Developing Skin Misfolded Protein-Based Diagnosis For Prion Disease And Other Neurodegenerative Disease

Background and Objectives: Prion diseases are associated with misfolded protein deposition in the central nervous system that similar to Alzheimer's disease(AD)and Parkinson's disease(PD),but it's specific that prion diseases are fatal and transmissible neurodegenerative diseases affectting both humans and animals.The current diagnosis of prion disease is mainly based on biopsy and autopsy of brain tissues for detection of Pr PSc deposition and spongiforms.However,the high risk of invasive biopsy makes it difficult to implement,and effective alternative clinical diagnostics have not been reported so far.And autopsy is not easy to achieve in many countries,making the diagnosis of these diseases more ambiguous.Since the skin is the most accessible organ that not only has a high degree of innervation,but also shares a common embryonic origin with the brain.Recent studies have shown the feasiblility of Pr PSc detection in the skin of 263 K infected animals and CJD patients at the clinical terminal stage.In a recent study,reseachers detected Pr PSc in the skin of CJD patients by RT-Qu IC assay.As it was shown,using CJD autopsy skin samples,RT-Qu IC specificity and sensitivity could both reach to 100%.It indicated that skin Pr PSc can be used as a reliable biomarker for the diagnosis of prion diseases.Other neurodegenerative diseases,such as AD and PD,are similar to prion diseases in the pathogenesis.?-synuclein aggregates are found in the epidermal peripheral nerve endings and skin appendages in PD patients,and phosphorylated tau protein aggregates are present in AD patients' skin fibroblasts.These studies indicate that skin misfolded proteins could be a possible diagnostic biomarker for prion diseases and other neurodegenerative diseases.The current study was designed to test the skin Pr PSc could be used as a biomarker for the early preclinical detection of prion diseases,and to efficiently detect specific misfolde protein in AD and PD patients' skin with extremely low level.Materials and Methods: Two amplification techniques with high sensitivity and high specificity were applied in this study: the first method is “Serial Protein Misfolding Cyclic Amplification”(sPMCA),incubating a small amount of Pr PSc with uninfected brain homogenate containing Pr Pc.The sensitivity of sPMCA of prion disease brain tissue can reach to 100%,but sPMCA is likely to be false positive after the multiple rounds of amplification.This study will optimize the sPMCA conditions to maximize the specificity and sensitivity.The second method is RealTime Quaking-Induced Conversion(RT-Qu IC),using Pr PSc as a seed and recombinant protein as a substrate,during which the recombinant protein will undergo a conformational change to form aggregates and aggregates will be fragmentated by shaking,then increase the index of amyloid formation,these aggregates are detected with the amyloid-sensitive Th T.To determine the feasibility of using skin for preclinical detection of prion disease,skin samples from two animal models were used in this study,including scrapie 263 K infected hamsters(prion disease rapid onset model)and s CJDMM1-infected humanized Tg40 h mice expressing human Pr P with polymorphism at residue 129 methionine/methionine,which is the closest model to the human prion disease.s CJDMM1 is the most common subtype of s CJD.At different time points after brain inoculation,the two highly sensitive techniques sPMCA and RT-Qu IC were used to determine Pr PSc in skin and brain.Clinical signs were observed at 10 weeks after inoculation(wpi)in infected hamster and 6 months after inoculation(mpi)in Tg40 h mice.In addition,specific misfolded proteins in skin samples from 20 PD patients and 20 AD patients could be detected by RT-Qu IC,while there were no false positive reactivities in those negative controls.Results: In this study,Pr PSc deposition and spongiform degeneration in the brain were detected in 263K-incoculated hamsters at 4 wpi and at 7 wpi by using conventional Western blot and histology tests,and detectable at 5 mpi in Tg40 h.However,Pr PSc could be detected at 2 wpi by sPMCA amplification,and in Tg40 h skin at 1 mpi.Skin prion-seeding activity was detectable by RT-Qu IC at 3 wpi in Pr PSc-infected hamsters and at 5 mpi in infected Tg40 h mice.This study also found that skin samples of negative control animals that co-housed with hamsters infected with 263 K could be amplified at terminal time point by sPMCA and RT-Qu IC.The above negative control animals were inhabited with the 263K-inoculated hamsters till the end stage.In contrast,the skin of PBS-inoculated hamster housed separately from 263K-infected hamsters exhibited negative for Pr PSc and prion-seeding activity.This suggests that the skin can be one of routes of prion transmission.Similar to the application of RT-Qu IC assay to prion disease,this study improved the skin detection of AD and PD patients by RT-Qu IC,and successfully detected abnormal ?-synuclein in the skin of 20 PD patients and tau protein in the skin of 20 AD patients in vitro.The positive rate of ?-synuclein detection could reach 100%,and Tau protein was 75%.Since no false positive reactivites occurred in the negative controlled samples,both of these two misfolded proteins were detected with the specificity of 100% in the RT-Qu IC assayConclusions: The current study confirmed that the skin misfolded proteins that could be used in detection for prion diseases,AD and PD with high specificity and sensitivity.Especially the skin Pr PSc can be an ideal preclinical detection biomarker for prion disease,and further confirmed its feasibility as a diagnostic biomarker for neurodegenerative diseases as well.It can be improved to develop as a standardized evaluation technique for neurodegenerative diseases.Since skin biopsy is clinically feasible with less trauma,the optimization of parameters of amplification techniques RT-Qu IC and sPMCA based on skin can be used not only for preclinical detection of prion diseases,but also other neurodegenerative diseases.It can also provide an opportunity for monitoring the disease progresses and assessing treatment efficacy of neurodegenerative diseases.The results of this study demonstated that develoing the skin biopsy of neurodegenerative diseases and optimization of misfolded protein amplification technology in vitro will have a highly broad clinical application prospect.