Molecular dissection of Alzheimer's disease neuropathology by depletion of serum amyloid P component

Abstract

New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Abeta amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated tau protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.

Fig. 1.

CPHPC administration in Alzheimer's disease. CPHPC concentration in cerebrospinal fluid (A) and SAP concentrations in the plasma (B) and cerebrospinal fluid (C) in 5 patients with Alzheimer's disease are shown before, during, and after treatment with CPHPC for 12 weeks.

Fig. 2.

Mass spectrometry of SAP under nondissociating conditions before (A) and after (B) cross-linking with CPHPC followed by bis(sulfosuccinimidyl) suberate (BS3). A very small amount of decamer is seen in the native SAP preparation, reflecting the known tendency of isolated pure human SAP to autoaggregate in this way (37). After cross-linking by CPHPC and BS3, >80% of the SAP was in the decameric assembly.

Fig. 3.

Structure of the SAP-CPHPC complex in solution. SAP decamer cross-linked by CPHPC and BS3 is shown, with (Inset) a close-up of the SAP dimer interface showing the position of residue Lys-143 in relation to the CPHPC binding site. The bis(sulfosuccinimidyl)suberate (BS3) cross-linker is shown as a yellow dashed line, residues 138 to 144 are shown in red, and the calcium atoms are shown in orange. The sequence of the unique peptide produced by proteolysis of the complex after covalent cross-linking by BS3 is shown below.

Fig. 4.

3D x-ray crystal structure of O-phospho-l-threonine bound by SAP. (A) There is clear electron density (Fo-Fc omit map contoured at 3σ) for 1 molecule of O-phospho-l-threonine bound in the double-calcium site of each subunit of SAP through the phosphate oxygens. The Cγ-methyl group of the amino acid side chain is positioned within the adjacent Leu-62/Tyr-64/Tyr-74 pocket that is also occupied by the proline ring of bound CPHPC (21). The amino and carboxylate components are positioned so that a turn with O-phospho-l-threonine at the apex might visit the site, with NH and CO forming hydrogen bonds with Tyr-74-OH and Gln-148. Calcium atoms are shown in yellow and phosphorus in orange. (B) SAP pentamer showing the 5 calcium binding sites occupied by O-phospho-l-threonine with calcium atoms shown in yellow.