Lei Zhou, Ph.D - US grants

DESCRIPTION (provided by applicant): Probing the complexity of UV induced apoptosis in Drosophila: Apoptosis is an evolutionarily conserved process that has been implicated in a variety of diseases, including cancer and neurodegenerative diseases. Regulation of apoptosis is under the control of a complex network of genes (proteins). Our preliminary data showed that UV induced apoptosis in Drosophila embryo can be mediated by different cell death regulatory genes. More interestingly, the cellular context (differentiation status) of the irradiated cell determines which cell death regulatory gene is activated to induce cell death upon UV irradiation, i.e. When embryos in differentiating stage were irradiated with UV, the reaper gene is induced by a DNA damage -dependent mechanism that involves Drosophila homologue of Ataxia Telangiectasia Mutated, mei-41 (and very likely dP53 as well). However, when embryos prior to differentiating stage were irradiated, the hac-1 (Homologue of Apaf-1 and Ced-4) gene is induced instead of reaper. The induction of hac-1 expression is required for UV induced apoptosis at this developmental stage. But in contrast to UV induction of reaper, UV induction of hac-1 appears to be independent of nuclear DNA damage and was not affected by mei-41 mutation (mei-41 [D5]). It has long been noticed that UV induced cell death can be mediated by DNA damage -dependent and -independent mechanisms. However, it remains unclear as to how are the mechanisms deployed and coordinated to mediate UV induced apoptosis. The aforementioned findings indicate that Drosophila embryo provides an excellent model for systematic analysis of the complexity of UV -induced cell death. The focus of this proposal is to characterize the molecular mechanism underlying UV induced hac- 1 expression and apoptosis in early stage embryos. In addition, we will apply genomic approaches to gain comprehensive understanding of UV -induced genomic response and apoptosis. The goal of this proposal is to elucidate in depth how different cell death regulatory pathways may be deployed to mediate genotoxic stimuli -induced cell death. The information provided by these investigations will contribute to our comprehensive understanding of cell death regulation and skin carcinogenesis. Molecular mechanisms uncovered through this project should provide insights for identifying alternative therapeutic targets, especially for cancers that are resistant to DNA-damage agents.

[unreadable] DESCRIPTION (provided by applicant): Infectious diseases transmitted through mosquitoes account for millions of death world wide each year. Cell death (apoptosis) is an important component of host/vector immuno-response mechanism and has been implicated in the life cycle of pathogens in mosquitoes. However the understanding of cell death regulation in mosquito was hindered greatly by the fact that major insect cell death regulators, namely the reaper/hid/grim (RHG) like lAP-antagonists identified in Drosophila, were not present in the annotated mosquito (Anopheles gambia) genome. Using a systematic bioinformatics approach, we have identified several candidates of reaper/hid/grim-like lAP-antagonist in both the Anopheles and Aedes mosquito genomes. One of them, michelob_x has been characterized as indeed an lAP-antagonist. This proposed project aims at further characterizing these lAP-antagonists and their role in mediating cell death during mosquito development as well as in response to pathogen infection. Findings generated from this study should significantly improve our understanding of immuno-response and cell death regulation in mosquitoes. It is also quite possible that some of these genes, because of their tissue-ablation activity, can be used for mosquito population control strategies. [unreadable] [unreadable] [unreadable]

Title: Systematic analysis of cell death regulation in mosquitoes. Cell death (apoptosis) is an important component of the vector/host immuno-response mechanism. It has been implicated in the infectious life cycle of a variety of pathogens, such as the malaria parasite and the West Nile virus, in mosquitoes. However, our understanding of cell death regulation in mosquitoes has been hindered greatly by the lacking of a complete list of major insect cell death regulators, i.e. the *Reaper/Hid/Grim -like IAP-antagonists, in the annotated mosquito genomes. IAPs (Inhibitor of Apoptosis Proteins) are the essential [unreadable]Brake[unreadable] of the core cell death machinery in insects. They bind to caspases and inhibit the activity of these [unreadable]deadly[unreadable] enzymes. Genetic studies in Drosophila have shown that most programmed cell death during development is mediated through specific expression of IAP-antagonists such as reaper, hid, and grim (RHG) in cells destined to die. Most of these IAP-antagonists are also required for mediating cell death in response to environmental stress. The genome project for the primary malaria vector, Anopheles gambiae, revealed a significant expansion of the IAP and caspase gene families when compared to Drosophila. This was postulated to reflect the functional requirement of fine-tuning cell death regulation in response to parasite and virus infection. It also strongly implies that the IAP-antagonist ?? IAP ?? Caspases pathway, as is in Drosophila, is fundamentally important for cell death regulation in mosquitoes. However, due to extensive sequence divergence, the annotated complete An. gambiae genome sequence did not initially include any ortholog of RHG-like IAP-antagonist. Using a systematic bioinformatics approach, we identified and subsequently characterized the first RHG-like IAPantagonists, michelob_x (mx), in Anopheles and Aedes genomes. To probe the involvement of these pro-apoptotic genes in mediating vector-pathogen interaction, we identified the mx ortholog in Culex and found that it is induced by CuniNPV (a mosquito baculovirus) infection to Cx.quinauifasciatus (section 4.5;Zhou et al, in preparation). In addition, we have identified another IAP-antagonist, IBMP6, as the ortholog of Drosophila Hid. Interestingly, ibmp6 is induced by MIV infection of the Aag2 cells (section 4.6). These data indicate that like in Drosophila, multiple IAP-antagonist are required for cell death regulation in mosquito. Further more, distinct IAP-antagonists is(are) utilized against different infections or in different tissues. Since the IAP-antagonist genes play pivotal roles in insect cell death regulation and our work revealed that they are involved in pathogen-induced vector response, elucidating fully this group of genes in mosquitoes will be essential for the comprehensive understanding of cell death regulation in response to pathogen challenge as well as during normal mosquito life cycle. This proposed project aims at (1) Systematic identification and functional verification of the IAP-antagonists in major mosquito genomes. (2)Elucidating the role of these IAP-antagonists in mediating vector-pathogen interaction using CuniNPV, MIV, and B.algerae. (3) Developing public resources for systematic analysis of cell death regulation in mosquitoes. Cell death (apoptosis) is implicated in the infectious life cycle of a variety of pathogens, such as the malaria parasite and the West Nile virus, in mosquitoes. Understanding cell death response will help us to develop strategy for controlling mosquito transmitted disease and for controlling mosquito population.

Arthropod-borne pathogens account for millions of death each year. Understanding the genetic basis of vector susceptibility to pathogens is pivotal to novel disease control strategies. The hypothesis that induction of apoptosis is a fundamental innate immune response has been supported by virology studies, which demonstrated that the anti- apoptotic activities of many viral genes are essential for their infectivity and/or virulence. However, the cellular mechanism mediating the induction of apoptosis following virus infection remained enigmatic. In addition, studies with cultured insect cells showed that either there is a lack of apoptosis, or the pro-apoptotic response happens relatively late, casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models mimicking native routes of viral infection, we found that there is a rapid induction of pro-apoptotic genes (RIPAG) within a few hours following exposure to DNA/RNA viruses. More importantly, using genetic tools in Drosophila, we showed that the RIPAG, and the ensuing apoptosis, is responsible for denying the expression of viral genes and blocking/limiting the infection. Animals with compromised RIAPG are much more susceptible to viral infection than wild type. In this proposal, we seek to unravel the transcriptional mechanisms and the regulatory pathway(s) controlling RIPAG using a combination of Drosophila genetics and comparative genomics. In addition, utilizing the information obtained through the mechanistic analysis, we will test the hypothesis that increased innate immunity against viral infection may be achieved by enhancing the RIPAG response to viral infection. Finally, we assess the fitness of the antiviral constructs we create in Drosophila, then translate the most powerful and most fit constructs to two mosquito vectors and perform preliminary evaluations of transgene effectiveness against Dengue.