Genome-wide association studies identified the BIN1 locus as a leading modulator of genetic risk in Alzheimer's disease (AD). One limitation in understanding BIN1's contribution to AD is its unknown function in the brain. AD-associated BIN1 variants are generally noncoding and likely change expression. Here, we determined the effects of increasing expression of the major neuronal isoform of human BIN1 in cultured rat hippocampal neurons. Higher BIN1 induced network hyperexcitability on multielectrode arrays, increased frequency of synaptic transmission, and elevated calcium transients, indicating that increasing BIN1 drives greater neuronal activity. In exploring the mechanism of these effects on neuronal physiology, we found that BIN1 interacted with L-type voltage-gated calcium channels (LVGCCs) and that BIN1–LVGCC interactions were modulated by Tau in rat hippocampal neurons and mouse brain. Finally, Tau reduction prevented BIN1-induced network hyperexcitability. These data shed light on BIN1's neuronal function and suggest that it may contribute to Tau-dependent hyperexcitability in AD.

Multi electrode array cultures

For 6-well multielectrode array recordings, neurons were plated at 100,000 per well in six-well MEA plates (ALA Scientific, ALAMEA-MEMMR5). For 48-well plate multielectrode array recordings, neurons were plated at 30,000 per well in 48-well MEA plates (Axion Biosystems, M768-tMEA-48B-5).

BIN1 constructs and vectors

A BIN1-mKate2 (GE Dharmacon, OHS5894-202501160) construct was developed to encode human BIN1 isoform 1 (593 AA, the major neuronal isoform) tagged with mKate2 (Evrogen, FP184, to allow for fluorescent visualization) at the C-terminus to allow for proper function of the N-terminal membrane-interacting BAR domain. A similar construct lacking the BAR domain (amino acids 32–273, BIN1-ΔBAR-mKate2) was produced as a BIN1 BAR domain deletion mutant. A construct encoding mKate2 only was used as a control. These constructs were then cloned into the CIGW vector (rAAV9-CBA-IRES-GFP-WPRE-rBG) (St Martin et al., 2007). Due to size limitations for efficient gene expression, the IRES-GFP was removed from the CIGW vector.

Tau anti-sense oligonucleotide (ASO) sequences were adapted from DeVos et al., 2013 and produced by Integrated DNA Technology (Tau ASO: 5-ATCACTGATTTTGAAGTCCC-3, Nontargeting control ASO: 5-CCTTCCCTGAAGGTTCCTCC-3). ASOs were dissolved to 100 μM in 10 mM Tris with 0.1 mM EDTA and stored at −20°C until use. At DIV 6, one week before testing for both MEA experiments and PLA, neurons were treated with ASO to a final concentration of 1 μM.

Axion Biosciences MEA

Single neuron electrophysiological activity was recorded using an Axion Maestero recording system as in Savell et al., 2019a. Briefly, neurons were plated on the 48-well MEA (Axion Biosystems, M768-tMEA-48B-5) with 16 extracellular recording electrodes and a ground electrode per well at a density of 30,000 neurons per well in Neurobasal medium (5 μL) with 10% FBS (Atlanta Biologicals, S11550) and placed in a 37°C incubator with 5% CO₂. After allowing neurons to attach to the plate for 2 hr, 300 μL serum-free Neurobasal (Life Technologies, 21103049) was added. The next day, AraC was added as with other experiments and a 50% medium change with BrainPhys (Stemcell Technologies Inc, 05790) supplemented with SM1 and L-glutamine was done at DIV 5. At DIV 6, neurons were treated with ASO to reduce Tau protein levels. At DIV 9, a 50% medium change was completed with supplemented BrainPhys, followed by a 50% medium change with supplemented Neurobasal at DIV 12. At DIV 13, neurons were recorded using Axion AxIS software for 15 min. Electrical activity was measured by an interface board at 12.5 kHz, digitized, and transmitted to an external computer for data acquisition and analysis in Axion AxIS Navigator software (Axion Biosystems). All data were filtered using dual 0.01 Hz (high pass) and 5,000 Hz (low-pass) Butterworth filters. Action potential thresholds were set automatically using an adaptive threshold for each electrode (>6 standard deviations from the electrode’s mean signal). Neuronal waveforms collected in Axion AxIS Navigator were exported to Offline Sorter (v. 4.0 Plexon). Offline Sorter automatically completes and plots PCA on waveforms for each electrode. Manual inspection of PCA, shape, inter-spike intervals, auto-correlograms, and cross-correlograms allowed us to distinguish between multiple units on a single electrode and to do per-neuron analyses. After waveforms were split into units, analysis of each unit’s action potential frequency and burst firing was completed in NeuroExplorer (v. 5.0, Plexon) using the built-in Burst Analysis function, with Poisson burst surprise = 5. Next, firing rates and bursting analysis were performed in NeuroExplorer (v. 5.0 Plexon). Researchers were blinded to experimental conditions performed in all MEA analyses.

Statistical distribution of data varied widely between data sets in this study, so we analyzed each data set for normality and analyzed using either parametric or non-parametric tests accordingly. The specific test used is indicated in the figure legend in each case. All statistical tests were performed with Prism 8 (GraphPad, v. 8.4.0).

An excel file with the plate layout is uploaded.