Hyperexcitability in young iPSC-derived C9ORF72 mutant motor neurons is associated with increased intracellular calcium release

A large hexanucleotide repeat expansion in the C9ORF72 gene is the most prevalent cause of amyotrophic lateral sclerosis (ALS). To better understand neuronal dysfunction during ALS progression, we studied motor neuron (MN) cultures derived from iPSC lines generated from C9ORF72 (C9) expansion carriers and unaffected controls. C9 and control MN cultures showed comparable mRNA levels for MN markers SMI-32, HB9 and ISL1 and similar MN yields (> 50% TUJ1/SMI-32 double-positive MNs). Using whole-cell patch clamp we showed that C9-MNs have normal membrane capacitance, resistance and resting potential. However, immature (day 40) C9-MNs exhibited a hyperexcitable phenotype concurrent with increased release of calcium (Ca2+) from internal stores, but with no changes to NaV and KV currents. Interestingly, this was a transient phenotype. By day 47, maturing C9-MNs demonstrated normal electrophysiological activity, displaying only subtle alterations on mitochondrial Ca2+ release. Together, these findings suggest the potential importance of a developmental component to C9ORF72-related ALS.

www.nature.com/scientificreports/ which also contained glial cells, Selvaraj et al. 18 used a 'pure' MN culture and did not observe any evoked excitability changes between C9 lines and control lines (CTRL). The authors of that study proposed a 'glial-mediated non-cell autonomous mechanism' accounting for the previously observed excitability changes 18 . Previous studies used various methods for measuring neuronal excitability and resulted in a lack of understanding of the proposed mechanisms underlying the changes in excitability. In this study, we used whole-cell patch clamp electrophysiology to evoke APs and compare the excitability of a physiologically relevant neuronal/glial mixed population of iPSC-derived C9-MNs at early (day 40) and late (day 47) stages of maturity. We demonstrate hyperexcitability is paralleled by increased intracellular Ca 2+ release without changes to Na V and K V currents in C9 cultures at day 40. The hyperexcitability phenotype is lost by day 47 and coincides with a normalization of intracellular Ca 2+ release, with parallel dysfunction in mitochondrial Ca 2+ emerging. Our work highlights a developmental component to ALS by showing an early disease-relevant disruption of neurophysiology which may impact on the pathophysiology of neurons later in life.

Results
Differentiation of C9ORF72 and control iPSCs to enriched spinal MN cultures in vitro. Three iPSC lines carrying the C9ORF72 expansion (C9-1, -2, and -3) and three CTRL (CTRL-1, -2 and -3) lines were previously generated by Sendai Virus reprogramming 19 ( Figure S1) with the respective presence or absence of the G 4 C 2 expansion confirmed using repeat primed PCR ( Figure S1). A modified version of the previously published MN differentiation protocol by Du et al. 20 was used to direct all lines towards a spinal MN lineage (Fig. 1a). Data presented in this study were obtained from multiple independent differentiations of C9 and CTRL iPSC lines.
Using immunocytochemistry to assess differentiation efficiency, we showed an enriched percentage of double positive SMI-32/TUJ1 MNs in all lines at day 40 and 47 ( Fig. 1b). Analysis by flow cytometry confirmed all lines produced > 80% TUJ1 positive neurons at day 40 and 47 ( Fig. 1ci). Over 50% of neurons also co-stained for the MN marker SMI-32, with a significant increase in co-staining noted for C9 lines vs CTRLs at day 40 (Fig. 1cii). Our protocol generates a mixed culture model enriched in MNs, with the majority of the remaining ~ 20% of non-neuronal cells represented by GFAP-positive astrocytes as measured by immunocytochemistry ( Figure S1). mRNA expression of SMI-32, as well as transient MN markers HB9 and ISL1, at day 40 and 47 showed no difference between the genotypes or time points (Fig. 1d).
C9ORF72 iPSC-derived MNs show early hyperexcitability. Whole-cell patch-clamp recordings at day 40 and 47 revealed no significant difference in passive properties between C9 or CTRL MNs (Fig. 2ac). However, at day 40 evoked AP firing frequency was significantly higher in C9 neurons vs CTRL neurons (Fig. 2di,ii, Figure S2a). Neurons were then further aged in vitro to day 47, but at this later timepoint the relative hyperexcitability of C9 neurons vs CTRL neurons was not observed (Fig. 2ei,ii, Figure S2b). This loss of hyperexcitability phenotype appeared to be the result of an increase in AP firing frequency in the CTRL neurons from day 40 to day 47 to match the rate of the C9 lines, rather than a decrease in the C9 neuron excitability. Thus, the hyperexcitability phenotype observed at day 40 is transient, with the high AP frequency in C9 lines reflecting a possible faster initial maturation rate in the C9 lines than the CTRL lines.
Increased excitability is not caused by altered voltage-gated currents. We recorded fast inactivating Na + and delayed rectifier K + voltage gated-channel currents, which are involved in the upstroke and recovery of APs, respectively 21,22 . Na V current and K V current recordings were normalised to capacitance giving current densities and allowing us to assess whether functional expression of ion channels contributed to the hyperexcitability phenotype. At neither time point was there a significant difference in either Na + or K + current density between genotypes ( Fig. 3a-d). However, a significant increase in peak current density was detected over time for both Na + or K + currents, suggesting a maturing population of neurons (Fig. 3ciii,diii). Overall, these results suggest that fast inactivating Na + or delayed rectifier K + channels do not play a significant role in the increased firing rate observed at day 40 in C9-MNs.
Early hyperexcitability correlates with aberrant increased intracellular calcium release. Ca 2+ release from internal neuronal stores supports multiple roles in the correct functioning of neurons and shapes neuronal output (e.g. AP frequency) through modulation of after-hyperpolarisations or after-depolarizations 23 .
We therefore investigated Ca 2+ release from intracellular stores using the Fura-2AM ratiometric dye. When MNs were activated with ionomycin, we observed a typical biphasic Ca 2+ response at both day 40 and 47 ( Fig. 3ei,ii), due to initial release of Ca 2+ from intracellular stores likely followed by store operated Ca 2+ entry (SOCE) 24 . A significant change in delta Ca 2+ release compared to baseline was detected in young neurons at day 40 ( Fig. 3ei, Figure S3a). Specifically, the delta peak magnitude of Ca 2+ release illustrated a significant increase in intracellular stored Ca 2+ release from C9-MNs vs CTRL MNs at day 40 (Fig. 3eiii). Importantly, as with the electrophysiological phenotype observed at day 40, the difference in internal Ca 2+ release was transient and no longer detected at day 47 ( Fig. 3eii,iv, Figure S3b). Finally, we found a significant increase in Ca 2+ release from the mitochondria in C9-MNs compared to CTRL MNs upon stimulation with FCCP. This difference in release properties was only observed at the later day 47 timepoint ( Figure S4a-d) and may represent a later stage in the disease process involving the mitochondria.

Discussion
This study focused on iPSC-derived MNs harbouring a C9ORF72 expansion to assess electrophysiological excitability changes during neuronal maturation as a potential mechanism underlying the earliest stages of disease. We sought to provide clarity to a field in which several studies have used different parameters to define neuronal excitability and shown a mixture of hypoexcitability, hyperexcitability transitioning phenotypes, or observed no excitability changes at all [15][16][17][18] . We demonstrated increased evoked AP firing in C9-MNs at an early stage (day 40) of iPSC-derived MN maturation. This effect was, however, transient, with the induced AP firing frequency in the CTRL MNs increasing from day 40 to 47 resulting in equivalent AP frequency in both CTRL and C9-MNs at day 47. These changes in excitability may result from different rates of neurophysiological maturation, with C9-MNs reaching greater maturity at day 40 compared to CTRL MNs, a genotype-specific difference lost by day 47. These data also provide a rationale for differing results in the field regarding C9 neuronal excitability. The data here confirm that hyperexcitability exists as an early stage phenotype, as also suggested by Devlin et al. 17 . Since Wainger et al. 15 and Devlin et al. 17 observed hyperexcitability at a similar timepoint to us (at approximately day 38 of differentiation, or 2 weeks post re-plating, respectively) our hyperexcitability data at day 40 confirm the idea of an early disease-associated hyperexcitability phenotype. Devlin et al. 17 noted hypoexcitability at 7-10 weeks post   16 and Devlin et al. 17 in their studies, thereby demonstrating the hyperexcitability phenotype in young neurons is robust and not simply the result of studying a particular set of iPSC lines. Both Na V and K V channels have been implicated in excitability changes in ALS patients and C9ORF72 iPSC models 11,[15][16][17]25,26 . However, in contrast, we found no change in fast inactivating Na V or delayed rectifier K V current. This does not rule out a contribution for Na + or K + current dysfunction as A-type K + channels and the Na + / K + pump could play a role. Our observations from day 40 to 47 show an increase in both Na V and K V current densities, indicating ongoing maturation of neuronal cultures in our study.
Ca 2+ dyshomeostasis has strong links to ALS pathogenesis 18,19,27,28 . MNs are particularly vulnerable to Ca 2+ dyshomeostasis due to high Ca 2+ influxes during neurotransmission as well as their low overall Ca 2+ buffering capability. Recent studies have shown AMPA receptors have increased Ca 2+ permeability in C9-MNs which may contribute to excitotoxicity via increased [Ca 2+ ] i and subsequent activated cellular stress pathways 18,29 . Using Fura-2AM, Dafinca et al. 28 showed iPSC-derived C9 neurons to have increased internal Ca 2+ release in response to ionomycin. In addition, the same study found that upon chemical neuronal depolarisation (KCl 50 mM), live cell imaging revealed increased Ca 2+ release and delayed Ca 2+ clearance from the cytosol in C9 vs CTRL MNs. We show that the dyshomeostasis in internal Ca 2+ release in response to ionomycin coincided with the presence of evoked hyperexcitability. One potential explanation may be that the effect of increased intracellular calcium during the period of neuronal maturation may impact expression of ion channel genes over time contributing to hyperexcitability.
Finally, in addition to perturbed Ca 2+ release from intracellular organelles, we observed a change in stimulated mitochondrial Ca 2+ release at day 47 in C9-MNs. Therefore, the loss of hyperexcitability at day 47 coincides with the loss of increased general intracellular organelle Ca 2+ release and with the appearance of mitochondrial Ca 2+ dysfunction. Mitochondria are important for ATP production as well as for Ca 2+ homeostasis, free radical formation, metabolite generation and apoptosis 30 . Mitochondrial dysfunction has been previously implicated in ALS, www.nature.com/scientificreports/ particularly in SOD1 models 31 . However, a variety of aberrations, including reduced ATP production as well as disrupted architecture, are also observed in different ALS mutations as reviewed by Smith et al. 32 . Ca 2+ overload is one mechanism proposed in ALS mitochondrial dysfunction and neuronal cell death pathway activation. Mitochondria rely on cross-talk with the ER for maintaining balanced Ca 2+ concentration and Ca 2+ signalling [31][32][33] . A shift in Ca 2+ storage from the ER to mitochondria could lead to reduced Ca 2+ in the ER, but increased Ca 2+ in the mitochondria. Too little Ca 2+ in the ER could result in protein misfolding, whereas too much Ca 2+ in the mitochondria leads to opening of the 'mitochondrial permeability transition pore' and subsequent necrosis or apoptosis 34 . The shift from increased general intracellular organelle calcium release at day 40 to increased Ca 2+ Figure 3. Transient calcium dyshomeostasis is evident in C9ORF72 MNs vs CTRL MNs. Example traces for Na V at − 20 mV (a) and K V at + 50 mV. (b) Current densities show no significant difference between the CTRL or C9 groups (Na V IV graph, ci; K V IV graph, di). The same measures also show no difference at day 47, (Na V IV graph, cii; K V IV graph, dii). However, a temporal comparison shows a significant increase of average peak current density of Na V current and K V current over time. Two-way RM ANOVA: Interaction Na + /K + p = ns, Time *Na + p = 0.0282, *K + p = 0.0145, Genotype Na + /K + p = ns with Sidak's multiple comparisons Na + /K + p = ns, N = 3 lines, 1-2 differentiations. Investigation of calcium store release (ei, eii; mean ± SEM) evoked by ionomycin injection showed a significant increase in the delta Fura-2 AM fluorescence of C9 versus CTRL at Day 40 (eiii; Two-way RM ANOVA: Interaction **** p = < 0.0001, Time p = < 0.0001, Genotype p = ns) but not Day 47 (eiv; Interaction p = ns, Time p = < 0.0001, Genotype p = ns.) Unpaired T-Test p * < 0.05, N = 3 lines, 2-3 differentiations. www.nature.com/scientificreports/ release specifically from the mitochondria at day 47 in C9-MNs compared to CTRL MNs could suggest the possibility of mitochondria Ca 2+ overload later in the process of cellular degeneration. The presence of astrocytes in iPSC-MN cultures and their impact on the biology of MNs carrying ALS mutations is an area of debate as highlighted by Zhao and colleagues 30 . In that paper the authors' showed astrocytes are affected by the presence of the C9ORF72 mutation which then contributes towards MN dysfunction. It should, therefore, be noted that the cultures used in our study include a mixed population of neurons and astrocytes as a result of the differentiation protocol used.
Overall, our study seeks to clarify the conflicting reports surrounding altered excitability in C9ORF72 neurons. Here, we report transient disease-related hyperexcitability in a mixed 'MN-Astrocyte culture' which is directly affected by neuronal maturity. Our work associates the hyperexcitability phenotype to a temporallylinked dyshomeostasis in Ca 2+ release, followed by a normalization of excitability and emergence of mitochondrial Ca 2+ dysfunction at later stages. Together, these data provide a Ca 2+ based neurophysiological phenotype which could be manipulated for therapeutic benefit in pre-symptomatic stages of disease. RT-qPCR. 1.25 × 10 6 cells were used per RNA sample. Trizol reagent and chloroform were used along with the RNase Easy Mini Columns (Qiagen) with DNase I treatment. cDNA was produced using SuperScript VILO MasterMix, using 1 μg of RNA. Fast SYBR green was used for the qPCR reaction. Primer sequences in Supplementary Table 1 and data analysed using dCT method.

Methods
Whole-cell patch clamp electrophysiology. 10 5 cells were plated per coverslip and selected for electrophysiology based on morphology. Neurons were selected for patch clamping both within and outside of neuronal clusters. Due to the enrichment of the culture for MNs the cells predominantly recorded were MNs. Although without live, cell-type specific labelling it cannot be excluded that some recordings may be from interneurons, the spread of passive properties data does not suggest a separation in cell types. Cells with R a greater than 40 MΩ were rejected and recordings with a change in R a or cell capacitance greater than 10% over the duration of the recording were omitted. Any cell lines post stained with less than 30% MNs were excluded.
Induced APs were recorded in current clamp by injecting 500 ms current steps in 5 pA increments from − 10 pA. Membrane potential was maintained around − 70 mV by injecting a hyperpolarizing bias current. Na V and K V currents were recorded in voltage clamp (sampling rate of 10,000 Hz) during a series of 10 mV voltage steps, 100 ms in duration, from a holding potential of − 70 mV. Leak subtraction was applied with 4 sub-sweeps and a settling time of 250 ms.
Organelle calcium release. Neurons were plated at a density of 10 5 cells per well on 96-well plates. Stimulation by ionomycin and recordings using Fura-2 AM were performed as described by Wallings et al. 35 .
Analysis. For the above techniques, data from multiple lines and differentiations have been pooled to allow more robust analysis with a higher number of cells and greater statistical power. Patch clamp electrophysiology is a low-throughput technique, and a much higher number of differentiations and patched neurons would be required to present separate data for individual lines. Key figures can be found in the supplementary data, displayed with each line separately for data transparency. Authors can confirm that all relevant data are included in the paper and/ or its supplementary information files.