Evaluation of CD33 as a genetic risk factor for Alzheimer's disease

Abstract

In 2011, genome-wide association studies implicated a polymorphism near CD33 as a genetic risk factor for Alzheimer's disease. This finding sparked interest in this member of the sialic acid-binding immunoglobulin-type lectin family which is linked to innate immunity. Subsequent studies found that CD33 is expressed in microglia in the brain and then investigated the molecular mechanism underlying the CD33 genetic association with Alzheimer's disease. The allele that protects from Alzheimer's disease acts predominately to increase a CD33 isoform lacking exon 2 at the expense of the prototypic, full-length CD33 that contains exon 2. Since this exon encodes the sialic acid ligand-binding domain, the finding that the loss of exon 2 was associated with decreased Alzheimer's disease risk was interpreted as meaning that a decrease in functional CD33 and its associated immune suppression was protective from Alzheimer's disease. However, this interpretation may need to be reconsidered given current findings that a genetic deletion which abrogates CD33 is not associated with Alzheimer's disease risk. Therefore, integrating currently available findings leads us to propose a model wherein the CD33 isoform lacking the ligand-binding domain represents a gain of function variant that reduces Alzheimer's disease risk.

Keywords: Alzheimer’s disease; CD33; Immunoreceptor tyrosine-based activation motif; Immunoreceptor tyrosine-based inhibitory motif; Inflammation; Molecular genetics; RNA splicing; SIGLEC.

Figure 1:. SIGLEC Expression in Human Microglia.

SIGLECs with microglial expression greater than 1 FPKM (Fragments Per Kilobase of transcript per Million mapped reads) are shown. Additional CD33-related SIGLECs expressed in the human brain include SIGLEC7 (0.9 FPKM), SIGLEC16 (0.8 FPKM), SIGLEC11 (0.4 FPKM) and SIGLEC12 (0.2 FPKM). Non-CD33-related SIGLECs with microglial expression include SIGLEC1 (0.5 FPKM) and CD22 (0.2 FPKM). Two SIGLECs expressed predominantly in oligodendrocytes include Myelin Associated Glycoprotein (SIGLEC4) at 1.5 FPKM, and CD22 (SIGLEC2) at 3.7 FPKM are not shown. These data (mean±SD, n=3) are derived from http://web.stanford.edu/group/barres_lab/brainseq2/brainseq2.html [77]. Other microglial RNAseq studies report similar SIGLEC expression profiles [78].

Figure 2.. Structures of SIGLEC Family Members Expressed in Human Brain Microglia.

SIGLECs expressed at >1 FPKM in microglia are depicted. The IgV (V) domain is amino-terminal while the IgC2 (C) domains are proximal to the membrane. While most brain SIGLECs contain an ITIM (IT) or ITIM-like domain (ITL), SIGLEC14 lacks these motifs and instead has a lysine in the transmembrane domain that is used to signal to DAP12 [25].

Figure 3.. CD33 ITIM and ITIM-like Sequences Aligned to Consensus ITIM sequence.

The CD33 ITIM and ITIM-like sequences (underlined) are shown relative to the ITIM consensus sequence of S/L/V/IxYxxL/V/I. Note that the CD33 ITIM-like sequence begins with a threonine, as opposed to the consequence S/L/V/I, and hence is relatively well-conserved with the consensus. The spacer between the ITIM and ITIM-like sequence in CD33 is 12 amino acids.

Figure 4.. Structure of CD33 Gene, Common CD33 mRNA Isoforms, and their Predicted Protein Domains.

The sites of genetic variants relevant to this review are depicted on the CD33 exon structure. The modular nature of SIGLEC exons and protein domains are reflect in the structures of full-length CD33, CD33 lacking exon 2 (D2-CD33), and a secreted IgV domain arising from a four bp indel in exon 3 (noted by carot). This diagram is not drawn to scale, e.g., full-length CD33 protein is 364 amino acids, exon 2 encodes 127 amino acids and exon 3 encodes 93 amino acids. The cytosolic tail consists of 82 amino acids. Alternative splicing produces an atypical exon 7 that is less abundant and does not include ITIM motifs (not depicted).

Figure 5.. Rs3865444 Allele Dose Dependent Association with CD33 Exon 2 Splicing Efficiency.

C is the major rs3865444 allele and A the minor allele. Each point represents the qPCR result from a different human brain RNA sample. Figure redrawn from Malik et al., with permission [56].

Figure 6.. CD33, D2-CD33 Homo- and Hetero-dimers, and IgV domain derived from CD33 indel.

CD33 homodimer model is based on CD33 x-ray crystallography (PDB:5J06 (www.rcsb.org/structure/5J06) [79]). D2-CD33 and indel models are derived from this CD33 model. Extracellular domains are shown. There are two monomers: one depicted as pink/red or the other as light blue/blue. The IgV domains are shown as pink or light blue and the IgC2 domains as red or dark blue. The arginine critical for sialic acid ligand binding is marked in green. Disulfide bonds are marked in yellow and circled in dotted orange. A CD33 monomer is stabilized by disulfide bonds within the IgV domain (aa41–101), within the IgC2 domain (aa163–212) and between the IgV and IgC2 domains (aa36–169, see dotted orange circles in CD33 homodimer). These three disulfide bonds are a common feature of all the SIGLECs [80]. D2-CD33 retains the disulfide bond within the IgC2 domain and has an unpaired cysteine that may be relevant to D2-CD33 gain of function. Conversely, the indel secreted IgV domain retains the disulfide bond within the IgV domain and also has an unpaired cysteine.

Figure 7.. Comparison of CD33 ITIM and ITIM-like Region with Consensus ITAM Sequence.

The consensus ITAM sequence is D/ExxYxxL//I x_6–12YxxL/V/I. Similar variations with the ITAM consensus sequence have been reported for functional ITAMs previously [–47, 49]. ITAM sequences with longer spacers have also been reported to bind and activate Syk [74, 81].

Figure 8:. Working model of D2-CD33 neuroprotection in AD.

Full-length CD33 recruits phosphatases such as SHP-1 and SHP-2. Targets of SHP-1 include spleen tyrosine kinase (Syk) and scaffolding proteins such as the B cell linker protein BLNK. SHP-1 dephosphorylates these proteins, thus acting as a negative regulator for microglial activation. Syk activation leads to multiple downstream cellular events, including calcium mobilization via phospholipase C γ activity, protein kinase B (AKT) signaling, and extracellular-related kinase (MAPK/ERK) signaling. These events lead to metabolic and transcriptional changes, along with context-dependent increases in activities such as phagocytosis and chemotaxis. D2-CD33 may be locked in a constitutively active or permissive conformation, leading to Syk recruitment at the membrane, and ultimately resulting in a higher propensity toward microglial activation through mechanisms similar to the well-recognized functions of TREM2 and its co-receptor, DAP12.