Data_Sheet_1_Modeling sporadic juvenile ALS in iPSC-derived motor neurons explores the pathogenesis of FUSR503fs mutation.doc

IntroductionFused in sarcoma (FUS) mutations represent the most common genetic etiology of juvenile amyotrophic lateral sclerosis (JALS), for which effective treatments are lacking. In a prior report, we identified a novel FUS mutation, c.1509dupA: p. R503fs (FUSR503fs), in a sporadic JALS patient.MethodsThe physicochemical properties and structure of FUSR503fs protein were analyzed by software: Multi-electrode array (MEA) assay, calcium activity imaging assay and transcriptome analysis were used to explore the pathophysiological mechanism of iPSC derived motor neurons.ResultsStructural analysis and predictions regarding physical and chemical properties of this mutation suggest that the reduction of phosphorylation and glycosylation sites, along with alterations in the amino acid sequence, may contribute to abnormal FUS accumulation within the cytoplasm and nucleus of induced pluripotent stem cell– derived motor neurons (MNs). Multi-electrode array and calcium activity imaging indicate diminished spontaneous electrical and calcium activity signals in MNs harboring the FUSR503fs mutation. Transcriptomic analysis reveals upregulation of genes associated with viral infection and downregulation of genes involved in neural function maintenance, such as the ATP6V1C2 gene. Treatment with ropinirole marginally mitigates the electrophysiological decline in FUSR503fs MNs, suggesting the utility of this cell model for mechanistic exploration and drug screening.DiscussioniPSCs-derived motor neurons from JALS patients are promising tools for drug screening. The pathological changes in motor neurons of FUSR503fs may occur earlier than in other known mutation types that have been reported.</p

Von Willebrand Factor Inhibits Mature Smooth Muscle Gene Expression through Impairment of Notch Signaling

<div><p>Von Willebrand factor (vWF), a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascular disease states. Previous studies have implicated potential non-hemostatic functions of vWF, but signaling mechanisms underlying its effects are currently undefined. We present evidence that vWF breaches the endothelium and is expressed in a transmural distribution pattern in cerebral small vessel disease (SVD). To determine the potential molecular consequences of vWF permeation into the vessel wall, we also tested whether vWF impairs Notch regulation of key smooth muscle marker genes. In a co-culture system using Notch ligand expressing cells to stimulate Notch in A7R5 cells, vWF strongly inhibited both the Notch pathway and the activation of mature smooth muscle gene promoters. Similar repressive effects were observed in primary human cerebral vascular smooth muscle cells. Expression of the intracellular domain of NOTCH3 allowed cells to bypass the inhibitory effects of vWF. Moreover, vWF forms molecular complexes with all four mammalian Notch ectodomains, suggesting a novel function of vWF as an extracellular inhibitor of Notch signaling. In sum, these studies demonstrate vWF in the vessel wall as a common feature of cerebral SVD; furthermore, we provide a plausible mechanism by which non-hemostatic vWF may play a novel role in the promotion of vascular disease.</p></div

Effect of vWF on Notch signaling.

<p>A co-culture system was used to assess the effects of vWF on Notch signaling in H460 (A) and A7R5 (B) cells reflected by ligand stimulation of HES-luciferase. Experiments were performed in either serum-containing or serum-free media supplemented with vWF (200 ng/ml). Representative results from four experiments done in triplicate are shown. Please note significant changes (p<0.05) induced by Notch ligands (+) or by vWF (*).</p

Arterial deposition of vWF in human cerebral small vessel disease.

<p>Pathologically thickened vessels of the cerebral white matter were examined by immunohistochemistry using antibodies against vWF in (A) vascular dementia with leukoencephalopathy and small vessel disease (73 year old man); (B) dementia and ischemic stroke (88 year old woman); (C) multiple infarct dementia (86 year old female); (D) sickle cell disease (25 year old female); (E) radiation necrosis following treatment for glioblastoma multiforme (63 year old female); (F) radiation necrosis following treatment for oligodendroglioma (36 year old man); (G) CADASIL (58 year old man); (H) control vessel with expected endothelial distribution of vWF. Photographs were taken at 400× magnification. Similar positive staining was seen with a mouse monoclonal antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075808#pone.0075808.s001" target="_blank">Supplemental Figure 1</a>). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075808#pone.0075808.s002" target="_blank">Supplemental Figure 2</a> for additional control studies which feature (1) omission of primary antibody, (2) addition of vWF protein to block staining, and (3) verification of staining pattern with an independent polyclonal antibody.</p

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The camera, installed on an unsecured gimbal without a fixed lock, experiences minor pose shifts with a gentle tap

Effect of vWF on cell autonomous, intracellular Notch activation.

<p>A7R5 cells were first transfected with vector or NICD (from NOTCH3) prior to co-culture with ligand expressing cells (with or without vWF 200 ng/ml). Transcript quantitation was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075808#pone-0075808-g006" target="_blank">Figure 6</a> for: smooth muscle actin (A; SMA), SM22 (B), smooth muscle myosin heavy chain (C; MHC), and calponin (D). Significant changes (p<0.05) induced by Jagged1 are marked (+). In the presence of vWF, there was no upregulation of mRNA by Jagged1; in addition, after NICD transfection, transcripts were unaffected by Jagged1, with or without vWF (NS; no differences between L and Jagged). All transcript levels were greater in NICD transfected cells compared to control cells cocultured with L cells, with or without vWF (p<0.05; not marked).</p

Effect of vWF on Notch-regulation of the SMA promoter.

<p>The effect of vWF on Notch regulation of a cloned SMA promoter driving firefly luciferase was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075808#pone-0075808-g004" target="_blank">Figure 4</a>. vWF was added at 200 ng/ml. Significant changes (p<0.05) induced by Notch ligands (+) or by vWF (*) are marked. No differences were induced by Notch ligand in DN-MAML or NICD transfected groups, which were constitutively repressed and activated, respectively.</p

Mature vWF, smooth muscle actin, and vWF precursor protein expression in human small vessel disease.

<p>The same CADASIL cortical brain artery was examined by immunohistochemistry for mature vWF (A), smooth muscle actin (B), and vWF-pp (C). This vessel demonstrated transmural staining for mature vWF, subendothelial reactivity for SMA, and intimal-specific vWF-pp immunoreactivity. As in other SVD tissues, the staining for vWF-pp was not observed beyond the endothelium, but many segments of arteries were devoid of vWF-pp, suggesting inhomogeneity of endothelial coverage. (D) Confirmatory double stains were performed in to localize vWF-pp (dark blue stain on in inside of the artery) mature vWF (brown stain that extends into the arterial wall). Mature vWF is found in regions that lack vWF-pp staining. Photographs were taken at 1000×.</p

Smooth muscle gene regulation by Notch and vWF.

<p>The effects of Notch signaling and vWF on expression of four core smooth muscle genes in A7R5 cells were determined by quantitative reverse transcriptase PCR. Gamma-secretase inhibitor DAPT was applied or purified vWF (200 ng/ml) was added to the media. Significant changes (p<0.05) induced by Jagged are marked (+). DAPT or vWF incubation fully repressed Jagged stimulated gene expression (*; p<0.05).</p

Interactions between vWF and Notch proteins in cultured cells.

<p>Co-immunoprecipitation assays were performed in transfected cultures to demonstrate molecular complex formation between vWF, Notch ectodomains, Jagged ectodomain, and full length NOTCH3 protein. (A–E) Cultured 293A cells were cotransfected with plasmids encoded vWF and/or Notch or Jagged1 ectodomains or full length NOTCH3, as indicated; vWF and ectodomain clones were C-terminally tagged to facilitate detection and pull down. Input cell lysates were immunoblotted (IB) to confirm expression. Monoclonal antibody-treated lysates were also immunoprecipitated with protein G-agarose. Precipitated proteins (IP) were analyzed by immunoblotting. Co-precipitation of vWF and CADASIL-causing NOTCH3 mutant proteins was assessed in (D–E). In negative control experiments (shown in each panel), IP of single transfections did not pull down proteins. Apparent molecular weights of proteins were >250 kDa (vWF; a doublet), approximately 180 kDa (Notch1-V5 and Notch2-V5), and 150 kDa (Notch3-HA and Notch4-V5). (F) vWF (200 ng/ml) was added to co-cultures of HRP- NOTCH3 and mouse fibroblasts to measure vWF-regulation of Notch3 transendocytosis. vWF reproducibly impaired trans-endocytosis of NOTCH3 into the signal-sending cells in three independent experiments (p<0.05).</p