Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are two distinctα-synucleinopathies traditionally differentiated through clinical symptoms.Early diagnosis of MSA is problematic,and seed amplificati...Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are two distinctα-synucleinopathies traditionally differentiated through clinical symptoms.Early diagnosis of MSA is problematic,and seed amplification assays(SAAs),such as real-time quaking-induced conversion(RT-QuIC),offer the potential to distinguish these diseases through their underlyingα-synuclein(α-Syn)pathology and proteoforms.Currently,SAAs provide a binary result,signifying either the presence or absence ofα-Syn seeds.To enhance the diagnostic potential and biological relevance of these assays,there is a pressing need to incorporate quantification and stratification ofα-Syn proteoform-specific aggregation kinetics into current SAA pipelines.Methods Optimal RT-QuIC assay conditions forα-Syn seeds extracted from PD and MSA patient brains were determined,and assay kinetics were assessed forα-Syn seeds from different pathologically relevant brain regions(medulla,substantia nigra,hippocampus,middle temporal gyrus,and cerebellum).The conformational profiles of diseaseand region-specificα-Syn proteoforms were determined by subjecting the amplified reaction products to concentration-dependent proteolytic digestion with proteinase K.Results Using our protocol,PD and MSA could be accurately delineated using proteoform-specific aggregation kinetics,includingα-Syn aggregation rate,maximum relative fluorescence,the gradient of amplification,and core protofilament size.MSA cases yielded significantly higher values than PD cases across all four kinetic parameters in brain tissues,with the MSA-cerebellar phenotype having higher maximum relative fluorescence than the MSA-Parkinsonian phenotype.Statistical significance was maintained when the data were analysed regionally and when all regions were grouped.Conclusions Our RT-QuIC protocol and analysis pipeline can distinguish between PD and MSA,and between MSA phenotypes.MSAα-Syn seeds induce faster propagation and exhibit higher aggregation kinetics than PDα-Syn,mirroring the biological differences observed in brain tissue.With further validation of these quantitative parameters,we propose that SAAs could advance from a yes/no diagnostic to a theranostic biomarker that could be utilised in developing therapeutics.展开更多
Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are classified asα-synucleinopathies and are primarily differentiated by their clinical phenotypes.Delineating these diseases based on their specific...Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are classified asα-synucleinopathies and are primarily differentiated by their clinical phenotypes.Delineating these diseases based on their specificα-synuclein(α-Syn)proteoform pathologies is crucial for accurate antemortem biomarker diagnosis.Newly identifiedα-Syn pathologies in PD raise questions about whether MSA exhibits a similar diversity.This prompted the need for a comparative study focusing onα-Syn epitope-specific immunoreactivities in both diseases,which could clarify the extent of pathological overlap and diversity,and guide more accurate biomarker development.Methods We utilised a multiplex immunohistochemical approach to detect multiple structural domains ofα-Syn proteoforms across multiple regions prone to pathological accumulation in MSA(n=10)and PD(n=10).Comparison of epitope-specificα-Syn proteoforms was performed in the MSA medulla,inferior olivary nucleus,substantia nigra,hippocampus,and cerebellum,and in the PD olfactory bulb,medulla,substantia nigra,hippocampus,and entorhinal cortex.Results N-terminus and C-terminus antibodies detected significantly moreα-Syn pathology in MSA than antibodies for phosphorylated(pS129)α-Syn,which are classically used to detectα-Syn.Importantly,C-terminus immunolabelling is more pronounced in MSA compared to PD.Meanwhile,N-terminus immunolabelling consistently detected the highest percentage ofα-Syn across pathologically burdened regions of both diseases,which could be of biological significance.As expected,oligodendroglial involvement distinguished MSA from PD,but in contrast to PD,no substantial astrocytic or microglialα-Syn accumulation in MSA occurred.These data confirm glial-specific changes between these diseases when immunolabelling the N-terminus epitope.In comparison,N-terminus neuronalα-Syn was present in PD and MSA,with most MSA neurons lacking pS129α-Syn proteoforms.This explains why characterisation of neuronal MSA pathologies is lacking and challenges the reliance on pS129 antibodies for the accurate quantification ofα-Syn pathological load acrossα-synucleinopathies.Conclusions These findings underscore the necessity of utilising a multiplex approach to detectα-Syn,most importantly including the N-terminus,to capture the entire spectrum ofα-Syn proteoforms inα-synucleinopathies.The data provide novel insights toward the biological differentiation of theseα-synucleinopathies and pave the way for more refined antemortem diagnostic methods to facilitate early identification and intervention of these neurodegenerative diseases.展开更多
基金JAW is funded through a Neurological Foundation W&B Miller Doctoral Scholarship(1951 MS)BVD is funded by a Health Research Council Hercus Fellowship(21/034)+2 种基金the School of Medical Science,University of Auckland,New Zealand,Catalyst(22-UOA-049-CSG,Royal Society of New Zealand Te Apārangi)Te Tītoki Mataora(3729090&3729858)GMH is supported by an NHMRC Senior Leadership Fellowship(1176607).
文摘Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are two distinctα-synucleinopathies traditionally differentiated through clinical symptoms.Early diagnosis of MSA is problematic,and seed amplification assays(SAAs),such as real-time quaking-induced conversion(RT-QuIC),offer the potential to distinguish these diseases through their underlyingα-synuclein(α-Syn)pathology and proteoforms.Currently,SAAs provide a binary result,signifying either the presence or absence ofα-Syn seeds.To enhance the diagnostic potential and biological relevance of these assays,there is a pressing need to incorporate quantification and stratification ofα-Syn proteoform-specific aggregation kinetics into current SAA pipelines.Methods Optimal RT-QuIC assay conditions forα-Syn seeds extracted from PD and MSA patient brains were determined,and assay kinetics were assessed forα-Syn seeds from different pathologically relevant brain regions(medulla,substantia nigra,hippocampus,middle temporal gyrus,and cerebellum).The conformational profiles of diseaseand region-specificα-Syn proteoforms were determined by subjecting the amplified reaction products to concentration-dependent proteolytic digestion with proteinase K.Results Using our protocol,PD and MSA could be accurately delineated using proteoform-specific aggregation kinetics,includingα-Syn aggregation rate,maximum relative fluorescence,the gradient of amplification,and core protofilament size.MSA cases yielded significantly higher values than PD cases across all four kinetic parameters in brain tissues,with the MSA-cerebellar phenotype having higher maximum relative fluorescence than the MSA-Parkinsonian phenotype.Statistical significance was maintained when the data were analysed regionally and when all regions were grouped.Conclusions Our RT-QuIC protocol and analysis pipeline can distinguish between PD and MSA,and between MSA phenotypes.MSAα-Syn seeds induce faster propagation and exhibit higher aggregation kinetics than PDα-Syn,mirroring the biological differences observed in brain tissue.With further validation of these quantitative parameters,we propose that SAAs could advance from a yes/no diagnostic to a theranostic biomarker that could be utilised in developing therapeutics.
文摘Background Parkinson’s disease(PD)and multiple system atrophy(MSA)are classified asα-synucleinopathies and are primarily differentiated by their clinical phenotypes.Delineating these diseases based on their specificα-synuclein(α-Syn)proteoform pathologies is crucial for accurate antemortem biomarker diagnosis.Newly identifiedα-Syn pathologies in PD raise questions about whether MSA exhibits a similar diversity.This prompted the need for a comparative study focusing onα-Syn epitope-specific immunoreactivities in both diseases,which could clarify the extent of pathological overlap and diversity,and guide more accurate biomarker development.Methods We utilised a multiplex immunohistochemical approach to detect multiple structural domains ofα-Syn proteoforms across multiple regions prone to pathological accumulation in MSA(n=10)and PD(n=10).Comparison of epitope-specificα-Syn proteoforms was performed in the MSA medulla,inferior olivary nucleus,substantia nigra,hippocampus,and cerebellum,and in the PD olfactory bulb,medulla,substantia nigra,hippocampus,and entorhinal cortex.Results N-terminus and C-terminus antibodies detected significantly moreα-Syn pathology in MSA than antibodies for phosphorylated(pS129)α-Syn,which are classically used to detectα-Syn.Importantly,C-terminus immunolabelling is more pronounced in MSA compared to PD.Meanwhile,N-terminus immunolabelling consistently detected the highest percentage ofα-Syn across pathologically burdened regions of both diseases,which could be of biological significance.As expected,oligodendroglial involvement distinguished MSA from PD,but in contrast to PD,no substantial astrocytic or microglialα-Syn accumulation in MSA occurred.These data confirm glial-specific changes between these diseases when immunolabelling the N-terminus epitope.In comparison,N-terminus neuronalα-Syn was present in PD and MSA,with most MSA neurons lacking pS129α-Syn proteoforms.This explains why characterisation of neuronal MSA pathologies is lacking and challenges the reliance on pS129 antibodies for the accurate quantification ofα-Syn pathological load acrossα-synucleinopathies.Conclusions These findings underscore the necessity of utilising a multiplex approach to detectα-Syn,most importantly including the N-terminus,to capture the entire spectrum ofα-Syn proteoforms inα-synucleinopathies.The data provide novel insights toward the biological differentiation of theseα-synucleinopathies and pave the way for more refined antemortem diagnostic methods to facilitate early identification and intervention of these neurodegenerative diseases.
