Methods

A.   Overview

B.   Clinical Evaluation and Cognition

Cognitive testing

The cognitive battery is outlined in the table above. Attention and executive function are assessed using the Trail Making Test A and B (TMT), the Symbol Digit Modalities Test (SDMT), and the Quick Test of Cognitive Speed (AQT). Visuospatial ability is measured using two subtests from the Visual Objects and Space Perception (VOSP) battery: Incomplete Letters and Cube Analysis. Memory is assessed with the Free and Cued Selective Reminding Test (FCSRT) in cohorts A and B. This assessment is complemented by the 10-word delayed recall test from ADAS-cog, including a recognition component. Verbal ability is evaluated with the animal and letter S fluency tests and the 15-item short version of the Boston Naming Test. Global cognition is assessed with the Mini-Mental State Examination (MMSE). In a subset of study participants, cognitive function is assessed using an application on a smartphone.

Assessments of symptoms and ADL

Cognitive symptoms are assessed using the Cognitive Impairment Questionnaire (CIMP-QUEST), filled out by an informant. Sleep-related symptoms and changes are assessed using the RBD screening questionnaire (RBDSQ) and the Epworth Sleepiness Scale (ESS). Other non-motor symptoms are assessed using The Non-Motor Symptoms Scale (NMSS), which is a 30-item rater-based scale to assess a wide range of non-motor symptoms in patients with Parkinson’s disease (PD). The NMSS measures the severity and frequency of non-motor symptoms across nine dimensions. Food and beverage intake is assessed using a Food Frequency Questionnaire (FFQ), which is a limited checklist of foods and beverages with a frequency response section for subjects to report how often each item was consumed over a specified period of time. Potential constipation is assessed using the Bristol stool scale & ROME-III and dysautonomia using SCOPA-AUT. Behavioral and psychiatric symptoms are assessed using the Questionnaire for Impulsive-Compulsive Disorders in Parkinson’s Disease (QUIP) and HADS. Smell function is tested using the 16-item Sniffin’ Sticks test. Objective measurement of orthostatic hypotension is included in the clinical assessment. Finally, quality of life is assessed using the EQ-5D.

C.  Assessment of Motor Function

Symptoms of Parkinson’s disease are evaluated using the MDS-UPDRS and assessment of Hoehn & Yahr stages. Subtle motor symptoms are examined by the use of the Purdue Pegboard, alternate tapping test, and the 3-m time up and go test. Questionnaires are used to reveal the presence of non-motor symptoms, including autonomic dysfunction that includes constipation and orthostatic hypotension. In addition, a more in-depth evaluation of motor symptoms by a physical therapist is performed for all participants, as described for the BioFINDER-2 study. Briefly, the assessments mainly address the level of physical activity, activity avoidance, gait- and balance problems (including falls and fear of falling) as well as dual-task performance, i.e., combining a motor and a cognitive task. The data collection consists of self-administered questionnaires, interview-administered questions, clinical assessments, and also using objective measures (i.e., using an electronic walkway or using six wearable sensors while conducting some of the tests).

D.  Cerebrospinal fluid and plasma sampling

Lumbar CSF samples are collected according to a standardized protocol aligned with clinical standards. Lumbar puncture is performed between 8-12 am, with 25 ml of CSF collected in Low Binding tubes, which are then stored on ice for 5-20 minutes before being centrifuged (2000g, +4°C, 10 min). Afterward, the CSF is aliquoted into approximately 1 ml portions into Low Binding tubes and stored at -80°C until batch analyses. CSF analyses include measurements of oligomeric and total α-synuclein, as well as α-synuclein SAA. CSF Aβ1–42, total tau, phosphorylated tau, and markers of inflammation and degeneration such as interleukin beta-1, TNF-alpha, and NO are also measured. Approximately 0.5 ml of CSF from all individuals at baseline and follow-up is analyzed directly as part of routine clinical practice to measure cells and signs of neuroinflammation. Plasma collection occurs at the same visit as the lumbar puncture. About 60 ml of blood is drawn into tubes (either EDTA or Lithium heparin tubes). After centrifugation (2000g, +4°C, 10 min), plasma samples are aliquoted into LowBind tubes and stored at -80°C pending biochemical analyses.

E.    Skin biopsy

Skin biopsies are taken from two sites using a 3 mm punch biopsy methodology as described in previous studies, with a local anesthetic applied prior to the biopsy. The biopsy sites include the paravertebral area at the level of C7-C8 and an area on the thigh, 10 cm proximal to the patella, previously identified as having the highest sensitivity for detecting pathological α-synuclein accumulation in patients. Various techniques, including real-time quaking-induced conversion assays, are used to detect the presence of pathological α-synuclein in skin biopsies.

F.    Dopamin PET

[18F]FE-PE2I is a highly selective imaging ligand for assessing dopamine transporters and provides an indirect measure of surviving dopaminergic neurons. Briefly, 185 MBq [18F]FE-PE2I is injected intravenously and a static PET scan of 25 minutes is performed on GE Discovery MI PET-CT cameras, starting 30 minutes post-injection. In a small subpopulation (10 controls and 10 PD patients), dynamic PET scans from 0-75 minutes post-injection are performed. The estimated radiation exposure is 4.3 mSv of radiation, and including a low-dose CT, the total radiation exposure per scanning session is 4.6 mSv. No arterial blood sampling is performed for this scan.

G. Magnetic resonance imaging; 3 Tesla and 7 Tesla

Magnetic resonance imaging (MRI) scans are performed on a 3T Siemens MAGNETOM Prisma scanner. A wide variety of MRI techniques are used to study regional brain volume (three-dimensional magnetization-prepared rapid acquisition with gradient echo (3D MPRAGE)), metabolism (MR spectroscopy (MRS)), structural and functional connectivity of different brain regions (diffusion tensor imaging (DTI) and functional MRI (fMRI)), regional blood flow (arterial spin labeling (ASL)), iron deposition (susceptibility-weighted imaging (SWI)), and the presence of small vessel disease (MPRAGE, SWI, and fluid-attenuated inversion recovery (FLAIR)), as well as neuromelanin-sensitive sequences that can be used as an indirect measure of dopaminergic and noradrenergic terminals. The protocol takes approximately 60 minutes to perform. No contrast-enhancing agent is used.

In a subsample, 7T MRI is performed. The hypothesis that degeneration of catecholaminergic brainstem nuclei and midbrain dopaminergic neurons can be better quantified using neuromelanin-sensitive sequences with high-field 7 Tesla rather than conventional 3 Tesla magnetic resonance imaging is tested in the current study.

H.  MIBG scintigraphy

[123I] MIBG scintigraphy is performed as previously published (Knudsen et al., 2018). Early and late images of the thorax are collected for 15 minutes, 210 minutes after the administration of 110MBq [123I] MIBG, respectively. Images are collected on a SPECT-gamma camera to estimate the mean heart-uptake/mean mediastinum-uptake ratios and to estimate washout rates as the differences in mean uptake between early and late images. A low dose SPECT/CT is performed after the late images. The estimated radiation exposure is 110MBq x 0.013 mSv/MBq ≈ 1.4 mSv, and including the low-dose SPECT/CT, the total is 1.7 mSv per scan session. [123I] MIBG scintigraphy is repeated at follow-up at 3 and 6 years after baseline.

Polysomnography

A comprehensive sleep registration is performed on the participants, which includes monitoring of breathing, pulse, saturation, and movement of limbs and chin, while simultaneously recording the electrical activity of the brain. All participants undergo polysomnography to detect any REM-sleep disorder. The examination takes place during a separate study visit that includes an overnight stay at the hospital.