In Situ Amplification and Spatial Mapping of α-Synuclein Seeding Activity

In Situ Amplification and Spatial Mapping of α-Synuclein Seeding Activity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results In Situ Amplification and Spatial Mapping of α -Synuclein Seeding Activity View ORCID Profile Hengxu Mao , View ORCID Profile Yaoyun Kuang , Wei Dai , Hao Lin , Jinkun Huang , Ping-Yi Xu doi: https://doi.org/10.1101/2025.03.26.645411 Hengxu Mao 1 Dept. of Neurology, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou, 510120, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hengxu Mao For correspondence: hengxu2010{at}qq.com 2009681002{at}gzhmu.edu.cn pingyixu{at}sina.com Yaoyun Kuang 1 Dept. of Neurology, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou, 510120, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yaoyun Kuang Wei Dai 1 Dept. of Neurology, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou, 510120, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hao Lin 1 Dept. of Neurology, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou, 510120, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jinkun Huang 1 Dept. of Neurology, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou, 510120, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: hengxu2010{at}qq.com 2009681002{at}gzhmu.edu.cn pingyixu{at}sina.com Ping-Yi Xu 1 Dept. of Neurology, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou, 510120, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: hengxu2010{at}qq.com 2009681002{at}gzhmu.edu.cn pingyixu{at}sina.com Abstract Full Text Info/History Metrics Preview PDF Abstract The prion-like propagation of proteopathic α-synuclein (αSyn) "seeds" is a central pathogenic mechanism in synucleinopathies. However, methods to directly visualize this "seeding activity" within intact tissue are lacking. Conventional antibody-based techniques provide only static snapshots of pathological deposits, while sensitive seed amplification assays lose spatial information via mechanical fragmentation. Here, we introduce the αSyn Quiescent Seed Amplification Assay (QSAA), a method for specific, robust, in situ amplification of αSyn seeds directly within tissue sections. QSAA demonstrates superior sensitivity and earlier detection than pS129 and ThS staining, revealing widespread seeding in a PD mouse model, and successfully identifies endogenous seeds in archival human brains. By integrating with immunofluorescence, IF-QSAA uncovered microglial seeding activity and established that pS129 phosphorylation and seeding activity represent distinct pathological states. This offers unprecedented spatial insights into synucleinopathy pathogenesis, holding significant promises for early diagnosis and therapeutic evaluation. Introduction Synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are progressive neurodegenerative disorders defined by the pathological accumulation of α-synuclein (αSyn) 1 , 2 . In these diseases, αSyn misfolds into proteopathic seeds capable of self-propagation through a prion-like mechanism, templating the conversion of native αSyn into insoluble aggregates 3 , 4 . This functional property, termed seeding activity, is a primary driver of pathology progression, leading to widespread neuronal dysfunction and neurodegeneration 5 , 6 . Consequently, visualizing the spatiotemporal distribution and propagation pathways of αSyn seeding activity is critical for understanding synucleinopathy pathogenesis and developing targeted therapies. Conventional antibody-based techniques, which often target pathological markers like αSyn phosphorylated at Serine 129 (pS129), have been invaluable in localizing αSyn aggregates in diseased brains 7 , 8 . However, these methods provide only a static snapshot of aggregate presence and fail to capture the dynamic seeding activity that underpins pathology 9 – 12 . To bridge this gap, seed amplification assays (SAAs) have been developed, exploiting the inherent ability of misfolded αSyn seeds to induce amplification in vitro by recruiting exogenous monomeric substrates 13 , 14 . Despite their high sensitivity, traditional SAAs depend on tissue homogenization and mechanical fragmentation (e.g., shaking or sonication) to sustain the amplification reaction 15 , 16 . This process disrupts tissue architecture and abolishes spatial information about seed localization. To address these challenges, we developed the αSyn Quiescent Seed Amplification Assay (QSAA), a method that enables in situ amplification of αSyn seeds within tissue sections without mechanical fragmentation. By optimizing reaction conditions, including the introduction of sulfate ions (SO₄²⁻), elevating temperature to 70°C, and using mouse-derived αSyn monomers as the substrate, QSAA achieves seed-dependent fibril elongation under quiescent conditions. This approach preserves spatial context and generates high-resolution propagation maps, revealing pathological spread patterns inaccessible by antibody staining alone. QSAA is compatible with a variety of samples, demonstrating broad applicability by successfully detecting endogenous seeds in human brain sections, including long-term formalin-fixed archival specimens. Furthermore, by integrating QSAA with immunofluorescence (IF), IF-QSAA can simultaneously visualize amplified seeds and specific cellular markers. This dual-labeling approach provides a comprehensive, cellular-level view of αSyn seeding activity, offering new insights into the mechanisms of synucleinopathies. Together, QSAA and IF-QSAA represent powerful tools to advance our understanding of αSyn propagation, holding great promise for improving diagnosis and therapeutic development. Results Development of a quiescent system for α Syn seed amplification A key prerequisite for in situ amplification of αSyn seeds is the ability to achieve robust and specific amplification under quiescent conditions. Traditional SAAs primarily rely on cyclic physical fragmentation to continuously generate new growth ends from fibrils that have entered the reaction plateau 17 , 18 , thereby sustaining rapid amplification ( Fig. 1a , solid line ). In contrast, a quiescent system necessitates an alternative mechanism to maintain high amplification efficiency ( Fig. 1a , dashed line ). To address this, our strategy focused on optimizing the biochemical environment to intrinsically accelerate fibril amplification in a seed-dependent manner, thereby circumventing the need for physical fragmentation ( Fig. 1b ). Download figure Open in new tab Fig. 1 Development of a quiescent system for α Syn seed amplification. a , Schematic illustrating the kinetics of ThT assays. Conventional SAAs rely on cyclic fragmentation (solid line) to sustain rapid amplification. In each quiescent phase without fragmentation, elongation would typically plateau. In contrast, QSAA is designed to maintain continuous amplification under quiescent conditions (dashed line). b, Schematic comparing the mechanisms of SAA (cyclic fragmentation) and QSAA (quiescent amplification). c, Amino acid sequence alignment of human wild-type (hWT), human A53T, and mouse wild-type (mWT) αSyn. The bar chart quantifies the lag phase. d, ThT fluorescence kinetics of αSyn aggregation under cyclic fragmentation, assessing the effect of different monomers (MM), sulfate ions (SO₄²⁻), and temperature (70℃). Reactions were seeded with 1 pg of PFFs (blue) or unseeded (orange). e, Quantification of the lag phase from experiments in d . f-g, Aggregation kinetics and specificity under cyclic fragmentation ( f ) versus quiescent conditions ( g ); bar charts show PAR (1 pg PFFs / no seed). h, Representative TEM images of the fibrillar products generated by QSAA under optimized quiescent conditions. Insert, unseeded control. Data are mean ± SD (n=3). *P < 0.05, ***P < 0.001, ****P < 0.0001; one-way ANOVA with Tukey’s post hoc test. Scale bars are indicated. We systematically examined several factors known to affect αSyn fibril growth, including the αSyn monomer substrate 13 , 19 , 20 , chemical additives 21 – 24 , and incubation temperature 25 , 26 . Initially, we constructed expression plasmids for human wild-type (hWT), human A53T mutant (hA53T), and mouse wild-type (mWT) αSyn, confirming their sequences ( Fig. 1c ). Notably, mWT αSyn naturally contains a threonine at position 53, analogous to the pathogenic A53T mutation in humans, which is known to accelerate αSyn aggregation 27 . Consistent with this, we demonstrated a progressively faster aggregation rate from hWT to hA53T and then to mWT, confirming that mWT αSyn serves as a more aggregation-prone substrate suitable for rapid amplification. Next, we explored the impact of additives and temperature on fibril amplification. Previous studies have shown that sulfate ions (SO₄²⁻) can promote αSyn fibril elongation by electrostatically stabilizing misfolded aggregates 21 , 22 and that an elevated temperature accelerates aggregation kinetics 25 , 26 . We evaluated these parameters individually to establish their baseline effects in the presence of αSyn pre-formed fibrils (PFFs) under cyclic fragmentation conditions ( Fig. 1d ). Each parameter (mWT αSyn monomer, SO₄²⁻, 70°C) independently shortened the lag phase of αSyn seed amplification ( Fig. 1e ). However, combining these factors under cyclic fragmentation resulted in rapid spontaneous amplification, leading to poor specificity ( Fig. 1f ). Remarkably, applying the same combination (mWT αSyn monomer, SO₄²⁻, 70°C) under quiescent conditions yielded rapid and specific amplification with a high fold separation of 7.547 ( Fig. 1g ). These results underscore that the optimized chemical environment and elevated temperature in QSAA effectively compensate for the lack of mechanical fragmentation, enabling continuous and robust fibril growth from existing seeds while minimizing spontaneous nucleation. To further characterize the amplification products, we performed Transmission Electron Microscopy (TEM) on samples generated by QSAA ( Fig. 1h ). TEM analysis revealed fibrillar morphologies characteristic of αSyn aggregates, including both densely packed and loosely associated fibrillar assemblies. In contrast, no fibrillar products were observed in the unseeded group, further demonstrating the specificity of the assay ( Fig. 1h , Inset). QSAA Enables In Situ Amplification and Mapping of α Syn Seeding Activity in Brain Sections Having established a quiescent amplification system, we sought to expand its application for in situ amplification on brain tissue sections. We developed a standardized operating procedure (SOP) involving tissue fixation, dehydration, sectioning, post-fixation, and incubation with the QSAA reaction buffer ( Fig. 2a ). To minimize artifacts caused by microscale fluid movement, a semi-solid agarose membrane was overlaid on the tissue sections during incubation 28 . Download figure Open in new tab Fig. 2 QSAA enables in situ amplification and mapping of α Syn seeding activity in mouse brain sections. a , Schematic of the standardized operating procedure for in situ QSAA on brain tissue, including tissue preparation, reagent preparation, and incubation on a temperature-controlled stage with an agarose overlay. b, Representative images of in situ QSAA on coronal, sagittal, and horizontal sections from a mouse brain 180 dpi with mPFFs. Green fluorescence (ThT) indicates αSyn seeding activity. Corresponding anatomical atlas plates show the signal distribution (red dots). Inset: a PBS-injected control. c, TEM of a QSAA-treated tissue showing fibrillar structures; arrowheads indicate fibrils adjacent to nuclei. d, Coronal sections imaged by fluorescence and brightfield after QSAA incubation; macroscopic aggregates are shown alongside ThT signal. Images are representative of at least three independent experiments. Scale bars are indicated. Brain sections from a synucleinopathy mouse model 29 , 30 at 180 days post-injection (dpi) of mouse αSyn pre-formed fibrils (mPFFs) revealed widespread αSyn seeding activity after 5 hours of incubation ( Fig. 2b ). The signal propagated along neuroanatomical tracts, such as the bilateral corpus callosum (CC) and anterior commissure (AC), into the contralateral hemisphere, whereas control sections from PBS-injected mice were negative ( Fig. 2b , Inset ). This pattern, consistently observed across coronal, sagittal, and horizontal sections, effectively mapped pathology routes directly within the tissue, with sagittal views clearly showing dissemination along the anterior commissure’s olfactory limb (ACO) towards the olfactory bulb (OB) ( Fig. 2b , sagittal ). To confirm the Thioflavin T (ThT) signal represented seed-dependent fibril formation, we used TEM on QSAA-treated tissue, which revealed typical αSyn amyloid fibrils within the tissue interstitium and on its surface ( Fig. 2c ). Brightfield microscopy further showed that fluorescent signals precisely co-localized with macroscopic aggregates, confirming that tangible amplification products were generated ( Fig. 2d ). These observations validate that QSAA amplifies αSyn seeds within intact tissue architecture. QSAA Provides Superior Sensitivity and Early Detection of α Syn Pathology By leveraging the ability of QSAA to preserve spatial information, we generated a brain-wide map of αSyn seeding activity in mPFF-injected mice (180 dpi) and compared it with pS129-αSyn immunofluorescence on consecutive sections ( Fig. 3a ). QSAA and pS129 signals showed broad concordance across multiple brain regions, including the striatum (STR), primary and secondary motor cortex (M1/M2), secondary somatosensory cortex (S2), and entorhinal cortex (ECT), confirming that QSAA faithfully localizes pathological seeds. Download figure Open in new tab Fig. 3 QSAA provides superior sensitivity and earlier detection of α Syn pathology in a mouse model. a , Brain-wide maps comparing the distribution of QSAA signal with pS129-IF on consecutive sections from mPFF-injected mouse brain (180 dpi). b, Direct comparison of QSAA, pS129-IF, and ThS staining on consecutive coronal sections from a mouse brain injected with one-tenth the standard mPFFs dose and analyzed at 30 dpi. Colored boxes indicate magnified regions. c, Quantification of relative fluorescence units (RFU) from the cortical regions shown in b . Data are mean ± SD (n=3). **P < 0.01, ****P < 0.0001; one-way ANOVA with Tukey’s post hoc test. d, Comparison of QSAA and pS129-IF in the mPFF-injected mouse model at 7, 10, and 14 dpi. e, Brain-wide map of αSyn seeding activity revealed by QSAA at 14 dpi in mPFF-injected mice compared to PBS-injected controls. Images are representative of at least three independent experiments. Scale bars are indicated. Importantly, QSAA revealed a much wider pathological landscape, particularly within white matter tracts such as the bilateral corpus callosum (CC), anterior commissure (ACA/ACP), ipsilateral internal capsule (IC), external capsule (EC), medial forebrain bundle (MFB), fasciculus retroflexus (FR), and nigrostriatal tract (NST). In these regions, pS129 immunoreactivity was relatively sparse, likely reflecting the lower density of neuronal soma. To further demonstrate the sensitivity of QSAA, we compared QSAA, pS129-IF, and Thioflavin S (ThS) staining on sections from mice injected with one-tenth the standard dose of mPFFs and analyzed at 30 dpi ( Fig. 3b ). QSAA revealed widespread fluorescent signals across the cortex, corpus callosum, and striatum of both hemispheres, whereas pS129 detected only sparse, neuritic-like structures, and ThS staining showed even weaker signals ( Fig. 3c ). We next examined the ability of QSAA to detect early-stage pathology. Because QSAA directly amplifies functional seeding activity rather than depending on downstream phosphorylation events, it confers a mechanistic advantage over pS129 immunostaining. Remarkably, QSAA detected distinct seeding activity as early as 7 dpi, a stage at which pS129 signals were undetectable ( Fig. 3d ). By 14 dpi, QSAA revealed widespread dissemination of αSyn seeding throughout the brain, whereas pS129 immunoreactivity was only beginning to emerge. Whole-brain QSAA mapping at 14 dpi confirmed broad propagation of seeding activity beyond the injection site, with no signal observed in PBS-injected controls ( Fig. 3e ). These findings demonstrate that extensive αSyn seeding precedes the detectable onset of pS129 pathology in the exogenous seeding model. QSAA is Applicable to Endogenous Seeds in Human Brain Tissue We next evaluated the applicability of QSAA to detect endogenous αSyn seeds in postmortem human brain tissue. Striatal sections from patients with PD, progressive supranuclear palsy (PSP), and healthy controls (HC) were analyzed. QSAA generated ThT fluorescence selectively in PD brains, with no signal observed in HC or PSP samples, demonstrating high specificity ( Fig. 4a ). Notably, QSAA was able to detect seeding activity in PD specimens that had been fixed in formalin for more than 20 years, in which pS129-IF failed to detect pathology ( Fig. 4a , FF-PD ). This finding indicates the stability of seeding-competent αSyn aggregates compared with antibody epitopes that deteriorate during prolonged fixation. To validate these results independently, we performed Real-Time Quaking-Induced Conversion (RT-QuIC) on lysates from the same brain specimens, which confirmed the seeding activity in both frozen and formalin-fixed PD samples ( Fig. 4b-c ). Download figure Open in new tab Fig. 4 QSAA detects endogenous seeding activity in human postmortem brain tissue. a , Representative pS129-IF and QSAA results on striatal sections from HC, PSP, PD, and long-term formalin-fixed PD (>20 years; FF-PD). b, RT-QuIC of brain lysates from the same cases. c, Quantification of the PAR from RT-QuIC data in b. Data are mean ± SD (n=3). ****P < 0.0001; one-way ANOVA with Tukey’s post hoc test. d, Schematic of brain section indicating the regions analyzed in e. e, Representative QSAA images of the corpus callosum, striatum, and cortex in HC and PD brains. L, left; R, right. f, Time-lapse QSAA imaging of a PD brain section during incubation (0-5 h). Scale bars are indicated. QSAA further revealed distinct spatial distributions of seeding activity in human PD brains ( Fig. 4d ). Seeding was detected predominantly in gray matter regions such as the striatum and cortex, whereas the corpus callosum showed no detectable activity ( Fig. 4e ). This contrasts with the mPFF-inoculated mouse model, where seeding also occurs within white matter tracts, suggesting differences in propagation dynamics between exogenously induced pathology and endogenously developed diseases. Finally, a time-course analysis of QSAA on PD brain sections revealed a clear, time-dependent increase in ThT fluorescence intensity at defined sites of αSyn seeding ( Fig. 4f ). Signals appeared as early as 1 h and continued to amplify through 5 h, highlighting both the sensitivity of the method and its ability to dynamically monitor in situ seeding activity. IF-QSAA Reveals Distinct Cellular Localization and Complementary Pathological States of Seeding Activity and pS129 We developed an integrated immunofluorescence-QSAA (IF-QSAA) protocol to determine the cellular localization of αSyn seeding activity. First, we confirmed the spectral compatibility of ThT with common IF fluorophores, finding that its excitation spectrum is distinct from DAPI, Cy3, and Cy5, but overlaps significantly with FITC ( Fig. 5a ). Next, a sequential optimization revealed that the QSAA reaction conditions (70°C, SO₄²⁻, 5h) degraded antibody epitopes, necessitating an "IF-first" workflow to preserve both signals ( Fig. 5b ). Applying this protocol with confocal microscopy on brain tissue from mPFF-injected mice (180 dpi), we visualized intraneuronal QSAA products ( Fig. 5c , arrows ) and larger products extending into the extracellular space ( Fig. 5c , arrowheads ). These results validated the capability of IF-QSAA to map seeding activity with subcellular precision, leading to our standardized IF-QSAA protocol ( Fig. 5d ). Download figure Open in new tab Fig. 5 IF-QSAA reveals cellular localization of seeding activity and its relationship with pS129 pathology. a , Normalized excitation spectra of ThT and common fluorophores (DAPI, FITC, Cy3, Cy5). b, Workflow comparison showing QSAA-first versus IF-first sequence with NeuN staining. c, Confocal image of IF-QSAA on striatal sections from mPFF-inoculated mice (180 dpi) co-labeled for NeuN (red) and QSAA (green). Arrow and arrowhead indicate selected cellular and extracellular structures. d, Schematic of the optimized IF-QSAA protocol. e, g, IF-QSAA images of mPFF-injected mouse S2 cortex sections (30 dpi) co-labeled with NeuN ( e ), IBA1 ( f ), and GFAP ( g ). Insets show magnified cells. h, i, Consecutive S2 cortex sections (30 dpi) stained for pS129 and either NeuN ( h ) or IBA1 ( i ); DAPI in blue. Arrows in i indicate selected pS129-positive neurons. j, High-resolution image showing QSAA (green) with pS129 (red) in the mPFF-model at the injected site. k, Schematic of core-halo arrangement of QSAA and pS129 signals in j . l, Conceptual four-quadrant classification model of αSyn aggregates. Images are representative of at least three independent experiments. Scale bars are indicated. To identify the specific cell types involved in αSyn seeding, we co-labeled sections from mPFF-injected mice (30 dpi) with markers for neurons (NeuN), microglia (IBA1), and astrocytes (GFAP). Strikingly, in addition to neuronal signals ( Fig. 5e ), we observed extensive co-localization between QSAA signals and IBA1-positive microglia ( Fig. 5f ), but not with GFAP-positive astrocytes ( Fig. 5g ). This finding contrasted with the cellular distribution of pS129-αSyn pathology 31 , which was predominantly found within neurons ( Fig. 5h ) and showed minimal co-localization with microglia ( Fig. 5i ). These findings suggest a previously underappreciated role for microglia in the uptake, processing, or propagation of αSyn seed species, differing from the traditional view that Lewy pathology primarily accumulates within neurons. To further investigate the relationship between αSyn seeding activity and pS129 pathology, we applied IF-QSAA to brain tissue sections from the mPFF mouse model. Through simultaneous imaging of the QSAA signal and pS129, we observed a heterogeneity among αSyn aggregates, with multiple subtypes coexisting in the same microspatial environment ( Fig. 5j ). Initially, we identified a large population of aggregates positive for both signals (QSAA+/pS129+), which exhibited a characteristic "core-halo" structure, comprising a dense, pS129-positive core enveloped by a more diffuse QSAA signal halo. This finding suggested that phosphorylated αSyn with seeding capacity is a significant component of the pathological landscape. However, this observation raises a critical question: is pS129 a reliable surrogate for αSyn seeding activity? A closer examination of the magnified regions in Figure 5j revealed that, in addition to the dual-positive aggregates, we could also identify: 1) aggregates that were exclusively pS129-positive but QSAA-negative (QSAA−/pS129+), appearing as discrete red puncta; and 2) aggregates that were exclusively QSAA-positive but pS129-negative (QSAA+/pS129−), appearing as distinct green puncta. This direct visual evidence demonstrates that αSyn seeding activity and its pS129 phosphorylation state are not strictly concordant or co-localized ( Fig. 5k ). We therefore propose a "four-quadrant model" to classify αSyn aggregates ( Fig. 5l ): QSAA+/pS129+ (Co-existent): Phosphorylated aggregates with seeding capacity, forming the characteristic core-halo structure. QSAA+/pS129− (Seeding-Dominant): Aggregates possessing seeding activity but with pS129 levels below the detection threshold or with a masked epitope. QSAA−/pS129+ (Phosphorylation-Dominant): Phosphorylated aggregates that have either lost or inherently lack seeding competency. QSAA−/pS129− (Pathology-Free): Regions devoid of detectable seeding activity and pS129 pathology. This model emphasizes that αSyn seeding activity and pS129 immunoreactivity represent two distinct yet complementary pathological states, rather than being equivalent disease markers. It provides a more precise framework for understanding the molecular heterogeneity and microenvironmental complexity of αSyn pathology in synucleinopathies. Discussion In this study, we established QSAA, a method that enables the in situ detection of αSyn seeding activity. Unlike antibody-based methods that capture pre-formed aggregates 32 , or conventional SAAs that require tissue homogenization 33 , 34 , QSAA achieves rapid, seed-dependent amplification under quiescent conditions. This methodology allows for the direct visualization of functional seeding activity within intact tissue sections. QSAA provided several key insights that extend beyond the capabilities of existing techniques. First, its amplification-based nature provided markedly superior sensitivity, enabling the detection of nascent αSyn seeds. Second, QSAA revealed a broader pathological landscape, particularly within white matter tracts and microglial populations, where antibody-based methods are often less effective. This is likely because conventional antibodies targeting post-translational modifications (PTMs) like pS129 rely on late-stage pathological events 9 , 11 , giving QSAA a distinct advantage in detecting seeding species that are either unmodified or at very early stages of aggregation. Together with the IF-QSAA protocol, these findings led us to propose a conceptual “four-quadrant model,” in which seeding activity and pS129 phosphorylation define both overlapping and distinct dimensions of αSyn pathology. This framework highlights early-stage QSAA+/pS129− seeds as a potentially critical driver of disease progression that is not captured by conventional markers. Importantly, QSAA also demonstrated strong translational potential. It successfully amplified endogenous seeds from postmortem PD brains, including archival tissue fixed for over 20 years, where pS129 immunoreactivity had deteriorated. This underscores the remarkable stability of seeding-competent αSyn structures and positions QSAA as a promising tool for both retrospective neuropathological studies and diagnostic applications. While QSAA offers the unique advantage of in situ assessment, it currently cannot differentiate between distinct αSyn strains, a property reported for some conventional SAAs 35 – 37 , nor can it directly identify PTMs as antibody-based methods can 38 , 39 . Future methodological refinements, such as fluorescence-based conformational fingerprinting of amplified fibrils 40 or the engineering of strain-specific monomer substrates, may broaden its utility. Although the high sensitivity of QSAA arises from controlled, seed-dependent amplification rather than from non-specific background signals, the risk of experimental false positives still requires rigorous control. Despite multiple layers of mitigation strategies including an agarose overlay, strict incubation time, parallel negative samples, and in situ TEM validation, further standardization will be essential for multi-center implementation. Uniform parameters such as section thickness, fixation protocols, and data normalization will be critical for ensuring inter-laboratory reproducibility 17 . In conclusion, QSAA provides a powerful platform for mapping αSyn seeding activity in situ with high sensitivity and specificity. By extending beyond static aggregate detection to capture the functional and spatial dimensions of pathogenic seeds, QSAA deepens our understanding of synucleinopathy pathogenesis and holds promise for clinical applications in early diagnosis and therapeutic evaluation. Moreover, the principle of quiescent amplification may be adaptable to other misfolded proteins, such as Tau and TDP-43, particularly those that have already demonstrated cyclic fragmentation-based amplification in vitro 41 , 42 , further broadening its relevance to neurodegenerative disease research. Methods Ethics and Oversight All animal procedures were conducted in strict accordance with the guidelines established by the Institutional Animal Care and Use Committee of Guangzhou Medical University. Human brain tissues were obtained from the Department of Neurology, First Affiliated Hospital of Guangzhou Medical University, with written informed consent from the next of kin and approval from the hospital Ethics Committee (MRE. No. 155). All experiments complied with relevant ethical regulations. Animals and Stereotaxic Injections Male C57BL/6J mice (10-12 weeks old, RRID: IMSR_JAX:000664) were purchased from the Experimental Animal Center of Guangzhou Medical University. Animals were maintained in a SPF facility on a 12-h light/dark cycle with ad libitum access to food and water. Sample size was chosen based on previous studies using similar models to ensure sufficient power for detecting inter-group differences. Animals were randomly assigned to experimental groups. All injections, data acquisition, and subsequent analyses were performed by experimenters blinded to the treatment conditions For stereotaxic surgery, mice were anesthetized with isoflurane (2.5% for induction, 1.5% for maintenance) and placed in a stereotaxic frame (Kopf Instruments, Model 940). Recombinant mouse αSyn pre-formed fibrils (mPFFs; 5 μg in 1 μL) or an equal volume of sterile PBS were injected unilaterally into the right striatum. Coordinates were determined relative to bregma according to the Paxinos & Franklin mouse brain atlas (3rd edition): AP: +0.8 mm, ML: +2.0 mm, DV: -3.5 mm. Injections were performed using a 10-μL Hamilton syringe with a 33-gauge needle connected to a microinfusion pump at a rate of 0.2 μL/min. The needle was left in place for 10 min post-injection to minimize backflow before being slowly withdrawn. For the low-dose sensitivity comparison, a one-tenth dose of mPFFs (0.5 μg in 1 μL) was used. At specified time points (7, 10, 14, 30, or 180 days post-injection), mice were deeply anesthetized and transcardially perfused with ice-cold PBS followed by 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4). Brains were post-fixed in the same fixative for 24 h at 4°C, then cryoprotected in 30% (w/v) sucrose in PBS until sectioning. Human Brain Specimens Postmortem human brain tissues (collected 2005-2022) were obtained from the First Affiliated Hospital of Guangzhou Medical University. The cohort included pathologically confirmed cases of Parkinson’s disease (PD; n=3), progressive supranuclear palsy (PSP; n=3), and age-matched healthy controls with no history of neurological disease (HC; n=3). Diagnoses were established postmortem based on consensus neuropathological criteria, including Braak staging for Lewy body pathology. The postmortem interval (PMI) for all fresh-frozen and standard-fixed samples was <12 h. For each case, the left hemisphere was fixed in 10% neutral buffered formalin (NBF) for 2-4 weeks, while the right hemisphere was coronally sliced and snap-frozen at -80°C. Along-term fixed PD specimen (FF-PD) had been stored in 10% NBF for over 20 years at room temperature. α -Synuclein Monomer Expression and PFF Preparation/QC Recombinant full-length mouse wild-type α-synuclein (mWT, UniProt ID: P58992) was expressed in E. coli BL21 (DE3) cells (TransGen Biotech, CD601) and purified as previously described with modifications 43 , 44 . Following IPTG induction (1 mM, 4 h), cells were lysed by sonication in high-salt buffer (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1x Protease inhibitor). The lysate was boiled for 15 min, followed by 45% ammonium sulfate precipitation and pH 3.0 acid precipitation. Endotoxin was removed using a high-capacity endotoxin removal resin (Sangon Biotech, B641718). The protein was further purified by anion exchange chromatography (HiTrap Q HP column, Cytiva, 17115401). The final monomer was dialyzed against TBS (10mM Tris-HCl pH 7.5, 50mM NaCl), filtered through a 50-kDa cutoff filter (Amicon Ultra), and stored at -80℃. To generate mPFFs, monomeric mouse αSyn (5 mg/mL in PBS) was incubated at 37°C with continuous agitation (1000 rpm) in an Eppendorf ThermoMixer C for 7 days. The resulting fibril suspension was fragmented into seeds using a non-contact water-bath sonicator (SCIENTZ, SCIENTZ08-III) for a total of 5 min using 300 pulses of 1-s on/1-s off at 90% power. PFF quality control was performed for each batch 45 . TEM confirmed fibrillar morphology. Dynamic light scattering (DLS) confirmed an average hydrodynamic diameter of 50-150 nm. Functional activity was validated using a standard SAA, with each batch required to produce a lag time within ±15% of a reference standard batch when seeded with 1 pg of PFFs. Plate-based Seed Amplification Assays Agitated SAA: Reactions were prepared in a 96-well black, clear-bottom plate (Thermo Scientific, 165305) in a final volume of 100 μL. Each well contained 1 mg/mL m-αSyn monomer, 40 μM Thioflavin T (ThT), and 1 pg PFFs (or PBS for no-seed controls) in Agitation Buffer (10 mM NaCl, 10 mM Tris-HCl, pH 7.5). Where indicated, 800 mM Na₂SO₄ was added. The plate was sealed with an optical adhesive film (Applied Biosystems, 4311971) and incubated in a plate reader (SpectraMax iD5, Molecular Devices) at 37°C. A cycle of 1 min orbital shaking (700 rpm, double orbital) followed by 14 min of rest was repeated. ThT fluorescence was measured every 15 min (Ex: 450 nm, Em: 490 nm, bottom-read). Quiescent SAA: Reactions were prepared in a 96-well polypropylene PCR plate (Thermo Scientific, AB0700) in a final volume of 20 μL. Each reaction contained 1 mg/mL mWT-αSyn monomer and 40 μM ThT in QSAA Reaction Buffer (800 mM Na₂SO₄, 25 mM NaCl, 10mM Tris-HCl, pH 6.0), seeded with 1 pg mPFFs or TBS. Each reaction was overlaid with 20 μL of mineral oil (Biosharp, BS927) to prevent evaporation. The plate was sealed and incubated in a real-time PCR instrument (Bio-Rad, CFX96) at 70°C without agitation. ThT fluorescence was monitored every 30 min via the FAM channel (Ex: 452-486 nm, Em: 505-545 nm). Lag phase was defined as the time to cross a threshold of the mean baseline fluorescence plus five standard deviations. In Situ QSAA on Tissue Sections Brains were sectioned at 14 μm thickness onto high-adhesion glass slides (CITOTEST, 188105W). Sections were air-dried for 30 min, then post-fixed in ice-cold acetone for 10 min at -20°C and air-dried again. For FF-PD tissues, 95°C PBS treatment for 1 h was performed prior to sectioning to reverse formalin cross-linking and restore seeding activity. To perform QSAA, a reaction mix was prepared containing 1 mg/mL m-αSyn monomer, 40 μM ThT in Reaction Buffer (800 mM Na₂SO₄, 25 mM NaCl, 10 mM Tris-HCl, pH 6.0). To prevent micro-convection and ensure even reagent distribution, a pre-warmed (∼50 °C) 1% (w/v) agarose solution (in ddH₂O) was overlaid onto the tissue section to form a gel approximately 1 mm thick. After gelling at room temperature (∼5min), the agarose was carefully trimmed flush with the tissue edges using a scalpel. Subsequently, 300 μL of the reaction mix was added to cover each section. The slide was then placed in a humidified, sealed chamber on a temperature-controlled stage (Ruicheng Instrument Co., TS400) and incubated at 70°C for 0-12 h unless otherwise specified. Following incubation, the agarose film was gently removed by immersing the slide in heated (95 °C) PBS, and the slide was washed 3 times for 1 min each in PBS before mounting with a coverslip. IF-QSAA Workflow An "IF-first" protocol was implemented to preserve antibody epitopes, which were found to be sensitive to the 70°C incubation. Sections were first processed for standard immunofluorescence. Briefly, sections were permeabilized with 0.5% Triton X-100 in PBS for 15 min and blocked with 5% normal goat serum in PBS for 1 h at room temperature. Primary antibodies (see Table 1 for details) were diluted in blocking buffer and incubated overnight at 4°C. After washing, sections were incubated with corresponding Alexa Fluor-conjugated secondary antibodies (1:1000, Invitrogen) for 1 h at room temperature. Following IF, sections were washed thoroughly in PBS and then subjected to the In Situ QSAA protocol as described above. View this table: View inline View popup Table 1: Key Reagents and Resources Thioflavin S Staining Sections were incubated in 0.05% ThS in 50% ethanol for 8 min, differentiated in 80% ethanol, and treated with TrueBlack® Lipofuscin Autofluorescence Quencher (Biotium, 23007) to suppress background fluorescence. After rinsing in PBS, slides were coverslipped with aqueous mounting medium and imaged. Microscopy and Image Quantification Fluorescence images were acquired using a NIKON ECLIPSE Ti2-E microscope equipped with a 4×, 10×, and 20× objectives. High-resolution confocal images were acquired on a Leica SP8 scanning confocal microscope using a 40× (NA 0.75) or a 63× oil-immersion (NA 1.40) objective. To prevent spectral bleed-through, a sequential scanning strategy was employed, acquiring signals in descending order of emission wavelength: Cy5/Alexa Fluor 647 (Ex: 633 nm, Em: 650-720 nm) → Cy3/Alexa Fluor555 (Ex: 561 nm, Em: 570-630 nm) → ThT/QSAA (Ex: 488 nm, Em: 495-550 nm) →DAPI (Ex: 405 nm, Em: 415-470 nm). Notably, because ThT emission (not excitation spectra) partially overlaps with DAPI, subtraction or spectral unmixing was applied during analysis. Z-stacks were acquired with a 2 μm step size over a 50 μm range. All images intended for comparison were acquired using identical microscope settings (laser power, gain, offset) and processed with the same linear adjustments for brightness and contrast. Image analysis was performed using Fiji/ImageJ (v2.3.0). For quantification of fluorescence intensity, regions of interest were manually drawn, and the mean gray value was measured. All experiments were performed with at least three independent biological replicates. Transmission Electron Microscopy For solution-state products, 10 μL of QSAA reaction mixture was adsorbed onto glow-discharged, 400-mesh carbon-coated copper grids for 2 min, washed with ddH₂O, and negatively stained with 2% (w/v) uranyl acetate. For in situ products, QSAA-treated tissue sections were scraped from the slide, fixed in 2.5% glutaraldehyde/2% PFA in 0.1 M cacodylate buffer, post-fixed with 1% osmium tetroxide, dehydrated in ethanol, and embedded in EMBed 812 resin. Ultrathin sections (70 nm) were cut, placed on grids, and stained with uranyl acetate and lead citrate. All grids were imaged on a FEI Talos L120C TEM at 120 kV. RT-QuIC Validation Brain homogenates (10% w/v) from human samples were prepared in ice-cold PBS using a bead beater. Homogenates were serially diluted from 10⁻² to 10⁻⁵ in PBS. RT-QuIC reactions were set up in a 96-well plate with each well containing 100 μL of reaction mix: 0.1 mg/mL human αSyn (1-140), 10 μM ThT, 10 mM Tris (pH 7.5), and 50 mM NaCl. 2 μL of diluted brain homogenate was added to seed the reactions. The plate was incubated in a BMG FLUOstar Omega plate reader at 37°C with cycles of 1 min shaking (700 rpm, double orbital) and 1 min rest. ThT fluorescence (Ex: 450 nm, Em: 490 nm) was recorded every 15 min. A sample was considered positive if the fluorescence of at least two of three technical replicates crossed a threshold calculated as the mean of negative control wells plus five standard deviations. Statistical Analysis All statistical analyses were performed using GraphPad Prism (v9.0). Data are presented as mean ± SD unless otherwise noted. Before applying parametric tests, data were assessed for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene’s test. Comparisons between two groups were performed using a two-tailed, unpaired Student’s t-test. Comparisons among three or more groups were performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. For data involving two independent variables, a two-way ANOVA with Tukey’s multiple comparisons test was used. A P value < 0.05 was considered statistically significant. Specific statistical details for each experiment, including the exact value of n and what n represents, are provided in the figure legends. Data availability Any additional information required to reanalyze the data reported in this report is available from the corresponding author upon reasonable request. All materials used in this study will be made available subject to a materials transfer agreement (MTA). This paper does not report the original code. Author contributions H.M. designed the study, performed experiments, and wrote the manuscript. Y.K. supervised experiments, analyzed data, and contributed to manuscript preparation. D.W. and H.L. assisted with experimental procedures and data collection. H.M., J.H., and P-Y.X. provided project oversight, funding, and critical revisions to the manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (grant numbers 81870856, 81870992, and 32171236). Funder Information Declared National Natural Science Foundation of China , 81870856 , 81870992 , 32171236 Footnotes This version (v4) has been substantially revised compared with the version (v3) posted on bioRxiv (March 2025). Major updates include: 1. Title and author information updated; added an additional corresponding author and clarified affiliations. 2. Expanded introduction to highlight conceptual advances and implications of QSAA and IF-QSAA. 3. New experimental data added: Enhanced sensitivity analysis of QSAA versus pS129 and ThS staining in early-stage PD mouse models. Demonstration of seeding activity in archival human brain tissues, including long-term formalin-fixed specimens. Discovery of microglial seeding activity through IF-QSAA dual labeling, revealing distinct cellular distributions. Establishment of a "four-quadrant model" to distinguish seeding activity and phosphorylation states. 4. Figures and supplementary materials extensively revised and expanded, including additional TEM validation, confocal imaging, and dynamic time-course data. 5. Methods section reorganized and expanded with full details on animal models, human brain tissues, stereotaxic injections, RT-QuIC validation, and imaging parameters. 6. Discussion updated to emphasize broader applicability of QSAA, technical limitations, risk-control strategies for false positives, and translational potential. 7. References updated and expanded. References ↵ Schweighauser , M. et al. Structures of alpha-synuclein filaments from multiple system atrophy . Nature 585 , 464 – 469 ( 2020 ). doi: 10.1038/s41586-020-2317-6 OpenUrl CrossRef ↵ Yang , Y. et al. Structures of alpha-synuclein filaments from human brains with Lewy pathology . Nature 610 , 791 – 795 ( 2022 ). doi: 10.1038/s41586-022-05319-3 OpenUrl CrossRef ↵ Rodriguez , J. A. et al. Structure of the toxic core of alpha-synuclein from invisible crystals . Nature 525 , 486 – 490 ( 2015 ). doi: 10.1038/nature15368 OpenUrl CrossRef PubMed ↵ Theillet , F. X. et al. Structural disorder of monomeric alpha-synuclein persists in mammalian cells . Nature 530 , 45 – 50 ( 2016 ). doi: 10.1038/nature16531 OpenUrl CrossRef PubMed ↵ Volpicelli-Daley , L. A. et al. Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death . Neuron 72 , 57 – 71 ( 2011 ). doi: 10.1016/j.neuron.2011.08.033 OpenUrl CrossRef PubMed Web of Science ↵ Thomzig , A. et al. Transmissible alpha-synuclein seeding activity in brain and stomach of patients with Parkinson’s disease . Acta Neuropathol 141 , 861 – 879 ( 2021 ). doi: 10.1007/s00401-021-02312-4 OpenUrl CrossRef PubMed ↵ Anderson , J. P. et al. Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease . J Biol Chem 281 , 29739 – 29752 ( 2006 ). doi: 10.1074/jbc.M600933200 OpenUrl Abstract / FREE Full Text ↵ Fujiwara , H. et al. alpha-Synuclein is phosphorylated in synucleinopathy lesions . Nat Cell Biol 4 , 160 – 164 ( 2002 ). doi: 10.1038/ncb748 OpenUrl CrossRef PubMed Web of Science ↵ Mahul-Mellier , A. L. et al. The process of Lewy body formation, rather than simply alpha-synuclein fibrillization, is one of the major drivers of neurodegeneration . Proc Natl Acad Sci U S A 117 , 4971 – 4982 ( 2020 ). doi: 10.1073/pnas.1913904117 OpenUrl Abstract / FREE Full Text Lashuel , H. A. et al. Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity . NPJ Parkinsons Dis 8 , 136 ( 2022 ). doi: 10.1038/s41531-022-00388-7 OpenUrl CrossRef PubMed ↵ Ghanem , S. S. et al. alpha-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity . Proc Natl Acad Sci U S A 119 , e2109617119 ( 2022 ). doi: 10.1073/pnas.2109617119 OpenUrl CrossRef PubMed ↵ Ramalingam , N. et al. Dynamic physiological alpha-synuclein S129 phosphorylation is driven by neuronal activity . NPJ Parkinsons Dis 9 , 4 ( 2023 ). doi: 10.1038/s41531-023-00444-w OpenUrl CrossRef ↵ Groveman , B. R. et al. Rapid and ultra-sensitive quantitation of disease-associated alpha-synuclein seeds in brain and cerebrospinal fluid by alphaSyn RT-QuIC . Acta Neuropathol Commun 6 , 7 ( 2018 ). doi: 10.1186/s40478-018-0508-2 OpenUrl CrossRef PubMed ↵ Shahnawaz , M. et al. Development of a Biochemical Diagnosis of Parkinson Disease by Detection of alpha-Synuclein Misfolded Aggregates in Cerebrospinal Fluid . JAMA Neurol 74 , 163 – 172 ( 2017 ). doi: 10.1001/jamaneurol.2016.4547 OpenUrl CrossRef PubMed ↵ Soto , C. , Saborio , G. P. & Anderes , L . Cyclic amplification of protein misfolding: application to prion-related disorders and beyond . Trends Neurosci 25 , 390 – 394 ( 2002 ). doi: 10.1016/s0166-2236(02)02195-1 OpenUrl CrossRef PubMed Web of Science ↵ Paciotti , S. , Bellomo , G. , Gatticchi , L. & Parnetti , L . Are We Ready for Detecting alpha-Synuclein Prone to Aggregation in Patients? The Case of "Protein-Misfolding Cyclic Amplification" and "Real-Time Quaking-Induced Conversion" as Diagnostic Tools . Front Neurol 9 , 415 ( 2018 ). doi: 10.3389/fneur.2018.00415 OpenUrl CrossRef ↵ Concha-Marambio , L. , Pritzkow , S. , Shahnawaz , M. , Farris , C. M. & Soto , C . Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid . Nat Protoc 18 , 1179 – 1196 ( 2023 ). doi: 10.1038/s41596-022-00787-3 OpenUrl CrossRef PubMed ↵ Rossi , M. et al. Ultrasensitive RT-QuIC assay with high sensitivity and specificity for Lewy body-associated synucleinopathies . Acta Neuropathol 140 , 49 – 62 ( 2020 ). doi: 10.1007/s00401-020-02160-8 OpenUrl CrossRef PubMed ↵ Sorrentino , Z. A. et al. Physiological C-terminal truncation of alpha-synuclein potentiates the prion-like formation of pathological inclusions . J Biol Chem 293 , 18914 – 18932 ( 2018 ). doi: 10.1074/jbc.RA118.005603 OpenUrl Abstract / FREE Full Text ↵ Ulamec , S. M. et al. Single residue modulators of amyloid formation in the N-terminal P1-region of alpha-synuclein . Nat Commun 13 , 4986 ( 2022 ). doi: 10.1038/s41467-022-32687-1 OpenUrl CrossRef PubMed ↵ Munishkina , L. A. , Henriques , J. , Uversky , V. N. & Fink , A. L . Role of protein-water interactions and electrostatics in alpha-synuclein fibril formation . Biochemistry 43 , 3289 – 3300 ( 2004 ). doi: 10.1021/bi034938r OpenUrl CrossRef PubMed ↵ Metrick , M. A ., 2nd et al. Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons . Proc Natl Acad Sci U S A 116 , 23029 – 23039 ( 2019 ). doi: 10.1073/pnas.1909322116 OpenUrl Abstract / FREE Full Text Giehm , L. , Oliveira , C. L. , Christiansen , G. , Pedersen , J. S. & Otzen , D. E . SDS-induced fibrillation of alpha-synuclein: an alternative fibrillation pathway . J Mol Biol 401 , 115 – 133 ( 2010 ). doi: 10.1016/j.jmb.2010.05.060 OpenUrl CrossRef PubMed Web of Science ↵ Giehm , L. & Otzen , D. E . Strategies to increase the reproducibility of protein fibrillization in plate reader assays . Anal Biochem 400 , 270 – 281 ( 2010 ). doi: 10.1016/j.ab.2010.02.001 OpenUrl CrossRef PubMed Web of Science ↵ Orru , C. D. et al. Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid . mBio 6 ( 2015 ). doi: 10.1128/mBio.02451-14 OpenUrl Abstract / FREE Full Text ↵ Bhumkar , A. et al. Single-Molecule Counting Coupled to Rapid Amplification Enables Detection of alpha-Synuclein Aggregates in Cerebrospinal Fluid of Parkinson’s Disease Patients . Angew Chem Int Ed Engl 60 , 11874 – 11883 ( 2021 ). doi: 10.1002/anie.202014898 OpenUrl CrossRef ↵ Ohgita , T. , Namba , N. , Kono , H. , Shimanouchi , T. & Saito , H . Mechanisms of enhanced aggregation and fibril formation of Parkinson’s disease-related variants of alpha-synuclein . Sci Rep 12 , 6770 ( 2022 ). doi: 10.1038/s41598-022-10789-6 OpenUrl CrossRef PubMed ↵ Lira , R. B. , Steinkuhler , J. , Knorr , R. L. , Dimova , R. & Riske , K. A . Posing for a picture: vesicle immobilization in agarose gel . Sci Rep 6 , 25254 ( 2016 ). doi: 10.1038/srep25254 OpenUrl CrossRef ↵ Uemura , N. et al. alpha-Synuclein aggregates amplified from patient-derived Lewy bodies recapitulate Lewy body diseases in mice . Nat Commun 14 , 6892 ( 2023 ). doi: 10.1038/s41467-023-42705-5 OpenUrl CrossRef PubMed ↵ Luk , K. C. et al. Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice . Science 338 , 949 – 953 ( 2012 ). doi: 10.1126/science.1227157 OpenUrl Abstract / FREE Full Text ↵ Tanriover , G. et al. Prominent microglial inclusions in transgenic mouse models of alpha-synucleinopathy that are distinct from neuronal lesions . Acta Neuropathol Commun 8 , 133 ( 2020 ). doi: 10.1186/s40478-020-00993-8 OpenUrl CrossRef ↵ Kumar , S. T. et al. How specific are the conformation-specific alpha-synuclein antibodies? Characterization and validation of 16 alpha-synuclein conformation-specific antibodies using well-characterized preparations of alpha-synuclein monomers, fibrils and oligomers with distinct structures and morphology . Neurobiol Dis 146 , 105086 ( 2020 ). doi: 10.1016/j.nbd.2020.105086 OpenUrl CrossRef PubMed ↵ Soto , C. alpha-Synuclein seed amplification technology for Parkinson’s disease and related synucleinopathies . Trends Biotechnol 42 , 829 – 841 ( 2024 ). doi: 10.1016/j.tibtech.2024.01.007 OpenUrl CrossRef PubMed ↵ Vascellari , S. , Orru , C. D. & Caughey , B . Real-Time Quaking-Induced Conversion Assays for Prion Diseases, Synucleinopathies, and Tauopathies . Front Aging Neurosci 14 , 853050 ( 2022 ). doi: 10.3389/fnagi.2022.853050 OpenUrl CrossRef PubMed ↵ Peng , C. et al. Cellular milieu imparts distinct pathological alpha-synuclein strains in alpha-synucleinopathies . Nature 557 , 558 – 563 ( 2018 ). doi: 10.1038/s41586-018-0104-4 OpenUrl CrossRef PubMed Peelaerts , W. et al. alpha-Synuclein strains cause distinct synucleinopathies after local and systemic administration . Nature 522 , 340 – 344 ( 2015 ). doi: 10.1038/nature14547 OpenUrl CrossRef PubMed ↵ Shahnawaz , M. et al. Discriminating alpha-synuclein strains in Parkinson’s disease and multiple system atrophy . Nature 578 , 273 – 277 ( 2020 ). doi: 10.1038/s41586-020-1984-7 OpenUrl CrossRef ↵ Zhang , S. et al. Post-translational modifications of soluble alpha-synuclein regulate the amplification of pathological alpha-synuclein . Nat Neurosci 26 , 213 – 225 ( 2023 ). doi: 10.1038/s41593-022-01239-7 OpenUrl CrossRef PubMed ↵ Altay , M. F. , Liu , A. K. L. , Holton , J. L. , Parkkinen , L. & Lashuel , H. A . Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders . Acta Neuropathol Commun 10 , 163 ( 2022 ). doi: 10.1186/s40478-022-01468-8 OpenUrl CrossRef PubMed ↵ Okuzumi , A. et al. Propagative alpha-synuclein seeds as serum biomarkers for synucleinopathies . Nat Med 29 , 1448 – 1455 ( 2023 ). doi: 10.1038/s41591-023-02358-9 OpenUrl CrossRef PubMed ↵ Scialo , C. et al. TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients . Brain Commun 2 , fcaa142 ( 2020 ). doi: 10.1093/braincomms/fcaa142 OpenUrl CrossRef ↵ Kraus , A. et al. Seeding selectivity and ultrasensitive detection of tau aggregate conformers of Alzheimer disease . Acta Neuropathol 137 , 585 – 598 ( 2019 ). doi: 10.1007/s00401-018-1947-3 OpenUrl CrossRef PubMed ↵ Volpicelli-Daley , L. A. , Luk , K. C. & Lee , V. M . Addition of exogenous alpha-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous alpha-synuclein to Lewy body and Lewy neurite-like aggregates . Nat Protoc 9 , 2135 – 2146 ( 2014 ). doi: 10.1038/nprot.2014.143 OpenUrl CrossRef PubMed ↵ Mao , H. et al. Optimal Preservation of PFFs in Glycerol Enhances Suitability for Modeling Parkinson’s Disease . Small 20 , e2401552 ( 2024 ). doi: 10.1002/smll.202401552 OpenUrl CrossRef PubMed ↵ Polinski , N. K. et al. Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents . J Parkinsons Dis 8 , 303 – 322 ( 2018 ). doi: 10.3233/JPD-171248 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 25, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following In Situ Amplification and Spatial Mapping of α-Synuclein Seeding Activity Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share In Situ Amplification and Spatial Mapping of α -Synuclein Seeding Activity Hengxu Mao , Yaoyun Kuang , Wei Dai , Hao Lin , Jinkun Huang , Ping-Yi Xu bioRxiv 2025.03.26.645411; doi: https://doi.org/10.1101/2025.03.26.645411 Share This Article: Copy Citation Tools In Situ Amplification and Spatial Mapping of α -Synuclein Seeding Activity Hengxu Mao , Yaoyun Kuang , Wei Dai , Hao Lin , Jinkun Huang , Ping-Yi Xu bioRxiv 2025.03.26.645411; doi: https://doi.org/10.1101/2025.03.26.645411 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41970) Biophysics (21463) Cancer Biology (18605) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19911) Epidemiology (2067) Evolutionary Biology (24329) Genetics (15615) Genomics (22514) Immunology (17743) Microbiology (40424) Molecular Biology (17194) Neuroscience (88650) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4827) Physiology (7648) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)