Harris Bell-Temin

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

Proteomic analysis of rat microglia establishes a high-confidence reference data set of over 3000 proteins.

Highly aggressively proliferating immortalized (HAPI) microglial cells have been used as an in vitro model for investigating key microglial functions including inflammatory, neurotoxic, and phagocytic activities. Through the use of offline strong cation‐exchange fractionation followed by inline reversed‐phase chromatographic separation and tandem mass spectrometric analysis on a hybrid linear ion trap‐Orbitrap instrument, the HAPI microglial proteome was characterized to a depth of 3006 unique protein groups. Upon bioinformatic analysis of the HAPI proteome data set, enrichment was observed for processes relevant to microglial function including those associated with immune system response. This study marks the most comprehensive reference data set generated to date for the rat microglial proteome.

PANDER transgenic mice display fasting hyperglycemia and hepatic insulin resistance

PANcreatic-DERived factor (PANDER, FAM3B) is a novel protein that is highly expressed within the endocrine pancreas and to a lesser degree in other tissues. Under glucose stimulation, PANDER is co-secreted with insulin from the β-cell. Despite prior creation and characterization of acute hepatic PANDER animal models, the physiologic function remains to be elucidated from pancreas-secreted PANDER. To determine this, in this study, a transgenic mouse exclusively overexpressing PANDER from the endocrine pancreas was generated. PANDER was selectively expressed by the pancreatic-duodenal homeobox-1 (PDX1) promoter. The PANDER transgenic (PANTG) mice were metabolically and proteomically characterized to evaluate effects on glucose homeostasis, insulin sensitivity, and lipid metabolism. Fasting glucose, insulin and C-peptide levels were elevated in the PANTG compared with matched WT mice. Younger PANTG mice also displayed glucose intolerance in the absence of peripheral insulin sensitivity. Hyperinsulinemic-euglycemic clamp studies revealed that hepatic glucose production and insulin resistance were significantly increased in the PANTG with no difference in either glucose infusion rate or rate of disappearance. Fasting glucagon, corticosterones, resistin and leptin levels were also similar between PANTG and WT. Stable isotope labeling of amino acids in cell culture revealed increased gluconeogenic and lipogenic proteomic profiles within the liver of the PANTG with phosphoenol-pyruvate carboxykinase demonstrating a 3.5-fold increase in expression. This was matched with increased hepatic triglyceride content and decreased p-AMPK and p-acetyl coenzyme A carboxylase-1 signaling in the PANTG. Overall, our findings support a role of pancreatic β-cell-secreted PANDER in the regulation of hepatic insulin and lipogenenic signaling with subsequent impact on overall glycemia.

Proteomic analysis of aged microglia: shifts in transcription, bioenergetics, and nutrient response

BackgroundAge is the primary risk factor for many diseases. As such, age is a critical co-factor for examination in order to understand the progression and potential intervention in disease progression. Studies examining both the phenotype and transcriptome of aged microglia demonstrated a propensity for the development of a pro-inflammatory phenotype. Less well studied is the concomitant blunting of anti-inflammatory aspects of microglial function with age which also impact plasticity and repair in the CNS.MethodsThis study utilizes mass spectrometry-based proteomics to compare primary microglia from young and aged animals.ResultsThis study revealed alterations in three clusters of inter-related proteins. The three pathways were inflammatory signaling, mitochondrial function, and cellular metabolism. Analysis of these clusters identified the protein rapamycin-insensitive companion of mTOR (RICTOR), a component of the mTORC2 complex, as a novel upstream regulator of several biological functions that are altered with age and potentially linked to phenotype development. A decrease in mTORC2-dependent AKT S473 phosphorylation, as assessed by insulin growth factor (IGF) treatment, was observed in aged microglia. This novel finding was confirmed by genetic manipulation of the microglial cell line. BV2 cells with diminished RICTOR displayed a phenotype that was strikingly similar to that of aged microglia. This finding is particularly relevant as the mTOR pathway already has a number of pharmacological modulators used clinically.ConclusionsThe results suggest that microglia from aged mice show changes in cellular metabolism and energy regulation that might underlie the alterations in inflammatory signaling. Modulation of one pathway identified in our bioinformatic analysis, RICTOR, may provide an avenue by which deleterious aspects of the aging microglia can be attenuated. If successful, this could mean potentially delaying or diminishing the progress of diseases for which progressive inflammation is involved.

SILAC-based proteomic analysis to investigate the impact of amyloid precursor protein expression in neuronal-like B103 cells.

Alzheimers disease (AD) is the most prevalent form of dementia in the elderly. Amyloid plaque formation through aggregation of the amyloid beta peptide derived from amyloid precursor protein (APP) is considered one of the hallmark processes leading to AD pathology; however, the precise role of APP in plaque formation and AD pathogenesis is yet to be determined. Using stable isotope labeling by amino acids in cell culture (SILAC) and MS, protein expression profiles of APP null, rat neuronal‐like B103 cells were compared to B103–695 cells that express the APP isoform, APP‐695. A total of 2979 unique protein groups were identified among three biological replicates and significant protein expression changes were identified in a total of 102 nonredundant proteins. Some of the top biological functions associated with the differentially expressed proteins identified include cellular assembly, organization and morphology, cell cycle, lipid metabolism, protein folding, and PTMs. We report several novel biological pathways influenced by APP‐695 expression in neuronal‐like cells and provide additional framework for investigating altered molecular mechanisms associated with APP expression and processing and contribution to AD pathology.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

The membrane protein PrsS mimics σS in protecting Staphylococcus aureus against cell wall-targeting antibiotics and DNA-damaging agents

Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.

Spike-In SILAC Approach for Proteomic Analysis of Ex Vivo Microglia

Stable isotope labeling by amino acids in cell culture (SILAC) is a versatile mass spectrometry-based proteomic approach that can achieve accurate relative protein quantitation on a global scale. In this approach, proteins are labeled while being synthesized by the cell due to the presence of certain amino acids exclusively as heavier mass analogs than their regular (light) counterparts. This differential labeling allows for the identification of heavy and light forms of each peptide corresponding to two or more different experimental groups upon mass spectrometric analysis, the intensities of which reflect their abundance in the sample analyzed. Relative quantitation is straightforward when SILAC labeling efficiency is high (>99%) and the same cell proteome is used as the quantitation reference, which is typically the case for immortalized cell lines. However, the SILAC methodology for the proteomic analysis of primary cells isolated after in vivo experimentation is more challenging given the low labeling efficiency that would be achieved post-isolation. Alternatively, a stable-isotope-labeled cell line representing the cell type can be used as an internal standard (spike-in SILAC); however, adequate representation of the primary cell proteome with the stable-isotope-labeled internal standard may limit overall protein quantitation, especially for cell types that exhibit a broad range of phenotypes such as microglia, the resident immune cells in the brain. Here, we present a way to circumvent this limitation by combining multiple phenotypes of a single-cell type (the immortalized mouse BV2 microglial cell line) into a single spike-in standard using primary mouse microglia as our model system. We describe the preparation of media, incorporation of labels, induction of four different activation states (plus resting), isolation of primary microglia from adult mice brains, preparation of lysates for analysis, and general guidelines for data processing.