18 Apr 2023

Signaling through the CD33 receptor counteracts the known beneficial microglial responses to plaques, while clusterin is thought to promote Aβ aggregation. Up until now, these two AD risk factors were thought to be going about their nefarious ways independently. Not anymore. According to data presented at the International Conference on Alzheimer’s and Parkinson’s Diseases, held March 28-April 1 in Gothenburg, Sweden, it appears the two have been working together all along. Peter St. George-Hyslop of Columbia University, New York City, reported that full-length CD33, the isoform favored by risk variants in the gene, forms dimers on the microglial surface—to which none other than clusterin tightly binds. This instigates inhibitory signaling through CD33, which ultimately spoils the cell's appetite for Aβ plaques.

What’s more, this cascade is maximally riled if clusterin is complexed with Aβ oligomers while binding CD33. The findings offer an intuitive explanation for how both genes drive AD risk, and support the therapeutic strategy of blocking CD33.

The data complement news on TREM2 signaling and therapeutic strategies presented at AD/PD (see Part 8 of this series). The two receptors share a yin-yang-like relationship, with TREM2 promoting beneficial microglial responses, and CD33 thwarting them (Chan et al., 2015; Jul 2019 news). As such, efforts are underway to beef up TREM2 signaling with agonistic antibodies and small molecules, while blocking CD33 might be a complementary, arguably more straightforward tactic (Apr 2019 conference news).

Longer Means Riskier. Alternative splicing produces two isoforms of CD33; the long isoform, aka CD33M, contains a ligand binding site. The short version, aka CD33m, lacks that site and is favored by the protective variant. [Courtesy of Peter St. George-Hyslop.]

CD33 was identified as an AD risk gene 15 years ago (Oct 2008 news). Also known as Siglec-3, the transmembrane protein binds sialic acid and regulates innate immunity through its inhibitory ITIM motifs. Alternative splicing yields two isoforms: a long, “CD33M” isoform containing an extracellular ligand binding domain; and a short, “CD33m” isoform sans this domain. Importantly, AD risk variants were found to favor production of the longer form, while protective variants skewed toward the short version. AD risk variants in CD33 reportedly inhibit helpful microglial functions including Aβ phagocytosis, while the protective variants counteract those effects (Griciuc et al., 2013; Aug 2013 news).

How might one inhibit CD33 signaling, and what are the ligands responsible for imparting AD risk through the receptor? St. George-Hyslop aimed to answer these questions. To understand how the receptor works, his group took a structural approach, crystallizing the extracellular domain of the receptor and resolving its structure at 2.4 angstrom resolution. At AD/PD, St. George-Hyslop showed that the extracellular portion of the receptor folds into two domains, each consisting of two β-sheet sandwiches separated by a flexible linker region. The ligand binding site resided in one of these sandwiches, with an arginine residue positioned to bind sialic acid.

St. George-Hyslop was surprised to realize that CD33 formed dimers—a coupling that was later found to be required for CD33 to travel to the membrane. In these dimers, the ligand binding sites from each molecule of CD33 were slightly offset from one another. “This arrangement, where you have two geometrically offset ligand binding sites with a flexible stalk, raises the possibility that CD33 dimers could potentially bind much larger polysialylated ligands,” he proposed.

What polysialylated ligands might those be? After testing out sialylated sugars, which only loosely bound CD33, as well as soluble sialylated ApoE, which bound CD33 not at all, the scientists hit paydirt with another sialylated protein: clusterin. Also known as ApoJ, clusterin joins ApoE as one of the two most abundant apolipoproteins in the brain.

Before it was pegged as an AD risk factor, clusterin was found to bind Aβ and bend its aggregation toward more toxic forms (Jul 2002 news). Now considered a top AD risk gene, clusterin has been implicated in all manner of mischief, ranging from Aβ aggregation to bungling communication between different regions of the brain (Jul 2010 news; Dec 2011 news). Elevated levels of clusterin in the blood or CSF associate with worse AD pathology and neurodegeneration (Apr 2011 news; Jan 2014 news).

So what are clusterin and CD33 doing together? First of all, St. George-Hyslop ascertained the rules of engagement: Only the long isoform of CD33 bound clusterin, and clusterin needed to be sialylated for binding to occur. In human AD brain samples, his team spotted the two proteins buddied up within microglia. Their partnership was most intense in microglia near plaques; in microglia distant from plaques, or microglia in control brains, the two proteins mostly kept to themselves.

In cultured human cell lines, and in peripheral blood mononuclear cells (PBMCs), clusterin binding set off signaling through CD33’s ITIM domains. It evoked an even stronger signaling cascade when complexed with Aβ oligomers, St. George-Hyslop reported. The functional consequences of clusterin binding to CD33 were borne out in subpar microglial phagocytosis of Aβ plaques. When the scientists let loose human PBMCs onto brain slices containing Aβ plaques, they found that PBMCs from CD33 risk variant carriers had a harder time mopping up plaques than did PBMCs from carriers of the protective CD33 variant. Adding clusterin to these cultures exacerbated the poor phagocytic performance of cells harboring the risk variant.

In toto, the findings unveiled a synergism between two Alzheimer's genes. Because the CD33 risk variant contains the ligand binding domain, it is open for stimulation by clusterin and other ligands. Conversely, the protective variant of CD33 lacks the ligand binding domain, making it less likely to nix beneficial microglial responses.

To what extent the interaction between clusterin and CD33 explains how these proteins contribute to AD risk remains unclear. “It is certainly possible that clusterin binding to CD33 is part of the reason that both genes/proteins contribute to risk,” commented David Holtzman of Washington University, St. Louis. “Clusterin also directly can interact with Aβ to influence its fibrillogenesis, and this may be another way that clusterin contributes to AD risk that is distinct from mechanisms relating to its interaction with CD33.”—Jessica Shugart

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References

News Citations

1. All Roads Lead to TREM2: Gearing Up to Target This Receptor 14 Apr 2023
2. Deleting CD33 Benefits Mice—If Their Microglia Express TREM2 11 Jul 2019
3. Could CD33 Be the Microglial Target for Stimulating Phagocytosis? 23 Apr 2019
4. Four New Acts Debut on GWAS Screen 31 Oct 2008
5. Protective Microglial Gene Variant Promotes Phagocytosis 22 Aug 2013
6. Stockholm: Clusterin (or Beware the Evil Chaperone!) 22 Jul 2002
7. Research Brief: Clusterin Grabs Spotlight Among Elite Few LOAD Genes 9 Jul 2010
8. Neuroimaging Offers a CLU to AD Risk Factor’s Functional Effects 13 Dec 2011
9. Research Brief: Link Tightens Between Plasma Clusterin and AD 10 Apr 2011
10. Does Clusterin Interact With Aβ to Kick Off Neurodegeneration? 10 Jan 2014

Paper Citations

1. Chan G, White CC, Winn PA, Cimpean M, Replogle JM, Glick LR, Cuerdon NE, Ryan KJ, Johnson KA, Schneider JA, Bennett DA, Chibnik LB, Sperling RA, Bradshaw EM, De Jager PL. CD33 modulates TREM2: convergence of Alzheimer loci. Nat Neurosci. 2015 Nov;18(11):1556-8. Epub 2015 Sep 28 PubMed.
2. Griciuc A, Serrano-Pozo A, Parrado AR, Lesinski AN, Asselin CN, Mullin K, Hooli B, Choi SH, Hyman BT, Tanzi RE. Alzheimer's disease risk gene CD33 inhibits microglial uptake of amyloid beta. Neuron. 2013 May 22;78(4):631-43. PubMed.

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