Absolute quantification of cholesterol from thin tissue sections by silver-assisted laser desorption ionization mass spectrometry imaging.

Cholesterol is essential to all animal life, and its dysregulation is observed in many diseases. For some of these, the precise determination of cholesterol's histological location and absolute abundance at cellular length scales within tissue samples would open the door to a more fundamental understanding of the role of cholesterol in disease onset and progression. We have developed a fast and simple method for absolute quantification of cholesterol within brain samples based on the sensitive detection and mapping of cholesterol by silver-assisted laser desorption ionization mass spectrometry imaging (AgLDI MSI) from thin tissue sections. Reproducible calibration curves were generated by depositing a range of cholesterol-D7 concentrations on brain homogenate tissue sections combined with the homogeneous spray deposition of a non-animal steroid reference standard detectable by AgLDI MSI to minimize experimental variability. Results obtained from serial brain sections gave consistent cholesterol quantitative values in very good agreement with those obtained with other mass spectrometry-based methods.

The many faces of vascular cognitive impairment.

This Preface introduces the articles of the special issue on "Vascular Dementia" in which several recognized experts provide an overview of this research field. The brain is a highly vascularized organ and consequently, vascular dysfunction and related pathways affect cognitive performance and memory. Vascular dementia or vascular cognitive impairment is the second most common type of dementia after Alzheimer's disease, and both disorders often occur in parallel. With this special issue, we hope to provide insight and a stimulating discussion for the future development of this research field. This article is part of the Special Issue "Vascular Dementia".

Characterization of intermediate steps in amyloid beta (Aß) production under near-native conditions.

Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aß peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aß and in particular Aß42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aß production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aß40 and Aß42 and an increase in shorter Aß peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aß51 to Aß31/Aß30 and from Aß49 to Aß30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aß48 to Aß38 compared with Aß49 to Aß37. These findings correlate with an increase in the Aß42/40 ratio. GSMs caused a decrease in Aß40 and Aß42 and an increase in Aß37 and Aß38 paralleled by an increase of the intermediates Aß40-38 and Aß42-39. Collectively, these data provide a thorough characterization of all intermediate steps in Aß production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aß generation.

Circulating plasma fibronectin affects tissue insulin sensitivity, adipocyte differentiation, and transcriptional landscape of adipose tissue in mice.

Plasma fibronectin (pFN) is a hepatocyte-derived circulating extracellular matrix protein that affects cell morphology, adipogenesis, and insulin signaling of adipocytes in vitro. In this study, we show pFN accrual to adipose tissue and its contribution to tissue homeostasis in mice. Hepatocyte-specific conditional Fn1 knockout mice (Fn1-/-ALB) show a decrease in adipose tissue FN levels and enhanced insulin sensitivity of subcutaneous (inguinal), visceral (epididymal) adipose tissue on a normal diet. Diet-induced obesity model of the Fn1-/-ALB mouse showed normal weight gain and whole-body fat mass, and normal adipose tissue depot volumes and unaltered circulating leptin and adiponectin levels. However, Fn1-/-ALB adipose depots showed significant alterations in adipocyte size and gene expression profiles. The inguinal adipose tissue on a normal diet, which had alterations in fatty acid metabolism and thermogenesis suggesting browning. The presence of increased beige adipocyte markers Ucp1 and Prdm16 supported this. In the inguinal fat, the obesogenic diet resulted in downregulation of the browning markers and changes in gene expression reflecting development, morphogenesis, and mesenchymal stem cell maintenance. Epididymal adipose tissue showed alterations in developmental and stem cell gene expression on both diets. The data suggests a role for pFN in adipose tissue insulin sensitivity and cell profiles.

The heat shock response is modulated by and interferes with toxic effects of scrapie prion protein and amyloid ß.

The heat shock response (HSR) is an evolutionarily conserved pathway designed to maintain proteostasis and to ameliorate toxic effects of aberrant protein folding. We have studied the modulation of the HSR by the scrapie prion protein (PrP(Sc)) and amyloid ß peptide (Aß) and investigated whether an activated HSR or the ectopic expression of individual chaperones can interfere with PrP(Sc)- or Aß-induced toxicity. First, we observed different effects on the HSR under acute or chronic exposure of cells to PrP(Sc) or Aß. In chronically exposed cells the threshold to mount a stress response was significantly increased, evidenced by a decreased expression of Hsp72 after stress, whereas an acute exposure lowered the threshold for stress-induced expression of Hsp72. Next, we employed models of PrP(Sc)- and Aß-induced toxicity to demonstrate that the induction of the HSR ameliorates the toxic effects of both PrP(Sc) and Aß. Similarly, the ectopic expression of cytosolic Hsp72 or the extracellular chaperone clusterin protected against PrP(Sc)- or Aß-induced toxicity. However, toxic signaling induced by a pathogenic PrP mutant located at the plasma membrane was prevented by an activated HSR or Hsp72 but not by clusterin, indicating a distinct mode of action of this extracellular chaperone. Our study supports the notion that different pathological protein conformers mediate toxic effects via similar cellular pathways and emphasizes the possibility to exploit the heat shock response therapeutically.

Model peptides uncover the role of the ß-secretase transmembrane sequence in metal ion mediated oligomerization.

The ß-secretase or ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-ß peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif. The sequence MxxxCxxxMxxxCxMxC spans the entire TMS, resembles metal ion binding motifs, and is highly conserved among homologues. We used a synthetic 31-mer model peptide comprising the TMS to study metal ion binding and oligomerization. Applying diverse biochemical and biophysical techniques, we detected dimer and trimer formation of the TMS peptide with copper ions. Replacement of the central Cys466 by Ala essentially abolished these effects. We show that the peptide undergoes a redox reaction with copper ions resulting in a disulfide bridge involving Cys466. Further, we find peptide trimerization that depends on the presence of monovalent copper ions and the sulfhydryl group of Cys466. We identified Cys466 as a key residue for metal ion chelation and to be the core of an oligomerization motif of the BACE1-TMS peptide. Our results demonstrate a novel metal ion controlled oligomerization of the BACE1 TMS, which could have an enormous therapeutic importance against Alzheimer disease.

Novel APP/Aß mutation K16N produces highly toxic heteromeric Aß oligomers.

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-ß (Aß) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aß. First, due to the K16N mutation APP secretion is affected and a higher amount of Aß peptides is being produced. Second, Aß peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells. Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject. Furthermore, Aß42 K16N inhibits fibril formation of Aß42 wild-type. Even more, Aß42 K16N peptides are protected against clearance activity by the major Aß-degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease.

Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.