Valentina Gelfanova

Macrophage-Mediated Degradation of β-Amyloid via an Apolipoprotein E Isoform-Dependent Mechanism

Recent studies suggest that bone marrow-derived macrophages can effectively reduce β-amyloid (Aβ) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Aβ, we cultured murine macrophages on top of Aβ plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP717V>F mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Aβ in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Aβ degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Aβ than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Aβ was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Aβ-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Aβ degradation by macrophages. These apoE isoform-dependent effects of macrophages on Aβ degradation suggest a novel “peripheral” mechanism for Aβ clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimers disease.

Proteomics of Cerebrospinal Fluid: Methods for Sample Processing

Cerebrospinal fluid (CSF) provides an important source of potential biomarkers for brain disorders and therapeutic drug development. Applications of proteomic technology to the identification and quantification of proteins in CSF are increasing rapidly. Key to obtaining reproducible and reliable data about protein levels in CSF are standardization of methods for sample collection, storage, and subsequent sample processing. Methods are described here for all steps of sample processing for a number of different proteomic approaches.

Identification, characterization, and comparison of amino-terminally truncated Aβ42 peptides in Alzheimer's disease brain tissue and in plasma from Alzheimer's patients receiving solanezumab immunotherapy treatment

observed in any Aß-degrading protease knockout mouse studied to date. Remarkably, this increase was selective for Aß42, resulting in elevated Aß42/ Aß40 ratios comparable to those induced by presenilin mutations that cause early-onset AD. Cultured neurons from CatD-KO mice also showed selective defects in the catabolism of Aß42, but not Aß40. Furthermore, relative to age-matched controls, the brains of CatD-KO mice showed pronounced increases in the levels of tau and phosphorylated forms thereof, while showing no increases in the levels of several other cytosolic proteins. Conclusions: Our findings demonstrate that, in vivo, CatD deficiency leads to large and highly selective increases in the two protein species most strongly linked to the pathogenesis of AD_Aß42 and tau. Combined with human molecular genetic data on the CatD gene (CTSD) to be presented at this meeting (Burgess et al.), our findings suggest that CatD plays a protective role in the pathogenesis of AD by mediating the catabolism of Aß42 and tau.

Epitope Mapping of Antibodies by Mass Spectroscopy: A Case Study

Epitope mapping of antibodies is the identification and characterization of binding sites of monoclonal antibodies (mAbs) on target antigens. This knowledge can be useful in generating novel antibodies to a particular target as well as elucidating an antibody mechanism of action. Several techniques are available to identify antibody epitopes among which are preliminary and simple ones like sequence homology analysis ELISA and Western blotting. However, the more widely used robust methods typically involve the use of mass spectrometry to fully analyze and interpret the data and accurately identify the binding site. Such methods include epitope extortion/excision, hydrogen deuterium exchange.

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

AbstractDe novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question. Figureᅟ

Characterization of amino-terminally truncated Abeta-42 peptides in plasma from Alzheimer's patients receiving solanezumab immunotherapy treatment

also assessed the specificity of E2012 for different gamma-secretase substrates. Results: E2012 was shown to decrease Ab(x-40) and Ab(x-42) and to increase Ab(1-38) without changing total Ab(1-x) levels. MALDITOF analysis also revealed that E2012 reduced Ab40 and Ab42 and increased shorter Ab peptides, such as Ab37 and Ab38. E2012 did not induce the accumulation of APP-CTFs, suggesting that E2012 modulates, but does not inhibit, the cleavage of APP-CTF by gamma-secretase. Production of Notch intracellular domain (NICD) was not inhibited by E2012. Conclusions: We have profiled E2012, a compound from a new chemical class of GSMs, and found that it preferentially reduced gamma-secretase mediated Ab40 and Ab42 production over Notch cleavage via a different mechanism from that of classical GSIs. These results provide the basis for the development of E2012 as a new therapeutic agent for Alzheimer’s disease.

Effects of solanezumab versus placebo administration on biomarkers in people with mild-to-moderate Alzheimer's disease: Results from two phase III clinical trials

ratiowas 4:1, consistent with the ratio in the general population; that for avagacestat was 1:1.75. NMSC incidence rates among avagacestat-treated patients were higher than those from the claims database. Conclusions: This is the first clinical report characterizing NMSC in patients treated with a GSI. The NMSCs observed were readily identified and easily managed without recurrence. Increased NMSC incidence (especially SCC) and inversion of BCC:SCC ratio suggest that Notch inhibition oncogenically effects SCC. Further evaluation is warranted to understand potential oncogenic or suppressor effects of similar agents on skin cancer subtypes. The algorithms utilized in this study may help accurately identify and manage skin malignancies in studies using agents that affect epidermal differentiation.