RESUMO
Generating effective therapies for neurodevelopmental disorders has remained elusive. An emerging drug discovery approach for neurodevelopmental disorders is to characterize transcriptome-wide dysregulation in an appropriate model system and screen therapeutics based on their capacity to restore functionally relevant expression patterns. We characterized transcriptomic dysregulation in a human model of HNRNPU-related disorder to explore the potential of such a paradigm. We identified widespread dysregulation in functionally relevant pathways and then compared dysregulation in a human model to transcriptomic differences in embryonic and perinatal mice to determine whether dysregulation in an in vitro human model is partially replicated in an in vivo model of HNRNPU-related disorder. Strikingly, we find enrichment of co-dysregulation between 45-day-old human organoids and embryonic, but not perinatal, mice from distinct models of HNRNPU-related disorder. Thus, hnRNPU deficient human organoids may only be suitable to model transcriptional dysregulation in certain cell types within a specific developmental time window.
RESUMO
Zika virus (ZIKV), a member of the Flaviviridae family, was brought into the spotlight due to its widespread and increased pathogenicity, including Guillain-Barré syndrome and microcephaly. Neural progenitor cells (NPCs), which are multipotent cells capable of differentiating into the major neural phenotypes, are very susceptible to ZIKV infection. Given the complications of ZIKV infection and potential harm to public health, effective treatment options are urgently needed. Betulinic acid (BA), an abundant terpenoid of the lupane group, displays several biological activities, including neuroprotective effects. Here we demonstrate that Sox2+ NPCs, which are highly susceptible to ZIKV when compared to their neuronal counterparts, are protected against ZIKV-induced cell death when treated with BA. Similarly, the population of Sox2+ and Casp3+ NPCs found in ZIKV-infected cerebral organoids was significantly higher in the presence of BA than in untreated controls. Moreover, well-preserved structures were found in BA-treated organoids in contrast to ZIKV-infected controls. Bioinformatics analysis indicated Akt pathway activation by BA treatment. This was confirmed by phosphorylated Akt analysis, both in BA-treated NPCs and brain organoids, as shown by immunoblotting and immunofluorescence analyses, respectively. Taken together, these data suggest a neuroprotective role of BA in ZIKV-infected NPCs.
Assuntos
Microcefalia , Células-Tronco Neurais , Infecção por Zika virus , Zika virus , Humanos , Triterpenos Pentacíclicos , Infecção por Zika virus/tratamento farmacológico , Ácido BetulínicoRESUMO
Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection, we used human neural progenitor cells (NPC), derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation, ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2+ cells was demonstrated by activation of caspases 3/7, 8 and 9, and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2+ cells was prevented by incubation with the pan-caspase inhibitor, Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities, including multipolar spindle, chromosome laggards, micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy, such as monosomy, trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain, including microcephaly.