Erythropoietin alleviates syndrome-associated intellectual disability and autism-like behavior in Zbtb20-haploinsufficient Primrose syndrome mouse model
Martin Hindermann, Justus B.H. Wilke, Yasmina Curto, Stefan N. Oline, Vinicius Daguano Gastaldi, Umer Javed Butt, Rakshit Dadarwal, Umut Çakır, Anja Ronnenberg, Kurt Hammerschmidt, Susann Boretius, Anastassia Stoykova, Anton B. Tonchev, Klaus-Armin Nave, Manvendra Singh, Hannelore Ehrenreich
Martin Hindermann, Justus B.H. Wilke, Yasmina Curto, Stefan N. Oline, Vinicius Daguano Gastaldi, Umer Javed Butt, Rakshit Dadarwal, Umut Çakır, Anja Ronnenberg, Kurt Hammerschmidt, Susann Boretius, Anastassia Stoykova, Anton B. Tonchev, Klaus-Armin Nave, Manvendra Singh, Hannelore Ehrenreich
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Research Article Clinical Research Neuroscience

Erythropoietin alleviates syndrome-associated intellectual disability and autism-like behavior in Zbtb20-haploinsufficient Primrose syndrome mouse model

Abstract

Among 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.

Authors

Martin Hindermann, Justus B.H. Wilke, Yasmina Curto, Stefan N. Oline, Vinicius Daguano Gastaldi, Umer Javed Butt, Rakshit Dadarwal, Umut Çakır, Anja Ronnenberg, Kurt Hammerschmidt, Susann Boretius, Anastassia Stoykova, Anton B. Tonchev, Klaus-Armin Nave, Manvendra Singh, Hannelore Ehrenreich

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Figure 2

Experimental outline and Zbtb20+/– impairments in ASD domains.

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Experimental outline and Zbtb20+/– impairments in ASD domains. (A and B)...
(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).