Understanding the Role of the Innate Immune System in Alzheimer’s and Parkinson’s Disease Pathogenesis

© 2021 Jasmina MarkulicAlzheimer’s (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases causing dementia. Microglia, a resident macrophage of the brain, have been implicated in disease pathogenesis, playing key roles in clearance of toxic protein aggregates and inflammation. Recently, genetic studies have identified microglial surface receptors as risk factors for AD and PD susceptibility. The focus of this thesis was to determine the structural and functional properties of two of those proteins: human cluster of differentiation 33 (hCD33) and human triggering receptor expressed on myeloid cells 2 (hTREM2). The structure of the ligand binding domain (also known as the V-set immunoglobulin (IgV) domain) of hCD33 was expressed and purified from a bacterial expression system and solved to 2.8 A resolution. Oligomerisation of hCD33 at the plasma membrane was dependent on the integrity of the ligand binding domain; mutation of a conserved arginine residue (R119A) responsible for ligand interaction compromised hCD33 oligomerisation. Activation of hCD33 signalling appeared to be dependent on a functional ligand binding domain; hCD33 wild-type (WT) expressing cells had elevated levels of SHP-2 protein, a key phosphatase recruited to activated hCD33, whereas this was lessened in hCD33 R119A and dIgV expressing cells. Activation of WT hCD33 by 6’-sialyllactose led to upregulation of SHP-1, SHP-2 and SYK expression over time; this was not observed in hCD33 R119A or dIgV expressing cells. Hence, these results support a mechanism by which hCD33 potentiates its own downstream intracellular signalling pathways, possibly resulting in amplification of hCD33 signalling. The functional activity of hCD33 may be dictated by its cellular localisation; qualitative results showed hCD33 WT and R119A presentation to the plasma membrane, while dIgV remained in intracellular compartments. These observations suggest that the IgV domain may be involved in controlling the trafficking of hCD33, and thus its ability to be available to ligand and activated. A bacterial expression system was used to purify the hTREM2 ligand binding domain, resulting in the production of natively folded, disulfide bonded protein and extensive biophysical analysis of the IgV domain. Ligand binding assays identified one novel low affinity binder of hTREM2. Disease-associated variants of hTREM2 altered its interactions with human DAP12 (hDAP12); a reduction in hDAP12 association was observed for the hTREM2 R47H mutant, a key residue involved in ligand binding, and for the K186N mutant, a critical residue responsible for association with hDAP12. The partnership with hDAP12 may also be affected by cellular localisation; qualitative results showed that while hTREM2 WT and R47H presented at the plasma membrane, K186N remained predominately intracellular. While hDAP12 appeared to co-localise at the plasma membrane with WT hTREM2, hDAP12 was predominately intracellular with both R47H and K186N co-expression. These observations suggest that hDAP12 translocation to the plasma membrane is dependent on functional native hTREM2. The cell surface presentation of hTREM2, and interaction with hDAP12, appeared to be essential for activation of hTREM2-hDAP12 signalling; activation of Akt, a downstream kinase in the hTREM2-hDAP12, was observed in both WT hTREM2 and WT hTREM2-hDAP12 expressing cells, but not for R47H or K186N expressing cells. Together, the results from these studies demonstrate that specific residues in the ligand binding domains of the microglial surface receptors, hCD33 and hTREM2, play key roles in intracellular signalling