The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis.

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

Tandem Mass Tag LC-MS/MS of Aqueous Humor From Individuals With Type 2 Diabetes Without Retinopathy Reveals Early Dysregulation of Synaptic Proteins.

Purpose: An early neurodegenerative component of diabetic retinal disease (DRD) that precedes the vascular findings of clinically diagnosed diabetic retinopathy (DR) is increasingly being recognized. However, the relevant molecular mechanisms and biomarkers for early DRD are poorly defined. The purpose of this study was to uncover novel potential mediators of early diabetic retinal neuronal dysfunction through analysis of the aqueous fluid proteome in preclinical DR. Methods: Aqueous fluid was collected from subjects with type 2 diabetes mellitus (DM) but no clinical DR and from nondiabetic controls undergoing routine cataract surgery. Preoperative spectral-domain optical coherence tomography of the macula was obtained. Tandem mass tag LC-MS/MS was performed to identify proteins differentially present in diabetic and control aqueous fluid, and proteins with >50% change and P < 0.05 were considered significant. Selected results were validated with western blot of human aqueous fluid samples. Results: We identified decreased levels of proteins implicated in neuronal synapse formation and increased levels of inflammatory proteins in the aqueous fluid from patients with type 2 DM but no DR compared with controls. Of the differentially present synaptic proteins that we identified and confirmed with western blot, the majority have not previously been linked with DRD. Conclusions: The proteomic profile of aqueous fluid from individuals with type 2 DM but no DR suggests that retinal neuronal dysfunction and inflammation represent very early events in the pathophysiology of DRD. These findings support the concept that diabetic retinal neurodegeneration precedes vascular pathology and reveal novel potential mediators and/or biomarkers warranting further investigation.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.

Pathologic α-synuclein plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. We report that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD.

An unbiased proteomic analysis of PAD4 in human monocytes: novel substrates, binding partners and subcellular localizations.

Peptidylarginine deiminase IV (PAD4) post-translationally converts arginine residues in proteins to citrullines and is implicated in playing a central role in the pathogenesis of several diseases. Although PAD4 was historically thought to be a nuclear enzyme, recent evidence has revealed a more complex localization of PAD4 with evidence of additional cytosolic and cell surface localization and activity. However, the mechanisms by which PAD4, which lacks conventional secretory signal sequences, traffics to extranuclear localizations are unknown. In this study, we show that PAD4 was enriched in the organelle fraction of monocytes with evidence of citrullination of organelle proteins. We also demonstrated that PAD4 can bind to several cytosolic, nuclear and organelle proteins that may serve as binding partners for PAD4 to traffic intracellularly. Additionally, cell surface expression of PAD4 increased with monocyte differentiation into monocyte-derived dendritic cells and co-localized with several endocytic/autophagic and conventional secretory pathway markers, implicating the use of these pathways by PAD4 to traffic within the cell. Our results suggest that PAD4 is expressed in multiple subcellular localizations and may play previously unappreciated roles in physiological and pathological conditions. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Development of an in situ cell-type specific proteome analysis method using antibody-mediated biotinylation.

Since proteins are essential molecules exerting cellular functions, decoding proteome changes is the key to understanding the normal physiology and pathogenesis mechanism of various diseases. However, conventional proteomic studies are often conducted on tissue lumps, in which multiple cell types are entangled, presenting challenges in interpreting the biological dynamics among diverse cell types. While recent cell-specific proteome analysis techniques, like BONCAT, TurboID, and APEX, have emerged, their necessity for genetic modifications limits their usage. The alternative, laser capture microdissection (LCM), although it does not require genetic alterations, is labor-intensive, time-consuming, and requires specialized expertise, making it less suitable for large-scale studies. In this study, we develop the method for in situ cell-type specific proteome analysis using antibody-mediated biotinylation (iCAB), in which we combined immunohistochemistry (IHC) with the biotin-tyramide signal amplification approach. Poly-horseradish peroxidase (HRP) conjugated to the secondary antibody will be localized at a target cell type via a primary antibody specific to the target cell type and biotin-tyramide activated by HRP will biotinylate the nearby proteins. Therefore, the iCAB method can be applied to any tissues that can be used for IHC. As a proof-of-concept, we employed iCAB for mouse brain tissue enriching proteins for neuronal cell bodies, astrocytes, and microglia, followed by identifying the enriched proteins using 16-plex TMT-based proteomics. In total, we identified ~8,400 and ~6,200 proteins from enriched and non-enriched samples. Most proteins from the enriched samples showed differential expressions when we compared different cell type data, while there were no differentially expressed proteins from non-enriched samples. The cell type enrichment analysis with the increased proteins in respective cell types using Azimuth showed that neuronal cell bodies, astrocytes, and microglia data exhibited Glutamatergic Neuron, Astrocyte and Microglia/Perivascular Macrophage as the representative cell types, respectively. The proteome data of the enriched proteins showed similar subcellular distribution as non-enriched proteins, indicating that the iCAB-proteome is not biased toward any subcellular compartment. To our best knowledge, this study represents the first implementation of a cell-type-specific proteome analysis method using an antibody-mediated biotinylation approach. This development paves the way for the routine and widespread use of cell-type-specific proteome analysis. Ultimately, this could accelerate our understanding of biological and pathological phenomena.

NAD metabolism modulates inflammation and mitochondria function in diabetic kidney disease.

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United -States. Despite the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies, we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion, and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing the progression of diabetic kidney disease.

NPTX2 in Cerebrospinal Fluid Predicts the Progression From Normal Cognition to Mild Cognitive Impairment.

OBJECTIVE: This study examined whether cerebrospinal fluid (CSF) baseline levels of the synaptic protein NPTX2 predict time to onset of symptoms of mild cognitive impairment (MCI), both alone and when accounting for traditional CSF Alzheimer's disease (AD) biomarker levels. Longitudinal NPTX2 levels were also examined. METHODS: CSF was collected longitudinally from 269 cognitively normal BIOCARD Study participants (mean baseline age = 57.7 years; mean follow-up = 16.3 years; n = 77 progressed to MCI/dementia). NPTX2 levels were measured from 3 correlated peptides using quantitative parallel reaction monitoring mass spectrometry. Levels of Aß42 /Aß40 , p-tau181 , and t-tau were measured from the same CSF specimens using Lumipulse automated electrochemiluminescence assays. RESULTS: In Cox regression models, lower baseline NPTX2 levels were associated with an earlier time to MCI symptom onset (hazard ratio [HR] = 0.76, SE = 0.09, p = 0.023). This association was significant for progression within 7 years (p = 0.036) and after 7 years from baseline (p = 0.001). Baseline NPTX2 levels improved prediction of time to MCI symptom onset after accounting for baseline AD biomarker levels (p < 0.01), and NPTX2 did not interact with the CSF AD biomarkers or APOE-ε4 genetic status. In linear mixed effects models, higher baseline p-tau181 and t-tau levels were associated with higher baseline levels of NPTX2 (both p < 0.001) and greater rates of NPTX2 declines over time. INTERPRETATION: NPTX2 may be a valuable prognostic biomarker during preclinical AD that provides additive and independent prediction of MCI onset among individuals who are cognitively normal. We hypothesize that NPTX2-mediated circuit homeostasis confers resilience during the early phase of AD. ANN NEUROL 2023;94:620-631.

An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function.

Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [Gde3 knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in Gde3 KOs, while EVs from Gde3 KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape.

Discovery of Biomarkers for Amyotrophic Lateral Sclerosis from Human Cerebrospinal Fluid Using Mass-Spectrometry-Based Proteomics.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of upper and lower motor neurons, which eventually may lead to death. Critical to the mission of developing effective therapies for ALS is the discovery of biomarkers that can illuminate mechanisms of neurodegeneration and have diagnostic, prognostic, or pharmacodynamic value. Here, we merged unbiased discovery-based approaches and targeted quantitative comparative analyses to identify proteins that are altered in cerebrospinal fluid (CSF) from patients with ALS. Mass spectrometry (MS)-based proteomic approaches employing tandem mass tag (TMT) quantification methods from 40 CSF samples comprising 20 patients with ALS and 20 healthy control (HC) individuals identified 53 proteins that are differential between the two groups after CSF fractionation. Notably, these proteins included both previously identified ones, validating our approach, and novel ones that have the potential for expanding biomarker repertoire. The identified proteins were subsequently examined using parallel reaction monitoring (PRM) MS methods on 61 unfractionated CSF samples comprising 30 patients with ALS and 31 HC individuals. Fifteen proteins (APOB, APP, CAMK2A, CHI3L1, CHIT1, CLSTN3, ERAP2, FSTL4, GPNMB, JCHAIN, L1CAM, NPTX2, SERPINA1, SERPINA3, and UCHL1) showed significant differences between ALS and the control. Taken together, this study identified multiple novel proteins that are altered in ALS, providing the foundation for developing new biomarkers for ALS.

Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis.

Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and hypothesized to drive the loss of immune tolerance to self-antigens in autoimmunity. Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines. Here, we investigate the hypothesis that citrullination exposes cryptic peptides by modifying protein structure and proteolytic cleavage. We show that citrullination alters processing and presentation of autoantigens, resulting in the generation of a unique citrullination-dependent repertoire composed primarily of native sequences. This repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than controls. The generation of this unique repertoire is achieved through altered protease cleavage and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA.

Mass Spectrometry-Based Proteomics Analysis of Human Substantia Nigra From Parkinson's Disease Patients Identifies Multiple Pathways Potentially Involved in the Disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) of the brain. Despite decades of studies, the precise pathogenic mechanism of PD is still elusive. An unbiased proteomic analysis of PD patient's brain allows the identification of critical proteins and molecular pathways that lead to dopamine cell death and α-synuclein deposition and the resulting devastating clinical symptoms. In this study, we conducted an in-depth proteome analysis of human SN tissues from 15 PD patients and 15 healthy control individuals combining Orbitrap mass spectrometry with the isobaric tandem mass tag-based multiplexing technology. We identified 10,040 proteins with 1140 differentially expressed proteins in the SN of PD patients. Pathway analysis showed that the ribosome pathway was the most enriched one, followed by gamma-aminobutyric acidergic synapse, retrograde endocannabinoid signaling, cell adhesion molecules, morphine addiction, Prion disease, and PD pathways. Strikingly, the majority of the proteins enriched in the ribosome pathway were mitochondrial ribosomal proteins (mitoribosomes). The subsequent protein-protein interaction analysis and the weighted gene coexpression network analysis confirmed that the mitoribosome is the most enriched protein cluster. Furthermore, the mitoribosome was also identified in our analysis of a replication set of ten PD and nine healthy control SN tissues. This study provides potential disease pathways involved in PD and paves the way to study further the pathogenic mechanism of PD.

Cntnap2-dependent molecular networks in autism spectrum disorder revealed through an integrative multi-omics analysis.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder in which patients present with core symptoms of social communication impairment, restricted interest, and repetitive behaviors. Although various studies have been performed to identify ASD-related mechanisms, ASD pathology is still poorly understood. CNTNAP2 genetic variants have been found that represent ASD genetic risk factors, and disruption of Cntnap2 expression has been associated with ASD phenotypes in mice. In this study, we performed an integrative multi-omics analysis by combining quantitative proteometabolomic data obtained with Cntnap2 knockout (KO) mice with multi-omics data obtained from ASD patients and forebrain organoids to elucidate Cntnap2-dependent molecular networks in ASD. To this end, a mass spectrometry-based proteometabolomic analysis of the medial prefrontal cortex in Cntnap2 KO mice led to the identification of Cntnap2-associated molecular features, and these features were assessed in combination with multi-omics data obtained on the prefrontal cortex in ASD patients to identify bona fide ASD cellular processes. Furthermore, a reanalysis of single-cell RNA sequencing data obtained from forebrain organoids derived from patients with CNTNAP2-associated ASD revealed that the aforementioned identified ASD processes were mainly linked to excitatory neurons. On the basis of these data, we constructed Cntnap2-associated ASD network models showing mitochondrial dysfunction, axonal impairment, and synaptic activity. Our results may shed light on the Cntnap2-dependent molecular networks in ASD.

c-Abl Regulates the Pathological Deposition of TDP-43 via Tyrosine 43 Phosphorylation.

Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl-deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.

Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet.

Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (PcxL-/-) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. PcxL-/- mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to PcxL-/- mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that PcxL-/- mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in PcxL-/- mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways.

Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy.

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.

Mass spectrometry-based proteomics analysis of human globus pallidus from progressive supranuclear palsy patients discovers multiple disease pathways.

BACKGROUND: Progressive supranuclear palsy (PSP) is a neurodegenerative disorder clinically characterized by progressive postural instability, supranuclear gaze palsy, parkinsonism, and cognitive decline caused by degeneration in specific areas of the brain including globus pallidus (GP), substantia nigra, and subthalamic nucleus. However, the pathogenetic mechanism of PSP remains unclear to date.Unbiased global proteome analysis of patients' brain samples is an important step toward understanding PSP pathogenesis, as proteins serve as workhorses and building blocks of the cell. METHODS: In this study, we conducted unbiased mass spectrometry-based global proteome analysis of GP samples from 15 PSP patients, 15 Parkinson disease (PD) patients, and 15 healthy control (HC) individuals. To analyze 45 samples, we conducted 5 batches of 11-plex isobaric tandem mass tag (TMT)-based multiplexing experiments. The identified proteins were subjected to statistical analysis, such as a permutation-based statistical analysis in the significance analysis of microarray (SAM) method and bootstrap receiver operating characteristic curve (ROC)-based statistical analysis. Subsequently, we conducted bioinformatics analyses using gene set enrichment analysis, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) protein-protein interaction (PPI) analysis, and weighted gene co-expression network analysis (WGCNA). RESULTS: We have identified 10,231 proteins with ∼1,000 differentially expressed proteins. The gene set enrichment analysis results showed that the PD pathway was the most highly enriched, followed by pathways for oxidative phosphorylation, Alzheimer disease, Huntington disease, and non-alcoholic fatty liver disease (NAFLD) when PSP was compared to HC or PD. Most of the proteins enriched in the gene set enrichment analysis were mitochondrial proteins such as cytochrome c oxidase, NADH dehydrogenase, acyl carrier protein, succinate dehydrogenase, ADP/ATP translocase, cytochrome b-c1 complex, and/or ATP synthase. Strikingly, all of the enriched mitochondrial proteins in the PD pathway were downregulated in PSP compared to both HC and PD. The subsequent STRING PPI analysis and the WGCNA further supported that the mitochondrial proteins were the most highly enriched in PSP. CONCLUSION: Our study showed that the mitochondrial respiratory electron transport chain complex was the key proteins that were dysregulated in GP of PSP, suggesting that the mitochondrial respiratory electron transport chain complex could potentially be involved in the pathogenesis of PSP. This is the first global proteome analysis of human GP from PSP patients, and this study paves the way to understanding the mechanistic pathogenesis of PSP.

Intracellular energy controls dynamics of stress-induced ribonucleoprotein granules.

Energy metabolism and membraneless organelles have been implicated in human diseases including neurodegeneration. How energy deficiency regulates ribonucleoprotein particles such as stress granules (SGs) is still unclear. Here we identified a unique type of granules induced by energy deficiency under physiological conditions and uncovered the mechanisms by which the dynamics of diverse stress-induced granules are regulated. Severe energy deficiency induced the rapid formation of energy deficiency-induced stress granules (eSGs) independently of eIF2α phosphorylation, whereas moderate energy deficiency delayed the clearance of conventional SGs. The formation of eSGs or the clearance of SGs was regulated by the mTOR-4EBP1-eIF4E pathway or eIF4A1, involving assembly of the eIF4F complex or RNA condensation, respectively. In neurons or brain organoids derived from patients carrying the C9orf72 repeat expansion associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the eSG formation was enhanced, and the clearance of conventional SGs was impaired. These results reveal a critical role for intracellular energy in the regulation of diverse granules and suggest that disruptions in energy-controlled granule dynamics may contribute to the pathogenesis of relevant diseases.

RNA-sequencing and mass-spectrometry proteomic time-series analysis of T-cell differentiation identified multiple splice variants models that predicted validated protein biomarkers in inflammatory diseases.

Profiling of mRNA expression is an important method to identify biomarkers but complicated by limited correlations between mRNA expression and protein abundance. We hypothesised that these correlations could be improved by mathematical models based on measuring splice variants and time delay in protein translation. We characterised time-series of primary human naïve CD4+ T cells during early T helper type 1 differentiation with RNA-sequencing and mass-spectrometry proteomics. We performed computational time-series analysis in this system and in two other key human and murine immune cell types. Linear mathematical mixed time delayed splice variant models were used to predict protein abundances, and the models were validated using out-of-sample predictions. Lastly, we re-analysed RNA-seq datasets to evaluate biomarker discovery in five T-cell associated diseases, further validating the findings for multiple sclerosis (MS) and asthma. The new models significantly out-performing models not including the usage of multiple splice variants and time delays, as shown in cross-validation tests. Our mathematical models provided more differentially expressed proteins between patients and controls in all five diseases. Moreover, analysis of these proteins in asthma and MS supported their relevance. One marker, sCD27, was validated in MS using two independent cohorts for evaluating response to treatment and disease prognosis. In summary, our splice variant and time delay models substantially improved the prediction of protein abundance from mRNA expression in three different immune cell types. The models provided valuable biomarker candidates, which were further validated in MS and asthma.