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Phenotypic complexities of rare heterozygous neurexin-1 deletions
Open accessPublished version of a preprint
Abstract
Given the large number of genes significantly associated with risk for neuropsychiatric disorders, a critical unanswered question is the extent to which diverse mutations-sometimes affecting the same gene-will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1, which encodes a presynaptic cell-adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain and are differentially affected by unique (non-recurrent) deletions 1. We contrast the cell-type-specific effect of patient-specific mutations in NRXN1 using human-induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Through distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1+/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Reciprocal isogenic manipulations causally demonstrate that aberrant splicing drives these changes in synaptic activity. For NRXN1 deletions, and perhaps more broadly, precision medicine will require stratifying patients based on whether their gene mutations act through LOF or GOF mechanisms, to achieve individualized restoration of NRXN1 isoform repertoires by increasing wild-type and/or ablating mutant isoforms. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disorders, our findings add nuance to future considerations of precision medicine.
Overview
CoChat Summary
TL;DR
Patient-specific heterozygous NRXN1 deletions act through distinct loss-of-function (5′-Del) or gain-of-function (3′-Del) splicing mechanisms, producing opposing synaptic phenotypes in human glutamatergic versus GABAergic neurons. The authors propose that precision medicine for NRXN1 deletions will require stratifying patients by LOF/GOF mechanism rather than by clinical diagnosis alone.
Key findings
- NRXN1+/− 5′-Del cases (≈115-kb deletion affecting the alpha-transcript promoter) showed reduced β→α splicing, consistent with LOF, while 3′-Del cases (≈136-kb deletion impacting SS4/SS5 across exons 21–23) expressed mutant exon 20–24 splice junctions, consistent with GOF.
- Spontaneous activity diverged by cell type: iGLUT neurons from both 5′-Del and 3′-Del showed >40% reduced weighted mean firing rate at WPI4 and WPI6, whereas immature iGABA neurons showed ~2-fold increased activity at ~WPI2 and mature iGABA neurons showed decreased activity at WPI6.
- Synaptic transmission was decreased in iGLUT neurons (increased sEPSC inter-event intervals for 5′-Del and 3′-Del) but enhanced in iGABA neurons (decreased sIPSC inter-event intervals in 3′-Del), with corresponding bidirectional changes in synapse number.
- Isogenic shRNA knockdown of wildtype NRXN1α recapitulated 5′-Del-like phenotypes, and knockdown of the mutant 20/24 splice junction reversed 3′-Del-like phenotypes; β-estradiol increased NRXN1α expression (p = 0.0297) and improved 5′-Del iGLUT activity/sEPSC phenotypes, while an ASO reduced mutant isoforms by ~55% (p < 0.001).
Methods
- Human induced pluripotent stem cells from patient-specific NRXN1+/− 5′-Del, 3′-Del, and healthy control donors were lineage-converted into iGLUT and iGABA neurons, and dorsal/ventral forebrain organoids were generated for scRNA-seq.
- Functional assays included multi-electrode array recordings, whole-cell patch-clamp electrophysiology for spontaneous and miniature synaptic currents, immunostaining for synaptic puncta, bulk RNA-seq, LeafCutter differential splicing analysis, and single-cell RNA-seq.
- Causal isogenic manipulations used shRNAs against wildtype exon 9 or the mutant exon 20/24 junction; therapeutic proof-of-principle experiments used β-estradiol and an antisense oligonucleotide targeting the mutant splice junction.
Limitations
- The paper states that the iGLUT and iGABA populations studied are immature and comprise diverse neuronal subtypes, so discrete effects on subtypes such as SST versus PV GABA neurons remain unresolved.
- The paper states that future studies in more physiologically relevant, circuit-like models may uncover non-autonomous and activity-dependent phenotypes, and that biochemical/proteomic interactions of NRXN1 remain to be probed, for example with proximity-labelling methods such as BioID.
- The paper states that it did not resolve whether mutant GOF isoforms shift alternative-splicing stoichiometry against wildtype isoforms or alter trans-synaptic protein–protein interactions; it also notes that the therapeutic findings are proof-of-principle in vitro results requiring in vivo and clinical validation.
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![Figure 6: Precise therapeutic targeting of stratified GOF- and LOF-NRXN1+/− in iGLUT neurons. (a) Model and proposed mechanism of rescue for LOF patients ameliorating loss of wildtype isoforms. (b) RT-qPCR of WT NRXN1 gene expression in 5-Del patients, post-acute treatment (3–5 days) (n: DMSO = 19/2; β-estradiol = 18/2 | 3 batches) compared via Student’s t-test t=2.293, df=35, p = 0.028. (c) Quantification of iGLUT neuronal activity at WPI3 across vehicle and treatment (n: DMSO = 72/3; β-estradiol = 74/3 | 3 batches) compared via Student’s t-test (t=2.804, df=144, p = 0.0057). (d) Splicing of β→α cluster and (e) volcano plot of DEGs in β-estradiol treated 5’-Del iGLUT neurons, compared to vehicle treated 5’-Del iGLUT neurons. (f) Representative patch-clamp traces and (h) cumulative probabilities of sEPSC IEI distributions, with insets of cell-averaged, IEI and amplitude measures (n: Control + DMSO = 18/1; 5’-Del + DMSO = 33/2 5’-Del + β-estradiol = 35/2). Curves were compared via Levene’s test with Bonferroni corrections (Control + DMSO v. 5’-Del + DMSO F = 13.151; df = 1, p = 8.763E-4; 5’Del + DMSO v 5’-Del + β-estradiol F = 14.359; df = 1, p = 4.62E-04; Control + DMSO v 5’-Del + β-estradiol, F = 0.0141; df = 1, p = 0.955). Insets were compared via Student’s t-test (IEI t=1.161, df=66, p = 0.2499). (h) Model and proposed mechanism of rescue for GOF patients expressing mutant isoforms. (i) Schematic of a NRXN1 ASO matrix, with the selective sequence targeting 20/24 splice junction. (j) RT-qPCR of MT NRXN1 gene expression in 3’-Del patients’ post-acute treatment for 72hrs (ASO-NT = 8/2; ASO-MT = 10/2 | batches), compared via Student’s t-test t=12.91, df=16 p < 0.0001. (k) Splicing of MT Exon 20→24 cluster and (l) volcano plot of DEGs in ASO-MT treated 3’-Del iGLUT neurons, compared to ASO-NT treated 3’-Del iGLUT neurons.(m) Biological process and (n) cellular compartment GO terms demonstrating enrichment (−log10[adj p value]) of selected synapse related pathways. Data represented as mean ± sem. n reported as samples/donors | independent batches.](/paper-figures/0d50ac9fba814e5cad17634121350dae/phenotypic-complexities-of-rare-heterozygous-neurexin-1-deletions-figure-6.jpg)
Verification
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In the paper
“Risk enrichments for schizophrenia, bipolar disorder, and autism spectrum disorder30,31 were most enriched in 5’-Del iGLUT neurons and 3’-Del iGABA neurons (Extended Data Fig. 2).”
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > NRXN1+/− mutations broadly impact synaptic and neurodevelopmental pathways in induced and organoid-derived glutamatergic and GABAergic neurons
What the sources say
- Check pendingcites [30] Paz, I., Kosti, I., Ares, M., Cline, M. & Mandel-Gutfreund, Y. RBPmap: a web server for mapping binding sites of RNA-binding proteins. Nucleic Acids Res. 42, W361–W367 (2014). source
- Check pendingcites [31] Ghanbarian, H., Aghamiri, S., Eftekhary, M., Wagner, N. & Wagner, K.-D. Small activating RNAs: towards the development of new therapeutic agents and clinical treatments. Cells 10, 591 (2021). source
plainstates an association- Asserted: passes — The authors put it forward as true.
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In the paper
“To stratify LOF and GOF genotypes, we confirmed exclusive MT NRXN1 isoforms in 3’-Del organoids (Extended Data Fig. 3d–e, k–l).”
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > NRXN1+/− mutations broadly impact synaptic and neurodevelopmental pathways in induced and organoid-derived glutamatergic and GABAergic neurons
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“In 5’Del iGLUT neurons, we also detected differential splicing at SS4 inclusion (ΔPSI −0.046, p = 0.006, Bonferroni corrected) (Fig. 1f,k).”
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > Patient-specific NRXN1+/− mutations produces differential NRXN1 splicing patterns across glutamatergic and GABAergic neurons
plainstates an associationSingle case- Asserted: passes — The authors put it forward as true.
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In the paper
“Differential splicing analysis between iGLUT and iGABA neurons produced a single significant cluster (out of nine identified), centered around SS3 (Extended Data Fig. 1a, p = 0.01960, Bonferroni corrected)”
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > Patient-specific NRXN1+/− mutations produces differential NRXN1 splicing patterns across glutamatergic and GABAergic neurons
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Splicing of NRXN1 transcript was perturbed at two locations in 5’-Del neurons.”
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > Patient-specific NRXN1+/− mutations produces differential NRXN1 splicing patterns across glutamatergic and GABAergic neurons
plainstates a causeSingle case- Asserted: passes — The authors put it forward as true.
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In the paper
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > Patient-specific alterations in spontaneous neural activity occur with minimal changes in passive and excitable membrane properties.
“We observed that immature NRXN1+/− iGABA neurons exhibited a robust ~2-fold increase in population-wide wMFR activity (~WPI2)”
§ Phenotypic complexities of rare heterozygous neurexin-1 deletions > RESULTS > Patient-specific alterations in spontaneous neural activity occur with minimal changes in passive and excitable membrane properties.
“Indeed, immature iGABA neurons expressed higher (10-fold increase compared to WPI2 neurons) levels of SLC12A5 (the gene encoding KCC2)”
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Metadata
- Authors
- Fernando, M. B., Fan, Y., Zhang, Y., Tokolyi, A., Murphy, A. N., Kammourh, S., Deans, P. J. M., Ghorbani, S., Onatzevitch, R., Pero, A., Padilla, C., Williams, S. E., Flaherty, E. K., Prytkova, I. A., Cao, L., Knowles, D. A., Fang, G., Slesinger, P. A., & Brennand, K. J.
- Affiliations
- Yale University (US)
- Icahn School of Medicine at Mount Sinai (US)
- New York Genome Center (US)
- Haukeland University Hospital (NO)
- Corresponding author
- Fang, Gang
- Slesinger, Paul A.
- Brennand, Kristen J.
- ORCID
- Venue
- Nature
- Published
- 2025-04-09
- Volume
- 642
- Issue
- 8068
- Pages
- 710-720
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC10634884
- OpenAlex
- W4409283565
- Cited by
- 30
- References
- 80
- Citations by year
- 2026: 16 · 2025: 11
- FWCI
- 10.54
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Nature (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (submittedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- Funders
- National Science Foundation (2146398)
- Icahn School of Medicine at Mount Sinai
- Johns Hopkins University
- National Institute on Alcohol Abuse and Alcoholism (F31AA027949)
- National Institute of Mental Health (R01MH121074, R01MH123155, RM1MH132648, R01MH125579)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomic variations and chromosomal abnormalities
- Keywords
- NRXN1, copy number variation, alternative splicing, neuropsychiatric disorders, loss-of-function mutations, gain-of-function mutations, human induced pluripotent stem cells, glutamatergic neurons, GABAergic neurons, synaptic activity, cell type-specific effects, precision medicine
- MeSH terms
- Loss of Function Mutation, Animals, Calcium-Binding Proteins, Female, Heterozygote, Humans, Male, Phenotype, Synapses, Cell Adhesion Molecules, Neuronal, Gene Deletion, Sequence Deletion, Alternative Splicing, Neural Cell Adhesion Molecules, Mice, DNA Copy Number Variations, Induced Pluripotent Stem Cells, GABAergic Neurons
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- The paper
- Fernando, M. B., Fan, Y., Zhang, Y., Tokolyi, A., Murphy, A. N., Kammourh, S., Deans, P. J. M., Ghorbani, S., Onatzevitch, R., Pero, A., Padilla, C., Williams, S. E., Flaherty, E. K., Prytkova, I. A., Cao, L., Knowles, D. A., Fang, G., Slesinger, P. A., & Brennand, K. J. (2025). Phenotypic complexities of rare heterozygous neurexin-1 deletions. Nature. https://doi.org/10.1038/s41586-025-08864-9
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