ABSTRACT
The creatine-phosphocreatine cycle serves as a crucial temporary energy buffering system in the brain, regulated by brain creatine kinase (CKB), in maintaining Adenosine triphosphate (ATP) levels. Alzheimer's disease (AD) has been linked to increased CKB oxidation and loss of its regulatory function, although specific pathological processes and affected cell types remain unclear. In our study, cerebral cortex samples from individuals with AD, dementia with Lewy bodies (DLB), and age-matched controls were analyzed using antibody-based methods to quantify CKB levels and assess alterations associated with disease processes. Two independently validated antibodies exclusively labeled astrocytes in the human cerebral cortex. Combining immunofluorescence (IF) and mass spectrometry (MS), we explored CKB availability in AD and DLB cases. IF and Western blot analysis demonstrated a loss of CKB immunoreactivity correlated with increased plaque load, severity of tau pathology, and Lewy body pathology. However, transcriptomics data and targeted MS demonstrated unaltered total CKB levels, suggesting posttranslational modifications (PTMs) affecting antibody binding. This aligns with altered efficiency at proteolytic cleavage sites indicated in the targeted MS experiment. These findings highlight that the proper function of astrocytes, understudied in the brain compared with neurons, is highly affected by PTMs. Reduction in ATP levels within astrocytes can disrupt ATP-dependent processes, such as the glutamate-glutamine cycle. As CKB and the creatine-phosphocreatine cycle are important in securing constant ATP availability, PTMs in CKB, and astrocyte dysfunction may disturb homeostasis, driving excitotoxicity in the AD brain. CKB and its activity could be promising biomarkers for monitoring early-stage energy deficits in AD.
Subject(s)
Alzheimer Disease , Astrocytes , Humans , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Astrocytes/metabolism , Astrocytes/pathology , Aged , Male , Female , Aged, 80 and over , Creatine Kinase, BB Form/metabolism , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Lewy Body Disease/metabolism , Lewy Body Disease/pathology , Creatine Kinase/metabolism , tau Proteins/metabolismABSTRACT
BACKGROUND: Developing and implementing new patient-centric strategies for drug trials lowers the barrier to participation for some patients by reducing the need to travel to research sites. In early chronic kidney disease (CKD) trials, albuminuria is the key measure for determining treatment effect prior to pivotal kidney outcome trials. METHODS: To facilitate albuminuria sample collection outside of a clinical research site, we developed 2 quantitative microsampling methods to determine the urinary albumin to creatinine ratio (UACR). Readout was performed by LC-MS/MS. RESULTS: For the Mitra device the within-batch precision (CV%) was 2.8% to 4.6% and the between-batch precision was 5.3% to 6.1%. Corresponding data for the Capitainer device were 4.0% to 8.6% and 6.7% to 9.0%, respectively. The storage stability at room temperature for 3 weeks was 98% to 103% for both devices. The recovery for the Mitra and Capitainer devices was 104% (SD 7.0%) and 95 (SD 7.4%), respectively. The inter-assay comparison of UACR assessment generated results that were indistinguishable regardless of microsampling technique. The accuracy based on LC-MS/MS vs analysis of neat urine using a clinical chemistry analyzer was assessed in a clinical setting, resulting in 102 ± 8.0% for the Mitra device and 95 ± 10.0% for the Capitainer device. CONCLUSIONS: Both UACR microsampling measurements exhibit excellent accuracy and precision compared to a clinical chemistry analyzer using neat urine. We applied our patient-centric sampling strategy to subjects with heart failure in a clinical setting. Precise UACR measurements using quantitative microsampling at home would be beneficial in clinical drug development for kidney therapies.