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10.1172/jci.insight.200021
1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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1Clinical Neuroscience, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
2Georg-August-University, Göttingen, Germany.
3Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
4Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, City Campus, Göttingen, Germany.
5Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
6Functional Imaging Laboratory, and
7Cognitive Ethology, German Primate Center, Göttingen, Germany.
8Rhythms - Beating Cilia and Ticking Clocks Group, Max Planck Institute for Multidisciplinary Sciences, Fassberg Campus, Göttingen, Germany.
9Anatomy and Cell Biology, Faculty of Medicine & Stem Cell Biology, Research Institute, Medical University-Varna, Varna, Bulgaria.
Address correspondence to: Hannelore Ehrenreich, Experimental Medicine, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, J 5, 68159 Mannheim, Germany. Phone: 49.160.97000331; Email: hannelore.ehrenreich@zi-mannheim.de.
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Published February 23, 2026 - More info
Published in Volume 11, Issue 4 on February 23, 2026
AbstractAmong the known genetic causes of syndromic autism spectrum disorders (ASDs) are transcription factor deficiencies. In this regard, haploinsufficiency of the zinc finger and broad complex, tramtrack, bric and brac domain–containing protein 20 (ZBTB20) leads to a prototypical clinical picture, referred to as Primrose syndrome, comprising severe ASD symptoms together with intellectual disability. Here, we present a comprehensive behavioral and phenotypical characterization of Zbtb20+/– mice, a construct valid model of this thus far untreatable human condition. Zbtb20+/– mice exhibited diminished sociability, reduced vocalization, distinct repetitive behaviors, impaired cognitive flexibility, hyperactivity, and hypoalgesia. Magnetic resonance imaging revealed increased volumes of hippocampus, cerebellum, brain matter, and whole brain, confirmed by postmortem brain weight measurements. Due to our previous observation of enhanced ZBTB20 expression in CA1 pyramidal neurons upon recombinant human erythropoietin (rhEPO) injections, we anticipated a mitigating effect through rhEPO treatment of Zbtb20 deficiency/Primrose syndrome. Indeed, after 3 weeks of alternate-day rhEPO injections, a remarkable improvement in the behavioral phenotype was observed. Our results highlight rhEPO as promising treatment for Primrose syndrome.
IntroductionAutism spectrum disorder (ASD), with a prevalence of approximately 1% across cultures, is characterized by 3 primary behavioral domains: impaired sociability, reduced communication, and repetitive behaviors (1). Additional symptoms are decreased cognitive flexibility (2), hyperactivity (3, 4), and pain hyposensitivity (5, 6). Despite multifactorial causes, including dysfunctions of chromatin and transcription factors, the final common pathway of ASD converges at the synapse (7). A transcription factor gene associated with ASD is the zinc finger and broad complex, tramtrack, bric and brac domain–containing protein 20 (ZBTB20), located at chromosome 3q13.31. The encoded protein is particularly abundant in the hippocampus (8). ZBTB20 contains 5 C2H2 zinc finger domains (ZnFI–ZnFV) and an N-terminal broad complex, tramtrack, bric and brac (BTB) domain, which facilitates interaction with the DNA (9).
ZBTB20 plays a crucial role in glucose metabolism, postnatal growth, neurogenesis (9–11), and the specification of the medial pallium, which forms the hippocampus (8, 11–14). Furthermore, ZBTB20 is instrumental for maturation of hippocampal cornu ammonis 1 (CA1) neurons, the development of dendritic and synaptic structures, olfactory bulb neurogenesis, and the generation of neuronal layers in the developing cortex (15–18).
In humans, a heterozygous pathogenic variant of ZBTB20 causes the extremely rare, autosomal dominant Primrose syndrome (19, 20), which is characterized by macrocephaly with developmental delay, intellectual disability, behavioral abnormalities, typical facial phenotypes, altered glucose metabolism, hypotonia, agenesis of the corpus callosum, hearing loss, ocular anomalies, cryptorchidism, and calcification of the ear cartilage (20–32). Behavioral deviations include attention deficit/hyperactivity disorder (ADHD), self-injurious behavior, sleep disturbances, tics, stereotypies, and ASD (23, 33–35).
Regarding animal models, only a few studies have reported on a limited number of behavioral tests performed with Zbtb20+/– mice. These tests examined visual capability, hippocampus-dependent learning and memory, anxiety, exploration, nociception, and circadian rhythm (12, 15, 36–38). Thus far, however, a comprehensive behavioral characterization or any kind of treatment approach are completely lacking.
The glycoprotein erythropoietin (EPO), named after its initially discovered effects on the hematopoietic system, has over the last decades attracted attention due to its neuroprotective, neuroregenerative, and cognition-enhancing properties. Moreover, EPO was just found to be a potent driver of neurodifferentiation (39). Recently, we detected an increase in ZBTB20 expression within the hippocampal CA1 region in mice following recombinant human EPO (rhEPO) treatment (40).
We thus hypothesized that rhEPO application might augment the residual expression of ZBTB20 in heterozygous mice, and thereby mitigate the overall phenotype. To test this hypothesis, we conducted an in-depth behavioral and phenotypical characterization of Zbtb20+/– mice following 3 weeks of rhEPO application. The results revealed multifaceted improvements, suggesting rhEPO as promising therapeutic approach for Primrose syndrome.
ResultsSynopsis
This study was designed with the objective of performing an exhaustive behavioral characterization of Zbtb20+/– mice, a construct-valid model of the thus far untreatable human Primrose syndrome. Based on our previous observations of EPO specifically targeting ZBTB20 expression, we incorporated a 3-week treatment trial using rhEPO. The findings unveiled impairments across all domains that are typically associated with ASD and intellectual disability. Importantly, improvements in several pathological parameters were observed following rhEPO administration (Figures 1–6, Supplemental Figures 1–3, and Supplemental Tables 1–4; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.200021DS1), thus potentially offering a promising therapeutic approach to Primrose syndrome.
Figure 1Histology. (A and B) Representative images of ZBTB20 expression (green) in the hippocampal region of Zbtb20+/+ (WT) and Zbtb20+/– (Het) mice. A1–A3 and B1–B3: higher magnifications of the outlined boxes in A and B, representing CA1, CA3, and DG areas. Scale bars: 100 μm for A and B; 20 μm for higher magnifications. (C) CA1 pyramidal layer of Zbtb20+/– (Het) including higher magnification reveals no double-labeling of Zbtb20 with PSD95.
Immunohistochemistry illustrates ZBTB20 expression in the hippocampus of Zbtb20+/+ versus Zbtb20+/– mice. Immunohistochemical images of ZBTB20 expression in the hippocampal regions CA1, CA3, and dentate gyrus (DG) of Zbtb20+/+ (WT) and Zbtb20+/– mice document the expectedly reduced expression/green signal in Zbtb20+/– mice (Figure 1, A and B). Interestingly, but still unclear in nature, are the small green puncta distributed all over the hippocampus exclusively of Zbtb20+/– mice. Exploratory costaining for PSD95 excluded a simple colocalization of these puncta with synaptic structures (Figure 1C). Furthermore, they were mainly located outside the nucleus and were not associated with astrocytic (GFAP) nor with neuronal processes (MAP2, data not shown).
Hematopoietic cell composition in lymphoid organs is largely unaffected in Zbtb20+/– mice. While ZBTB20 has been most extensively studied in the central nervous system, accumulating evidence, derived from cell-type-specific Zbtb20-knockout mice, highlights important roles for this transcription factor in several immunological processes (41–44). To assess whether ZBTB20 haploinsufficiency impacts the hematopoietic cell composition, we performed high-parameter flow cytometric immunophenotyping to quantify 6 major immune cell types, namely B cells, CD4+ and CD8+ T cells, NK cells, monocytes, and granulocytes, as well as 19 functionally distinct immune cell subsets across several lymphoid compartments, including blood, bone marrow, lymph nodes, and spleen (Supplemental Figure 3A and Supplemental Table 4). Qualitative comparison of immune cell clusters expectedly revealed major differences between the immune cell compartments but no gross abnormalities in Zbtb20+/– versus WT mice (Supplemental Figure 3B). Subsequent quantitative permutational multivariate analysis of variance (PERMANOVA) comparison of the immune cell compositions within each lymphoid compartment confirmed the absence of major changes in the immune cell composition of Zbtb20+/– mice (all P > 0.05, Supplemental Figure 3C). Detailed analysis of individual immune cell populations revealed a subtle but significant increase in CD4+ T cells in lymph nodes of Zbtb20+/– mice (FDR = 0.023, fold change = 1.1) as well as an increase in splenic NK cells (FDR = 0.0275, fold change = 1.2). No significant differences between Zbtb20+/– and WT mice were observed for distinct subsets of NK cells, T cells, B cells, and monocytes (all FDR > 0.05, Supplemental Table 4).
Zbtb20+/– males display impairments in all primary domains of ASD-like behavior. An in-depth behavioral characterization of Zbtb20+/– males (Figure 2, A and B) revealed impairments across all primary domains of an ASD phenotype. The compromised social interaction became evident in the 3-chamber sociability test. Here, placebo-treated Zbtb20+/– males failed to differentiate between the chamber housing a stimulus mouse and the opposite chamber containing an empty cage (P = 0.1261). In contrast, both placebo- and rhEPO-treated WT and rhEPO-treated Zbtb20+/– demonstrated a preference for the chamber of the stimulus mouse over the empty chamber (P = 0.0008, P < 0.0001, P = 0.0080; Figure 2C and Supplemental Table 1).
Figure 2Experimental outline and Zbtb20+/– impairments in ASD domains. (A and B) Graphical experimental outlines for main (A) and additional (B) cohort of male Zbtb20+/– mice and their Zbtb20+/+ littermates, used as control. After 11 consecutive placebo resp. rhEPO injections, behavior experiments were performed. PND, postnatal day; DPI, days post injection. (C–F) Assessment of social characteristics shows impaired social preference in the 3-chamber sociability test (C), since placebo-treated Zbtb20+/– mice did not distinguish between stimulus mouse (S) and empty cage (E). This impairment is rescued by rhEPO treatment (t test, mean ± SEM). Communication assessed with ultrasound vocalization (D and E) shows that Zbtb20+/– mice exhibit an increased latency to first call (D) with improvement after rhEPO treatment and a reduced number of calls (E) compared with controls (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). Nest building (F) results in a reduced score in Zbtb20+/– mice regarding quality of constructed nests overnight (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). (G and H) Repetitive behavior was assessed in home cages via LABORAS and shows increased duration (G) and events (H) of climbing in Zbtb20+/– mice (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). (I) Hot plate test reveals hyposensitivity toward heat-mediated nociception in Zbtb20+/– mice, with an increased latency of the first reaction to heat perception (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). (J–M) Spatial learning, memory, as well as reversal learning and memory were assessed in a Morris water maze. Hidden training over 8 days shows time and genotype effect in escape latency (J, 3-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). Probe trial for hidden learning task (K) reveals impaired spatial memory in Zbtb20+/– mice compared with controls, shown by reduced time spent in target quadrant (TQ, 2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). Reversal hidden training over 4 days (L) displays impaired spatial reversal learning in placebo-treated Zbtb20+/– mice and a rescue effect after rhEPO treatment, indicated by escape latency to platform (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). Reversal probe trial (M) demonstrates impaired reversal spatial memory in Zbtb20+/– mice, indicated by the time spent in target quadrant (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). (N–Q) Hyperactivity of Zbtb20+/– mice was assessed in probe trials for both hidden (N) and reversal (O) tasks of the Morris water maze, neophobia test (P) (all 2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM), and 4-hour-complex wheel running (Q, 3-way ANOVA with Bonferroni’s post hoc test, mean ± SEM).
Communication deficits were discernible in the vocalization test. Both placebo- and rhEPO-treated Zbtb20+/– males exhibited an increased latency to their first vocalization upon exposure to an anesthetized female stimulus mouse (genotype effect: P = 0.0106). Interestingly, rhEPO treatment of both WT and Zbtb20+/– mice reduced the latency to their first vocalization (treatment effect: P = 0.0295). Furthermore, both Zbtb20+/– groups emitted significantly fewer calls toward the stimulus mice throughout the 3-minute recording, compared with their respective controls (genotype effect: P < 0.0001, Figure 2, D and E, and Supplemental Table 1). In nest building, another social skill test, Zbtb20+/– males constructed nests of inferior quality compared with WT controls and rhEPO had no effect (genotype effect: P = 0.0279, Figure 2F and Supplemental Table 1).
This result was also mirrored in their female littermates, where both Zbtb20+/– groups scored lower in nest building compared with their respective WT controls (genotype effect: P < 0.0001, Supplemental Figure 1A and Supplemental Table 2). Intriguingly, the diminished quality of nests constructed by both Zbtb20+/– sexes was already obvious in their home cages, an observation that experimenters became aware of only after being unblinded at project conclusion.
Assessment by the Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS) revealed enhanced repetitive behavior, with Zbtb20+/– males displaying increased duration of climbing and number of climbing events (genotype effects: P = 0.0099 and P = 0.0008, respectively; Figure 2, G and H, and Supplemental Table 1) compared with WT controls. In females, the result was similar (genotype effects: P = 0.0145 and P = 0.0002, respectively; Supplemental Figure 1, B and C, and Supplemental Table 2).
Additional abnormalities of Zbtb20+/– males linked to ASD-like behavior. In the hot-plate test, an augmented tolerance for heat-mediated pain became aware. Both placebo- and rhEPO-treated Zbtb20+/– males exhibited a heightened latency to their initial visible reaction toward the stimulus (genotype effect: P = 0.0002, Figure 2I and Supplemental Table 1). In females, placebo-treated Zbtb20+/– mice also demonstrated an increased latency compared with their WT control (genotype effect: P = 0.0003). However, unlike in males, female rhEPO-treated Zbtb20+/– mice displayed a latency to the first reaction that was on par with WT controls and therefore lower than placebo-treated Zbtb20+/– females (treatment effect: P = 0.0093, Supplemental Figure 1D and Supplemental Table 2).
The Morris water maze was employed to evaluate spatial learning and memory. Zbtb20+/– males exhibited visual abilities equivalent to their WT controls (Supplemental Table 1). A genotype effect was observed during 8 days of hidden training (P = 0.0387, Figure 2J). During both hidden and reversal probe trials, Zbtb20+/– males spent significantly less time in the target quadrant that previously held the escape platform (both genotype effects: P < 0.0001, Figure 2, K–M, and Supplemental Table 1). Between both probe trials, 4 days of reversal training were conducted, revealing that placebo-treated Zbtb20+/– males were the only group not showing significant improvement in escape latency over time (P = 0.5629). In contrast, both placebo- and rhEPO-treated WT males (P = 0.0476, P = 0.0265) and rhEPO-treated Zbtb20+/– males improved significantly (P = 0.0339, Figure 2L and Supplemental Table 1). In females, a phenotype was only observable during both probe trials of hidden and reversal task where Zbtb20+/– mice spent less time in the target quadrant compared with controls, independent of treatment (genotype effects: P = 0.0153 and P = 0.0040, respectively; Supplemental Figure 1, F–H, and Supplemental Table 2). Both male and female Zbtb20+/– mice displayed increased locomotion across a variety of tests. In males, hidden and reversal probe trials (genotype effects: P < 0.0001), the neophobia test (genotype effect: P = 0.0001), and 4-hour complex wheel running (genotype effect: P = 0.0007, Figure 2, N–Q, and Supplemental Table 1) revealed significantly increased track lengths. In females, Zbtb20+/– mice demonstrated increased locomotion in the open field test (genotype effect: P = 0.0006), both hidden and reversal probe trials (genotype effects: P = 0.0009 and P = 0.0004, respectively), and in the LABORAS test (genotype effect: P = 0.0076, Supplemental Figure 1, L–O, and Supplemental Table 2).
Zbtb20+/– phenotypes in working memory, sensorimotor gating, and motivation. The Y-maze test was employed to assess working memory by contrasting the frequency of alternating and non-alternating entries into the maze arms. Despite all 4 male groups performing significantly more alternating than non-alternating entries, a comparison of the deltas from these 2 readouts revealed that placebo-treated Zbtb20+/– mice had the lowest delta of all groups, significantly lower than rhEPO-treated Zbtb20+/– males, thus demonstrating a treatment effect (P = 0.0314, Figure 3, A and B, and Supplemental Table 1). The open field and hole board tests were utilized to assess exploratory behavior and the motivation to engage in such. Zbtb20+/– males spent more time within the periphery and less time within the intermediate zone in the open field compared with their WT controls (genotype effects: P = 0.0085 and P = 0.0040, respectively; Figure 3C and Supplemental Table 1). The hole board experiment revealed a reduction in revisits to the most recently visited hole in placebo-treated Zbtb20+/– males compared with their WT controls (genotype effect: P = 0.0148). Additionally, an increase toward control levels was observed in rhEPO-treated Zbtb20+/– males (treatment effect: P = 0.0139, Figure 3D and Supplemental Table 1). A similar result was obtained in the female groups, where placebo-treated Zbtb20+/– mice showed fewer revisits compared with WT control, and rhEPO-treated Zbtb20+/– females exhibited a tendency of increasing the number of revisits to control level (genotype effect: P = 0.0982, treatment effect: P = 0.0526, Supplemental Figure 1I and Supplemental Table 2).
Figure 3Working memory, exploration, pre-pulse inhibition, obsessive-compulsive readouts, and related control testing in Zbtb20+/– mice. (A and B) Working memory was assessed in the Y-maze test and shows significantly more alternating (a) than non-alternating (na) entries in all 4 groups (A, t test, mean ± SEM). Comparing Δ of entries (B) reveals further improved preference of alternating entries in rhEPO-treated Zbtb20+/– mice compared with placebo-treated controls (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). (C) Placebo-treated Zbtb20+/– mice show increased time spent in peripheral zone and reduced time spent in intermediate zone in the open field test (2-way ANOVA with Bonferroni’s post hoc test, mean ± SEM). (D)
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