Exercise, Brain, Cognition, OMICs, Molecular Markers and Functionality in People at Risk of Mild Cognitive Impairment

Brain magnetic resonance images will be acquired in a Philips-Ingenia 3-Tesla scanner with a 32-channel digital coil exclusively dedicated to skull (Philips, The Netherlands) with the anatomical sequences:

• Sequence T1-3D in sagittal orientation. This sequence will quantify the cortical thickness and perform morphometric studies of cortical and subcortical structures.

The pattern of cortical thickness changes and the volume of different brain structures will be obtained with the tools implemented in the software Freesurfer v5.3 and SPM12.

Brain magnetic resonance images will be acquired in a Philips-Ingenia 3-Tesla scanner with a 32-channel digital coil exclusively dedicated to skull (Philips, The Netherlands) with the anatomical sequences:

• DTI (Diffusion Tensor Imaging) sequence in axial orientation. This sequence will allow to evaluate the properties of the microstructure of the white matter and its nerve fiber tracts.
• T2 / FLAIR sequences in axial orientation. Both sequences will be used to determine the degree of involvement of the deep and periventricular white matter using the Fazekas scale.

An overall SCORE (z-score values or similar;higher value for better outcome) of the 8 measurements on cognitive status will be developed to arrive at one reported value:

• - Clinical Dementia Rating -CDR: This is a valid and reliable instrument for staging dementia.
• - Mini Mental State Examination to detect cognitive deterioration.
• - Trail Making Test -TMT: This is an indicator of the speed of cognitive processing and executive functioning. 2 parts.
• - Boston Naming Test to assess the ability to name objects.
• - Clock Drawing Test -CDT: valid test for evaluation of cognitive deterioration.
• - Rey Auditory Verbal Learning Test -RAVLT: learning and verbal episodic memory.
• - The Stroop Color and Word Test -SCWT: examines basic psychological processes (cognitive flexibility, selective attention, resistance to interference from external stimuli and cognitive inhibition).
• -Controlled Oral Word Association Test -COWAT: designed to assess verbal fluency.

To carry out the analysis of the OMICs a full untargeted profiling will be carried out in the pre and post-intervention and retest measurements for different OMICS including metabolomics, proteomics,etc. For these analyzes, the following large equipment and techniques will be used, ultrahigh performance liquid chromatography / mass spectrometry (LC-quadrupole-time of flight, QTPF-MS, Agilent 1200-Agilent 6520); capillary electrophoresis / mass spectrometry (CE, TOF-MS, Agilent 7100-Agilent 6210) and gas chromatography / mass spectrometry (GC, EI-GC), mass spectrometry (capillary electrophoresis / mass spectrometry, CE-TOF-MS; Q-MS, (Quadrupolo, Agilent 7890A-Agilent 5965C) In addition, data processing platforms will be used, both mass databases and statistical tools for multivariate analysis: Mass Hunter, Mass Profiler Professional, SIMCA, MATLAB.

The telomere length will be measured by quantitative real-time PCR (RT-qPCR) following the method previusly described. Briefly, the DNA from the cell fraction of the blood will be extracted with the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's specifications. The DNA concentration of the sample, as well as its integrity, will be confirmed in a NanoDrop 2000. For the RT-qPCR the telomeric sequence will be amplified as well as a single copy gene sequence to normalize. Calibration curves will be made with serial dilutions of known concentration of both the gene sequence as well as an oligo specially designed to contain 114 copies of the TTAGGG telomeric sequence. The RT-qPCR will be carried out with 20 ng of DNA using myScript SYBR Green PCR Kit (ROCHE). The PCR conditions are: 10 min at 95 ° C, followed by 40 cycles of 95 ° C for 15 sec and 60 ° C for 1 min, followed by the corresponding dissociation curve.

Consist in a Standardized field test battery including:

1.Muscle-skeletal (number of repetitions)

Consist in a Standardized field test battery including:

2.Cardiorespiratory fitness (metres and estimated maximal oxygen uptake-VO2max)

Consist in a Standardized field test battery including:

3.Agility-motor coordination (seconds)

Consist in a Standardized field test battery including:

4.Flexibility (centimetres)

Consist in a Standardized field test battery including:

5.Handgrip strength (kilograms)

All subjects performed a graded exercise test on a treadmill (Lode Valiant, Groningen, Netherlands) until exhaustion. Specifically, a modified Bruce protocol previously used in a similar sample and designed for a geriatric population. Participants were asked if they had ever walking on a treadmill and instructed of the incremental test. If necessary, we familiarized them with the treadmill by walking slowly until the initial protocol speed was achieved. Before start, subjects were instructed not to talk during the test because this is known to affect the breathing and gas exchange. Participants began walking to 2.7 km/h at 0% inclination grade, every 2 minutes the speed or/and inclination were increased according the modified Bruce protocol. VO2peak was considered as the highest observed value of oxygen consumption obtained in the last three intervals of 10 seconds.

This scale measures the possibility of carrying out daily instrumental activities in daily life (Total score 0 to 8; higher value for better outcome).

the hemodynamic status will be measured twice after 5 minutes at rest using a upper blood pressure monitor (M6 Omron):

• -Systolic and diastolic Blood pressure (mmHg)
• - Resting heart rate (beats per minute)

Body Composition will be measured using a bio-impedanciometer (BIA; Tanita-MC-780MA MF 8-elc) including:

1.Weight (in kilograms)

Body Composition will be measured using a Stadiometer (SECA):

2.Height (in meters)

Body Composition will be measured using an anthopometric flexible tape (SECA):

3.Waist circumference (in centimetres)

Body Composition will be measured as:

4.Calculated body mass index (weight and height will be combined to report BMI in kg/m^2)

Body Composition will be measured using a bio-impedanciometer (BIA; Tanita-MC-780MA MF 8-elc) including:

• Total body fluids (litres).

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

1.Glucose (mg/dl)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

2.Total cholesterol (mg/dl)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

3.HDL cholesterol (mg/dl)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

4.LDL cholesterol (mg/dl)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

5.triglycerides (mg/dl)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

6.Apolipoprotein B (g/l)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

7.Insulin (μlU/mL)

The Cardio-metabolic Risk Factors Profile will be measured by several biochemical parameters including:

8.Insulin resistance (HOMA is calculated as fasting insulin [pmol/l]/6.945).

The Antioxidant Capacity will be measured by the following markers:

• - The 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS)
• Oxygen radical absorption capacity (micromol/100 milliliter)
• Ferric reducing antioxidant potential (mmol/l).

The Neurotrophic Factor Derived from the Brain will be measured as followed:

1.-The brain-derived neurotrophic factor (BDNF in ug/L) will be measured in serum using the ELISA Kit (Enzyme-linkerd immunosorbent Assay) of RAyBio Human BDNF (RAFER). This marker is highly related to brain function and could mediate or explain to a certain extent the results of the EFICCOM project.

The Cerebrospinal fluid will be measured as followed:

1.- Lumbar punctures will be performed accordinly with clinical standards and procedures manual at the specific unit of Neurology from the Puerta del Mar Hospital. CSF samples will be frozen on dry-ice soon after collection (~1 hour) and shipped to the university unit responsible for the analysis. This marker is highly relevant for the identifciation of the real risk for mild cognitive impairment using gold standard biomarkers of the EFICCOM project.

The Short-Form Health Survey 36 -SF36 includes 8 dimensions scored from 0 to 100 (higher value for better outcome).

The Depression will be measured using the Geriatric Depression Scale-GDS which is composed of 15 questions with dichotomous answers (yes / no) specifically designed for the elderly population and will be used to rule out depression. The GDS score from 0 to 15, where higher value means a worse outcome.

The Sociodemographic Characteristics will be measured by several items including:

• Age (years)

The Sociodemographic Characteristics will be measured by several items including:

• Sex (male or female)

The Sociodemographic Characteristics will be measured by several items including:

3.Marital status (Categorical variable: Single, Married or with a partner, Widowed and without partner, legally separated or divorced)

The Sociodemographic Characteristics will be measured by several items including:

• Educational level and socio-economic status (Ordinal variables where the higher the better: low, middle, high for Educational level; Low and high for socio-economic status)

The Sociodemographic Characteristics will be measured by several items including:

5.Family history of dementia (yes/no and details),

The Sociodemographic Characteristics will be measured by several items including:

6.Number and type of Medications (register)

The Sociodemographic Characteristics will be measured by several items including:

7.Presence of other pathologies (list of diseases).