Brain cells derived from Alzheimer’s disease patients have multiple specific innate abnormalities in energy metabolism

Altered energy metabolism has been implicated both in aging and the pathogenesis of late-onset Alzheimer’s disease (LOAD). However, it is unclear which anomalies are acquired phenotypes and which are inherent and predispose to disease. We report that neural progenitor cells and astrocytes differentiated from LOAD patient-derived induced pluripotent stem cells exhibit multiple inter-related bioenergetic alterations including: changes in energy production by mitochondrial respiration versus glycolysis, as a consequence of alterations in bioenergetic substrate processing and transfer of reducing agents, reduced levels of NAD/NADH, diminished glucose uptake and response rates to insulin (INS)/IGF-1 signaling, decreased INS receptor and glucose transporter 1 densities, and changes in the metabolic transcriptome. Our data confirm that LOAD is a “multi-hit” disorder and provide evidence for innate inefficient cellular energy management in LOAD that likely predisposes to neurodegenerative disease with age. These processes may guide the development and testing of diagnostic procedures or therapeutic agents.

min. Cells were then treated with 1 mM 2-deoxyglucose (2DG) and incubated for 20 min at room temperature. Subsequently, Stop Buffer and Neutralization Buffer were added sequentially. Before reading the luminescence using a Synergy HT BioTek plate reader (BioTek), 2DG6P Detection Reagent was added and incubated for 1 hr at room temperature. After assay performance, proteins were measured using the Bio-Rad Protein Assay for data normalization.

MitoTracker
NPCs were plated at a density of 2 x 10 5 and astrocytes 1 x 10 5 cells in VTN-coated black 96 well culture dishes. The next day, cells were stained with MitoTracker TM Green FM (Invitrogen) and Hoechst and fluorescence was measured with 485 nm excitation and 520 nm emission using a Synergy HT BioTek plate reader (BioTek).

Biolog Assays
Bioenergetics substrate metabolism was measured using Biolog MitoPlate S-1 assays (Biolog, Hayward, CA) according to the manufacturer's instructions. The MitoPlates were preincubated with 70 µg/ml saponin (Sigma), Biolog Mitochondrial assay solution, and Redox dye for 1 hr at room temperature. After the incubation, 60,000 NPCs and 40,000 astrocytes per well were added to each plate and the OD 590 was measured at various time points (0, 1, 2, 3, 4, 5, 6, and 24 hrs) using a Synergy HT BioTek plate reader (BioTek). Measurements were normalized to and calculated as percent change from no substrate control.

APOE genotyping
DNA from cells was extracted and a portion of the APOE gene containing the polymorphic single nucleotides rs429358 and rs7412 amplified by PCR using the primers FW: 5'-CTGATGGACGAGACCATGAAG-3' and RV: 5'-GGCTCGAACCAGCTCTTGAG-3'. PCR reactions were according to the Platinum™ Taq DNA Polymerase protocol (Invitrogen, Thermo Fisher) in 25 µl volumes containing 1 U Platinum™ Taq DNA Polymerase, 0.2 μM of each primer, 8 % KB Extender and 50 ng genomic DNA. The PCR cycling conditions were as follows: Initial denaturation at 94 °C for 2 min followed by 35 cycles with denaturation at 94 °C for 30 sec, annealing at 55 °C for 50 sec, extension at 72 °C for 50 sec; then a final extension at 72 °C for 10 min. All PCR products were purified by ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems) and sent for sequencing at the MGH Center for Computational and Integrative Biology (CCIB) DNA Core using the same primer pair.

RNA isolation
RNA samples were isolated from cultured cells using TRI-Reagent (Sigma) according to the manufacturer's instructions. Briefly, the cells were homogenized in 1 ml of TRI reagent and incubated for 5 min at room temperature (RT). Then, 200 μl of chloroform (Merck, Darmstadt, Germany) was added, vigorously mixed, and incubated for 15 min at RT. After the mixture was centrifuged at 12,000 g for 15 min at 4 °C, the separated aqueous phase was transferred to a new tube. To precipitate RNA, 500 μl of 2propanol (Merck) per ml of TRI Reagent was added and inverted gently, incubated for 10 min at RT, and centrifuged at 12,000 g for 10 min at 4 °C. After centrifugation, the supernatant was removed, and the RNA pellet was washed with 1 ml of 75 % ethanol (Merck) per ml of TRI Reagent. The samples were centrifuged at 7,500 g for 5 min at 4 °C, then the supernatants removed again, and the RNA pellet was dried for 20 min. After drying, total RNA pellets were dissolved in nuclease-free water. RNA quality and quantity were assessed using Nano Drop 8000 spectrophotometer (Thermo Scientific) and total RNA integrity was checked using Agilent Bioanalyzer 2100 (Agilent) with an RNA Integrity Number (RIN) value.

CellProfiler
CellProfiler is a free program and can be downloaded and installed from the following website (www.cellprofiler.org). For the current study we used version CellProfiler 3.1.8. Cells were imaged at 100 x using oil-immersion and 10 images from each slide were analyzed per subject. We focused on the following modules: (a) IdentifyPrimaryObjects, which recognizes biological objects of interest and from which used the following data; Count: Total number of primary objects identified in each image. (b) MeasureObjectSizeShape, which measures the area and shape of identified objects and from which we used; Area: The number of pixels in a given region. We then normalized the total numbers of speckles (counts) to the cell area using green for INSR or IGF1-R and red for GLUT-1.

Statistical analysis
Data were plotted as mean ± standard error of the mean (SEM) from at least 2 independent experiments performed in triplicates (n = 3), unless otherwise stated. One-way analysis of variance (ANOVA) tests for independent measures were performed using the Social Science Statistics software (http://www.socscistatistics.com/Default.aspx) or PRISM 8 for macOS Version 8.1.0. Differences of comparison were considered statistically significant when p-values were less than 0.05, while p-values between 0.05 and 0.1 were considered trend data.   Fig. 1 Human iPSC lines are pluripotent. a Immunostaining of iPSC lines AD7, AD8, and AD9 with common pluripotency markers OCT4, SOX2, TRA-60, TRA1-81, NANOG, and SSEA-4. b TaqMan hPSC scorecard assays for lines C3, C4, C5, AD7, AD8, and AD9. c Upon injection into SCID mice, Control-and LOAD-iPS cells form teratomas in vivo, which contain tissues of all three embryonic germ layers, such as epidermis and neural epithelium (ectoderm), bone tissue (mesoderm), and epithelium duct (endoderm).

Basal
Resp.     Supplementary Fig. 8 Profiling of cell bioenergetic metabolism in Biolog assays. Data from Biolog experiments on LOAD (n = 9) and Control (n = 5) NPCs or LOAD (n = 9) and Control (n = 5) astrocytes, human astrocytes, and HEK293 cells, depicting kinetic measurements at 1 to 6 and 24 hrs. Shown are percent changes of no-substrate normalized values of 31 metabolites.   Supplementary Fig. 9 Differences in bioenergetic functions between Control and LOAD cells. Data from Biolog experiments on LOAD (n = 9) and Control (n = 5) NPCs or LOAD (n = 9) and Control (n = 5) astrocytes for kinetic measurements at 1 to 6 and 24 hrs. Shown are percent changes of no-substrate control normalized values as percent astrocytes versus NPCs and LOAD versus Control cells of 31 metabolites organized by association with metabolic function. or Control (n = 5) NPCs and astrocytes. Genes (rows) for individual samples (columns) were transformed to z-scores and plotted as heatmaps. b qRT-PCR results for GPDH1/2, MDH1/2, IDH3A, and OGDH, in LOAD (n = 9, red bars) and Control (n = 5, black bars) NPCs or LOAD (n = 9, orange bars) and Control (n = 5, grey bars) astrocytes plotted as 2 -DCT values. Data are means +/-SEM from duplicate measurements in two repeat experiments. *p < 0.1; **p < 0.05; ***p < 0.01 using one-way ANOVA. c Comparison of fold changes from qRT-PCR with RNA-Seq data.