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A C i ical Commen a y on “Syn he ic α-synuclein
ib ils eplica e in mice causing MSA-like pa hology”.
Bu ge e al., Na u e 2025; doi: 10.1038/s41586-
025-09698-1.
Mengxi Zhu and Shu-Feng Zhou*
College o Chemical Enginee ing, Huaqiao Uni e si y, Xiamen, China
*Co espondence: [email p o ec ed]
In oduc ion
Bu ge e al.1 (2025) epo ha syn he ic α-synuclein ib ils (Syn-Fibs) a e capable o
eplica ing in i o and p oducing mul iple-sys em-a ophy (MSA)-like pa hology in mice.
The a icle aims o esol e a long-s anding deba e abou whe he mis olded α-synuclein
alone is su icien o seed and p opaga e MSA- ype inclusion pa hology cha ac e is ic o
glial cy oplasmic inclusions (GCIs). While he s udy ep esen s an ambi ious and
echnically sophis ica ed e o , i p esen s se e al concep ual inconsis encies,
me hodological blind spo s, quan i ica ion weaknesses, and in e p e a ion o e - each.
Below, we p o ide a sys ema ic, igu e-by- igu e c i ique—including Ex ended Da a
Figu es and Supplemen a y Figu es—highligh ing issues o expe imen al design,
s a is ical analysis, image in eg i y, model alidi y, and claims exceeding e idence.
Main Commen a y
1. In oduc ion and Concep ual F aming
The au ho s p opose ha syn he ic α-synuclein ib ils, assembled in i o, can sel -
p opaga e in i o and eplica e an MSA-like pheno ype—a guing ha his demons a es
he "in ec ious p ion-like na u e" o Syn-Fibs. This aming has h ee weaknesses:
1. Assump ion o pu i y – Syn he ic α-synuclein ib ils gene a ed in i o o en
con ain s uc u al polymo phs. The au ho s do no demons a e ha he ib il
ba ches used ac oss expe imen s a e homogeneous o s able ac oss
p epa a ions.
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2. O e ex ension o “ eplica ion” e minology – Fib il elonga ion o seeding does
no inhe en ly cons i u e “ eplica ion” in he biological o p ion sense; he
concep ual leap is unjus i ied wi hou biochemical kine ic e idence.
3. Absence o longi udinal, in i o biochemical cha ac e iza ion – Wi hou se ial
biochemical acing o ib il s uc u e in mouse b ains, he au ho s canno
con incingly a gue ha he ib ils main ain hei MSA-like con o ma ional s a e.
These ounda ional issues pe mea e he en i e manusc ip and a ec he in e p e a ion
o all igu es.
Figu e-by-Figu e C i ique
Figu e 1 – S uc u al and biochemical cha ac e iza ion o
syn he ic α-synuclein ib ils
O e all assessmen : The au ho s claim ha he ib ils adop ed a “dis inc MSA-like
con o ma ion,” bu he s uc u al analyses p esen ed a e insu icien o subs an ia e
ha claim.
Majo c i icisms:
1.1 C yo-EM esolu ion inconsis encies
• The epo ed map esolu ion (~3.2–3.6 Å) is no adequa e o con iden ly assign
sidechain o ame s dis inguishing MSA-polymo phs om PD-polymo phs.
• Sha pening and il e ing pa ame e s a e poo ly desc ibed.
• The igu e panels lack local esolu ion hea maps, p e en ing eade s om
e alua ing he e ogeneous egions.
1.2 Polymo ph he e ogenei y no add essed
The s udy does no p o ide:
• 2D class dis ibu ions,
• classi ica ion o polymo ph sub-popula ions,
• ib il wis ing pe iodici y a iabili y.
This omission unde mines he claim ha he ib ils ep esen a single de ined s uc u al
species.
1.3 Missing biochemical cha ac e iza ion
The au ho s do no p o ide:
• p o ease esis ance p o iles,
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• sedimen a ion assays,
• con o ma ional s abili y cu es.
These a e s anda d p ion- eplica ion me ics. Wi hou hem, he s uc u al asse ions
a e weak.
1.4 Lack o compa ison o bona ide human MSA ib ils
Panels show supe icial simila i y, bu :
• alignmen me ics,
• RMSD alues,
• c oss-sec ions,
• backbone o e lays
a e missing. The igu e hus o e s a es simila i ies.
Figu e 2 – In i o inocula ion and dis ibu ion o pa hology
O e all assessmen :
The igu e claims widesp ead p opaga ion o ib il seeds and esul ing pa hology, bu
bo h he expe imen al design and quan i ica ion a e insu icien ly igo ous.
2.1 Injec ion pa ame e s poo ly con olled
C i ical omissions:
• Exac coo dina es o all injec ion eplica es.
• Needle gauge, in usion a e, dwell ime.
• Ba ch a iabili y o ib ils.
This p e en s ep oducibili y.
2.2 Pa hology quan i ica ion lacks s e eology
The au ho s use:
• % a ea co e age,
• ela i e luo escence uni s,
• a bi a y h esholds.
No unbiased s e eology (e.g., op ical ac iona o ), which is he gold s anda d o
neu odegene a ion quan i ica ion.
2.3 Missing con ols
The c ucial con ol g oups a e ei he absen o unde de eloped:
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• Hea -inac i a ed ib ils
• Monome ic α-synuclein
• Sc ambled ib ils
• PBS wi h iden ical injec ion auma
A igu e ha pu po s p opaga ion bu lacks c i ical nega i e con ols is no con incing.
2.4 Ambiguous immunos aining
An ibodies used (pS129 α-synuclein) a e no o ious o nonspeci ic binding when issue
ixa ion is subop imal. The au ho s do no epo :
• ixa ion du a ion,
• an igen e ie al me hod,
• backg ound sub ac ion p ocedu es.
Thus he pa hology dis ibu ion is di icul o in e p e .
Figu e 3 – MSA-like glial cy oplasmic inclusion (GCI) o ma ion
This is he key igu e suppo ing he cen al claim. I is also he weakes .
3.1 Failu e o show ul as uc u al GCIs
While he au ho s display immunos ained inclusions:
• No elec on mic oscopy e idence is p o ided o ue GCI mo phology.
• GCIs ha e dis inc ul as uc u al signa u es (compac , ing-like s acking); none
a e shown.
Thus, he claim “MSA-like GCI o ma ion” is no suppo ed.
3.2 Poo di e en ia ion be ween neu onal and glial inclusions
The au ho s ely on ma ke s:
• Olig2,
• GFAP.
Howe e :
• Olig2 labels p ogeni o s, no exclusi ely ma u e oligodend ocy es.
• GFAP is up egula ed by s ess and is no speci ic o inclusion-bea ing glia.
Co-localiza ion is insu icien wi hou :
• high- esolu ion z-s acks,
• cell- ype- es ic ed epo e s.
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3.3 Missing quan i ica ion o glial-only s neu onal-only inclusion
equencies
The au ho s claim glial p edilec ion, bu :
• nume ic compa ison is missing,
• ela i e bu den is no epo ed,
• sample sizes a e small (n = 3 pe g oup).
3.4 Possible image o e -con as o mis-adjus men
Some panels display:
• blown-ou highligh s,
• halos a ound inclusions,
• possible o e -sha pening.
This isks a i icial enhancemen o pa hology.
Figu e 4 – Beha io al pheno ypes
The claim: Syn-Fibs induce mo o and au onomic de ici s esembling MSA.
4.1 Choice o beha io al es s is insu icien
MSA in ol es:
• au onomic ailu e,
• pa kinsonism,
• ce ebella a axia.
The au ho s only es :
• o a od,
• pole es ,
• open ield.
These e lec gene al mo o de ici s, no MSA-speci ic impai men s. Missing:
• au onomic assays (blood p essu e a iabili y),
• ce ebella es s,
• espi a o y pa e ning,
• u odynamic assessmen s.
Thus, he beha io al e idence is nonspeci ic.
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4.2 Sample sizes oo small
n = 6–7 pe g oup is inapp op ia e o :
• o a od pe o mance (high a iabili y),
• complex mo o es s.
S a is ical powe is no e alua ed.
4.3 Blinding p ocedu es unclea
No s a emen con i ming:
• beha io al sco ing blinding,
• andomiza ion o ea men alloca ions.
4.4 Missing longi udinal ajec o ies
Acu e s ch onic de ici s a e indis inguishable om his igu e.
Figu e 5 – Biochemical p opaga ion and s ain labeling
This igu e a emp s o show ha ib ils ex ac ed om mouse b ains e ain hei "s ain
iden i y".
5.1 P o einase K diges ion insu icien
P ion s ain iden i ica ion ia PK esis ance equi es:
• mul i-concen a ion diges s,
• ime-cou se cu es,
• mass spec ome y o clea age agmen s.
The au ho s p o ide only:
• single ime poin ,
• single concen a ion,
• low- esolu ion Wes e n blo s.
This does no es ablish s ain e en ion.
5.2 Ampli ica ion assays lack igo
The au ho s use PMCA-like assays, bu :
• c oss-con amina ion is a majo isk and is no add essed.
• nega i e con ols a e ain ly shown o missing.
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5.3 Missing quan i a i e kine ics
P opaga ion cu es lack:
• lag phase es ima ion,
• g ow h a e calcula ion,
• endpoin sa u a ion beha io s.
This is equi ed o de ine “ eplica ion”.
Figu e 6 – Neu odegene a ion and oligodend ocy e dys unc ion
This igu e claims ha Syn-Fibs induce selec i e oligodend ocy e ulne abili y
esembling MSA.
6.1 Cell coun ing no s e eologically alida ed
Coun s a e made using:
• h esholded segmen a ion,
• egion o in e es (ROI) selec ion.
ROIs appea inconsis en ly placed ac oss animals.
6.2 Myelin s aining inconsis en
MBP s aining b igh ness:
• d as ically di e s ac oss panels,
• appea s possibly adjus ed pos -acquisi ion,
• lacks in e nal luo escence s anda ds.
6.3 Failu e o demons a e ue demyelina ion
T ue demyelina ion equi es:
• g- a io analysis ia EM,
• pa anodal dis up ion (Casp s aining),
• axonal swelling measu es.
None a e shown.
6.4 Possible ci cula easoning
The au ho s assume α-synuclein inclusions → oligodend ocy e dys unc ion. Bu he
images me ely show colocaliza ion, no causa ion.
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Figu e 7 – The apeu ic in e en ion expe imen s
This igu e es s an i-α-synuclein an ibodies o inhibi o s.
7.1 Sho ea men window
The apy is ini ia ed e y ea ly a e inocula ion—be o e pa hology seeds s abilize. Thus,
he s udy es s p e en ion, no ea men , hough he au ho s claim he apeu ic e icacy.
7.2 Dosing unclea
The igu e does no epo :
• mg/kg,
• dosing schedule,
• b ain pene a ion es ima es.
7.3 Pa hology educ ion me ics un eliable
Figu es ely on:
• h eshold-based quan i ica ion,
• non-linea luo escen signals,
• po en ially biased ROI sampling.
7.4 O e in e p e a ion
F om minimal educ ions in s aining in ensi y, he au ho s conclude “ escue o
pa hology,” which is an exagge a ion.
Ex ended Da a Figu e C i iques
The Ex ended Da a Figu es con ain se e al me hodological inconsis encies.
Ex ended Da a Figu e 1 – Addi ional ib il s uc u al analyses
• No ep oducibili y ac oss mul iple ba ches.
• No compa ison o wild- ype s mu an α-synuclein ib ils.
• Insu icien epo ing o alignmen pa ame e s in c yo-EM econs uc ions.
Ex ended Da a Figu e 2 – Addi ional pa hology maps
• Hea maps a e low esolu ion and possibly smoo hed excessi ely.
• Colo scales di e be ween samples, p e en ing compa ison.
• Missing aw coun s and slice numbe s.
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Ex ended Da a Figu e 3 – An ibody alida ion
• No do -blo o pep ide compe i ion es s.
• An ibody speci ici y o ib illa s monome ic α-syn no demons a ed.
Ex ended Da a Figu e 4 – Cell- ype esolu ion
• Single-plane images only; no 3D econs uc ions.
• Canno dis inguish engul ed ib ils (by mic oglia) s ue in acellula inclusions.
Ex ended Da a Figu e 5 – Fib il p epa a ion QC
• Dynamic ligh sca e ing esul s appea inconsis en wi h ib il leng hs obse ed
unde EM.
• Missing endo oxin analysis—endo oxin con amina ion can gene a e glial
pheno ypes esembling MSA.
Ex ended Da a Figu e 6 – Beha io al aw sco es
• La ge a iance indica es insu icien s a is ical powe .
• No sex-speci ic analysis.
• Con ol mice unde pe o m compa ed o s anda d colony a e ages, aising
gene al wel a e conce ns.
Ex ended Da a Figu es 7–10 – Blo quan i ica ion and eplica ion
expe imen s
• Densi ome y lacks linea ange alida ion.
• Blo s appea o e exposed.
• Some lanes show a i ac s esembling ho izon al smea ing—possible ans e
issues.
Supplemen a y Figu e C i iques
Supplemen a y Figu e 1 – Full c yo-EM p ocessing pipeline
• Missing 2D/3D classi ica ion low cha s.
• Pa icle numbe educ ion om ini ial picking o inal map is unusually la ge (~85%
loss), sugges ing ins abili y o ib ils.
Supplemen a y Figu e 2 – Raw beha io al ideos ( ames)
• Selec ed ames do no illus a e de ici s clea ly.
• No blinded sco ing shown.
Supplemen a y Figu e 3 – PMCA assay aw aces
• Baseline d i and noise a e no co ec ed.
