RESUMO
Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.
Assuntos
Alelos , Inativação Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Inibidores da Topoisomerase/farmacologia , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/tratamento farmacológico , Síndrome de Angelman/genética , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Pai , Feminino , Impressão Genômica/efeitos dos fármacos , Impressão Genômica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mães , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores da Topoisomerase/administração & dosagem , Inibidores da Topoisomerase/análise , Inibidores da Topoisomerase/farmacocinética , Topotecan/administração & dosagem , Topotecan/farmacocinética , Topotecan/farmacologia , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
BACKGROUND: Prefrontal deficits in gamma-aminobutyric acid (GABA)ergic gene expression, including neuropeptide Y (NPY), somatostatin (SST), and parvalbumin (PV) messenger RNAs (mRNAs), have been reported for multiple schizophrenia cohorts. Preclinical models suggest that a subset of these GABAergic markers (NPY/SST) is regulated by brain-derived neurotrophic factor (BDNF), which in turn is under the inhibitory influence of small noncoding RNAs. However, it remains unclear if these mechanisms are important determinants for dysregulated NPY and SST expression in prefrontal cortex (PFC) of subjects with schizophrenia. METHODS: Using a postmortem case-control design, the association between BDNF protein, NPY/SST/PV mRNAs, and two BDNF-regulating microRNAs (miR-195 and miR-30a-5p) was determined in samples from the PFC of 20 schizophrenia and 20 control subjects. Complementary studies were conducted in cerebral cortex of mice subjected to antipsychotic treatment or a brain-specific ablation of the Bdnf gene. RESULTS: Subjects with schizophrenia showed deficits in NPY and PV mRNAs. Within-pair differences in BDNF protein levels showed strong positive correlations with NPY and SST and a robust inverse association with miR-195 levels, which in turn were not affected by antipsychotic treatment or genetic ablation of Bdnf. CONCLUSIONS: Taken together, these results suggest that prefrontal deficits in a subset of GABAergic mRNAs, including NPY, are dependent on the regional supply of BDNF, which in turn is fine-tuned through a microRNA (miRNA)-mediated mechanism.