This project is structured in 3 different work packages (WP), addressing different tasks, performed by all research units in an integrated way, each task being coordinated by one research unit (RU) and run in parallel to the other ones. The three years duration of the project will allow a longitudinal follow-up of patients to highlight the prognostic significance of the investigated clinical and molecular parameters.
WP1. Patient recruitment and characterization
A total of 50 patients with Mild Cognitive Impairment (MCI) and 50 patients with mild AD will be enrolled in this study at the Neurological Department of San Gerardo Hospital, University of Milano-Bicocca. The diagnoses will be defined according to standardised criteria for Mild Cognitive Impairment (MCI) (Petersen 1999) and AD (McKhann 2011) and all subjects will be characterized by structural brain imaging and neuropsychological profiling. Furthermore, all enrolled patients will undergo to cerebral amyloid PET and/or lumbar puncture to measure AD CSF biomarkers (amyloid beta-42, phosphorylated tau and total tau): only those with at least one of these two tests indicative of AD will be considered. On the basis of the clinical and neuropsychological presentation, MCI patients will be subdivided into amnesic single domain (a-MCI), amnesic multi-domain (a-md-MCI), non-amnesic single domain (na-MCI) and non amnesic multi-domain (na-md-MCI).
Baseline frailty (F) will be defined by the presence of three or more of the following components, as proposed by Fried and colleagues (2001): (1) Shrinking: weight loss, unintentional, of 10 pounds in prior year or, at follow-up, of 5% of body weight in prior year (by direct measurement of weight); (2) Weakness: grip strength in the lowest 20% at baseline, adjusted for gender and body mass index; (3) Poor endurance and energy: as indicated by self-report of exhaustion. Self-reported exhaustion, identified by two questions from the Center for Epidemiologic Studies-Depression (CES-D) scale; (4) Slowness: Walking time/15 feet: slowest 20% (by gender, height); (5) Low physical activity level: Kcals/week: lowest 20% males: ics of the enrolled population; in fact, all patients will be evaluated by an experiences team including: a behavioural neurologists, a geriatrician and a neuropsychologist, with a structured clinical interview, a full general and neurological examination, and a standard neuropsychological battery.
To be included in the study all subjects will have to give informed consent according to a protocol approved by the local ethics committee.
WP1a. clinical and short neuropsychological follow-up visits every 6 months in order to evaluate possible conversion to dementia, for MCI, or the progression rate of dementia in AD will be performed. MMSE, CDR and the Neuropsychiatric Inventory (NPI) will be included, besides drug history (AChEi and memantine; neuroleptic equivalent dose (Tremolizzo 2013).
WP2. Monocyte studies: role of the inflammasome
This WP will focus on ex vivo monocytes and in particular on the role of activated inflammasome and related cytokine production, as well as autophagic pathway and signal transduction markers.
Moreover, the eventual conversion to dementia (MCI) or the progression of dementia itself (Mild AD) will be related to the presence of inflammatory state evaluated at the time of enrollment.
Blood samples will be collected in K2EDTA to obtain peripheral blood mononuclear cells (PBMC) and monocytes. Isolation will be performed using a lymphocyte separation medium and magnetic beads respectively. Serum/plasma samples will be isolated by cooled centrifugation and stored in aliquots at – 20°C. Monocytes will be cultured in RPMI 1640 supplemented with 10% human serum, 2mM L-glutamine, and 1% penicillin (Invitrogen Ltd, Paisley, UK) and will be un-stimulated (medium) or will be stimulated with: 1) 2ug/ml Lipopolysaccharide (LPS) for 2 hours; 2) 10ug/ml of Abeta peptide oligomer for 24 hours 3) LPS (2ug/ml) for 2 hours before stimulation with 10ug/ml of Abeta peptide oligomer for 24 hours at 37°C in a humidified 5% CO2 atmosphere. LPS pre-incubation is required because either NLRP3 or pro-IL-1 is not constitutively expressed and requires transcriptional induction (Mariathasan 2006; Martinon 2006; Dostert 2008; Halle 2008).
Cultured monocytes will be analyzed by Amnis FlowSight flow cytometer to determine the percentage of cell co-expressing NLRP3 or ASC or caspase-1 or IL-1b and by confocal microscopy to evaluate the co-localization of inflammasome proteins. Quantikine immunoassay will be used to measure the following cytokine: Caspase-1, TNFa, IL-1b, IL-6, IL-18, IL-33 and IL-37 in supernatants of monocyte. In cytoplasmic protein extracts from monocytes, macroautophagy LC3 and beclin1 markers will be evaluated by western blot, while ERK, p38 and AKT signaling pathways by phospho-ELISA kits.
RNA extraction and reverse transcription: RNA will be extracted from monocytes by using the acid guanidium thiocyanate–phenol–chloroform method. cDNA will be evaluated for GAPDH expression by Real Time PCR to test the quality of RNA. Real Time quantitative Reverse Transcription PCR (RQPCR) will be performed with gene specific primers and the SybrGreen chemistry. All primers, casp-1, ASC, IL-1b, IL-18, IL-33, IL-37 and NLRP3 employed will be cDNA specific and will be purchased from Qiagen. Amplification of specific PCR products will be detected using the RT2 SYBR Green Fluor. Results will be expressed as deltaCt and will be presented as ratios between the target gene and the GAPDH housekeeping mRNA.
The functional characteristics of the “cellular” arm of the immune reflex, i.e., monocyte crossing the BBB will be assessed on ex vivo monocyte samples: chemotaxis and phagocytosis targets will include TSPO-18 and TREM2 mRNA expression. Finally, the TSPO-18 rs6971 SNP (sequencing of the rs6971 polymorphism, determining HH, HL and LL genotypes (Kreisl 2013) and the APOE genotype will be analyzed.
WP3. Serum studies: soluble peripheral biomarkers
Serum sample are easily accessible and are excellent candidates for studies with longitudinal sampling and in view of creating future standardized measurements for analytical laboratories.
In these samples, disease-specific biomarkers, i.e., Abeta 40, Abeta42, anti-Abeta 1-42 antibodies, will be initially evaluated by ELISA assay (Conti 2010; Conti 2016).
Furthermore, humoral mediators of inflammation, i.e., cytokines, representing the “soluble” afferent arm of the immune reflex, will be assessed: TNFa, IL-1b, IL-6 (Sala 2003). The TSPO-18 endogenous ligand, DBI, will be assessed in serum by ELISA.