Detecting and Validating MAPT Mutations in Neurodegeneration Patients and Analysis of Exon Splicing Consequences.

Mutation of MAPT has been observed in patients with parkinsonism, progressive supranuclear palsy, and corticobasal degeneration and is a significant cause of frontotemporal dementia. In this chapter, we discuss considerations for next-generation sequencing analysis to identify MAPT mutations in patient genomic DNA and describe the validation of these mutations by Sanger sequencing. One of the most common effects of MAPT mutations is differential splicing of exon 10, which leads to an imbalance in the proportion of 3-repeat and 4-repeat tau isoforms. We describe how to investigate the effect of novel DNA variants on the splicing efficiency of this exon in vitro using the exon-trapping technique, also known as the splicing reporter minigene assay.

ReTimeML: a retention time predictor that supports the LC-MS/MS analysis of sphingolipids.

The analysis of ceramide (Cer) and sphingomyelin (SM) lipid species using liquid chromatography-tandem mass spectrometry (LC-MS/MS) continues to present challenges as their precursor mass and fragmentation can correspond to multiple molecular arrangements. To address this constraint, we developed ReTimeML, a freeware that automates the expected retention times (RTs) for Cer and SM lipid profiles from complex chromatograms. ReTimeML works on the principle that LC-MS/MS experiments have pre-determined RTs from internal standards, calibrators or quality controls used throughout the analysis. Employed as reference RTs, ReTimeML subsequently extrapolates the RTs of unknowns using its machine-learned regression library of mass-to-charge (m/z) versus RT profiles, which does not require model retraining for adaptability on different LC-MS/MS pipelines. We validated ReTimeML RT estimations for various Cer and SM structures across different biologicals, tissues and LC-MS/MS setups, exhibiting a mean variance between 0.23 and 2.43% compared to user annotations. ReTimeML also aided the disambiguation of SM identities from isobar distributions in paired serum-cerebrospinal fluid from healthy volunteers, allowing us to identify a series of non-canonical SMs associated between the two biofluids comprised of a polyunsaturated structure that confers increased stability against catabolic clearance.

Ranking the risk factors for Alzheimer's disease; findings from the UK Biobank study.

Background: The cause of the most common form of dementia, sporadic Alzheimer's disease (AD), remains unknown. This may reflect insufficiently powered studies to date for this multi-factorial disorder. The UK Biobank dataset presents a unique opportunity to rank known risk factors and determine novel variables. Methods: A custom machine learning approach for high dimensionality data was applied to explore prospectively associations between AD in a sub-cohort of 156,209 UK Biobank participants aged 60-70 including more than 2,090 who were subsequently diagnosed with AD. Results: After the possession of the APOE4 allele, the next highest ranked risk factors were other genetic variants within the TOMM40-APOE-APOC1 locus. When stratified by their apolipoprotein epsilon 4 (APOE4) carrier status, the most prominent risk factors in carriers were AST:ALT ratio, the "number of treatments/ medications" taken as well as "time spent in hospital" while protection was conferred by "Sleeplessness/Insomnia". In non-APOE carriers, lower socioeconomic status and fewer years of education were highly ranked but effect sizes were small relative to APOE4 carriers. Conclusions: Possession of the APOE4 allele was confirmed as the most important risk factor in AD. Other TOMM40-APOE-APOC1 locus variants further moderate the risk of AD in APOE4 carriers. Liver pathology is a novel risk factor in APOE4 carriers while "Sleeplessness/Insomnia" is protective in AD irrespective of APOE4 status. Other factors such as "Number of treatments/ medications" suggest that multimorbidity is an important risk factor for AD. Future treatments aimed at co-morbidities, including liver disease, may concomitantly lower the risk of sporadic AD.

Machine learning analysis of the UK Biobank reveals IGF-1 and inflammatory biomarkers predict Parkinson's disease risk.

INTRODUCTION: Parkinson's disease (PD) is the most common movement disorder, and its prevalence is increasing rapidly worldwide with an ageing population. The UK Biobank is the world's largest and most comprehensive longitudinal study of ageing community volunteers. The cause of the common form of PD is multifactorial, but the degree of causal heterogeneity among patients or the relative importance of one risk factor over another is unclear. This is a major impediment to the discovery of disease-modifying therapies. METHODS: We used an integrated machine learning algorithm (IDEARS) to explore the relative effects of 1,753 measured non-genetic variables in 334,062 eligible UK Biobank participants, including 2,719 who had developed PD since their recruitment into the study. RESULTS: Male gender was the highest-ranked risk factor, followed by elevated serum insulin-like growth factor 1 (IGF-1), lymphocyte count, and neutrophil/lymphocyte ratio. A group of factors aligned with the symptoms of frailty also ranked highly. IGF-1 and neutrophil/lymphocyte ratio were also elevated in both sexes before PD diagnosis and at the point of diagnosis. DISCUSSION: The use of machine learning with the UK Biobank provides the best opportunity to explore the multidimensional nature of PD. Our results suggest that novel risk biomarkers, including elevated IGF-1 and NLR, may play a role in, or are indicative of PD pathomechanisms. In particular, our results are consistent with PD being a central manifestation of a systemic inflammatory disease. These biomarkers may be used clinically to predict future PD risk, improve early diagnosis and provide new therapeutic avenues.

Machine learning analysis of the UK Biobank reveals prognostic and diagnostic immune biomarkers for polyneuropathy and neuropathic pain in diabetes.

AIMS: We assessed the health data of 11,047 people with diabetes in the UK Biobank to rank 329 risk factors for diabetic polyneuropathy (DPN) and DPN with chronic neuropathic pain without a priori assumption. METHODS: The Integrated Disease Explanation and Risk Scoring (IDEARS) platform applies machine learning algorithms to multimodal data to determine individual disease risk, and rank risk factor importance using mean SHapley Additive exPlanations (SHAP) score. RESULTS: IDEARS models showed discriminative performances with AUC > 0.64. Lower socioeconomic status, being overweight, poor overall health, cystatin C, HbA1C, and immune activation marker, C-reactive protein (CRP), predict DPN risk. Neutrophils and monocytes were higher in males and lymphocytes lower in females with diabetes that develop DPN. Neutrophil-to-Lymphocyte Ratio (NLR) was increased and IGF-1 levels decreased in people with type 2 diabetes that later develop DPN. CRP was significantly elevated in those with DPN and chronic neuropathic pain compared to DPN without pain. CONCLUSIONS: Lifestyle factors and blood biomarkers predict the later development of DPN and may relate to DPN pathomechanisms. Our results are consistent with DPN as a disease involving systemic inflammation. We advocate for the use of these biomarkers clinically to predict future DPN risk and improve early diagnosis.

Polygenic risk of paclitaxel-induced peripheral neuropathy: a genome-wide association study.

BACKGROUND: Genetic risk factors for chemotherapy-induced peripheral neuropathy (CIPN), a major dose-limiting side-effect of paclitaxel, are not well understood. METHODS: We performed a genome-wide association study (GWAS) in 183 paclitaxel-treated patients to identify genetic loci associated with CIPN assessed via comprehensive neuropathy phenotyping tools (patient-reported, clinical and neurological grading scales). Bioinformatic analyses including pathway enrichment and polygenic risk score analysis were used to identify mechanistic pathways of interest. RESULTS: In total, 77% of the cohort were classified with CIPN (n = 139), with moderate/severe neuropathy in 36%. GWAS was undertaken separately for the three measures of CIPN. GWAS of patient-reported CIPN identified 4 chromosomal regions that exceeded genome-wide significance (rs9846958, chromosome 3; rs117158921, chromosome 18; rs4560447, chromosome 4; rs200091415, chromosome 10). rs4560447 is located within a protein-coding gene, LIMCH1, associated with actin and neural development and expressed in the dorsal root ganglia (DRG). There were additional risk loci that exceeded the statistical threshold for suggestive genome-wide association (P < 1 × 10-5) for all measures. A polygenic risk score calculated from the top 46 ranked SNPs was highly correlated with patient-reported CIPN (r2 = 0.53; P = 1.54 × 10-35). Overlap analysis was performed to identify 3338 genes which were in common between the patient-reported CIPN, neurological grading scale and clinical grading scale GWAS. The common gene set was subsequently analysed for enrichment of gene ontology (GO) and Reactome pathways, identifying a number of pathways, including the axon development pathway (GO:0061564; P = 1.78 × 10-6) and neuronal system (R-HSA-112316; adjusted P = 3.33 × 10-7). CONCLUSIONS: Our findings highlight the potential role of axon development and regeneration pathways in paclitaxel-induced CIPN.

Vascular Dysfunction Is Central to Alzheimer's Disease Pathogenesis in APOE e4 Carriers.

Alzheimer's disease (AD) is the most common form of dementia and the leading risk factor, after age, is possession of the apolipoprotein E epsilon 4 allele (APOE4). Approximately 50% of AD patients carry one or two copies of APOE4 but the mechanisms by which it confers risk are still unknown. APOE4 carriers are reported to demonstrate changes in brain structure, cognition, and neuropathology, but findings have been inconsistent across studies. In the present study, we used multi-modal data to characterise the effects of APOE4 on the brain, to investigate whether AD pathology manifests differently in APOE4 carriers, and to determine if AD pathomechanisms are different between carriers and non-carriers. Brain structural differences in APOE4 carriers were characterised by applying machine learning to over 2000 brain MRI measurements from 33,384 non-demented UK biobank study participants. APOE4 carriers showed brain changes consistent with vascular dysfunction, such as reduced white matter integrity in posterior brain regions. The relationship between APOE4 and AD pathology was explored among the 1260 individuals from the Religious Orders Study and Memory and Aging Project (ROSMAP). APOE4 status had a greater effect on amyloid than tau load, particularly amyloid in the posterior cortical regions. APOE status was also highly correlated with cerebral amyloid angiopathy (CAA). Bulk tissue brain transcriptomic data from ROSMAP and a similar dataset from the Mount Sinai Brain Bank showed that differentially expressed genes between the dementia and non-dementia groups were enriched for vascular-related processes (e.g., "angiogenesis") in APOE4 carriers only. Immune-related transcripts were more strongly correlated with AD pathology in APOE4 carriers with some transcripts such as TREM2 and positively correlated with pathology severity in APOE4 carriers, but negatively in non-carriers. Overall, cumulative evidence from the largest neuroimaging, pathology, and transcriptomic studies available suggests that vascular dysfunction is key to the development of AD in APOE4 carriers. However, further studies are required to tease out non-APOE4-specific mechanisms.

The Key Factors Predicting Dementia in Individuals With Alzheimer's Disease-Type Pathology.

Dementia affects millions of individuals worldwide, yet there are no effective treatments. Alzheimer's disease, the most common form of dementia, is characterized by amyloid and tau pathology with amyloid accumulation thought to precipitate tau pathology, neurodegeneration, and dementia. The Religious Orders Study and Memory and Aging Project (ROSMAP) cohort is a unique resource with quantitative pathology from multiple brain regions, RNA sequencing, and longitudinal cognitive data. Our previous work applying machine learning to the RNA sequencing data identified lactoferrin (LTF) as the gene most predictive of amyloid accumulation with a potential amyloidogenic mechanism identified in vitro and with cell-culture models. In the present study, we examined which pathologies and genes were related to cognitive status (dementia, mild impairment, and no cognitive impairment) and rate of cognitive decline. Tau load in the anterior cingulate and ADAMTS2, encoding a metallopeptidase, were the respective regional pathology and gene most associated with cognitive decline, while PRTN3, encoding a serine protease, was the key protective feature. ADAMTS2, but not PRTN3, was related to amyloid and tau load in the previous study while LTF was not related to cognitive decline here. These findings confirm a general relationship between tau pathology and dementia, show the specific importance of tau pathology in the anterior cingulate cortex and identify ADAMTS2 as a potential target for slowing cognitive decline.

Overlap between Central and Peripheral Transcriptomes in Parkinson's Disease but Not Alzheimer's Disease.

Most neurodegenerative disorders take decades to develop, and their early detection is challenged by confounding non-pathological ageing processes. Therefore, the discovery of genes and molecular pathways in both peripheral and brain tissues that are highly predictive of disease evolution is necessary. To find genes that influence Alzheimer's disease (AD) and Parkinson's disease (PD) pathogenesis, human RNA-Seq transcriptomic data from Brodmann Area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC), whole blood (WB), and peripheral blood mononuclear cells (PBMC) were analysed using a combination of differential gene expression and a random forest-based machine learning algorithm. The results suggest that there is little overlap between PD and AD, and the AD brain signature is unique mainly compared to blood-based samples. Moreover, the AD-BA9 was characterised by changes in 'nervous system development' with Myocyte-specific enhancer factor 2C (Mef2C), encoding a transcription factor that induces microglia activation, a prominent feature. The peripheral AD transcriptome was associated with alterations in 'viral process', and FYN, which has been previously shown to link amyloid-beta and tau, was the prominent feature. However, in the absence of any overlap with the central transcriptome, it is unclear whether peripheral FYN levels reflect AD severity or progression. In PD, central and peripheral signatures are characterised by anomalies in 'exocytosis' and specific genes related to the SNARE complex, including Vesicle-associated membrane protein 2 (VAMP2), Syntaxin 1A (STX1A), and p21-activated kinase 1 (PAK1). This is consistent with our current understanding of the physiological role of alpha-synuclein and how alpha-synuclein oligomers compromise vesicle docking and neurotransmission. Overall, the results describe distinct disease-specific pathomechanisms, both within the brain and peripherally, for the two most common neurodegenerative disorders.

The cytokines interleukin-6 and interferon-α induce distinct microglia phenotypes.

BACKGROUND: Elevated production of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is implicated in the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Transgenic mice with CNS-targeted chronic production of IL-6 (GFAP-IL6) or IFN-α (GFAP-IFN) recapitulate important clinical and pathological features of these human diseases. The activation of microglia is a prominent manifestation found both in the human diseases and in the transgenic mice, yet little is known about how this contributes to disease pathology. METHODS: Here, we used a combination of ex vivo and in situ techniques to characterize the molecular, cellular and transcriptomic phenotypes of microglia in GFAP-IL6 versus GFAP-IFN mice. In addition, a transcriptomic meta-analysis was performed to compare the microglia response from GFAP-IL6 and GFAP-IFN mice to the response of microglia in a range of neurodegenerative and neuroinflammatory disorders. RESULTS: We demonstrated that microglia show stimulus-specific responses to IL-6 versus IFN-α in the brain resulting in unique and extensive molecular and cellular adaptations. In GFAP-IL6 mice, microglia proliferated, had shortened, less branched processes and elicited transcriptomic and molecular changes associated with phagocytosis and lipid processing. In comparison, microglia in the brain of GFAP-IFN mice exhibited increased proliferation and apoptosis, had larger, hyper-ramified processes and showed transcriptomic and surface marker changes associated with antigen presentation and antiviral response. Further, a transcriptomic meta-analysis revealed that IL-6 and IFN-α both contribute to the formation of a core microglia response in animal models of neurodegenerative and neuroinflammatory disorders, such as Alzheimer's disease, tauopathy, multiple sclerosis and lipopolysaccharide-induced endotoxemia. CONCLUSIONS: Our findings demonstrate that microglia responses to IL-6 and IFN-α are highly stimulus-specific, wide-ranging and give rise to divergent phenotypes that modulate microglia responses in neuroinflammatory and neurodegenerative diseases.

New perspectives on cytoskeletal dysregulation and mitochondrial mislocalization in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington's disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.

The acute phase protein lactoferrin is a key feature of Alzheimer's disease and predictor of Aß burden through induction of APP amyloidogenic processing.

Amyloidogenic processing of the amyloid precursor protein (APP) forms the amyloid-ß peptide (Aß) component of pathognomonic extracellular plaques of AD. Additional early cortical changes in AD include neuroinflammation and elevated iron levels. Activation of the innate immune system in the brain is a neuroprotective response to infection; however, persistent neuroinflammation is linked to AD neuropathology by uncertain mechanisms. Non-parametric machine learning analysis on transcriptomic data from a large neuropathologically characterised patient cohort revealed the acute phase protein lactoferrin (Lf) as the key predictor of amyloid pathology. In vitro studies showed that an interaction between APP and the iron-bound form of Lf secreted from activated microglia diverted neuronal APP endocytosis from the canonical clathrin-dependent pathway to one requiring ADP ribosylation factor 6 trafficking. By rerouting APP recycling to the Rab11-positive compartment for amyloidogenic processing, Lf dramatically increased neuronal Aß production. Lf emerges as a novel pharmacological target for AD that not only modulates APP processing but provides a link between Aß production, neuroinflammation and iron dysregulation.

Periodontitis induces endothelial dysfunction in mice.

The treatment of periodontitis has numerous positive effects on established chronic health conditions, including cardiovascular disease and diabetes. However, ethical considerations do limit the establishment of human trials to investigate whether periodontitis promotes the early stages of chronic conditions. Therefore, the aim of this study was to investigate whether periodontitis induces endothelial dysfunction in hyperlipidemic apolipoprotein E gene-deficient (ApoE-/-) mice. Forty-five 8-week-old ApoE-/- mice were challenged by oral lavage with Porphyromonas gingivalis and Streptococcus gordonii for 4 weeks. A subgroup of animals (n = 15-17/group) was placed in a metabolic chamber immediately before euthanasia at 4 weeks to measure VO2/CO2 concentrations and voluntary locomotion. In infected and control animals alveolar bone levels were measured by x-ray imaging and endothelial function was determined by measuring endothelial-dependent vasorelaxation of aortic rings. The mRNA expression levels of serum amyloid A and tumor necrosis factor were determined in liver tissues by qRT PCR and protein concentrations in serum by ELISA. Caecal contents were analysed by sequencing to determine changes to the gut microbiota to investigate linkages between microbiome and systemic changes. The results showed that oral lavage of P. gingivalis and S. gordonii for 4 weeks, initiated periodontitis in ApoE-/- mice, similar to the human situation. The oral inflammation was accompanied by a significant increase in mRNA expression of pro-inflammatory mediators serum amyloid A1 and tumor necrosis factor in the liver. Mice with periodontitis also exhibited impaired endothelial-dependent vasorelaxation responses to acetylcholine. This systemic response was connected to increased energy expenditure, locomotion and respiratory quotient. No differences were detected in caecal microbiota between the infected and control animals. Overall, this is the first report that provide evidence that periodontitis induces endothelial dysfunction in mice. Other systemic responses observed in response to the local reaction need further investigation. The study suggests that early prevention of periodontitis may help limit the early stages of endothelial dysfunction that is linked to atherogenesis in humans.

Author Correction: A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

A Correction to this paper has been published: https://doi.org/10.1038/s41590-021-00929-x.

Defining early changes in Alzheimer's disease from RNA sequencing of brain regions differentially affected by pathology.

Tau pathology in Alzheimer's disease (AD) spreads in a predictable pattern that corresponds with disease symptoms and severity. At post-mortem there are cortical regions that range from mildly to severely affected by tau pathology and neuronal loss. A comparison of the molecular signatures of these differentially affected areas within cases and between cases and controls may allow the temporal modelling of disease progression. Here we used RNA sequencing to explore differential gene expression in the mildly affected primary visual cortex and moderately affected precuneus of ten age-, gender- and RNA quality-matched post-mortem brains from AD patients and healthy controls. The two regions in AD cases had similar transcriptomic signatures but there were broader abnormalities in the precuneus consistent with the greater tau load. Both regions were characterised by upregulation of immune-related genes such as those encoding triggering receptor expressed on myeloid cells 2 and membrane spanning 4-domains A6A and milder changes in insulin/IGF1 signalling. The precuneus in AD was also characterised by changes in vesicle secretion and downregulation of the interneuronal subtype marker, somatostatin. The 'early' AD transcriptome is characterised by perturbations in synaptic vesicle secretion on a background of neuroimmune dysfunction. In particular, the synaptic deficits that characterise AD may begin with the somatostatin division of inhibitory neurotransmission.

A regulatory T cell Notch4-GDF15 axis licenses tissue inflammation in asthma.

Elucidating the mechanisms that sustain asthmatic inflammation is critical for precision therapies. We found that interleukin-6- and STAT3 transcription factor-dependent upregulation of Notch4 receptor on lung tissue regulatory T (Treg) cells is necessary for allergens and particulate matter pollutants to promote airway inflammation. Notch4 subverted Treg cells into the type 2 and type 17 helper (TH2 and TH17) effector T cells by Wnt and Hippo pathway-dependent mechanisms. Wnt activation induced growth and differentiation factor 15 expression in Treg cells, which activated group 2 innate lymphoid cells to provide a feed-forward mechanism for aggravated inflammation. Notch4, Wnt and Hippo were upregulated in circulating Treg cells of individuals with asthma as a function of disease severity, in association with reduced Treg cell-mediated suppression. Our studies thus identify Notch4-mediated immune tolerance subversion as a fundamental mechanism that licenses tissue inflammation in asthma.

Novel immune biomarkers in complex regional pain syndrome.

We investigated serum levels of 29 cytokines and immune-activated kynurenine and tetrahydrobiopterin pathway metabolites in 15 complex regional pain syndrome (CRPS) subjects and 14 healthy controls. Significant reductions in interleukin-37 and tryptophan were found in CRPS subjects, along with positive correlations between kynurenine/tryptophan ratio and TNF-α levels with kinesiophobia, tetrahydrobiopterin levels with McGill pain score, sRAGE, and xanthurenic acid and neopterin levels with depression, anxiety and stress scores. Using machine learning, we identified a set of binary variables, including IL-37 and GM-CSF, capable of distinguishing controls from established CRPS subjects. These results suggest possible involvement of various inflammatory markers in CRPS pathogenesis.