APP modulates KCC2 expression and function in hippocampal GABAergic inhibition
- South China Normal University, China
- University of Texas Health Sciences Center, United States
- Baylor College of Medicine, United States
Figures
Figure 1 with 1 supplement
GABA reversal potential shifts toward depolarization in hippocampus of App-/- mice.
(A) Slice preparations were made from the hippocampus and neurons were recorded in CA1 (inset). Pipettes were positioned to clamp neurons (Rec) while puffing molecules onto the cell (Puff). Scale …
Figure 1—figure supplement 1
GABA reversal potential shifts toward depolarization in App-/- hippocampal cultures.
(A) Sample traces showing perforated patch recordings of EGABA in WT and App-/- mice hippocampal cultures. Currents were recorded at the indicated holding potentials shown to the left of each trace. …
Figure 2 with 2 supplements
Decreased inhibitory IPSC amplitude and GABAAR α1 protein levels in App-/- mice.
(A) Images of 12–14 DIV hippocampal cultures of Gad67+/GFP mice. In the upper panel, under fluorescent illumination, GABAergic neurons appear green, and were stimulated (Sti) while recording from …
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Figure 2—source data 1
Contains source data for Figure 2 and all accompanying Figure 2—figure supplements 1,2.
- https://doi.org/10.7554/eLife.20142.006
Figure 2—figure supplement 1
Similar numbers of GABAergic interneurons and mIPSC in WT and App-/- hippocampus.
(A) Images of hippocampal sections from App-/--Gad67+/GFP and WT-Gad67+/GFP littermates. Scale bar: 400 µm; Enlarge: 20 µm. (B) Quantification shows no difference in the number of GABAergic …
Figure 2—figure supplement 2
Identical mEPSC and GluRs levels in between WT and App-/- hippocampus.
(A) Sample traces showing mEPSCs from App-/- and WT littermates. (B) Quantification of the amplitude and frequency of mEPSCs shows no significant difference between WT and App-/- mice (WT, n = 7 …
Figure 3
Full length APP is required for maintaining normal total and surface KCC2 protein levels.
(A) Representative immunoblots of hippocampal extracts from WT and App-/- littermates. (B) Quantification of the immunoblots reveals a significant decrease in surface and total KCC2 protein levels …
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Figure 3—source data 1
Contains source data for Figure 3.
- https://doi.org/10.7554/eLife.20142.010
Figure 4 with 1 supplement
APP interacts with KCC2 to limit KCC2 tyrosine phosphorylation and degradation.
(A) KCC2 interacts with APP in HEK293 cells by co-IP with both KCC2 and APP antibodies. Rabbit IgG (IP: IgG) was used as a negative control. (B) GABAAR α1 subunit does not coimmunoprecipitate with …
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Figure 4—source data 1
Contains source data for Figure 4.
- https://doi.org/10.7554/eLife.20142.012
Figure 4—figure supplement 1
Coomassie-stained SDS-PAGE gel showing KCC2 binding proteins after immunoprecipitated with an anti-KCC2 antibody (IP: KCC2).
https://doi.org/10.7554/eLife.20142.013
Figure 5
Restoring KCC2 expression and function in App-/- mice rescues hippocampal EGABA, GABAAR expression and IPSC amplitude.
(A) Sample traces showing perforated patch recording of EGABA in CLP257 or vehicle treated App-/- hippocampal slices. Currents were recorded at the indicated holding potentials shown to the left of …
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Figure 5—source data 1
Contains source data for Figure 5.
- https://doi.org/10.7554/eLife.20142.015
Figure 6 with 1 supplement
GABAAR α1 subunit is regulated by KCC2 expression through affecting intracellular Cl-.
(A) Representative immunoblots of GABAAR α1+KCC2, GABAAR α1+pcDNA3, GABAAR β2+KCC2, GABAAR β2+pcDNA3, transfected HEK293 cells blotted with anti-GABAAR antibodies. (B) Quantification of the …
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Figure 6—source data 1
Contains source data for Figure 6.
- https://doi.org/10.7554/eLife.20142.017
Figure 6—figure supplement 1
Immunofluorescent staining of hippocampal KCC2 in P14 and P35 WT mice shows increased KCC2 levels in P35 WT mice.
Scale bar: 100 µm.
Figure 7
APP interacts with mKCC2 to limit mKCC2 ubiquitination and degradation.
(A) Representative immunoblots of HEK293 cells transfected with mKCC2 and hAPP695. (B) KCC2 protein levels are elevated when HEK293 cells are transfected with both constructs. (C) Representative …
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Figure 7—source data 1
Contains source data for Figure 7.
- https://doi.org/10.7554/eLife.20142.020
Figure 8
APP deficiency causes an increase in KCC2 ubiquitination leading to a reduction of KCC2 levels.
(A) Representative immunoblots of HEK293 cells transfected with mKCC2 alone, mKCC2 and hAPP695, incubated with MG132 or vehicle. (B) Quantification of the immunoblots reveals a significantly …
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Figure 8—source data 1
Contains source data for Figure 8.
- https://doi.org/10.7554/eLife.20142.022
Figure 9
Reduced tonic GABA current in CA1 of App-/- hippocampus.
(A) Sample traces of tonic GABA current in WT, App-/- and App-/- incubated with 100 μM CLP257. (B) Quantification of tonic GABA current amplitude shows a significant decrease in App-/- mice and CLP …
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Figure 9—source data 1
Contains source data for Figure 9.
- https://doi.org/10.7554/eLife.20142.024
Figure 10
A working model shows the mechanism underlying APP regulation of GABAAR mediated inhibition.
KCC2 transports Cl- outside the neuron maintaining low [Cl-]i in mature neurons and therefore insures an Cl- influx upon GABA binding to the GABAARs. APP and KCC2 physically interacts to limit …
