Methods

Overview

Table 1. Overview of baseline assessments in the different cohorts

Assessments of motor aspects and visuospatial navigation are explained in Tables 2-3.
*only a subgroup ^†only participants with parkinsonian disorder (cohort E) at inclusion or if suspicion of parkinsonian disorder during follow-up for other cohorts

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 will be 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 will be evaluated with the animal and letter S fluency tests and the 15-item short version of the Boston Naming Test. Global cognition will be assessed with the Mini-Mental State Examination (MMSE). Computerized cognitive batteries focusing on memory, attention, visuospatial ability, and navigation is also administered (CANTAB and an in-house developed battery – BioCog).

Assessments of symptoms and ADL

Cognitive symptoms

All subjects rate their memory and attention/executive function relative to others of the same age according to the Brief Anosognosia Scale (BAS). We have also added similar questions to cover the other cognitive domains. These questions have been validated against neuropsychological testing but there are data indicating that self-reported cognitive complaints are only valid in a lesser degree of cognitive impairment. To assess a broader range of cognitive complaints, the Cognitive Function Instrument (CFI) is administered to participants and informants in cohorts A and B (controls). Subjects from Cohorts C, D, and E are assessed using the Cognitive Impairment Questionnaire (CIMP-QUEST), filled out by an informant.

Functional ability

This is evaluated with the informant-based Functional Activities Questionnaire (FAQ) focusing on instrumental activities of daily living (IADL).

Global function

Participants are classified as having SCD, MCI or dementia based on the neuropsychological battery, clinical symptoms and loss of independence in ADL.

Behavioral and psychological symptoms in dementia (BPSD)

Behavioral and Psychological Symptoms of Dementia (BPSD) are assessed using the Mild Behavioral Impairment Checklist (MBI-C). Mood and anxiety levels are further assessed using the Hospital Anxiety and Depression Scale (HADS).

Quality of Life (QoL)

The overall health status will be rated by the subjects using the EQ-5D from Euro-QoL. In patients with dementia, this is also rated by an informant, such as a spouse or close relative.

Sleep

The presence of REM sleep behavior disorder will be evaluated with a single validated composite question derived from the Mayo Sleep Questionnaire. Sleep quality is also assessed using questions from the Sleep Scale from the Medical Outcomes Study (MOS).

Cognitive reserve

Premorbid cognition and cognitive reserve are estimated using the Cognitive Reserve Index questionnaire (CRI-q), specifically the subitems “Education” and “Working Activity” (excluding “Leisure Time”).

Cerebrospinal fluid (CSF) and blood sampling and analyzes

Lumbar CSF samples are collected according to a standardized protocol, adhering to the Alzheimer’s Association Flow Chart for CSF biomarkers. Lumbar punctures are performed between 8-12 am. 20 ml of CSF is collected in low binding tubes and stored on ice for 5-20 minutes before being centrifuged (2000g, +4°C, 10 min). Subsequently, the CSF is aliquoted in approximately 1 ml portions into low binding tubes and then stored at -80°C until batch analyses.

About 0.5 ml of CSF from each individual at baseline is analyzed directly as part of routine clinical measurements for Aβ42, Aβ40, Tau, P-tau, and NfL using Lumipulse.

Plasma is collected during the same visit as the lumbar puncture. Blood is drawn into tubes containing either EDTA or Lithium heparin. Following centrifugation (2000g, +4°C, 10 min), plasma samples are aliquoted into low binding tubes and stored at -80°C pending biochemical analyses. Further, EDTA-blood will also be obtained for genetic DNA analyses.

In a subset of approximately 30 individuals, repeated full blood and plasma collections are performed over an 8-week period to analyze short-term variability in blood-based biomarkers.

Beyond measuring Aβ, Tau, and P-tau, we are investigating other markers for their roles in the pathogenesis of neurocognitive disorders and their potential to enhance early diagnosis. We analyze both candidate markers such as α-synuclein seed amplification assay and MTBR-tau243, and employ proteomic approaches. Additionally, RNA and DNA analyses, including genome-wide association studies and whole genome sequencing, are conducted to identify markers potentially linked to neurodegenerative diseases.

Skin biopsy

Accumulation of pathological alpha-synuclein in skin tissue can be detected in patients with synucleinopathies. Up to three skin biopsies are performed from two sites using a 3 mm punch biopsy tool. A local anesthetic is administered prior to each biopsy. The biopsy sites include the paravertebral area at the C7-C8 level and a thigh area 10 cm proximal to the patella. Various techniques, including real-time quaking-induced conversion assays, are used to detect the presence of aggregated α-synuclein in the skin biopsies.

Amyloid PET

PET imaging of Aβ aggregates, including 18F-flutemetamol PET, is approved for clinical routine use in Sweden. In this study, 18F-flutemetamol PET is conducted in non-demented cases only. For most patients with dementia, CSF Aβ is sufficient to determine the presence or absence of brain amyloid pathology. However, in cognitively healthy individuals, those with SCD or MCI, and a subgroup of AD dementia patients, we are exploring how amyloid pathology spreads throughout the brain during the preclinical stages of AD and its spatial relationship with tau pathology. Consequently, Amyloid PET is conducted according to clinical routine procedures alongside CSF Aβ measurements in these groups. Amyloid PET is performed using 18F-flutemetamol, with GE Healthcare providing the precursor. A 20-minute scan is carried out 90-110 minutes after the injection of 18F-flutemetamol.

Tau PET

In this study, Tau PET imaging is conducted using 18F-RO948, developed by Hoffmann-La Roche, which also provides the precursor for this PET ligand. This imaging agent accurately detects tau pathology in Alzheimer’s cases compared to controls. We conduct a 20-30 minute PET scan about 60 minutes after the intravenous injection of 18F-RO948. The impact of this investigation on clinical diagnostic accuracy and patient care is also being evaluated.

Dopamine PET

Dopamine PET is performed with FE-PE2I PET (initially conducted using F-DOPA). This imaging is carried out in participants with PD, PDD, DLB, MSA, PSP, and CBD (Cohort E). It is also performed in other cohorts for patients suspected of having a parkinsonian disorder during follow-up.

Magnetic Resonance Imaging

A 3 Tesla MRI (Siemens Prisma) is conducted in all study cohorts. We employ a variety of MRI techniques to assess regional brain volume (3D MPRAGE), metabolism (MRS), structural and functional connectivity (DTI and fMRI), regional blood flow (ASL), iron deposition (SWI), and the presence of small vessel disease (using MPRAGE, SWI, and FLAIR). This comprehensive protocol takes approximately 60 minutes to complete, and no contrast agent is used.

Assessment of Motor Function

This part is performed on healthy cases (Cohort B), patients with SCD/MCI (Cohort C), and patients with parkinsonian disorders (PD, PDD, DLB, CBD, MSA, PSP; from Cohort E). It addresses motor symptoms, gait, balance, visuospatial navigation, and level of physical activity, and is administered by a physical therapist trained specifically for this project.

Motor symptoms

The motor part of the Unified Parkinson’s Disease Rating Scale (UPDRS, part III) includes 14 items; one item specifically captures the postural response in relation to an external perturbation (scored 0-4). The total score of the UPDRS part III ranges from 0-108 points (higher scores = worse). Hoehn & Yahr staging is also done for patients with PD, PDD, and DLB in Cohort E.

Lower extremity function

The chair-stand test assesses lower extremity function. The person rises (arms folded) from sitting to erect standing (5 times) as fast as possible. The time (seconds) is registered.

Mobility

The Timed Up & Go (TUG) test assesses basic mobility skills. It includes the following tasks: to rise from a chair, walk 3 m, turn around and walk back to sit down again. TUG is performed in comfortable and fast gait speed; the time (seconds) is registered.

Gait

An electronic walkway (GAITRite®, version: Platinum, 5.8 meters in total length) with over 18,000 sensors is used to conduct objective temporo-spatial gait analysis. Walking in comfortable and fast gait speed will be assessed. Functional Gait Assessment (FGA, 10 items) assesses both balance and gait. The total score ranges from 0-30 (higher scores= better). The generic Walk-12 (Walk-12G, 12 items) assesses perceived walking difficulties in daily life. The total score ranges from 0-42 (higher scores= worse). Dichotomous (yes/no) questions are used for perceived changes in walking ability, unsteadiness while walking and while turning.

Visuo-spatial navigation

The two-dimensional Floor Maze Test (FMT) assesses spatial navigation while walking. The following are registered: planning time (initial and total), number of stops and errors, and total Maze time. A qualitative performance rating is also done.

Balance

The Tandem Gait Test includes 10 consecutive tandem steps along a straight line, performed without walking aids or support, and with eyes open. The total number of side steps is registered; performance is also scored as follows: score 0, no side steps; 1, single side step; 2, multiple side steps; 3, unable to take more than 4 consecutive steps. Turning (on the spot, 360 degrees, in both directions) is timed and scored from 0-4, with higher scores indicating better performance.

Dual tasking

Assessments such as TUG, gait (i.e., GAITRite), and Turning are performed both with and without a concurrent dual task, such as a subtraction task.

Falls

Questions about the history of falls or near falls are included, along with Yes/No questions concerning fear of falling and activity avoidance due to fall risk. Falls Efficacy Scale-International (FES-I, 16 items, i.e. activities) assesses concerns about falling. The total score ranges from 16 to 64 (higher = worse). The Modified Survey of Activities and Falls in the Elderly (mSAFFE, 17 items, i.e. activities) assesses activity avoidance due to the risk of falling. The total score ranges from 17-51 (higher = worse).

Physical activity

The National Board of Health and Welfare (Socialstyrelsen, a government agency) and the Public Health Agency of Sweden have specific questions that target a person’s level of physical activity and sedentary time, based on recommendations by the World Health Organization (WHO).

APDM MobilityLab (wearable sensors: gyroscopes and accelerometers) is used during certain assessments in the test battery to analyze aspects like anticipatory postural adjustments, postural sway, range of motion, and gait variables.