This project aimed to validate an hiPSC-derived cortical neuron system for monitoring AD- relevant functional deficits to enable the evaluation of the effectiveness of AD therapeutics. Two functional assays were employed: patch clamp analysis on neurons cultured on coverslips and LTP analysis of neural circuits cultured on patterned MEAs. To evaluate the response of Aβ oligomer treatment, cells on coverslips were treated with the Aβ42 oligomers, control scrambled Aβ oligomers (Aβscr), or Aβ42 oligomers plus AD therapeutics for 24hr and then subjected to patch clamp analysis. Those on MEAs were recorded for baseline activity, high frequency stimulation for LTP induction, then treated with Aβ42 oligomers, Aβscr, or Aβ42 oligomers plus AD therapeutics for 1 hr and the LTP activity was measured. For patch clamp electrophysiology, the parameters analyzed were: amplitude of the sodium current, amplitude of the Action Potentials (AP), number of spontaneous firrings with a 30 sec duration, and amplitude of spontaneous firing. For the MEA assay, the parameter analyzed was the neural activity at 1 hr post LTP induction normalized by baseline activity. For each drug evaluated, there were three experimental groups: cells treated with Aβscr as the control, those treated with Aβ42 oligomers as the AD pathological group, and those treated with Aβ42 oligomers plus AD drug as the therapeutic treated group. The AD deficit was evaluated by comparing between the Aβ42 group and the control, and the drug’s effect was evaluated by comparing between the Aβ42 + drug group with the Aβ42 group. The number of systems tested was determined statistically so as to detect a significant difference between the Aβ42 group and the control, with a type I error rate (α) of 0.05 using a Dunnetts’s multiple-comparisons test to the control condition.
3.1 Neural Cells: Human cortical neurons were used to assess the effects of multiple classes of AD therapeutics on Aβ oligomer induced neuronal cell dysfunction. The cells were derived from human iPSCs from healthy individuals and were either purchased from Cellular Dynamics International (CDI, iCell GlutaNeurons, Cat. #: C1033, Madison,WI) or defferentiated directly in our lab as described previously [53–55]. Human primary astrocytes were purchased from ScienCell (Cat. # 1800).
3.2 Surface chemistry: Neuronal surface patterning on custom MEA chips was prepared using a surface coating protocol of polyethylene glycol (PEG), followed by laser ablation and backfill coating with DETA (N-1(3-[trimethoxysilyl]propyl)-diethylenetriamine) as detailed in Wilson et al ([56]). Subsequently, neuronal attachment was further encouraged with a protein adsoprtion coating of poly-L-ornithine (PLO) and laminin, which consisted of a 1hr RT incubation of 0.01%
PLO solution, followed by 3X rinses of 1X PBS before incubating overnight at 4°C with 10 µg/ml laminin solution ([55]). The laminin solution was removed prior to cell seeding.
3.3 Cell Culture: The cells were seeded on the patterned MEA surfaces and on coverslips. A monoculture of cortical neurons were plated at a density of 150 cells/mm2 on coverslips for immunostaining (ICC) analysis and patch-clamp electrophysiology as decribed previously [35, 53]. For the MEA cultures, a co-culture of cortical neurons and astrocytes were seeded directly onto patterned MEAs to promote cell adhesion of separated cell-clusters on the individual electrodes, as well as the formation of synaptic connections between two adjacent electrodes. The neuronal cells were plated at a density of 500 cells/mm2 and the astrocytes at 250 cells/mm2. The cells were were first plated in the manufacture’s (CDI) recommended medium for the first 24h before they were switched to a serum-free medium. The cells were maintained in culture in the serum-free medium for 28–35 days prior to dosing and testing [53].
3.4 Aβ Oligomers Preparation: The Aβ oligomers were prepared using peptides from rPeptide (Aβ1–42 catalog number A-1002-2; Aβ-scrambled A-1004-2) as described previously [57]. First, the peptides were resuspended in 500 µL of HFIP (catalog number AC445820100; Fisher Scientific) and left to dry overnight under a ventilated hood. The next day the samples were spun in a SpeedVac until dry and stored desiccated at -20°C until use. Prior to using, the solution was sonicated for 5 minutes and centrifuged at 1400 x g for 5 minutes.
3.5 AD Drugs. Several classes of drugs used in the treatment of AD were used in this study, including Rolipram (Cayman Chemicals, Cat. # 10011132), Saractinib (Cayman Chemicals, Cat. # 379231-04-6), Memantine (Cayman Chemicals, Cat # 14184)and Donepezil (Sigma, Cat. # D6821). Memantine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that binds to the receptor and blocks the binding of glutamate and thus cell overexcitotoxicity caused by glutamate [58]. Donepezil is an FDA-approved drug for AD, it is an acetylcholinesterase inhibitor which prevents the breakdown of acetylcholine in the synapse by the acetylcholinesterase enzyme [59, 60]. Saracatinib inhibits the action of Fyn, a member of the tyrosine kinase family that phosphorylates other proteins including tau and NMDAR, resulting in tau hyperphosporylation and synaptoxicity/or cells excitotoxicity from NMDAR activation [61–64]. Rolipram is a selective phosphodiesterase-4 (PDE4) inhibitor that helps to restore cAMP level which is affected (i.e., reduced) in AD as a reslust of adenylate cyclase (synthesizes cAMP) inactivation by Aβ42 peptides [65]. All drug stocks were prepared by reconstitution in 1X PBS.
3.6 Drug Treatment of Cortical Neurons: Neuronal cultures were treated for patch clamp via half medium exchange containing either Aβscr or Aβ42 oligomers with or without AD drugs 24 hours prior to testing. For MEA analysis, cells were fed at 24 hours prior to treatment by performing a half medium change to prepare the cells for the intensive activity testing the next day. Upon testing time, following the 5 min baseline testing, LTP induction and 5 min recording right after the induction, the cells were either dosed with a final concentration of 5 µM Aβscr, 5 µM of Aβ1–42 alone, or 5 µM of Aβ1–42 plus drug (either 10 nM of saractinib, 1 µM of donepezil, or 5 µM of memantine, 1 µM of rolipram). To examine the neurotoxic effects of Aβ42 oligomers on cell electrophysiological function, the cells or cultures were tested at 1 hour (MEAs) following treatment by conducting another 5 min recording. At the end of the activity testing, a dose of 1mM Lidocaine ) was always applied to silence all the neural activity so as to confirm the biological source of the recorded signals. Those signals that were not abolished by Lidocane would be considered as noise and will be excluded from MEA analysis.
3.7 Immunocytochemistry and Confocal Microscopy: To analyze the cells for gene expression of the drug targets, the cells were fixed in 4% paraformaldehyde (PFA), followed by cell permeabilization and incubation in primary and secondary antibodies solutions (diluted in BSA/NGS/T20 buffer) for each specific marker. Following antibodies staining, the cells were counterstained with DAPI (4’,6-diamidino-2-phenylindole) and mounted on glass slides for analysis. The cells were imaged using a confocal microscopy (Zeiss, Axioskop 2, Germany). The following primary antibodies (at 1/1000 dilution) were used: Rabbit Anti-Microtubule-Associated Protein 2 (Millipore, Cat. #: AB5622), Mouse Anti-MAP2 (Abcam, Cat. #: ab11257), Mouse Anti-Fyn (ThermoFisher, Cat. #: MA1-15865), Mouse Anti-NMDAR2B (ThermoFisher, Cat. #: MA1-2014), Mouse Anti-Acetylcholinesterase (Abcam, Cat. #: ab2803), Mouse Anti- muscarinic Acetylcholine Receptor (Abcam, Cat. #: ab90805), Rabbit Anti-nicotinic Acetylcholine Receptor (Abcam, Cat. #: ab221868), Rabbit Anti-Phosphodiesterase Type 4 (Abcam, Cat. #: ab14628). The secondary antibodies used: Alexa-Fluor 488 goat anti-rabbit (ThermoFisher, Cat. # A11008), AlexFluor 488 Goat anti-mouse (ThermoFisher Cat. #: A11001), AlexFluor 568 Goat anti-Rabbit (ThermoFisher Cat. #: A11036), and AlexFluor 568 Goat anti-mouse (ThermoFisher Cat. #: A11004).
3.8 Patch-Clamp Electrophysiology Recording of Cortical Neurons: To measure individual neuronal activity, whole cell patch-clamp recordings were taken using a Zeiss, upright microscope (Axioscope, FS2, Carl Zeiss, Germany) equiped with a multiclamp 700B amplifier and an intracellular solution consisting of 140 mM K-gluconate, 4 mM NaCl, 0.5 mM CaCl2, 1 mM MgCl2, 1 mM EGTA, 5 mM HEPES Acid, 5 mM HEPES base and 5 mM Na2ATP as described previously [53]. Depolarization-evoked inward and outward currents were examined in voltage-clamp mode while induced action potentials (APs) were recorded in current-clamp mode. Sucessive analysis of the data were carried out using pClamp 10 software (Axon Instrument, Foster City, CA, USA) followed by quantification using Microsoft Excel and GraphPad Prism.
3.10 Long-Term Potentiation Induction on MEAs: To induce LTP on cortical neurons cultured on MEAs, a high frequency stimulation (HFS) protocol was used as described previously [53]. Test stimuli were delivered to all the electrodes in the form of 80 pulses at 100Hz. The evoked response or induced cell activity was then analyzed using Anaconda with Python (i.e., Monday.com) software. The waveforms (e.g., action potential spikes and frequency) were thresholded at -5 standard deviations from the noise and high-pass filtered at 100Hz. Any electrodes with firing frequency post-stimulation less than or equal to baseline levels were excluded from the data analysis.
3.11 Analysis of Cortical Neuron Firing Activity on MEAs: The cellular activity of the cells was measured extracellularly on MEAs as described previously [35, 53, 66]. The cells were plated directely onto the MEA chips in housing and maintained in culture for 28–35 days before LTP experimentswith Aβ oligomers and AD drugs. Prior to treatment, spontaneous activity (baseline) of the neurons was recorded (for 5 minutes) followed immediately by electrical stimulation (LTP induction) and another 5 minutes recording post-stimulation. Immediately following LTP induction and recording, the cultures were treated with Aβ oligomers and/or Aβ oligomers AD drugs and incubated for 1 hour at 37°C and 5% CO2. Next, the neuronal firing potential (e.g., firing frequency) was once again recorded for 5 minutes before analysis. The data was analyzed using Anaconda with Python software.
3.12 Statistical Analysis: Comparison of the mean of at least three or more replicates and more than 15 electrodes between groups was performed. For computational analyses, Microsoft Excel software and GraphPad Prism were used. Student t-tests were used for statistical comparison analysis between two experimental groups, whereas one-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for multiple experimental groups. The SEM was used with statistical significance taken at p ≤ .05.