Periodic Reporting for period 1 - Spindle Brain Organoid (Understanding cellular mechanisms of human brain development using cerebral organoids)
Okres sprawozdawczy: 2017-09-01 do 2019-08-31
The human brain is the centre of human cognitive abilities. It is made of billions of neurons that are produced during fetal development. Human brain development differs substantially from rodents and the underlying mechanisms of its development and neurodevelopmental disorders are poorly understood. Human brain organoids are novel 3D stem cells-derived human models developed that recapitulate human-specific features of brain development with great accuracy. These emerging systems offer the opportunity to perform functional studies of human brain development and investigate mechanisms underlying human brain disorders with unprecedented accuracy. The goals of the project was to establish assays for a better experimental manipulation in brain organoid models and to gain insights into the mechanisms that control human brain development and that, when defective, lead to a smaller brain, a condition called microcephaly.
One objective of the project was to establish assays for a better experimental manipulation in brain organoid models. Specifically, I have established a protocol for a live imaging assay of organoid cultures. This method could be very useful for the study of a variety cellular phenotypes, such as cell-cycle, proliferation and neuronal activity, in living cells and with great detail. This assay could be applied for the analysis of these features in both healthy and disease conditions, thereby contributing to our understanding of cellular mechanisms underlying neurodevelopmental disorders.
The other main goal of the project was to gain insights into the mechanisms that control human brain development and that when defective, they lead to a smaller brain, a condition called microcephaly. A variety of genetic and non-genetic (external) factors regulate the production of human neurons but the underlying mechanisms are poorly understood. Although we initially proposed to investigate how genetic factors contribute to human brain expansion, we focused on external factors and in particular on a group of viruses, referred to as TORCH pathogens. During pregnancy, these pathogens can infect the fetal brain and cause severe brain malformations and neurological defects in newborns. Recently, Zika virus (ZIKV) has been associated with an increase risk of microcephaly in human fetuses and is now considered a new TORCH member. TORCH infections represent the major cause of morbidity and mortality in children and treatment options are very limited.
Here we have used organoid models to investigate how TORCH pathogens impair human brain development and to develop potential antiviral strategies. To model TORCH exposure during early stages of human brain development, we incubated young organoid cultures with the TORCH pathogens ZIKV and Herps Simplex (HSV) diluted in the organoid medium. We have found that ZIKV and HSV infected human brain stem cells, the precursors that give rise to human neurons. By using immunohistochemical analysis to analyze the effect of viral exposure on organoid cultures, we have found that ZIKV and HSV are very destructive, as they severely inhibit organoid growth and reduce organoid size, a feature reminiscent of microcephaly. We concluded that TORCH infections severely impair human brain development by targeting human brain stem cells, thereby underscoring the importance of human brain stem cells in human brain development. Next, we have investigated the reasons why organoids cultures are so vulnerable to these infections. We have found that vulnerability is due to poor antiviral defenses, as organoid cultures cannot mount potent antiviral responses against these pathogens. However, we have uncovered that we can augment these antiviral defenses by supplying organoid cultures with antiviral cytokines called interferons. In fact, treatment with these cytokines is sufficient to prevent both ZIKV- and HSV-induced organoid defects. Importantly, we demonstrate the efficacy of these treatment also in a mouse model of ZIKV infection. Remarkably, these treatments conferred neuroprotection in a virus-specific manner, thereby unraveling an unprecedented selectivity of these interferons. These findings highlight key roles of antiviral signaling in defending the human fetal brain against invading viruses and pave the way for the development of better antiviral compounds against neurotropic infections.Together this research provides novel insights into the mechanisms underlying virus-induced microcephaly and in the etiology of brain disorders.
The other main goal of the project was to gain insights into the mechanisms that control human brain development and that when defective, they lead to a smaller brain, a condition called microcephaly. A variety of genetic and non-genetic (external) factors regulate the production of human neurons but the underlying mechanisms are poorly understood. Although we initially proposed to investigate how genetic factors contribute to human brain expansion, we focused on external factors and in particular on a group of viruses, referred to as TORCH pathogens. During pregnancy, these pathogens can infect the fetal brain and cause severe brain malformations and neurological defects in newborns. Recently, Zika virus (ZIKV) has been associated with an increase risk of microcephaly in human fetuses and is now considered a new TORCH member. TORCH infections represent the major cause of morbidity and mortality in children and treatment options are very limited.
Here we have used organoid models to investigate how TORCH pathogens impair human brain development and to develop potential antiviral strategies. To model TORCH exposure during early stages of human brain development, we incubated young organoid cultures with the TORCH pathogens ZIKV and Herps Simplex (HSV) diluted in the organoid medium. We have found that ZIKV and HSV infected human brain stem cells, the precursors that give rise to human neurons. By using immunohistochemical analysis to analyze the effect of viral exposure on organoid cultures, we have found that ZIKV and HSV are very destructive, as they severely inhibit organoid growth and reduce organoid size, a feature reminiscent of microcephaly. We concluded that TORCH infections severely impair human brain development by targeting human brain stem cells, thereby underscoring the importance of human brain stem cells in human brain development. Next, we have investigated the reasons why organoids cultures are so vulnerable to these infections. We have found that vulnerability is due to poor antiviral defenses, as organoid cultures cannot mount potent antiviral responses against these pathogens. However, we have uncovered that we can augment these antiviral defenses by supplying organoid cultures with antiviral cytokines called interferons. In fact, treatment with these cytokines is sufficient to prevent both ZIKV- and HSV-induced organoid defects. Importantly, we demonstrate the efficacy of these treatment also in a mouse model of ZIKV infection. Remarkably, these treatments conferred neuroprotection in a virus-specific manner, thereby unraveling an unprecedented selectivity of these interferons. These findings highlight key roles of antiviral signaling in defending the human fetal brain against invading viruses and pave the way for the development of better antiviral compounds against neurotropic infections.Together this research provides novel insights into the mechanisms underlying virus-induced microcephaly and in the etiology of brain disorders.
TORCH infections represent the major cause of morbidity and mortality in children. This research has important implications for understanding the etiology and for the treatment of congenital TORCH infections and their associated neurological complications. Our results validate the use of organoids as human models for studying TORCH-induced pathogenesis and provide novel insights into the mechanisms underlying virus-induced microcephaly. Also, this work suggests a potential therapeutic intervention for pregnant women at risk and lays the groundwork for further development of novel antiviral compounds. For example, the infection models described could serve as platforms for antiviral testing and guide the development of more specific antiviral compounds. Furthermore, these models could be used for further study of immunity in the brain and viral pathogenesis, thereby leading to the discovery and study of novel targets for antiviral therapy.