Astrocytes contribute to olanzapine-mediated reversal of kleefstra syndrome-associated neurodevelopmental regression
Karlijn Vermeulen-Kalk, Shan Wang, Joost Kummeling, Britt Mossink, Kim Wijnant, Carlos Gonzales Jiménez, Zoe Frazier, Brian Rozumny, Anne O’Donnell-Luria, Ellen Hanson, Monica Frega, Katrin Linda, Moritz Negwer, Bas Lendemeijer, Astrid Oudakker, Monica Pop-Purceleanu, Joost Janzing, Linde van Dongen, Femke M.S. de Vrij, Steven A. Kushner, Ilse van der Werf, Chantal Schoenmaker, Wouter Oomens, Siddharth Srivastava, Jos I.M. Egger, Hans van Bokhoven, Dirk Schubert, Nael Nadif Kasri, Tjitske Kleefstra
Karlijn Vermeulen-Kalk, Shan Wang, Joost Kummeling, Britt Mossink, Kim Wijnant, Carlos Gonzales Jiménez, Zoe Frazier, Brian Rozumny, Anne O’Donnell-Luria, Ellen Hanson, Monica Frega, Katrin Linda, Moritz Negwer, Bas Lendemeijer, Astrid Oudakker, Monica Pop-Purceleanu, Joost Janzing, Linde van Dongen, Femke M.S. de Vrij, Steven A. Kushner, Ilse van der Werf, Chantal Schoenmaker, Wouter Oomens, Siddharth Srivastava, Jos I.M. Egger, Hans van Bokhoven, Dirk Schubert, Nael Nadif Kasri, Tjitske Kleefstra
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Research Article Development Neuroscience

Astrocytes contribute to olanzapine-mediated reversal of kleefstra syndrome-associated neurodevelopmental regression

Abstract

Kleefstra syndrome (KLEFS1) results from EHMT1 haploinsufficiency and is characterized by variable neurodevelopmental delays and psychopathology. Developmental regression, marked by the sudden loss of previously acquired daily life skills during late puberty or early adulthood, has emerged as a severe complication in individuals with KLEFS1. To investigate the clinical and molecular mechanisms underlying developmental regression and assess the therapeutic potential of olanzapine, we conducted a sequential study in an international cohort of 54 individuals with KLEFS1. Among 16 individuals treated with olanzapine, 10 exhibited a beneficial response based upon improvement of their adaptive functioning, and 4 showed temporary improvement. These clinical findings informed preclinical studies using human induced pluripotent stem cell-derived and ex-vivo cortical slices from a mouse model of KLEFS1. We identified hyperactivity in EHMT1+/– neuronal networks cocultured with EHMT1+/– astrocytes, a dysfunction reversible by olanzapine. Mechanistically, EHMT1+/– astrocytes displayed elevated levels of S100B, a neuroinflammatory marker contributing to neuronal network hyperactivity. Notably, olanzapine treatment reduced S100B levels, and pharmacological inhibition or genetic knockdown of S100B in EHMT1+/– astrocytes was sufficient to rescue the neuronal hyperactivity phenotype. These findings underscore a critical role for astrocytes in KLEFS1 pathophysiology and identify a potential cellular target for olanzapine in mitigating developmental regression.

Authors

Karlijn Vermeulen-Kalk, Shan Wang, Joost Kummeling, Britt Mossink, Kim Wijnant, Carlos Gonzales Jiménez, Zoe Frazier, Brian Rozumny, Anne O’Donnell-Luria, Ellen Hanson, Monica Frega, Katrin Linda, Moritz Negwer, Bas Lendemeijer, Astrid Oudakker, Monica Pop-Purceleanu, Joost Janzing, Linde van Dongen, Femke M.S. de Vrij, Steven A. Kushner, Ilse van der Werf, Chantal Schoenmaker, Wouter Oomens, Siddharth Srivastava, Jos I.M. Egger, Hans van Bokhoven, Dirk Schubert, Nael Nadif Kasri, Tjitske Kleefstra

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Figure 2

EHMT1+/– human astrocytes show increased calcium response to glutamate and altered transcriptomic profile.

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EHMT1+/– human astrocytes show increased calcium response to glutamate ...
(A) IPS cells generated through CRIPSR/cas9 used in this study. (B) Western blot showing the EHMT1 protein levels in control and EHMT1+/– astrocytes. (C) Glutamate uptake ability of control and EHMT1+/– astrocytes, n = 4 independent cultures (D) Representative image of astrocytes for calcium imaging measure using Fluo8-AM. (E) Representative traces of control and EHMT1+/– astrocytes showing spontaneous calcium transients. (F) Quantification of AUC of spontaneous calcium transients. n = 139 for control and n = 197 for EHMT1+/–, 3 biological replicates (G) Representative traces of control and EHMT1+/– astrocytes showing evoked activity upon 50 μM glutamate stimulation. Thick black trace represents the average trace. (H) Quantification of AUC of evoked calcium events. n = 21 for control and n = 30 for EHMT1+/-, 3 biological replicates. (I) Principal component analysis (PCA) of RNA-seq data from control (Ctrl) and EHMT1+/– astrocytes, n = 3 independent cultures. (J) Volcano plots showing differentially expressed genes (DEGs) between EHMT1+/– and control human astrocytes. Relative to the control, significantly up- or downregulated genes are shown above the black dashed line in red and blue (FDR < 0.05; log2(FC) > 1; log2(FC) < –1).(K) Heatmap showing relative normalized gene expression across samples. Normalized counts were log2-transformed and scaled per gene to Z-scores, representing relative expression levels. Both genes and samples were hierarchically clustered based on Z-scores. (L) GO term analysis of molecular function. Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired Student’s t test (C, F and H).