The discovery that heterozygous missense mutations in the gene encoding triggering receptor expressed on myeloid cells 2 (TREM2) are risk factors for Alzheimer’s disease (AD), with only the apolipoprotein E (APOE) ε4 gene allele conferring a higher risk, has led to increased interest in immune biology in the brain. TREM2 is expressed on microglia, the resident immune cells of the brain and has been linked to phagocytotic clearance of amyloid β (Aβ) plaques. Soluble TREM2 (sTREM2) has previously been measured in cerebrospinal fluid (CSF) by ELISA but in our hands commercial kits have proved unreliable, suggesting that other methods may be required. We developed a mass spectrometry method using selected reaction monitoring for the presence of a TREM2 peptide, which can be used to quantify levels of sTREM2 in CSF. We examined CSF samples from memory clinics in Sweden and the UK. For all samples the following were available: clinical diagnosis, age, sex, and measurements of the CSF AD biomarkers Aβ42, T-tau and P-tau181. AD patients (n = 37) all met biomarker (IWG2) criteria for AD. Control individuals (n = 22) were cognitively normal without evidence for AD in CSF. We found significantly higher sTREM2 concentration in AD compared to control CSF. There were significant correlations between CSF sTREM2 and T-tau as well as P-tau181. CSF sTREM2 increase in AD was replicated in a second, independent cohort consisting of 24 AD patients and 16 healthy volunteers. CSF concentrations of sTREM2 are higher in AD than in controls, and correlate with markers of neurodegeneration. CSF sTREM2 may be used to quantify glial activation in AD.

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.

Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer’s Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer’s and Parkinson’s disease (PD). Thirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2−activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r2 ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r2 ≥ 0.4, p ≤ 0.03) with phosphorylated–tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups. Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

‘Long Covid’, or medical complications associated with post SARS-CoV-2 infection, is a significant post-viral complication that is being more and more commonly reported in patients. Therefore, there is an increasing need to understand the disease mechanisms, identify drug targets and inflammatory processes associated with a SARS-CoV-2 infection. To address this need, we created a targeted mass spectrometry based multiplexed panel of 96 immune response associated proteins. We applied the multiplex assay to a cohort of serum samples from asymptomatic and moderately affected patients. All patients had tested positive for a SARS-CoV-2 infection by PCR and were determined to be subsequently positive for antibodies. Even 40-60 days post-viral infection, we observed a significant remaining inflammatory response in all patients. Proteins that were still affected were associated with the anti-inflammatory response and mitochondrial stress. This indicates that biochemical and inflammatory pathways within the body can remain perturbed long after SARS-CoV-2 infections have subsided even in asymptomatic and moderately affected patients.

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.

‘Long Covid’, or medical complications associated with post SARS-CoV-2 infection, is a significant post-viral complication that is being more and more commonly reported in patients. Therefore, there is an increasing need to understand the disease mechanisms, identify drug targets and inflammatory processes associated with a SARS-CoV-2 infection. To address this need, we created a targeted mass spectrometry based multiplexed panel of 96 immune response associated proteins. We applied the multiplex assay to a cohort of serum samples from asymptomatic and moderately affected patients. All patients had tested positive for a SARS-CoV-2 infection by PCR and were determined to be subsequently positive for antibodies. Even 40-60 days post-viral infection, we observed a significant remaining inflammatory response in all patients. Proteins that were still affected were associated with the anti-inflammatory response and mitochondrial stress. This indicates that biochemical and inflammatory pathways within the body can remain perturbed long after SARS-CoV-2 infections have subsided even in asymptomatic and moderately affected patients.