Complement C1q-dependent excitatory and inhibitory synapse elimination by astrocytes and microglia in Alzheimer's disease mouse models

Microglia and complement can mediate neurodegeneration in Alzheimer's disease (AD). By integrative multi-omics analysis, here we show that astrocytic and microglial proteins are increased in Tau P301S synapse fractions with age and in a C1q-dependent manner. In addition to microglia, we identified that astrocytes contribute substantially to synapse elimination in Tau P301S hippocampi. Notably, we found relatively more excitatory synapse marker proteins in astrocytic lysosomes, whereas microglial lysosomes contained more inhibitory synapse material. C1q deletion reduced astrocyte-synapse association and decreased astrocytic and microglial synapses engulfment in Tau P301S mice and rescued synapse density. Finally, in an AD mouse model that combines β-amyloid and Tau pathologies, deletion of the AD risk gene Trem2 impaired microglial phagocytosis of synapses, whereas astrocytes engulfed more inhibitory synapses around plaques. Together, our data reveal that astrocytes contact and eliminate synapses in a C1q-dependent manner and thereby contribute to pathological synapse loss and that astrocytic phagocytosis can compensate for microglial dysfunction

The UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec Processing Pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications

Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers

The Nrf2 pathway is frequently activated in human cancers through mutations in Nrf2 or its negative regulator KEAP1. Using a cell-line-derived gene signature for Nrf2 pathway activation, we found that some tumors show high Nrf2 activity in the absence of known mutations in the pathway. An analysis of splice variants in oncogenes revealed that such tumors express abnormal transcript variants from the NFE2L2 gene (encoding Nrf2) that lack exon 2, or exons 2 and 3, and encode Nrf2 protein isoforms missing the KEAP1 interaction domain. The Nrf2 alterations result in the loss of interaction with KEAP1, Nrf2 stabilization, induction of a Nrf2 transcriptional response, and Nrf2 pathway dependence. In all analyzed cases, transcript variants were the result of heterozygous genomic microdeletions. Thus, we identify an alternative mechanism for Nrf2 pathway activation in human tumors and elucidate its functional consequences

Lack of a functional MntABC system renders <i>S</i>. <i>aureus</i> more sensitive to killing by methyl viologen and human neutrophils but not murine macrophages, unless <i>S</i>. <i>aureus</i> is pre-exposed to oxidative stress.

<p>(A, B) Survival of wild-type and <i>mntC</i> mutant strains within neutrophils harvested from heparin-treated human blood (A) and INF-γ-activated murine macrophages (B). Bacteria were either untreated or pre-exposed to 1 μM methyl viologen for 1 hour. Neutrophils (A) and macrophages (B) were lysed after 90 min and 24 hours of infection, respectively, to enumerate CFU. Bars represent the mean value of triplicate samples and error bars are standard deviation. <i>P-</i>values were determined using one-way ANOVA with multiple comparisons between samples via Tukey’s post-test.</p

Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q Antibodies

Unbiased proteomics reveals multiple molecular changes at hippocampal synapses that occur prior to neurodegeneration in a taupathy/Alzheimer's disease mouse model. Complement C1q labels phospho-Tau-containing synapses and drives microglia-mediated synapse loss that can be rescued by a C1q-blocking antibody

The <i>nrdEF</i> genes are highly upregulated in the <i>mntC</i> mutant after exposure to murine macrophages and human neutrophils <i>ex vivo</i>.

<p>Expression of genes involved in oxidative stress response (filled bars) and DNA repair (striped bars) in the <i>mntC</i> mutant strain relative to the wild-type strain that were phagocytosed by murine macrophages for 2 hours (A) or human neutrophils for 45 minutes (B). Expression levels were determined via qRT-PCR. Bars represent the mean value of triplicate samples and error bars are standard deviation. <i>P</i>-values (ns = non-significant, * = <0.05, *** = <0.001, **** = <0.0001) were determined using student’s t-test.</p

Improved Quantitative Mass Spectrometry Methods for Characterizing Complex Ubiquitin Signals

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages