Microdissected Pyramidal Cell Proteomics of Alzheimer Brain Reveals Alterations in Creatine Kinase B-Type, 14-3-3-γ, and Heat Shock Cognate 71

Abstract

Novel insights on proteins involved in Alzheimer’s disease (AD) are needed. Since multiple cell types and matrix components are altered in AD, bulk analysis of brain tissue maybe difficult to interpret. In the current study, we isolated pyramidal cells from the cornu ammonis 1 (CA1) region of the hippocampus from five AD and five neurologically healthy donors using laser capture microdissection (LCM). The samples were analyzed by proteomics using 18O-labeled internal standard and nano-high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for relative quantification. Fold change between AD and control was calculated for the proteins that were identified in at least two individual proteomes from each group. From the 10 cases analyzed, 62 proteins were identified in at least two AD cases and two control cases. Creatine kinase B-type (CKB), 14-3-3-γ, and heat shock cognate 71 (Hsc71), which have not been extensively studied in the context of the human AD brain previously, were selected for further studies by immunohistochemistry (IHC). In hippocampus, semi-quantitative measures of IHC staining of the three proteins confirmed the findings from our proteomic analysis. Studies of the same proteins in the frontal cortex revealed that the alterations remained for CKB and 14-3-3-γ but not for Hsc71. Protein upregulation in CA1 neurons of final stage AD is either a result of detrimental, pathological effects, or from cell-specific protective response mechanisms in surviving neurons. Based on previous findings from experimental studies, CKB and Hsc71 likely exhibit protective effects, whereas 14-3-3-γ may represent a detrimental pathway. These new players could reflect pathways of importance for the development of new therapeutic strategies.

Characterization of reduced astrocyte creatine kinase levels in Alzheimer's disease

“…In contrast to earlier studies, we did not observe CKB expression in inhibitory neurons (Burbaeva et al, 1992;Lowe et al, 2013;Yoshimine et al, 1983) nor did we observe CKB-ir cells that do not co-stain for astrocyte markers. However, our results cannot completely rule out expression of CKB in neurons, as previous single nuclei transcriptomic (Hodge et al, 2019) and single cell mass spectrometry (Sandebring-Matton et al, 2021) studies have identified CKB in neurons. These discrepancies might be due to differences in CKB translation, intercellular transport, or post translational state of CKB in neurons and astrocytes.…”

Alzheimer’s disease biomarker profiling in a memory clinic cohort without common comorbidities

“…Deparaffinization and antigen retrieval of the brain tissue sections were performed as previously described. 30 After washing with phosphate-buffered saline (PBS), sections were blocked in 0.1% Triton X-100 (Sigma-Aldrich), 2% normal goat serum (Thermo Fisher Scientific, MA, USA) and 1% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies diluted in blocking buffer overnight at +4°C.…”

Tenofovir Activation Is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice

“…In subcellular regions where energy is being rapidly consumed (e.g., surrounding ATPases), CKB transfers the phosphate group from phosphocreatine to ADP, increasing local concentrations of ATP . The role of CKB in the creatine/phosphocreatine cycle has been studied in various tissues, and dysregulation of CKB via mutations, oxidation, or abundance alterations is associated with several disease states, including Alzheimer’s and Huntington’s diseases, and cancer metastases. ,− However, the contribution of CKB to xenobiotic biotransformation remains underexplored, as most studies have only investigated the contribution of CKM. − …”