TSC2 patient iPSCs-derived neurons exhibit higher neuronal excitability but decreased synchronicity
In previous work, we reported enhanced excitability of neurons derived iPSC generated from ASD patients 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 differentiated patient iPSC cells lacking functional TSC2, due to a single nucleotide duplication (1563dupA) leading to a frameshift mutation H522T . Multiple 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 plated onto MEA plates at ~day 60 and developed further to form activity neuronal networks. 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). 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, 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), Significantly, this is accompanied by a large increase in the interval between SB for TSC2 neurons compared to controls; at 40DPP there was an approx. 5.8-fold increase of SB interval in TSC2 cultures (151.5 ± 50.21 v 26.67 ± 4.37 s, p < 0.01, unpaired t-test), (Fig. 1f). When SBs do occur in the TSC2 neurons, they persisted for 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). Finally, we investigated the variation inherent to the dataset representing the SB intervals in TSC2 neurons as compared to the control. Whilst, the SBs in control cells exhibited a regular defined pattern with intervals that are tightly clustered around the mean, the distribution of the intervals in TSC2 neurons are more dispersed (Fig. 1g). Combined these data indicate that although the SBs of TSC2 neurons have more persistent and have higher firing rates, their spontaneous frequency is significantly reduced and disorganised. This pattern is well illustrated in Fig. 1b.
The reduced synchronicity seen in the TSC2 neuronal networks is suggestive of a lower 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 spatial 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 pharmacological agents that modulate glutamate or GABA signalling, applied after 50DPP when SBs patterns had fully established. In agreement with that 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. This was also seen in TSC2 patient derived cultures (Fig. 2C and Fig. S2). Probing the TSC2 cultures with kainic acid (KA), a drug that mimics the effect of glutamate, resulted in a 3-fold increase in the number of SBs also consistent with what has been previously described in control neurons  (Fig. 3C, Fig. S2). Probing the culture with drugs that antagonise GABA receptors, bicuculline or DMCM (6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), a negative allosteric modulator of GABAA receptors  increased the number of SBs (Fig. 2C and Fig. S2). We also examined the effect of GABA and a GABA positive allosteric modulator on this culture. Previously we showed that application of GABA completely suppressed the SBs in control neurons, here we showed that application of GABA or diazepam, also supressed the SBs in TSC2 neurons (Fig. 2C and fig. S2). Whereas, exposure to diltiazem, resulted in increasing the number of the SBs, as also seen consistent previously seen for control neurons  (Fig. 2C and fig. S2). In all of the previous cases, the patterns of the SBs rapidly recovered after washing the drug out. Taken together, these findings indicate that the SB patterns detected in TSC2 neurons arise via synaptic activity and that Glutamate (AMPA, NMDA) and GABA signalling are all required for their neuronal network activity.
Synaptic gene of TSC2 patient iPSC-derived neurons indicates an excitatory/inhibitory imbalance
Having demonstrated that the SB firing patterns in TSC2 neurons arise from intact synaptic activity, we interrogated whether their abnormal network phenotype could be due an imbalance of excitatory/inhibitory synaptic signalling of the cells. We tested this hypothesis by looking at the expression of a panel of inhibitory and excitatory synaptic markers in TSC2 and control neurons via qPCR. At the end of the MEA recording period ~60DPP, RNA samples were collected from TSC2 and control neurons and the expression of various synaptic markers was quantified (Fig. 3).
We quantiﬁed mRNA levels for the inhibitory synaptic genes GAD1 and GAD2, which encode distinct GAD (glutamic acid decarboxylase) proteins GAD67 and GAD65, respectively . Our analysis showed a statistically signiﬁcant increase of approximately 5- and 20-fold changes, respectively in GAD1 and GAD2 mRNA levels in TSC2 neurons as compared to the controls (Fig. 3a, 3b), (p < 0.05, unpaired t-test). Likewise, expression of the postsynaptic GABAA receptor subunits α1 and α2 (GABA-α1 and -α2) were also elevated by 2- and 6-fold respectively in TSC2 neurons (Fig. 3c, 3d), (p < 0.001, unpaired t-test). The expression levels of GRIN2A and GRIN2B which encode the NMDA receptor subunits NR2B and NR2A, respectively ; the glutamatergic marker VGLUT1 which constitutes the main presynaptic vesicular glutamate transporter; the postsynaptic density proteins PSD95 and Homer1, and the presynaptic marker synaptophysin  were determined. In contrast to the elevation in the mRNA levels of the inhibitory synaptic genes, the levels for the glutamatergic marker GRIN2A, GRIN2B and VGLUT1 showed a clear reduction by approximately 2-, 2- and 14-fold, respectively (Fig. 3e-3g), (p < 0.01, unpaired t-test). Genes encoding presynaptic and postsynaptic density proteins were not significantly altered (Fig. 3h-3j), an observation that is consistent with previous reports that showed no significant changes in neuronal morphology or synaptic number in these TSC2 patient neurons . These findings suggest that at the signalling level there may be an imbalance of excitatory/inhibitory signalling in TSC2 neurons and is consistent with the effects of decreased glutamate and increased GABA signalling on neuronal network behaviour seen previously.
Chronic inhibition of mTORC1 pathway has no effect on the neuronal network behaviour in TSC2 patient neurons
Loss of TSC2 increases mTORC1 activity, we therefore 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 40 post plating on MEAs. We did not detect significant alterations in the overall neuronal activity or synchronicity in control neurons treated with rapamycin (fig. S1A), whereas, significant reductions in the basal neuronal activity were seen in TSC2-derived neuronal cultures (Fig. 4a). Although rapamycin decreased neuronal hyperactivity of TSC2 neurons to 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, it had no significant effect on SB frequency, SB length or interval (Fig. 4b, 4c, 4d). This indicates that incubation with rapamycin did not rescue the neuronal synchronicity deficit in TSC2 neurons. 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. Treatment with rapamycin had also no effect on the number of SBs while it decreased the basal excitability of TSC2 neurons (Fig. S1B). We concluded that both long- and short-term rapamycin treatment was not able to rescue of the abnormal network phenotype of TSC2 neurons.
Suppression of mTORC1 via ULK1 enhances the neuronal synchronicity in TSC2 patient-derived neuronal networks
A possible insensitivity to rapamycin treatment could be due insufficient drug activity to effectively restore energy and nutrient homeostasis. In many cell types, rapamycin is reported as being a poor inducer of autophagy, likely due to an incomplete allosteric inhibition of mTORC1 . To explore this notion further, we employed activators of AMPK and ULK1 to better target homeostatic regulation of energy sensing and autophagy, which are both downstream processes that are dysregulated in TSC-diseased cells. TSC2 cultures were probed with AICAR (an AMPK activator) for 24h and the SB patterns were then determined. Unlike rapamycin, AICAR significantly increased the number of SBs and decreased the SB firing rate in TSC2 neurons (Fig. 5Bb). However, it did not alter the SB length or interval (Fig. 5Bd) and did not decrease the number of firing spikes outside the SBs (Fig. 5Bc).
As an alternative route to rescue the aberrant TSC2 phenotype we used an activator of ULK1, LYN-1604 to inhibit mTORC1 through direct phosphorylation of Raptor [34, 35]. Following LYN-1604 treatment for 24 hours and we observed a significant increase in the number of SBs (Fig. 5Bb). The SB number in the presence of LYN-1604 was closely comparable to that observed in control neurons. LYN-1604 also reduced both the SB length and interval, and significantly decreased the number of firing spikes outside the SBs (Fig. 5Bc, 5Bd). Taken together these results show that the defective TSC2 neuronal networks can be reversed by induction of AMPK and ULK1, two kinases that are fundamentally involved in the regulation of autophagy to restore energy and nutrient homeostasis.
As AICAR and LYN-1604 both improved the 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. 5C), 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. 5C). These findings indicate that the aberrant neuronal connectivity seen in TSC2 neurons can be restored by activation of ULK1 by LYN-1604 administration.