Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise when dysregulated. Here we consider X-linked spinal and bulbar muscular atrophy (SBMA), a repeat disorder caused by polyglutamine-expanded androgen receptor (polyQ-AR). We found that polyQ-AR reduced long-term protein turnover and impaired autophagic flux in motor neuron–like cells. Ultrastructural analysis of SBMA mice revealed a block in autophagy pathway progression. We examined the transcriptional regulation of autophagy and observed a functionally significant physical interaction between transcription factor EB (TFEB) and AR. Normal AR promoted, but polyQ-AR interfered with, TFEB transactivation. To evaluate physiological relevance, we reprogrammed patient fibroblasts to induced pluripotent stem cells and then to neuronal precursor cells (NPCs). We compared multiple SBMA NPC lines and documented the metabolic and autophagic flux defects that could be rescued by TFEB. Our results indicate that polyQ-AR diminishes TFEB function to impair autophagy and promote SBMA pathogenesis.
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The authors wish to thank L.I. Macedo de Souza, A.C. Smith and H. Burke for technical assistance, N. Mizushima (University of Tokyo) and Z. Yue for (Icahn School of Medicine at Mount Sinai) providing GFP-LC3 transgenic mice, J.P. Taylor (St. Jude's Children's Research Hospital) for supplying the mCherry-GFP-LC3 MEFs and T. Johansen (Arctic University of Norway) for the gift of the mCherry-EGFP-LC3 construct. This work was supported by grants from the US National Institutes of Health (R01 NS041648 to A.R.L., R01 AG033082 to A.R.L. and DP2-OD006495-01 to A.R.M.), the Muscular Dystrophy Association (Basic Research Grant to A.R.L., Development Grant to C.J.C. and Basic Research Grant to A.R.M.), and the California Institute for Regenerative Medicine (CIRM TR2-01814 to A.R.M.).
The authors declare no competing financial interests.
Integrated supplementary information
After labeling of newly synthesized proteins with L-azidohomoalanine (AHA), an analog of methionine, we measured rates of protein turnover over 100 hrs after the AHA pulse. We noted a significantly reduced rate of long-term protein turnover for MN-1 AR65Q cells in comparison to wild-type MN-1 cells (n = 3 independent experiments, ANOVA with post-hoc Tukey test, *P <.05). MN-1 AR24Q cells displayed a rate of protein turnover intermediate to wild-type MN-1 cells and MN-1 AR65Q cells, but the MN-1 AR24Q protein turnover rate was not significantly different from either cell line. MN-1 WT cells treated with leupeptin served as a positive control. Data are presented as mean + s.e.m.
Here we see representative images of autophagosomes (yellow arrowheads) and autolysosomes (red arrowhead). Autophagosomes were identified as double-membrane bound vacuoles containing heterogeneous cytoplasmic material within the double membrane, based upon well-established criteria (Eskelinen et. al., 2004, Mol Biol Cell 15: 3132–3145; Nixon et al., 2005, Neuropathol Exp Neurol 64: 113–122). Magnification of autophagosomes is at 12,460x. Autolysosomes were identified as vacuoles with homogeneous contents that were associated with electron-dense lysosomes, as delineated in previous studies (Eskelinen et. al., 2004, Mol Biol Cell 15: 3132–3145; Nixon et al., 2005, Neuropathol Exp Neurol 64: 113–122). The autolysosome panel is at 10,150x magnification.
Supplementary Figure 3 Increased expression of TFEB target genes in skeletal muscle of SBMA YAC AR100 mice.
We measured expression levels of TFEB target genes in quadriceps muscle from non-transgenic (Nt), YAC AR20, and YAC AR100 transgenic mice. RT-PCR analysis of isolated RNAs revealed significant increases in TFEB target gene expression in YAC AR20 and YAC AR100 mice: Lamp1 (lysosomal-associated membrane protein 1) [F=2,12]=8.15, Atp6v1h (vesicular ATPase V1 subunit H) [F(2,12)=49.95]; Gla (galactosidase-α) F(2,12)=8.63]; Ctsd (cathepsin D) [F(2,12)=25.57]; Mcoln1 (mucolipin1) [F(2,12)=31.164]; Ctsf (cathepsin F) [F(2,12)=264.43]; Sqstm1 (sequesterome 1, p62) [F(2,12)=280.48], and Tfeb [F(2,12)=8.32]; n = 3 independent experiments, *P <.05, **P <.01, ***P <.001, ANOVA with post-hoc Tukey test. Data are presented as mean + s.e.m.
Quantification of autophagic vesicle type in MN-1 WT cells that were transfected with BFP-empty, or transfected with BFP-TFEB. Note marked increase in autolysosomes in MN-1 WT cells expressing BFP-TFEB; n = 3 independent experiments, F(2,87)=4.97, ANOVA with post-hoc Tukey test. **P <.01. Data are presented as mean + s.e.m
Here we see a diagram delineating the number of different human patients lines and corresponding number of induced pluripotent stem cell (iPSC) lines and their clonal derivatives utilized in this study. As shown here, we derived three unique clonal lines per iPSC line, and we created neuron precursor cell (NPC) lines for EACH unique clone shown here. Hence, all NPC experimentation involved the use of three unique clones per human patient sample. This was done to dramatically reduce the likelihood of random variation creating artifact results in affected samples when compared to control samples. The CAG repeat allele size for each patient sample is indicated.
(a) We confirmed expression of the pluripotency markers Oct4, Nanog, Lin28, and Sox2 in all iPSC lines. Representative images, with the nuclei stained with DAPI, are shown here. Scale bar = 40 μM. (b) We confirmed that embryoid bodies generated from iPSC lines display expression of markers from all three germ layers based upon RT-PCR analysis of isolated RNAs. Representative results for endoderm markers (ASP, ESG), mesoderm markers (BRACH, GATA4), and ectoderm markers (NESTIN, PAX6) are shown. (c) We confirmed normal chromosome numbers for all six iPSC lines by karyotyping analysis. Representative normal karyotyping results for one iPSC line are shown here. (d) We confirmed expression of the neural progenitor markers Sox2 and Nestin in NPC lines derived from iPSC lines. Representative images, with the nuclei stained with DAPI, are shown here. Scale bar = 40 μm.
We cultured control and SBMA NPCs in the presence of JC-1 dye, a ratiometric probe that fluoresces differentially based upon whether it is taken up and retained by normally polarized mitochondria (red) or remains in the cytosol (green), and sorted cells based upon the red: green ratio by FACS. Here we see a representative run demonstrating an increased number of SBMA NPCs with a decreased red: green ratio.
Quantification of autophagic vesicle type in control NPCs that were transfected with BFP-empty, or transfected with BFP-TFEB. Note the trend toward an increase in autolysosomes in control NPCs expressing BFP-TFEB, but lack of significant increase; n = 3 independent experiments. Data are presented as mean + s.e.m.
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Cortes, C., Miranda, H., Frankowski, H. et al. Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA. Nat Neurosci 17, 1180–1189 (2014). https://doi.org/10.1038/nn.3787
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