Search for Molecular Biomarkers of Parkinson’s Disease. New Tissues and Methods
Pubblicato online: 20 apr 2023
Pagine: 15 - 22
Ricevuto: 10 feb 2023
Accettato: 08 mar 2023
DOI: https://doi.org/10.2478/acm-2023-0003
© 2023 Racay P., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Parkinson’s disease (PD) is the second most frequent chronic neurodegenerative disorder that is clinically manifested by motor and non-motor symptoms. At the pathological level, PD is characterised by the loss of dopaminergic neurons within the substantia nigra pars compacta (1) and the deposits of α-synuclein in a misfolded state that aggregates and forms intracellular inclusions within the cell bodies (Lewy bodies) and processes (Lewy neurites) of affected neurons (2). Other pathophysiologic features include mitochondrial dysfunction, impaired lysosomes or vesicle transport, impaired synaptic transport, and neuroinflammation (3). Finally, recent molecular genetic studies have revealed that genetic factors, in addition to aging and environmental factors, play an important role in the development of PD (4).
At the early stage of the disease, PD often presents similar clinical manifestations as multiple system atrophy (MSA) that is also categorized as α-synucleinopathy and often makes precise differentiation and diagnosis of these conditions difficult. PD can also be misdiagnosed with atypical conditions such as progressive supranuclear paralysis (PSP) due to overlapping clinical features. In addition, on the basis of converging results from clinical, neuropathological, and imaging research, it has been suggested that PD-specific pathology is initiated several years prior to the appearance of classical motor symptoms (5). This latent phase of neurodegeneration in PD characterised at cellular level by a preservation of significant fraction of dopaminergic neurones is of particular interest with respect to the development of disease-modifying or neuroprotective therapies which would require intervention at the earliest stages of the disease with an aim to slow down or reverse the disease progression. Therefore, there are two main challenges related to the molecular biomarkers of PD:
To find and validate a biomarker or set of biomarkers that would reliably differentiate PD from other neurologic diseases namely from atypical parkinsonian syndromes (APS), such as MSA, PSP, and corticobasal degeneration (CBD).
To identify and validate a biomarker or set of biomarkers that would reveal preclinical/prodromal stage of PD.
In addition, some effort is devoted to the identification of biomarkers that would monitor the efficiency of treatment and/or would be indicative for personalised treatment (6).
This short review is focused on the recent findings related to the validation of molecular biomarkers of PD with some focus on new methods of analysis or new analysed tissues. This review does not deal with biomarkers from a genetic point of view, which play a role in juvenile-onset and early-onset as well as late-onset adult PD caused by mutations in certain genes. Such mutations representing monogenically inherited PD are suitable biomarkers for an early detection of PD (4).
Alpha-synuclein is a small protein expressed at the nerve terminals where it is involved in the regulation of synaptic functions and neurotransmitter release (7). Its structure and function are extensively modulated by posttranslational modifications including phosphorylation as well as conformational changes that might result in the formation of protein aggregates and consequent death of neurones (8). Due to its central role in PD pathophysiology, α-synuclein represents the main protein biomarker related to PD (9). In terms of search for PD biomarkers, previous effort was focused on sensitivity and specificity of detection of either α-synuclein or α-synuclein phosphorylated at serine 129 in different tissues including gut, skin,
Recent meta-analysis showed a high degree of association between α-synuclein detected in biopsy samples from gastrointestinal tract (GIT) and PD (12). But it was also suggested that the measurement of GIT α-synuclein alone could result in the underdiagnosis of PD and combination GIT α-synuclein with other biochemical markers could increase specificity and sensitivity of examination (12). Another meta-analysis, comparing different α-synuclein antibodies as well as GIT and skin samples, has shown that antibodies against phosphorylated α-synuclein exhibit higher specificity than anti-native α-synuclein antibody (13). In addition, the same study revealed that the skin biopsy examination using antibodies against phosphorylated α-synuclein has the best diagnostic accuracy (13). With respect to the new methods, both RT-QuIC and PMCA assays of phosphorylated α-synuclein aggregation seeding activity in skin can differentiate synucleinopathies (PD, Lewy body dementia (LBD), and MSA) from non-synucleinopathies (Alzheimer disease, PSP, CBD, and non-neurodegenerative controls) with high sensitivity and specificity (14). Sensitivity and specificity of α-synuclein determination in skin biopsy as a potential diagnostic tool in PD as well as current methodological problems were summarised recently (15). Deposit patterns of either α-synuclein or phosphorylated α-synuclein in skin correlate well with clinical phenotypes in PD patients and can serve as a reference for the diagnosis and classification of PD (16).
Deposits of either α-synuclein or phosphorylated α-synuclein were found in CSF, however, a meta-analysis did not confirm a significant difference in total α-synuclein between patients with PD and other synucleinopathies or APS (17). The combinations of either total α-synuclein with NFL (18) or total α-synuclein with amyloid beta 1-42 peptide and NFL (19) could be promising approach for the differential diagnosis of PD and APSs.
Recent studies have revealed abnormal deposits of phosphorylated α-synuclein in both CSF (20) and dermal nerve fibres (21, 22) in persons with REM sleep behaviour disorder (RBD). Thus, phosphorylated α-synuclein seems to be a reliable candidate biomarker to screen for prodromal PD during enrolment in trials of disease-modifying, α-synuclein-based therapies (10). Deposits of α-synuclein can also be analysed in biopsy samples of other peripheral tissues, such as submandibular or minor salivary glands and submucosal enteral tissue, however, these biopsies are more invasive than skin biopsy (23, 24, 25).
With respect to invasiveness of sample collection, blood represents the most suitable tissue. Since α-synuclein is also expressed in erythrocytes (26), conflicting data about the level of α-synuclein in blood of PD patients were obtained (6). Detection of exosomal α-synuclein or determination of α-synuclein oligomers as well as α-synuclein phosphorylated at serine 129 re presents possibilities to increase sensitivity and specificity of blood α-synuclein as a biomarker for PD (8). Currently there is not a report focused on determination of blood α-synuclein seeding-capacity determined by either RT-QuIC or PMCA (10, 11). Saliva represent another body fluid that can be easily collected. Recent pilot study showed promising results indicating association between salivary α-synuclein seeding-capacity determined by RT-QuIC and PD (27).
Neurofilament light chain (NFL) is a structural protein that is expressed at high levels in axons, therefore NFL is a sensitive biomarker of axonal injury; however, its specificity is lower (28).
Recently performed meta-analysis suggested that the level of NFL in CSF could provide important information for the differential diagnosis of PD and APS, but NFL levels in CSF were found comparable between PD and control samples (19, 29).
Blood concentration of NFL is also increased in APS (30, 31, 32, 33) compared to PD. Determination of blood NFL has high potential to serve as biomarker with high accuracy levels to differentiate APS from PD, even in early stages of APS, when clinical symptoms are not yet conclusive (31, 32). Serum NFL concentrations correlate with age (30, 32, 33) in PD and controls, but not in APS (32). They seem to be higher in more advanced PD patients compared to controls, while controversial data were obtained for early disease stages (6). Heterogeneous data were obtained with respect to association of blood NFL concentrations with motor impairment in PD, revealing both positive (30, 34, 35) and negative results (36) while consistent positive association between baseline blood NFL levels and negative cognitive outcome have been reported (30, 36, 37).
Tyrosine hydroxylase (TH) as a key regulatory enzyme involved in dopamine synthesis was considered as a good marker of degeneration of dopaminergic neurones. Despite this fact, the utilisation of TH as a biomarker of PD was investigated in a few studies several decades ago. Recent studies unexpectedly revealed that monocytes isolated from blood of PD patients express significantly higher levels of TH in comparison to age-matched healthy controls (38). It was suggested that the dopaminergic machinery on peripheral immune cells displays an association with human PD, with some implications in facilitating diagnosis. Higher expression of TH in monocytes was attributed to high levels of tumour necrosis factor α (TNFα) (39). Therefore, diagnostic accuracy of monocyte TH is not yet clear, since other diseases characterised with higher TNFα expression like multiple sclerosis might be associated with higher expression of TH in monocytes.
MicroRNAs (miRNAs) are endogenous small (18–25 nucleotides), single-stranded, non-coding RNAs that are playing a key role in post-transcriptional regulation of gene expression. Recently, different types of miRNA are considered to play a role in pathophysiology of PD (40), therefore significant effort was focused on identification and validation of miRNAs that can serve as biomarker of PD.
One of the first study revealed that miR-195 was up-regulated, and miR-185, miR-15b, miR-221 and miR-181a were down-regulated in plasma of PD patients thus this set of five miRNAs can precisely distinguish PD patients from healthy individuals (41). Three plasma miRNAs (miR-671-5p, miR-19b-3p, and miR-24-3p) were found to be differently presented in MSA and PD having potential to become markers that could reflect the pathophysiology or symptoms of PD and MSA (42).
A group of three exosomal miRNAs (miR-21-3p, miR-22-3p, and miR-223-5p) discriminated PD from control while another group of exosomal miRNAs (miR-425-5p, miR-21-3p, and miR-199a-5p) discriminated PSP from PD with good diagnostic accuracy. A combination of the levels of six exosomal miRNAs (miR-21-3p, miR-199a-5p, miR-425-5p, miR-483-5p, miR-22-3p, and miR-29a-3p) discriminated PSP from PD with a relatively high sensitivity and specificity (43).
Marques and collaborators have identified two miRNAs in CSF (miR-24 and miR-205) that accurately discriminated PD from controls and four miRNAs (miR-19a, miR-19b, miR-24, and miR-34c) that differentiated MSA from controls (44).
Groups of three microRNAs in CSF discriminated PD (miR-7-5p, miR-331-5p, and miR-145-5p) and MSA (miR-7-5p, miR-34c-3p, and miR-let-7b-5p) from controls with good diagnostic accuracy. The combination that best distinguished MSA and PD consisted of two microRNAs (miR-9-3p and miR-106b-5p) while a single microRNA in the CSF (miR-106b-5p) exhibited the best discrimination between PD and PSP (45).
Since the panel of several miRNAs can be easily analysed via specific miRNA qPCR array, the determination of target miRNAs represents a promising diagnostic approach.
Mitochondrial dysfunction is often implicated as an early event in pathophysiology of PD although the cause-consequence relationships are not fully clear (46). Mitochondrial dysfunction is often a result of endoplasmic reticulum (ER) stress (47) that is also suggested to be involved in the pathophysiology of PD. Unlike molecular determinants of mitochondrial dysfunction, alterations of some critical molecular components of ER stress pathways were explicitly detected in brains of PD patients (48, 49) previously and ER stress-regulated proteins were recently suggested as blood biomarkers to confirm the diagnosis of PD (50). Recent study has identified several proteins associated with mitochondrial functions that are significantly altered in brains of PD patients (46) but their use as biomarkers for PD will require additional experiments and validations.
Despite some critical view (51), the content of mtDNA in leukocytes could reflect the relative content of mitochondria in the affected peripheral tissue. For example, in the largest investigation of mtDNA copy number associated with PD, the reduced mtDNA copy number was documented exclusively in the substantia nigra pars compacta of PD patients but not in other brain regions. The same study has shown that the reduced mtDNA documented in brain was also reflected by the decreased mtDNA copy number in the peripheral blood cells (52). However, the decreased mtDNA in leukocytes was also documented in other diseases such as Alzheimer’s disease (53), bipolar disorder (54), and schizophrenia (55). Finally, the decreased mtDNA copy number could be a useful biomarker of mitochondrial dysfunction associated with a number of different pathological conditions including PD (56). Although, the reduction in mtDNA copy number found in substantia nigra dopaminergic neurons of idiopathic PD patients was related to the dysfunction of mitochondrial respiratory chain (57), the recent pilot study has documented increased both maximal respiration and respiratory capacity in leukocytes of PD patients (58). Thus, the potential of PD biomarker based on mitochondria seems to be questioning.
In addition to the above discussed molecules, some other molecules were investigated as potential biomarkers of PD including inflammatory cytokines, amyloid peptide, and tau protein (6). With respect to the high throughput methods, OMICS approach involving metabolomics (59) or proteomics (60) were investigated but their clinical use is hampered due to technologic and financial demands. Apart from molecular biomarkers, methods/protocols based on tissue imaging (e.g. fMRI, PET), allowing functional examination of brains of PD patients, are investigated in terms of differential diagnosis (61, 62).
Despite strong effort, a reliable biomarker that exhibit a high sensitivity and specificity for diagnostic and/or therapeutic purposes in PD does not currently exist. Such situation is probably a result of the high heterogeneity of PD caused by both phenotype and genotype or epigenetic factors. Further analyses of molecules in larger cohorts and in different samples are required for identification and validation of useful and reliable clinical biomarkers. In order to increase discrimination power, a combination of different biomarkers or a combination of molecular methods with imaging methods represents the possibility how to increase accuracy of PD diagnosis and management.