RESUMEN
Parkinson's disease is increasingly prevalent. It progresses from the pre-motor stage (characterised by non-motor symptoms like REM sleep behaviour disorder), to the disabling motor stage. We need objective biomarkers for early/pre-motor disease stages to be able to intervene and slow the underlying neurodegenerative process. Here, we validate a targeted multiplexed mass spectrometry assay for blood samples from recently diagnosed motor Parkinson's patients (n = 99), pre-motor individuals with isolated REM sleep behaviour disorder (two cohorts: n = 18 and n = 54 longitudinally), and healthy controls (n = 36). Our machine-learning model accurately identifies all Parkinson patients and classifies 79% of the pre-motor individuals up to 7 years before motor onset by analysing the expression of eight proteins-Granulin precursor, Mannan-binding-lectin-serine-peptidase-2, Endoplasmatic-reticulum-chaperone-BiP, Prostaglaindin-H2-D-isomaerase, Interceullular-adhesion-molecule-1, Complement C3, Dickkopf-WNT-signalling pathway-inhibitor-3, and Plasma-protease-C1-inhibitor. Many of these biomarkers correlate with symptom severity. This specific blood panel indicates molecular events in early stages and could help identify at-risk participants for clinical trials aimed at slowing/preventing motor Parkinson's disease.
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Biomarcadores , Enfermedad de Parkinson , Proteómica , Humanos , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/diagnóstico , Biomarcadores/sangre , Masculino , Proteómica/métodos , Femenino , Anciano , Persona de Mediana Edad , Aprendizaje Automático , Trastorno de la Conducta del Sueño REM/sangre , Trastorno de la Conducta del Sueño REM/diagnóstico , Estudios de Casos y Controles , Espectrometría de MasasRESUMEN
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is defined clinically by pathological left ventricular hypertrophy. We have previously developed a plasma proteomics biomarker panel that correlates with clinical markers of disease severity and sudden cardiac death risk in adult patients with HCM. The aim of this study was to investigate the utility of adult biomarkers and perform new discoveries in proteomics for childhood-onset HCM. METHODS: Fifty-nine protein biomarkers were identified from an exploratory plasma proteomics screen in children with HCM and augmented into our existing multiplexed targeted liquid chromatography-tandem/mass spectrometry-based assay. The association of these biomarkers with clinical phenotypes and outcomes was prospectively tested in plasma collected from 148 children with HCM and 50 healthy controls. Machine learning techniques were used to develop novel pediatric plasma proteomic biomarker panels. RESULTS: Four previously identified adult HCM markers (aldolase fructose-bisphosphate A, complement C3a, talin-1, and thrombospondin 1) and 3 new markers (glycogen phosphorylase B, lipoprotein a and profilin 1) were elevated in pediatric HCM. Using supervised machine learning applied to training (n=137) and validation cohorts (n=61), this 7-biomarker panel differentiated HCM from healthy controls with an area under the curve of 1.0 in the training data set (sensitivity 100% [95% CI, 95-100]; specificity 100% [95% CI, 96-100]) and 0.82 in the validation data set (sensitivity 75% [95% CI, 59-86]; specificity 88% [95% CI, 75-94]). Reduced circulating levels of 4 other peptides (apolipoprotein L1, complement 5b, immunoglobulin heavy constant epsilon, and serum amyloid A4) found in children with high sudden cardiac death risk provided complete separation from the low and intermediate risk groups and predicted mortality and adverse arrhythmic outcomes (hazard ratio, 2.04 [95% CI, 1.0-4.2]; P=0.044). CONCLUSIONS: In children, a 7-biomarker proteomics panel can distinguish HCM from controls with high sensitivity and specificity, and another 4-biomarker panel identifies those at high risk of adverse arrhythmic outcomes, including sudden cardiac death.
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Biomarcadores , Cardiomiopatía Hipertrófica , Proteómica , Humanos , Cardiomiopatía Hipertrófica/sangre , Cardiomiopatía Hipertrófica/diagnóstico , Biomarcadores/sangre , Niño , Femenino , Masculino , Preescolar , Adolescente , Pronóstico , Proteómica/métodos , Lactante , AdultoRESUMEN
Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Células Epiteliales , Mucosa Nasal , SARS-CoV-2 , Serina Endopeptidasas , Humanos , COVID-19/virología , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Adulto , Persona de Mediana Edad , Anciano , Células Epiteliales/virología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Mucosa Nasal/virología , Niño , Factores de Edad , Replicación Viral , Preescolar , Tropismo Viral , Masculino , Femenino , Anciano de 80 o más Años , Células Cultivadas , Adolescente , LactanteRESUMEN
A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.
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ARN , Tetrahidrofolato Deshidrogenasa , Humanos , Línea Celular , Péptidos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.
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Aciduria Argininosuccínica , Hepatopatías , Adulto , Humanos , Animales , Ratones , Aciduria Argininosuccínica/genética , Aciduria Argininosuccínica/terapia , Cisteína , Glutatión , MetabolómicaRESUMEN
SARS-CoV-2 initially infects cells in the nasopharynx and oral cavity. The immune system at these mucosal sites plays a crucial role in minimizing viral transmission and infection. To develop new strategies for preventing SARS-CoV-2 infection, this study aimed to identify proteins that protect against viral infection in saliva. We collected 551 saliva samples from 290 healthcare workers who had tested positive for COVID-19, before vaccination, between June and December 2020. The samples were categorized based on their ability to block or enhance infection using in vitro assays. Mass spectrometry and enzyme-linked immunosorbent assay experiments were used to identify and measure the abundance of proteins that specifically bind to SARS-CoV-2 antigens. Immunoglobulin (Ig)A specific to SARS-CoV-2 antigens was detectable in over 83% of the convalescent saliva samples. We found that concentrations of anti-receptor-binding domain IgA >500 pg/µg total protein in saliva correlate with reduced viral infectivity in vitro. However, there is a dissociation between the salivary IgA response to SARS-CoV-2, and systemic IgG titers in convalescent COVID-19 patients. Then, using an innovative technique known as spike-baited mass spectrometry, we identified novel spike-binding proteins in saliva, most notably vimentin, which correlated with increased viral infectivity in vitro and could serve as a therapeutic target against COVID-19.
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COVID-19 , Humanos , Anticuerpos Antivirales , Antígenos Virales , Inmunoglobulina A , SARS-CoV-2 , VimentinaRESUMEN
As disease-modifying therapies are now available for Alzheimer's disease (AD), accessible, accurate and affordable biomarkers to support diagnosis are urgently needed. We sought to develop a mass spectrometry-based urine test as a high-throughput screening tool for diagnosing AD. We collected urine from a discovery cohort (n = 11) of well-characterised individuals with AD (n = 6) and their asymptomatic, CSF biomarker-negative study partners (n = 5) and used untargeted proteomics for biomarker discovery. Protein biomarkers identified were taken forward to develop a high-throughput, multiplexed and targeted proteomic assay which was tested on an independent cohort (n = 21). The panel of proteins identified are known to be involved in AD pathogenesis. In comparing AD and controls, a panel of proteins including MIEN1, TNFB, VCAM1, REG1B and ABCA7 had a classification accuracy of 86%. These proteins have been previously implicated in AD pathogenesis. This suggests that urine-targeted mass spectrometry has potential utility as a diagnostic screening tool in AD.
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Enfermedad de Alzheimer , Sistema Urinario , Humanos , Enfermedad de Alzheimer/diagnóstico , Proteómica , Aprendizaje Automático , Biomarcadores , Proteínas de Neoplasias , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls (n = 10) and Gaucher (n = 4), Fabry (n = 10), Pompe (n = 2), mucopolysaccharidosis types I-VI (n = 52), and Niemann-Pick disease type C (NPC) (n = 5) patients. We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96-97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.
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Enfermedad de Fabry , Enfermedades por Almacenamiento Lisosomal , Humanos , Glucosilceramidas , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Enfermedad de Fabry/diagnóstico , Biomarcadores , Isoformas de ProteínasRESUMEN
Fabry disease stems from a deficiency of alpha-galactosidase and results in the accumulation of globotriaosylceramide (Gb3). However, the production of its deacylated form globotriaosylsphingosine (lyso-Gb3) is also observed and its plasma levels have closer association with disease severity. Studies have shown that lyso-Gb3 directly affects podocytes and causes sensitisation of peripheral nociceptive neurons. However, little is understood of the mechanisms of this cytotoxicity. To study the effect on neuronal cells, we incubated SH-Sy5y cells with lyso-Gb3 at low (20 ng/mL) and high (200 ng/mL) levels, to mimic mild and classical FD serum levels. We used glucosylsphingosine as a positive control to determine specific effects of lyso-Gb3. Proteomic analyses revealed that cellular systems affected by lyso-Gb3 included cell signalling particularly protein ubiquitination and protein translation. To confirm ER/proteasome perturbations, we performed an immune enrichment of ubiquitinated proteins and demonstrated specific increased protein ubiquitination at both doses. The most ubiquitinated proteins observed included the chaperone/heat shock proteins, cytoskeletal proteins and synthesis/translation proteins. To detect proteins that interact directly with lyso-Gb3, we immobilised lyso-lipids, then incubated them with neuronal cellular extracts and identified bound proteins using mass spectrometry. Proteins that specifically bound were chaperones and included HSP90, HSP60 and the TRiC complex. In conclusion, lyso-Gb3 exposure affects pathways involved in protein translation and folding. This response is observed as increased ubiquitination and changes in signalling proteins which may explain the multiple biological processes, particularly cellular remodelling, often associated with FD.
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Enfermedad de Fabry , Neuroblastoma , Humanos , Enfermedad de Fabry/genética , Proteínas Ubiquitinadas , Proteómica , alfa-Galactosidasa/genética , Esfingolípidos/metabolismo , Glucolípidos/metabolismo , Glucolípidos/farmacologíaRESUMEN
BACKGROUND: Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of scales, often affecting the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterized; however, recent publications suggest that common mechanisms are active in ichthyotic tissue and in analogous models of ichthyosis. OBJECTIVES: To determine common mechanisms of hyperkeratosis that may be easily targeted with small-molecule inhibitors. METHODS: We combined gene expression analysis of gene-specific short hairpin RNA (shRNA) knockdowns in rat epidermal keratinocytes (REKs) of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Tgm1 and Alox12b, and proteomic analysis of skin scale from patients with ARCI, as well as RNA sequencing data from rat epidermal keratinocytes treated with the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. RESULTS: We identified common activation of the TLR2 pathway. Exogenous TLR2 activation led to increased expression of important cornified envelope genes and, in organotypic culture, caused hyperkeratosis. Conversely, blockade of TLR2 signalling in keratinocytes from patients with ichthyosis and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time course of TLR2 activation in REKs revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread upregulation of epidermal differentiation-related proteins. Both nuclear factor kappa B phosphorylation and GATA3 upregulation was associated with this switch, and GATA3 overexpression was sufficient to increase keratin 1 expression. CONCLUSIONS: Taken together, these data define a dual role for TLR2 activation during epidermal barrier repair that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction.
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Ictiosis , Receptor Toll-Like 2 , Animales , Ratas , Ictiosis/genética , Queratina-1/genética , Mutación , Fenotipo , Proteómica , ARN Interferente Pequeño , Receptor Toll-Like 2/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by pathogenic variations in the DMD gene. There is a need for robust DMD biomarkers for diagnostic screening and to aid therapy monitoring. Creatine kinase, to date, is the only routinely used blood biomarker for DMD, although it lacks specificity and does not correlate with disease severity. To fill this critical gap, we present here novel data about dystrophin protein fragments detected in human plasma by a suspension bead immunoassay using two validated anti-dystrophin-specific antibodies. Using both antibodies, a reduction of the dystrophin signal is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD.
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Distrofia Muscular de Duchenne , Proteómica , Femenino , Humanos , Anticuerpos , Biomarcadores , Cromatografía Liquida , Distrofia Muscular de Duchenne/genética , Proteómica/métodos , Distrofina/sangreRESUMEN
Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease.
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Enfermedad de Fabry , Terapia Genética , Animales , Humanos , Ratones , Células Cultivadas , Enfermedad de Fabry/genética , Enfermedad de Fabry/terapia , Fibroblastos , Vectores Genéticos , Hígado/metabolismo , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismoRESUMEN
OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.
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COVID-19 , SARS-CoV-2 , Humanos , Pandemias , COVID-19/diagnóstico , Prueba de COVID-19 , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida , Estudios Prospectivos , Técnicas de Laboratorio Clínico/métodos , Sensibilidad y Especificidad , PéptidosRESUMEN
BACKGROUND: The majority of those infected by ancestral Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) during the UK first wave (starting March 2020) did not require hospitalisation. Most had a short-lived mild or asymptomatic infection, while others had symptoms that persisted for weeks or months. We hypothesized that the plasma proteome at the time of first infection would reflect differences in the inflammatory response that linked to symptom severity and duration. METHODS: We performed a nested longitudinal case-control study and targeted analysis of the plasma proteome of 156 healthcare workers (HCW) with and without lab confirmed SARS-CoV-2 infection. Targeted proteomic multiple-reaction monitoring analysis of 91 pre-selected proteins was undertaken in uninfected healthcare workers at baseline, and in infected healthcare workers serially, from 1 week prior to 6 weeks after their first confirmed SARS-CoV-2 infection. Symptom severity and antibody responses were also tracked. Questionnaires at 6 and 12 months collected data on persistent symptoms. FINDINGS: Within this cohort (median age 39 years, interquartile range 30-47 years), 54 healthcare workers (44% male) had PCR or antibody confirmed infection, with the remaining 102 (38% male) serving as uninfected controls. Following the first confirmed SARS-CoV-2 infection, perturbation of the plasma proteome persisted for up to 6 weeks, tracking symptom severity and antibody responses. Differentially abundant proteins were mostly coordinated around lipid, atherosclerosis and cholesterol metabolism pathways, complement and coagulation cascades, autophagy, and lysosomal function. The proteomic profile at the time of seroconversion associated with persistent symptoms out to 12 months. Data are available via ProteomeXchange with identifier PXD036590. INTERPRETATION: Our findings show that non-severe SARS-CoV-2 infection perturbs the plasma proteome for at least 6 weeks. The plasma proteomic signature at the time of seroconversion has the potential to identify which individuals are more likely to suffer from persistent symptoms related to SARS-CoV-2 infection. FUNDING INFORMATION: The COVIDsortium is supported by funding donated by individuals, charitable Trusts, and corporations including Goldman Sachs, Citadel and Citadel Securities, The Guy Foundation, GW Pharmaceuticals, Kusuma Trust, and Jagclif Charitable Trust, and enabled by Barts Charity with support from University College London Hospitals (UCLH) Charity. This work was additionally supported by the Translational Mass Spectrometry Research Group and the Biomedical Research Center (BRC) at Great Ormond Street Hospital.
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COVID-19 , SARS-CoV-2 , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios de Casos y Controles , Proteoma , ProteómicaRESUMEN
BACKGROUND: The molecular drivers of early sporadic Parkinson's disease (PD) remain unclear, and the presence of widespread end stage pathology in late disease masks the distinction between primary or causal disease-specific events and late secondary consequences in stressed or dying cells. However, early and mid-stage Parkinson's brains (Braak stages 3 and 4) exhibit alpha-synuclein inclusions and neuronal loss along a regional gradient of severity, from unaffected-mild-moderate-severe. Here, we exploited this spatial pathological gradient to investigate the molecular drivers of sporadic PD. METHODS: We combined high precision tissue sampling with unbiased large-scale profiling of protein expression across 9 brain regions in Braak stage 3 and 4 PD brains, and controls, and verified these results using targeted proteomic and functional analyses. RESULTS: We demonstrate that the spatio-temporal pathology gradient in early-mid PD brains is mirrored by a biochemical gradient of a changing proteome. Importantly, we identify two key events that occur early in the disease, prior to the occurrence of alpha-synuclein inclusions and neuronal loss: (i) a metabolic switch in the utilisation of energy substrates and energy production in the brain, and (ii) perturbation of the mitochondrial redox state. These changes may contribute to the regional vulnerability of developing alpha-synuclein pathology. Later in the disease, mitochondrial function is affected more severely, whilst mitochondrial metabolism, fatty acid oxidation, and mitochondrial respiration are affected across all brain regions. CONCLUSIONS: Our study provides an in-depth regional profile of the proteome at different stages of PD, and highlights that mitochondrial dysfunction is detectable prior to neuronal loss, and alpha-synuclein fibril deposition, suggesting that mitochondrial dysfunction is one of the key drivers of early disease.
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Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Mitocondrias/metabolismo , Enfermedad de Parkinson/patología , Proteoma/metabolismo , Proteómica , alfa-Sinucleína/metabolismoRESUMEN
Determining the protection an individual has to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VoCs) is crucial for future immune surveillance, vaccine development, and understanding of the changing immune response. We devised an informative assay to current ELISA-based serology using multiplexed, baited, targeted proteomics for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. Serum from individuals collected after infection or first- and second-dose vaccination demonstrates this approach and shows concordance with existing serology and neutralization. Our assays show altered responses of both immunoglobulins and complement to the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.1) VoCs and a reduced response to Omicron (B1.1.1529). We were able to identify individuals who had prior infection, and observed that C1q is closely associated with IgG1 (r > 0.82) and may better reflect neutralization to VoCs. Analyzing additional immunoproteins beyond immunoglobulin (Ig) G, provides important information about our understanding of the response to infection and vaccination.
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Formación de Anticuerpos , COVID-19 , Humanos , Proteómica , SARS-CoV-2/genética , Inmunoglobulina G , Anticuerpos AntiviralesRESUMEN
AIM: Using Niemann-Pick type C disease (NPC) as a paradigm, we aimed to improve biomarker discovery in patients with neurometabolic disorders. METHOD: Using a multiplexed liquid chromatography tandem mass spectrometry dried bloodspot assay, we developed a selective intelligent biomarker panel to monitor known biomarkers N-palmitoyl-O-phosphocholineserine and 3ß,5α,6ß-trihydroxy-cholanoyl-glycine as well as compounds predicted to be affected in NPC pathology. We applied this panel to a clinically relevant paediatric patient cohort (n = 75; 35 males, 40 females; mean age 7 years 6 months, range 4 days-19 years 8 months) presenting with neurodevelopmental and/or neurodegenerative pathology, similar to that observed in NPC. RESULTS: The panel had a far superior performance compared with individual biomarkers. Namely, NPC-related established biomarkers used individually had 91% to 97% specificity but the combined panel had 100% specificity. Moreover, multivariate analysis revealed long-chain isoforms of glucosylceramide were elevated and very specific for patients with NPC. INTERPRETATION: Despite advancements in next-generation sequencing and precision medicine, neurological non-enzymatic disorders remain difficult to diagnose and lack robust biomarkers or routine functional testing for genetic variants of unknown significance. Biomarker panels may have better diagnostic accuracy than individual biomarkers in neurometabolic disorders, hence they can facilitate more prompt disease identification and implementation of emerging targeted, disease-specific therapies. WHAT THIS PAPER ADDS: Intelligent biomarker panel design can help expedite diagnosis in neurometabolic disorders. In Niemann-Pick type C disease, such a panel performed better than individual biomarkers. Biomarker panels are easy to implement and widely applicable to neurometabolic conditions.
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Enfermedad de Niemann-Pick Tipo C , Masculino , Femenino , Niño , Humanos , Recién Nacido , Enfermedad de Niemann-Pick Tipo C/diagnóstico , BiomarcadoresRESUMEN
Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.
Asunto(s)
Liasas de Carbono-Carbono/genética , Lipidómica/métodos , Peroxisomas/metabolismo , Fitol/administración & dosificación , Proteómica/métodos , Animales , Encéfalo/metabolismo , Familia 2 del Citocromo P450/metabolismo , Familia 4 del Citocromo P450/metabolismo , Ácidos Grasos/metabolismo , Femenino , Técnicas de Inactivación de Genes , Riñón/metabolismo , Hígado/metabolismo , Masculino , Ratones , Oxidación-Reducción , Ácido Fitánico/análogos & derivados , Ácido Fitánico/metabolismo , Fitol/farmacologíaRESUMEN
Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene that causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (n = 4) compared to infant controls (n = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease.