Dale Chaput

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.

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.

Metabolome and proteome changes with aging in Caenorhabditis elegans.

To expand the understanding of aging in the model organism Caenorhabditis elegans, global quantification of metabolite and protein levels in young and aged nematodes was performed using mass spectrometry. With age, there was a decreased abundance of proteins functioning in transcription termination, mRNA degradation, mRNA stability, protein synthesis, and proteasomal function. Furthermore, there was altered S-adenosyl methionine metabolism as well as a decreased abundance of the S-adenosyl methionine synthetase (SAMS-1) protein. Other aging-related changes included alterations in free fatty acid levels and composition, decreased levels of ribosomal proteins, decreased levels of NADP-dependent isocitrate dehydrogenase (IDH1), a shift in the cellular redox state, an increase in sorbitol content, alterations in free amino acid levels, and indications of altered muscle function and sarcoplasmic reticulum Ca(2+) homeostasis. There were also decreases in pyrimidine and purine metabolite levels, most markedly nitrogenous bases. Supplementing the culture medium with cytidine (a pyrimidine nucleoside) or hypoxanthine (a purine base) increased lifespan slightly, suggesting that aging-induced alterations in ribonucleotide metabolism affect lifespan. An age-related increase in body size, lipotoxicity from ectopic yolk lipoprotein accumulation, a decline in NAD(+) levels, and mitochondrial electron transport chain dysfunction may explain many of these changes. In addition, dietary restriction in aged worms resulting from sarcopenia of the pharyngeal pump likely decreases the abundance of SAMS-1, possibly leading to decreased phosphatidylcholine levels, larger lipid droplets, and ER and mitochondrial stress. The complementary use of proteomics and metabolomics yielded unique insights into the molecular processes altered with age in C. elegans.

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.

Isolation and Proteomic Analysis of Microvesicles and Exosomes from HT22 Cells and Primary Neurons

Exosomes and microvesicles are extracellular vesicles (EVs) released by most cell types. The role of EVs as a method of intercellular communication has led to these vesicles becoming a major area of interest in a variety of scientific fields including neuroscience. Emerging evidence is now demonstrating that the biomolecular composition of EVs, especially exosomes, can play a role in the progression of disease including various neurodegenerative diseases and cancer. In addition to the miRNA profiles of EVs, these vesicles also show interesting changes in protein expression profiles under different physiological and pathological conditions. Characterization of these profiles could prove valuable for both understanding disease pathogenesis and for the discovery of new biomarkers of disease. In this chapter, we describe a protocol for isolation of exosomes and microvesicles from immortalized HT22 cells and primary cortical neurons with sufficient yield and low serum contamination required for downstream analysis and label-free relative quantitation by mass spectrometry.

Potential role of PCTAIRE-2, PCTAIRE-3 and P-Histone H4 in amyloid precursor protein-dependent Alzheimer pathology

Amyloid Precursor Protein (APP) is regulated in a mitosis-specific manner and plays a role in proliferative signaling in cells. Though APP-derived Aβ generation has a well-established role in neurodegeneration, the mechanistic role of APP in this process is not fully understood. Here, we performed an unbiased, comprehensive analysis of the phosphoproteome signature in APP-null neuroblastoma cells (B103) compared to those expressing APP-695 isoform (B103-695) to determine if APP expression affects protein phosphorylation. Stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry-based phosphoproteomic analysis with PolyMAC identified a total of 2,478 phosphopeptides in the B103 and B103-695 cell culture model system. We observed that phosphorylation of PCTAIRE-2 (CDK17), PCTAIRE-3 (CDK18), and Histone H4 are significantly elevated in B103-695 cells; western blot analysis confirmed overexpression of PCTAIREs and increased phosphorylation of Histone H4. More importantly, analysis of primary neurons treated with Aβ, as well as brain samples from MCI (mild cognitive impaired) and AD patients recapitulated these results, showing increased levels of PCTAIREs and P-Histone H4. These novel findings identify a hitherto uncharacterized mechanism by which APP and/or Aβ may promote AD neurodegeneration, and raises the possibility that their inhibition may protect against pathology development in AD.

Quantitative proteomic profiling reveals hepatic lipogenesis and liver X receptor activation in the PANDER transgenic model.

PANcreatic-DERived factor (PANDER) is a member of a superfamily of FAM3 proteins modulating glycemic levels by metabolic regulation of the liver and pancreas. The precise PANDER-induced hepatic signaling mechanism is still being elucidated and has been very complex due to the pleiotropic nature of this novel hormone. Our PANDER transgenic (PANTG) mouse displays a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased, a phenomena observed in type 2 diabetes. To examine the complex PANDER-induced mechanism of SHIR, we utilized quantitative mass spectrometry-based proteomic analysis using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) to reveal the global hepatic proteome differences within the PANTG under the metabolic states of fasting, fed and insulin-stimulated conditions. Proteomic analysis identified lipid metabolism as one of the top cellular functions differentially altered in all metabolic states. Differentially expressed proteins within the PANTG having a lipid metabolic role included ACC, ACLY, CD36, CYP7A1, FASN and SCD1. Central to the differentially expressed proteins involved in lipid metabolism was the predicted activation of the liver X receptor (LXR) pathway. Western analysis validated the increased hepatic expression of LXRα along with LXR-directed targets such as FASN and CYP7A1 within the PANTG liver. Furthermore, recombinant PANDER was capable of inducing LXR promoter activity in-vitro as determined by luciferase reporter assays. Taken together, PANDER strongly impacts hepatic lipid metabolism across metabolic states and may induce a SHIR phenotype via the LXR pathway.

Identification of Novel Cdc37 Interacting Proteins and Pathways in Human Alzheimers Disease Brain Tissue Using Mass Spectrometry

Alzheimer’s disease (AD) is the most common form of dementia and the 6th leading cause of death in the United States. The major pathological hallmarks observed in AD include the formation of intracellular neurofibrillary tangles comprised of phosphorylated forms of the microtubule associated protein tau, and the deposition of extracellular plaques composed of amyloid beta. Cdc37 is a co-chaperone of Hsp90, which recruits client kinases to the Hsp90 complex for folding and stabilization. It has been previously shown that Cdc37 can not only bind and preserve tau, but also stabilize kinases that can phosphorylate tau. The goal of the current study was to identify novel Cdc37- interacting proteins in human AD tissue compared to normal tissue using an immunoprecipitation-based approach combined with mass spectrometry. We identified 39 unique proteins that interacted with Cdc37 in AD samples only and 7 proteins that interacted with Cdc37 in normal samples only. 39 proteins were found to bind Cdc37 in both AD and normal tissue. Of these, 18 showed increased interaction in AD tissue, 10 showed increased interaction in normal tissue and 11 showed equal nteraction in both samples. Ingenuity Pathway Analysis of the data indicates that these Cdc37-interacting proteins could signal through the p70S6K, PI3K / Akt, TGFs, ErbB, NF- kB, calmodulin, p38 MAPK and JNK pathways. Identification of these novel proteins and pathways linked to Cdc37 may indicate its role both as a non-kinase co-chaperone and in other pathways in the AD brain.

Proteomic and mutant analysis of the CO2 concentrating mechanism of hydrothermal vent chemolithoautotroph Thiomicrospira crunogena

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.

Individual Amino Acid Supplementation Can Improve Energy Metabolism and Decrease ROS Production in Neuronal Cells Overexpressing Alpha-Synuclein

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with l-serine, l-proline, l-aspartate, or l-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or l-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.