Moreover, RanBP9 TG neurons also showed significantly reduced red JC-1 aggregates and increased green JC-1 monomers (Figures 7e and f), indicative of reduced MMP. Open in a separate window Figure 7 p73 is essential for RanBP9/A(FITC) intensities were quantified using Nikon NIS-Elements-AR software (for 24?h and subjected to staining with Annexin V-FITC and DAPI. Alocalizes to mitochondrial membrane and impairs mitochondrial functions through interacting with mitochondrial proteins, disrupting electron-transport chain and increasing mitochondrial ROS products.7, 8, 9 A recent study also demonstrated early deficits in synaptic mitochondria, Aaccumulation within mitochondria prior to extracellular Adeposition, and impaired axonal transport of mitochondria in mutant APP transgenic mice.10 Mitochondria-mediated apoptosis is the best known intrinsic apoptotic pathway. Impaired mitochondrial function is usually associated with the aging process and prevalent age-related diseases including AD.11, 12 Conversely, perturbation in mitochondria-mediated apoptosis has a critical role in oncogenic processes and downstream effects of tumor suppressor proteins such as p53 and p73. Cellular stress from DNA damage, loss of cell survival factors or defective cell cycle promotes the accumulation of pro-apoptotic proteins, Gap 26 such as Bax, Bak, Noxa, and puma.13 Meanwhile, anti-apoptotic proteins such as Bcl-2 and Bcl-xl prevent apoptosis by inhibiting the action of pro-apoptotic proteins.14, 15 Accordingly, when the Gap 26 balance of activity between pro- and anti-apoptotic users is upset, the permeability of mitochondrial membrane is lost and mitochondrial reactive oxygen species (ROS) is induced.16, 17 Apoptogenic proteins like cytochrome or apoptotic inducing factors are then released to the cytosol, which activate pro-caspases to induce apoptosis.18 We recently demonstrated that this scaffolding protein RanBP9 interacts with the cytoplasmic tails of LRP, APP BCL2 and BACE1, and functions as a scaffold upon which APP is brought together with BACE1 and LRP. Such interactions of RanBP9 promote the endocytosis of APP and strongly increase BACE1 cleavage of APP to generate Ain cultured cells and generation via BACE1 processing of APP.21 We also recently demonstrated that RanBP9 functions to inhibit cell adhesion by accelerating the endocytosis of modulates exogenously expressed p73levels and nuclear translocation of RanBP9.25 Moreover, it has been shown that p73 can induce apoptosis via nuclear and non-nuclear pathways, the latter involving direct translocation into mitochondria.26 However, the mechanism of RanBP9-induced apoptosis, the involvement of mitochondria in such course of action, and the functional role of the RanBP9/p73 complex are not well understood. In this study, we found that RanBP9 together with p73 induce aberrant changes in mitochondria Gap 26 (MMP, superoxide levels, apoptotic proteins & fission) and induce apoptosis that depend on their cooperative actions. Such results implicate the crucial role of the RanBP9/p73 pathway in the regulation of mitochondria-mediated apoptosis during neurodegenerative processes. Results Excessive RanBP9 induces mitochondrial membrane permeability and promotes apoptosis in mouse hippocampal HT22 cells It has been reported that overexpression of RanBP9 can increase the activation of caspases and induce cell death in Hela cells.13 Consistent with this observation, we also showed that RanBP9 induces neurodegeneration and mediates Avector-transfected cells, indicating increased production of mitochondrial ROS (Determine 1d, upper panels). Further examination of MitoSox Reddish by FACS analysis also demonstrated comparable results, with RanBP9-transfected cells displaying Gap 26 median fluorescence intensity of 111 91 in vector-transfected cells (Physique 1d, lower panels). These results taken together indicate that RanBP9 increases the vulnerability of cells to undergo apoptosis and mitochondrial dysfunction even under conditions where overt cell death is not readily detectable. Overexpression of RanBP9 alters Bax/Bcl2 protein ratio, promotes Bax oligomerization, and induces cytochrome release It has been shown that knockdown of RanBP9 decreases mitochondrial Bax and increases Bcl2 in Hela cells.13 To determine whether corresponding changes are similarly seen after RanBP9 overexpression in brain-derived cells, we analyzed Bax and Bcl2 protein levels after control vector or RanBP9 transfection in HT22 cells. Indeed, Bcl2 levels were markedly decreased after RanBP9 transfection either under 10 or 2% FBS culture conditions, and Bax levels were moderately increased in 10% FBS and further increased in 2% FBS (Physique 2a). As transfection efficiency could dilute the effects of RanBP9 observed from total cell lysates, we also transfected EGFP or EGFP-RanBP9 and visualized Bax.