Toward Molecular Profiling of Parkinson's Disease in Easily Accessible Biological Matrices

Summary description Parkinson's disease (PD) is the most common neurodegenerative disease involving disabling motor deficits. PD is clinically heterogeneous, thus biomarkers reflecting different pathophysiological aspects, are needed for the stratification and selection of patients in clinical trials. In this project we aim at measuring biomarkers associated to different molecular pathways (e.g., synucleinopathy, neuroinflammation, neurodegeneration, amyloidosis and tauopathy) in different biological samples (cerebrospinal fluid, plasma, olfactory mucosa and skin) collected from a cohort of well-characterized patients affected by PD, other neurological/neurodegenerative diseases and controls. Among the samples considered, olfactory mucosa and plasma are easily accessible and thus suitable for repeated sampling in clinical trials. The different biomarkers will be associated with motor, neuropsychological and functional scores to understand their ability to predict different clinical outcomes.

Background / State of the art Parkinson's disease (PD) is a synucleinopathy and the most common neurodegenerative disease involving disabling motor deficits. PD is clinically heterogeneous; motor symptoms may be accompanied by non-motor symptoms such as cognitive impairment. Many molecular processes may underlie the phenotypic heterogeneity of PD, among which synaptic and axonal degeneration, neuroinflammation, and the co-occurrence of different proteinopathies are among the most characterized ones and those that have the best chance of being reliably reflected by currently existing biomarkers. In fact, the definition of robust biomarkers that reflect distinct pathophysiological pathways taking place in PD may favor the selection of more homogeneous cohorts of patients in clinical trials, thus increasing the chance of success of a disease modifying therapy that acts on one or more of these pathways. Moreover, the possibility of measuring these markers in biological matrices suitable for repeated sampling could provide objective measures of the effectiveness of the therapeutic approach. In this proposal we will combine the expertise of three different Italian medical research centers to verify the clinical utility of biomarkers reflecting different biological pathways, among which synucleinopathy, neuroinflammation, neurodegeneration, amyloidosis and tauopathy, in different biological samples by applying immunoassays, proximity extension assays (PEA) and seed amplification assays (SAA).

Description and distribution of activities of each operating unit The research group consists of three operating units (OU): Azienda Ospedaliera di Perugia (AOPG), Fondazione IRCCS Istituto Neurologico Carlo Besta (FINCB) and Centro Neurolesi Bonino Pulejo (CNBP). Each OU will be responsible for the selection of retrospective biological samples and for the recruitment of patients and controls in the first year of the study. Two easily accessible biological samples, i.e. plasma and OM, have been/will be collected using standardized procedures by each OU from PD patients, controls (healthy subjects and patients affected by minor neurological disturbances, e.g., headache, psychiatric disorders, subjective memory complaints) and patients affected by Alzheimer's disease (AD) as other neurodegenerative disease controls. Skin biopsies and cerebrospinal fluid (CSF) paired to plasma and OM samples are being collected in PD patients and controls at FINCB and AOPG, respectively. The project will include a total number of 200 PD patients (AOPG n = 80, FINCB n = 80, CNBP n = 40), 100 controls (AOPG n = 40, FINCB n = 40, CNBP n = 10) and 40 AD (AOPG n = 40). All PD patients that will be recruited will undergo cognitive evaluation including (but not limited to) Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA), according to the Movement Disorder Society criteria for assessment of mild cognitive impairment in PD. The Movement Disorders Society Unified Parkinson's Disease Rating Scale (UPDRS) will be completed including the UPDRS-III examination, which will be used to determine motor severity in the "on" motor state. Also, motor severity of disease-related disability will be assessed by means of the Hoehn and Yahr scale (H\&Y). PD patients will also undergo smell tests before OM sampling. The control group will be constituted by healthy volunteers for OM and plasma collection as well as by cognitively unimpaired subjects affected by subjective memory complaints by minor neurological symptoms. The above-mentioned clinical information has been already collected for the retrospective cohort. AOPG will take care of the measurement of plasma amyloid-ß (Aß) 42/Aß40 ratio and phosphorylated at threonine 181 tau (p-tau) with the Lumipulse automated platform. In a subset of 80 PD patients and 40 AD, the CSF levels of this markers have been already measured and will be used to assess the correlation between their CSF and plasma levels. FINCB will perform SAAs analysis of OM and skin samples for the detection of prone to aggregation a-synuclein and immunohistochemical analysis of skin samples for the detection of a-synuclein phosphorylated at serine 129 (p-a-syn). FINCB has extensive expertise in SAA analysis of OM samples and IHC analysis of skin biopsies. Here, the center will extend SAA analysis to skin punch biopsies with the aim of comparing the diagnostic accuracy of p-a-syn with the corresponding SAA findings. In a subset of 30 PD and 30 AD/CTRL with paired OM and CSF samples the CSF SAA protocol of Amprion Inc. will be applied at AOPG to verify the concordance of the results between CSF and OM. CNBP will be specifically responsible for collecting clinical and biomarker data from the three centers and for assessing their relationships. For the sake of measurements uniformity, the PEA analyses will be instead centralized at Olink (Uppsala, Sweden), at which the Olink Explore 384 Inflammation and Olink Explore 384 Neurology panels will be measured in all plasma samples.

Specific Aims and Experimental Design

1. To determine a fingerprint of different clinical phenotypes of PD by combining biomarkers measured in OM and plasma.
2. To evaluate the correlation between clinical scores (smell tests, MMSE, MoCA, UPDRS-III) and measured biomarkers in plasma and OM.
3. To validate synucleinopathy, amyloidosis, tauopathy markers in paired OM, plasma, CSF and skin samples.

To accomplish the above-described aims 1, 2 and 3, the biomarkers that will be measured are:

• The ones measured in plasma by applying the Olink Explore 384 Inflammation and Neurology panels the people responsible for these analyses are Dr. Rossella Ciurleo and Dr. Santina Caliri for CNBP, Dr. Lorenzo Gaetani and Prof. Massimiliano Di Filippo for AOPG and Dr. Fabio Moda for FINCB.
• The a-synuclein seeding activity in OM and skin samples obtained by means of SAA. These analyses will be centralized at FINCB and will be performed under the supervision of Dr. Fabio Moda and Dr. Chiara Maria Giulia De Luca.
• Plasma levels of Aß42/Aß20 and p-tau. These analyses will be centralized at AOPG by using the Lumipulse full automated platform under the supervision of Prof. Lucilla Parnetti and Dr. Lorenzo Gaetani.
• Detection and characterization of p-a-syn deposits in skin biopses, using immunohistochemical methods. Dr. Grazia Devigili will be responsible for this analysis. The responsible for clinical data collection in each center are Prof. Lucilla Parnetti and Prof. Massimiliano Di Filippo for AOPG, Dr. Paola Caroppo for FINCB and Dr. Davide Cardile for CNBP. The data analysis task, for each of the three described aims, will be instead coordinated by prof. Lucilla Parnetti with the help of Dr. Santina Caliri and Dr. Davide Cardile at CNBP.