Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling

BACKGROUND: Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain. RESULTS: Cohorts of mice were pulse labeled with (13) C(6)-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid β (Aβ). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aβ clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aβ deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain. CONCLUSIONS: Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound

The accumulation of amyloid beta (Aβ) in Alzheimer’s disease is caused by an imbalance of production and clearance, which leads to increased soluble Aβ species and extracellular plaque formation in the brain. Multiple Aβ-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Aβ, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Aβ physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a γ-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Aβ (β- and γ-secretase) is that precursors of Aβ may accumulate and cause a rapid increase in Aβ production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable isotope labeling, and dose-dependently reduced newly generated CNS Aβ. In contrast to systemic Aβ metabolism, CNS Aβ production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Aβ, including C-terminal truncated forms of Aβ: 1–14, 1–15 and 1–16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during γ-secretase inhibition

In Vivo Human Apolipoprotein E Isoform Fractional Turnover Rates in the CNS

Apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer’s disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis

Baseline [18F]GTP1 tau PET imaging is associated with subsequent cognitive decline in Alzheimer’s disease

BackgroundThe role and implementation of tau PET imaging for predicting subsequent cognitive decline in Alzheimer's disease (AD) remains uncertain. This study was designed to evaluate the relationship between baseline [18F]GTP1 tau PET and subsequent longitudinal change across multiple cognitive measures over 18 months.MethodsOur analyses incorporated data from 67 participants, including cognitively normal controls (n = 10) and β-amyloid (Aβ)-positive individuals ([18F] florbetapir Aβ PET) with prodromal (n = 26), mild (n = 16), or moderate (n = 15) AD. Baseline measurements included cortical volume (MRI), tau burden ([18F]GTP1 tau PET), and cognitive assessments [Mini-Mental State Examination (MMSE), Clinical Dementia Rating (CDR), 13-item version of the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog13), and Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)]. Cognitive assessments were repeated at 6-month intervals over an 18-month period. Associations between baseline [18F]GTP1 tau PET indices and longitudinal cognitive performance were assessed via univariate (Spearman correlations) and multivariate (linear mixed effects models) approaches. The utility of potential prognostic tau PET cut points was assessed with ROC curves.ResultsUnivariate analyses indicated that greater baseline [18F]GTP1 tau PET signal was associated with faster rates of subsequent decline on the MMSE, CDR, and ADAS-Cog13 across regions of interest (ROIs). In multivariate analyses adjusted for baseline age, cognitive performance, cortical volume, and Aβ PET SUVR, the prognostic performance of [18F]GTP1 SUVR was most robust in the whole cortical gray ROI. When AD participants were dichotomized into low versus high tau subgroups based on baseline [18F]GTP1 PET standardized uptake value ratios (SUVR) in the temporal (cutoff = 1.325) or whole cortical gray (cutoff = 1.245) ROIs, high tau subgroups demonstrated significantly more decline on the MMSE, CDR, and ADAS-Cog13.ConclusionsOur results suggest that [18F]GTP1 tau PET represents a prognostic biomarker in AD and are consistent with data from other tau PET tracers. Tau PET imaging may have utility for identifying AD patients at risk for more rapid cognitive decline and for stratification and/or enrichment of participant selection in AD clinical trials. Trial registration ClinicalTrials.gov NCT02640092 . Registered on December 28, 2015

Safety and Efficacy of Semorinemab in Individuals With Prodromal to Mild Alzheimer Disease: A Randomized Clinical Trial

Importance: Neurofibrillary tangles composed of aggregated tau protein are one of the neuropathological hallmarks of Alzheimer disease (AD) and correlate with clinical disease severity. Monoclonal antibodies targeting tau may have the potential to ameliorate AD progression by slowing or stopping the spread and/or accumulation of pathological tau. Objective: To evaluate the safety and efficacy of the monoclonal anti-tau antibody semorinemab in prodromal to mild AD. Design, Setting, and Participants: This phase 2 randomized, double-blind, placebo-controlled, parallel-group clinical trial was conducted between October 18, 2017, and July 16, 2020, at 97 sites in North America, Europe, and Australia. Individuals aged 50 to 80 years (inclusive) with prodromal to mild AD, Mini-Mental State Examination scores between 20 and 30 (inclusive), and confirmed β-amyloid pathology (by positron emission tomography or cerebrospinal fluid) were included. Interventions: During the 73-week blinded study period, participants received intravenous infusions of placebo or semorinemab (1500 mg, 4500 mg, or 8100 mg) every 2 weeks for the first 3 infusions and every 4 weeks thereafter. Main Outcomes and Measures: The primary outcomes were change from baseline on the Clinical Dementia Rating-Sum of Boxes score from baseline to week 73 and assessments of the safety and tolerability for semorinemab compared with placebo. Results: In the modified intent-to-treat cohort (n = 422; mean [SD] age, 69.6 [7.0] years; 235 women [55.7%]), similar increases were seen on the Clinical Dementia Rating-Sum of Boxes score in the placebo (n = 126; Δ = 2.19 [95% CI, 1.74-2.63]) and semorinemab (1500 mg: n = 86; Δ = 2.36 [95% CI, 1.83-2.89]; 4500 mg: n = 126; Δ = 2.36 [95% CI, 1.92-2.79]; 8100 mg: n = 84; Δ = 2.41 [95% CI, 1.88-2.94]) arms. In the safety-evaluable cohort (n = 441), similar proportions of participants experienced adverse events in the placebo (130 [93.1%]) and semorinemab (1500 mg: 89 [88.8%]; 4500 mg: 132 [94.7%]; 8100 mg: 90 [92.2%]) arms. Conclusions and Relevance: In participants with prodromal to mild AD in this randomized clinical trial, semorinemab did not slow clinical AD progression compared with placebo throughout the 73-week study period but did demonstrate an acceptable and well-tolerated safety profile. Additional studies of anti-tau antibodies may be needed to determine the clinical utility of this therapeutic approach. Trial Registration: ClinicalTrials.gov Identifier: NCT03289143

Amyloid positron emission tomography and cerebrospinal fluid results from a crenezumab anti-amyloid-beta antibody double-blind, placebo-controlled, randomized phase II study in mild-to-moderate Alzheimer’s disease (BLAZE)

Abstract Background We investigated the effect of crenezumab, a humanized anti-amyloid-beta (Aβ) immunoglobulin (Ig)G4 monoclonal antibody, on biomarkers of amyloid pathology, neurodegeneration, and disease progression in patients with mild-to-moderate Alzheimer’s disease (AD). Methods This double-blind, placebo-controlled, randomized phase II study enrolled patients with mild-to-moderate AD and a Mini-Mental State Examination (MMSE) score of 18–26. In part 1 of the study, patients were 2:1 randomized to receive low-dose subcutaneous (SC) 300 mg crenezumab every 2 weeks (q2w) or placebo for 68 weeks; in part 2, patients were 2:1 randomized to receive high-dose intravenous (IV) 15 mg/kg crenezumab every 4 weeks (q4w) or placebo for 68 weeks. The primary endpoint was change in amyloid burden from baseline to week 69 assessed by florbetapir positron emission tomography (PET) in the modified intent-to-treat population. Secondary endpoints were change from baseline to week 69 in cerebrospinal fluid (CSF) biomarkers and fluorodeoxyglucose PET, and change from baseline to week 73 in 12-point Alzheimer’s Disease Assessment Scale cognitive subscale (ADAS-Cog12) and Clinical Dementia Rating Sum of Boxes (CDR-SB). Safety was assessed in patients who received at least one dose of study treatment. Results From August 2011 to September 2012, 91 patients were enrolled and randomized (low-dose SC cohort: crenezumab (n = 26) or placebo (n = 13); high-dose IV cohort: crenezumab (n = 36) or placebo (n = 16)). The primary endpoint was not met using a prespecified cerebellar reference region to calculate standard uptake value ratios (SUVRs) from florbetapir PET. Exploratory analyses using subcortical white matter reference regions showed nonsignificant trends toward slower accumulation of plaque amyloid in the high-dose IV cohort. In both cohorts, a significant mean increase from baseline in CSF Aβ(1–42) levels versus placebo was observed. Nonsignificant trends toward ADAS-Cog12 and CDR-SB benefits were identified in a mild (MMSE 20–26) subset of the high-dose IV cohort. No amyloid-related imaging abnormalities due to edema/effusion were observed. Conclusion The primary endpoint was not met. Exploratory findings suggest potential Aβ target engagement with crenezumab and possible slower accumulation of plaque amyloid. Studies investigating the effects of higher doses of crenezumab on amyloid load and disease progression are ongoing. Trial registration ClinicalTrials.gov, NCT01397578. Registered on 18 July 2011