Cognitive and Functional Connectivity Effects of Methylene Blue in Healthy Aging, Mild Cognitive Impairment and Alzheimer's Disease

Healthy aging and aging human subjects with mild cognitive impairment and mild Alzheimer's
disease from the TARCC cohort and South Texas will be studied using a double-blind,
placebo-controlled design. After informed consent and familiarity with the tasks and the MRI
environment, the subject will enter an MRI scanner and perform the following 6 tasks.

fMRI and behavioral data will be collected simultaneously while inside the scanner.

Delayed match-to-sample task: The subject views a pattern for a few seconds and then is
prompted to recall the memorized pattern using a response system (approx. 10 mins).

Face-name task: The subject is shown blocks of stimuli where a novel or familiar face is
paired with a name. In a later run, the subjects are asked whether the correct name is
matched with the correct face. (approx. 10 mins).

Psychomotor vigilance task: The subject receives a visual cue that alerts them to press a
button as fast as possible. (approx. 10 mins).

Cerebral Blood Flow and Resting State fMRI: Subject scanned with eyes closed and told to not
think about a particular topic, each lasting about 10 minutes.

fMRI data acquisition: fMRI and neuropsychological battery measurements will be made before
the intervention. These measurements will then be repeated after 2 weeks and 12 weeks.

fMRI will image changes in regional brain activity associated with these tasks. The MRI pulse
sequences include diffusion tensor imaging, standard and non-invasive anatomical and
quantitative MRI for coregistration and blood-oxygen-level dependent (BOLD) fMRI.

CO2 challenge: Cerebral blood flow measurements will be obtained while the subject rests in
the scanner after administration of medical-grade 5% CO2 in air for 3-5 minutes. This will be
repeated on weeks 2 and 12.

Data analysis: Standard fMRI analysis will be analyzed using established fMRI software.
Statistical parametric analysis will be performed to generate activation maps. fMRI data will
be corrected for multiple comparisons using a false discovery rate (q < 0.05) and threshold
for cluster values to conservatively control for type I error. Behavioral data will be
analyzed with paired t-test and ANOVA calculations used for group comparison with p < 0.05
(with Bonferroni correction) considered statistically significant.

Expected results: The investigators predict that, compared to placebo, MB will: i) improve
working memory retention in a delayed match-to-sample task by memory performance and enhanced
fMRI responses in the prefrontal cortex and parietal lobes, ii) improve episodic memory as
determined by fMRI activation in the hippocampus, medial temporal lobes and prefrontal cortex
iii) reduce reaction time in a psychomotor vigilance test and enhance fMRI responses within a
cortical sustained attention network iv) improve CBF and v) improve fMRI connectivity in
default mode and visuospatial and memory networks/subnetworks. The fMRI and behavioral
performance effects on memory will be greater in the MCI and mild AD groups than in the
healthy aging group. The effects will be greater in the MCI and AD groups than in the control
groups.

Power analysis: Sample sizes were calculated using an fMRI power tool based on pilot data
from the current study for a power of 80%, alpha = 0.05, False Discovery Rate < 0.05, to
detect statistical difference between MB and placebo23. The investigators estimate they will
need 20-25 subjects per arm of group (complete studies) and thus will recruit 200-240
subjects to account for potential failed studies.

Inclusion Criteria for all subjects:

1. 45-89 years old

2. All genders

3. All minorities

4. English, Spanish, or multilingual speakers

5. Postmenopausal or surgically sterile females only.

6. Inclusion for MCI group only: participants will meet the criteria for amnestic and
non-amnestic MCI such as those currently used by Texas Alzheimer's Research and Care
Consortium (TARCC) consensus diagnosis

7. Inclusion for AD group only: Alzheimer's Early-stage, sporadic-type

Exclusion Criteria:

1. Pregnancy or breastfeeding

2. Contraindication for MRI (Claustrophobia and magnetic metal implants)

3. Glucose-6-phosphate deficiency, methemoglobinemia

4. Allergy to MB

5. Color-blindness

6. Craniotomy, craniectomy or endovascular neurosurgery

7. A current diagnosis of stroke, transient ischemic attack (TIA), any primary
neurodegenerative disorder, or any other causes of neuropsychologic disturbances or
secondary dementia (MCI or AD does not exclude subject)

8. A serious intercurrent illness likely to cause death within the next 5 years, such as
terminal cancer

9. Alcohol and/or drug abuse

10. Any detection of an unknown disease process (eg. new tumor) on the study's
neuroimaging at the discretion of the investigators

11. A systolic blood pressure ≥180 mmHg and/or a diastolic blood pressure ≥105 mmHg

12. Severe difficulty or an inability to perform any one of the 6 Katz Activities of Daily
Living

13. Patients who are unlikely to comply with trial visit schedule or with trial
medication,

14. On any psychiatric serotonergic antidepressant medication or psychotropic medication
within the last 5 weeks

15. Diagnosis of epilepsy, traumatic brain injury with loss of consciousness, psychosis,
panic attacks,

16. Chronic kidney disease, cirrhosis, liver or renal transplants

17. Known hypersensitivity to thiazide diuretics and phenothiazines

18. Any other condition, which in the opinion of the investigator, would put the
participant at risk and warrant exclusion from the study