Complement-mediated STAT3 activation suppresses TFEB-driven autophagy in aging retinal pigment epithelium

Hypothesis

Chronic complement activation in the aging retinal pigment epithelium (RPE) actively suppresses autophagy via C3aR/C5aR‑STAT3 signaling, which blocks TFEB nuclear translocation and lysosomal biogenesis.

Mechanistic Model

1. Complement ligand‑receptor engagement – Age‑related rise in C3a and C5a binds C3aR and C5aR on RPE, triggering JAK‑STAT3 phosphorylation {1}.
2. STAT3‑dependent transcriptional program – Phospho‑STAT3 induces SOCS3 and suppresses MITF, leading to sustained mTORC1 activity that keeps TFEB phosphorylated and retained in the cytosol {2}.
3. Impaired lysosomal biogenesis – Cytosolic TFEB fails to activate CLEAR network genes, reducing lysosomal acidification, hydrolase content, and autophagosome‑lysosome fusion capacity.
4. Direct lysosomal damage by complement – Deposition of C3b and membrane attack complex (C5b‑9) on lysosomal membranes generates oxidative stress, compromises LAMP2 integrity, and causes lysosomal membrane permeabilization, further hindering autophagic degradation {3}.
5. Feedback amplification – Accumulated lipofuscin and oxidative lipids act as danger‑associated molecular patterns that stimulate complement production, creating a self‑reinforcing loop of complement activation and autophagy suppression.

Experimental Plan

• Animal studies – Use aged Cfh‑/‑ mice (exhibit complement hyperactivation) and administer either a C3aR antagonist (SB 290157) or a STAT3 inhibitor (Stattic) via intravitreal injection for four weeks. Assess:
  • Autophagic flux: LC3‑II/I ratio with and without bafilomycin A1 by western blot.
  • TFEB localization: immunofluorescence quantification of nuclear vs cytoplasmic TFEB.
  • Lysosomal function: LysoTracker intensity and cathepsin D activity.
  • Lipofuscin load: autofluorescence imaging and ELISA for A2E.
  • Complement deposition: C3b and MAC immunostaining.
• Cell‑culture studies – Differentiate human iPSC‑derived RPE and treat with recombinant C3a (100 ng/mL) or C5a (50 ng/mL) for 24 h. Measure:
  • STAT3 phosphorylation (p‑STAT3 Y705) by western blot.
  • TFEB subcellular fractionation followed by immunoblot.
  • Lysosomal pH using LysoSensor dyes.
  • Autophagosome formation: mCherry‑GFP‑LC3 reporter ratio.
  • Include siRNA knock‑down of C3aR, C5aR, or STAT3 to confirm specificity.
• Rescue experiments – Overexpress a constitutively active TFEB (TFEB‑S142A/S146A) in complement‑treated RPE to test whether restoring lysosomal gene expression bypasses the block.

Expected Outcomes

If complement drives active autophagy suppression, blocking C3aR/C5aR or STAT3 will:

• Increase nuclear TFEB and lysosomal gene expression.
• Enhance autophagic flux (higher LC3‑II turnover).
• Reduce lipofuscin accumulation and restore lysosomal acidification. Conversely, activating STAT3 in young RPE should phenocopy the aged autophagy defect, confirming sufficiency.

Potential Pitfalls

Complement inhibition may affect inflammation and cell survival independently of autophagy; therefore, parallel assays for cytokine release and cell viability are essential. Additionally, redundant pathways (e.g., NF‑κB) could compensate for STAT3 loss, necessitating combined genetic approaches to isolate the autophagy‑specific effect.