HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE

Abstract

Objective: Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice.

Methods: Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology.

Results: Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (-49%; p=0.01) and mouse lymphocytes (-41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (-86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001).

Conclusions: These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.

Keywords: Autoimmune Diseases; Autoimmunity; Systemic Lupus Erythematosus.

Figure 1

Depletion of Drp1 by HRES-1/Rab4. (A) Effect of HRES-1/Rab4 on Drp1 protein levels, phosphorylation of Drp1 at Ser 637, and levels of AKAP10, which regulates phosphorylation of Drp1, were evaluated in Jurkat human T cells carrying doxycyclin-inducible vectors that express wild-type HRES-1/Rab4 or dominant-negative HRES-1/Rab4^S27N (HRES-1/Rab4-DN). Bafilomycin reversed the depletion of Drp1 by HRES-1/Rab4 and depletion of AKAP10 by HRES-1/Rab4-DN. Left panel, representative western blots; right panel, mean±SEM of 4–6 experiments. (B) Upregulation of Drp1 protein levels by siRNA-mediated knock-down of HRES-1/Rab4. Left panel, HRES-1/Rab4 and Drp1 were analysed relative to β-actin in HeLa cells 48 h after transfection with siRNA specific for HRES-1/Rab4 nucleotides 377–399 or scrambled siRNA. Right panel, mean±SEM of five independent experiments. (C) Effect of Drp1 deletion in MEFs on Rab4 protein levels assessed by western blot and on mitochondrial mass measured by MTG fluorescence. Representative western blot (left panel) and bar chart reflect mean±SEM of 4 experiments (right panel).

Figure 2

HRES-1/Rab4A promotes the accumulation of mitochondria in human PBL. (A) Effect of HRES-1/Rab4 overexpression on mitochondrial mass assessed in healthy control PBL stimulated with 5 μg/mL ConA for 24 h. Left panel, expression levels of adeno-associated virus (AAV)-transduced HRES-1/Rab4 was monitored by western blot. Control reflects PBL infected with AAV lacking HRES-1/Rab4 cDNA. Right panel, Assessment of mitochondrial mass by MitoTracker Deep Red (MTDR) fluorescence using flow cytometry. Data represent mean±SEM in five healthy donors. p Values reflect comparison of MTDR fluorescence in PBL infected with HRES-1/Rab4-producing AAV relative to PBL infected with control AAV using paired two-tailed t test. (B) Partitioning of HRES-1/Rab4 between the cytosol and mitochondria. Left panel, western blot analysis of HRES-1/Rab4, TAL, and VDAC in cytosolic and mitochondrial fractions of negatively isolated T cells from SLE and matched healthy subjects. Right panel, cumulative data represent mean±SE of HRES-1/Rab4 partitioning between the cytosol and mitochondria in nine lupus patients relative to the mean mitochondria/cytosol ratio of nine healthy controls normalised to 1.0.

Figure 3

Assessment of Drp1, Beclin 1, and LC3 expression relative to β-actin by western blot analysis of protein lysates from PBL of healthy donors (n=12) and lupus patients treated without (n=14) or with rapamycin (n=10). Representative western blots are shown in left panels and cumulative analyses are shown in right panels. p Values <0.05 are indicated.

Figure 4

Increased expression of Rab4A and depletion of Drp1 in MRL/lpr and NZB/W F1 mice. (A) Western blot analysis of Rab4A and Rab5 expression in thymocytes from 4-week-old C57BL/6 (B6), MRL, C57BL/6/Lpr (B6/Lpr), and MRL/lpr mice. Representative blots are shown in the left panel, while cumulative analyses are shown in the right panel. p Values <0.05 are indicated. (B) Western blot analysis of Drp1 in negatively isolated T cells of 4-week-old mice. (C) Western blot analysis of Rab4A and Rab5 expression in splenocytes from 8-week-old mice. (D) Western blot detection of VDAC and flow cytometry of mitochondrial mass using MTG fluorescence in splenocytes of 8 week-old mice. For each parameter 4 or more mice were analysed; p values <0.05 are indicated. (E) Western blot analysis of Drp1 protein levels in B6 mouse splenocytes transduced with adeno-associated virus (AAV) expressing HRES-1/Rab4 (left panel). Data represent mean±SEM of five experiments; p values reflect comparison to cells infected with control AAV using paired two-tailed t test (right panel).

Figure 5

Effect of 3-PEHPC and rapamycin (Rapa) relative to solvent controls PBS and CMC, respectively, on autoimmunity and nephritis in MRL/lpr mice treated three times weekly, between ages of 4–14 weeks. (A) Body weight, ANA production, and proteinuria. (B) Renal pathology of 14-week-old mice. Upper and lower panels represent 200-fold and 400-fold original magnifications of identical sections; size bars represent 50 microns. PBS: GN is characterised by circumferential, predominantly cellular crescents with segmental sclerosis and hyalinosis. There are prominent interstitial lymphocytic infiltrates, tubular atrophy and protenaceous casts. 3-PEHPC: glomeruli are free of crescents. There is minimal tubular atrophy and interstitial fibrosis. CMC: glomeruli show cellular or fibrocellular crescents. Glomerular tufts are hypercellular with lobular accentuation. There is tubular atrophy and interstitial lymphocytic infiltrates and fibrosis. Rapa: normocellular glomeruli; minimal tubular atrophy and interstitial fibrosis. (C) Renal disease scores in 14-week-old MRL/lpr mice. Left panel, for each kidney, the severity of GN, GS, and IN was graded in a 0–4 semiquantitative scale. Right panel, numbers of glomeruli with sclerosis and/or crescent formation were determined and expressed as a percentage of total glomeruli observed in the entire cortical field. p values <0.05 reflect two-way analysis of variance.

Figure 6

Effect of 3-PEHPC and Rapa relative to solvent controls PBS and CMC, respectively, on the immune system and mitochondrial homeostasis of MRL/lpr mice treated weekly between ages of 4–14 weeks, as described under figure 5. (A) Effect of 3-PEHPC and Rapa on spleen size, B and T cells, as well as CD4, CD8 and DN T-cell frequencies. (B) Effect of 3-PEHPC and Rapa treatment on IFN-γ, IL-10, and IL-17A levels in serum of 14-week-old MRL/lpr mice. (C) Effect of 3-PEHPC and Rapa treatment on mTOR activity in spleen subsets of 14-week-old MRL/lpr mice. (D) Effect of 3-PEHPC and Rapa treatment on Foxp3 expression in CD4, CD4/CD25 and CD8 T cells. (E) Prevention of Drp1 depletion by 3-PEHPC in 14-week-old MRL/lpr mice. Representative (left panel) and cumulative western blot analyses of negatively isolated T cells from MRL/lpr mice-treated PBS, CMC, 3-PEHPC and Rapa (right panel). (F) Effect of 3-PEHPC and Rapa treatment on mitochondrial mass (MTG) in spleen lymphocyte subsets of 14-week-old MRL/lpr mice. p Values <0.05 reflect comparison with unpaired two-tailed t test.