Methods

Overview

*only in a subset

Clinical Evaluation and Cognition

Clinical Evaluation

At baseline individuals included in these cohorts are assessed by a physician experienced in dementia disorders or with special interest in movement disorders. The assessing physician conducts a psychiatric, physical and neurological examination, including a thorough interview with the patient and relative/spouse that focus on ADL function and cognitive impairment. Experienced nurses administer the tests.

Basic 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), Stroop Test, Clock Test, Month backwards, and the Quick Test of Cognitive Speed (AQT). Visuospatial ability is measured using Cube-copying Test and in part Clock Drawing Test. Memory is assessed with the 10-word delayed recall test from ADAS-cog. Verbal ability will be evaluated with the animal and letter S fluency tests. Global cognition will be assessed with the Mini-Mental State Examination (MMSE).

Neuropsychological assessment – Extended cognitive testing

Patients with mild cognitive complaints also undergo a thorough neuropsychological assessment with a large test battery that evaluates memory and learning, verbal and visuospatial functions, attention, and executive ability as well as premorbid function. The results from this assessment are used in the classification of patients in subjective cognitive decline (SCD) and different MCI subgroups as well as in analyses with other data including the different biomarkers. The patients are assessed by means of a test-battery covering four broad cognitive domains:

Verbal ability is examined through a multiple-choice vocabulary test (SRB:1) and a semantic Verbal Fluency test.

Episodic memory functions are examined with Rey Auditory Verbal Learning Test (RAVLT) and Rey Complex Figure Test (RCFT): free immediate and delayed recall.

Visuospatial construction ability is examined using Block design and the copy trial of RCFT.

Attention and executive functions are tested by means of Trail Making Test and letter Verbal Fluency.

Assessments of symptoms and ADL

Cognitive symptoms

In patients with cognitive symptoms or impairment, degree of symptoms is assessed by informants using the Cognitive Impairment Questionnaire (CIMP-QUEST).

Functional ability

This is evaluated in patients with cognitive symptoms or impairment, using 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. For healthy controls and patients with mild cognitive complaints this is also scored using the Global Deterioration Scale (GDS).

Behavioral and psychological symptoms in dementia (BPSD)

Mood and anxiety levels are further assessed using the Hospital Anxiety and Depression Scale (HADS). In addition, apathy prevalence is assess using Apathy Evaluation Scale (AES) both self-rated (-S) and informant-rated (-I). In healthy elderly depression is further assessed using Cornell Scale for Assessing Depression in Dementia and A Comprehensive Psychopathological Rating Scale – Self Assessment (CPRS-S).

Quality of Life (QoL)

The overall health status is rated by the subjects using the EQ-5D from Euro-QoL. In two cohorts, parkinsonian symptoms and healthy elderly, self-rating 12-item Short Form Health Survey (SF-12) is also used.

Sleep, smell and autonomic function

Presence of symptoms associated with neuronal alpha-synuclein disease (such as sleep, smell, and autonomic dysfunction) is assessed using self-rating SCOPA-SLEEP and SCOPA-AUT questionnaires in the parkinsonian symptoms cohort and the healthy elderly cohort. These two cohorts also perform the 12-item Brief Smell Identification Test.

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 polypropylene 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 polypropylene tubes and then stored at -80°C until batch analyses.

About 0.5 ml of CSF from each individual with cognitive symptoms or impairment at baseline is analyzed directly as part of routine clinical measurements for Aβ42, Tau, P-tau, and NfL.

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 polypropylene tubes and stored at -80°C pending biochemical analyses. Further, EDTA-blood will also be obtained for genetic DNA analyses.

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.

Magnetic Resonance Imaging

The participants (except parkinsonian symptoms cohort) undergo MR imaging (MRI) on the same 3T Siemens Trio MRI scanner to avoid inter-scanner variabilities in the results. Participants with parkinsonian symptoms are instead imaged with a 3T Siemens Skyra MRI scanner.

The protocol at the Siemens Trio includes: sagittal T1-weighted spin echo; axial T2-weighted FLAIR for assessment of focal pathology; coronal T2-weighted GRE for assessment of siderosis and microbleeds; T1-weighted 3D MP-RAGE for volumetric measurements;  diffusion tensor imaging (DTI, 64 directions) and diffusion kurtosis imaging (DKI, 64 directions) for white matter alterations and structural connectivity; resting state functional MRI (rsfMRI) for estimation of functional  connectivity; arterial spin labelling (ASL) to evaluate cerebral blood flow; single voxel 1H MR spectroscopy for assessment of metabolite concentrations in the precuneus.

The protocol at the Siemens Skyra includes: sagittal T2-weighted spin echo; axial T2-weighted FLAIR for assessment of focal pathology; axial SWI for assessment of susceptibility changes; T1-weighted 3D MP-RAGE for volumetric measurements;  diffusion tensor imaging (DTI, 32 directions) and diffusion kurtosis imaging with 3-shell HARDI data acquisition (DKI, 64 directions, 2500) for white matter alterations and structural connectivity; resting state functional MRI (rs-fMRI) for estimation of functional  connectivity; arterial spin labelling (ASL) to evaluate cerebral blood flow.

Amyloid PET

PET imaging of Aβ aggregates, including 18F-flutemetamol PET, is approved for clinical routine use in Sweden. In a subset of participants 18F-flutemetamol PET is conducted according to clinical routine procedures. A 20-minute scan is carried out 90-110 minutes after the injection of 18F-flutemetamol. All subjects are examined on the same type of scanner (a Philips Gemini TF 16) to avoid technical biases in the results. The precursor is provided by GE Healthcare.

Tau PET

Detection of tau accumulation and measurement of tau deposits is done using 18F-AV1451 in a subset of participants. It is conducted in collaboration with AVID Radiopharmaceuticals/Lilly (USA). All subjects are examined on the same type of scanner (a Philips Gemini TF 16) to avoid technical biases in the results. The participants undergo repeated TauPET examinations every 2^nd year for up to 6 years.

Assessment of Motor Function

A wide range of assessments addressing motor aspects, gait, balance, and visuospatial navigation are performed within the study (not in the dementia cohort).

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 and Yahr staging is also performed.

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. In addition, Figure of Eight Test is performed to assess mobility skills.

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.

Speed: Ten-meter walk test is used to assess walking speed.

The TUG and the ten-meter walk tests are performed in both comfortable and fast gait speed. In comfortable gait speed, subsequent trials of these tests include a dual task (motor and cognitive, respectively). All tests are videotaped for detailed analysis.