Rodent β-amyloid (Aβ) staining following cortical photothrombosis.

<p>Digital photomicrographs showed atypical, Aβ deposits in the thalamus ipsilateral to the cortical lesion (<b>A–D</b>). The inserts <b>A1–D1</b> at higher magnification are taken from the same brain sections. The genotype had a significant effect on rodent Aβ load (two-way ANOVA, <i>P</i><0.05) being more pronounced in non-transgenic mice (<b>E</b>). Values are presented as mean±s.e.m. Scale bar: 500 µm (<b>A–D</b>), 20 µm (<b>A1–D1</b>).</p

Confirmation of the transgenic Aβ phenotype.

<p>Human specific W0-2 staining against Aβ showed a relative high staining intensity in transgenic mice due to intracellular Aβ typical to young transgenic animals (A). A few plaques were observed (arrows). Only some non-specific staining was observed in non-transgenic mice due to degenerative processes (<b>B</b>). Scale bar: 400 µm.</p

Rose Bengal induced cortical photothrombosis.

<p>(<b>A</b>) The cold light was positioned to illuminate the skull 2.4 mm right from Bregma. (<b>B</b>) Nissl-stained sections show a typical photothrombotic lesion in the right sensorimotor cortex (arrows). (<b>C</b>) The genotype had a significant effect on lesion size (two-way ANOVA, <i>P</i><0.05) with smaller lesions in transgenic mice. Values are presented as mean±s.e.m. Scale bar: 1 mm (<b>B</b>).</p

Calcium staining (Alizarin Red) following cortical photothrombosis.

<p>Digital photomicrographs showed atypical, calcium deposits in the thalamus ipsilateral to the cortical lesion (<b>A–D</b>). The inserts <b>A1–D1</b> at higher magnification are taken from the same brain sections. Treatment had a significant effect on calcium accumulation (two-way ANOVA, <i>P</i><0.001) (<b>E</b>). Values are presented as mean±s.e.m. Scale bar: 500 µm (<b>A–D</b>), 20 µm (<b>A1–D1</b>).</p

Cellular localization of rodent Aβ deposits in the thalamus following cortical photothrombosis.

<p>Double immunofluorescence staining for rodent Aβ and NeuN <b>(A)</b> or rodent Aβ and GFAP <b>(B)</b> did not show co-localization in AD transgenic mice (arrows).</p

mTh1 driven expression of hTDP-43 results in typical ALS/FTLD neuropathological symptoms

<div><p>Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models.</p></div

Additional file 1: of Phosphorylation of different tau sites during progression of Alzheimer’s disease

Online Source 9 Double labeling of pSer262 and pSer202/Thr205 tau in the temporal cortex at Braak stage V/VI. Images show different labeling pattern of pSer262 (arrows) and pS202 (white arrowheads) as well as their overlay (yellow arrowheads) (a1) and single fluorescence images (a2,3,4) of case 17. AF: autofluorescence. Scale bar: 20 μm. Online Source 10 Example of measurement procedure of tau pSer262. Objects in the unlabeled autofluorescence channel were detected by thresholding (red in a1). The resulting mask images (a2) were then subtracted from tau pSer262 images to remove autofluorescence (a3). The resulting images were Edge+ filtered (a4) to facilitate threshold-based detection of tau pSer262-positive objects (red outline in a5). These outlines were then loaded onto the raw images to quantify original tau pSer262 signal (red outline in a6). AF: autofluorescence. Scale bar: 20 μm. Online Source 11 Example of detecting ThioS-positive amyloid-β but not NFTs. Image a displays the co-labeling of ThioS (green) and HT7 (red), while images b and c, respectively, show single channel images. ThioS shows intense labeling of plaque-associated β-sheets (b, asterisk) whereas tangles are only weakly labeled (c, arrows) (c). A combination of threshold-based identification of ThioS and size restriction (d‘, green rectangle) enables quantification of ThioS+ plaque labeling (red highlighted) but not tangles (d). ThioS: ThioflavinS. Scale bar: 20 μm. (PDF 599 kb

Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.

<p>(A) Brain homogenates, (E) hippocampal and (I) spinal cord homogenates from ntg, TAR6 and TAR6/6 mice at the age of 1.5, 3 and 6 months were analyzed by Western blotting and probed with the indicated antibodies (long exp. = long time ECL exposure for 30 min). One representative example of 3 is shown. Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 levels normalized to β-tubulin levels of brain (B- D), hippocampal (F-H) and spinal cord (J-L) homogenates. Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between age groups. *p<0.05, **p<0.01, ***p<0.001.</p

Posttranslational changes in TAR6/6 mice.

<p>(A-D) Localization of tTDP-43, CTF-35 and hTDP-43 after cellular fractionation of 3 months old TAR6/6 mice. (A) Cell fractions of midbrain and cortex samples of 3 months old ntg, TAR6 and TAR6/6 mice were analyzed by Western blotting and probed with the indicated antibodies (GAPDH as cytoplasmic and HDAC3 as nuclear marker; long exp. = long time ECL exposure for 30 min). One representative example of 4 is shown (C: cytoplasm, N: nucleus). (B- D) Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 levels in cell fractions (cyt: cytoplasm, nuc: nucleus). Nuclear tTDP-43 and CTF-35 levels of ntg and hTDP-43 of TAR6/6 were set as 100%. (E-H) Soluble and insoluble TDP-43 protein levels in brains of 3 months old TAR6/6 mice. Brain samples were separated into RIPA soluble (R) and RIPA insoluble / UREA fractions (U). Three representative examples of 5 are shown. Fractions were analyzed by Western blotting and probed with the indicated antibodies (long exp. = long time ECL exposure for 30 min). Coomassie Blue staining was performed as loading control for UREA fraction. (F-H) Densitometric analysis of tTDP-43, CTFs and hTDP-43 levels in RIPA and UREA fractions. For tTDP-43 and CTF evaluation, RIPA fraction of ntg was set as 100%, whereas for hTDP-43 analysis, RIPA sample of TAR6/6 was set as 100%. (B-D; F-H) Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between fractions. *p<0.05, **p<0.01, ***p<0.001.</p

Quantification of hTDP-43 expression in ntg, TAR6 and TAR6/6 mice.

<p>Quantitative hTDP-43 expression in hypothalamus (A), medulla oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS samples. (D, E) Representative overview image and magnifications of hTDP-43 labeling of a brain section (olfactory bulb (1), cortex (2), hippocampus (3), thalamus (4), hypothalamus (5) and medulla oblongata (6)) of 3 months old TAR6/6 (D) and ntg (E) mice. Scale bars: Overview images = 1000 μm; magnification images = 20 μm. (A, B) 1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5; TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (C) n as in A, B exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by Bonferroni‘s <i>post-hoc</i> test. Mean+SEM. *significances between genotypes, ^#significances between age groups. *p<0.05, **p<0.01, ***p<0.001.</p