Pathological disruption of CELF2 shuttling causes neuronal hyperactivity, learning deficits, and seizures
Michelle Hua, et al.
Michelle Hua, et al.
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Research Article Clinical Research Development Genetics

Pathological disruption of CELF2 shuttling causes neuronal hyperactivity, learning deficits, and seizures

Abstract

De novo heterozygous variants in CUGBP Elav-like family member 2 (CELF2) have recently been associated with a rare neurodevelopmental disorder, yet the mechanisms linking specific variants to distinct clinical phenotypes remain poorly understood. Here, we reported a cohort of 18 individuals and provided evidence that variants causing CELF2 mislocalization, but not protein-null variants, were associated with seizures. Using proband-derived human cortical neurons and transgenic mouse models, we demonstrated that CELF2 underwent activity-dependent nucleocytoplasmic shuttling in excitatory neurons and that its cytoplasmic retention caused neuronal hyperactivity, elevated seizure susceptibility, and learning and memory deficits. We further found that cytoplasmic CELF2 regulated mRNAs critical for synaptic function and neuronal excitability and implicated in epileptic seizures and intellectual disability. Drug screening further identified AKT signaling as a key regulator of CELF2 nucleocytoplasmic shuttling and a candidate target for reversing neuronal hyperactivity. Together, our findings expand the clinical and genetic spectrum of CELF2-related neurodevelopmental disorders and establish a variant-specific mechanism that links CELF2 mislocalization to neuronal hyperactivity, seizures, and cognitive impairment.

Authors

Michelle Hua, Mohamad-Reza Aghanoori, Melissa J. MacPherson, Yi Ren, Shehani V. Siripala, Yifan Yang, Yvonne Yan Yan Or, Malea Nguyen, Robert Duba-Kiss, Daniel Feng, Laura Williams, Christopher J. Gafuik, GengYi Wang, Chloe Quelin, Boris Keren, Sarah Schuhmann, Georgia Vasileiou, Alexia Bourgois, Antonio Vitobello, Christophe Philippe, Zornitza Stark, Richard J. Leventer, George McGillivray, Frederic Tran Mau-Them, Marine Tessarech, Clément Prouteau, Phillis Lakeman, Mahdi M. Motazacker, Donald R. Latner, Raymond C. Caylor, Yvette van Ierland, Eloise Prijoles, Angie Lichty, Evangelos Theodorou, David A. Sweetser, Edward Steel, Jan Cobben, Majed J. Dasouki, Daniel G. Calame, Bertrand Isidor, Benjamin Cogné, Mitchell Kesler, Brooke Rackel, Isabel Clark, Deborah M. Kurrasch, G. Campbell Teskey, James Ellis, Guiqiong He, Scott D. Ryan, Douglas J. Mahoney, A. Micheil Innes, Jonathan R. Epp, Guang Yang

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Figure 2

The p.Arg493His variant causes CELF2 mislocalization and neuronal hyperactivity.

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The p.Arg493His variant causes CELF2 mislocalization and neuronal hypera...
(A) Confocal images of control (Ctrl), p.R493H, and isogenic control (Iso. ctrl) hiPSCs, immunostained for CELF2 (red) and SOX2 (green). (B) Quantifications of CELF2 distribution, from A. n = 12 (600 cells each). (C) WB images and (D) quantifications of CELF2 levels in total lysates, cytoplasmic (Cyto.), and nuclear (Nuc.) fractions in hiPSCs. (E) Schematic and confocal images showing hiPSC induction into hNPCs for 7 days followed by differentiation into hCNs for 29 days, immunostained for indicated markers. diff., differentiation; ind., induction; mat., maturation. (F) Confocal images and (G) quantifications of βIII+ (green) and c-FOS+ (red) hCNs. n = 5 (2,000 cells each). (H–L) MEA of hCNs showing Raster plots of spike distribution on day 29 before and after electrical stimulations (H), weighted mean firing rate (I), and single-electrode burst frequency (J) over time and network burst frequency (K) and AUCC poststimulation (L). n = 17–24. ***P < 0.001. (M) Sanger sequencing of Celf2KI/+ mice. (N) WB images and (O) quantifications of CELF2 levels in cortical lysates from E17.5 embryos. GAPDH serves as a loading control. (P) Confocal images of E17.5 cortex, immunostained for CELF2 (green) and SATB2 (red). Arrows highlight CELF2 signals in the cytoplasm. (Q) Confocal images and (R) quantification of MAP2+ (green) and c-FOS+ (red, arrows) neurons in mouse cortical cultures. Nuclei were counterstained with Hoechst 33258 (blue, A, F, P, and Q) and are outlined with dashed white lines in (A and P) with “N” denoting the nucleus. Means ± SEM. Each dot represents 1 experiment. One-way ANOVA, Tukey’s test (B and D); 2-way ANOVA, Šídák’s multiple comparisons test (I and J); Mann-Whitney U test (K and L); 1-sample 2-tailed t test (O); unpaired 2-tailed t test (R). Scale bars: 2.5 μm (P), 10 μm (A and E), 50 μm (F and Q).