Hyperactive somatostatin interneurons contribute to excitotoxicity in neurodegenerative disorders

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping neurodegenerative disorders whose pathogenesis remains largely unknown. Using TDP-43A315T mice, an ALS and FTD model with marked cortical pathology, we found that hyperactive somatostatin interneurons disinhibited layer 5 pyramidal neurons (L5-PNs) and contributed to their excitotoxicity. Focal ablation of somatostatin interneurons efficiently restored normal excitability of L5-PNs and alleviated neurodegeneration, suggesting a new therapeutic target for ALS and FTD.

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Figure 1: Reduced GABAergic transmission, hyperexcitability and excitotoxicity of L5-PNs in TDP mice.
Figure 2: Hyperactive Sst neurons lead to sustained disinhibition of L5-PN in TDP mice.
Figure 3: Bilateral ablation of Sst interneurons diminished excitotoxicity of L5-PN in TDP mice.

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Acknowledgements

This research was supported by Jackson Lab Startup Funds (D.-T.L.), 1R21 NS075382-01A1 (Y.L. and G.A.C.), and the Intramural Research Program of the National Institute on Drug Abuse (D.-T.L.).

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Contributions

Y.L., D.-T.L., W.Z. and L.Z. designed the study and wrote the manuscript. W.Z. and L.Z. performed all of the experiments and analysis. B.L. helped with data analysis. D.S. maintained some mouse colonies. G.A.C. and Z.Z. advised on experiments and manuscript preparation.

Corresponding authors

Correspondence to Yun Li or Da-Ting Lin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Selective impairments in GABAergic transmission in L5-PNs of TDP mice.

(a) L5-PNs in TDP mice (3 weeks age) exhibited significantly reduced eIPSC. Left: overlay of the average of maximal response of eIPSC recorded from WT and TDP mice. Right: quantitative analysis of the amplitude (WT, 5.1 ± 0.3 nA; TDP, 3.1 ± 0.4 nA; Mann-Whitney U-test, p = 0.0007, z(20) = 2.6) of the maximal response of eIPSC (WT and TDP, n = 11 neurons, 3 mice each group). (b) TDP mice (3 weeks age) showed reduced GABAergic synapse densities around somatic areas of L5-PN. TDP mice were crossed with Thy1-YFPH (YFP) mice to label L5-PNs and GABAergic synapses were identified through immunostaining of presynaptic vesicular GABA transporter (VGAT). Left: representative overlaid (Red, VGAT; Green, YFP) and separated images of VGAT staining alone (scale bar represents 5 μm). Right: quantitative analyses of puncta densities (TDP-43::YFP, 79.5 ± 6.0% of YFP controls, unpaired t-test, p = 0.0326, t(85) = 2.173; n = 45, 42 neurons from 4 mice each group). (c) mEPSC was similar in 3-week-old TDP and WT littermate controls. Top: representative mEPSC traces. Bottom: quantitative analyses of amplitude (WT, 11.3 ± 0.7 pA; TDP, 11.5 ± 0.9 pA; Mann-Whitney U-test, p = 0.99, z(42) = 0.01) and frequency (WT, 5.6 ± 0.5 Hz; TDP, 5.9 ± 1.1 Hz; Mann-Whitney U-test, p = 0.47, z(42) = 0.72) of mEPSC (WT and TDP, n = 24 and 20 neurons, 3 mice each group). (d) Intracellular application of picrotoxin (PTX, 350 μM) enhanced AP firing of L5-PNs from both TDP and WT mice, and abolished the AP firing differences between TDP and WT mice. Mann-Whitney U-test, p < 0.0001, z(91) = 5.23 for WT (n = 53, 40 neurons from 6 mice without PTX and 3 mice with PTX); Mann-Whitney U-test, p = 0.0024, z(106) = 3.02 for TDP (n = 58, 50 neurons from 6 mice without PTX and 3 mice with PTX). See also Supplementary Table 1. (e) Intrinsic membrane properties of L5-PNs in M1 of TDP mice were not different from WT mice, including resting membrane potential (WT, –62.5 ± 1.6 mV; TDP, –61.6 ± 1.0 mV; Mann-Whitney U-test, p = 0.65, z(52) = 0.39), action potential (AP) threshold (WT, –40 ± 0.9 mV; TDP, –41.6 ± 1.3 mV, Mann-Whitney U-test, p = 0.53, z(52) = 0.66), and input resistance (WT, 92.6 ± 5.0 MΩ; TDP, 92.1 ± 4.7 MΩ, Mann-Whitney U-test, p = 0.96, z(52) = 0.05). Recordings are from 3 TDP mice (n = 29 neurons) and 3 WT mice (n = 25 neurons). Data are presented as mean ± s.e.m. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant, p > 0.05.

Supplementary Figure 2 Sustained impairments in GABAergic transmission and hyperexcitability of L5-PNs at late disease stage of TDP mice.

(a) Survival curve of TDP mice. Males (n = 22) and females (n = 11) TDP mice on a congenic C57BL/6J genetic background were monitored for mortality. Survival curve showed an average survival of 105 days for TDP males, and 182 days for TDP females (Log-rank or Mantel-Cox test, p < 0.0001, Chi square = 28.12). (b) TDP mice (15-week-old) showed significantly reduced VGAT puncta in perisomatic L5-PNs. Left: representative images of VGAT staining (red, VGAT immunostaining; green, YFP; scale bar represents 5 μm). Right: quantitative analyses of puncta densities (TDP-43::YFP, 68.3 ± 3.2 % of 15-week-old YFP controls, Mann-Whitney U-test, p < 0.0001, z(191) = 5.69; n = 101 and 92 neurons for YFP and TDP-43::YFP, 4 mice each group). See also Supplementary Table 2. (c) Hyperexcitability of L5-PNs in 15-week-old TDP mice. Left: representative AP responses to 600 pA current injection. Right: F-I plots of WT (n = 21 neurons, 3 mice) and TDP (n = 20 neurons, 3 mice; Unpaired t-test, p = 0.0317, t(39) = 2.228 for 600 pA current injection). Data are presented as mean ± s.e.m. *, p < 0.05; ***, p < 0.001.

Supplementary Figure 3 Two-photon in vivo imaging and neurodegeneration in M1 cortex of TDP-43::YFP mice.

(a) Schematic top view of cranial bone mapping showing the location of cranial window on M1 cortex. (b) Schematic cartoon of thin-skulled cranial window. Skull (black) was thinned and polished to 15-20 μm thickness. A glass coverslip (red) was attached to the thinned skull with cyanoacrylate cement (yellow). Dental cement (pink) was used to seal the edge of the glass coverslip against the remaining skull. This optical window allowed imaging of fluorescent neurons (green) with a long-working distance water immersion (blue) with 20x (NA 1.0) objective. (c) Representative vascular images beneath the cranial window across the experimental period. Vascular patterns were used as land marker for in vivo repetitive imaging. Scale bar represents 50 μm. (d) Dynamic appearance of dendritic blebs in M1 cortex of TDP mice viewed through in vivo imaging. Arrows (red) in both panels showed that blebs on dendrites (left, 6-week-old) disappeared (right, 15-week-old). Arrowhead (green) indicated the appearance of new dendritic blebs in 15-week-old TDP mouse image. Scale bar represents 20 μm. (e) Neurodegeneration of L5-PNs at late stage of TDP mice. TDP mice were crossed with Thy1-YFPH (YFP) mice to label L5-PNs. YFP positive neurons were counted and compared in 15-week-old mice. TDP-43::YFP mice showed a significantly reduced YFP positive neuron density at 15-week-old age, compared to their YFP littermate controls (YFP, 3348 ± 107.6 mm−3; TDP-43::YFP, 2048 ± 170.2 mm−3; n = 36, 25 slices from 5 mice each group; Mann-Whitney U-test, p < 0.0001, z(59) = 4.85). Data are presented as mean ± s.e.m. ***, p < 0.001.

Supplementary Figure 4 Analysis of Pv interneurons in TDP mice.

(a) Pv interneurons show normal mIPSCs in 3-week-old TDP mice. Left: representative mIPSC traces. Right: quantitative analyses of frequency (WT, 24.2 ± 1.9 Hz; TDP, 25.5.8 ± 1.8 Hz; Mann-Whitney U-test, p = 0.55, z(59) = 0.6) and amplitude (WT, 32.7 ± 1.9 pA; TDP, 37.0 ± 2.0 pA; Mann-Whitney U-test, p = 0.11, z(59) = 1.58) of mIPSC (WT and TDP, n = 30 and 31 neurons, 3 mice each group). (b) The excitability of Pv interneurons appeared normal in 8-week-old TDP mice. Left: representative AP firings of Pv interneurons in response to 300 pA current injection from PvCre (control) and TDP-43::PvCre (TDP) mice injected with Cre dependent eYFP virus. Right: quantitative analyses of resting membrane potential (control, –68.4 ± 0.8 mV; TDP, –68.9 ± 0.8 mV; Mann-Whitney U-test, p = 0.32, z(55) = 0.47) and AP firing frequency (control, 94.0 ± 21.6 Hz; TDP, 84.9 ± 14.4 Hz; Mann-Whitney U-test, p = 0.68, z(55) = 0.42; n = 29 and 28 neurons for control and TDP, 3 mice each group). (c) There were no significant changes in the number of Pv interneurons in M1 cortex of 3-week-old TDP mice. Left: representative images of Pv immunostaining (scale bar represents 100 μm). Right: quantitative analyses (WT and TDP, n = 16 counts from each side of 8 slices, 4 mice each group; Unpaired t-test, p = 0.5197, t(30) = 0.651). (d) There were no significant changes in the number of Pv interneurons in M1 cortex of 15-week-old TDP mice. Left: representative images of Pv immunostaining (scale bar represents100 μm). Right: quantitative analyses (WT and TDP, n = 24 counts from each side of 12 slices, 4 mice each group; Unpaired t-test, p = 0.393, t(46) = 0.863). Data are presented as mean ± s.e.m. N.S., not significant, p > 0.05.

Supplementary Figure 5 Analysis of Sst interneuron densities in different disease stages of TDP mice.

(a) Sst interneurons were reduced in M1 cortex of 3-week-old TDP mice. Left: representative images from SstCre::tdTomato (control, scale bar represents 100 μm) and TDP-43::SstCre::tdTomato mice (TDP). Right: quantitative analyses (TDP, 83.1 ± 3.2% of age matched control, Unpaired t-test, p = 0.0003, t(44) = 3.961; n =24, 22 counts from 12 slices, 4 mice each group). (b) Sst interneurons were significantly increased in M1 cortex of 15-week-old TDP mice. Left: representative images from SstCre::tdTomato (control, scale bar represents 100 μm) and TDP-43::SstCre::tdTomato mice (TDP). Right: quantitative analyses (TDP, 109.5 ± 2.5% of age matched control, Unpaired t-test, p = 0.013, t(45) = 2.586; n = 23, 24 counts from 12 slices, 4 mice each group). Data are presented as mean ± s.e.m. *, p < 0.05; ***, p < 0.001.

Supplementary Figure 6 Sst interneurons disinhibit L5-PNs, while Pv interneurons directly inhibit L5-PNs.

(a) Photo-stimulation of ChR2 or eNpHR3.0 induced changes in membrane properties. Top: photo-stimulation (1 ms) led to firing of YFP positive neuron in PvCre mice injected with Cre dependent ChR2 virus. Middle: photo-stimulation (1 ms) led to hyperpolarization of YFP positive neuron in PvCre mice injected with Cre dependent eNpHR3.0 virus. Bottom: the paradigm of photo-stimulation. (b) Sst interneurons disinhibit L5-PNs. Long-pulse (5 second each pulse) photo-stimulation of Sst interneurons modulates AP firing frequency of L5-PNs. Plot shows population (open circles) and average results (filled circles, black = light off; blue or yellow = light on) of ChR2 (light off, 1.0 ± 0.3 Hz; light on, 1.5 ± 0.3 Hz, Wilcoxon matched-pairs signed rank test, p = 0.004, z(13)= 2.72; n = 14, 3 mice) and eNpHR3.0 (light off, 2.8 ± 0.6 Hz; light on, 2.0 ± 0.5 Hz, Wilcoxon matched-pairs signed rank test p < 0.0001, z(19) = 3.91; n = 20, 3 mice). (c) Sst interneurons disinhibit L5-PNs. Short-pulse (10 ms, 20 Hz for blue light and 40 ms, 20 Hz for yellow light) photo-stimulation of ChR2 (left) and eNpHR3.0 (right) on Sst interneurons of M1 cortex induced changes in L5-PNs AP firing frequency. Top: representative traces of L5-PNs recordings before and after photoactivation of Sst interneurons by brief pulses of blue light (10 ms, 20 Hz, blue bars), or photo-inhibition of Sst interneurons by brief pulses of yellow light (40 ms, 20 Hz, yellow bars). Middle: plots show population (open circles) and average results (filled circles, black = light off; blue or yellow = light on) of ChR2 (light off, 1.80 ± 0.44 Hz; light on, 2.20 ± 0.47 Hz, Paired T-test, p = 0.0039, t(13) = 3.45; n = 14, 3 mice) and eNpHR3.0 (light off, 6.07 ± 0.98 Hz; light on, 4.09 ± 0.81 Hz, Paired T-test, p = 0.0016, t(17) = 3.75; n = 18, 3 mice). Bottom: plots show population (open circles) and average (filled circles, black = light off; blue or yellow = light on) of normalized ratio in AP frequency of ChR2 (Ratio of “light on” over “light off”, 1.58 ± 0.24; Wilcoxon Signed Rank Test, p = 0.0064, z(13) = 2.57; n = 14 neurons, 3 mice) and eNpHR3.0 (Ratio of “light on” over “light off”, 0.65 ± 0.09; Wilcoxon Signed Rank Test, p = 0.002, z(17) = 3.1; n = 18 neurons, 3 mice). (d) Short-pulse (10 ms, 20 Hz for blue light and 40 ms, 20 Hz for yellow light) photo-stimulation of Sst interneurons modulates charges of sIPSC of L5-PNs. Plot shows population (open circles) and average sIPSC charges (filled circles, black = light off; blue or yellow = light on) of ChR2 (light off, 1.34 ± 0.21 pC S−1; light on, 1.24 ± 0.18 pC S−1, Paired T-test, p = 0.019, t(14) = 2.64, n = 15 neurons, 3 mice) and eNpHR3.0 (light off, 1.07 ± 0.13 pC S−1; light on, 1.30 ± 0.21 pC S−1, Wilcoxon Signed Rank Test, p = 0.001, z(19) = 3.09, n = 20 neurons, 3 mice). (e) Pv interneurons inhibit L5-PNs. Long-pulse (5 second) photo-activation of ChR2 (left) and eNpHR3.0 (right) of Pv interneurons in M1 cortex induced changes in L5-PNs spontaneous firing frequency. Top: representative traces of L5-PNs recordings before and after photoactivation. Bottom: population (open circles) and average results (filled circles, black = light off; blue or yellow = light on) of ChR2 (light off, 3.6 ± 0.5 Hz; light on, 0.5 ± 0.3 Hz, Wilcoxon matched-pairs signed rank test, p < 0.0001, z(20) = 4.01; n = 21 neurons, 3 mice) and eNpHR3.0 (light off, 1.4 ± 0.4 Hz; light on, 2.5 ± 0.4, Wilcoxon matched-pairs signed rank test, p = 0.0005, z(21) = 3.24; n = 22 neurons, 3 mice). Data are presented as mean ± s.e.m. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Supplementary Figure 7 Schematic design of rescue experiments.

Sst interneurons were genetically labeled with diphtheria toxin receptor (DTR) and diphtheria toxin (DT) was bilateral focal injected into the M1 cortex of 6-week-old TDP mice. Two weeks after DT injection, mIPSC and spiking activity of L5-PNs were measured. Six weeks after DT injection, various immunostainings were carried out. VGAT staining was used for detect GABAergic synapses formed on L5-PNs. Ubiquitin staining was used to detect neuropathology. And NeuN staining was used for total neuron count to study the neurodegeneration.

Supplementary Figure 8 Sst immunostaining conformed the bilateral ablation of Sst interneurons after DT injection.

Left images (scale bar represents 1 mm) showing the Sst immunostaining from the entire coronal sections in WT and DTR::SstCre mice. Middle and right images are high magnification images (scale bar represents 100 μm) within the boxed areas.

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Zhang, W., Zhang, L., Liang, B. et al. Hyperactive somatostatin interneurons contribute to excitotoxicity in neurodegenerative disorders. Nat Neurosci 19, 557–559 (2016). https://doi.org/10.1038/nn.4257

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