Age-dependent changes in the expression of ADNP: Differences between Tau-Tg and control mice.

<p>A] Tau-Tg mice western blot analyses. Western blot analyses for ADNP were performed on 3-10.5-month-old-mice (3 or 4 replicates/age group, with 4–5 mice/group). One blot is shown, including extracts from 3-10.5-month-old-mice with actin as a control protein (each lane represents 1 mouse and 4 mice are shown per group). B] Age-dependent changes in the expression of ADNP at the protein level in the cerebral cortex: Differences between Tau-Tg and control mice. The ADNP/actin ratio in each immunoreactive band (in A) was calculated as the ADNP percentage from the total amount of all bands, and then divided by the correlating actin amount of the same sample. ADNP/actin ratios for each group were averaged (n = 5/group, average of 3–4 gel replicates). Quantitative densitometry is shown for four ages: 3, 5.5, 9 and 10.5 months (black bars, Tau-Tg; white bars, littermates not expressing the 4R mutated tau as outlined in the method section, *** <i>p</i><0.001, Tau-Tg vs. control mice, see results section). The 10.5-month point shows only the Tau-Tg ADNP expression. Additional experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Gozes5" target="_blank">[54]</a> compared Tau-Tg and controls revealing no differences at the actin level (densitometry results of 8.4±3.4, vs. 8.3±2.6). In contrast, ADNP levels were significantly decreased in the Tau-Tg 5.5±0.9, vs. controls 11.2±1.35, <i>p</i><0.01 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Gozes5" target="_blank">[54]</a>. The insert is showing a western blot comparing ADNP expression in Tau-Tg (n = 4) and controls (n = 3), at the age of 3 months, each lane represents 1 mouse. C] Age-dependent changes in the expression of ADNP mRNA in the cerebral cortex: Differences between Tau-Tg and control mice. The ADNP mRNA amount in each sample was calculated using the correlated HPRT mRNA amount of the same sample. ADNP mRNA amounts for each group were averaged. [Tg: 1-month-old 9.422+<u>0.92</u>, 3-month-old 2.788±0.407, 5.5-month-old 1.242±0.314, 9-month-old 1.402±0.642. Non-Tg: 1-month-old 2.<u>78+0.28, 3</u>-month-old 1±0.357, 5.5-month-old 1.222±0.535, 9-month-old 2.18±0.27, n = 5/age group]. RQ  =  relative quantity (<a href="http://de-de.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-Profiling/PDFs.Par.83765.File.dat/relative-quant-ct.pdf" target="_blank">http://de-de.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-Profiling/PDFs.Par.83765.File.dat/relative-quant-ct.pdf</a>), (*** <i>p</i><0.01).</p

ADNP expression in the cerebellum is not subject to regulation by transgene expression in other brain areas.

<p>A] Western blot analysis. Expression of nuclear ADNP in the cerebellum of Tau-Tg mice (1) and non-Tg mice (2) is shown by gel electrophoresis and western blot analysis coupled to quantitative densitometry. Results show an increase in ADNP expression at 3 months of age in both transgenic and non-transgenic mice, followed by a decrease at 5.5 and 9 months of age (each experimental group included 4 mice, 3–4 representatives are shown). B] ADNP quantification in the cerebellum. The ADNP amount in each band was calculated as its percentage from the total amount of all bands, and was then divided by the correlated actin amount of the same sample. ADNP/actin amounts of each group were averaged [Tg: 1-month-old 0.43+0.078, 3-month- old 1.794+0.228, 5.5-month-old 1.009+0.08, 9-month-old 0.998+0.195. Non-Tg: 1-month-old 0.532+0.125, 3-month-old 1.798+0.264, 5.5-month-old 0.903+0.196, 9-month-old 0.406+0.166]. Four mice were used for each experimental point and gel electrophoresis was repeated twice, average results are shown. Statistical analysis is shown in the text.</p

Neuropathology of 25 month old iTDP-43^8A mice.

<p>Immunohistochemical detection of ubiquitin revealed rare cells bearing increased ubiquitin staining in the cortex of iTDP-43^8A mice (arrows, A) that was absent in NT animals (B, scale bar = 200 µm). Staining was detected in both nucleus and cytoplasm of affected cells (inset in A, scale bar = 10 µm). (C) In iTDP-43^8A animals hTDP-43 was predominantly nuclear, some cells displaying cytoplasmic localization without aggregation. Cytoplasmic localization was observed in NT and iTDP-43^8A mice using antibodies to total TDP-43 (tTDP-43 Ab1) and phosphorylated forms of TDP-43 (p403/404, p409/410).</p

Early degenerative phenotype in iTDP-43^14A mice at P5 in the absence of FTLD-like TDP-43 aggregation.

<p>(A) Monoclonal antibody to human TDP-43 showed expression at P5 remained restricted to previously characterized regions of hippocampus, cortex and striatum. (B) Western blotting of brain lysate of P5 non-transgenic (NT) and iTDP-43^14A demonstrated increased levels of activated caspase 3 in iTDP-43^14A mice. (C) Abundant caspase 3 immunoreactivity in the cortex of iTDP-43^14A mice that was virtually absent in NT mice, suggestive of elevated cell death in iTDP43^14A compared to NT mice. iTDP-43^14A mice were also characterized by increased ubiquitin staining in the upper layers of the cortex compared to NT mice, which upon higher magnification appeared to be completely diffuse and cytoplasmic. (D) Immunohistochemistry for hTDP-43 and p403/404 and immunofluorescence using antibodies to total TDP-43 and p409/410 TDP-43. Significant amounts of cytoplasmic hTDP-43 were observed in iTDP-43 mice (arrowheads). Note that this cytoplasmic staining was also observed in NT mice (arrowheads) with antibodies to total TDP-43 (tTDP-43 Ab1) and TDP-43 phosphorylated at 409/410 (p409/410). Scale bars in D = 50 µm.</p

ADNP co-immunoprecipitates with splicing factors.

<p>A] Tau-Tg mice western blot analyses. Western blot analyses for Brm and ADNP were performed on 1- and 3-month-old-mice (3 or 4 replicates/age group, n = 5). One blot is shown, using actin as a control protein (results from 3–4 mice/experimental group are shown). B] Brm. Immunoprecipitation, gel electrophoresis (8% polyacrylamide) and western blot analysis was carried out as described in the method section. Increasing concentrations of nuclear protein extracts from the thalamus and subthalamic nucleus of 11-month-old C57BL/129 male mice were subjected to immunoprecipitation with Brm antibody (Sigma), 8% polyacrylamide gel electrophoresis and immunoblotting with either ADNP (BD) (lanes 1–3) or Brm antibody (4–6). The immunoprecipitates included the following gel lanes. Lane 1 & 4: 100 µg protein, 1 µg Brm antibody; lane 2 & 5: 200 µg protein, 1 µg Brm antibody; lane 3 & 6: 300 µg protein, 2 µg Brm antibody. C] PSF. As PSF apears in two molecular weights ∼100 KD and ∼49 KD (a proteolytic cleavage product <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Lee1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-ShavTal1" target="_blank">[34]</a>, we opted to use an immunoprecipitation kit (Pierce) that separates most antibodies used from the antigen that is then subjected to futher protein separation and immunodetection. Mouse cerebellar nuclear-enriched extracts (500 µg) from 8-month-old C57BL/129 male mice were subjected to immunoprecipitation with either 10 µg monoclonal PSF antibody (Sigma), (lanes 1) or 10 µg ADNP antibody (Bethyl, lane 2), followed by western blot analysis (10% polyacrylamide gel) with PSF antibody (1:2000 dilution; Sigma). As a control (lane 3), 500 µg of nuclear-enriched fraction from the thalamus and subthalamic nuclei were immunoprecipitated with 10 µg PSF antibody and subjected to ADNP western blot analysis (1:500 dilution; Bethyl, lane 3). D] Negative controls. Mouse nuclear protein extracts from the thalamus and subthalamic nucleus of 11-month-old C57BL/129 male mice (20 µg) were subjected only to western blot analysis to verify antibody binding (lane 1), the same nuclear-enriched extracts (500 µg) were further subjected to immunoprecipitation in absence of antibody (lane 2), for ADNP and Brm, western analysis was performed as in Fig. 5B PSF antibody (1:2000 dilution; Sigma) and ADNP antibody (1:500 dilution; Bethyl, lane 2). Mouse cerebellar nuclear-enriched extracts, (20 µg, lane 1, and 500 µg, lane 2) were further subjected to immunoprecipitation in absence of antibody, followed by western analysis with Brm antibody (1:1000 dilution; Sigma, as in Fig. 5C). Gel electrophoresis and extracts were as in 5B and C). E, F] Comparison between ADNP+/+ and ADNP+/- mice for Brm and PSF, ADNP interactions, respectively. Immunoprecipitation experiments were carried out as described in the materials and methods section. Western blot analysis was performed on the following samples: 1,5 Positive control (20 µg cortical lysate). 2,6 Negative control (IP without ADNP antibody). 3,7 First elution from the antibody affinity binding and specific antibody detection (as marked on the figure). 4,8 Second elution from the antibody affinity binding and specific antibody detection (as marked). 8% polyacrylamide gels were used for ADNP and Brm and 10% polyacrylamide was used for PSF. G] A suggested model of interaction.</p

Doxycycline treatment which shuts down transgene expression normalizes ADNP and tau 3R expression, while ADNP deficiency may regulate tau isotype expression.

<p>A] ADNP expression with doxycycline: Western blot analysis is depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone-0087383-g002" target="_blank">Fig. 2A</a>, on nuclear extracts with ADNP antibodies. When the ADNP/actin ratios were calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone-0087383-g001" target="_blank">Fig. 1</a>, a significant reduction of ADNP/actin ratio was observed following doxycycline treatment, correlating ADNP expression to tau transgene expression (*<i>p</i><0.05). Five mice were used per an experimental group; each gel lane includes one mouse extract with 3-4 mice representing an experimental group. B] ADNP mRNA quantification. The ADNP mRNA amount in each sample (n = 5/experimental group) was calculated using the correlated HPRT mRNA amount of the same sample. A comparison of Tg and non-Tg as well as Dox+ treatment is shown as for the proteins (panel A, above), (* <i>p</i><0.05). C] Trend toward deregulation of tau expression in the cortex of 2-month-old ADNP+/− mice. The expression of mouse tau 3R and mouse tau 4R (4–5 female mice, cerebral cortex/experimental group) was compared by quantitative real time PCR and results are depicted on the graph, comparing 2-month-old to 9-month-old. There was a significant difference (P<0.05, one tailed, Student's t-test) in tau 4R expression in the 2-month-old ADNP+/- mice compared to ADNP+/+ mice. Data is presented as fold-change (2^{−Δ<i>C</i>}_T), <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Schmittgen1" target="_blank">[55]</a>.</p

Biochemistry of iTDP-43^14A brain lysates at P5.

<p>(A) Western blotting using two antibodies to total TDP-43 (tTDP-43 Ab1 and tTDP-43 Ab2) demonstrated increased levels of low molecular weight species at 35 kDa (arrow) and 25 kDa (arrowhead) in iTDP-43^14A mice relative to NT mice. These species were not observed using antibodies to the C-terminus (405–414) or N-terminus (3–12) of TDP-43. (B) Western blot analysis of high salt (HS), myelin floatation buffer (MFB), sarkosyl (SARK) and urea fractions using antibody to human TDP-43. Note that human TDP-35 (arrow) is present in the urea fraction but is absent from MFB and SARK fractions, N = non-transgenic, T = iTDP-43^14A. (C) Antibody to murine Tdp-43 demonstrated reduction of mTdp-43 in brain compared to NT mice. (D) Quantification of blot in (C), **<i>p</i><0.01, unpaired two tailed <i>t-</i>tes<i>t</i>.</p

Expression of human TDP-43 in iTDP-43^14A and iTDP-43^8A mice in the postnatal period.

<p>Immunohistochemical detection of hTDP-43 expression in cortex (CTX), hippocampus (HIP) and striatum (STR) in iTDP-43^14A (A) and iTDP-43^8A (B). Western analysis of organs demonstrated specificity of hTDP-43 expression to the brain in both iTDP-43^14A (C) and iTDP-43^8A (D) (SC = spinal cord, He = heart, Lu = lung, Li = liver, Ki = kidney, St = stomach, SM = skeletal muscle, Sp = spleen, Br = brain). (E) Brain weight measurement of non-transgenic (NT) and iTDP-43^14A mice at postnatal stages until 2 months of age (P60) (*<i>p</i><0.05, **<i>p</i><0.01, *** p<0.001, unpaired two tailed <i>T-test</i>). (F) Expression of hTDP-43 at indicated postnatal time points for iTDP-43^14A. (G) Expression of hTDP-43 at indicated postnatal time points for iTDP-43^14A (14) compared to iTDP-43^8A (8).</p

Additional file 7: of Differential induction of mutant SOD1 misfolding and aggregation by tau and α-synuclein pathology

Figure S7. Quantification of G85R-SOD1:YFP levels between G85R-SOD1:YFP and rTg4510/G85R-SOD1:YFP mice using direct fluorescence and immunoblot densitometric analysis. Quantification of fluorescence intensity reveals a significantly more intense YFP fluorescence in rTg4510/G85R-SOD1:YFP mice (abbreviated rTg4510-SOD1) compared to G85R-SOD1:YFP controls (abbreviated SOD1) (n = 4) (a). However, immunoblot analysis using an antibody to both mouse and human SOD1 demonstrates no statistical difference between levels of G85R-SOD1:YFP in the two mouse groups (b, c) (n = 3 per genotype). Endogenous mouse SOD1 (mSOD1) was used as a loading control, and was detected on the same blot shown. Statistical analysis was conducted using GraphPad Prism (version 7.0 h). Error bars show mean ± S.D.; unpaired, two tailed, T-test revealed a significant difference in fluorescence intensity in forebrain by genotype (p < 0.01). n.s.; not significant. (TIF 172 kb

Additional file 2: of Differential induction of mutant SOD1 misfolding and aggregation by tau and Îą-synuclein pathology

Figure S2. Low power views of G85R-SOD1:YFP pathology in the spinal cord of bigenic JNPL3-G85R-SOD1:YFP mice (a). The box marks the position of the image shown in Fig. 1d of the main text. Low power view of fluorescence in mice expressing G85R-SOD1:YFP alone (c). Images shows midsagittal brain section (b). Nuclei were stained with DAPI (blue). The left and right arrows are drawn to magnified regions that are shown in the top left and top right of (b), respectively. Images shown are representative of 8 JNPL3-G85R-SOD1:YFP mice and 3 G85R-SOD1:YFP mice. (TIF 8095 kb