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 5

Pharmacological inhibition and genetic knockdown of S100B in EHMT1+/– astrocytes rescue the neuronal network hyperactivity in EHMT1+/– astrocyte-neuron cocultures.

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Pharmacological inhibition and genetic knockdown of S100B in EHMT1+/– as...
(A) Representative traces and (B) quantification of EHMT1+/– astrocytes with ONO2506 showing reduced evoked activity upon 50 μM glutamate stimulation. n = 57 for EHMT1+/– and n = 63 for EHMT1+/– with ONO2506. (C) Representative raster plots and (D) quantification of mean firing rate exhibited by EHMT1+/– astrocytes cocultured with EHMT1+/– neurons on MEAs at DIV21 after 24-hour treatment of 100 μM ONO2506. n = 12. (E) EHMT1+/– astrocytes infected lentivirus expressing mcherry and scrambled or S100B shRNA. (F) Relative expression of mRNA S100B in EHMT1+/– astrocytes infected with S100B shRNA compared with astrocytes transfected with scrambled shRNA. n = 6 for each group. (G) Representative traces and (H) quantification of calcium wave from EHMT1+/– astrocytes transfected with scrambled shRNA and S100B shRNA upon 50 μM glutamate stimulation. n = 29 for EHMT1+/– + scrambled shRNA and n = 21 for EHMT1+/– + S100B shRNA. (I) Representative raster plots and (J) quantification of mean firing rate exhibited by EHMT1+/– neurons cocultured with EHMT1+/– astrocytes transfected with scrambled shRNA or S100B shRNA on MEAs. Dashed line represents mean of untreated control cells, n = 6 for scrambled shRNA. n = 15 for S100B shRNA. Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired Student’s t test (B, F, H, and J) and paired Student’s t test (D).