Toward Molecular Profiling of Parkinson's Disease in Easily Accessible Biological Matrices
Toward Molecular Profiling of Parkinson's Disease in Easily Accessible Biological Matrices
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 nonmotor 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. 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 targeted disease-modifying therapy. The possibility of measuring these markers in biological matrices suitable for repeated sampling could provide objective measures of the effectiveness of a therapeutic approach. In this proposal we will combine the expertise of three different Italian medical research centers to establish a molecular profile of PD based on biomarkers reflecting different biological pathways, in different biological matrices, by applying immunoassays, proximity extension assays (PEA) and seed amplification assays (SAA). Two easily accessible biological matrices, i.e., blood plasma and olfactory mucosa (OM), has been/will be collected in each center for PD patients, controls and patients affected by Alzheimer's disease (AD) as other neurodegenerative disease controls. OM will be collected by a non-invasive procedure known as nasal brushing which is already operational and standardized among the three participating centers. The project will include both a prospective and retrospective cohort composed of 200 PD patients, 100 controls and 40 AD. All PD patients that will be recruited will undergo a thorough clinical and neuropsychological evaluation. The control group will be constituted by healthy volunteers as well as by cognitively unimpaired subjects with subjective memory complaints or patients affected by minor neurological symptoms (i.e., headache, peripheral neuropathy, etc.). The above-mentioned clinical information has been already collected for the retrospective cohort. Plasma amyloid-ß (Aß) 42/Aß40 ratio and phosphorylated at threonine 181 tau (p-tau) will be measured with the Lumipulse automated platform for all collected plasma samples. 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 robustness of these markers as well as the correlation between their CSF and plasma levels. SAA will be applied in OM for the detection of misfolded a-synuclein. Phosphorylated a-synuclein (p-a-syn) will be measured in a subset of PD patients and controls to compare the diagnostic accuracy of p-a-syn in skin biopsies and a-synuclein seeding activity in OM. 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 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.
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
To accomplish the above-described aims 1, 2 and 3, the biomarkers that will be measured are:
Inclusion Criteria:
patients with clinical diagnosis of Parkinson's disease, atypical parkinsonisms, Alzheimer's disease, healthy volunteers, people with subjective cognitive decline.
Exclusion Criteria:
Other acute neurological diseases or neurodegenerative diseases such as Frontotemporal dementia, vascular dementia, and stroke
Relevant comorbidities: chronic kidney disease (significant alteration of blood biomarkers)
Skin biopsy: local skin infection at biopsy site, severe dermatologic disease at sampling area, known bleeding disorder, significant thrombocytopenia, anticoagulation not safely manageable, allergy to local anesthetics, poor wound healing, severe peripheral vascular disease, high-risk immunosuppression, inability to consent or cooperate, extensive scarring at intended site
Magnetic resonance spectroscopy: non-MRI-compatible implanted device, ferromagnetic intracranial clip, metallic foreign body, severe claustrophobia not manageable, inability to remain still, MRI-conditional device not meeting safety conditions, pregnancy per institutional policy, severe agitation or confusion, body size exceeding scanner limits, severe motion disorder affecting acquisition
Venipuncture: refusal or inability to consent, local infection at puncture site, severe coagulopathy, marked thrombocytopenia, high bleeding risk anticoagulation, severe anemia relative to planned blood volume, difficult venous access, history of severe vasovagal syncope
Eligibility Criteria
Inclusion Criteria:
Adults able to provide informed consent
Clinical diagnosis of Parkinson's disease
Clinical diagnosis of atypical parkinsonism
Clinical diagnosis of Alzheimer's disease
Individuals with subjective cognitive decline
Healthy volunteers
Exclusion Criteria:
Acute neurological diseases
Other neurodegenerative diseases not included in the study groups (e.g., frontotemporal dementia, vascular dementia)
History of stroke with significant neurological sequelae
Chronic kidney disease with significant alteration of blood biomarkers
Procedure-specific exclusion criteria
Skin biopsy:
Local skin infection at biopsy site
Severe dermatologic disease at sampling area
Known bleeding disorder
Significant thrombocytopenia
Anticoagulation not safely manageable
Allergy to local anesthetics
Poor wound healing
Severe peripheral vascular disease
High-risk immunosuppression
Inability to consent or cooperate
Extensive scarring at intended biopsy site
Magnetic resonance spectroscopy (MRS):
Non-MRI-compatible implanted device
Ferromagnetic intracranial clip
Metallic foreign body incompatible with MRI
Severe claustrophobia not manageable with standard procedures
Inability to remain still during MRI acquisition
MRI-conditional device not meeting safety conditions
Pregnancy according to institutional MRI safety policy
Severe agitation or confusion preventing examination
Body size exceeding scanner limits
Severe motion disorder affecting acquisition
Venipuncture:
Refusal or inability to provide consent
Local infection at puncture site
Severe coagulopathy
Marked thrombocytopenia
High bleeding risk due to anticoagulation
Severe anemia relative to planned blood draw volume
Difficult venous access
History of severe vasovagal syncope during venipuncture
Lumbar puncture:
Signs of increased intracranial pressure due to mass lesion
Local infection at puncture site
Uncorrected coagulopathy
Severe thrombocytopenia
Anticoagulation not safely interruptible
Spinal cord compression
Unstable spine
Refusal or inability to cooperate with the procedure
Severe spinal deformity
Prior lumbar surgery complicating access
Severe anxiety or intolerance to the procedure
Pregnancy requiring procedural precautions
Nasal brushing:
Significant nasal polyposis
Severe septal deviation preventing access to the olfactory cleft
Active acute or chronic rhinosinusitis
Recent epistaxis or bleeding-prone nasal mucosa
Nasal lesions, ulcers, or tumors
Recent nasal surgery Nasal brushing: Significant nasal polyposis, Severe septal deviation preventing access to the olfactory cleft, Active acute or chronic rhinosinusitis, Recent epistaxis or bleeding-prone nasal mucosa, Nasal lesions, ulcers, tumors, or recent nasal surger