10.5061/DRYAD.RBNZS7H8Z
Voskobiynyk, Yuliya
0000-0003-4169-3002
University of Alabama at Birmingham
Roth, Jonathan
University of Alabama at Birmingham
Cochran, J Nicholas
University of Alabama at Birmingham
Rush, Travis
University of Alabama at Birmingham
Carullo, Nancy
University of Alabama at Birmingham
Mesina, Jacob
University of Alabama at Birmingham
Waqas, Mohhamad
0000-0002-4633-4167
University of Alabama at Birmingham
Vollmer, Rachael
University of Alabama at Birmingham
Day, Jeremy
University of Alabama at Birmingham
McMahon, Lori
University of Alabama at Birmingham
Roberson, Erik
0000-0002-1810-9763
University of Alabama at Birmingham
Alzheimer’s disease risk gene BIN1 induces Tau-dependent network
hyperexcitability — MEA Axion Biosciences Maestro Recordings, Figure 6
Dryad
dataset
2020
National Institute on Aging
https://ror.org/049v75w11
RF1AG059405
Alzheimer's Association
https://ror.org/0375f4d26
Weston Brain Institute
http://dx.doi.org/10.13039/100012479
2020-08-19T00:00:00Z
2020-08-19T00:00:00Z
en
https://doi.org/10.7554/eLife.57354
2137782720 bytes
2
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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. Antisense oligonucleotide application 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. Statistics 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.