Increased Ca²⁺ signaling in NRXN1α^+/− neurons derived from ASD induced pluripotent stem cells

Autism spectrum disorder (ASD) is a chronic neurodevelopmental disorder characterized by repetitive behavior and deficits in social interaction and communication skills. Epilepsy, intellectual disabilities, language delay, anxiety, and hyperactivity are highly comorbid with ASD [1]. An increased ratio of synaptic excitation/inhibition (E/I) affecting neuroplasticity has been proposed as a common pathway for ASD [2]. This has been linked to altered functional and structural connectivity. Additional evidence from post-mortem neuropathology also showed reduced parvalbumin and altered density/abundance of glutamatergic receptors including GRM5 and GRIA1 in ASD [3‐5]. On the other hand, overproduction of GABAergic neurons with FOXG1 overexpression and accelerated cell cycle were also reported in induced pluripotent stem cells (iPSCs) of sporadic ASD with macrocephaly [6].

For synaptic excitation, rare mutations in NRXN, NLGN, and SHANK are reported in individuals with ASD and intellectual disability, further supporting the E/I imbalance hypothesis [7]. NRXN1 and SHANK2 are in fact the commonest rare genetic factors identified by a meta-analysis of multiple genetic studies [8, 9]. Notably, NRXN1 deletions are shared by ASD [10‐15], schizophrenia [16‐20], intellectual disability [21], ADHD [22], and epilepsy [14, 23‐26]. Whereas most human deletions involve in 5′ of NRXN1α^+/− with diverse clinical phenotypes, mouse Nrxn1α^−/− mutants display only mild behavioral deficit in nest building but are otherwise viable, fertile, and indistinguishable from wild-type littermates [27]. This suggests that mouse and human may have different sensitivity to NRXN1α gene lesions.

Three NRXN1 family members (NRXN1-3) exist in the genome, and Nrxn1α^−/−/Nrxn2α^−/−/Nrxn3α^−/− triple knockout mice are impaired in Ca²⁺-triggered neurotransmitter release with altered expression of synaptic Ca²⁺ channels and die of lung dysfunction [28]. Ca²⁺ concentration in neurons is tightly controlled by distinct influx/efflux mechanisms. Ca²⁺ influx occurs commonly through voltage-gated calcium channels (VGCCs) on membrane [29, 30], which facilitate a Ca²⁺ rise during neuronal firing. The influx of Ca²⁺ triggers vesicle exocytosis and neurotransmitter release. The long form of Nrnx1α has been shown to couple release-ready vesicles with metabotropic receptors, facilitating Ca²⁺-triggered exocytosis of neurons [31].

In addition to the long NRXN1α isoforms, which interact with post-synaptic Neuroligins and influence both excitation and inhibition through coupling to GABAergic or NMDA/AMPA receptors [31] and VGCCs [32], NRXN1 also encodes short NRXN1β isoforms by an alternative promoter, which is largely associated with creation of the scaffolding for excitation [33‐35]. NRXN1α is therefore proposed to influence E/I balance in both directions, whereas NRXN1β primarily mediates excitation. Indeed, conditional knockdown of NRXN1β severely impaired the neurotransmitter release at excitatory synapses [36]. It is likely that NRXN1α deletion may display increased neuronal excitability, as a result of reduced ratio of NRXN1α to NRXN1β isoforms, and/or a compensatory increase of NRXN1β expression if it happens. Pak et al. showed a reduced mEPSC frequency in human ESC-derived neurons after disrupting shared exon 19 or 24 of NRXN1 gene, which knocked out an entire NRXN1 allele with all NRXN1α/β isoforms [37]. However, this is different from the genetics in the majority of patients who carry heterozygous deletion at 5′ of NRXN1 gene which affect NRXN1α only, and to date, there have been no patient models to investigate the effects of isoform deletion and/or genetic background. Moreover, it has been shown that common pathophysiological social and cognitive deficits in autism can be linked to gain of function of synaptic proteins and ion channels [7]. These include hyperactivity in frontal brain regions, high-frequency oscillation in cortical regions, and the presence of clinically apparent seizures in 30% of autistic individuals [38‐42]. In addition, mutation in neuronal adhesion molecule CNTN5 has also shown hyper-excitability and increased excitation in iPSC-derived neurons of ASD individuals [43]. These studies show the presence of hyper-excitability and hyperactivity in some of the ASD patients.

The iPSC technology now offers significant benefits for disease modeling [44‐46], which can be derived from patient somatic tissues. They resemble embryonic stem (ES) cells and can be differentiated into disease cell types, so to provide human models for investigating disease progression and testing therapeutic drugs, in particular for organs such as the brain and heart, which are impossible to culture by conventional methods. We therefore derived iPSCs from controls and ASD patients carrying NRXN1α^+/− and differentiated them into cortical excitatory neurons, as altered cortical regions, thickness, folding, surface, columnar lamination, and the number excitatory neurons have been reported in ASD [1, 47‐51]. We investigated Ca²⁺ signaling and the transcriptome in day 100 neurons and provided novel phenotype with increased Ca²⁺ transients and upregulated VGCCs in ASD NRXN1α^+/− neurons.

NRXN1^+/− deletions are the most frequent single-gene disruptions associated with ASD [10, 12, 14, 15, 59, 60], schizophrenia [16‐20], intellectual disability [21], ADHD [22], and epilepsy [14, 23‐26]. Little is known about the consequences of NRXN1^+/− lesions in patients’ neurons or why the same heterozygous NRXN1^+/− deletions lead to diverse clinical phenotypes. We are the first to report derivation of human iPSCs from ASD patients carrying NRXN1α^+/−. The cortical excitatory neurons from NRXN1α^+/− iPSCs displayed a novel phenotype of increased frequency, duration, and amplitude of Ca²⁺ transients. This is supported by transcriptome analyses, which revealed an upregulation of VGCCs (CACNA1A, CACNA2D1, CACNG2, and CACNG3) and Ca²⁺ pathways in NRXN1α^+/− neurons.

Typically in neurons, calcium influx is facilitated by the opening of the α1 subunit in the tetrameric VGCCs in response to membrane depolarizations. The α1 subunit is encoded by CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, and CACNA1S genes. Consistent with ASD NRXN1α^+/− phenotype, gain-of-function of VGCCs are implicated in neurodevelopmental disorders (Additional file 1: Table S6). For example, Cav1.2 G406R (CACNA1C) causes Timothy syndrome with ASD by delayed inactivation and prolonged opening [61, 62]. Knock-in of the G406R to mice results in autistic phenotype [61, 62]. Exome sequencing has identified various CACNA1D mutations (encoding Cav1.3) in ASD [63‐66], epilepsy [67], and developmental delay [67]. A CACNA1D paralog, CACNA1F (Cav1.4), also is linked to New Zealand autistic males with excessive Ca²⁺ influx [61, 62].

We have identified CACNA1A encoding P/Q-type and CACNA2D1 encoding L-type VGCC as the most interactive NRXN1α^+/− targets. CACNA1A is predominantly expressed in neurons and involved in NRXN1α signaling which triggers the release of fusion-ready vesicles [68]. CACNA1A polymorphisms are associated with Chinese ASD [68], and CACNA1A mutations with epileptic encephalopathy [68]. Additionally, mutations in other VGCCs are also identified as a major pathway in schizophrenia [68, 69], the common risks across seven brain diseases [70, 71], and in ASD (Additional file 1: Table S6) [72, 73]. In addition, loss-of-function mutations in some VGCCs are also reported, i.e., CACNA1H R212C, R902W, W962C, and A1874V reduce their activity in ASD [74]; CACNA2D1 is deleted in epilepsy and intellectual disability [74]; CACNG2 V143L decreases its binding to GLUR1 or GLUR2 [75]; and Cacng2 hypomorph results in epileptic phenotype [74]. This evidence supports altered VGCCs as a mechanism in ASD NRXN1α^+/− neurons.

The human NRXN1α^+/− phenotype reported here differs from some of the data reported previously. Pak et al. created a mutant human H1 ES cell line with disruption of exon 19 or 24, which are shared by all NRXN1 isoforms (Additional file 1: Figure S1A), and showed reduced frequency of mEPSCs [37]. NRXN1 consists of 2 promoters and 11 differentially spliced exons which may result in 2048 NRXN1α and 4 NRXN1β isoforms. The human H1 ES cells (NRXN1^+/−) from Pak et al. are genetically different from the ASD patients here, who carry 1 copy of NRXN1α^+/−. However, qRT-PCR using primer pairs from exons 9–10 or 15–16 demonstrate 24 or 26% (not 50%) reduction. This is likely due to the complex exon usage of differential NRXN1 splicing. Meanwhile, we observe 262% compensational increase in NRXN1β expression; therefore, the phenotype in this study is likely to result from combinational effects of reduced NRXN1α and overexpression of NRXN1β. This may also re-enforce the concept that NRXN1α and NRXN1β isoforms play differential roles in neuronal E/I.

Sudhoff et al. propose that Neurexin variants from alternative splicing may perform the same canonical functions but may have different patterns of redundancy [76‐78]. Nrxn1α homozygous knockout presented no apparent phenotype, and Pak et al. also showed that mouse Nrxn1 knockout cells differed from H1 ES cells and displayed no phenotype [79]. Mice with triple knockout of Nrxn1α, Nrxn2α and Nrxn3α genes were shown to produce different phenotypes in different neurons or synapses [76, 77]. In hippocampal presynaptic cells, the Ca²⁺ influx was reduced in conjunction with lower Cav2.1-mediated transients and elevated axonal mobility of α2δ1 [80]. Although overexpression of Nrxn1α and α2δ1 is shown to rescue Ca²⁺ currents in Nrxn1α^−/−Nrxn2α^−/−Nrxn3α^−/− triple knockout mouse neurons, this is yet to be investigated in human cells [80]. In addition, species differences also exist: i.e., Nrxn1 at Caenorhabditis elegans acetylcholine neuromuscular synapse is located postsynaptically, not presynaptically [32], and approximately > 20% of human essential genes are nonessential in mice [37].

The penetrance of human NRXN1a^+/− is not 100%, and clinical conditions of NRXN1a^+/− are diverse. Therefore, co-factors in the genetic background may play a part in clinical phenotype. Investigations of patient-derived samples are essential for understanding roles of NRXN1a^+/− in different human conditions. The ASD NRXN1a^+/− phenotype here is consistent with the proposal that NRXN1β triggers excitation, and NRXN1α regulates both excitation and inhibition [33‐35]. NRXN1α deletions are therefore anticipated to weaken neuronal inhibition and increase excitation. A recent publication has shown that ASD neurons derived from autism CNTN5^+/− or EHMT2^+/− human iPSCs develop hyperactive neuronal networks [43]. This suggests indirect effects of NRXN1α on Ca²⁺ transients. The upregulated CACNA1A, CACNA2D2, and CACNG2 are linked to “the presynaptic depolarization and calcium channel opening” by STRING (Additional file 1: Table S5). Direct interactions of NRXNs with VGCCs are reported but limited. Mouse Nrxn1α is shown to positively modulate Ca²⁺ influx through Cav2.1-α2δ1 interaction [80]. On the other hand, human NRXN1α may also form NRXN1α-Cav2.2-αδ3 complex and negatively regulate Cav2.2 currents in transfected cells [32]. Furthermore, Neuroligins contain Ca²⁺-binding EF-hand domains, and Neuroligin-NRXN1β interaction is dependent on Ca²⁺ [81]. Elevated Ca²⁺ transients in human NRXN1α^+/− neurons may therefore also enhance excitation through increased Neuroligin-NRXN1β interactions. Furthermore, we have observed an increase in the expression of few members of SNARE complexes, i.e., synaptotagmins, suggesting an interaction of the cytoplasmic membrane of neurexins with synaptotagmins [82]. It seems likely that NRXN1α may regulate the level of synaptotagmins or other members of SNARE proteins, which might be critical for neurotransmitter and vesicle release [83]. Interestingly, two of our ASD patients had a history of seizures. While the patient numbers were small, it appeared that the increase of the frequency was more prominent in two ASD probands with seizure (ND1, ND4) than the ASD without seizure (ND2, Additional file 1: Figure S11). This concurs with disrupted Ca²⁺ signaling implicated in a range of neurodevelopmental disorders including ASD and epilepsy [79, 84‐88].

The DEGs in NRXN1α^+/− neurons may arise from Ca²⁺ influx and voltage-dependent conformational changes of VGCCs. For example, Cav1.2 may interact with αCaMKII, and βCaMKII is then recruited by Ca²⁺ mobilization. Voltage-dependent conformational changes can lead to α/βCaMKII activation, CREB phosphorylation and nuclear accumulation [89], and activation of transcription factors NFAT and MEF2 [90‐94]. Therefore, the transcriptomic changes may reflect both the activity-driven alterations and functional features of ASD NRXN1α^+/− neurons.

There are several limitations which may be addressed in the follow-up studies. (1) While we provide strong evidence for the role of VGCCs as a contributor to alterations in NRXN1α^+/− neurons, in this study, we employed the non-ratiometric calcium reporter Fluo-4 AM to represent intracellular calcium dynamics in the absence of ground-truth electrophysiological recordings and direct measurements of VGCCs. Future studies will be required to directly measure the channel activation and kinetics in NRXN1α^+/− neurons. (2) The NRXN1 deletions are associated with different clinical symptoms; therefore, NRXN1 deletion iPSCs from different neurodevelopmental/neuropsychiatric diseases may be investigated through collaborative research (3). The heterogeneity of iPSCs is common. Although the current data are conducted with statistically viable numbers and vigorously justified with different statistical methods, experiments with a larger cohort of iPSC lines will be desirable to confirm the commonality of the phenotype. (4) Genetic rescue will be important to validate genotype-phenotype correlation, but this is technically challenging, given that the NRXN1 deletion sizes of chromosomal regions are beyond the limit of conventional rescue constructs. In addition, the NRXN1 non-coding sequences are evolutionally conserved, and NRXN1 gene expression is highly regulated; therefore, no single cDNA-based construct may be able to rescue the phenotype with the right dose, isoform, and/or developmental regulation of the NRXN1 expression. (5) As the clinical penetrance of NRXN1 deletion is incomplete, a second hit may be required for different clinical phenotypes. Creation of isogenic lines with large chromosomal deletions is under the way, albeit technically challenging. It remains to see if the isogenic lines on healthy genetic background will have the same cellular phenotype as from the ASD individuals.

NRXN1α^+/− neurons derived from ASD patients’ iPSCs revealed alteration in calcium transients’ properties, leading to increased calcium activity. These findings may suggest an alteration in neurotransmitter release and a possible higher excitability in neurons. The NRXN1α^+/− iPSCs may be offered as a human model with translatable phenotype for drug screening and testing of ASD.