ABSTRACT
Immunofluorescence for phosphorylated alpha‐synuclein in skin biopsy samples is an emerging biomarker in synucleinopathies comprising Dementia with Lewy bodies. In this pilot study, 19 patients with recent‐onset (≤ 18 months) cognitive impairment underwent skin biopsy at baseline, with follow‐up clinical re‐evaluation. Five patients fulfilled the Dementia with Lewy bodies diagnosis, all of them positive for skin phosphorylated alpha‐synuclein. The remaining 14 patients were all negative. Skin tissue immunofluorescence may represent a promising technique in identifying synuclein pathology in recent‐onset cognitive decline: larger cohorts are needed to confirm our preliminary data.
Keywords: dementia, immunofluorescence, Lewy bodies, skin biopsy, synucleinopathy
1. Introduction
Synucleinopathies are disorders in which the abnormally phosphorylated protein alpha‐synuclein (p‐syn) accumulates in different parts of the nervous system, driving neurodegeneration. Cognitive impairment, either mild or major, is frequent in synucleinopathies and represents a key clinical issue with an impact on the quality of life of both patients and caregivers. Cognitive decline is observed in two main forms: Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB).
DLB is estimated to represent the second most common cause of cognitive decline after Alzheimer's Disease (AD) [1]. As outlined in the most recent criteria [2], DLB diagnosis requires the presence of cognitive decline plus core and supportive criteria. Nonetheless, differential diagnosis with other disorders characterized by parkinsonism and autonomic failure, such as Parkinson's Disease (PD), can be challenging and later disproven by pathological post‐mortem studies, as highlighted in a recent meta‐analysis describing diagnostic accuracy < 80% in early phases [3].
Reliable biomarkers to improve diagnostic capabilities are urgently needed. Identification of p‐syn in different tissues could represent a significant strategy in this sense, based on the pattern of synuclein deposition in both central and peripheral nervous system [4]. More specifically, the skin is an easily accessible, non‐invasive target of diagnostic biopsy, due to the presence of intraepidermal nerve fibers affected by p‐syn. One of the main approaches for analyzing skin biopsy samples is immunofluorescence, with PD and Multiple System Atrophy (MSA) being among the most studied synucleinopathies in this context [5, 6, 7, 8].
Regarding DLB, skin biopsy has been explored as a diagnostic tool in fewer patients: a 2017 study by Donadio et al. identified p‐syn via immunofluorescence in skin samples from 18 patients with full‐fledged DLB, while yielding negative results in all 23 patients with nonsynucleinopathy dementia and all 25 healthy controls [9]. Moreover, a recent 2024 study by Gibbons et al. identified cutaneous p‐syn in 48 out of 50 DLB patients from a cohort of 223 patients with synucleinopathy [10], while detecting it in only 4 out of 120 controls.
We present a pilot study on p‐syn identification via skin biopsy immunofluorescence in a cohort of patients presenting with recent‐onset (< 18 months) cognitive decline, then clinically followed for at least 18 months. The aim of this study was to establish the diagnostic accuracy of skin p‐syn in autonomic nerves in identifying DLB among patients with recent onset of cognitive decline.
2. Methods
2.1. Study Participants and Clinical Criteria
A multi‐center, prospective follow‐up study was conducted with patients recruited from 3 different tertiary centers for neurological disorders. Inclusion criteria were: 1) recent onset (< 18 months) of cognitive impairment, defined as either major or mild (MCI) according to the DSM‐5 definitions [11]; 2) onset at age > 45 years.
2.2. Clinical Examination and Diagnosis
Cognitive status was first screened using the Mini‐Mental State Examination (MMSE) by the attending neurologist, followed by a comprehensive neuropsychological assessment conducted by neuropsychologists, consisting of Mini‐Mental State Examination, Frontal Assessment Battery, barrage tests, Rey's 15‐word test, immediate visual memory testing, simple analogy testing, Stroop test, verbal fluency testing, and figure copying tests.
Subsequently, to better delineate the clinical picture, every patient underwent a full neurological examination, including the MDS‐UPDRS part III if extrapyramidal impairment was detected.
Imaging was performed at baseline using brain MRI or CT scan, in order to exclude central nervous lesions (e.g., expansive, vascular, or inflammatory). Additional imaging studies (i.e., FDG‐PET, cerebral SPECT with Datscan, MIBG scintigraphy) were performed according to clinical practice and when consented by the patient. Still at baseline, when required by the clinical picture of the patient, lumbar puncture was proposed and performed when consented for CSF collection and analysis for markers of amyloid and tau pathology, namely beta‐amyloid 1–42/1–40 ratio, phosphorylated tau, total tau protein dosing (Lumipulse G600II—Fujirebio).
Specific diagnostic criteria were employed to diagnose DLB [2], ad [ 12], FTD [13, 14]. Patients presenting with self‐referred cognitive decline but no evidence of any disease from a clinical, neuroradiological, and biochemical (e.g., CSF biomarkers of neurodegeneration) point of view, a diagnosis of subjective cognitive impairment was formulated.
After the baseline evaluation, patients returned for a follow‐up visit (≥ 18 months later), during which the same clinical assessment and scales were administered again. All analyses and diagnoses were performed from December 2017 to February 2024.
At baseline, a skin biopsy was performed for detection of p‐syn via immunofluorescence using the method described below.
2.3. Skin Biopsy Immunofluorescence Confocal Microscopy
The technique has been previously described in earlier studies [15]: 3 mm (mm) punch biopsies were taken bilaterally from proximal (cervical C7 paravertebral area 5 cm from the midline) and distal (10 cm above the lateral malleolus) skin sites. A second biopsy was taken 1–2 cm from the first sample to increase the rate of p‐syn positivity. The total number of skin samples taken for each patient was 8. Skin samples were immediately fixed in cold Zamboni's fixative and kept at 4°C overnight. 10 μm sections were obtained using a cryostat (CM 1950; Leica, Deerfield, IL), vertically along the depth of the skin biopsy. 3 skin sections (10 μm of thickness) were routinely analyzed for each skin sample. The sections were triple‐immunostained overnight with a panel of primary antibodies, including rabbit monoclonal p‐syn (1:500, Abcam, Cambridge, UK, cat. no. ab‐51253), and mouse pan‐neuronal marker protein gene product 9.5 (PGP, 1:750; Abcam, Cambridge, UK, cat. no. ab72911). Sections were then washed, and secondary antibodies were added for an incubation of 1 h. As secondary antibodies, an anti‐mouse Alexa Fluor(R) 488 (1:400; Jackson ImmunoResearch, West Grove, PA, USA, cat. no. 715‐545‐150) and rabbit cyanine dye fluorophores 3.18 (1:800, Jackson ImmunoResearch, West Grove, PA, USA; cat. no. 711–165‐152) were used. Nucleic acid counterstaining was achieved with 4′, 6‐diamidino‐2‐phenylindole (DAPI, D9542—Sigma Aldrich). The microscopy analysis and criteria to define p‐syn positivity have been previously described. Shortly, sections were analyzed using a laser‐scanning confocal microscope (Nikon confocal microscopy, Eclipse Ti A1, Japan) by two authors with expertise in immunoflorescent analysis (DV and IA). Digital images were collected in successive frames of 1–2 μm increments on a Z‐stack plan at the appropriate wavelengths for secondary antibodies with a ×40 plan apochromat objective and subsequently projected to obtain a double‐stained digital image by a computerized system. The correspondence between rabbit p‐syn and mouse PGP staining helped to verify the intraneuronal deposits excluding possible non‐specific staining arising from the background. This analysis has previously been proven to have a high intra‐laboratory reproducibility [16] and was made in a blinded fashion by authors with expertise in immunofluorescence analysis (VD, AI, and CD). A single p‐syn positive section was considered sufficient to classify the patient as positive.
3. Results
19 patients who presented with a suspicion of cognitive decline at baseline were included in this study. All patients underwent neuropsychological testing, which confirmed cognitive impairment, except for 2 patients whose severe cognitive symptoms prevented them from completing the tests.
The overall demographic and clinical characteristics are presented in Tables 1, 2, 3, 4.
TABLE 1.
Clinical characteristics, results of skin biopsy, and final diagnosis of patients.
| Pt number | Skin biopsy | Dementia onset (years) | Motor onset (years) | Sex | Final diagnosis | cMMSE | Neuropsychological testing |
|---|---|---|---|---|---|---|---|
| DLB | |||||||
| 1 | POS | 74 | 73 | M | DLB | 19 | ND |
| 2 | POS | 78 | 79 | M | DLB | 26 | Impaired long‐term memory |
| 3 | POS | 73 | 78 | F | DLB | 19 | ND |
| 4 | POS | 68 | 69 | M | DLB | 13.2 | Multidomain impairment |
| 5 | POS | 73 | F | AD‐LBD co‐occurrence | 23.7 | Impaired visuospatial skills and memory | |
| Non‐synucleinopathies | |||||||
| 6 | NEG | 61 | 62 | F | Amnestic MCI and secondary parkinsonism | 26 | Impaired memory and fluency |
| 7 | NEG | 79 | F | Amnestic MCI | 26 | Impaired visuospatial skills | |
| 8 | NEG | 66 | F | Amnestic MCI | 26.6 | Impaired memory and attention | |
| 9 | NEG | 67 | M | Amnestic MCI | 21.53 | Multidomain impairment | |
| 10 | NEG | 51 | M | FTD | 30 | Apathy, disinibition | |
| 11 | NEG | 73 | M | FTD | 11.86 | Apathy | |
| 12 | NEG | 63 | F | Subjective cognitive impairment | 30 | Depression | |
| 13 | NEG | 59 | F | Subjective cognitive impairment | 27.9 | Depression | |
| 14 | NEG | 55 | F | Subjective cognitive impairment | 23.8 | Depression | |
| 15 | NEG | 71 | F | AD (logopenic PPA) | 13.7 | Multidomain impariment | |
| 16 | NEG | 80 | F | AD | 26.1 | Impaired memory and fluency | |
| 17 | NEG | 79 | F | AD | 23 | Impaired memory and fluency | |
| 18 | NEG | 70 | F | AD | 23 | Multidomain impairment | |
| 19 | NEG | 63 | F | AD | 15.2 | Multidomain impairment | |
TABLE 2.
Core clinical features and results of neuropsychological testing of patients.
| Pt number | cMMSE | Neuropsychology | Parkinsonism | RBD | Hallucinations | Fluctuations |
|---|---|---|---|---|---|---|
| DLB | ||||||
| 1 | 19 | ND | + | + | − | − |
| 2 | 26 | Impaired long‐term memory | + | − | − | − |
| 3 | 19 | ND | + | − | + | + |
| 4 | 13.2 | Multidomain impairment | + | + | − | − |
| 5 | 23.7 | Impaired visuospatial skills, memory | − | + | − | − |
| Non‐synucleinopathies | ||||||
| 6 | 26 | Impaired memory and fluency | + | − | − | − |
| 7 | 26 | Impaired visuospatial skills | − | − | − | − |
| 8 | 26.6 | Impaired memory and attention | − | − | − | − |
| 9 | 21.53 | Multidomain impairment | − | − | − | − |
| 10 | 30 | Apathy, disinibition | − | − | − | − |
| 11 | 11.86 | Apathy | − | − | − | − |
| 12 | 30 | Depression | − | − | − | − |
| 13 | 27.9 | Depression | − | − | − | − |
| 14 | 23.8 | Depression | − | − | − | + |
| 15 | 13.7 | Multidomain impariment | − | − | − | − |
| 16 | 26.1 | Impaired memory and fluency | − | − | − | − |
| 17 | 23 | Impaired memory and fluency | − | − | − | − |
| 18 | 23 | Multidomain impairment | − | − | − | − |
| 19 | 15.2 | Multidomain impairment | − | − | − | − |
TABLE 3.
Indicative and supportive biomarkers of patients.
| Pt number | SPECT‐DatScan | Cardiac MIBG | FDG‐PET hypocaptation | Brain atrophy at MRI |
|---|---|---|---|---|
| DLB | ||||
| 1 | ND | ND | ND | Bilateral parieto‐temporal |
| 2 | + | + | ND | Diffuse |
| 3 | − | ND | ND | Bilateral mesial temporal (cingulus spared) |
| 4 | ND | ND | ND | Diffuse |
| 5 | ND | ND | Left temporo‐parietal | Hippocampal |
| Non‐synucleinopathies | ||||
| 6 | ND | ND | Bilateral mesial temporal | − |
| 7 | ND | ND | ND | Frontal |
| 8 | ND | ND | ND | Diffuse cortical |
| 9 | ND | ND | ND | Fronto‐temporal |
| 10 | ND | ND | ND | Fronto‐temporal |
| 11 | ND | ND | ND | Frontal and hippocampal |
| 12 | ND | ND | ND | − |
| 13 | ND | ND | ND | − |
| 14 | ND | ND | ND | − |
| 15 | ND | ND | Left temporoparietal cingulum involved | Bilateral fronto‐temporo‐parietal |
| 16 | ND | ND | Right temporo‐parietal, bilateral frontal | ND |
| 17 | ND | ND | ND | Diffuse |
| 18 | ND | ND | ND | Bilateral fronto‐insular, parieto‐temporal |
| 19 | ND | ND | Left fronto‐parietotemporal | Fronto‐temporal, insular |
TABLE 4.
Amyloid and tau biomarkers in patients, as well as evidence of neurogenic orthostatic hypotension.
| Pt number | CSF 1–42/1–40 amyloid ratio | CSF p‐tau | t‐tau | Neurogenic orthostatic hypotension |
|---|---|---|---|---|
| DLB | ||||
| 1 | ND | ND | ND | − |
| 2 | ND | ND | ND | + |
| 3 | 0.46 | ND | ND | − |
| 4 | 4.8 | 10 | 159 | + |
| 5 | 0.57 | 91 | 566 | − |
| Non‐synucleinopathies | ||||
| 6 | 0.92 | 26 | 236 | − |
| 7 | ND | ND | ND | − |
| 8 | 3.6 | 32 | 252 | − |
| 9 | 4.6 | 57 | 228 | − |
| 10 | 5.4 | 27 | 239 | − |
| 11 | 4.6 | 25 | 395 | − |
| 12 | 1.15 | 27 | 182 | − |
| 13 | 0.99 | 35 | 288 | − |
| 14 | 1.02 | 33 | 211 | − |
| 15 | 0.39 | 179 | 1013 | − |
| 16 | 0.62 | 113 | 663 | − |
| 17 | 0.42 | 139 | 945 | − |
| 18 | 0.41 | 219 | 1507 | − |
| 19 | 0.68 (limite) | 25 | 229 | − |
Final diagnosis was as follows: DLB (N = 5, 26%), comprising one case of DLB‐AD co‐occurrence (N = 1, 5%), AD (N = 5, 26%, including one patient with primary progressive aphasia, logopenic variant), subjective cognitive impairment (N = 3, 16%), amnestic mild cognitive impairment of undetermined origin (N = 4, 21%) and Frontotemporal Dementia (FTD), behavioral variant (N = 2, 11%). Skin biopsy yielded a value of 100% for both sensitivity and specificity. The case of DLB‐AD co‐occurrence initially presented with a suspicion of amnestic MCI. During subsequent investigations, the patient presented positive markers for amyloid (reduced Aβ42/40 ratio) and tau (elevated p‐tau181) pathology at CSF examination, as well as brain MRI imaging consistent with AD. However, she also presented with marked visuospatial impairment at neuropsychological testing. Over the following year, she developed RBD and hypomimia, leading to a final diagnosis of AD with DLB co‐occurrence.
Immunofluorescence on skin biopsy samples was positive for p‐syn in all 5 DLB patients, while negative in all other patients. P‐syn positivity was found in skin nerves depicted by PGP 9.5 that innervate the autonomic structures, as exemplified by (Figure 1).
FIGURE 1.
Confocal microscope analysis (×400) of skin tissue from the cervical (C7) region of patient 5, showing innervation of arterioles. Signals from nerve fibers (PGP9.5, green), nuclei (DAPI, blue) and phosphorylated α‐synuclein (red) are merged in (A), showing PGP9.5 in (B) and p‐syn signals in (C). P‐syn accumulates in nerve fibers, as shown by co‐localization of p‐syn (red) and PGP9.5 (green) signal.
4. Discussion
In our cohort of patients with recent‐onset cognitive decline, immunofluorescence for p‐syn detected in autonomic nerve fibers identified all individuals who either presented with or subsequently developed a clinical picture of DLB.
Notably, skin biopsy was negative for p‐syn in all cases of non‐synucleinopathic cognitive decline, suggesting very high specificity. Thus, these preliminary data suggest a useful role of this technique also in differential diagnosis, especially when the clinical picture is complex and makes it difficult to distinguish DLB from other disorders characterized by cognitive decline (e.g., AD or FTD).
Skin immunofluorescence has provided promising results for synucleinopathies, with current evidence suggesting high sensitivity and specificity [15]. However, differences in the execution of this technique hinder the comparison of studies, warranting refinement of an agreed‐upon protocol. Preliminary data on DLB are promising, albeit based on a small number of patients [9, 10].
Limitations of this study include the small number of patients included. For this reason, the study must be considered preliminary. To confirm the data currently presented, a study with a larger cohort of patients recruited is therefore necessary; moreover, the absence of post‐mortem pathological data definitely confirming diagnosis is another limitation of this current study.
In conclusion, our preliminary data showed that immunofluorescence for p‐syn detection in synucleinopathies could represent a viable and valuable diagnostic technique for the identification of cognitive decline related to Lewy body pathology.
Author Contributions
All authors contributed to the study conception and design. Data collection was performed by Alessandro Furia, Giovanni Rizzo, Salvatore Bonvegna, Marco Piatti, Enrica Olivola, Francesco Ventruto, Veria Vacchiano, Enrico Fileccia, Roberto Cilia, Nicola Modugno, Rocco Liguori, and Vincenzo Donadio. Vincenzo Donadio performed skin biopsy on all patients: sample preparation and reading analysis for detection of p‐syn was performed by Vincenzo Donadio, Alex Incensi, and Cecilia Delprete. The first draft of the manuscript was written by Alessandro Furia, and authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Ethics Statement
The study was approved by a local Ethical Committee (Comitato Etico di Area Vasta Emilia Centro della Regione Emilia‐Romagna, CE‐AVEC) and followed the Helsinki Declaration regarding clinical research involving human beings.
Consent
Written informed consent was obtained from all participants included in the study.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgment
Open access publishing facilitated by Universita degli Studi di Bologna, as part of the Wiley ‐ CRUI‐CARE agreement.
Funding: This work was supported by Ricerca Finalizzata Ministero della Salute Grant RF‐2016‐02362047 to V.D. and A.I.
Funding Statement
This work was funded by Ministero della Salute grant RF‐2016‐02362047.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.