Supplemental document 1
Transcript analyses
Meaning of Sqstm1 and Tdp-43 levels for SIL1 patho-physiology in wz muscle
Increased levels of Sqstm1 may either indicate increased autophagic activity or reduced autophagic
clearance. Therefore, we performed quantitative PCR studies of Sqstm1 transcript levels in 26 week
old mice (see below) and found similar levels in wz and wt animals (Fig. 5f), indicating impaired
autophagic clearance of SqStm1. This observation is in line with the formation of autophagic vacuoles
observed by electron microscopy (see results section of main document).
We also asked whether TAR DNA-binding protein-43 (Tdp-43) is involved in these processes. Tdp-43
is a nuclear protein functioning in the regulation of transcription and mRNA splicing with emerging
roles in neurodegeneration [37]. Accumulation of TDP-43 is a common hallmark of myopathies with
rimmed vacuoles [36] and could also be linked to ER stress [6]. Efficient degradation of TDP-43
aggregates is mediated by autophagy [81]. Our quantitative PCR studies (see below) showed slightly
elevated levels (2 fold increase) (Fig. 5f) and immunoblots of Tdp-43 protein revealed also a significant
increase in wz mouse quadriceps muscles (Figs. 5c, 5f). Prima facie, simultaneously increased Tdp-43
transcript and proteins levels do not support the assumption that autophagic clearance is impaired.
However, considering the above described pattern of Sqstm1 expression, the increase in Tdp-43
transcript levels – presumably leading to elevated protein levels – could be related to the known
function of TDP-43 in RNA decay and transcriptional repression of DNA [37]. Insufficient autophagic
clearance of the TDP-43 protein could also lead to this build-up.
RNA isolation, Xbp1 splicing assay and quantitative PCR studies of Sqstm1 and Tdp-43:
Total RNA was extracted from quadriceps muscles using RNeasy Mini Kit (Qiagen) according to
manufacturer´s instructions. First strand cDNA was synthesized with the iScript cDNA Synthesis Kit
from BioRad in accordance with the supplier’s instructions. 100 ng of the cDNA were used to amplify
an Xbp1 amplicon harbouring the splice site. For PCR conditions see Suppl. Tab. 2a. Primer
sequences for the amplification were as follows: Xbp1_F: 5´-AAACAGAGTAGCAGCGCAGACTGC-3´;
Xbp1_R: 5´-TCCTTCTGGGTAGACCTCTGGGAG-3´. The PCR products were tested on a 1 %
agarose gel and digested with PstI restriction endonuclease (New England Biolabs) according to the
manufacturer’s instructions for 16 hours at 37°C. Restriction products were separated on a 2 %
agarose gel to visualize differences between unspliced (Xbp1u) and spliced (Xbp1s) fragments.
Real-time quantification of Sqstm1 and Tdp-43 relative to Gapdh mRNA was performed using
SsoAdvanced SYBR Green Supermix (BioRad) and a CFX Connect Real-Time PCR Detection
System (BioRad). Real-Time PCR primers for the indicated genes were designed with Primer 3 and
synthesized by Metabion (Martinsried, Germany). Quantitative RT-PCR was performed in triplicate in
20 μl reaction volumes consisting of 10 µL SsoAdvanced SYBR Green Supermix, 50 pmol forward and
reverse primers, 8 µl RNAse free water and 1 µl cDNA. For PCR conditions see Suppl. Tab. 2b.
Primer sequences for the amplification were as follows: Sqstm1_F 5´- GACACCCACTACCCCAGAAA
-3´; Sqstm1_R 5´- ATCTGTTCCTCTGGCTGTCC -3´; Tdp-43_F 5´- CCCCTGGAAAACAACTGAGC 3´; Tdp-43_R 5´- ACCCTTTCGAGTGACCAGTT -3´; Gapdh_F 5´- AACTTTGGCATTGTGGAA -3´;
Gapdh_R 5´- ACACATTGGGGGTAGGAA -3´. The analysis of the gene expression data were made
with the CFX96 analysis software which use the 2-ΔΔCT method [43].
a
Temperature
Time
Cycles
95°C
30 sec
95°C
5 sec
58°C
30 sec
72°C
10 min
final
Temperature
Time
Cycles
95°C
30 s
95°C
5s
55°C
30 s
35x
b
65°C – 95°C
PCR conditions: (a) Xbp1 splicing studies. (b) qPCR studies
c
40x
0,5°C per step
d
Analyses of Xbp1 splicing in 16-, 26-, 52- and 84-week old animals (c) as well as in three animal pairs with the
age of 26 weeks (d). Enhanced splicing of Xbp1 is detectable in quadriceps muscles of wz animals compared to
wt littermates.
e
Sqstm1
1,4
1,2
1
0,8
0,6
0,4
0,2
0
26 week control animals 1,2,3
f
26 week woozy animals 1,2,3
Tdp-43
3
2,5
2
1,5
1
0,5
0
26 week control animals 1,2,3
26 week woozy animals 1,2,3
Quantitative PCR studies of Sqstm1 (e) and Tdp-43 (f) in three animal pairs with the age of 26 weeks. Whereas
Sqstm1 transcript levels show a low grade decrease, the transcript levels of Tdp-43 are more than 2-fold
increased in wz quadriceps muscles.