3.1 Neuropathologic features of LBD
In most cases of LBD without cognitive deficits, the macroscopic findings are comparable to age- and sex-matched controls, except for loss of neuromelanin pigment in the substantia nigra and locus coeruleus (Fig. 2). Dopaminergic neuronal loss in the substantia nigra, particularly in the ventrolateral part, is a pathologic hallmark of PD [86, 87]. The severity of neurodegeneration of the substantia nigra correlates with severity of extrapyramidal motor symptoms and the degree of striatal dopaminergic deficiency [88, 89]. Neuronal loss is moderate to marked in PD and PDD, but more variable in DLB. In fact, a subset of DLB patients lack parkinsonism and have preserved neuronal population in the substantia nigra. The locus coeruleus is a major noradrenergic nucleus, and neuronal loss leads to deficiency of noradrenaline, which may contributes to various symptoms, including cognitive impairment, affective symptoms, RBD, and gait difficulties [90].
Lewy bodies are round, eosinophilic inclusions in neuronal perikarya. There are two types of Lewy bodies: classical (or brainstem) type and cortical type. Classical-type Lewy bodies have a dense hyaline appearance with a peripheral clear halo and are easily visible on hematoxylin and eosin (H&E) stained sections (Fig. 3). Cortical type Lewy bodies have a less compact appearance and are more difficult to detect with histologic methods (Fig. 3) [5]. Both types of Lewy bodies are strongly immunoreactive with antibodies against α-synuclein, especially antibodies targeting phospho-Ser129 (Fig. 3) [53]. In addition to α-synuclein, more than 90 components of Lewy bodies have been reported, based mostly upon immunohistochemical colocalization with brainstem type Lewy bodies, including sequestration of neurotransmitter enzymes of cholinergic and dopaminergic neurons [91–93]. Accumulation of phosphorylated α-synuclein also occurs within cell processes (mostly axonal), so-called Lewy neurites (Fig. 3). Lewy neurites in the CA2/3 sectors of the hippocampus (Fig. 3) are a characteristic histopathologic finding in many cases of PD and most cases of PDD and DLB [94]. Deposits of phosphorylated α-synuclein are observed less frequently in oligodendroglia, and rarely in astrocytes in the midbrain and basal ganglia [95].
In addition to Lewy bodies and neuronal loss in the substantia nigra, a subset of cases have spongiform change or neuropil microvacuolation that is often most severe in the amygdala, but also seen in limbic and superior temporal cortices (Fig. 3) [96–98]. The most important co-pathology in LBD is Alzheimer-type pathology. In initial reports of DLBD, the main neuropathologic features included not only numerous cortical Lewy bodies, but also numerous senile plaques and neurofibrillary tangles in the cerebral cortex [6]. The majority of LBD cases, not exclusively DLBD, have some degree of Alzheimer-type pathology (i.e., neocortical senile plaques and neurofibrillary tangles) [99–102]. Senile plaques in the cerebral cortex are common, and they often are characterized by non-neuritic diffuse amyloid deposits [103]. Of note, 28% of DLB and 10% of PDD cases have sufficient pathology for a secondary neuropathologic diagnosis of AD [104].
Lewy bodies are also found frequently in cases with advanced AD, particularly in the amygdala [105–108]. Hamilton screened α-synuclein pathology in 145 cases of AD and found that 88 cases (61%) had LBD [106]. The amygdala was the most affected region; Lewy bodies were present in all cases of AD with LBD. Interestingly, some of the cases had numerous Lewy bodies in the amygdala but rare or absent in the brainstem in some cases (amygdala-predominant Lewy bodies [ALB]). Uchikado and colleagues also screened Lewy-related pathology in 347 cases of AD and found that 62 cases (18%) were consistent with ALB [108]. Of those, Lewy bodies were only found in the amygdala (“amygdala-only” LBD) in 32 cases (9%). The clinical significance of ALB in AD remains uncertain [109], but evidence suggests that they may be associated with increased frequency of visual hallucinations compared to AD without amygdala Lewy bodies [110].
Lewy bodies are widely distributed not only in the central nervous system but also in the peripheral autonomic nervous system, including the nerve terminals and autonomic ganglia in the heart, submandibular glands, enteric nervous system, adrenal glands, skin, and cutaneous nerve [111–118]. Interestingly, phosphorylated-α-synuclein deposits in cutaneous autonomic nerves can be detected by immunohistochemistry in 56–82% of patients with IRBD, a prodromal phase of synucleinopathies [119–123]. The presence of α-synuclein was associated with greater autonomic dysfunction in IRBD [122]. A biopsy taken from these peripheral tissues might be a feasible biomarker in the prodromal phase of synucleinopathies, and increasingly sensitive and specific methods to detect abnormal α-synuclein in peripheral tissues (e.g., real-time quaking-induced conversion [RT-QuIC] or protein misfolding cyclic amplification [PMCA], see below) offer hope for peripheral biomarkers for the disease. [124].
3.2 Classification of LBD
The term “Lewy body disease” was coined by Kosaka and colleagues to refer to neurodegenerative diseases with numerous Lewy bodies in the central nervous system [20]. He classified LBD into three groups: groups A, B, and C, which were later named diffuse type (DLBD), transitional type (TLBD), and brainstem type (BLBD) [125]. BLBD corresponded to PD in their scheme, and DLBD was thought to be an extension of PD pathology into the limbic lobe and the neocortex. DLBD was separated into two forms: a common form and pure form. The common form had not only numerous Lewy bodies, including neocortical Lewy bodies, but also many senile plaques and variable neurofibrillary tangles. In contrast, the pure form had few or no Alzheimer-type changes [99]. Kosaka later added a “cerebral type” of LBD, which had numerous Lewy bodies in the cerebral cortex and amygdala, but minimal or no Lewy bodies in the brainstem and diencephalon [126].
The First International Consortium for Lewy Body Dementia (ICDLB) proposed criteria for clinical and pathologic diagnosis of DLB [7]. Subtypes of Lewy-related pathology were categorized based upon the severity and topographical distribution of Lewy bodies [127]: diffuse neocortical, limbic (transitional), and brainstem-predominant, which corresponded roughly to Kosaka’s classification of DLBD, TLBD, and BLBD. The Third ICDLB report developed a diagnostic scheme that was devised to predict likelihood that the pathology would be associated with DLB. The criteria took into account both the extent of Lewy-related pathology and Alzheimer’s-type pathology to assign a probability that the pathology would be associated with the clinical presentation (Table 1). The severity of Lewy-related pathology was semi-quantitatively assessed on a five-scale: 0 = none, 1 = mild, 2 = moderate, 3 = severe, and 4 = very severe. Recommended brain regions for assessment included the dorsal motor nucleus of vagus, locus coeruleus, and substantia nigra in the brainstem regions, nucleus basalis of Meynert, amygdala, transentorhinal cortex, and cingulate cortex in the limbic regions, and temporal, frontal, and parietal cortices (Fig. 4). The likelihood of DLB clinical was directly related to severity of Lewy body pathology and indirectly related to severity of Alzheimer pathology. It was recognized from studies of prospective cohorts that when Alzheimer pathology was severe, most patients had Alzheimer type dementia, rather than DLB [128]. A recent study validates this approach in that the diagnostic sensitivity for probable DLB was significantly higher in TLBD and DLBD without neocortical tangles than in those with neocortical tangles [129]. These findings indicate that the phenotypic expression of DLB is associated directly related to the extent of Lewy bodies and inversely related to the extent of neurofibrillary tangles [129].
Table 1
Likelihood of a typical clinical presentation of dementia with Lewy bodies
Lewy-related pathology | ADNC based on NAI-AA |
None/low | Intermediate | High |
Diffuse neocortical | High | High | Intermediate |
Limbic (transitional) | High | Intermediate | Low |
Brainstem-predominant | Low | Low | Low |
Amygdala-predominant | Low | Low | Low |
Olfactory bulb only | Low | Low | Low |
Abbreviations: ADNC, Alzheimer’s disease neuropathological change; NIA-AA, National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer disease. |
Table 2
Studies on α-synuclein RT-QuIC using CSF
Author | Autopsy | Disease | N | N Control | Sensitivity | Specificity |
Fairfoul [209] | Yes | Pure DLB | 12 | 20 | 92% | 100% |
DLB with AD | 17 | 20 | 65% | 100% |
AD with ILBD | 13 | 20 | 15% | 100% |
PD | 2 | 20 | 100% | 100% |
No | PD | 20 | 15 | 95% | 100% |
IRBD | 3 | 15 | 100% | 100% |
Manne [220] | No | PD | 15 | 11 | 94% | 100% |
Shahnawaz [210] | No | PD | 76 | 65 | 88% | 94% |
DLB | 10 | 65 | 100% | 94% |
MSA | 10 | 65 | 80% | 94% |
Groveman [211] | Yes | PD | 12 | 31 | 92% | 100% |
DLB | 17 | 32 | 94% | 100% |
Bongianni [212] | Yes | DLB | 7 | 49 | 100% | 96% |
MSA | 1 | 50 | 100% | 96% |
mixed LBD *2 | 20 | 51 | 90% | 96% |
No | DLB | 26 | 10 | 65% | 100% |
Kang [213] | No | PD | 105 | 79 | 96% | 82% |
Van Rumund [214] | Yes *1 | PD | 53 | 52 | 84% | 98% |
MSA | 17 | 52 | 35% | 98% |
DLB | 1 | 52 | 100% | 98% |
Garrido [215] | No | PD | 10 | 10 | 90% | 80% |
LRRK2-PD | 15 | 10 | 40% | 80% |
LRRK2-NMC | 16 | 10 | 19% | 80% |
Rossi [216] | Yes | DLB | 14 | 101 | 100% | 98% |
mixed LBD *3 | 7 | 102 | 86% | 98% |
No | DLB | 34 | 166 | 97% | 94% |
PD | 71 | 167 | 94% | 94% |
IRBD | 18 | 168 | 100% | 94% |
PAF | 28 | 169 | 93% | 94% |
Iranzo [217] | No | IRBD | 52 | 40 | 90% | 90% |
Shahnawaz [218] | No | PD | 94 | 56 | 94% | 100% |
MSA | 75 | 56 | 85% | 100% |
PD vs MSA | 88 | 65 *4 | 97% | 94% |
Rossi [219] | No | MCI-LB | 81 | 58 | 95% | 97% |
*1: 98% of cases are not autopsy-confirmed. *2 This includes LBD with AD (n = 15), LBD with PART (n = 2), and CJD with LBD (n = 3). *3 This includes CJD with DLB (n = 2), CJD with brainstem LBD (n = 3), and other primary diagnoses with limbic LBD (n = 1) or brainstem LBD (n = 1). *4 This number indicates MSA patients. The sensitivity and specificity are for PD against MSA. Abbreviations: AD, Alzheimer’s disease; CJD, Creutzfeldt-Jakob disease; DLB, dementia with Lewy bodies; ILBD, incidental Lewy body disease; IRBD, isolated rapid eye movement sleep behavior disorder; LRRK2, ; MCI, mild cognitive impairment; MSA, multiple system atrophy; PD, Parkinson’s disease; |
Braak and colleagues proposed a staging scheme for Lewy-related pathology in PD [130], which was anatomically more detailed and specified than Kosaka’s classification of LBD (Fig. 4). Lewy-related pathology initially occurred in the medulla oblongata (dorsal motor nucleus of vagus and the glossopharyngeal nucleus), and in the anterior olfactory nucleus of stage 1. In stage 2, α-synuclein pathology ascends to the pontine tegmentum, while stage 3 is associated with involvement of midbrain, stage 4 with limbic regions, and stages 5 and 6 with neocortical involvement. This staging scheme has largely been confirmed in several studies, but some exceptions have also been pointed out, such as cases with Lewy bodies restricted to the olfactory bulb or to the amygdala, particularly in cases with advanced AD pathology (i.e., ALB). [108, 131, 132].
The BrainNet Europe Consortium assessed 33 LBD cases by 22 pathologists and pointed out low inter-rater agreement (65%) for the Braak staging scheme. Only four cases of Braak stage 6 reached 100% agreement [133]. They proposed a new protocol to improve the inter-rater reliability by (1) selecting nine blocks for α-synuclein immunohistochemistry, (2) using a dichotomous approach to assess Lewy-related pathology (i.e., present vs. absent), and (3) incorporating an amygdala-predominant category (Fig. 4). This new protocol achieved a high inter-rater agreement for both Braak Lewy body stage, as well as assignment of brainstem, limbic, neocortical, and amygdala-predominant categories.
Beach and colleagues applied Braak Lewy body staging to 216 cases of LBD, and found about half of their cases were unclassifiable [132]. About two-thirds of unclassifiable cases had limbic system involvement without significant brainstem pathology, and the remaining one-third had Lewy-related pathology only in the olfactory bulb. Based on their findings, they devised a staging scheme to include LBD confined to the olfactory bulb and limbic predominant cases with variable or no brainstem involvement [132]. Given that the olfactory bulb is often initially affected in both ILBD and ALB, they defined cases of having Lewy-related pathology confined to the olfactory bulb as stage I. Stage II was divided along two branches: brainstem predominant (stage IIa) and limbic predominant (stage IIb). In the majority of cases, Lewy-related pathology passed through the brainstem stage prior to the limbic stage (stage IIa), while most cases of ALB showed abundant Lewy-pathology in the limbic system, particularly in the amygdala, before affecting the brainstem. Following the two pathways of stage II, both the brainstem and limbic systems converged at stage III, and the neocortical regions are affected in stage IV. Subsequently, Adler and colleagues applied this staging system to 280 cases of LBD and found that parkinsonism, cognitive impairment, hyposmia, and RBD were significantly correlated with increasing stage [134]. Based on these various schemes, the ICDLB criteria were revised in 2017 to include amygdala-predominant and olfactory bulb only types (Fig. 4) [28].
Semi-quantitative evaluation has been used for diagnosing and classifying neurodegenerative disorders, but there are inherent weaknesses in semi-quantitative measures when it comes to inter-rater reliability [135]. Braak Lewy body stages, ICDLB criteria, and other staging schemes have been widely used, but not all cases fit well into the specified stages, or they have features that overlap between more than one stage. Inter-rater agreement is not optimal. Recently, Attems and colleagues devised neuropathological consensus criteria for Lewy pathology, which they named Lewy pathology consensus criteria [136]. This new system is based on the ICDLB criteria modified to use a dichotomous approach for assessing Lewy-related pathology (i.e., present vs. absent), rather than semi-quantitative scores. In this scheme, a case that had very sparse Lewy bodies or Lewy neurites (i.e., only one Lewy neurites in a neocortical section) without Lewy-related pathology in other brain regions, such as medial temporal lobe, would be still assigned neocortical subtype. Nevertheless, in the select and relatively small study cohort, all cases with neocortical Lewy pathology had dementia. They also demonstrated that all cases were successfully classified and that there was high inter-rater reliability. In parallel they assessed other criteria on the same sections, and up to 30% were unclassifiable with Braak staging or ICDLB criteria. Although this simple approach for Lewy pathology classification might be useful in routine diagnostic practice, clinicopathologic correlations of this classification, particularly neocortical type, need to be validated. They need to be compared critically to ICDLB neuropathologic criteria, which have been shown to “predict” the DLB clinical syndrome [137, 138].
3.3. Underlying pathology of PD, PDD, and DLB
PD, PDD, and DLB are considered distinct clinical entities that have varying degrees and distributions of Lewy body pathology. PD and PDD are disorders associated extrapyramidal clinical syndrome of parkinsonism, which correlates less well with distribution of Lewy bodies, and better with nigrostriatal dopaminergic deficiency. Given this fact, ICDLB criteria were proposed to capture this important clinical correlate, namely, moderate-to-severe neuronal loss in the ventrolateral substantia nigra [86]. The specification of the ventrolateral region was driven by the fact that ventrolateral dopaminergic neurons are the origin of the nigro-striatal dopaminergic pathway (projecting to putamen and caudate nucleus), while ventromedial substantia nigra dopaminergic neurons are the origin of the mesolimbic pathway (projecting to the ventral striatum and nucleus accumbens).
While most patients with PD have BLBD (or TLBD) at autopsy, a “pure form” of DLBD can also be found in a few patients with PD [99, 139]. In contrast, most patients with PDD and DLB have DLBD. It has proven challenging to differentiate DLB from PDD based on the neuropathologic findings alone [104]. Although DLB patients tend to have more severe Alzheimer-type pathology, and PDD patients tend to have more severe neuronal loss in the substantia nigra [140], there are no clear neuropathologic distinctions between DLB and PDD [141]. A clinical diagnosis of PDD or DLB requires information about not only about the nature of clinical findings, but also the timing of dementia relative to motor signs and symptoms. Patients with PD who develop dementia later in the disease course (“more than one-year” after onset of parkinsonism) are considered to have PDD. On the other hand, patients who develop dementia, with (or without) parkinsonism after one year of other symptoms are diagnosed with DLB [28].Some patients with DLB have minimal or no parkinsonism, but they have other typical features of DLB including progressive cognitive impairment, RBD, fluctuations in level of consciousness, spontaneous visual hallucinations, delusions, and misidentifications (e.g., Capgras syndrome) [142].
3.4 Neuropathologic features of MSA
Macroscopic findings in MSA vary with the subtype of MSA. In MSA-P there is loss of neuromelanin pigment in the ventrolateral substantia, as well as atrophy and discoloration of the posterolateral putamen. In contrast, MSA-C has atrophy of the pontine base and the middle cerebellar peduncle with attenuation and discoloration of the cerebellar white matter (Fig. 5). The neocortex and limbic structures are usually macroscopically unremarkable.
System-specific neuronal loss and gliosis are observed in both striatonigral and olivopontocerebellar systems (Fig. 6). In addition to these macroscopically affected brain regions, immunohistochemistry for α-synuclein reveals more widespread α-synuclein pathology, characterized by GCI and variable NCI. GCI are argyrophilic inclusions (Gallyas silver stain positive) in the cytoplasm of oligodendrocytes [10]. They are visible on routine H&E stains, but immunohistochemistry for phosphorylated α-synuclein is far more sensitive for visualization of GCI [16]. The density of GCI correlates with both neuronal loss and disease duration [80]. The neuropathologic diagnostic criteria for MSA require “widespread and abundant GCI in association with neurodegenerative changes in striatonigral or olivopontocerebellar structures” for the definite diagnosis of MSA. Thus, GCI is the pathologic hallmark of MSA [143]. In addition to accumulation of α-synuclein in the cytoplasm of NCI and dystrophic neurites, many affected neurons also have intranuclear α-synuclein inclusions [18]. The distribution of NCIs is distinct from that of Lewy bodies, and they are most often observed in the putamen, pontine nuclei, and inferior olivary nuclei, which are not susceptible to Lewy bodies. NCI can also be observed in the substantia nigra, cingulate cortex, amygdala, hippocampus, entorhinal cortex, hypothalamus, and neocortex [144, 145]. Some MSA cases may have concurrent LBD [137]. In such cases, Gallyas-Braak silver staining is helpful in distinguishing NCI from Lewy bodies; the former are positive in Gallyas-Braak silver staining, but the latter are not [146]. The clinicopathologic significance of NCIs has been reviewed by Cykowski and colleagues in a large cohort of MSA cases. They found that presence of NCI in neocortex was associated with cognitive impairment [145]. Other studies have reported that the burden of NCI is associated with cognitive impairment or memory loss in MSA [37, 38, 147].
3.5 Subtypes of MSA
MSA has been divided into two major pathologic subtypes: SND and OPCA, although virtually all cases had microscopic involvement in both systems. Wenning and colleagues proposed a grading system for SND based upon semi-quantitative assessment of atrophy, neuronal loss, astrogliosis, and GCI: In this scheme, Grade 1 = has neuronal loss confined to the substantia nigra; Grade 2 = has neuronal loss extending to the putamen; and Grade 3 = has involvement of the caudate nucleus and globus pallidus [148]. Ozawa and colleagues proposed three grades of SND and OPCA based upon semi-quantitative assessment of neuronal loss in regions of interest: putamen, globus pallidus, and substantia nigra for SND; pontine nuclei, cerebellar hemisphere and vermis, inferior olivary nucleus, and substantia nigra for OPCA [80]. This classification showed good correlation with clinical features; patients with SND had more severe bradykinesia, and those with OPCA had more frequent cerebellar signs. Jellinger and colleagues extended the Wenning grading system of SND for both SND and OPCA to assign MSA-P and MSA-C [149], which correlated with initial symptoms and clinical features of both subtypes [149].
Although the diagnostic criteria for MSA require neurodegeneration in striatonigral or olivopontocerebellar systems, or both, some MSA cases do not have significant neurodegeneration in even though they have widespread GCI. This rare subtype is referred to as “minimal change” MSA [150–154]. Cases of minimal change MSA suggest that GCI formation precedes neuronal loss. Clinical presentations of minimal change MSA vary. Some patients are asymptomatic (“preclinical MSA”) [151, 154], but in a case series from the UK, all minimal change MSA patients had respiratory dysfunction and early orthostatic hypotension [152]. Rarely, minimal change MSA has been reported with limbic-predominant distribution of α-synuclein pathology [153].
MSA was previously considered three distinct disorders. Clinical presentations of each disorder have been considered typical features of MSA (i.e., autonomic dysfunction [Shy-Drager syndrome], parkinsonism [MSA-P], and cerebellar ataxia [MSA-C]). Several case series, however, have reported atypical clinical presentations of MSA [155–158]. Aoki and colleagues coined the term “frontotemporal lobar degeneration (FTLD)-synuclein” for a rare subtype of MSA, based on a series of four patients with atypical MSA [159]. These patients had clinical features consistent with an FTLD, including corticobasal syndrome, progressive nonfluent aphasia, or behavioral variant frontotemporal dementia. Neuropathologic assessment revealed widespread GCI with striatonigral degeneration consistent with MSA. Furthermore, abundant NCI, including Pick body-like inclusions, and severe neuronal loss were observed in limbic structures and the neocortex [159].
More recently, Ando and colleagues reported that 12 of 146 (8%) of MSA had abundant NCI in the hippocampus and parahippocampal gyrus (“hippocampal MSA”). Severe neuronal loss with gliosis in the hippocampus and the medial temporal atrophy were also observed. Patients with hippocampal MSA had short disease duration and higher prevalence of cognitive impairment compared to typical MSA, but they lacked clinical features of FTLD-synuclein [160]. Despite differences in clinical presentations, hippocampal MSA and FTLD-synuclein share pathologic features; therefore, a subset of MSA may have a vulnerability to the limbic structures, and these subtypes can be within the same spectrum of this limbic-predominant MSA.
3.6 α-Synuclein oligomers
α-Synuclein exists as a monomer, but as aggregation proceeds, it forms oligomeric species and then fibrils [161]. Mounting evidence suggests that α-synuclein oligomers are toxic [162–164], and that they interact with lipids and disrupt cellular membranes, resulting in aberrant ion fluxes and neuronal toxicity [163, 164]. α-Synuclein oligomers can also generate reactive oxygen species, leading to oxidation of lipids in membranes and mitochondrial proteins, which can culminate in cell death [165, 166]. It is not possible to confidently detect α-synuclein oligomers with routine histologic methods, but Roberts and colleagues applied proximity ligation assay (PLA) methods to detect α-synuclein oligomers in histologic sections [167]. The PLA technique was originally developed to increase sensitivity for protein detection and it also has been applied to detect protein-protein interactions [168, 169]. In this method, combination of specific antibody binding to antigen and amplification of signal with polymerase chain reaction enables in situ detection of specific antigens.
The mechanism of homotypic PLA for α-synuclein oligomers is shown in Fig. 7. Two forms of oligonucleotides are linked to α-synuclein antibodies, which interact at a close distance only when α-synuclein oligomerizes. The oligonucleotides are joined by ligase and serve as templates for formation of circular DNA, which serves as a template in rolling-circle amplification to produce thousands of single-stranded products at the site of the oligomers. The oligonucleotides are finally labeled with horseradish peroxidase to produce at the site of the oligomers.
Using this emerging technique, Roberts and colleagues showed α-synuclein oligomers in the cingulate cortex and reticular formation of the medulla in PD [167]. They also detected α-synuclein oligomers in morphologically intact neurons and in the periphery of Lewy bodies. Sekiya and colleagues studied PD and MSA brains and showed widespread and abundant α-synuclein oligomers, especially in cortical neurons and Purkinje cells of MSA (Fig. 7) [170]. They also found that most α-synuclein oligomers were localized in neurons and that α-synuclein oligomer accumulation progressed in neurons but in oligodendrocytes. This technique has also been used to detect α-synuclein oligomers in peripheral tissues, such as gastrointestinal or skin biopsies [171, 172]. Ruffmann and colleagues eluated gastrointestinal tissues with α-synuclein-PLA and found two staining patterns – cellular and diffuse [171]. Mazzetti and colleagues showed α-synuclein oligomers in skin biopsies of PD [172]. These results suggest that α-synuclein-PLA has potential as diagnostic biomarker. Homotypic PLA for α-synuclein oligomers also has been used to characterize α-synuclein animal experiments [173, 174], permitting detection of early pathological changes.