Subcellular TDP-43 distribution in MNs of mutant <i>hSOD1</i>^<i>G93A</i> mice <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A-C’</b>) Representative photomicrographs depicted in the ventral cervical spinal cord of control <b>(A, A’)</b>, presymptomatic <b>(B, B’)</b> and diseased <b>(C, C’)</b> <i>hSOD1</i>^<i>G93A</i> transgenic mice. In MNs (green), the cytoplasmic TDP-43 signal (red) did not exceed background levels, whereas the nuclear TDP-43 signal was strong in specimens of both control and <i>hSOD1</i>^<i>G93A</i> mice. Similar to <i>in vivo</i> conditions, cultured <b>(D-E’)</b> non-transgenic <b>(D-D’)</b> and transgenic <b>(E, E’)</b> MNs displayed a comparably low cytoplasmic TDP-43 signal, which was not increased in MNs carrying the <i>hSOD1</i>^<i>G93A</i> mutation. Nuclear TDP-43 location was verified by DAPI counterstaining (blue). <b>(F)</b> Representative western blot of TDP-43 in cytoplasmic (CP), soluble nuclear (sNE) and chromatin-bound nuclear (cNE) subcellular extracts derived from ventral cervical and thoracic spinal cords of control (ctrl), presymptomatic (PS) and diseased (DS) hSOD1^G93A mice. Under resting conditions, detection of cytoplasmic p65 (RelA) was utilized as a loading control and for purity validation of subcellular fractions. The cytoplasmic fraction was free of TDP-43, whereas TDP-43 was present in both nuclear extracts, with a higher abundance in the cNE than the sNE fraction. Scale bars depict 20 μm (A-C’) and 10 μm (D-E’).</p

TE regulation in diseased <i>hSOD1</i>^<i>G93A</i> mutants <i>in vivo</i>.

<p>The relative levels of the TE <i>L1</i>_<i>orl</i> transcripts were significantly increased in the spinal cord of diseased <i>hSOD1</i>^<i>G93A</i> mice compared with controls. In contrast, spinal transcripts of <i>Ago2</i>, as part of the Ago2-RISC TE silencing complex, did not differ significantly between control and fALS-like conditions. **, <i>p</i> < 0.01. n.s., not significant.</p

DNA strand breaks and TDP-43 mislocation are absent in the murine <i>hSOD1^G93A</i> model of amyotrophic lateral sclerosis <i>in vivo</i> and <i>in vitro</i>

<div><p>Mutations in the human <i>Cu/Zn superoxide dismutase type-1</i> (<i>hSOD1</i>) gene are common in familial amyotrophic lateral sclerosis (fALS). The pathophysiology has been linked to, e.g., organelle dysfunction, RNA metabolism and oxidative DNA damage conferred by SOD1 malfunction. However, apart from metabolically evoked DNA oxidation, it is unclear whether severe genotoxicity including DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), originates from loss of function of nuclear SOD1 enzyme. Factors that endogenously interfere with DNA integrity and repair complexes in <i>hSOD1</i>-mediated fALS remain similarly unexplored. In this regard, uncontrolled activation of transposable elements (TEs) might contribute to DNA disintegration and neurodegeneration. The aim of this study was to elucidate the role of the fALS-causing <i>hSOD1</i>^<i>G93A</i> mutation in the generation of severe DNA damage beyond well-characterized DNA base oxidation. Therefore, DNA damage was assessed in spinal tissue of <i>hSOD1</i>^<i>G93A</i>-overexpressing mice and in corresponding motor neuron-enriched cell cultures <i>in vitro</i>. Overexpression of the <i>hSOD1</i>^<i>G93A</i> locus did not change the threshold for severe DNA damage <i>per se</i>. We found that levels of SSBs and DSBs were unaltered between <i>hSOD1</i>^<i>G93A</i> and control conditions, as demonstrated in post-mitotic motor neurons and in astrocytes susceptible to replication-dependent DNA breakage. Analogously, parameters indicative of DNA damage response processes were not activated <i>in vivo</i> or <i>in vitro</i>. Evidence for a mutation-related elevation in TE activation was not detected, in accordance with the absence of TAR DNA binding protein 43 (TDP-43) proteinopathy in terms of cytoplasmic mislocation or nuclear loss, as nuclear TDP-43 is supposed to silence TEs physiologically. Conclusively, the superoxide dismutase function of SOD1 might not be required to preserve DNA integrity in motor neurons, at least when the function of TDP-43 is unaltered. Our data establish a foundation for further investigations addressing functional TDP-43 interaction with ALS-relevant genetic mutations.</p></div

DSB events in spinal MNs <i>in vivo</i> and <i>in vitro</i>.

<p><b>(A-C’)</b> Representative photomicrographs of 53BP1 immunoreactivity in the ventral cervical spinal cord of control <b>(A, A’)</b>, presymptomatic <b>(B, B’)</b> and diseased <i>hSOD1</i>^<i>G93A</i> transgenic mice <b>(C, C’)</b>. <b>(A-C’)</b> In MNs (green), 53BP1-positive foci (red) occurred at a very low frequency (arrowheads), independently of the underlying condition. <b>(D-E’)</b> Similarly, <i>in vitro</i> nuclear 53BP1 foci (arrowheads) were rarely detected both in control <b>(D, D’)</b> and <i>hSOD1</i>^<i>G93A</i> transgenic MNs <b>(E, E’)</b>. <b>(F-I)</b> Representative photomicrographs of γH2AX immunoreactivity <i>in vitro</i>. <b>(F-G’)</b> Nuclear γH2AX-positive foci (red; arrowheads) remained as single events both in MNs of non-transgenic <b>(F, F’)</b> and <i>hSOD1</i>^<i>G93A</i> donors <b>(G, G’)</b>. <b>(H, I)</b> <i>In vitro</i>, γH2AX-positive foci exhibited an apparently higher abundance in astrocytes both in control <b>(H)</b> and transgenic <b>(I)</b> cultures compared with MNs. <b>(J)</b> The amount of γH2AX foci in astrocytes <i>in vitro</i> did not differ significantly between control and transgenic astrocytes as quantified by means of integrated density per nucleus. Cell nuclei were counter-stained with DAPI (blue) to confirm the nuclear signal locations. n.s., not significant. Scale bars depict 20 μm (A-C’) and 10 μm (D-I).</p

Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis-1

<p><b>Copyright information:</b></p><p>Taken from "Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis"</p><p>BMC Neurology 2006;6():17-17.</p><p>Published online 25 Apr 2006</p><p>PMCID:PMC1459868.</p><p>Copyright © 2006 Grosskreutz et al; licensee BioMed Central Ltd.</p>tter atrophy in the precentral and postcentral gyrus bilaterally, which extended from the primary motor cortex to premotor, parietal and frontal regions bilaterally (displayed at p = 0.001, uncorrected, extended threshold 100 voxels). The color bar represents the T-score. The differences between the groups are superimposed on a standard normalized T1-weighted image. Images are shown in neurological convention

Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis-0

<p><b>Copyright information:</b></p><p>Taken from "Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis"</p><p>BMC Neurology 2006;6():17-17.</p><p>Published online 25 Apr 2006</p><p>PMCID:PMC1459868.</p><p>Copyright © 2006 Grosskreutz et al; licensee BioMed Central Ltd.</p>rt was mildly affected by disease. Note that some patients had barely gone into rapid progression whereas others have remained stable at a high score

Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis-2

<p><b>Copyright information:</b></p><p>Taken from "Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis"</p><p>BMC Neurology 2006;6():17-17.</p><p>Published online 25 Apr 2006</p><p>PMCID:PMC1459868.</p><p>Copyright © 2006 Grosskreutz et al; licensee BioMed Central Ltd.</p>l grey matter volumes (modulated data, B) in ALS patients versus controls are displayed within a 'glass brain' view. Note that only the comparison of modulated data survived the correction for multiple comparisons specified a priori. Images are displayed at p = 0.001, uncorrected, extended threshold 100 voxels and shown in neurological convention

Additional file 1: of Dysregulation of chemokine receptor expression and function in leukocytes from ALS patients

ALS patient information and supplemental material and methods. (DOCX 97 kb

International Survey of ALS Experts about Critical Questions for Assessing Patients with ALS

<p><i>Objective</i>: To define an applicable dataset for ALS patient registries we weighted specific clinical items as scored by worldwide ALS experts. <i>Methods</i>: Sixty participants were invited based on relevant clinical work, publications and personal acquaintance. They rated 160 clinical items consensually agreed by the members of our project, incorporating specialists from five European Centres. Scoring scheme was defined as: 1 – essential; 2 – important; 3 – not very important. A mixed effect model was applied to rank items and to find possible correlations with geographical region (Europe vs. outside Europe). <i>Results</i>: We received 40 responses, 20 from Europe and 20 from outside; 42/160 data were scored as essential by >50% of the respondents, including: date of birth, gender, date of disease onset, date of diagnosis, ethnicity, region of onset, predominant upper neuron (UMN) or lower motor neuron (LMN) impairment, proximal versus distal weakness, respiratory symptoms, dysarthria, weight loss, signs of LMN/UMN involvement, emotional incontinence, cognitive changes, respiratory signs, neck weakness, body mass index, ALSFRS-R at entry, ALSFRS-R subscores at entry, timing and pattern of spreading and staging, electromyography, spirometry, MRI, CK level, riluzole intake, genetic background, history of physical exercise and previous and current main occupation. Four components were scored as non-relevant, including place of birth, blood pressure and pain at onset. There was no significant difference between regions (European vs. non-European countries). <i>Conclusions</i>: Our study identified a consensual set of clinical data with 42 specific items that can be used as a minimal data set for patient registers and for clinical trials.</p

Intensity response in key CST areas and control regions.

<p>Mean intensity inside ROIs along the CST and in ALS independent white matter regions are shown. The absolute difference of the mean in patients versus controls is referenced to the total white matter intensity spread to allow comparability to other study settings. Significances are given as * (p<0.05), ** (p<0.01), ***(P<0.001) or as not significant (n.s.). The highest response was found in the left PLIC (8.7% higher intensity in patients than in controls). The non ALS disease related white matter regions did not significantly differ between patients and controls.</p