Could the key to detecting synucleinopathies be no more than skin-deep? In the March 20 JAMA, scientists led by Christopher Gibbons and Roy Freeman at Harvard Medical School in Boston detected phosphorylated synuclein in the skin of people clinically determined to have four such disorders. Skin p-serine 129 synuclein identified synucleinopathies with 93 to 100 percent accuracy. People at more advanced stages of disease harbored more p-synuclein. “The sensitivity and specificity of the procedure are both high (more than 90 percent), which is very encouraging," wrote Oskar Hansson of Lund University in Sweden (comment below). He was not involved in the work. "The method might, therefore, potentially be used to improve the diagnostic work-up in clinical practice but also when selecting appropriate patients for trials targeting α-synuclein.”

Synucleinopathies are typically diagnosed by their symptoms, which overlap with other diseases or can sometimes seem unrelated, such as REM sleep disorder or orthostatic hypotension. As such people often go undiagnosed or misdiagnosed for years, in the search for robust diagnostic marker, some scientists are using seed amplification assays. They pick up minuscule amounts of α-synuclein aggregates in tissue or cerebrospinal fluid samples, pegging Parkinson’s disease and Lewy body disorders with over 85 percent accuracy (Apr 2023 conference news; Aug 2023 conference news).

For their part, Gibbons and colleagues use skin samples taken via punch biopsy (image below). During this typically painless procedure, skin on the leg or back is numbed with local anesthetic. The physician pushes a tool with a hollow circle at the end into the skin, twists it to cut through the dermal layers, and then removes the 3mm circle of tissue. The patient is bandaged and sent on his or her way.

Punch Biopsy. Schematic of a punch biopsy to collect a skin sample. [Courtesy of Bruce Blaus on Wikimedia.]

Previously, the scientists detected phosphorylated α-synuclein in skin samples from 54 people with PD and 31 with multiple system atrophy (MSA) with 94 and 100 percent accuracy, respectively (Gibbons et al., 2023).

Now, Gibbons and colleagues turned to a larger sample size, including people with two other types of synucleinopathy—dementia with Lewy bodies (DLB) and pure autonomic failure (PAF), a degenerative disorder of the autonomic nervous system characterized by synuclein aggregates confined to those nerves. They analyzed skin samples from 343 older adults recruited from 30 academic and community neurology clinics across the U.S. Among participants, 120 were controls, 96 had PD, 50 DLB, 55 MSA, and 22 had PAF. Participants were 70, on average, and half were women.

The scientists measured p-synuclein in dermal neurons immunohistochemically using two antibodies, one that binds p-129 synuclein, and another that targets nerve fibers. The latter binds protein gene product 9.5 (PGP 9.5), aka UCH-L1, an abundant cytoplasmic protein within neurons and nerves. The authors took co-localization of the two antibodies to indicate p-S129-synuclein within neurons, and a sample with any overlap was deemed positive.

The assay detected cutaneous nerve p-S129-synuclein in 93 percent of people with PD. The assay did slightly better for DLB and MSA, picking out all but two of the former and one of the latter. It pegged all 22 cases of PAF. Gibbons chalked up negative tests to some forms of the diseases possibly having truncated α-synuclein or synuclein phosphorylated at a different residue. These are also not pathologically confirmed cases of synucleinopathy, so Gibbons suspects that a few might actually have another neurodegenerative disease, such as drug- or vascular-induced Parkinsonism, progressive supranuclear palsy, corticobasal degeneration, or Alzheimer’s disease.

Intriguingly, four of the 120 controls tested positive for p-synuclein. It is unclear if these represent false positives or if these people might be in the early stages of a synucleinopathy. In fact, upon closer review, Gibbons said three of four positives had signs of such a disorder, i.e., mild cognitive impairment or orthostatic hypotension.

Time since diagnosis did not sway biopsy results. For example, 93.5 or 92 percent of people with PD came up positive whether they had been diagnosed within the past three years or more than five years ago, respectively.

However, the amount of skin p-S129-synuclein did seem to change as the diseases progressed. People at a more advanced stage, as measured by symptom and neurological scale scores, had more p-synuclein.

The authors acknowledge that these results will need to be validated with larger sample sizes and in people without a clinical diagnosis of synucleinopathy to fully understand the potential of this biopsy test in clinical care.—Chelsea Weidman Burke


  1. This study confirms previous smaller studies showing that the levels of phosphorylated α-synuclein are increased in peripheral nerve terminals in the skin in neurological diseases characterized by accumulation of α-synuclein aggregates in the brain. The sensitivity and specificity of the procedure are both high (more than 90 percent), which is very encouraging. The method might therefore potentially be used to improve the diagnostic work-up in clinical practice, but also when selecting appropriate patients for trials targeting α-synuclein. Still, the method relies on immunohistochemistry and visual inspection of the stained sections using confocal microscopy to manually quantify the number of α-synuclein–positive nerve fibers, which is time-consuming and potentially user-dependent.

    Future advancements will likely include automated methods for actual quantification of the levels of phosphorylated α-synuclein in the tissue, potentially normalizing those levels to a marker representing the total amount of nerve fibers in the same tissue. Another interesting advancement would be to determine if there is a subtype of α-synuclein disease that starts in the periphery or not. In the BioFINDER study, we collect skin biopsies and CSF from many hundreds of healthy people, and it will be interesting to see whether there are cases with abnormal α-synuclein in the skin who do not yet have abnormal α-synuclein in the CSF, and if such individuals become positive in the CSF during follow-up. Alternatively, we might find that all study participants will be positive in CSF before skin, implying that the α-synuclein diseases always start in the brain before affecting peripheral nerves.  

  2. In Parkinson’s as in Alzheimer’s disease, biological markers are important to improve early diagnostics. This paper provides strong evidence that cutaneous phosphorylated α-synuclein may serve as diagnostic biomarker for Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and pure autonomic failure.

    The large cohort is convincing, and the numbers of patients testing positive (more than 90 percent for PD, DLB, and MSA) are impressive. Even though the collection method (skin punch) is invasive, it is well-tolerated by most participants. The read-out evaluation is immunohistochemistry by an experienced histopathologists, is not that simple or automated for now, but automation can be foreseen. It will be relevant to know if one punch would suffice: They took three at different locations, and positivity was defined by at least one being positive.

    It is also unclear why no sensitivity or specificity—let alone PPV or NPV—has been provided. Such measures would allow easier comparison with other biomarker studies.

    As the authors also indicated, comparison between CSF seeding assay outcomes and skin biopsy studies within the same persons is relevant to determine if the reported more accurate diagnostic results obtained by skin biopsies can be replicated. It will become even more interesting if additional information can be obtained from these sections, for prognosis for example, analogous to being able to measure panels of biomarkers from one drop of CSF. 

  3. The publication of Gibbons et al., like most clinical biomarker studies, suffers first and foremost from the use of an inadequate gold standard. The clinical diagnoses of Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are all quite poor when compared to the autopsy diagnoses. The clinical diagnostic accuracy of PD, against autopsy, has been published (Adler et al., 2021) as 70.6 percent for early PD and 89.1 percent for late PD (defined as in Gibbons et al. as less than five years, versus five or more years of disease duration), as approximately half of the subjects had early and half had late PD, the diagnostic accuracy against autopsy was probably the average of those figures, hence 78.8percent. Autopsy confirmation of  a clinical diagnosis of MSA, across multiple North American centers, is about 61 percent (Koga et al., 2015; Sekiya et al., 2023). For dementia with Lewy bodies, it is only about 45 percent (Fujishiro et al., 2008). Therefore, the findings of Gibbons et al. only appear to show that their skin biopsy diagnoses of these disorders are equally as inaccurate as the clinical diagnoses.

    What is the point of a biomarker that only confirms an inaccurate clinical diagnoses? Amyloid PET (Clark et al., 2012) in Alzheimer’s disease (AD) is an instructive example. Prior to amyloid PET, the clinical diagnostic accuracy against autopsy ranged widely across centers, while the accuracy as compared to autopsy in a definitive multicenter study was only 70 percent (Beach et al., 2012). The low clinical diagnostic accuracy was soon verified by the realization that PET amyloid was only positive in about two-thirds of those clinically diagnosed as having AD (Johnson et al., 2013; Sevigny et al., 2016). The discordance between the clinical diagnosis and a positive PET scan was a result of the greater accuracy of the latter for identifying “true” AD. Amyloid PET has subsequently become the clinical gold standard for AD diagnosis (Jack et al., 2018). If the methods of Gibbons et al. were truly accurate for identifying underlying synucleinopathy, then their results should have substantially diverged from the clinical categorizations.

    A second, and perhaps more important, concern is that the highest standards for scientific rigor in a diagnostic study are not met. The most critical aspect is that third-party blinding to diagnosis (triple blinding) was not used. This despite both tissue slide readers (Gibbons and Levine) being founders of CND, a for-profit company that sells this test, who are poised to financially benefit from these study results if they are used to market the CND test.

    The only third-party, blinded, multicenter study of a skin biopsy-based diagnostic test for PD was done as part of the Michael J. Fox-sponsored Systemic Synuclein Sampling Study (S4) (Chahine et al., 2020), which found that skin biopsies stained for pathological a-synuclein had only 24.1 percent sensitivity (100 percent specificity) for PD across 60 early, mid-, and late-stage subjects and 20 controls. This remains the most rigorous study of any biopsy-based diagnostic test for synucleinopathies. An immunoperoxidase method was employed on formalin-fixed, thin (i.e. 5-10 microns) paraffin-embedded (FFPE) sections. Gibbons’ group has argued that their methods are more sensitive due to the greater section thickness and lack of treatment with formalin and paraffin embedding. This used to be true 30-40 years ago, but the development of antigen unmasking methods, such as the protease pretreatment used in the MJFF S4 study, has made immunostaining of FFPE sections for many protein targets across many diseases equivalent to that obtained with cryostat or free-floating sections such as those used by Gibbons et al. Gibbons et al. have claimed that their methods, using dual-color immunofluorescence on thick free-floating sections, are inherently more sensitive, but others using essentially the same methods have reported sensitivities as low as 61 percent and 47 percent (Doppler, 2021; Brumberg et al., 2021; Donadio et al., 2019).  

    Ultimately we concluded, as has at least one other group (Doppler, 2021), that IHC of skin biopsies is unlikely to have sufficient sensitivity for diagnosing idiopathic PD and therefore we have been concentrating on applying RT-QuIC methods (seeding amplification assay) to skin biopsies, because of their apparent greater sensitivity.  

    Other issues with the Gibbons et al. report that are of concern are:

    • What was done to minimize “bleed-through” of one fluorophore signal to the other?
    • Was there standardized “packaging” of sample, slides, and images so that diagnosis could not be surmised?
    • Was there randomized presentation of slides and/or images, that mixed control and patient material?

    To their credit, the authors admit other limitations:

    • Test results on early and prodromal disease conditions were not evaluated.
    • Important “disease mimics,” including PSP and CBD were not assessed. In our center, PSP is the most common condition to be clinically confused with PD.
    • The population is highly selected, screened first by highly specialized neurologists and thus probably enriched with subjects more likely to have the targeted diagnoses.
    • One of the two clinical experts (RF) is affiliated with CND.
    • Controls were much younger than the patients, median age 59. In our center, these subjects have only about a 5 percent likelihood to have incidental Lewy body disease, while an age-similar control group to their study group (60-80) is about 15 percent likely to have ILBD. Because of this, and because of the lack of more suitable disease controls such as PSP and CBD, the specificity of the test was not adequately tested.


    . Clinical Diagnostic Accuracy of Early/Advanced Parkinson Disease: An Updated Clinicopathologic Study. Neurol Clin Pract. 2021 Aug;11(4):e414-e421. PubMed.

    . When DLB, PD, and PSP masquerade as MSA: an autopsy study of 134 patients. Neurology. 2015 Aug 4;85(5):404-12. Epub 2015 Jul 2 PubMed.

    . Validation Study of the MDS Criteria for the Diagnosis of Multiple System Atrophy in the Mayo Clinic Brain Bank. Neurology. 2023 Dec 12;101(24):e2460-e2471. Epub 2023 Oct 10 PubMed.

    . Validation of the neuropathologic criteria of the third consortium for dementia with Lewy bodies for prospectively diagnosed cases. J Neuropathol Exp Neurol. 2008 Jul;67(7):649-56. PubMed.

    . Cerebral PET with florbetapir compared with neuropathology at autopsy for detection of neuritic amyloid-β plaques: a prospective cohort study. Lancet Neurol. 2012 Aug;11(8):669-78. PubMed.

    . Accuracy of the clinical diagnosis of Alzheimer disease at National Institute on Aging Alzheimer Disease Centers, 2005-2010. J Neuropathol Exp Neurol. 2012 Apr;71(4):266-73. PubMed.

    . Florbetapir (F18-AV-45) PET to assess amyloid burden in Alzheimer's disease dementia, mild cognitive impairment, and normal aging. Alzheimers Dement. 2013 Oct;9(5 Suppl):S72-83. PubMed.

    . Amyloid PET Screening for Enrichment of Early-Stage Alzheimer Disease Clinical Trials: Experience in a Phase 1b Clinical Trial. Alzheimer Dis Assoc Disord. 2016 Jan-Mar;30(1):1-7. PubMed.

    . NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease. Alzheimers Dement. 2018 Apr;14(4):535-562. PubMed.

    . In vivo distribution of α-synuclein in multiple tissues and biofluids in Parkinson disease. Neurology. 2020 Sep 1;95(9):e1267-e1284. Epub 2020 Aug 3 PubMed.

    . Detection of Dermal Alpha-Synuclein Deposits as a Biomarker for Parkinson's Disease. J Parkinsons Dis. 2021;11(3):937-947. PubMed.

    . Dermal and cardiac autonomic fiber involvement in Parkinson's disease and multiple system atrophy. Neurobiol Dis. 2021 Jun;153:105332. Epub 2021 Mar 17 PubMed.

    . Abnormal α-synuclein deposits in skin nerves: intra- and inter-laboratory reproducibility. Eur J Neurol. 2019 Feb 15; PubMed.

Make a Comment

To make a comment you must login or register.


News Citations

  1. Synuclein Assay Passes the Sniff Test—What of Other Seeds?
  2. Finally, a Diagnostic Marker for Lewy Body Disease?

Paper Citations

  1. . Cutaneous α-Synuclein Signatures in Patients With Multiple System Atrophy and Parkinson Disease. Neurology. 2023 Apr 11;100(15):e1529-e1539. Epub 2023 Jan 19 PubMed. Correction.

Further Reading

No Available Further Reading

Primary Papers

  1. . Skin Biopsy Detection of Phosphorylated α-Synuclein in Patients With Synucleinopathies. JAMA. 2024 Apr 16;331(15):1298-1306. PubMed.