G-protein coupled estrogen receptor 1, amyloid-ß, and tau tangles in older adults.

Accumulation of amyloid-ß (Aß) and tau tangles are hallmarks of Alzheimer's disease. Aß is extracellular while tau tangles are typically intracellular, and it is unknown how these two proteinopathies are connected. Here, we use data of 1206 elders and test that RNA expression levels of GPER1, a transmembrane protein, modify the association of Aß with tau tangles. GPER1 RNA expression is related to more tau tangles (p = 0.001). Moreover, GPER1 expression modifies the association of immunohistochemistry-derived Aß load with tau tangles (p = 0.044). Similarly, GPER1 expression modifies the association between Aß proteoforms and tau tangles: total Aß protein (p = 0.030) and Aß38 peptide (p = 0.002). Using single nuclei RNA-seq indicates that GPER1 RNA expression in astrocytes modifies the relation of Aß load with tau tangles (p = 0.002), but not GPER1 in excitatory neurons or endothelial cells. We conclude that GPER1 may be a link between Aß and tau tangles driven mainly by astrocytic GPER1 expression.

Unconventional Source of Neurotoxic Protein Aggregation from Organelle Off-Target Bax∆2 in Alzheimer's Disease.

Protein aggregates are a hallmark of Alzheimer's disease (AD). Extensive studies have focused on ß-amyloid plaques and Tau tangles. Here, we illustrate a novel source of protein aggregates in AD neurons from organelle off-target proteins. Bax is a mitochondrial pore-forming pro-death protein. What happens to Bax if it fails to target mitochondria? We previously showed that a mitochondrial target-deficient alternatively spliced variant, Bax∆2, formed large cytosolic protein aggregates and triggered caspase 8-mediated cell death. Bax∆2 protein levels were low in most normal organs and the proteins were quickly degraded in cancer. Here, we found that 85% of AD patients had Bax∆2 required alternative splicing. Increased Bax∆2 proteins were mostly accumulated in neurons of AD-susceptible brain regions. Intracellularly, Bax∆2 aggregates distributed independently of Tau tangles. Interestingly, Bax∆2 aggregates triggered the formation of stress granules (SGs), a large protein-RNA complex involved in AD pathogenesis. Although the functional domains required for aggregation and cell death are the same as in cancer cells, Bax∆2 relied on SGs, not caspase 8, for neuronal cell death. These results imply that the aggregation of organelle off-target proteins, such as Bax∆2, broadens the scope of traditional AD pathogenic proteins that contribute to the neuronal stress responses and AD pathogenesis.

Associations of VGF with Neuropathologies and Cognitive Health in Older Adults.

OBJECTIVE: VGF is proposed as a potential therapeutic target for Alzheimer's (AD) and other neurodegenerative conditions. The cell-type specific and, separately, peptide specific associations of VGF with pathologic and cognitive outcomes remain largely unknown. We leveraged gene expression and protein data from the human neocortex and investigated the VGF associations with common neuropathologies and late-life cognitive decline. METHODS: Community-dwelling older adults were followed every year, died, and underwent brain autopsy. Cognitive decline was captured via annual cognitive testing. Common neurodegenerative and cerebrovascular conditions were assessed during neuropathologic evaluations. Bulk brain RNASeq and targeted proteomics analyses were conducted using frozen tissues from dorsolateral prefrontal cortex of 1,020 individuals. Cell-type specific gene expressions were quantified in a subsample (N = 424) following single nuclei RNASeq analysis from the same cortex. RESULTS: The bulk brain VGF gene expression was primarily associated with AD and Lewy bodies. The VGF gene association with cognitive decline was in part accounted for by neuropathologies. Similar associations were observed for the VGF protein. Cell-type specific analyses revealed that, while VGF was differentially expressed in most major cell types in the cortex, its association with neuropathologies and cognitive decline was restricted to the neuronal cells. Further, the peptide fragments across the VGF polypeptide resembled each other in relation to neuropathologies and cognitive decline. INTERPRETATION: Multiple pathways link VGF to cognitive health in older age, including neurodegeneration. The VGF gene functions primarily in neuronal cells and its protein associations with pathologic and cognitive outcomes do not map to a specific peptide. ANN NEUROL 2023;94:232-244.

Estrogen Receptor Genes, Cognitive Decline, and Alzheimer Disease.

BACKGROUND AND OBJECTIVES: Lifetime risk of Alzheimer disease (AD) dementia is twofold higher in women compared with men, and low estrogen levels in postmenopause have been suggested as a possible contributor. We examined 3 ER (GPER1, ER2, and ER1) variants in association with AD traits as an indirect method to test the association between estrogen and AD in women. Although the study focus was on women, in a comparison, we separately examined ER molecular variants in men. METHODS: Participants were followed for an average of 10 years in one of the 2 longitudinal clinical pathologic studies of aging. Global cognition was assessed using a composite score derived from 19 neuropsychological tests' scores. Postmortem pathologic assessment included examination of 3 AD (amyloid-ß and tau tangles determined by immunohistochemistry, and a global AD pathology score derived from diffuse and neurotic plaques and neurofibrillary tangle count) and 8 non-AD pathology indices. ER molecular genomic variants included genotyping and examining ER DNA methylation and RNA expression in brain regions including the dorsolateral prefrontal cortex (DLPFC) that are major players in cognition and often have AD pathology. RESULTS: The mean age of women (N = 1711) at baseline was 78.0 (SD = 7.7) years. In women, GPER1 molecular variants had the most consistent associations with AD traits. GPER1 DNA methylation was associated with cognitive decline, tau tangle density, and global AD pathology score. GPER1 RNA expression in DLPFC was related to cognitive decline and tau tangle density. Other associations included associations of ER2 and ER1 sequence variants and DNA methylation with cognition. RNA expressions in DLPFC of genes involved in signaling mechanisms of activated ERs were also associated with cognitive decline and tau tangle density in women. In men (N = 651, average age at baseline: 77.4 [SD = 7.3]), there were less robust associations between ER molecular genomic variants and AD cognitive and pathologic traits. No consistent association was seen between ER molecular genomic variations and non-AD pathologies in either of the sexes. DISCUSSION: ER DNA methylation and RNA expression, and to some extent ER polymorphisms, were associated with AD cognitive and pathologic traits in women, and to a lesser extent in men.

Cortical proteins may provide motor resilience in older adults.

Motor resilience proteins may be a high value therapeutic target that offset the negative effects of pathologies on motor function. This study sought to identify cortical proteins associated with motor decline unexplained by brain pathologies that provide motor resilience. We studied 1226 older decedents with annual motor testing, postmortem brain pathologies and quantified 226 proteotypic peptides in prefrontal cortex. Twenty peptides remained associated with motor decline in models controlling for ten brain pathologies (FDR < 0.05). Higher levels of nine peptides and lower levels of eleven peptides were related to slower decline. A higher motor resilience protein score based on averaging the levels of all 20 peptides was related to slower motor decline, less severe parkinsonism and lower odds of mobility disability before death. Cortical proteins may provide motor resilience. Targeting these proteins in further drug discovery may yield novel interventions to maintain motor function in old age.

Identification of novel genes associated with dysregulation of B cells in patients with primary Sjögren's syndrome.

BACKGROUND: The aim of this study was to identify the molecular mechanism of dysregulation of B cell subpopulations of primary Sjögren's syndrome (pSS) at the transcriptome level. METHODS: We enrolled patients with pSS (n = 6) and healthy controls (HCs) (n = 6) in the discovery cohort using microarray and pSS (n = 14) and HCs (n = 12) in the validation cohort using quantitative PCR (qPCR). Peripheral B cells acquired from these subjects were separated by cell sorting into four subsets: CD38-IgD+ (Bm1), CD38+IgD+ (naive B cells), CD38highIgD+ (pre-germinal centre B cells) and CD38±IgD- (memory B cells). We performed differentially expressed gene (DEG) analysis and weighted gene co-expression network analysis (WGCNA). RESULTS: Expression of the long non-coding RNA LINC00487 was significantly upregulated in all B cell subsets, as was that of HLA and interferon (IFN) signature genes. Moreover, the normalized intensity value of LINC00487 significantly correlated with the disease activity score of all pSS B cell subsets. Studies of human B cell lines revealed that the expression of LINC00487 was strongly induced by IFNα. WGCNA revealed six gene clusters associated with the B cell subpopulation of pSS. Further, SOX4 was identified as an inter-module hub gene. CONCLUSION: Our transcriptome analysis revealed key genes involved in the dysregulation of B cell subpopulations associated with pSS. TRIAL REGISTRATION: Not required.

Phosphoproteomics-based systems analysis of signal transduction networks.

Signal transduction systems coordinate complex cellular information to regulate biological events such as cell proliferation and differentiation. Although the accumulating evidence on widespread association of signaling molecules has revealed essential contribution of phosphorylation-dependent interaction networks to cellular regulation, their dynamic behavior is mostly yet to be analyzed. Recent technological advances regarding mass spectrometry-based quantitative proteomics have enabled us to describe the comprehensive status of phosphorylated molecules in a time-resolved manner. Computational analyses based on the phosphoproteome dynamics accelerate generation of novel methodologies for mathematical analysis of cellular signaling. Phosphoproteomics-based numerical modeling can be used to evaluate regulatory network elements from a statistical point of view. Integration with transcriptome dynamics also uncovers regulatory hubs at the transcriptional level. These omics-based computational methodologies, which have firstly been applied to representative signaling systems such as the epidermal growth factor receptor pathway, have now opened up a gate for systems analysis of signaling networks involved in immune response and cancer.

Phosphoproteomics-based modeling defines the regulatory mechanism underlying aberrant EGFR signaling.

BACKGROUND: Mutation of the epidermal growth factor receptor (EGFR) results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling. METHODOLOGY/PRINCIPAL FINDINGS: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992), one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics. CONCLUSIONS/SIGNIFICANCE: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling.

Temporal perturbation of tyrosine phosphoproteome dynamics reveals the system-wide regulatory networks.

Signal transduction systems are known to widely regulate complex biological events such as cell proliferation and differentiation. Because phosphotyrosine-dependent networks play a key role in transmitting signals, a comprehensive and fine description of their dynamic behavior can lead us to systematically analyze the regulatory mechanisms that result in each biological effect. Here we established a mass spectrometry-based framework for analyzing tyrosine phosphoproteome dynamics through temporal network perturbation. A highly time-resolved description of the epidermal growth factor-dependent signaling pathways in human A431 cells revealed a global view of their multiphase network activation, comprising a spike signal transmission within 1 min of ligand stimulation followed by the prolonged activation of multiple Src-related molecules. Temporal perturbation of Src family kinases with the corresponding inhibitor PP2 in the prolonged activation phase led to the down-regulation of the molecules related to cell adhesion and receptor degradation, whereas the canonical cascades as well as the epidermal growth factor receptor relatively maintained their activities. Our methodology provides a system-wide view of the regulatory network clusters involved in signal transduction that is essential to refine the literature-based network structures for a systems biology analysis.

Modeling and estimation of dynamic EGFR pathway by data assimilation approach using time series proteomic data.

Cell Illustrator is a model building tool based on the Hybrid Functional Petri net with extension (HFPNe). By using Cell Illustrator, we have succeeded in modeling biological pathways, e.g., metabolic pathways, gene regulatory networks, microRNA regulatory networks, cell signaling networks, and cell-cell interactions. The recent development of tandem mass spectrometry coupled with liquid chromatography (LC/MS/MS) technology has enabled researchers to quantify the dynamic profile of a wide range of proteins within the cell. The proteomic data obtained by using LC/MS/MS has been considerably useful for introducing dynamics to the HFPNe model. Here, we report the first introduction of the time-series proteomic data to our HFPNe model. We constructed an epidermal growth factor receptor signal transduction pathway model (EFGR model) by using the biological data available in the literature. Then, the kinetic parameters were determined in the data assimilation (DA) framework with some manual tuning so as to fit the proteomic data published by Blagoev et al. (Nat. Biotechnol., 22:1139-1145, 2004). This in silico model was further refined by adding or removing some regulation loops using biological background knowledge. The DA framework was used to select the most plausible model from among the refined models. By using the proteomic data, we semi-automatically constructed a well-tuned EGFR HFPNe model by using the Cell Illustrator coupled with the DA framework.