Mechanistic Study Of The Role Of NMNAT In Glioma

Background:Gliomas are highly malignant brain tumors with poor prognosis and short survival.NAD⁺has been shown to impact multiple processes that are dysregulated in cancer;however,anti-cancer therapies targeting NAD⁺synthetic pathway have been unsuccessful due to insufficient mechanistic understanding.Significance:Mechanistic study of the role of NMNAT in glioma.Methods:1.Established an in vivo glioma model in Drosophila（1）Detected the protein expression level of NMNAT in gliomaAccording to the literature about glioma model in Drosophila,we took advantage of the transgenic tools and established a glioma model in fly by overexpressing Ras^v12 in glia to induce cell over-growth and mimic glioma formation.Through the high resolution of immunofluorescence confocol imaging,we marked glioma location and quantified the volume and number of gliomas.Detected protein expression of NMNAT in glioma by probing NMNAT antibody.（2）Detected the growth of glioma when modulating NMNAT expressionIn the glioma model of glial Ras^v12 overexpression,knock down NMNAT or overexpressing NMNAT（nuclear,nuclear without enzyme activity,cytoplasmic）.Dissected fly brains at the same development stage,marked glial cell and NMNAT by immunofluorescence,quantified volume and number of gliomas,and recorded survival rate.（3）Mechanistic study of NMNAT in gliomaBased on the literature,detected the effect of NMNAT on DNA damage-p53-Caspase-3signaling pathway.Used glioma model,dissected fly brains at same development stage,and probed DNA damage marker,p53 and Caspase-3 in immunofluorescence.Quantified and analyzed staining location and intensity.2.Study on effect and mechanism of NMNAT in vitro（1）The effect on glioma cell proliferation when knocking down or overexpressing NMNATExamined the effects of NMNAT1/2 on glioma cell proliferation after modulating the expression level.Determined cell proliferation by MTT assay,colony formation and growth curve after siRNA knockdown or overexpressing NMNAT.（2）Mechanistic study of NMNAT in gliomaBased on the results in vivo,examined effect of NMNAT on cell apoptosis.Probed Caspase-3 after siRNA knocking down or overexpression NMNAT,and quantified staining intensity.When overexpressing NMNAT,pulled down p53 by immunocoprecipitation assay then probed for NMNAT,PARP1 and PAR.Marked NMNAT,PARP1 and p53 in cells using immunofluorescence,detected location and quantified staining intensity.Results:1.The results from in vivo show that the endogenous NMNAT was upregulated in glioma.The volume and number were inhibited when RNAi knockdown NMNAT in glial cell;The overexpression result show that overexpressing nuclear NMNAT promoted glioma progression and reduced the survival rate.2.The human glioma cell results show that knockdown NMNAT1 or NMNAT2 inhibited cells proliferation,while overexpressing NMNAT1 or NMNAT2 promoted cells growth.3.The mechanistic study results show that NMNAT1 colocalized with p53 and PARP1.They becamed a trimeric complex in the nucleus and p53 protein formed clusters.The total nuclear Caspase-3 was decreased.Conclusion:We optimized a Drosophila glioma model and discovered the genetic requirement for NAD⁺synthase nicotinamide mononucleotide adenylyltransferase（NMNAT）in glioma progression in vivo and in human glioma cells.Overexpressing enzymatically active NMNAT significantly promotes glioma growth and reduces animal viability.Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD⁺-dependent poly（ADP-ribosyl）ation（PARylation）and deacetylation of p53.Interestingly,NMNAT forms a complex with p53 and PARP1 to facilitate PARylation.As PARylation and deacetylation reduce p53 pro-apoptotic activity,our results demonstrate that NMNAT promotes glioma progression through regulating p53 post-translational modification.Innovation:Our findings reveal NMNAT-mediated NAD⁺synthetic pathway as a novel therapeutic target for glioma.