Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner

Amyloid-precursor-protein-lowering small molecules for disease modifying therapy of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia in the elderly with progressive cognitive decline and memory loss. According to the amyloid-hypothesis, AD is caused by generation and subsequent cerebral deposition of β-amyloid (Aβ). Aβ is generated through sequential cleavage of the transmembrane Amyloid-Precursor-Protein (APP) by two endoproteinases termed beta- and gamma-secretase. Increased APP-expression caused by APP gene dosage effects is a risk factor for the development of AD. Here we carried out a large scale screen for novel compounds aimed at decreasing APP-expression. For this we developed a screening system employing a cell culture model of AD. A total of 10,000 substances selected for their ability of drug-likeness and chemical diversity were tested for their potential to decrease APP-expression resulting in reduced Aβ-levels. Positive compounds were further evaluated for their effect at lower concentrations, absence of cytotoxicity and specificity. The six most promising compounds were characterized and structure function relationships were established. The novel compounds presented here provide valuable information for the development of causal therapies for AD

The lectin OS-9 delivers mutant neuroserpin to endoplasmic reticulum associated degradation in familial encephalopathy with neuroserpin inclusion bodies

A feature of neurodegenerative diseases is the intraneuronal accumulation of misfolded proteins. In familial encephalopathy with neuroserpin inclusion bodies (FENIB), mutations in neuroserpin lead to accumulation of neuroserpin polymers within the endoplasmic reticulum (ER) of neurons. Cell culture based studies have shown that ER-associated degradation (ERAD) is involved in clearance of mutant neuroserpin. Here, we investigate how mutant neuroserpin is delivered to ERAD using cell culture and a murine model of FENIB. We show that the ER-lectin OS-9 but not XTP3-B is involved in ERAD of mutant neuroserpin. OS-9 binds mutant neuroserpin and the removal of glycosylation sites leads to increased neuroserpin protein load whereas overexpression of OS-9 decreases mutant neuroserpin. In FENIB mice, OS-9 but not XTP3-B is differently expressed and impairment of ERAD by partial inhibition of the ubiquitin proteasome system leads to increased neuroserpin protein load. These findings show that OS-9 delivers mutant neuroserpin to ERAD by recognition of glycan side chains and provide the first in vivo proof of involvement of ERAD in degradation of mutant neuroserpin

Amyloid-precursor-protein-lowering small molecules for disease modifying therapy of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia in the elderly with progressive cognitive decline and memory loss. According to the amyloid-hypothesis, AD is caused by generation and subsequent cerebral deposition of β-amyloid (Aβ). Aβ is generated through sequential cleavage of the transmembrane Amyloid-Precursor-Protein (APP) by two endoproteinases termed beta- and gamma-secretase. Increased APP-expression caused by APP gene dosage effects is a risk factor for the development of AD. Here we carried out a large scale screen for novel compounds aimed at decreasing APP-expression. For this we developed a screening system employing a cell culture model of AD. A total of 10,000 substances selected for their ability of drug-likeness and chemical diversity were tested for their potential to decrease APP-expression resulting in reduced Aβ-levels. Positive compounds were further evaluated for their effect at lower concentrations, absence of cytotoxicity and specificity. The six most promising compounds were characterized and structure function relationships were established. The novel compounds presented here provide valuable information for the development of causal therapies for AD

Amyloid-Precursor-Protein-Lowering Small Molecules for Disease Modifying Therapy of Alzheimer's Disease

<div><p>Alzheimer's disease (AD) is the most common form of dementia in the elderly with progressive cognitive decline and memory loss. According to the amyloid-hypothesis, AD is caused by generation and subsequent cerebral deposition of β-amyloid (Aβ). Aβ is generated through sequential cleavage of the transmembrane Amyloid-Precursor-Protein (APP) by two endoproteinases termed beta- and gamma-secretase. Increased APP-expression caused by APP gene dosage effects is a risk factor for the development of AD. Here we carried out a large scale screen for novel compounds aimed at decreasing APP-expression. For this we developed a screening system employing a cell culture model of AD. A total of 10,000 substances selected for their ability of drug-likeness and chemical diversity were tested for their potential to decrease APP-expression resulting in reduced Aβ-levels. Positive compounds were further evaluated for their effect at lower concentrations, absence of cytotoxicity and specificity. The six most promising compounds were characterized and structure function relationships were established. The novel compounds presented here provide valuable information for the development of causal therapies for AD.</p></div

Characterization of the six best compounds.

<p>A) Flowchart of the screening of the 10,000 compound library DIVER Set. 10,000 compounds were screened and hits were analyzed by serial dilutions (100 µM, 50 µM, 10 µM, 1 µM). Compounds effective at lower concentrations were checked for cytotoxicity and the non-cytotoxic ones were further analyzed by western blot, ELISA and RT-PCR. B) Structures of the highly potent 5 compounds (A, C–F) in the DIVER Set library which have an specific effect on APP/Aβ-production at a concentration of a minimum of 10 µM and are not cytotoxic. The structure of compound B is added below.</p

Identification of six compounds lowering APP levels at lower concentrations are non cytotoxic.

<p>A) Toxicity assays. Trypan Blue Assay was performed after 3 days of compound incubation. The percentage of surviving cells was calculated. 1%DMSO was used as a negative and 10% DMSO as a positive control. For the MTT assay absorbance of formazan was measured at 570 nm. All experiments were performed in triplets. Results are shown as mean±S.D., n = 3, ***p<0.001. B) The effect of different concentrations was assessed using serial dilutions (100 µM, 50 µM, 10 µM, 1 µM) in four independent experiments. Untransfected HEK 293 cells and solvent-treated APPsw cells were used as controls. A representative example (n = 4) of one blot of the 6 non-cytotoxic compounds is shown.</p

Characterization of a new cell-based assay for screening of APP-lowering small molecules.

<p>A) Expression of APP/Aβ in APPsw transfected HEK 293 cells and in HEK 293 cells. APPsw cells and HEK cells were fixed, labelled with 6E10 antibody and stained with Cy3 anti-mouse IgG for detection of APP/Aβ (red). Nuclei were stained with DAPI (blue). Scale bar 25 µm. B) Supernatants of HEK and APPsw cells were characterized via dot blot with the 6E10 antibody. APPsw cells produce a higher level of sαAPP than control HEK cells. C) Representative example of a dot blots from the screening stage of the study. Supernatants of compound-treated APPsw cells and controls (solvent-treated APPsw, extreme left and right lane). With this approach 80 compounds could be assessed in parallel. Only compounds reducing the signal in four independent experiments were evaluated as “positive”.</p