Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Deficiency of Both Farnesoid X Receptor and Takeda G Protein-Coupled Receptor 5 Exacerbated Liver Fibrosis in Mice.

Activation of the nuclear bile acid receptor farnesoid X receptor (FXR) protects against hepatic inflammation and injury, while Takeda G protein-coupled receptor 5 (TGR5) promotes adipose tissue browning and energy metabolism. Here, we examined the physiological and metabolic effects of the deficiency of these two bile acid receptors on hepatic metabolism and injury in mice. Fxr/Tgr5 double knockout mice (DKO) were generated for metabolic phenotyping. Male DKO mice fed a chow diet had reduced liver lipid levels but increased serum cholesterol levels. Liver cholesterol 7α-hydroxylase (Cyp7a1) activity and sterol 12α-hydroxylase mRNA levels were induced, while ileum FXR target genes were suppressed in DKO mice compared to wild-type (WT) mice. Bile acid pool size was increased in DKO mice, with increased taurocholic acid and decreased tauromuricholic acids. RNA sequencing analysis of the liver transcriptome revealed that bile acid synthesis and fibrosis gene expression levels are increased in chow-fed DKO mice compared to WT mice and that the top regulated pathways are involved in steroid/cholesterol biosynthesis, liver cirrhosis, and connective tissue disease. Cholestyramine treatment further induced Cyp7a1 mRNA and protein in DKO mice and increased bile acid pool size, while cholic acid also induced Cyp7a1 in DKO mice, suggesting impaired bile acid feedback regulation. A Western diet containing 0.2% cholesterol increased oxidative stress and markers of liver fibrosis but not hepatic steatosis in DKO mice. Conclusion: FXR and TGR5 play critical roles in protecting the liver from inflammation and fibrosis, and deficiency of both of these bile acid receptors in mice increased cholic acid synthesis and the bile acid pool, liver fibrosis, and inflammation; FXR and TGR5 DKO mice may be a model for liver fibrosis.

A minimally Invasive Biomarker for Sensitive and Accurate Diagnosis of Parkinson's Disease.

Importance: Parkinson's disease (PD), the second most common neurodegenerative disease, is pathologically characterized by intraneuronal deposition of misfolded alpha-synuclein aggregates (αSyn D ). αSyn D seeding activities in CSF and skin samples have shown great promise in PD diagnosis, but they require invasive procedures. Sensitive and accurate αSyn D seed amplification assay (αSyn-SAA) for more accessible and minimally invasive samples (such as blood and saliva) are urgently needed for PD pathological diagnosis in routine clinical practice. Objective: To develop a sensitive and accurate αSyn-SAA biomarker using blood and saliva samples for sensitive, accurate and minimally invasive PD diagnosis. Design Setting and Participants: This prospective diagnostic study evaluates serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC) without PD at an academic Parkinson's and Movement Disorders Center from February 2020 to March 2024. Patients diagnosed with non-PD parkinsonism were excluded from this analysis. A total of 124 serum samples (82 PD and 42 HC) and 131 saliva samples (83 PD and 48 HC) were collected and examined by αSyn-SAA. Out of the 124 serum donors, a subset of 74 subjects (48 PD and 26 HC) also donated saliva samples during the same visits. PD patients with serum samples had a mean age of 69.21 years (range 44-88); HC subjects with serum samples had a mean age of 66.55 years (range 44-81); PD patients with saliva samples had a mean age of 69.58 years (range 49-87); HC subjects with saliva samples had a mean age of 64.71 years (range 30-81). Main Outcomes and Measures: Serum and/or saliva αSyn D seeding activities from PD and HC subjects were measured by αSyn-SAA using the Real-Time Quaking-Induced Conversion (RT-QuIC) platform. These PD patients had extensive clinical assessments including MDS-UPDRS. For a subset of PD and HC subjects whose serum and saliva samples were both collected during the same visits, the αSyn D seeding activities in both samples from the same subjects were examined, and the diagnostic accuracies for PD based on the seeding activities in either sample alone or both samples together were compared. Results: RT-QuIC analysis of αSyn D seeding activities in the 124 serum samples revealed a sensitivity of 80.49%, a specificity of 90.48%, and an accuracy of 0.9006 (AUC of ROC, 95% CI, 0.8472-0.9539, p <0.0001) for PD diagnosis. RT-QuIC analysis of αSyn D seeding activity in 131 saliva samples revealed a sensitivity of 74.70%, a specificity of 97.92%, and an accuracy of 0.8966 (AUC of ROC, 95% CI, 0.8454-0.9478, p <0.0001). When aSyn D seeding activities in the paired serum-saliva samples from the subset of 48 PD and 26 HC subjects were considered together, sensitivity was 95.83%, specificity was 96.15%, and the accuracy was 0.98 (AUC of ROC, 95% CI, 0.96-1.00, p <0.001), which are significantly better than when αSyn D seeding activities in either serum or saliva were used alone. For the paired serum-saliva samples, when specificity was set at 100% by elevating the αSyn-SAA cutoff values, a sensitivity of 91.7% and an accuracy of 0.9457 were still attained. Detailed correlation analysis revealed that αSyn D seeding activities in the serum of PD patients were correlated inversely with Montreal Cognitive Assessment (MoCA) score ( p =0.04), positively with Hamilton Depression Rating Scale (HAM-D) ( p =0.03), and weakly positively with PDQ-39 cognitive impairment score ( p =0.07). Subgroup analysis revealed that the inverse correlation with MoCA was only seen in males ( p =0.013) and weakly in the ≥70 age group ( p =0.07), and that the positive correlation with HAM-D was only seen in females ( p =0.04) and in the <70 age group ( p =0.01). In contrast, αSyn D seeding activities in the saliva of PD patients were inversely correlated with age at diagnosis ( p =0.02) and the REM sleep behavior disorder (RBD) status ( p =0.04), but subgroup analysis showed that the inverse correlation with age at diagnosis was only seen in males ( p =0.04) and in the <70 age group ( p =0.01). Conclusion and Relevance: Our data show that concurrent RT-QuIC assay of αSyn D seeding activities in both serum and saliva can achieve high diagnostic accuracies comparable to that of CSF αSyn-SAA, suggesting that αSyn D seeding activities in serum and saliva together can potentially be used as a valuable biomarker for highly sensitive, accurate, and minimally invasive diagnosis of PD in routine clinical practice. αSyn D seeding activities in serum and saliva of PD patients correlate differentially with some clinical characteristics and in an age and sex-dependent manner. KEY POINTS: Question: Are αSyn D seeding activities in serum and saliva together a more sensitive and accurate diagnostic PD biomarker than αSyn D seeding activities in either sample type alone? Are αSyn D seeding activities in either serum or saliva correlated with any clinical characteristics? Findings: Examinations of αSyn D seeding activities in 124 serum samples and 131 saliva samples from PD and heathy control subjects show that αSyn D seeding activities in both serum and saliva samples together can provide significantly more sensitive and accurate diagnosis of PD than either sample type alone. αSyn D seeding activities in serum or saliva exhibit varied inverse or positive correlations with some clinical features in an age and sex-dependent manner. Meaning: αSyn D seeding activities in serum and saliva together can potentially be used as a valuable pathological biomarker for highly sensitive, accurate, and minimally invasive PD diagnosis in routine clinical practice and clinical studies, and αSyn D seeding activities in serum or saliva correlate with some clinical characteristics in an age and sex-dependent manner, suggesting some possible clinical utility of quantitative serum/saliva αSyn-SAA data.

Seeding Activity of Skin Misfolded Tau as a Biomarker for Tauopathies.

Background: Tauopathies are a group of age-related neurodegenerative diseases characterized by the accumulation of pathologically phosphorylated tau protein in the brain, leading to prion-like propagation and aggregation. They include Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Currently, reliable diagnostic biomarkers that directly reflect the capability of propagation and spreading of misfolded tau aggregates in peripheral tissues and body fluids are lacking. Methods: We utilized the seed-amplification assay (SAA) employing ultrasensitive real-time quaking-induced conversion (RT-QuIC) to assess the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies, including AD, PSP, CBD, and PiD, compared to normal controls. Results: We found that the skin prion-SAA demonstrated a significantly higher sensitivity (75-80%) and specificity (95-100%) for detecting tauopathy, depending on the tau substrates used. Moreover, increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients examined. Analysis of the end products of skin-tau SAA confirmed that the increased seeding activity was accompanied by the formation of tau aggregates with different physicochemical properties related to two different tau substrates used. Conclusions: Overall, our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of misfolded tau in the skin could serve as a diagnostic biomarker for tauopathies.