Gene Expression Profiling in Tibial Muscular Dystrophy Reveals Unfolded Protein Response and Altered Autophagy

Peer reviewe

Summary of patient and control muscle biopsies.

<p><i>Tibialis anterior</i> (TA), <i>tibialis posterior</i> (TP), <i>gastrocnemius lateralis</i> (GL), <i>gastrocnemius medialis</i> (GM), <i>soleus</i> (SOL), <i>extensor hallucis longus</i> (EHL), <i>extensor digitorum longus</i> (EDL), thigh posterior (THP), immunofluorescence microscopy (IF), western blotting (WB) and immunohistochemistry (IHC). The expression array controls (C1, C2 & C5) and (C3 & C4) were collected from amputation material from the distal lower limb muscles of two individuals.</p

Immunofluorescent microscopy of TMD and control biopsies.

<p>(A) HSPA5 staining showed the presence of cytoplasmic granular dots in non-vacuolated TMD muscle fibres. The LC3B channel showed the area has no vacuolated fibres and no autofluorescent material present. (B) A control of HSPA5/LC3B double staining was included for comparison. (C) LAMP2 and LC3B double staining of a TMD muscle fibre showed a representative rimmed vacuole with massive accumulation of LC3B inside and negligible signal from LAMP2 around the edge of the fibre. The top right corner of the LC3B image showed a rimmed vacuole in bright field. (D) A control of LAMP2/LC3B double staining was included for comparison. All images are nuclear counterstained with DAPI (blue signal). Sections are from samples TMD 6 and Ctrl 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090819#pone-0090819-t001" target="_blank">Table 1</a>. Scale bar 50 µm.</p

Histology and immunohistochemical analysis of TMD muscle biopsies.

<p>(A) Herovici histology staining of TMD muscle sections showing rimmed vacuolar regions within the fibres. (B) Immunohistochemical (IHC) analysis of TMD muscle sections stained for ubiquitin showing localised cytoplasmic ubiquitin containing inclusions in atrophic and rimmed vacuolated fibres and immunoreactivity at the edge of rimmed vacuoles. (C) Autophagosome marker LC3B staining showed strong immunoreactivity within the rimmed vacuolar regions. (D) VCP staining showed immunoreactivity in bodies within a large subset of rimmed vacuoles. (E) p62 staining showed immunoreactivity in rimmed vacuoles and granular immunoreactive dots (shown by an arrow) in an atrophic fibre. (F) Lysosomal protein LAMP2 staining showed no overall increase in rimmed vacuolated fibres. Two separate TMD fibres (A–C and D–F) with rimmed vacuoles in serial sections are shown. Controls of (G) Herovici staining, (H) ubiquitin, (I) LC3B, (J) VCP, (K) p62 and (L) LAMP2 IHC are included for comparison. Sections are from samples TMD 6 and Ctrl 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090819#pone-0090819-t001" target="_blank">Table 1</a>. Scale bar 50 µm.</p

Quantitative real-time PCR analysis of key components of selected pathways.

<p>Expression of (A) <i>HSPA5</i>, (B) <i>HSPB1</i>, (C) <i>ERN1</i>, (D) spliced (s) <i>XBP1</i>, and (E) <i>JUN</i> normalised to <i>GAPDH</i> by quantitative real-time PCR from muscle biopsy total RNA (arbitrary units). *Indicates statistical significance in an unpaired <i>t</i>-test (<i>P</i><0.05) comparison to the control group. The average and standard deviation of five TMD and three control biopsies each performed in triplicate are shown.</p

Protein level analysis of UPR and ERAD in TMD biopsies.

<p>(A) Western blotting showed HSPA5 increases in two in five samples when compared to controls. (B) Immunoblotting VCP showed an increase in full length VCP in two TMD samples and an extra band (∼70 kDa) just below the full length VCP in a third sample. Bands at approx. 25 kDa (red boxes) are observed in three of the TMD samples. (C) Samples were also blotted with p97D179 antiserum specific for VCP cleaved at D179 and show bands at approx. 25 kDa in three of the same samples. Total protein content is shown by the myosin band in Coomassie blue staining and showed equal sample loading. A graph of the quantification of the VCP cleavage product (25 kDa) signals normalised to the Coomassie myosin band showed a trend towards being increased in TMD in a <i>t-</i>test (<i>P</i> = 0.077).</p

Hierarchal clustering of TMD and control expression profiles.

<p>Clustering of differentially expressed probes using centroid linkage and euclidean similarity in GeneSpring GX v11.0. The expression profiles of TMD (T1–T5) and controls (C1–C5) are represented as a heatmap (blue = low expression, red = high expression). A list of significantly changed probes is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090819#pone.0090819.s002" target="_blank">Table S2</a>.</p

Mutant (CCTG)n Expansion Causes Abnormal Expression of Zinc Finger Protein 9 (ZNF9) in Myotonic Dystrophy Type 2

The mutation that underlies myotonic dystrophy type 2 (DM2) is a (CCTG)n expansion in intron 1 of zinc finger protein 9 (ZNF9). It has been suggested that ZNF9 is of no consequence for disease pathogenesis. We determined the expression levels of ZNF9 during muscle cell differentiation and in DM2 muscle by microarray profiling, real-time RT-PCR, splice variant analysis, immunofluorescence, and Western blotting. Our results show that in differentiating myoblasts, ZNF9 protein was localized primarily to the nucleus, whereas in mature muscle fibers, it was cytoplasmic and organized in sarcomeric striations at the Z-disk. In patients with DM2, ZNF9 was abnormally expressed. First, there was an overall reduction in both the mRNA and protein levels. Second, the subcellular localization of the ZNF9 protein was somewhat less cytoplasmic and more membrane-bound. Third, our splice variant analysis revealed retention of intron 3 in an aberrant isoform, and fourth quantitative allele-specific expression analysis showed the persistence of intron 1 sequences from the abnormal allele, further suggesting that the mutant allele is incompletely spliced. Thus, the decrease in total expression appears to be due to impaired splicing of the mutant transcript. Our data indicate that ZNF9 expression in DM2 patients is altered at multiple levels. Although toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for the differences in DM1