TSC2 patient iPSCs-derived neurons exhibit higher neuronal excitability but decreased synchronicity
In previous work, we reported enhanced neuronal excitability of iPSCs derived from ASD with TSC1 or TSC2 mutations . In these experiments, mature neurons exhibited increased spontaneous calcium influx frequencies and an increased firing rate of TSC patient-derived neurons plated on MEAs as compared to control neurons . In the current study, we have investigated the synchronisation and connectivity of neuronal network activity of two independent control lines and one patient that was lacking functional TSC2, possessing a single nucleotide duplication (1563dupA) leading to a frameshift mutation H522T . Several iPSC clones were obtained from each patient and control fibroblast line and verified by sequencing and characterised by immunostaining as previously shown .
Control and patient iPSCs were differentiated into functional neurons, and at ~day 60 of differentiation neurons were plated on the MEA plate to record their activities. Spike detection from filtered raw voltage recording were generated from each electrode via the threshold-based method of QuianQuiroga and colleagues  and following quality control spikes were time-stamped for subsequent analysis. As previously reported, spontaneous activity of cultures on MEAs differed between control and TSC2 mutated patient neurons (Fig. 1A, B) with increased spontaneous firing rates and higher total numbers of single unit bursts in TSC2 neurons, with 0.9 ± 0.2 Hz, 273.1 ± 68.81 bursts, respectively compared to the control values of 0.16 Hz ± 0.07 Hz, 57.67 ± 46.76 bursts, respectively (p < 0.01, p < 0.05, unpaired t-test, Fig. 1C).
After 20 days on MEAs, synchronised bursting emerges, where multiple electrodes across the array simultaneously detect burst firing. Control neuronal cultures develop a regular pattern of synchronised bursts (SBs) separated by intervals of similar length, consistent with that previously reported  (Fig. 1A, B, S1). In TSC2 mutated patient neurons, there was a significant reduction in SB frequency (Fig. 1A, B). For example, at 40DPP (days post plating) the number of SBs was significantly lower in TSC2 neurons, 4.5 ± 1.55 SBs as compared to control neurons, 22.67 ± 4.4 SBs (p < 0.01, unpaired t-test, Fig. 1D). Consistent with elevated general spontaneous activity in TSC2 neurons, the firing rate within the TSC2 SBs was higher; for example, at 40DPP the SB firing rate in TSC2 neurons was 282.3 ± 77.04 Hz as compared to 13.67 ± 2.33 Hz in control neurons (p < 0.05, unpaired t-test, Fig. 1D). This suggests that although a higher intrinsic neuronal activity is present in TSC2 neurons, it is not reflected in increased synchronised activity within the neuronal network, and in fact there exists a previously undetected deficit in network behaviour of the patient neurons.
To probe further we investigated how the pattern of neuronal firing differed between control and TSC2 neurons. As would be predicted in the case of increased neuronal activity but decreased synchronisation, the percentage of firing spikes occurring outside SBs was significantly higher in TSC2 neurons (78.44 ± 8.5% in TSC2 neurons as compared to 38.5 ± 9.8% in control neurons (p < 0.05, unpaired t-test, Fig. 1E). However, when SBs did occur in the TSC2 neurons, they persisted for a longer period compared to those in control neurons; approx. 2.9-fold longer at 40 DPP than those in the control (1.25 ± 0.13 s, 3.65 ± 0.54 s (p < 0.01, unpaired t-test), (Fig. 1F). This is accompanied by a substantial increase, approx. 5.8-fold, in the time interval between SB for TSC2 neurons compared to controls (151.5 ± 50.21 v 26.67 ± 4.37 s at 40DPP, p = 0.09, unpaired t-test), (Fig. 1F). Due to higher variation in SB interval observed forTSC2 neurons, the increased SB interval did not reach a p<0.05 threshold, conventionally considered as statistically significant. To assess the increased variation with the TSC2 dataset in comparison to the control, we plotted the range of SB interval lengths for both control and TSC2 neurons (Fig. 1G). SBs in control cells exhibited a regular defined pattern with intervals that are tightly clustered around the mean interval length, in contrast the distribution of the interval times in TSC2 neurons were widely dispersed with a range approx. 5-fold greater than control (Fig. 1G). Combined these data indicate that although the SBs of TSC2 neurons have more persistent and higher firing rates, their spontaneous frequency is significantly reduced and a disorganised. This pattern is well illustrated in Fig. 1B.
This loss of synchronicity seen in the TSC2 neuronal networks is suggestive of a reduced connectivity between groups of neurons. To pursue this observation, we interrogated how the neuronal spatial connectivity may differ between the control and TSC2 neurons plated on MEAs by plotting correlation matrices between all electrodes in the MEA. In control neurons, we observed a high firing correlation between the majority of the electrodes in the cultures, represented as red and dark red pixels in Fig. 1H, indicative of high level of neuronal connectivity. The firing correlation is substantially reduced for TSC2 plated neurons, showing a loss of neuronal connectivity.
Pharmacological profiling of TSC2 patient-derived neuronal networks
To establish whether the SB firing patterns observed in TSC2 neurons arise due to changes in synaptic activity as previously reported in our control neurons , we probed our cultures with agents that modulate glutamate or GABA signalling. The agents were applied after 50DPP when SBs patterns had fully established. In agreement with what we found previously in control neurons inhibition of glutamate signalling via an AMPA receptor antagonist (CNQX) or an NMDA receptor antagonist (APV) lead to a complete abolition of the SB in TSC2 cultures (Fig. S2). Previously, it has been reported that the glutamate mimic, kainic acid (KA) increased the number of SBs , we found that any increase in control or TSC2 neurons did not reach statistical significance (Fig. 2A, 2B). Probing TSC2 or control cultures with bicuculline or DMCM (6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), drugs that antagonise GABA receptors  increased the number of SBs (Fig. 2A, 2B). As in all of the previous cases, the patterns of the SBs rapidly recovered after washing out the drug (data not shown). Taken together, these findings indicate that consistent with observations in control iPSCs-derived neurons, the SB patterns detected in TSC2 neurons arise via synaptic activity and that AMPA, NMDA and GABA signallings are all required for their neuronal network activity.
TSC2 patient iPSC-derived neurons show an excitatory/inhibitory synaptic marker imbalance
As the SB firing patterns in TSC2 neurons are dependent on synaptic activity, we interrogated whether the abnormal network phenotype observed in TSC2 neurons correlated with a potential excitatory/inhibitory imbalance. We used transcription analysis to probe possible modes of change in 1) excitatory-inhibitory cell ratio, 2) synaptic gene expression and 3) mediators of glutamate-GABA signalling. At the end of the MEA recording period ~60DPP, mRNA levels in TSC2 and control neuron cultures were quantified by qRT-PCR (Table1, Fig. 3, Fig S3).
Expression of cell and regional specific markers for GABAergic cells, DLX1, DLX2, LHX6 and VGAT was used to probe the proportion of inhibitory neuron cells. In TSC2 cultures elevated GABA signalling would be expected to suppress SB formation and frequency. There was no significant expression change of these cell markers between TSC and control neurons (Table1, Fig. S3), suggesting that major changes in cell-type proportions is unlikely. Previously, we observed no significant differences in neuronal morphology or synaptic number in TSC2 patient neurons , and consistent with these earlier studies there was no significant differences in expression of the postsynaptic density protein genes PSD95 and Homer1 or the presynaptic marker synaptophysin  (Table1, Fig. S3).
In contrast, significant differences were observed in expression of genes encoding enzymes and receptor proteins associated with glutamate-GABA signalling. GAD1 and GAD2, which encode the glutamic acid decarboxylases GAD67 and GAD65  showed a statistically signiﬁcant increase of approximately 5- and 20-fold changes, respectively in TSC2 neurons as compared to the controls (Table 1, Fig. 3A,B), (p < 0.05, p < 0.01, unpaired t-test). Likewise, expression of the postsynaptic GABAA receptor subunits α1, β2 and γ1 (GABAα2, GABAβ1 and GABAγ1) were also elevated by 6-, 4- and 4-fold respectively in TSC2 neurons (Table-1, Fig. 3D-F), (p < 0.05, p < 0.001, unpaired t-test). In addition, expression of the glutamate receptor genes GRIN1, GRIN3A, and GRIA1 [33,34] and the presynaptic vesicular glutamate transporter VGLUT2, also showed a clear increase (Table 1, Fig. S3), (p < 0.05, p < 0.01, unpaired t-test). GRIN2A and GRIN2B genes had no significant change, and VGLUT1 was very strongly decreased, although its mRNA abundance is relatively low (~0.05) even in control neurons. These results are indicative of disruption of both GABA and glutamate signalling. Elevation of glutamate signalling could explain the neuronal hyperexcitability of TSC2 model, while increased GABA signalling would be expected to suppress synchronised neuronal network behaviour .
Chronic inhibition of mTORC1 pathway has no effect on the neuronal network behaviour in TSC2 patient neurons
As loss of TSC2 increases mTORC1 activity, we interrogated whether longer-term inhibition of mTORC1 by rapamycin may rescue the neuronal network defects phenotype detected in TSC2 neurons. Control neurons and TSC2 neurons were treated with 10nM rapamycin from day 45 of differentiation, and neuronal activity of control and TSC2 mutant cells was examined at day 60 on MEAs (Fig. 4A,B). As seen previously, there was a significant decrease of neuronal hyperactivity in the basal neuronal activity (spike firing rate and total number of bursts) in TSC2-derived neuronal cultures, reaching a comparable level seen in the control firing rate, i.e. a decrease from 0.52 ± 0.09 Hz to 0.13 ± 0.02 in the presence of rapamycin, p < 0.05, unpaired t-test. However, there was no significant alteration in the overall neuronal activity or synchronicity in TSC2 patient-derived neurons, or even those of the control, with rapamycin (Fig. 4B). Given that chronic inhibition of the mTORC1 pathway did not increase the number of SBs, we tested whether short-term treatment with rapamycin may have an effect. TSC2 neurons were treated with rapamycin for 24h and then the neuronal network activity was recorded. Similar to that see in TSC2 neurons chronically treated with rapamycin, short-term rapamycin treatment decreased the basal excitability of TSC2 neurons, but again it had no effect on the SB number, SB length or percentage of spikes outside SB, although we did observe a small change in the mean interval between SB (Fig. S4). In conclusion, neither long- nor short-term rapamycin treatment had a major effect on the abnormal network pattern seen for TSC2 neurons.
Suppression of mTORC1 via ULK1 enhances the neuronal synchronicity in TSC2 patient-derived neuronal networks
In many cell types, rapamycin is reported as being a poor inhibitor of mTORC1 due to incomplete allosteric inhibition . However, TORC1 activity is also inhibited by the action of the two kinases AMPK and ULK1 (Fig. 5A). TSC2 cultures were probed with AICAR, an AMPK activator, for 24h and found to decreaseTSC2 neuronal excitability (Fig. 5B,D). Unlike rapamycin, AICAR significantly increased the number of SBs and SB length and decreased the SB firing rate in TSC2 neurons (Fig. 5Db, 5Dc). However, it did not alter the SB interval (Fig. 5Dc) or decrease the number of firing spikes outside the SBs (Fig. 5Dd). In control neurons, AICAR had no effect on the neurons basal activity and in fact reduced the number of SB following 24h of treatment (Fig. S5).
To seek a more effective reversal of the aberrant phenotype of TSC2 neurons we investigated LYN-1604, a small molecule which is a potent and selective ULK1 activator [37, 38] and should inhibit mTORC1 through direct phosphorylation of Raptor [23, 39]. A preliminary screen of LYN-1604 showed that in control neurons it reduced expression of GABAα2 and GRIA1 with 24 hours of treatment (Fig. 5C), an indication that it may target both GABA and glutamate signalling in the TSC2 patient-derived neurons. Consistent with this possibility, MEA cultures of TSC2 patient-derived neurons treated with LYN-1604 for 24h showed a significant increase in the number of SB, comparable to that observed in control neurons (Fig. 5Db). It also reduced the SB length and interval, and significantly decreased the number of firing spikes outside the SBs (Fig. 5Dc, 5De). Whereas the effect of LYN-1604 on the number of SBs in the control neurons is yet to be determined.
As AICAR and LYN-1604 both showed degrees of improvement of synchronicity in TSC2 neurons, we examined whether probing the culture with any of these drugs would alter the neuronal spatial connectivity. We analysed the correlation matrices between all electrodes of the MEAs for TSC2 neurons before and after treatment with AICAR and LYN-1604. While treatment with AICAR had no detectable effect on connectivity matrices of the TSC2 neurons plated on MEAs (Fig. S5c), treatment with LYN-1604 improved the correlation between several electrodes. This effect was presented by increasing the red and dark red pixels between several electrodes in the presence of LYN-1604, an indicative of increasing the neuronal connectivity (Fig. 5E). Taken together these results show that the defective TSC2 neuronal networks may be partially restored by induction of AMPK but are substantially restored by ULK1 activation by LYN-1604.