A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dst(dt)) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dst(dt-Tg4) mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dst(dt-Tg4) sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dst(dt-Tg4) mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dst(dt-Tg4) mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dst(dt-Tg4) dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dst(dt) pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis.
Keywords: ANOVA, analysis of variance; BPAG1; CASP3, caspase 3, apoptosis-related cysteine peptidase; DMC; DMC, dynein/dynactin motor complex; DMEM, Dulbecco's modified Eagle's medium; DNAIC1, dynein, axonemal, intermediate chain 1; DRG, dorsal root ganglion; DST, dystonin; Dstdt, dystonia musculorum; EM, electron microscopy; FBS, fetal bovine serum; HSAN-VI; HSAN-VI, hereditary sensory and autonomic neuropathy type VI; MACF1, microtubule-actin crosslinking factor 1; MAP1B; MAP1B, microtubule-associated protein 1B; MAP1LC3/LC3, microtubule associated-protein 1 light chain 3; MT, microtubule; P, postnatal day; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PrP, prion protein; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQTSM1/p62, sequestosome 1; TCA, trichloroacetic acid; TUBB3, tubulin, β, 3 class III; WT, wild type; autophagosome; dynein; dystonin; microtubules; trafficking.