An antisense oligonucleotide-based strategy to ameliorate cognitive dysfunction in the 22q11.2 Deletion Syndrome

Our genetic material is ‘packaged’ into chromosomes, which are compact structures made of DNA found in every cell. Chromosomal abnormalities occur either when a person has the wrong number of chromosomes, or when parts of a chromosome are deleted or duplicated. This can cause a wide range of health problems, including psychiatric and cognitive symptoms.

Individuals with ‘22q11.2 deletion syndrome’ are missing a small DNA segment on chromosome 22. This results in cognitive impairment and a high risk of disorders like schizophrenia. Recent research in mice has shown that the DNA deletion in 22q11.2 deletion syndrome disrupts tiny molecules called microRNAs, which help control the activity of many genes (usually by ‘switching’ them off).

The gene for EMC10 is affected by these changes in microRNAs. Normally, the EMC10 protein plays a role in maintaining the health of brain cells. However, further studies in mice have shown that a DNA deletion equivalent to the one in humans with 22q11.2 deletion syndrome leads to excessive production of EMC10 – suggesting that too much EMC10 can be harmful. Reducing the amount of EMC10 in these mice restored normal brain function and behaviour.

Based on these results, Thakur, Lackinger et al. investigated if the same connection between disrupted microRNAs and abnormally high EMC10 levels also occurred in humans with 22q11.2 deletion syndrome. Analysis of cultured brain cells derived from patients with the condition confirmed that problems similar to those previously observed in mice emerged: disruption of microRNAs led to the cells accumulating too much EMC10, resulting in abnormal cell behaviour such as defective growth.

Thakur, Lackinger et al. then tested if gene-targeting tools called antisense oligonucleotides (ASOs) could be used to treat 22q11.2 syndrome. Similarly to microRNAs, ASOs work by turning off specific genes. When adult mice with the deletion were given ASOs targeting the gene for EMC10, their levels of EMC10 protein decreased, and their ‘cognitive function’ (including performance in simple memory tests) improved. Importantly, these benefits lasted over two months after a single treatment.

These results shed new light on the molecular mechanisms behind the effects of 22q11.2 deletion in humans. They also highlight ASOs targeting the EMC10 gene as a potential treatment for the condition, even when treatment begins in adulthood. In the future, Thakur, Lackinger et al. hope that this work will help to develop therapies that improve quality of life for those affected by the syndrome.

Adults and children with the 22q11.2 Deletion Syndrome (22q11.2DS) demonstrate cognitive, social, and emotional impairments (Morrison et al., 2020; Woodin et al., 2001; McCabe et al., 2013). 22q11.2 deletions are also one of the strongest genetic risk factors for schizophrenia (SCZ) (Xu et al., 2008). There are currently no targeted therapies that address the underlying molecular mechanisms of 22q11.2DS. Previous work in a model of the 22q11.2 deletion, carrying a hemizygous 1.3 Mb deficiency on mouse chromosome 16 [Df(16)A], which is syntenic to the 1.5 Mb 22q11.2 deletion [Df(16)A^+/–mice] revealed a distinct behavioral and cognitive profile (Stark et al., 2008; Piskorowski et al., 2016). Molecular analysis of the Df(16)A^+/–strain provided compelling evidence for abnormal processing of brain-enriched microRNAs (miRNAs) (Stark et al., 2008; Xu et al., 2013). The Df(16)A^+/–related miRNA dysregulation is due to (i) hemizygosity of Dgcr8, a component of the “microprocessor” complex that is essential for miRNA production (Gregory et al., 2004) and (ii) hemizygosity of miRNA genes residing within the deletion, including Mir185. Reduction of Mir185 levels and to a lesser degree of miRNAs residing outside the deletion {such as Mir485 Xu et al., 2013} result in a de-repression of Emc10 gene (alias Mirta22), whose expression is under the repressive control of miRNAs Xu et al., 2013. Indeed, comprehensive RNA profiling of Df(16)A^+/–mice found that postnatal elevation in the expression of the Emc10 gene represents a key transcriptional effect of the 22q11.2 deletion (Xu et al., 2013). Increased brain expression of Emc10 is recapitulated in Df(16)A^+/−primary neurons (Sun et al., 2018) as well as in mouse models of the more common 3 Mb 22q11.2 deletion (Saito et al., 2020). Other miRNA targets are dysregulated, but their levels of change are subtler and more variable. Emc10 encodes for a component of the ER membrane complex (EMC), which promotes membrane insertion and maturation of a subset of polytopic and tail-anchored membrane proteins including neurotransmitter receptors, channels, and transporters (Guna et al., 2018; Chitwood et al., 2018; Richard et al., 2013; Satoh et al., 2015; Bircham et al., 2011; Louie et al., 2012; Shurtleff et al., 2018; Tian et al., 2019). Emc10 is a prenatally biased gene with high expression in embryonic life that gradually subsides after birth (Xu et al., 2013), a developmental pattern of expression conserved between mice, humans and nonhuman primates (Diamantopoulou et al., 2017). Emc10 Loss-of-Function (LoF) mutation that leads to reduction of Emc10 levels rescues key cellular, cognitive and behavioral alterations in the Df(16)A^+/–mice (Diamantopoulou et al., 2017). However, whether similar beneficial effects could be achieved in human neurons and whether Emc10 normalization in the adult brain could reverse established cognitive deficits remained unknown.

Here, we show that 22q11.2 deletion results in abnormal processing of miRNAs in human neurons and in turn drives misexpression of EMC10 as previously described in animal models (Stark et al., 2008). Human EMC10 expression is elevated in neurons derived from 22q11.2 deletion carriers and reversal of EMC10 expression leads to restoration of key morphological and functional alterations linked to 22q11.2 deletions, supporting normalization of EMC10 expression as a disease-modifying intervention. Toward this end, we also show that antisense oligonucleotide (ASO)-mediated Emc10 normalization in the adult mouse brain is effective at reversing cognitive alterations. Improvements in cognition are sustained for over 2 months post ASO administration, underscoring the potential of this approach for providing durable therapeutic benefits. The observations that ASO-mediated Emc10 reduction in adult mouse brain rescues cognitive deficits linked to 22q11.2 deletion strongly support a key contribution of Emc10 and Emc10-dependent membrane protein trafficking in mediating the effects of 22q11.2 deletions on cognitive function and pave the way toward translating these observations into potential disease-modifying therapeutic interventions.

To investigate whether miRNA dysregulation and upregulation of EMC10 is also prominent in cortical neurons from patients carrying 22q11.2 deletions (Figure 1A), we used hiPSC lines obtained from three independent 22q11.2DS/SCZ donors carrying a 3 Mb deletion and diagnosed with SCZ, along with matched healthy controls (Supplementary file 1) to ensure the robustness and generalizability of our findings across different genetic backgrounds. The first patient/control pair is derived from dizygotic twins discordant for the 22q11.2DS and SCZ [Q6 (22q11.2) and Q5 (Ctrl)] (Figure 1—figure supplement 1A–D). The second patient/control pair is derived from siblings [Q1 (22q11.2) and Q2 (Ctrl)] while the third patient/control pair is a case and age/sex-matched unrelated control pair from the NIMH Repository and Genomic Resource [QR27 (22q11.2) and QR20 (Ctrl)].

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We examined whether 22q11.2 deletion results in abnormal processing of miRNAs in human neurons as we have previously described in animal models (Stark et al., 2008). We performed parallel small RNA/miRNA sequencing on DIV8 differentiated human cortical neurons from the sibling (Q5/Q6) pair derived using an approach that combines small-molecule inhibitors to repress SMAD and WNT signaling pathways to promote CNS fate (Qi et al., 2017). This protocol has been extensively validated and is known to robustly generate cortical neurons while actively suppressing glial differentiation. We confirmed the efficiency of differentiation using immunohistochemistry (IHC) and gene expression assays, which indicated the anticipated increase of TUJ1/TBR1-positive derived neurons and downregulation of embryonic stem cell marker OCT4 (Figure 1B and Figure 1—figure supplement 1E–G). We identified a number of mature miRNAs dysregulated in response to the 22q11.2 deletion (Figure 1C, Figure 1—figure supplement 2A, B Supplementary file 2). As a validation of our approach, we observed the expected reduction of expressions of miRNA genes MIR185, MIR1286, and MIR1306 that reside in the 22q11.2 locus (expression of three other predicted 22q11.2 miRNA genes, MIR649, MIR3618, and MIR4761 were not detected in DIV8 neurons) (Figure 1—figure supplement 2A). Among miRNAs located outside the 22q11.2 region, we note downregulation of mature miRNAs such as miR-137, as well as miR-134 and several other members from the largest placental mammal-specific miRNA gene cluster miR379-410 (Figure 1—figure supplement 2B) that have been previously implicated in neuronal development, differentiation, and function (Stark et al., 2008; Siegert et al., 2015; Thomas et al., 2017; Schratt et al., 2006; Fiore et al., 2009; Rago et al., 2014; Lackinger et al., 2019; Gardiner et al., 2012; Tomasello et al., 2022; Whipple et al., 2020). We used the miRNA-target interaction network tool miRNet 2.0 (Chang et al., 2020) to perform target enrichment and network analysis for the dysregulated miRNAs and conducted GO term enrichment analysis on this target interaction network. Affected biological processes were prominently centered on cell division and intracellular protein transport (Figure 1—figure supplement 3A) whereas cellular components were associated with the nucleus and the perinuclear region (endoplasmic reticulum and Golgi apparatus) of the cytoplasm (Figure 1—figure supplement 3B).

In addition to 22q11.2 deletion region miRNAs, lower abundance of miRNAs in cases is likely due to haploinsufficiency of the DGCR8 gene and is expected to result in upregulation of target genes. To identify candidate miRNA target genes, we performed an unbiased evaluation of the transcriptional responses using bulk RNA sequencing on RNA collected from DIV8 differentiated cortical neurons derived from the patient (Q6) and the corresponding healthy dizygotic twin (Q5) line (Figure 1—figure supplement 4A). We observed the expected downregulation of genes within the 22q11.2 locus in patient neurons (Supplementary file 3). Further, RNA and protein expression characterization confirmed the reductions in the abundance of the 22q11.2 locus residing genes DGCR8 and RANBP1 (Figure 1—figure supplement 4B, C). Among the differentially expressed genes (DEGs) 2094 were downregulated and 1937 were upregulated. As expected EMC10 expression was elevated in patient neurons while expression of other EMC subunits (EMC1-4, EMC6-9) detected in our DIV8 sequencing data did not show significant differences. GO term enrichment analysis on downregulated DEGs identified significantly altered biological processes centered on neurogenesis, neuronal development, and differentiation (Figure 1—figure supplement 4D). Among the upregulated DEGs, the GO terms enriched were related to neuronal development as well as neuronal cilia assembly and structure (Figure 1—figure supplement 4E).

Intersection of predicted targets of downregulated miRNAs and upregulated DEGs identified 774 predicted targets of downregulated miRNAs (Figure 1—figure supplement 4F, Supplementary file 4) including EMC10. Notably, functional annotation revealed that predicted targets of downregulated miRNAs include genes that modulate neuronal development and are associated with GO terms such as endoplasmic reticulum and endomembrane system of neurons (Figure 1—figure supplement 4G, H).

qRT-PCR assays confirmed a robust and significant upregulation of EMC10 levels in RNA extracted from cortical neurons derived from hiPSCs of the Q5/Q6 pair through SMAD/WNT signaling inhibition, at three distinct stages of in vitro maturation (Figure 1D–F). Additionally, this upregulation was confirmed in protein extracts from cortical neurons at day 8 of differentiation (Figure 1G). To examine whether transcriptional EMC10 upregulation is independent of the neuronal derivation method, we generated neurons via inducible expression of Neurogenin-2 (NGN2), a widely used protocol that generates a robust population of excitatory neurons (NGN2-iNs) within 3 weeks (Zhang et al., 2013; Yi et al., 2016; Ho et al., 2016; Pak et al., 2018). MAP2 staining was used to demonstrate the successful neuronal differentiation of the hiPSC lines (Figure 1H). qRT-PCR assay of EMC10 mRNA expression level in NGN2-iNs at DIV21 confirmed transcriptional EMC10 upregulation in three independent pairs of patient and sex-/age matched healthy control lines (Figure 1I–K). Taken together our results highlight a reproducible and robust upregulation of EMC10 in neurons derived from patients with 22q11.2 deletions, which is independent of the derivation method. It is noteworthy that in addition to monolayer cultures, EMC10 shows significant upregulation along the excitatory neuron lineage (radial glia, intermediate progenitors and excitatory neurons) but not in astrocytes, choroid or interneuron lineage cells, in patient forebrain organoids generated by the same hiPSCs lines used in the present study (Rao et al., 2023).

We have previously shown that upregulation of the murine orthologue of Emc10 is primarily due to downregulation of miR-185 and to a lesser degree of miR-485 (Xu et al., 2013). Both conserved and non-conserved binding sites at the 3’UTR of human EMC10 are predicted in silico for both miRNAs (Figure 2—figure supplement 1A). Consistently, the observed upregulation in the levels of EMC10 gene is accompanied by a robust reciprocal decrease in the levels of the miRNA precursor of miR-185 at DIV8 as indicated both by our miRNA sequencing analysis (Figure 1C, Supplementary file 2) and follow-up qRT-PCR assays (Figure 2A). The miRNA precursor of miR-485 exhibited a modest but non-significant reduction in abundance (Figure 2B), consistent with our miRNA sequencing profile (Figure 1—figure supplement 2B). This may be attributed to the early developmental stage of the neurons, as miR-485 expression increases during neuronal maturation (Cohen et al., 2011; Soutschek et al., 2023). Collectively, these findings demonstrate a strong inverse correlation between EMC10 upregulation and miR-185 downregulation, while suggesting that miR-485 may play a less prominent role at this early stage of neuronal development. Notably, overexpression of miR-185 and miR-485 using miRNA mimics in human cortical neurons at DIV10 resulted in a reduction of EMC10 expression levels in both the healthy control (Q5, Figure 2C) and patient line (Q6, Figure 2D). Furthermore, inhibition of endogenous miR-185 and miR-485 in the control line by using specific miRNA inhibitors increased EMC10 expression level (Figure 2E) confirming the predicted conserved functionality of miR-185 and miR-485 miRNA binding sites in EMC10. It is worth noting that in addition to miR-185, non-conserved binding sites at the 3’UTR of human EMC10 are predicted in silico for two additional downregulated miRNA genes residing within the 22q11.2 locus, MIR1286 and MIR1306 (Supplementary file 5). The functionality of these miRNA binding sites in EMC10 and whether they contribute to the observed elevation of its expression in human neurons remains to be determined. Taken together, our results confirm that miRNA dysregulation emerges in human neurons as a result of the 22q11.2 deletion and in turn drives misexpression of genes primarily involved in intracellular membrane and protein trafficking-related processes required for neuronal development and maturation. Among them, EMC10 represents a major downstream effector of the 22q11.2-linked miRNA dysregulation.

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To investigate the relevance of EMC10 de-repression in the development and function of patient neurons, we generated derivatives of the Q6 patient hiPSC line carrying either heterozygous (Q6/EMC10^HET) or homozygous (Q6/EMC10^HOM) EMC10 LoF mutations using standard CRISPR/Cas9 editing approaches (Figure 3—figure supplement 1A). Mutations were confirmed by sequencing (Figure 3—figure supplement 1A, lower panel) and karyotyping confirmed normal chromosome complement (Figure 3—figure supplement 1B). We confirmed reduced expression levels of 22q11.2 gene RANBP1 by western blot in both derivative hiPSC lines (Figure 3—figure supplement 1C) whereas stem-cell markers NANOG and OCT4 were equally expressed in all lines assayed by qRT-PCR (Figure 3—figure supplement 1D, E). EMC10 mRNA and protein levels were reduced by ~50% in the Q6/EMC10^HET hiPSC line and abolished in the Q6/EMC10^HOM line (Figure 3—figure supplement 1F, G). It is noteworthy that we did not observe an upregulation of EMC10 mRNA levels in the Q6 hiPSC lines (Figure 3—figure supplement 1F), a finding likely attributed to the general low expression level of miR-185 and miR-485 in hiPSCs (Wilson et al., 2009). Indeed both miRNAs are developmentally regulated and show increased expression levels during neuronal development (https://ethz-ins.org/igNeuronsTimeCourse/) (Soutschek et al., 2023). Additional characterization of hiPSC-derived NGN2-iNs (Figure 3A), conclusively demonstrated a reduction (Q6/EMC10^HET) or elimination (Q6/EMC10^HOM) of EMC10 mRNA (Figure 3B). Expression assays of a panel of cell type-specific markers did not reveal significant differences between NGN2-iNs from the Q6 patient line and both derivative lines, indicating that gene editing has no adverse effect on neuronal differentiation (Figure 3—figure supplement 1H).

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Df(16)A^+/−mice show impaired formation of dendrites in deep layer cortical neurons, which are faithfully recapitulated in primary neuronal cultures and are partially reversed by reduction of Emc10 levels (Xu et al., 2013). We asked whether impaired dendritic formation is also observed in human neuronal cultures from patients with 22q11.2 deletions and whether reduction of EMC10 levels could prevent such morphological alterations during neuronal maturation. We employed monolayer neuronal cultures of NGN2-iNs. Neuronal cells were fixed at DIV21 of differentiation, immuno-stained, traced and key indices of dendritic architecture were quantified (see Materials and methods). Our analysis confirmed a reduced dendritic complexity in mutant neurons as reflected in total neuronal length, the number of branch points and the total number of dendrites per cell (Figure 3C–F). The number of primary dendrites per cell was unchanged (Figure 3G) in accordance with previous findings from the murine 22q11.2 deletion model where only subtle changes were detected in the number of primary neurons (Xu et al., 2013). Importantly, we found that reduction or elimination of EMC10 expression restored to WT levels neuronal length and branch points. Importantly, we found that reducing or eliminating EMC10 expression restored neuronal length and branch points to WT levels. Notably, the number of branch points in Q6/EMC10^HOM neurons exceeded those in WT neurons (Figure 3E) likely suggesting that reduced (or abolished) Emc10 expression can alter normal neurite growth, resulting in excessive responses, potentially triggered upon gene restoration by the mutant system’s adaptation to dysfunction, leading to altered receptor sensitivity or signaling dynamics. This highlights the critical importance of precise Emc10 expression for maintaining proper neuronal development and function.

Our previous evaluation of Ca²⁺ homeostasis perturbations caused by 22q11.2 deletions using Ca²⁺ imaging on primary neurons from Df(16)A^+/–mice revealed a significantly lower amplitude of Ca²⁺ elevation following KCl evoked depolarization (Sun et al., 2018). This impairment was replicated in human cortical neurons from patients with 22q11.2 deletions (Khan et al., 2020) and shown to be partially restored by exogenous expression of DGCR8, indicating a potential role of miRNA dysregulation. Using the green-fluorescent calcium indicator Fluo-4 and time-lapse microscopy, we confirmed a decrease in the amplitude of Ca²⁺ rise following KCl evoked depolarization, in patient (Q6) derived NGN2-iNs at DIV37/38 compared to the healthy twin (Q5) (Figure 3—figure supplement 2A, B). We asked whether reduction of EMC10 levels could reverse such alterations. Notably, the observed defect in Ca²⁺ signaling were reversed in both Q6/EMC10^HET and Q6/EMC10^HOM NGN2-iNs as demonstrated by the increased amplitude of Ca²⁺ rise following depolarization (Figure 3H, I). Interestingly, the amplitudes of Ca²⁺ rise in Q6/EMC10^HET and Q6/EMC10^HOM were slightly elevated compared to the WT control group, consistent with the effects on neurite outgrowth (Figure 3H). The observation that reduction of EMC10 levels fully restores the Ca²⁺ signaling deficits observed in patient neurons suggests that miRNA-dependent elevation of EMC10 may interfere with one or more sources of intracellular Ca²⁺ and a wide range of calcium-dependent processes.

DEGs are often organized into functional groups or pathways based on their known biological roles. We used transcriptional profiling as an indirect measure of cellular pathways affected by the reduction of EMC10 levels by identifying genes differentially expressed between the parental Q6/Q5 lines whose expression differences are abolished or nearly abolished (‘rescued’) in either Q6/EMC10^HET or Q6/EMC10^HOM NGN2-iNs (Figure 3J and K). In the Q6/EMC10^HET line, 237 DEGs (6%) were rescued (111 downregulated and 126 upregulated), while in the Q6/EMC10^HOM line, 382 DEGs (11%) were rescued (193 downregulated and 189 upregulated; Supplementary file 6). In both cases, functional annotation analysis indicated highest enrichment scores for terms related to nervous system development as well as an enrichment in GO terms relevant to neuronal generation and differentiation. Intersection of “rescued” genes in Q6/EMC10^HET and Q6/EMC10^HOM NGN2-iNs identified 103 shared DEGs (Figure 3L and Supplementary file 7). To assess the significance of the overlap between the rescued DEGs in Q6/EMC10^HET and Q6/EMC10^HOM NGN2-iNs, we performed a hypergeometric test, which calculates the probability of observing the degree of overlap between two gene groups under the null hypothesis that the overlap occurs by chance. Our analysis revealed a significant overlap (enrichment factor = 1.7; p<0.0001), indicating that the overlap is much greater than expected by chance. These 103 shared DEGs likely play a key role in pathways influenced by EMC10 levels, particularly those involved in nervous system development, as suggested by our functional annotation analysis. To further investigate the functional relationships among these shared DEGs, we conducted a protein-protein interaction (PPI) network analysis. This analysis highlighted a functional cluster including 30 of these genes, such as the SCZ-linked genes PCDHA2 (Shao et al., 2019), RBFOX1 (O’Leary et al., 2022) and RGS4 (Mirnics et al., 2001; Erdely et al., 2006; Figure 3—figure supplement 3A), involved in nervous system development (Figure 3—figure supplement 3B). It should be noted that the beneficial effect of elimination of EMC10 expression is consistent with previous findings that lack of EMC10 does not compromise EMC assembly (Volkmar et al., 2019) and implying an auxiliary or modulatory role of EMC10 in the EMC function.

Taken together, our analysis of neurons from 22q11.2 deletion carriers indicate that elevation of EMC10 expression disrupts their development and maturation in a way similar to observations in murine neurons, and support normalization of EMC10 expression as a disease-modifying intervention. While our previous work has shown that constitutive genetic reduction of Emc10 levels rescues key cognitive and behavioral alterations in the Df(16)A^+/–mice, translating these observations into therapeutic interventions requires demonstration that it is the sustained elevation of EMC10 throughout the adult life that interferes with the underlying neural processes rather than an irreversible impact on brain maturation during early development. Toward this end, we first investigated whether restoration of Emc10 levels in the brain of adult (2–4 month-old) Df(16)A^+/–mice is effective at reversing cognitive alterations (Stark et al., 2008; Diamantopoulou et al., 2017). Specifically, we examined the effects of Emc10 reduction in adult brain on social memory (SM), a cognitive domain robustly and reproducibly affected in adult Df(16)A^+/–mice (Piskorowski et al., 2016; Diamantopoulou et al., 2017; Donegan et al., 2020). Notably, SM deficits are also present in juvenile Df(16)A^+/–mice as early as postnatal day 22 (Figure 4—figure supplement 1A, B), underscoring the severity of this phenotype, which emerges during early adulthood. In humans, SM, a key component of social cognition, involves encoding, storing, and retrieving information about social experiences, such as recognizing familiar individuals and recalling past interactions and emotions. Social cognition, the broader ability to perceive, interpret, and respond to social cues, is essential for forming relationships and understanding others' behavior. Disruptions in SM can impair social cognition, contributing to the functional deficits commonly observed in schizophrenia (Green et al., 2015). Deficits in social cognition are present in individuals with 22q11.2 deletions (Jalal et al., 2021) and use of rodent tasks that evaluate SM can serve as a useful proxy of the human condition. Impaired SM in Df(16)A^+/–mice is fully restored by constitutive genetic reduction of Emc10 levels (Diamantopoulou et al., 2017).

To manipulate the expression of the Emc10 gene in adult Df(16)A^+/–mice, we used an Emc10 conditional ‘knockout-first’ design by conducting a Flp- and Cre-dependent genetic switch strategy (Figure 4—figure supplement 2A). Parental Emc10^{+/- tm1a} mice were crossed to a germline Flp mouse line that activates global Flp function and leads to the deletion of the frt-flanked sequence(s) in the offspring. The Emc10^tm1c offspring from this cross carry a loxP flanked WT Emc10 allele and are essentially WT. To achieve temporal control of Emc10 expression, we used an inducible UBC-Cre/ERT2 mouse line that activates global Cre function upon tamoxifen (TAM) treatment. This approach enables postnatal normalization of Emc10 expression at its endogenous locus preserving Emc10 expression within its physiological levels. We used UBC-Cre/ERT2 mice in crosses to generate compound Emc10^tm1c+/-; UBC-cre/ERT2; Df(16)A^+/–mice. These mice have two WT Emc10 copies upregulated, as expected in the Df(16)A background, until TAM-induced Cre expression deletes the tagged Emc10 allele. We used oral gavage to deliver TAM and implement Cre-mediated Emc10 deletion during adulthood (postnatal day 56–70). Corn oil treatment served as a control. Behavioral analysis was performed on the following four groups: Emc10^tm1c+/-; UBC-cre/ERT2; Df(16)A^+/+ mice treated with TAM (WT +TAM), Emc10^tm1c+/-; UBC-cre/ERT2; Df(16)A^+/–mice treated with TAM (Df(16)A^+/–+TAM), Emc10^tm1c+/-; UBC-cre/ERT2; Df(16)A^+/+ mice treated with corn oil vehicle (WT +oil), and Emc10^tm1c+/-; UBC-cre/ERT2; Df(16)A^+/–mice treated with corn oil vehicle (Df(16)A^+/–+oil). Investigation of the efficiency of Cre-mediated deletion in brain lysate preparations from prefrontal cortex (PFC) (Figure 4—figure supplement 2B, C), hippocampus (HPC) (Figure 4—figure supplement 2D, E) and cerebellum (CB; Figure 4—figure supplement 2F) confirmed that upon TAM treatment, Emc10 mRNA and protein levels were restored to near WT levels in the adult brain of Df(16)A^+/–mice. As expected, Df(16)A^+/–+oil mice showed impaired SM performance compared to WT +oil control littermates, which was fully rescued upon TAM treatment. Specifically, upon reintroduction of a familiar juvenile mouse Df(16)A^+/–+TAM mice showed a strong reduction in social interaction, indicative of intact SM, comparable to TAM-treated WT littermates and significantly different from Df(16)A^+/–mice treated with corn oil (Figure 4—figure supplement 2G). The intact SM of the Df(16)A^+/–+TAM mice was further evident in analysis of difference score (Figure 4—figure supplement 2H) compared to the corn oil-treated Df(16)A^+/–mice. Interaction times during the first trial of the SM assay, which measures general social interest, were unaffected by TAM treatment. In contrast to SM, Df(16)A^+/–mice hyperactivity in the open field was not affected upon TAM treatment consistent with our previous results from constitutive genetic reduction of Emc10 levels (Figure 4—figure supplement 2I). Notably, TAM treatment did not alter the time spent in the center area of the open field, indicating an absence of changes in anxiety-related behavior. These findings demonstrate that restoring Emc10 levels in adult Df(16)A^+/–mice can significantly improve cognitive deficits, underscoring a broad therapeutic window and establishing Emc10 as a promising target for postnatal interventions.

We explored the translation potential of this finding by employing transient injection of single-stranded ASOs targeting the mouse gene as dictated by their demonstrated efficacy as a therapeutic modality in preclinical models (Becker et al., 2017; DeVos et al., 2017; Kordasiewicz et al., 2012; Korobeynikov et al., 2022; Scoles et al., 2017; Sztainberg et al., 2015) and clinical studies of neurodevelopmental disorders (NDDs) or neurodegenerative disorders (Tabrizi et al., 2019; Winkelsas and Fischbeck, 2020; Rinaldi and Wood, 2018). Over 300 chimeric 2′-O-methoxyethyl (2’MOE)/DNA gapmer ASOs were generated and screened for Emc10 mRNA reduction in 4T1 cells via electroporation (Figure 4—figure supplement 3A). Lead ASOs were then confirmed in a dose-response assay (Figure 4—figure supplement 3B), including the lead ASO (1081815, herein referred to as Emc10^ASO1), which targets intron 2 of mouse Emc10 (Figure 4A). Emc10^ASO1 was selected for subsequent studies, as it was effective in lowering Emc10 expression both in vitro and in vivo. Specifically, following transient intracerebral ventricular (ICV) injection in the posterior ventricle of 8 weeks old WT mice, Emc10^ASO1 effectively suppressed the levels of Emc10 mRNA (Figure 4—figure supplement 3C) and protein (Figure 4—figure supplement 3D) in both left and right HPC compared to WT mice treated with a control ASO (Ctrl^ASO1) without complementarity in the mouse. Analysis of Gfap and Aif1 expression did not reveal any changes (Figure 4—figure supplement 3E, F) suggesting lack of astroglial and microglial activation upon Emc10^ASO1 injection. Emc10^ASO1 injected mice showed normal gait and no signs of behavioral toxicity. IHC analysis using an antibody that selectively recognizes the phosphorothioate backbone verified a robust diffusion primarily in HPC and to a lesser degree in surrounding brain areas. Colocalization with the neuronal marker NeuN and glial fibrillary acidic protein (GFAP) confirmed accumulation in hippocampal neurons as well as GFAP-expressing astrocytes (Figure 4B). Analysis of Df(16)A^+/–mice treated by intraventricular injection at 8 weeks of age showed that Emc10^ASO1 effectively lowered hippocampal Emc10 mRNA to nearly WT levels 3 weeks post-injection resulting in normalization of Emc10 expression (Figure 4C, left panel). By contrast, consistent with the pattern of ASO distribution, we did not observe a significant reduction of Emc10 expression levels in the PFC of Df(16)^+/– mice treated with Emc10^ASO1 (Figure 4C, right panel). In addition to targeted assays, we performed bulk RNA-sequencing analysis of Ctrl^ASO1 and Emc10^ASO1-treated Df(16)A^+/–mice and WT littermates to evaluate the effect of Emc10^ASO1 treatment on the hippocampal transcriptome profile. In the Ctrl^ASO1-treated group (Figure 4D, left panel), we observe the tripartite differential gene expression signature characteristic of Df(16)A^+/–mice: upregulation of Emc10 and non-coding RNAs (pri-forms of miRNAs and long non-coding RNAs (Figure 4D, left panel) and inset, Log2Fold Change = 0.5) as well as the expected downregulation of genes included within the Df(16)A deficiency. In the Emc10^ASO1-treated group (Figure 4D, right panel and inset), Emc10 is no longer upregulated in Df(16)A^+/–mice while non-coding RNAs remain upregulated, and genes included in the deficiency are robustly downregulated. Apart from Emc10, seven other genes (Mir9-3hg, Plxnd1, Cd68, Mir22hg, Gm28439, Adgre1, and Tnn) are significantly upregulated in Df(16)A^+/–mice in the Ctrl^ASO1 but not in the Emc10^ASO1-treated group (Supplementary file 8). We used the Bowtie mapping tool (Langmead et al., 2009) to align short sequencing reads on both genomic and transcript sequence to assess whether these expression changes represent potential off-target effects of the Emc10^ASO1 in the mouse transcriptome. Emc10^ASO1 exclusively aligned with full complementarity to an intronic region in the Emc10 gene (Figure 4A) providing additional support for high target specificity. The observed changes might represent downstream effects of Emc10 level reduction or reflect expression variability due to low expression levels of the upregulated genes.

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Eight-week-old Df(16)A^+/–mice and WT littermates were treated by ICV injection of Emc10^ASO1 and Ctrl^ASO1 and tested 3 weeks later in SM assays. Df(16)A^+/–mice treated with Ctrl^ASO1 showed the expected deficits in SM as reflected in the sustained high interaction time with the reintroduced familiar juvenile mouse. By contrast, Df(16)A^+/–mice injected with Emc10^ASO1 had significantly improved memory performance to levels indistinguishable from Emc10^ASO1-treated WT littermates, consistent with improvement of function arising from adult restoration of Emc10 levels (Figure 4E and F). Interaction times during the first trial of the SM assay, which measures general social interest, were unaffected by ASO treatment. Rescue was observed in both sexes and no significant differences were seen in treatment across sexes. In control experiments, we did not observe any effects of genotype or treatment upon reintroduction of a novel juvenile mouse in trial 2 (Figure 4—figure supplement 4A, B), strongly indicating that SM deficits are not driven by a simple task fatigue.

To evaluate the consistency and reproducibility of the ASO-mediated SM rescue we generated additional ASOs targeting different regions within the Emc10 transcript and screened them for Emc10 mRNA reduction in vivo (Figure 5—figure supplement 1A–C). One of these ASOs (1466182, herein referred to as Emc10^ASO2), which targets intron 1 of mouse Emc10 (Figure 5A) was selected as the lead ASO candidate for the replication analysis based on its efficacy in reducing Emc10 levels, distribution pattern, as well as the lack of any signs of astroglial/microglial activation or behavioral toxicity (see Materials and methods). IHC analysis showed robust ASO distribution in both the hippocampus (HPC, Figure 5—figure supplement 2A, first and second panels) and prefrontal cortex (PFC, Figure 5—figure supplement 2A, third and fourth panels), as well as diffusion into both neuronal and non-neuronal cells. This was indicated by colocalization with the neuronal marker NeuN and the glial marker GFAP (Figure 5—figure supplement 2A, bottom panel). Higher-magnification (40 x) analysis revealed extensive overlap between the ASO signal and NeuN staining, demonstrating that the vast majority (>97%) of neurons exhibited ASO uptake. qRT-PCR analysis of Df(16)A^+/–mice treated by intraventricular injection at 8 weeks of age showed that Emc10^ASO2 effectively lowered Emc10 mRNA to nearly WT levels 3 weeks post-injection resulting in normalization of Emc10 expression in the HPC (Figure 5B), PFC (Figure 5C) and somatosensory cortex (SSC) (Figure 5D). To study the effects of Emc10^ASO2-mediated Emc10 reduction on SM performance, 8-week-old Df(16)A^+/-male mice and WT littermates were treated by ICV injection of Emc10^ASO2 and Ctrl^ASO2 and tested 3 weeks later. Df(16)A^+/-mice injected with Emc10^ASO2 had significantly improved SM performance to levels indistinguishable from Emc10^ASO2-treated WT littermates (Figure 5E and F).

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In addition to SM deficits, mouse models of the 22q11.2 deletion show a spectrum of cognitive impairments in episodic and spatial memory as reflected, for example, in impaired performance in an Y-maze-based delayed alternations task that probes short-term spatial memory (Tripathi et al., 2020) and contextual fear conditioning a form of associative learning test used for studying episodic learning and spatial memory (Stark et al., 2008). We investigated the impact of ASO-mediated Emc10 reduction in the adult brain on both of these cognitive tasks. First, we confirmed that adult male Df(16)A^+/–mice exhibit impaired short-term spatial memory during novelty exploration in a two-trial delayed alternation Y-maze task (Figure 5G) as previously described for another mouse model of the 22q11.2 deletion (Benger et al., 2018). The total number of arm entries remained unchanged, indicating no alterations in locomotor activity (Figure 5—figure supplement 2B). To determine whether reducing Emc10 expression in the brain via ASO treatment could rescue short-term spatial memory deficits, we tested a new cohort of Df(16)A^+/–mice and WT littermates 3 weeks following ASO administration (Figure 5H). ASO-treated Df(16)A^+/–mice exhibited a significant improvement in delayed alternations compared to Df(16)A^+/–mice treated with control ASO (Figure 5H). Furthermore, no significant differences in total number of arm entries were observed between the groups (Figure 5—figure supplement 2C). We confirmed the reduction of Emc10 levels in the ASO-treated animals through qRT-PCR assays of the HPC, PFC, and SSC brain regions (Figure 5—figure supplement 2D–F). In the contextual fear conditioning task, while ASO treatment was not sufficient to fully rescue the learning deficit in Df(16)A^+/–mice to WT levels (Figure 5I, right panel), there was a modest improvement in fear memory of ASO-treated Df(16)A^+/–mice, since these mice did not differ significantly from the ASO-treated WT littermates. Interestingly, we have previously shown that genetic reduction of Emc10 levels in Df(16)A^+/–mice resulted in only partial restoration of deficits in contextual fear memory (Diamantopoulou et al., 2017). Thus, our finding faithfully recapitulates results from our previous constitutive genetic rescue assays (Diamantopoulou et al., 2017) and likely indicates a more limited role of Emc10 upregulation in the 22q11.2-linked fear memory deficits rather than requirement for additional treatment time or for earlier onset of Emc10 normalization.

The application of ASOs as a novel therapeutic strategy has seen a significant rise in recent years, in part due to their versatility in durably modifying RNA transcripts. Therefore, we investigated the longevity of ASO-mediated repression of Emc10 as an indicator of future therapeutic relevance for the treatment of 22q11.2DS. To this end, we conducted the SM and Y-maze assays on a new cohort of Df(16)A^+/–mice at 3–4 weeks and 8–9 weeks post-injection with Emc10^ASO2 (Figure 6A). We observed behavioral rescue in Emc10^ASO2-treated Df(16)A^+/–mice in the SM assay at 3e weeks (Figure 6B, left panel) and in the Y-maze assay at 4 weeks post ASO-administration (Figure 6C, left panel) in accordance to our previous findings (Figure 5F and H). Remarkably, we replicated these results at 8–9 weeks post-injection, demonstrating sustained behavioral rescue of SM (Figure 6B, right panel) and spatial memory deficits (Figure 6C, right panel). Importantly, locomotor activity remained unchanged in the Y-maze assays at both, 4 weeks (Figure 6—figure supplement 1A) and 9 weeks (Figure 6—figure supplement 1B) post-injection. Finally, we confirmed the downregulation of Emc10 in ASO-treated animals via qRT-PCR assays of the HPC, PFC, and SSC brain regions at 10 weeks post-treatment (Figure 6D–F). These results suggest that normalizing Emc10 expression in the brain can ameliorate social and spatial memory deficits in adult Df(16)A^+/–mice in a time period of at least 2 months.

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Despite an understanding of the molecular mechanisms of 22q11.2DS, especially ones related to abnormal expression and processing of miRNAs (Stark et al., 2008; Xu et al., 2013; Diamantopoulou et al., 2017; Khan et al., 2020; Paranjape et al., 2023), we still do not have a promising therapeutic avenue for the cognitive and neuropsychiatric symptoms associated with the 22q11.2 deletion. By leveraging our recent understanding of the molecular, cellular and behavioral consequences of 22q11.2-linked miRNA dysregulation, the present study represents an advancement towards developing a potential therapeutic strategy in two ways:

First, we show that 22q11.2 deletion results in abnormal processing of miRNAs in human neurons and in turn drives upregulation of EMC10 levels as previously described in mouse models (Stark et al., 2008). Effective reduction to near WT levels or even complete depletion of EMC10 leads to restoration of key alterations in morphological and functional neuronal maturation emerging due to 22q11.2 deletions. The miRNA regulatory mechanism underlying EMC10 upregulation and the restoration of cellular deficits are very similar to the ones we previously described in Df(16)A^+/-mice, highlighting the robustness of this molecular alteration as well as the translational value of using animal models to probe the link between Emc10 upregulation and 22q11.2-linked behavioral dysfunction.

Second, we show that normalization of Emc10 levels in adult Df(16)A^+/-mouse brain, by ASO-mediated targeted knockdown, is effective in rescuing SM deficits (which emerge during postnatal development and are present as early as postnatal day 22) as well as short-term spatial memory deficits. Significantly, these improvements in cognition were sustained for over 2 months post-ASO administration. These findings strongly suggest that at least for a subset of 22q11.2-associated cognitive deficits it is the sustained miRNA dysregulation and elevation of Emc10 throughout the adult life that interferes with the underlying neural processes rather than an irreversible impact on brain maturation during early development and demonstrate the therapeutic potential for treating a wide range of cognitive symptoms associated with 22q11.2DS.

In vivo delivery of ASOs offers a potential venue for emerging treatments of genetically driven and postnatally reversible symptoms of neurodevelopmental disorders (NDDs) focusing in reduction of culprit gene expression via sequence-specific knockdown of mRNA transcripts (Benger et al., 2018). A common challenge in efforts to employ gene-knockdown therapies for dosage-sensitive genes such as EMC10 is restricting target gene expression within optimal levels to avoid potential toxicity due to target hyper-knockdown or complete elimination (Shao et al., 2021b; Kaiyrzhanov et al., 2022; Shao et al., 2021a; Umair et al., 2020). A large number of relatively rare LoF variants or potentially damaging missense variants have been identified in the human EMC10 gene among likely healthy individuals in gnomAD (Shao et al., 2021a; Karczewski et al., 2020) which is depleted of individuals known to be affected by severe NDDs. Taken together with our previous analysis of constitutive heterozygous Emc10 knockout mice (Diamantopoulou et al., 2017) that showed no evidence of motor deficits or anxiety-like behavior, these observations strongly suggest that partial reductions in Emc10 levels of approximately 50% are well tolerated at the organismal level and do not result in behavioral abnormalities. The highest dose for both Emc10-specific ASOs used in mutant Df(16)A^+/–mice was limited to ~300 µg and reduced Emc10 mRNA to either normal or below normal (30–50% of WT) expression while attaining full behavioral rescue. While higher dose may be required to ameliorate other behavioral deficits (Shao et al., 2021b; Shao et al., 2021a), it should be noted that an acute injection of 700 µg Emc10^ASO in WT mice that resulted in ~50% reduction of Emc10 levels (Figure 5—figure supplement 1) did not cause secondary cellular and behavioral toxicity. Thus, available evidence indicates that therapeutically effective ASO-mediated normalization of EMC10 levels can be up- or down-titrated within a well-tolerated range. However, further work is needed to establish a comprehensive safety profile, including the evaluation of non-cognitive phenotypes, to fully validate the therapeutic potential of Emc10-targeting approaches. Additionally, the long-term effects of Emc10 reduction beyond 2 months post-injection require further investigation to determine the full extent of its therapeutic benefits. Future studies will also explore whether ASO-mediated normalization of adult Emc10 levels can restore additional 22q11.2 behavioral and cognitive alterations and whether earlier postnatal ASO treatment could prevent the onset of behavioral deficits or mitigate those resistant to adult interventions.

In addition to reduction of Emc10 expression, our findings have implications for therapeutic interventions aiming to manipulate its downstream targets. In that respect, it is attractive to speculate that different EMC10 upregulation-linked phenotypes and their developmental requirements may be driven by dysregulation of distinct, individually or in combinations, downstream EMC targets. Such targets are typically multi-transmembrane domain (TMD) proteins (Shurtleff et al., 2018; Tian et al., 2019; Wideman, 2015) that contain low-hydrophobicity TMDs which are hard to insert into ER membrane and thus require the aid of EMC as a membrane insertase (Guna et al., 2018; Chitwood et al., 2018; Shurtleff et al., 2018). Identification of neurotransmitter receptors, channels, and transporters whose biogenesis, trafficking and membrane insertion are affected by EMC10 upregulation could help establish a link between such targets and 22q11.2-related behavioral dysfunction and guide efforts to develop treatments for specific 22q11.2 deletion symptoms.

Overall, by highlighting the manipulation of EMC10 expression and activity, as well as its downstream targets, as a promising alternative or augmentation to currently available treatments, and emphasizing a broad temporal window for therapeutic and preventive intervention in 22q11.2 deletion-associated cognitive and behavioral symptoms, our results lay the foundation for developing mechanism-based therapeutic strategies. These strategies aim to leverage insights from both human and animal models to improve clinical outcomes in precision medicine for neuropsychiatric disorders. Furthermore, our findings may have broader implications for understanding the role of miRNA dysregulation and ER-mediated membrane protein translocation in other neurodevelopmental and neuropsychiatric disorders with overlapping genetic or phenotypic features.

The sequencing data described in this manuscript were deposited into the Gene Expression Omnibus database under accession number GSE236596 and are available at the following URL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE236596.

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Author details

1. Pratibha Thakur

   Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing, Designed the in vivo ASO screening strategy; Performed ASO injections/surgeries; Conducted mouse-related RNA expression studies, immunohistochemistry, and behavioral assays/analysis in ASO-treated animals

   Contributed equally with

   Martin Lackinger

   Competing interests

   No competing interests declared

2. Martin Lackinger

   1. Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States
   2. Stavros Niarchos Foundation Center for Precision Psychiatry and Mental Health, Columbia University, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing, Designed, performed, and analyzed all human iPSC and human iPSC-derived-neuronal experiments (including RNAseq/miRNAseq); Designed in vivo ASO screening strategy and conducted behavioral assays in (longitudinal) ASO-treated and Emc10 conditional knockout mice; Contributed to mouse-related RNA/protein expression assays, coordinated all behavioral and molecular experiments

   Contributed equally with

   Pratibha Thakur

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7946-5366

3. Anastasia Diamantopoulou

   Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States

   Present address

   Institute of Neurophysiology, Goethe University, Theodor-Stern-Kai 7, Frankfurt am Main, Germany

   Contribution

   Conceptualization, Validation, Investigation, Writing – review and editing, Contributed to the design/implementation of in vivo ASO screening strategy

   Competing interests

   No competing interests declared

4. Sneha Rao

   Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States

   Contribution

   Formal analysis, Visualization, Writing – review and editing, Analyzed mouse RNA-sequencing data

   Competing interests

   No competing interests declared

5. Yijing Chen

   1. Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States
   2. Department of Genetics and Development, Columbia University Irving Medical Center, New York, United States

   Contribution

   Validation, Investigation, Writing – review and editing, Contributed to ASO-related immunohistochemistry assays

   Competing interests

   No competing interests declared

6. Khakima Khalizova

   1. Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States
   2. Stavros Niarchos Foundation Center for Precision Psychiatry and Mental Health, Columbia University, New York, United States

   Contribution

   Investigation, Writing – review and editing, Contributed to ASO injections/surgeries and qRT-PCR assays

   Competing interests

   No competing interests declared

7. Annie Ferng

   Ionis Pharmaceuticals, Inc, Carlsbad, United States

   Contribution

   Resources, Validation, Visualization, Methodology, Writing – review and editing, Contributed to the identification and characterization of lead ASOs

   Competing interests

   Affiliated with Ionis Pharmaceuticals, Inc; no other competing interests to declare

8. Curt Mazur

   Ionis Pharmaceuticals, Inc, Carlsbad, United States

   Contribution

   Resources, Validation, Visualization, Methodology, Writing – review and editing, Contributed to the identification and characterization of lead ASOs

   Competing interests

   Affiliated with Ionis Pharmaceuticals, Inc; no other competing interests to declare

9. Holly Kordasiewicz

   Ionis Pharmaceuticals, Inc, Carlsbad, United States

   Contribution

   Resources, Validation, Visualization, Methodology, Writing – review and editing, Contributed to the identification and characterization of lead ASOs

   Competing interests

   Affiliated with Ionis Pharmaceuticals, Inc; no other competing interests to declare

10. Robert J Shprintzen

    The Virtual Center for Velo-Cardio-Facial-Syndrome, Inc, Manlius, United States

    Contribution

    Resources, Writing – review and editing, Provided patient referrals

    Competing interests

    Affiliated with The Virtual Center for Velo-Cardio-Facial-Syndrome; no other competing interests to declare

    "This ORCID iD identifies the author of this article:" 0000-0001-8940-4973

11. Sander Markx

    1. Stavros Niarchos Foundation Center for Precision Psychiatry and Mental Health, Columbia University, New York, United States
    2. Department of Psychiatry, Vagelos College of Physicians & Surgeons, Columbia University, New York, United States

    Contribution

    Resources, Validation, Investigation, Writing – original draft, Writing – review and editing, Contributed to the generation and initial characterization of human iPSC lines

    Competing interests

    No competing interests declared

12. Bin Xu

    1. Stavros Niarchos Foundation Center for Precision Psychiatry and Mental Health, Columbia University, New York, United States
    2. Department of Psychiatry, Vagelos College of Physicians & Surgeons, Columbia University, New York, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing, Contributed to the generation and initial characterization of human iPSC lines and the design of human neuron-related assays

    Competing interests

    No competing interests declared

13. Joseph A Gogos

    1. Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, United States
    2. Stavros Niarchos Foundation Center for Precision Psychiatry and Mental Health, Columbia University, New York, United States
    3. Department of Psychiatry, Vagelos College of Physicians & Surgeons, Columbia University, New York, United States
    4. Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, United States
    5. Department of Neuroscience, Columbia University, New York, United States

    Contribution

    Conceptualization, Data curation, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing, Designed the study

    For correspondence

    jag90@columbia.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7491-4476

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