Article Text

  1. R. Towns1,
  2. C. Guo1,
  3. Y. Shangguan1,
  4. S. Hong1,
  5. I. Song1,
  6. T. Yoshimori1,
  7. J. W. Wiley1
  1. 1University of Michigan, Ann Arbor


Background We reported previously that diabetic neuropathy is associated with mitochondrial dysfunction and that sera from type 2 diabetic patients with neuropathy induces complement-independent apoptosis in cultured human neuroblastoma (SY5Y) neurons. Mitochondrial dysfunction is associated with engulfment of the injured organelles in autophagosomes, e.g. autophagy. We hypothesized that sera from type 2 diabetic patients with neuropathy would induce autoantibody-mediated, Fas-dependent activation of autophagy and cell death in SY5Y neurons.

Methods Complement-inactivated sera were obtained from 12 patients with type 2 diabetes and neuropathy (D + N), 6 DM patients without neuropathy (D - N) and 6 healthy age- and gender-matched controls (C). IgG and IgM autoantibody binding to the cell surface was detected using specific FITC-tagged antibodies. Immunoglobulins (Igs) were removed from sera with protein L agrose beads. Fas activation was monitored using Northern and Western detection of Fas and Fas-activated Death Domain (FADD) ± gene silencing with si-FADD. Autophagy was measured using Western and immunofluoresence (IF) detection of the specific marker for autophagosomes, LC3 and mitochondrial colocalization with MitoTracker. Apoptosis was measured by activated caspase-3 and TUNEL assays. Oncosis was measured by mu-calpain immunoreactivity.

Results Sera from the D + N subjects but not D - N or C demonstrated significant increase in IgG and IgM autoantibodies. These sera induced a marked increased in Fas and FADD immunoreactivity detected by Western and IF methods after 2 hours treatment and a parallel increase in autophagy associated with engulfment of mitochondria compared to sera from DM - N subjects and C. Treatment with purified Fas ligand (10 ng/mL) produced similar results at 2 h. After 24 hours treatment with D + N sera there was a significant increase in apoptosis (1.6 ± 0.3%) and oncosis (2.5 ± 0.4%) compared to D - N (0.4 ± 0.2, p<0.05). Treatment with si-FADD reduced D + N induction of FADD and autophagy by 72 ± 8% (p<0.01) and decreased the induction of apoptosis by 48 ± 8% (p<0.05). Induction of Fas, FADD, autophagy, apoptosis and oncosis were absent after removal of Igs.

Summary These results suggest that autoimmune, Fas-dependent activation of autophagy and cell death participate in the pathophysiology of diabetic neuropathy.

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