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Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice

Nature Neuroscience volume 17, pages 694–703 (2014)Cite this article

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Abstract

Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K+ channel functional expression, leading to elevated in vivo striatal extracellular K+, which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K+, ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K+ homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.

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Figure 1: No evidence for astrogliosis at symptomatic ages in R6/2 mice.
Figure 2: Striatal astrocytes from R6/2 mice display nuclear mHTT inclusions, depolarized membrane potentials and lower membrane conductances.
Figure 3: Striatal astrocytes from R6/2 mice display reduced Ba2+-sensitive Kir4.1 currents at symptomatic ages (P60–80).
Figure 4: Mechanistic studies of Kir4.1 in R6/2 mice and HEK-293 cells.
Figure 5: Kir4.1 immunostaining is reduced in individual striatal astrocytes that contain nuclear mHTT inclusions.
Figure 6: AAV2/5-mediated Kir4.1-GFP expression rescues deficits observed in striatal astrocytes from R6/2 mice at P60–80.
Figure 7: Elevated striatal extracellular K+ levels in R6/2 mice in vivo and their effects on WT MSN excitability in vitro.
Figure 8: AAV2/5-mediated Kir4.1-GFP expression attenuates a motor phenotype in R6/2 mice at P92.

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Acknowledgements

The authors thank P. Kofuji (University of Minnesota) for sharing Kir4.1-GFP plasmids, R. Korsak for coordinating the genotyping, and V. Beaumont, I. Munoz-Sanjuan, M.S. Levine, C. Cepeda, E. Shigetomi, and all current and past members of the Khakh and Sofroniew laboratories for discussions. This work was supported by the CHDI Foundation (B.S.K., M.V.S., X.T., Y.A.) and the US National Institutes of Health (NS060677, MH104069 to B.S.K.).

Author information

Author notes

  1. Xiaoping Tong and Yan Ao: These authors contributed equally to this work.

  2. Michael V Sofroniew and Baljit S Khakh: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Xiaoping Tong, Ji Xu, Martin D Haustein, Istvan Mody, Michael V Sofroniew & Baljit S Khakh

  2. Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Yan Ao, Mark A Anderson & Baljit S Khakh

  3. Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Guido C Faas & Istvan Mody

  4. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA

    Sinifunanya E Nwaobi & Michelle L Olsen

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Contributions

X.T., J.X., M.D.H. and B.S.K. carried out the molecular biology, imaging and electrophysiology experiments. Y.A., M.A.A. and M.V.S. performed immunohistochemistry, western blot and behavior experiments. G.C.F. and I.M. carried out the potassium concentration measurements. S.E.N. and M.L.O. performed the qPCR experiments. B.S.K. and M.V.S. directed the work. B.S.K. wrote the first draft of the paper. All of the authors contributed to the final version of the manuscript.

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Correspondence to Michael V Sofroniew or Baljit S Khakh.

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Integrated supplementary information

Supplementary Figure 1 Basic characterization of R6/2 mice.

a-b. Nissl (cresyl violet) stained brain sections from WT and R6/2 mice at P30 (a) and P60 (b). Note, at P60, when R6/2 mice display HD-like symptoms, the volume of the caudate putamen (CP) is reduced leading to enlarged ventricles (V). c. Quantification of the reduced CP area in R6/2 mice relative to WT and P60. d. R6/2 mice weighed less than their WT littermates at P60. Significance was assessed with an unpaired Student's t test; p values are shown.

Supplementary Figure 2 Ki67 IHC in WT and R6/2 mice.

Ki67 IHC in WT and R6/2 mice at P110 shows that many proliferating cells were seen in the subventricular zone (arrow heads), but few were seen in the striatum (arrows). In this regard, there were no differences between WT and R6/2 mice at P60-70 or P100. Some, but not all, Ki67 positive cells in the striatum colocalized with S100β, indicating that we were able to detect proliferating reactive astrocytes but that almost none were present.

Supplementary Figure 3 Western blot analysis of protein expression in striatal tissue at P104-110 for WT and R6/2 mice.

Statistical significance was assessed with an unpaired Student's t test. n indicates the number of animals from each group. P values are shown.

Supplementary Figure 4 Labeling of striatal astrocytes with four commonly used astrocyte markers.

GFAP (a), Aldh1L1(b), Glutamine synthetase (GS) (c) and S100β (d) in WT mouse at P60. As shown in a, only a few astrocytes were labeled with GFAP antibodies compared with the other three astrocyte markers. The images in the lower panels show higher magnification views from the boxes marked in the upper panels in each group.

Supplementary Figure 5 Colocalization of astrocyte markers in striatum of R6/2 mice at P60.

(a) Shows glutamine synthetase (GS; green) colocalized with Aldh1 (red) in an astrocyte shown with an arrow. (b) As in (a), but for Aldh1L1 (green) colocalized with S100β (red).

Supplementary Figure 6 Basic properties of medium spiny neurons and astrocytes.

Medium spiny neurons (MSN) and astrocytes in the striatum were easily distinguished during whole-cell recordings with Alexa 488 fluorescent dye in the pipette solution. (a) Shows a typical dendritic morphology of a MSN dialyzed with Alexa 488 (50 μM) for 30 min and the electrophysiological traces below show a negative resting membrane potential (-81 mV) and action potential firing during small depolarizing current injections. (b) Shows an astrocyte identically dialyzed with Alexa 488: a few short processes can be seen with a generally bushy appearance. The electrophysiological traces below show a negative resting membrane potential (-80 mV), but no action potential firing even for large depolarizing current injections.

Supplementary Figure 7 Barium sensitive currents.

Representative traces showing total macroscopic currents (i), Ba2+- insensitive currents (in 100 μM Ba2+ in the bath; ii) and Ba2+-sensitive currents (i-ii) recorded by whole-cell voltage clamp (from -120 mV to 40 mV) for striatal astrocytes from WT (a) and R6/2 mice (b) at P60. c. Note on average that the Ba2+-sensitive current is smaller in R6/2 astrocytes than that in WT astrocytes at P60-80.

Supplementary Figure 8 qPCR analyses.

a. Kir4.1 (KCNJ10) qPCR data for R6/2 and WT mice at P60-80 relative to three control mRNAs (GAPDH, ATP5B, and UBC). There were no significant differences when the data were assessed using un paired Student's t tests. P values are shown. b. qPCR data for Q175 mice aged 10 months for Kir4.1, GFAP and GLT-1. Only mRNA levels for GLT-1 were significantly different to WT controls. Statistical significance was assessed using an unpaired Student's t test; P values are shown. The experiments were done on 4-5 mice. c. Kir4.1 (KCNJ10) qPCR data for Q175 and WT mice at 10 months age relative to three control mRNAs (GAPDH, ATP5B, and UBC). There were no significant differences when the data were assessed using un paired Student's t tests. P values are shown.

Supplementary Figure 9 Characterization of Q175 mice.

Representative images show there no CP volume change in wild type mice and heterozygous Q175 mice by Nissl staining at 6-7 months. Bar graph in right summaries the striatal area between two groups. (B) Representative images show decrease of the striatal volume in heterozygous Q175 mice at 12 months compared with comparative wild type mice. Bar graph in right shows the significant decrease of striatal areas in heterozygous Q175 mice. (C) Summary of western blots of Kir4.1, glutamine synthetase (GS), GLT-1 and GFAP protein expression levels in striatums of wild type, heterozygous and homozygous Q175 mice at 6-7 months and 9-12 months. Statistical tests were done using un paired Student's t tests; p values are shown. n indicates the number of animals in each group.

Supplementary Figure 10 Representative images for IHC using two antibodies against mHTT (mEM48 and MW8).

Both labeled nuclear inclusions in S100β labeled astrocytes to an equivalent degree. Neither is able to label cytoplasmic mHTT.

Supplementary Figure 11 Representative images show S100β immunostaining in striatal tissue from R6/2 mice injected with AAV2/5 gfaABC1D Kir4.1-GFP virus (a) or AAV2/5 gfaABC1D tdTomato virus (c) at P56.

Slice sections were collected for immunohistochemistry two weeks after viral injections at P56. Panels (a) and (c) show colocalization of S100β labeled astrocytes with Kir4.1-GFP and tdTomato positive cells, respectively. Controls from mice that received no virus injections (b and d) showed no colocalisation, but still showed S100b staining.

Supplementary Figure 12 Controls for AAV2/5 Kir4.1-GFP.

a. Astrocyte current-voltage (IV) relations. AAV2/5 Kir4.1-GFP expression in R6/2 mice increases the slope of the astrocyte IV relations and restores them to WT levels. However, Kir4.1-GFP expression in WT astrocytes did not boost the IV relations higher than WT levels. b. Ba2+ sensitive currents under the conditions shown. c-e. Western blot analysis from R6/2 mice injected with AAV2/5 for Kir4.1-GFP or AAV2/5 tdTomato. The Western blot data are for GFP (c) total Kir4.1 (d) and Glt-1 (e). Statistical differences were assessed using un paired Student's t tests. P values are shown.

Supplementary Figure 13 Assessment of inflammation.

a. Representative images for Iba1 and CD68 IHC under the conditions shown. b-c. Summary data for Iba1 and CD68 IHC for experiments such as those shown in a. No qualitative or quantitative differences are detectable in staining intensity for inflammatory markers when comparing mice that received AAV2/5Kir4.1-GFP or AAV2/5-tdTomato.

Supplementary Figure 14 Representative trace and average data for the astrocyte membrane potential in different concentrations of bath K+.

The slope of the line shows a 55 mV depolarization for a 10-fold change in K+ concentration. Additional Note: A salient feature across mouse models of HD is that MSNs display depolarized membrane potentials by as much as ∼12 mV. A vaguely formulated idea suggests that reducing astrocyte Kir4.1 function with Ba2+ in WT slices may reveal secondary consequences for MSNs, i.e., this approach will elevate K+ near neurons, depolarize MSNs and thus reveal astrocyte Kir4.1 contributions to altered MSN properties frequently observed in mouse models of HD. However, this view has problems. First, although Ba2+ is a useful tool to isolate astrocyte Kir4.1 currents, it also blocks MSN K+ channels, meaning that it will be impossible to tell if any measured effects are due to Ba2+ actions on astrocytes or MSNs directly. Second, the suggested experiment can only work if brain slices can buffer K+ locally, i.e. that local K+ is not set by the bath K+ concentration. We tested for this by measuring the astrocyte membrane potential in bath solutions of different K+ concentration, exploiting the fact that astrocytes function as K+ microelectrodes because of their high K+ conductance. The Nernst equation predicts that the membrane potential of an astrocyte with a high K+ conductance should change by ∼56 mV for a ten-fold change in bath K+ concentration. If brain slices can in fact buffer K+ in the vicinity of a cell, then we would predict a deviation from this, because the local K+ concentration would be set by buffering and not by the bath K+ concentration. We found that striatal astrocytes showed a near perfect Nernst-like ∼55 mV depolarization for a 10-fold change in bath K+ concentration. These data show that isolated preparations such as brain slices cannot buffer K+. Thus, the idea of blocking astrocyte Kir4.1 channels with Ba2+ and looking for downstream effects on MSNs because of disrupted local K+ buffering is flawed.

Supplementary Figure 15 Average data for behavioral analysis.

R6/2 mice injected with AAV2/5 for Kir4.1-GFP were not different to R6/2 mice or to R6/2 mice injected with AAV2/5 for tdTomato in assays that measured motor function (forelimb clasp time and the time to fall off a rotarod). The clasp time experiments were conducted over 1 min. Since the WT mice did not clasp in this time period, their data values are all at 60 s.

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Tong, X., Ao, Y., Faas, G. et al. Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice. Nat Neurosci 17, 694–703 (2014). https://doi.org/10.1038/nn.3691

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