Widespread remodeling of proteome solubility in response to different protein homeostasis stresses.

The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington's disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.

Protein aggregation in cell biology: An aggregomics perspective of health and disease.

Maintaining protein homeostasis (proteostasis) is essential for cellular health and is governed by a network of quality control machinery comprising over 800 genes. When proteostasis becomes imbalanced, proteins can abnormally aggregate or become mislocalized. Inappropriate protein aggregation and proteostasis imbalance are two of the central pathological features of common neurodegenerative diseases including Alzheimer, Parkinson, Huntington, and motor neuron diseases. How aggregation contributes to the pathogenic mechanisms of disease remains incompletely understood. Here, we integrate some of the key and emerging ideas as to how protein aggregation relates to imbalanced proteostasis with an emphasis on Huntington disease as our area of main expertise. We propose the term "aggregomics" be coined in reference to how aggregation of particular proteins concomitantly influences the spatial organization and protein-protein interactions of the surrounding proteome. Meta-analysis of aggregated interactomes from various published datasets reveals chaperones and RNA-binding proteins are common components across various disease contexts. We conclude with an examination of therapeutic avenues targeting proteostasis mechanisms.

A Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress.

Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.

When proteostasis goes bad: Protein aggregation in the cell.

Protein aggregation is a hallmark of the major neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and motor neuron and is a symptom of a breakdown in the management of proteome foldedness. Indeed, it is remarkable that under normal conditions cells can keep their proteome in a highly crowded and confined space without uncontrollable aggregation. Proteins pose a particular challenge relative to other classes of biomolecules because upon synthesis they must typically follow a complex folding pathway to reach their functional conformation (native state). Non-native conformations, including the unfolded nascent chain, are highly prone to aberrant interactions, leading to aggregation. Here we review recent advances in knowledge of proteostasis, approaches to monitor proteostasis and the impact that protein aggregation has on biology. We also include discussion of the outstanding challenges. © 2017 IUBMB Life, 69(2):49-54, 2017.

Hypoxia-induced tau phosphorylation and memory deficit in rats.

Hypoxia was shown to be associated with an increased risk of Alzheimer's disease (AD). The effects of hypoxia on the development of AD pathology and spatial memory ability and the possible molecular mechanisms remain poorly understood. In this study, we demonstrate that rats exposed to a hypoxic condition (10% oxygen concentration) for 1, 2, 4 and 8 weeks (6 h each day) displayed spatial memory impairment and increased tau phosphorylation at Ser198/199/202, Thr205, Ser262, Ser396 and Ser404 in the hippocampus. Concomitantly, the levels of Tyr216-phosphorylated glycogen synthase kinase (GSK)-3ß (activated form of GSK-3ß) and Tyr307-phosphorylated protein phosphatase 2A (inactivated form of PP2A) were significantly increased in the hippocampus of the rats with 1, 2, 4 and 8 weeks of hypoxia exposure, while the levels of methylated PP2A (activated form of PP2A) were significantly decreased in the hippocampus of the rats with 4 and 8 weeks of hypoxia exposure. In addition, the content of malondialdehyde, an indicator of oxidative stress, was elevated, whereas the activity of superoxide dismutase was not significantly changed in the hippocampus of the rats exposed to hypoxia. Taken together, these data demonstrated that hypoxia induced tau hyperphosphorylation and memory impairment in rats, and that the increased tau phosphorylation could be attributed to activation of GSK-3ß and inactivation of PP2A. These data suggest that interventions to improve hypoxia may be helpful to prevent the development of AD pathology and cognitive impairment.

Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H2O2-activated calcium signaling pathway in mouse hippocampal neurons.

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H(2)S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O(2), 5% CO(2) and 93% N(2) at 37 °C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H(2)S) for 30 min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca(2+)](i)), and pretreatment with NaHS prevented the increase in [Ca(2+)](i). Additionally, polyethylene glycol (PEG)-catalase (a H(2)O(2) scavenger) but not PEG-SOD (an O(2)(-) scavenger) conferred an inhibitory effect similar to H(2)S on the hypoxia-induced increase in [Ca(2+)](i). Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP(3)) receptor blocker xestospongin C. Taken together, these findings suggest that H(2)S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H(2)O(2))-activated Ca(2+) signaling pathway in mouse hippocampal neurons.

Probing Protein Solubility Patterns with Proteomics for Insight into Network Dynamics.

Proteome solubility contains latent information on the nature of protein interaction networks in cells and changes in solubility can provide information on rewiring of networks. Here, we report a simple one-step ultracentrifugation method to separate the soluble and insoluble fraction of the proteome. The method involves quantitative proteomics and a bioinformatics strategy to analyze the changes that arise. Because protein solubility changes are also associated with protein misfolding and aggregation in neurodegenerative disease, we also include a protocol for isolating disease-associated protein aggregates with pulse shape analysis (PulSA) by flow cytometry as a complementary approach that can be used alongside the more general measure of solubility or as a stand-alone approach.

Comparative Serum Proteomic Analysis of the Effects of Sodium Selenate on a Mouse Model of Alzheimer's Disease.

Selenium (Se), as a nutritionally essential trace element, has been shown to decrease with age and is closely related to Alzheimer's disease (AD). To probe the effects of Se on AD pathology, two-dimensional fluorescence difference gel electrophoresis was applied to the serum samples collected from the wild-type (WT) mice and the triple transgenic (PS1M146V/AßPPSwe/TauP301L) AD mice (3xTg-AD), treated with or without sodium selenate in drinking water for 4 months beginning at 2 months of age. Proteomics results revealed 17 differentially expressed proteins between WT and 3xTg-AD mice. It was found that the administration of selenate reversed the alterations of the differentially expressed serum proteins by up-regulating 13 proteins and down-regulating 2 proteins which were reported to be involved in the key pathogenesis of AD, including regulation of Aß production, lipid metabolism regulation, and anti-inflammation. These results suggested that a dietary supplement with selenate is effective for prevention and treatment of AD, and the mechanism was maybe related to its role in Aß regulation, lipid metabolism, and anti-inflammation. Moreover, we also presented that α-2 macroglobulin, transthyretin, haptoglobin, alpha-2-HS-glycoprotein, and alpha-1-antitrypsin in the serum can be used to evaluate the effect of selenate on AD pathology.

Identification of Novel Key Molecules Involved in Spatial Memory Impairment in Triple Transgenic Mice of Alzheimer's Disease.

The molecular mechanisms underlying cognitive impairment in Alzheimer's disease (AD) remain largely unclear. In the present study, we were aimed to identify the potential key molecules involved in spatial memory impairment in a triple transgenic (3xTg-AD) mouse model of AD. By employing two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, we revealed a total of 24 differentially expressed proteins in hippocampus of 9-month-old 3xTg-AD mice with significant spatial memory impairment in comparison to the age-matched controls. These differentially expressed proteins can be categorized into several functional classifications that are related to synaptic/memory-, energy metabolism-, intracellular transport-, cell cycle-, cellular defense and structure, and stress response. To further verify the target proteins that may underlie the memory deficits, we pre-treated the 3xTg-AD mice for 3 months with coenzyme Q10 (CoQ10) (800 mg/kg body weight/day), a powerful endogenous antioxidant that has been shown to be able to prevent memory deficits in several AD mouse models. We found that administration of CoQ10 altered the expression levels of nine proteins in hippocampus of 3xTg-AD mice with simultaneous improvement of spatial memory. Interestingly, complexin-1/2, two molecules which were shown to alter LTP, were modulated (i.e., the levels were reduced in 3xTg-AD mice and CoQ10 restored the levels) in response to CoQ10 treatment among these nine proteins. Furthermore, we found that adeno-associated virus serotype 9 (AAV-9)-mediated overexpression of complexin-1/2 prevented memory impairment in the AD mouse model. Taken together, this study has identified a number of differentially expressed proteins in hippocampus of 3xTg-AD mice and the control in presence or absence of CoQ10. The modulation of complexin-1/2 expression by CoQ10 may contribute to the amelioration of memory impairment in the AD transgenic mice.

Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis.

Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

Chronic copper exposure causes spatial memory impairment, selective loss of hippocampal synaptic proteins, and activation of PKR/eIF2α pathway in mice.

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

Cerebrospinal fluid biomarkers of Alzheimer's disease.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder that takes about a decade to develop, making early diagnosis possible. Clinically, the diagnosis of AD is complicated, costly, and inaccurate, so it is urgent to find specific biomarkers. Due to its multifactorial nature, a panel of biomarkers for the multiple pathologies of AD, such as cerebral amyloidogenesis, neuronal dysfunction, synapse loss, oxidative stress, and inflammation, are most promising for accurate diagnosis. Highly sensitive and high-throughput proteomic techniques can be applied to develop a panel of novel biomarkers for AD. In this review, we discuss the metabolism and diagnostic performance of the well-established core candidate cerebrospinal fluid (CSF) biomarkers (ß-amyloid, total tau, and hyperphosphorylated tau). Meanwhile, novel promising CSF biomarkers, especially those identified by proteomics, updated in the last five years are also extensively discussed. Furthermore, we provide perspectives on how biomarker discovery for AD is evolving.

Immobilization of trypsin onto multifunctional meso-/macroporous core-shell microspheres: a new platform for rapid enzymatic digestion.

A simple, fast, efficient, and reusable microwave-assisted tryptic digestion system which was constructed by immobilization of trypsin onto porous core-shell Fe3O4@fTiO2 microspheres has been developed. The nanostructure with magnetic core and titania shell has multiple pore sizes (2.4 and 15.0 nm), high pore volume (0.25 cm(3) g(-1)), and large surface area (50.45 m(2) g(-1)). For the proteins, the system can realize fast and efficient microwave-assisted tryptic digestion. Various standard proteins (e.g., cytochrome c (cyt-c), myoglobin (MYO), ß-lactoglobulin (ß-LG), and bovine serum albumin (BSA)) used can be digested in 45 s under microwave radiation, and they can be confidently identified by mass spectrometry (MS) analysis; even the concentration of substrate is as low as 5 ng µL(-1). Furthermore, the system for the 45 s microwave-assisted tryptic digestion is still effective after the trypsin-immobilized microspheres have been reused for 5 times. Importantly, 1715 unique proteins from 10 µg mouse brain proteins can be identified with high confidence after treatment of 45 s microwave-assisted tryptic digestion.

Uptake of silica nanoparticles: neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells.

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 µg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular ß-amyloid 1-42 (Aß(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-ß-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3ß at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3ß. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.

Proteomic analysis of serum proteins in triple transgenic Alzheimer's disease mice: implications for identifying biomarkers for use to screen potential candidate therapeutic drugs for early Alzheimer's disease.

Alzheimer's disease (AD) is the most common fatal neurodegenerative disease affecting the elderly worldwide. There is an urgent need to identify novel biomarkers of early AD. This study aims to search for potential early protein biomarkers in serum from a triple transgenic (PS1M146V/APPSwe/TauP301L) mouse model. Proteomic analysis via two-dimensional fluorescence difference gel electrophoresis was performed on serum samples from wild-type (WT) and triple transgenic mice that were treated with or without coenzyme Q10 (CoQ10) (800 mg/kg body weight/day), a powerful endogenous antioxidant displaying therapeutic benefits against AD pathology and cognitive impairment in multiple AD mouse models, for a period of three months beginning at two months of age. A total of 15 differentially expressed serum proteins were identified between the WT and AD transgenic mice. The administration of CoQ10 was found to alter the changes in the differentially expressed serum proteins by upregulating 10 proteins and down-regulating 10 proteins. Among the proteins modulated by CoQ10, clusterin and α-2-macroglobulin were validated via ELISA assay. These findings revealed significant changes in serum proteins in the AD mouse model at an early pathological stage and demonstrated that administration of CoQ10 could modulate these changes in serum proteins. Our study suggested that these differentially expressed serum proteins could serve as potential protein biomarkers of early AD and that screening for potential candidate AD therapeutic drugs and monitoring of therapeutic effects could be performed via measurement of the changes in these differentially expressed serum proteins.