Amyloid β-Protein (1-15)

Amyloid-β(1-15/16) as a marker for γ-secretase inhibition in Alzheimer's disease

Amyloid-β (Aβ) producing enzymes are key targets for disease-modifying Alzheimer's disease (AD) therapies since Aβ trafficking is at the core of AD pathogenesis. Development of such drugs might benefit from the identification of markers indicating in vivo drug effects in the central nervous system. We have previously shown that Aβ(1-15) is produced by concerted β-and α-secretase cleavage of amyloid-β protein precursor (AβPP). Here, we test the hypothesis that this pathway is more engaged upon γ-secretase inhibition in humans, and cerebrospinal fluid (CSF) levels of Aβ(1-15/16) represent a biomarker for this effect. Twenty healthy men were treated with placebo (n = 5) or the γ-secretase inhibitor semagacestat (100 mg [n = 5], 140 mg [n = 5], or 280 mg [n = 5]). CSF samples were collected hourly over 36 hours and 10 time points were analyzed by immunoassay for Aβ(1-15/16), Aβ(x-38), Aβ(x-40), Aβ(x-42), sAβPPα, and sAβPPβ. The CSF concentration of Aβ(1-15/16) showed a dose-dependent response over 36 hours. In the 280 mg treatment group, a transient increase was seen with a maximum of 180% relative to baseline at 9 hours post administration of semagacestat. The concentrations of Aβ(x-38), Aβ(x-40), and Aβ(x-42) decreased the first 9 hours followed by increased concentrations after 36 hours relative to baseline. No significant changes were detected for CSF sAβPPα and sAβPPβ. Our data shows that CSF levels of Aβ(1-15/16) increase during treatment with semagacestat supporting its feasibility as a pharmacodynamic biomarker for drug candidates aimed at inhibiting γ-secretase-mediated AβPP-processing.

124I/125I-Fibril-reactive monoclonal antibody

¹²⁴I-Fibril-reactive monoclonal antibody (¹²⁴I-11-1F4 MAb) is a radiolabeled antibody that was developed for positron emission tomography (PET) imaging of primary systemic amyloid light –chain (AL) amyloidosis (1). ¹²⁴I is a positron emitter with a physical half-life (t_?) of 4.2 days. ¹²⁵I-11-1F4 MAb is used for small animal studies. ¹²⁵I-11-1F4 MAb was also used in the animal investigation for small animal single-photon emission computed tomography (SPECT) studies, but ¹²⁵I is a low-energy gamma emitter with a long physical half-life (t_?) of 60 days.

Amyloidosis describes a heterogeneous group of diseases with a common feature of the extracellular deposition of amyloid proteins in various tissues (2). Amyloid is an amorphous, homogeneous, hyaline-like, eosinophilic substance. Despite its homogeneous appearance, it has a fibrillar structure that consists of linear, non-branching, aggregrated fibrils. The abnormal deposition of this relatively inert fibrillar material in the extracellular compartment of various organs leads to interference with normal functions and destruction of involved tissues and organs. At least 13 different proteins have been identified as human amyloid fibril precursors (2, 3). Primary or idiopathic systemic AL amyloidosis is one of the most common subtypes of amyloidosis. It is a pathologic condition of monoclonal plasma cell dyscrasia (4-6). It is frequently characterized by an abnormal neoplastic proliferation of plasma cells, which synthesize amyloidogenic immunoglobulin light chains. The variable region of the light chain is highly mutated, and this translates into structural heterogeneity and several possible mechanistic pathways of fibril formation (7). The disease may involve the heart, kidney, liver, spleen, tongue, nerves, as well as the vascular system and other body organs/tissues (8)

Monitoring the pathogenesis of these diseases and their responses to treatments is difficult and has traditionally relied on the histologic evaluation of tissue biopsies, and surveys of surrogate physiologic markers. Thus, a specific and noninvasive method for the estimation of amyloid deposition is needed. Hawkins et al. (9-11) demonstrated the possibility of imaging pathologic deposits in vivo with ¹²³I-labeled human serum amyloid P component (SAP). Antiproteases are also known to be present in amyloid deposits, and ^99mTc-labeled aprotinin (an anti-serine protease) has been studied for AL amyloid imaging with some success in Europe (12, 13).

Solomon et al. (4, 14) developed a murine amyloid-reactive monoclonal antibody, IgG_1к, 11-1F4 MAb (m11-1F4 MAb), for immunotherapy of AL amyloidosis. This MAb was prepared against a к4 Bence Jones protein. It appeared to react specifically with AL fibrils, regardless of their к or λ constant region isotype or variable region subgroup but did not recognize native (soluble) light chains. The 11-F4 MAb has now been chimerized (c11-1F4 MAb) and is undergoing good manufacturing practice production for phase I and II clinical trials. Wall et al. (1, 15) successfully radioiodinated 11-1F4 MAb with ¹²⁴I and ¹²⁵I for in vivo imaging and provided the basis for future clinical trials of ¹²⁴I-11-1F4 MAb as a potential diagnostic tool in patients with AL amyloidosis and other systemic amyloidoses.

Serum amyloid A and C-reactive protein levels may predict microalbuminuria and macroalbuminuria in newly diagnosed type 1 diabetic patients

Background: In this study we evaluated the association of baseline levels of six different candidate proteins for the development of microalbuminuria and macroalbuminuria in type 1 diabetic patients, who were followed for approximately 30 years. Two of the proteins are markers of inflammation: serum amyloid A (SAA) and C-reactive protein (CRP), three are involved in lipid metabolism: apolipoprotein A1, apolipoprotein E and adiponectin and the last protein, fibronectin, is related to structural changes.
Methods: A nested case control study population of 60 patients from an inception cohort of type 1 diabetic patients where 20 developed microalbuminuria followed by macroalbuminuria and 40 stayed normoalbuminuric during approximately 30 years of follow-up time was used to evaluate baseline levels of the six candidate biomarkers. The proteins were quantified by multiplexed immunoassays.
Results: Log SAA levels were borderline predictor of microalbuminuria, HR 2.31 (1-5.4) p=0.053 in a univariate Cox regression model and predicted the development of macroalbuminuria HR 2.432 (1-6) p=0.049, also univariate. When adjusting for covariates, log SAA predicted the development of microalbuminuria with an HR 4.131 (1.1-15) p=0.03. Log CRP predicted the development of microalbuminuria, HR 2.928 (1.4-6.1) p=0.004, and macroalbuminuria, HR 2.785 (1.3-5.8) p=0.007 in univariate models. When adjusting for covariates, log CRP predicted the development of microalbuminuria with an HR 5.882 (1.7-20.9) p=0.006 and macroalbuminuria with an HR 3.233 (1.1-9.8) p=0.038. Apolipoprotein A1, apolipoprotein E, fibronectin and adiponectin were not associated with development of elevated albumin excretion rate.
Conclusions: SAA and CRP baseline levels predicted development of micro- and macroalbuminuria during 30 years of follow up, supporting the theory that inflammation is involved in the progression of diabetic nephropathy. Further studies are needed to fully establish the two proteins' potential as additional biomarkers for the development of diabetic nephropathy.

Synthesis aided structural determination of amyloid-β(1-15) glycopeptides, new biomarkers for Alzheimer's disease

Unique tyrosine glycosylated amyloid-β(1-15) glycopeptides were synthesized with well-defined stereochemistry at the glycosidic linkages. Aided by these glycopeptides and tandem mass spectrometry analysis, the naturally existing amyloid-β glycopeptides, isolated from Alzheimer's disease patients, were determined to contain an α-linked N-acetyl galactosamine at the modified tyrosine 10 residue. Glycosylation can significantly impact the properties of amyloid-β as the glycopeptide has much lower affinity for Cu(+) ions.

Association of Cortical β-Amyloid Protein in the Absence of Insoluble Deposits With Alzheimer Disease

Importance: β-Amyloid deposits are a pathologic hallmark of Alzheimer disease (AD). However, the extent to which cortical β-amyloid protein in the absence of insoluble deposits is associated with classic features of AD appear to be unknown.
Objective: To examine the associations of cortical β-amyloid protein in the absence of insoluble deposits with cognitive decline, neurofibrillary tangles, other age-associated neuropathologic conditions, and APOE.
Design, setting, and participants: This analysis combines data from 2 community-based clinicopathologic cohort studies of aging. The Religious Orders Study started in 1994, and the Rush Memory and Aging Project started in 1997. Both studies are ongoing. Participants without known dementia were enrolled and agreed to annual clinical evaluations and brain donation after death. Primary analyses focused on individuals without β-amyloid deposits. Data analyses occurred in mid-September 2018.
Main outcomes and measures: β-Amyloid protein abundance was measured by targeted proteomics using selected reaction monitoring. β-Amyloid deposits were detected using immunohistochemistry. Other neuropathologic indices were quantified via uniform structured evaluation. Linear mixed models were used to examine the association of β-amyloid protein with cognitive decline. Regression models examined the protein associations with neuropathologic outcomes and the APOE genotype.
Results: By mid-September 2018, 3575 older persons were enrolled, and 1559 had died and undergone brain autopsy. Proteomic data were collected in 1208 individuals, and 5 with missing cognitive scores were excluded. Of the remaining 1203, primary analyses focused on 148 individuals (12.3%) without β-amyloid deposits. In this group, the mean (SD) age at death was 87.0 (7.0) years, and 84 individuals (56.8%) were women. In the absence of β-amyloid deposits, we did not observe an association of β-amyloid protein with decline in episodic memory, but the protein was associated with faster rates of decline in processing speed (mean [SE] change, -0.014 [0.005]; P = .008) and visuospatial abilities (mean [SE] change, -0.013 [0.005]; P = .006). We did not observe protein association with paired helical filament tau tangle density. The protein was associated with amyloid angiopathy (odds ratio, 1.38 [95% CI, 1.15-1.67]; P < .001) but no other brain pathology. The associations with cognitive decline were unchanged after controlling for amyloid angiopathy. Neither APOE ε4 nor a polygenic Alzheimer risk score was associated with β-amyloid protein.
Conclusions and relevance: Cortical β-amyloid protein was associated with faster cognitive decline in the absence of β-amyloid deposits, which supports the role of cortical soluble β-amyloid as a neurotoxic agent in aging. The lack of protein association with paired helical filament tau tangles, episodic memory decline, or strong genetic drivers of deposited β-amyloid suggests an underlying neuropathologic change that may differ from that of AD.