Pathological disruption of CELF2 shuttling causes neuronal hyperactivity, learning deficits, and seizures
Michelle Hua, et al.
Michelle Hua, et al.
View: Text | PDF
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

×

Figure 4

CELF2 shows dynamic subcellular localization in response to neuronal activity in mice.

Options: View larger image (or click on image) Download as PowerPoint
CELF2 shows dynamic subcellular localization in response to neuronal act...
(A) Confocal images of cortical and hippocampal regions from P45 mouse brain sections, immunostained for CELF2 (green). (B) Confocal images showing CELF2 expression (green) in SATB2+ (red) cortical neurons from P0, P7, and P14. (C) Quantifications of CELF2 nucleocytoplasmic distribution in cortical neurons at the indicated developmental stages. P < 0.001 except for P6 (P = 0.03) and P21 (*P = 0.01). Mann-Whitney U test with Bonferroni-Holm correction, E16 as reference. n ≥ 30 cells each. Box plots show the interquartile range (IQR), median (line), and 1.5× IQR (whiskers). (D) WB images of total lysates, cytoplasmic (Cyto.), and nuclear (Nuc.) fractions from P7 cortical tissues, probed for GAPDH (cytoplasmic marker) and LMNB1 (nuclear marker). (E) WB images and (F) quantifications of CELF2 levels in cytoplasmic and nuclear fractions from P0 and P7 cortical tissues. (G) Confocal images and (H) quantifications of CELF2 (green) distribution in cortical neurons cultured for 2 or 13 DIV. n = 3 (≥30 cells each). (I) Confocal images and (J) quantification of CELF2 (green) distribution in MAP2+ (red) 13 DIV neurons treated with or without TTX for 48 hours. Magnified views of the white boxed area are shown below. n = 3 (≥35 cells each). (K) Confocal images and (L) quantification of CELF2 distribution in hippocampal CA1 regions ipsilateral or contralateral to AAV injection sites, with or without blue light stimulation. Sections were immunostained for EGFP-ChR2 (green) and c-FOS or CELF2 (both red). Magnified boxed areas are shown to the right. n = 3 (≥25 cells each). Means ± SEM. Each dot represents 1 sample. Hoechst-stained nuclei (“N,” blue, A, B, G, I, and K) are outlined with dashed white lines. Arrows and arrowheads (A, G, I, and K) highlight predominant cytoplasmic and nuclear CELF2, respectively. Unpaired 2-tailed t test (F, H, J, and L). Scale bars: 5 μm (A and G), 10 μm (B and K), 25 μm (I).