Background/Purpose: T cells from patients with systemic lupus erythematosus (SLE) exhibit accumulation of mitochondria and activation of the mammalian target of rapamycin (mTOR). Although mTOR has been implicated in blocking of autophagy and may account for increased mitochondrial mass, paradoxically, blockade of mTOR with rapamycin failed to reduce mitochondrial mass in lupus T cells. mTOR  interacts and co-localizes with the small GTPase HRES-1/Rab4 on endosomes that carry autophagocytic cargo, including mitochondria, to lysosomes. Therefore, we investigated whether HRES-1/Rab4, that is over-expressed in lupus T cells, may mediate the accumulation of mitochondria and whether its inhibition by geranylgeranyl transferase inhibitor, 2-[3-pyridinyl]-1-hydroxyethylidene-1,1-phosphonocarboxylic acid (3-PEHPC) can influence the development of disease in lupus-prone mice.

Methods: Microarray and confirmatory western blot as well as glutathione-S-transferase pull-down assays were used to map the interactome of HRES-1/Rab4 in human peripheral blood lymphocytes. Expression of Rab4, the murine homolog of HRES-1/Rab4, and its interacting partners were assessed with respect to mitochondrial mass and mTOR activity in the thymus and spleen cell subsets of lupus-prone MRL/lpr and C57BL/6, MRL/MpJ, and Black 6/lpr control mice at ages of 4 and 8 weeks. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin in comparison to solvent controls for 10 weeks, beginning at 4 weeks of age. Disease development was monitored by antinuclear antibody (ANA) production and proteinuria.  At the time of sacrifice, nephritis was assessed by histopathology, serum cytokine levels were measured by ELISA, gene expression in splenocyte subsets was measured by western blot, and mitochondrial homeostasis was evaluated by flow cytometry. Statistical analyses were done by t-tests and ANOVA, with p<0.05 considered significant.

Results: HRES-1/Rab4 was found to directly interact with Drp1and its overexpression caused the depletion of Drp1 in human Jurkat cells (p=0.01) and mouse splenocytes (p=0.03).  Rab4A was over-expressed in MRL/lpr thymocytes at 4 weeks (p=0.0002).  Drp1 is reduced in MRL/lpr T cells at 4 weeks (p=0.007). MRL/lpr mice exhibited increased mitochondrial mass in thymocytes (p=0.02) at 4 weeks and in splenocytes (p=0.01) at 8 weeks of age. At 14 weeks of age, treatment with 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in MRL/lpr T cells (p=0.02), reduced ANA production (p=0.021), reduced proteinuria (p=0.00004), and reduced nephritis scores in MRL/lpr mice (p<0.001). Unlike 3-PEHPC, rapamycin reduced mTOR activity (p<0.05) but failed to affect mitochondrial mass. IL-10 production was reduced by 3-PEHPC (p=0.04), while rapamycin reduced production of IFN-γ (p=1 x 10^-5) and IL-17A (p=0.01). 

Conclusion: These data reveal a pathogenic role of Drp1 depletion and identify the regulation of mitophagy by HRES-1/Rab4 as a promising target for treatment in SLE.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/hres-1rab4-mediated-loss-of-drp1-inhibits-mitophagy-promotes-accumulation-of-mitochondria-and-serves-as-target-for-treatment-in-sle/