were reported by Koch et al. (Koch et al., 2011). In that study, SDS-insoluble exp.-ataxin-3 buy HG-9-91-01 could be induced by stimulation with glutamate. This aggregation could be prevented by calpain inhibition, supporting the hypothesis that glutamate induced calcium influx activates calpains, which subsequently mediates the cleavage and aggregation of exp-ataxin-3. In the present study, we aimed at creating iPSCs from SCA3 patient fibroblasts by a non-integrating method, as integration of reprogramming vectors might lead to insertional mutagenesis and has been shown to decrease the differentiation capacity of iPSCs (Sommer et al., 2010). We strived to obtain a model system with increased resemblance to the affected regions of SCA3 brains by applying a differentiation method, which favored the development of hindbrain neurons. Furthermore, we sought to obtain mature neurons to approximate the situation in SCA3 patients where pathology develops in late childhood at earliest (Kieling et al., 2007). Finally, we investigated whether glutamate-induced SDS-insoluble exp-ataxin-3 aggregates similar to those detected by Koch et al. could be observed in our neurons.
Materials and methods
Results
Discussion In general, studies of human neurodegenerative disease mechanisms are limited by the lack of human neuronal in vitro models. In this study, we report iPSC lines that can form the basis for developing a new improved model system for studying SCA3 disease mechanisms. iPSCs generated from two SCA3 patients (four lines) and one healthy individual (three lines) by reprogramming of dermal fibroblasts with an integration-free method were differentiated into neurons. As the hindbrain is particularly affected by neurodegeneration in SCA3, we applied a neuronal differentiation protocol, which has been reported to give rise to NSCs expressing hindbrain genes such as HOXA2 and HOXB2 (Yan et al., 2013). The applied neuronal differentiation method generated large amounts of NSCs from a single neural induction (1× 6-well of iPSCs can generate 400 vials of 4 million p4 NSCs) which could be easily obtained and frozen. Differentiations from these NSCs have proven to be highly reproducible in our laboratory. Expression of the hindbrain genes HOXA2 and GBX2 were increased in all derived NSCs and neurons to levels comparable to fetal hindbrain. In addition, the forebrain markers FOXG1 and LHX2 were also expressed at the level of fetal hindbrain. Together, these data indicate that the applied differentiation paradigm resulted in neurons with caudal identity. It has to be kept in mind that the regional markers are transiently expressed during embryo development and that further analysis is need to confirm protein expression and determine the percentage of neurons with hindbrain identity. Investigation of the neuronal subtype indicated cholinergic identity based on mRNA expression when comparing to adult whole brain. Furthermore low expression of GABAergic and dopaminergic markers were also observed. Overall, these results suggest the presence of cholinergic neurons of hindbrain origin. Neuronal cultures from all iPSC lines expressed a range of pan-neuronal markers (TUBB3 (βIIItub), MAP2 (MAP2), MAPT (Tau), RBFOX3 (NeuN)) and SYN1 (Synapsin 1) with only minor differences in mRNA expression levels between the neuronal cultures. The neurons seemed to have reached a certain state of maturity as the postmitotic marker NeuN and the presynaptic marker Synapsin 1 were expressed in subsets of neurons. The gene expression levels of RBFOX3 (NeuN) and SYN1 (Synapsin 1) in iPSC-derived neurons were at the level of fetal whole brain but lower than in adult whole brain indicating that some or all of the cells were not fully mature. In line with these observations, only the embryonic isoform of Tau 0N3R (Goedert and Jakes, 1990) was expressed. So far, only one study has detected all adult Tau isoforms in iPSC-derived neurons. Noticeably, they did not arise until day 365 of cell culture (Sposito et al., 2015).