Skin Biopsy as a Diagnostic Tool for Synucleinopathies

Studies published in the last decade identified skin biopsies as a promising source of material for detecting alpha-synuclein (αSN). Alpha-synuclein gets deposited in the skin of patients with synucleinopathies, and therefore, a skin biopsy can be used to diagnose and confirm these diseases histopathologically. A skin biopsy can also be helpful for studies focusing on the nature of αSN deposits. The most important aspects of a biomarker are sensitivity, specificity, and technical feasibility. The potential for a skin biopsy to become the clinical tool of choice as a reliable biomarker for diagnosing synucleinopathies appears to be high, with consistently high sensitivity (>80%) and specificity approaching 100%. The review aims to provide an overview of the factors impacting skin biopsy's sensitivity, specificity, and feasibility in detecting dermal αSN deposits.


Introduction And Background
Skin biopsies are recommended for various dermatological diseases where histopathologic knowledge is required to diagnose the underlying skin disease [1].Besides skin disorders, interestingly, it can also be used to identify neurological conditions called "synucleinopathies."Synucleinopathies include Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and rapid eye movement (REM) sleep behavior disorder (RBD) [2].
More than two million people in the United States are affected by synucleinopathies [3].Depending on the location of the lesions, synucleinopathies are characterized by a chronic and progressive decrease in motor, cognitive, behavioral, and autonomic abilities [2].These disorders and atypical parkinsonism, such as progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), share the cardinal parkinsonian symptoms [4].This clinical overlap makes differential diagnosis sometimes exceedingly challenging [2].Consensus clinical criteria are currently used to diagnose Parkinson's disease [3].Even among specialists, early diagnosis of synucleinopathies has only moderate accuracy, especially in atypical and complex presentations [3].The pathogenesis of the disease starts many years before its clinical manifestations.Motor symptoms in PD are reported to first develop when >50% of substantia nigra dopamine neurons are destroyed.Therefore, there is a much higher need for an earlier or prodromal-stage diagnostic tool that is more precise [5].
In synucleinopathies, fibrillary deposits of the alpha-synuclein (αSN) protein start to deposit in the submucosal neurons of the colon in the early phases of synucleinopathies.Besides the colon, the skin, genitourinary tract, salivary glands, and heart are also involved [5].The skin is the most accessible organ among the peripheral tissues that have been investigated and can be used for both single and repetitive sampling.Furthermore, a skin biopsy is risk-free and minimally invasive.As a result, as various researchers have suggested, detecting and quantifying αSN deposition in skin biopsy samples may be useful for synucleinopathies [5].This article will explore the recent publications on cutaneous αSN deposition in synucleinopathies to define its significance as a prospective biomarker for these disorders.

Review Methods
We have done a thorough literature search in PubMed, Google Scholar, and ResearchGate.The medical subject headings (MeSH) terms and strategy used for PubMed search were (("alpha-synuclein"[MeSh] AND "synucleinopathies"[MeSh]) AND ("biopsy"[MeSh] OR "pathology"[MeSh] )) AND "skin" [MeSh].All articles published in PubMed in English since 2008 are included in the search criteria.For other databases, keywords used were synucleinopathies, Parkinson's disease, REM sleep behavioral disorder, multiple system atrophy, dementia with Lewy bodies, pure autonomic failure, and skin biopsy.

Discussion
After describing the importance of α-synuclein in the pathogenesis of synucleinopathies, we will discuss the role of skin biopsy in diagnosing these disorders.

Role of α-Synuclein
One of the important molecules in developing synucleinopathies is αSN.It is a classic protein found in soluble cytosolic fractions of the brain and primarily at presynaptic terminals [5].Recent research suggests that it cooperates with the cysteine-string protein, which has a characteristic domain for heat shock protein (HSP) 40-type molecular cochaperones, to play an important role in synaptic functions [6].In its natural (or native) state, αSN affects presynaptic signaling, membrane trafficking, and neuronal membrane integrity [7].Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions are pathological markers for the misfolding of αSN.This misfolding causes it to polymerize into fibrils and accumulate throughout the nervous system due to environmental factors like neurotoxins, low pH, high temperature, and genetic mutations [5].
Alpha-synuclein within Lewy bodies goes through different posttranslational modifications, for example, phosphorylation, cross-linking, or ubiquitination.These modifications may cause the αSN to aggregate and contribute to disease pathogenesis in vivo [8,9].When phosphorylated at the serine 129 residue phosphorylated alpha-synuclein (p-αSN)), it leads to αSN aggregation, which has increased toxicity and leads to the development of PD [9].Wang et al. (2011) reported novel methods for studying cutaneous autonomic innervation in skin biopsies from patients with peripheral nerve disease [10,11].Based on the prominent autonomic manifestations of Parkinson's disease, Wang et al. (2013) hypothesized that αSN deposition would be elevated in cutaneous structures with autonomic innervation [12].They concluded that in PD patients, αSN deposition increases in cutaneous sympathetic adrenergic and sympathetic cholinergic fibers but not in the sensory fibers.Higher αSN deposition is linked to more autonomic dysfunction and advanced PD.These findings suggest that measuring αSN deposition in cutaneous autonomic nerves could be a useful biomarker in Parkinson's disease patients [12].Figure 1 shows a photomicrograph of a double immunostained skin biopsy [13].

Studies Evaluating Skin Biopsies
Ikemura et al. obtained the first impressive outcomes on using p-αSN detection in skin biopsies as a biomarker.This study investigated the samples from autopsies and showed 70% sensitivity and 100% specificity for patients with PD compared to patients without central nervous system (CNS) Lewy body pathology [14].Miki et al. found a very low sensitivity (10%) in biopsy samples of living patients with PD [15].In the last decade, more studies have determined 100% specificity and 55% to 100% sensitivity compared to controls [16,17].Table 1 summarizes the studies done since 2010 using living subjects' skin biopsies to detect dermal p-αSN [4,12,.

Effect of Fixation and Staining Techniques on the Sensitivity of the Test
Table 1 shows that some studies detected dermal αSN in skin biopsies and demonstrated very low sensitivities for this diagnostic tool [20,21].Discussing potential causes, including using various biopsy protocols, fixation, immunostaining, and neuropathological evaluation, revealed the need for methodological studies comparing various protocols [17,45].Only a few studies compare fixation and staining techniques [17,45] despite the successful demonstration of the inter-and intra-laboratory reproducibility of skin section analysis [46].An improved protocol for paraffin sections was developed recently in the Systemic Synuclein Sampling Study (S4).This study used paraffin-embedded samples and reported skin biopsies to be 24.1% sensitive.This sensitivity is significantly lower than determined in previous studies.The highest sensitivity, however, was primarily reported in studies using cryosections when examining published studies more closely (Table 1).Unfortunately, formalin-fixed paraffin-embedded tissue was the only type of biopsy procedure systematically compared in the S4 study, even though it appears to be more practical in clinical practice [47]; this could lead to lower sensitivity (Table 1).
However, a recent study showed 70% sensitivity in PD, proving that moderate to high sensitivity is achievable when using paraffin sections [33].This study used double-staining with an axonal marker and protease anine phosphatase pretreatment.It also analyzed more sections in cases with a fewer number of positive fibers, all of which could potentially lead to increased sensitivity [33].

Effect of Sample Thickness on Sensitivity of the Test
In various studies, the detection rate of cutaneous p-αSN in patients with PD ranged from 30% to 100% [44].Therefore, Wang et al. (2020) hypothesized that these variations happen due to variations in the thickness of the tissue sections used for testing [44].So, in this study, the samples were cut into 10-, 20-, and 50-µmthick sections.50 µm double-immunostained skin biopsy tissue sections outperformed 20 and 10 µm in detecting p-αSN in PD patients.This result can be attributed to increased tissue volume for analysis and better visualization of nerve fiber architecture [44].

Selecting the Ideal Biopsy Location
The best biopsy site selection is another factor that might directly impact sensitivity.The leg and the C7 and C8 paravertebral regions were the sites selected in most studies (Table 1).However, only a few extensive studies systematically compare various biopsy sites.In PD, proximal locations may be involved more than distal ones [27,36,37].However, MSA affects the distal locations more than the proximal ones [27].The distribution of p-αSN in MSA may differ from that in PD, but larger studies are required [13].

Specificity of Dermal P-αSN as a Diagnostic Marker
Studies that used p-αSN-specific antibodies reported a specificity of 100% compared to controls, indisputably [34,36,37].Discrete p-αSN was only found in patient samples.Diffuse or granular p-αSN staining was reported in controls, possibly because no alkaline phosphatase pretreatment was done.[33].Wang et al. (2013) reported that skin biopsies from patients with PD had higher immunoreactivity when using an antibody against native αSN than controls.However, native αSN was also found in dermal nerve fibers from healthy subjects [12].In other studies, native αSN was similarly found in the dermal annexes' innervation of patients with synucleinopathies and controls [16,27].Contrarily, it has also been reported that using protein K digestion or antibodies directed specifically against aggregated αSN (5G4) can distinguish between biopsies from patients with PD and controls [30,32,47].
Dermal p-αSN deposition, on the other hand, is not a specific marker of idiopathic PD and has been observed in other synucleinopathies.Phosphorylated alpha-synuclein was found primarily in somatosensory nerve fibers in MSA, as opposed to autonomic fibers in idiopathic PD [27].Phosphorylated alpha-synuclein is frequently found in dermal autonomic nerve fibers in patients with PAF and DLB [16,24].

Feasibility of Skin Biopsy as a Diagnostic Tool
The usability of skin biopsy in diagnosing idiopathic PD is frequently debated.It is less expensive and widely available than iodine-123-radiolabeled 2β-carbomethoxy-3β-4-iodophenyl-N-3-fluoropropyl nortropane with single-photon emission computed tomography (FP-CIT-SPECT).A skin biopsy is easier than endoscopic gastrointestinal and salivary gland biopsies; patients generally tolerate it well [48].However, laboratory equipment is required for biopsies' processing and cryoconservation.Serial sections and analyses of multiple biopsy sites are needed due to the low number of p-αSN deposits, which is time-consuming.Evaluating skin sections under the microscope requires experienced examiners and takes time, too [47].
As a result, we need high-throughput analyzing techniques that can easily be repeated on many samples in a short time.The most promising approach is real-time quaking-induced conversion (RT-QuIC), an aggregation assay created to detect prions in Creutzfeldt-Jakob disease [49].Because alpha-synuclein contains prion-like seeding activity, small amounts of dermal αSN can be detected using RT-QuIC [49].
Several studies [49][50][51] found αSN aggregates in patients with idiopathic PD, dementia with Lewy bodies, and idiopathic RBD in the cerebrospinal fluid.This detection method has shown promising results on skin tissues [40,52].We need more extensive studies with a larger sample size to fully analyze the feasibility of dermal RT-QuIC in assessing idiopathic PD.
A recent study compared immunofluorescence with RT-QuIC and found that both have high diagnostic accuracy.This study concluded that immunofluorescence has optimal reproducibility compared to RT-QuIC [42].This review provides an update on the studies done over the last decade to detect p-αSN in skin biopsies for diagnosing synucleinopathies.These studies are done on limited subjects; therefore, more studies with large sample sizes and standardized methodological protocols are needed to provide an accurate answer to the sensitivity, specificity, accuracy, and precision of skin biopsy as a diagnostic marker for synucleinopathies.

Conclusions
The discovery of p-αSN in skin biopsies from patients with synucleinopathies occurred more than 10 years ago.Several studies have confirmed that autonomic nerve fibers are involved in αSN pathology, and they can be detected in the nerve fibers with high sensitivity and specificity even in the early stages of the disease.Most of these studies were performed on Parkinson's disease patients.Current challenges are that serial skin biopsy sections must be used and stained with an immunofluorescence staining technique.
Multiple skin sites must be biopsied to gain higher sensitivity on the test.All the studies have used different fixation, embedding, and sectioning techniques.Larger studies comparing these different techniques can help develop a more standardized protocol for skin biopsy to be used as a reliable biomarker for detecting synucleinopathies.
Reprinted from Journal of Parkinson's Disease, vol.11, Kathrin Doppler, Detection of Dermal Alpha-Synuclein Deposits as a Biomarker for Parkinson's Disease, pp.937-947, Copyright 2021, with permission from IOS Press.