Background/Purpose: Background/Purpose

            The human GTPase HRES-1/Rab4 has been identified as a susceptibility locus for systemic lupus erythematosus (SLE) and is overexpressed in T cells of SLE patients ¹. HRES-1/Rab4 depletes DRP-1, a mitochondrial protein that initiates mitophagy, thereby increasing the mass of mitochondria ². Rab4A is the mouse orthologue of HRES-1/Rab4, which is overexpressed in lupus-prone mice ². Therefore, the role of Rab4A in disease pathogenesis has been modeled in Jurkat cells and investigated in lupus-resistant and lupus-prone strains carrying constitutively active Rab4A^Q72L alleles or lacking Rab4A in T cells.

Methods: Methods             HRES-1/Rab4 was overexpressed in Jurkat cells using a doxycycline-inducible GFP-encoding bi-cistronic expression vector system. Mitochondrial function was assessed by Seahorse metabolic analyzer. The effect of Rab4A was examined in lupus-prone triple congenic SLE123 (SLE) mice and autoimmune resistant C57Bl/6 mice carrying wildtype (WT), constitutive active Rab4AQ72L, or Rab4-deficient alleles (Rab4KO) created through crossing of floxed Rab4AQ72L and CD4Cre mice. Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer was used for targeted assessment of >800 metabolites. Mouse behavior was evaluated by the elevated plus maze and open field tests. Statistical analyses were performed by Student’s T-test or Tukey’s HSD ANOVA using GraphPad software and considered significant at p< 0.05 for hypothesis testing.

Results: Results

Overexpression of HRES-1/Rab4 led to a significant increase in ATP production (fold change, FC=1.2, p= 0.03), % spare respiratory capacity (FC=1.2, p= 0.002), increased glutathione disulfide (GSSG, FC=4.3, p=0.04) and malondialdehyde (MDA, FC=1.2, p=0.01) in Jurkat cells. The Rab4A^Q72L mice traveled a significantly further distance than the WT mice (FC=1.5, p=0.03). The Rab4A^Q72L mice also traveled further than the SLE mice, SLE Rab4A^Q72L and SLE Rab4A^Q72L CD4Cre. Interestingly the deletion of Rab4A in the T cells in SLE mice increased the distance traveled significantly relative to SLE Rab4A^Q72L mice (FC=1.7, p=0.01). The Rab4A^Q72L mice entered the open arms of the maze more often than WT (FC=1.6, p=0.008). Rab4A^Q72L mice entered the open arm significantly more times than all SLE mice. Similar trends were seen in the open field test.

The brain metabolomes of Rab4A^Q72L mice showed a significant increase in GSSG (FC=1.4, p=0.02) and MDA (FC=1.6, p=0.04). SLE and SLE Rab4A^Q72L mice also had more GSSG (SLE: FC=1.5, p=0.04; SLE Rab4A^Q72L: FC=1.7, p=0.01) and MDA (SLE: FC=1.9, p=0.12; SLE Rab4A^Q72L: FC=2.5, p=0.02) than WT controls.

Conclusion: Conclusion

Activation of Rab4A increases mitochondrial oxidative stress and GSSG accumulation, which apparently underlie neurobehavioral changes in lupus-resistant and lupus-prone mice.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/rab4a-increases-mitochondrial-oxidative-stress-and-glutathione-disulfide-accumulation-that-underlie-neurobehavioral-changes-in-lupus-prone-mice/