The SAUNA Study: a blood- and stool-based investigation of markers correlating with hallmarks of dementia

Study design: A cross-sectional study. The exposure will be participants who test positive for amyloid plaques, and the comparator group will be participants who test negative for amyloid plaques. This study will be utilising an existing workflow/recruitment regime, and will not be assigning any form of treatment to individuals. Rather, the study will be recruiting participants as they enroll into the ADNeT program. Primary aim: to understand if a cellular recycling process called autophagy is different in people who have the molecular hallmarks of Alzheimer’s disease compared with people who do not. Secondary aim: To determine if the gut microbiome composition and structure, measured in stool samples, is altered in people who have the molecular hallmarks of Alzheimer’s disease. Setting: The study is a cross-sectional study that will recruit participants from the Australian Dementia Network (ADNet) - Screens and Trials Ready Cohort (TRC). The ADNet TRC is a phase I, multiple site open label study that aims to recruit individuals who have expressed an interest in being involved in dementia research and/or clinical trials to test interventions for Alzheimer's disease and other neurodegenerative diseases causing dementia. Only participants from the South Australian Health and Medical Research Institute (SAHMRI) site in South Australia will be invited to participate in this study. Recruitment/screening: 160 male and female individuals, of at least 40 years of age, who have been recruited into the ongoing ADNet TRC study. These people will be cognitively intact and will not meet criteria for mild cognitive impairment or dementia. Blood collection: Venepuncture will be performed as part of the ADNet TRC study. An additional blood collection will be sampled for the SAUNA study during the same venepuncture procedure. Stool collection: Faecal samples will be entirely optional and not a requirement following blood collection. Willing participants will be provided with a stool collection kit, and will be instructed on how to collect a stool sample by an experienced and qualified staff member. It will be stressed to participants that they are not required to provide a stool sample if they choose to provide blood samples, and that it is an entirely optional and additional investigation. Willing participants will also be provided with a pre-paid postage envelope, so that they may collect their stool sample independently, within the privacy and comfort of their own home, and securely and privately post their samples to the research laboratory, within one week of blood collection. Randomisation: No randomisation will be performed as this is a cross-sectional study and group membership will be determined according to whether participants have positive amyloid beta PET scans during the ADNet TRC. Autophagic flux measurement: Blood samples will be processed immediately after collection for measurement of autophagic flux according to the method developed by our group. Autophagic flux will be determined by the difference in the accumulation of LC3BII over time between blocked and unblocked samples (i.e. what should have been degraded during the one-hour incubation) using the equation deltaLC3II = LC3II+CQ – LC3II-CQ. Autophagic flux will be compared between groups of amyloid positive and amyloid negative individuals. Plasma hormones, amino acids and lipids: Plasma will be snap frozen at -80C, for later assessment of blood glucose, insulin, blood lipids, free fatty acids, glucose regulating hormones (e.g.: insulin, C-peptide, adiponectin), inflammatory cytokines (e.g.: C-reactive protein), and appetite-regulating hormones (eg: GLP-1, GIP, Ghrelin) using commercially available kits. Plasma amino acids may be analysed by mass spectrometry. Samples will be compared between groups of amyloid positive and amyloid negative individuals. Gut microbiome characterisation: Stool samples will be provided by participants in the comfort and privacy of their own home through the use of stool nucleic acid collection and preservation tubes (Norgen, Biotek, Canada). Stabilised samples will be collected from participants via pre-paid post for sample processing, metagenomic sequencing, and whole genome sequencing, which will be performed at the SAHMRI Microbiome Research Laboratory, using established protocols. Briefly, DNA extraction will be performed, prior to 150 bp paired-end metagenomic shotgun sequencing using the Illumina HiSeq system at the SAHMRI Genomics Core. Sequences will be filtered, human-derived reads removed and destroyed. Importantly, no human DNA sequence that is generated by microbiome characterisation will be analysed as a part of this study. Microbiome characteristics will be compared between groups of amyloid positive and amyloid negative individuals. Additional data from ADNet TRC Data collected from ADNet may be used by the SAUNA study to aid interpretation of autophagy measurement in this cohort. This may include age, sex, weight/BMI, data taken from medical history, eligibility assessment, alcohol use tests, memory complaint instruments, mood indexes and scales, sleep indexes and questionnaires, functional independence questionnaires, physical activity questionnaires, diet questionnaires, neuropsychological and cognitive assessment, MRI, amyloid beta PET, and tau PET. Data collected will from recruitment up to 2 years post-enrolment.