Alzheimer’s disease risk gene BIN1 induces Tau-dependent network hyperexcitability

  1. Yuliya Voskobiynyk
  2. Jonathan R Roth
  3. J Nicholas Cochran
  4. Travis Rush
  5. Nancy VN Carullo
  6. Jacob S Mesina
  7. Mohammad Waqas
  8. Rachael M Vollmer
  9. Jeremy J Day
  10. Lori L McMahon
  11. Erik D Roberson  Is a corresponding author
  1. Center for Neurodegeneration and Experimental Therapeutics, Alzheimer’s Disease Center, and Evelyn F. McKnight Brain Institute, Departments of Neurology and Neurobiology, University of Alabama at Birmingham, United States
  2. Department of Neurobiology, University of Alabama at Birmingham, United States
  3. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, United States
6 figures, 3 tables and 1 additional file

Figures

BIN1 increases action potential and burst frequency in primary hippocampal neurons cultured on microelectrode arrays (MEAs).

(A) ICC experimental timeline: neurons were plated on day in vitro (DIV) 0, virally transduced on DIV 2, and immunostained at DIV 15, DIV 22, or DIV 27. (B) Representative images of primary …

BIN1 increases both excitatory and inhibitory synaptic transmission.

(A) Synaptic transmission recordings experimental timeline: neurons were plated on DIV 0, virally transduced on DIV 2, electrophysiologically recorded on DIV 19–21. (B) Representative traces of …

Higher BIN1 levels in mature neurons increase calcium influx in primary hippocampal neuronal cultures.

(A) Calcium imaging experimental timeline: neurons were plated on DIV 0, co-transfected on DIV 14 with GCaMP6f calcium indicator and either BIN1-mKate2, BIN1-ΔBAR-mKate2, or mKate2 control …

BIN1 interacts with LVGCC-β1 subunits in neurons.

(A) Localization of endogenous BIN1, LVGCC-β1, and endogenous BIN1-LVGCC-β1 interaction detected by PLA. Scale bar = 10 µm. (B) Experimental timeline: neurons were plated on DIV 0, transduced with …

Tau-dependent BIN1–LVGCC interaction.

(A) Model of Tau-dependent BIN1–LVGCC interaction. BIN1’s BAR domain localizes BIN1 to the plasma membrane, and PxxP motifs in Tau’s central proline-rich domain interact with the SH3 domains of BIN1 …

Tau reduction prevents network hyperexcitability induced by BIN1.

(A) MEA experimental timeline: neurons were plated on DIV 0, virally transduced on DIV 2, treated with Tau or scrambled ASO on DIV 5, and electrophysiologically recorded on DIV 12. (B) The number of …

Tables

Table 1
Resting membrane potential (RMP) and input resistance (Rin) in patched hippocampal neurons did not differ across untransduced, AAV-mKate2, and AAV-BIN1-mKate2 groups.
RMP, mVRin, MΩN, Cells
Untransduced–60.43 ± 5.36843.04 ± 55.114
AAV-mKate2–59.90 ± 3.70834.83 ± 41.096
AAV-BIN1-mKate2–62.40 ± 2.24790.18 ± 15.747
One-way ANOVA, p0.850.36
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Homo sapiens)BIN1NCBIGene ID 274
AntibodyAnti-NeuN
Rabbit polyclonal
abcamCat# ab104225; RRID:AB_10711153ICC (1:500), Lot #GR3321966-1
AntibodyAnti-GAD67
Mouse monoclonal
Millipore SigmaCat# MAB5406; RRID:AB_2278725ICC (1:500), Lot #3015328
AntibodyAnti-BIN1
Rabbit polyclonal
Santa CruzCat# sc-30099; RRID:AB_2243399ICC/PLA/IP (1:500), Lot #K1605; H-100
AntibodyAnti-LVGCC-β1
Mouse monoclonal
abcamCat# ab85020; RRID:AB_1861569ICC/PLA/IP (1:1000), Lot #413-8RR-52
AntibodyAnti-Tau
Rabbit polyclonal
DAKOCat# A0024; RRID:AB_10013724ICC/IP (1:1000), Lot #20031827
AntibodyAnti-BIN1
Mouse monoclonal
Santa CruzCat# sc-13575; RRID:AB_626753ICC/IP (1:1000), Lot #L3014; 99D
Cell line
(Rattus norvegicus)
Primary neuronCharles RiverFresh from E19 albino Sprague Dawley rats
Genetic reagentAAV-BIN1-mKate2UPenn Vector CoreAAV2
Genetic reagentAAV-mKate2UPenn Vector CoreAAV2
Sequence-based reagentTau ASOPMID:23904623
IDT
5-ATCACTGATTTTGAAGTCCC-3
Sequence-based reagentScrambled ASOPMID:23904623
IDT
5-CCTTCCCTGAAGGTTCCTCC-3
Commercial assay, kitDuolink PLA kitMillipore SigmaCat#s DUO92014; DUO92002; DUO92004
Transfected constructGCaMP6fAddgeneRRID:Addgene_40755
Transfected constructmKate2PMID:25156556
Evrogen
Cat# FP184Actin was removed from the construct obtained
Transfected construct (Homo sapiens)BIN1Horizon Discovery ORFeome Collaboration ClonesOHS5894-202501160Isoform 1
Cell line
(Cricetulus griseus)
CHO-K1Millipore SigmaCat# 85051005-1VLChinese Hamster Ovary cell line
Transfected constructmKate2-Tau-mKate2PMID:25156556
Transfected constructFyn-SH3-CBGPMID:25156556BIN1-SH3 or LVGCC-β1-SH3 was cloned in replacing Fyn-SH3
Transfected construct (Homo sapiens)BIN1-SH3IDTAAC28646.1Codon optimized
Transfected construct (Homo sapiens)LVGCC-β1-SH3IDTAAA35632.1Codon optimized
Author response table 1
Calculated E/I ratios of spontaneous postsynaptic current frequencies and amplitudes.
mKate2 Frequency, mean ±SEMBIN1-mKate2 Frequency, mean ±SEMmKate2 Amplitude, mean ±SEMBIN1-mKate2 Amplitude, mean ±SEM
sEPSCs1.65 ± 26.59 Hz3.53 ± 65.58 Hz31.15 ± 0.40 pA29.02 ± 0.53 pA
sIPSCs6.82 ± 37.61 Hz7.57 ± 15.25 Hz83.07 ± 1.49 pA67.73 ± 0.55 pA
E/I ratio0.24 ± 0.710.47 ± 4.300.37 ± 0.270.43 ± 0.97

Additional files

Download links