Entorhinal cortex vulnerability to human APP expression promotes hyperexcitability and tau pathology

Abstract

Preventative treatment for Alzheimer's Disease (AD) is dire, yet mechanisms underlying early regional vulnerability remain unknown. In AD, one of the earliest pathophysiological correlates to cognitive decline is hyperexcitability, which is observed first in the entorhinal cortex. Why hyperexcitability preferentially emerges in specific regions in AD is unclear. Using regional, cell-type-specific proteomics and electrophysiology in wild-type mice, we uncovered a unique susceptibility of the entorhinal cortex to human amyloid precursor protein (hAPP). Entorhinal hyperexcitability resulted from selective vulnerability of parvalbumin (PV) interneurons, with respect to surrounding excitatory neurons. This effect was partially replicated with an APP chimera containing a humanized amyloid-beta sequence. EC hyperexcitability could be ameliorated by co-expression of human Tau with hAPP at the expense of increased pathological tau species, or by enhancing PV interneuron excitability in vivo. This study suggests early interventions targeting inhibitory neurons may protect vulnerable regions from the effects of APP/amyloid and tau pathology.

Fig. 1. PV-INTs in an AD vulnerable region display reduced baseline firing.

Graphical summary of AAV.E2.tdTom stereotactic injection in either the Lateral Entorhinal Cortex or Somatosensory cortex. PV-interneurons were fluorescently targeted for whole-cell current clamp recordings (d) as well as nearby excitatory cells (a). AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in excitatory cells (a) and PV-interneurons (d) mice from L2 LEC (left) and L5 SS Ctx (Right) at 12 pA/pF. b Group data summary of AP firing frequency in WT mice. Excitatory cells between LEC and SS Ctx showed no difference in AP Frequency (Hz) Ctx (LEC: Max: 50.42 ± 5.63 Hz, SS Ctx: Max: 46.35 ± 5.51 Hz, p = 0.46). c AP waveforms of excitatory cells were compared at 12 pA/pF square pulse injections in WT mice from L2 LEC and L5 SS Ctx. Aps from the 1st spike in the train are superimposed for comparison. e Group data summary of AP firing frequency in WT mice. PV interneurons in L2 LEC show a strong reduction in AP max firing frequency at higher current densities when compared to PV interneurons of L5 SS Ctx (LEC: Max: 131.6 ± 11.48 Hz, SS Ctx: Max: 301.1 ± 27.59 Hz, p = <0.0001 for 16 pA/pF and above). f AP waveforms of tdTom+ PV interneurons were compared at 12 pA/pF square pulse injections in WT mice from L2 LEC and L5 SS Ctx. Aps from the 1st spike in the train are superimposed for comparison. g Summary data of AP properties. L2 LEC PV interneurons display a significantly increased AP peak (LEC: 41.86 ± 2.66 pA, minimum: 21.33, 25% percentile: 33.47, Median: 44.53, 75% percentile: 49.12, maximum: 56.17, range: 34.84; SS Ctx: 28.75 ± 1.30 pA, minimum: 23.57, 25% percentile: 23.73, Median: 30.34, 75% percentile: 31.76, maximum: 34.89, range: 11.33; p = 0.0002, t = 4.83, df = 24, two-tailed unpaired t-test) and AP Hwdt (LEC: 0.48 ± 0.02 ms, minimum: 0.37, 25% percentile: 0.43, median: 0.47, 75% percentile: 0.55, maximum: 0.60, range: 0.23; SS: 0.33 ± 0.01 ms, minimum: 0.25, 25% percentile: 0.32, median: 0.33, 75% percentile: 0.37, maximum: 0.40, range: 0.15; p = <0.0001, t = 6.10, df = 25) for the first AP of the spike train. Individual data points (technical replicates, LEC n = 15, SS Ctx n = 11) and box plots are displayed. Significance is defined as p < 0.05, unpaired t-test. h Relationship between AP peak or width, in WT mice and AP # during spike trains elicited with a 12 pA/pF current injection. Data are averages of technical replicates, LEC n = 15, SS Ctx n = 11. For AP peak p < 0.0001, AP hwdt p < 0.0001. i Experimental approach for Regional-specific PV-interneuron Proteomes: E2 enhancer Cre AAV was retro-orbitally delivered to WT (Control) or Rosa26TurboID/wt (PV-CIBOP) mice (n = 3 per genotype, including males and females) followed by 3 weeks of Cre-mediated recombination, and 2 additional weeks of biotin supplementation (drinking water). The brain was then microdissected into LEC and SS Ctx and prepared for biochemical studies. j STRING analysis of PV-enriched proteins for LEC PV-INTs (left) and SS Ctx PV-INTs (right) (>2-fold enriched over other region) shows synaptic receptors, synaptic vesicle and exocytosis related proteins including GAD1/2, GABAb2/3, and complexins. k Enrichment of PWAS-identified proteins associated with cognitive slope in LEC (left) or SS Ctx (right) PV-enriched proteomic signatures. Cognitive slope was estimated in ROSMAP cases. Positive slope indicates cognitive stability or resilience when proteins are present while a negative slope indicates cognitive decline when proteins are present. Proteins positively correlated with cognitive slope are referred to as pro-resilience proteins while those negative correlated with cognitive slope are anti-resilience proteins. Enrichment of previously identified ‘pro’ and ‘anti’ resilience proteins within the PV protein dataset identified by CIBOP were assessed after weighting based on strength of association between proteins and cognitive slope. (LEC: 0.13 ± 0.03 and SS Ctx: −0.01 ± 0.03; p = 0.001, t = 3.71, df = 53, two-tailed Mann Whitney test). Proteins are derived from four biological replicates for each group. For b, e, and h: For all summary graphs, data are expressed as mean ( ± SEM). Statistical significance is denoted as *p < 0.05, as determined by Two-way ANOVA with Sidak’s multiple comparison test. For g, k: For all summary graphs, data are expressed as mean ( ± SEM). Also see Supplementary Fig. 1 for related analyses and datasets. Source data are provided as a Source Data File.

Fig. 2. Adult-onset human APP expression reduces LEC PV interneuron excitability.

a Graphical summary of AAV.E2.tdTom and AAV.EF1a.hAPP (or for Ctrl, saline) stereotactic injection in the Lateral Entorhinal Cortex. PV-interneurons were fluorescently targeted (tdTom + ) for whole-cell current clamp recordings. b AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in tdTom+ PV-INT from L2 LEC at 12 pA/pF. c Group data summary of AP firing frequency in L2 LEC from Ctrl (black) and hAPP injected mice (magenta). LEC PV interneurons from hAPP injected mice show a significant reduction in AP Frequency (Hz) when compared to Ctrl(Ctrl: Max: 156.6 ± 13.52 Hz, hAPP: Max: 91.84 ± 8.74 Hz, p < 0.0001, Two-Way ANOVA). d Summary data of AP properties. L2 LEC PV interneurons after hAPP injection display a significantly decreased input resistance (Ctrl: 145.7 ± 11.61 MΩ, minimum: 82.33, 25% percentile: 125.8, median: 148.4, 75% percentile: 175.3, maximum: 192.9, range: 110.6; hAPP: 88.78 ± 15.11 MΩ, minimum: 43.02, 25% percentile: 56.95, median: 80.93, 75% percentile: 139.1, maximum: 189.9, range: 146.8; p = 0.01, t = 2.73, df = 21) and an insignificant increase in membrane capacitance (Ctrl: 68.83 ± 5.34 pF, minimum: 40.49, 25% percentile: 60.14, median: 75.62, 75% percentile: 86.40, maximum: 99.78, range 59.26, hAPP: 90.21 ± 9.77 pF, minimum: 39.90, 25% percentile: 66.88, median: 90.51, 75% percentile: 115.2, maximum: 152.4, range: 112.5; p = 0.07, t = 1.92, df = 21). e. Graphical summary of AAV.E2.tdTom and AAV.EF1a.hAPP (or for Ctrl, saline) stereotactic injection in the Somatosensory Cortex. PV-interneurons were fluorescently targeted (tdTom + ) for whole-cell current clamp recordings. f AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in tdTom+ PV-INT from L5 SS Ctx at 12 pA/pF. g Group data summary of AP firing frequency in L5 SS Ctx from Ctrl (black) and hAPP injected mice (magenta). SS Ctx PV interneurons from hAPP injected mice show no significant change in AP Frequency (Hz) when compared to Ctrl (Ctrl: Max: 301.1 ± 27.59 Hz, hAPP: Max: 257.2 ± 24.06 Hz). h Summary data of AP properties. SS Ctx interneurons after hAPP injection display an unchanged Membrane Capacitance (Ctrl: 71.91 ± 9.514, minimum: 34.82, 25% percentile: 46.09, median: 67.31, 75% percentile: 104.9, maximum: 124.2, range 89.40; hAPP: 73.14 ± 7.327, minimum: 44.84, 25% percentile: 50.37, median: 68.69, 75% percentile: 98.70, maximum: 117.7, range: 72.83; p = 0.9180, t = 0.1041, df = 23) and input resistance (Ctrl: 121.2 ± 17.14, minimum: 65.09, 25% percentile: 68.17, median: 112.1, 75% percentile: 137.5, maximum: 240.6, range: 175.5; hAPP: 109.1 ± 10.56, minimum: 64.34, 25% percentile: 80.07, median: 89.76, 75% percentile: 148.6, maximum: 177.9, range: 113.6; p = 0.5475, t = 0.6106, df = 23). i RNAscope representative images at 40x magnification for Ctrl injected (top) and hAPP injected mice (bottom): mAPP mRNA (cyan), Parvalbumin mRNA (gold), human APP mRNA (magenta), and a final merged image. j RNAscope quantification for hAPP copies per PV+ cell comparing control to hAPP injected. hAPP injected show a significant increase in hAPP copies per PV+ cell (p = 0.0039, t = 5.987, df = 4; two-tailed paired t-test). For all summary graphs, data are expressed as mean ( ± SEM). For c, g, and i: Statistical significance is denoted as *p < 0.05, as determined by Two-way ANOVA with Sidak’s multiple comparison test. For d, h: Individual data points (technical replicates, LEC: Ctrl n = 10, hAPP n = 13; SS: Ctrl n = 12, hAPP n = 13; all data collected from 3 biological replicates each) and box plots are displayed. Statistical significance is denoted as *p < 0.05, as determined by two-tailed unpaired t-test. Source data are provided as a Source Data File.

Fig. 3. Murine APP does not affect PV interneuron physiology, but mAPP/hAB chimera replicates partial findings of hAPP-induced deficits.

a Pictorial representation of differing amino acids between murine APP and human APP proteins; 26 different amino acids in total, 3 of which are in the amyloid-beta segment of the protein. b Graphical summary of AAV.E2.tdTom and AAV.EF1a.mAPP (or for Ctrl, saline) stereotactic injection in the Lateral Entorhinal Cortex. PV interneurons were fluorescently targeted for whole-cell current clamp recordings. AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in PV interneurons from Ctrl (left) and mAPP injected (right) L2 LEC at 12 pA/pF. c Group data summary of AP firing frequency in Ctrl and mAPP injected mice. PV interneurons between Ctrl and mAPP injected showed no difference in AP Frequency (Hz) (Ctrl: Max: 122.3 ± 11.11 Hz, mAPP: Max: 120.6 ± 11.50 Hz, p = 0.95). Statistical significance is denoted as *p < 0.05, as determined by Two-way ANOVA with Sidak’s multiple comparison test. d RNAscope quantification for APP copies per PV+ cell with APP injected (mAPP or hAPP) each normalized to their contralateral hemisphere average endogenous murine APP copy per PV+ cell. mAPP injected and hAPP injected mice show similar increases in increased APP expression. copies per PV+ cell (p = 0.84, t = 0.21, df = 9; two-tailed unpaired t-test), data points represent biological replicates; mAPP n = 6, hAPP n = 5. e Pictorial representation of the resultant Chimera protein; murine APP with a humanized amyloid-beta segment. f Graphical summary of AAV.E2.tdTom and AAV.EF1a.mAPP/hAB Chimera (or for Ctrl, saline) stereotactic injection in the Lateral Entorhinal Cortex. PV interneurons were fluorescently targeted for whole-cell current clamp recordings. AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in PV interneurons from Ctrl (left) and Chimera injected (right) L2 LEC at 12 pA/pF. g Group data summary of AP firing frequency in Ctrl and Chimera injected mice. PV interneurons between Ctrl and mAPP injected showed no difference in AP Frequency (Hz) (Ctrl: Max: 193.6 ± 19.47 Hz, mAPP: Max: 145.4 ± 14.05 Hz, p < 0.0001; for 12 pA p = 0.0378, for 14 pA p = 0.0368, for 16 pA p = 0.0426). Statistical significance is denoted as *p < 0.05, as determined by Two-way ANOVA with Sidak’s multiple comparison test. h Comparison of PV interneuron firing frequencies expressing mAPP, mAPP/hAB Chimera, or hAPP normalized to their dataset controls at 12 pA/pF. Statistical significance is denoted as *p < 0.05, as determined by Ordinary one-way ANOVA with Tukey’s multiple comparisons test. (mAPP vs. Chimera: p = 0.0011, mAPP vs. hAPP: <0.0001, Chimera vs. hAPP: p = 0.0335; df = 35), data points represent technical replicates (cells) normalized to their own group controls, mAPP n = 11, Chimera n = 16, hAPP n = 11. Box plots (mAPP: minimum: 0.6925, 25% percentile: 0.8446, median: 1.065, 75% percentile: 1.420, maximum: 1.541, range: 0.8487; Chimera: minimum: 0.1538, 25% percentile: 0.5879, median: 0.7143, 75% percentile: 0.8571, maximum: 1.033, range: 0.8791; hAPP: minimum: 0.0000, 25% percentile: 0.1022, median: 0.3292, 75% percentile: 0.6360, maximum: 0.9407, range: 0.9407). For all summary graphs, data are expressed as mean ( ± SEM). Source data are provided as a Source Data File.

Fig. 4. Adult-onset human APP expression does not alter excitatory neuron physiology.

a Graphical summary of AAV.EF1a.hAPP (or for Ctrl, saline) stereotactic injection in the Lateral Entorhinal Cortex. Excitatory cells were targeted for whole-cell current clamp recordings. b AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in Ctrl and hAPP injected L2 LEC excitatory cells from at 12 pA/pF. c Group data summary of AP firing frequency in L2 LEC from Ctrl (black) and hAPP injected mice (magenta). Excitatory neurons in L2 LEC from hAPP injected mice show no alteration in AP Frequency (Hz) when compared to Ctrl (Ctrl: Max: 50.42 ± 5.63 Hz, hAPP: Max: 59.43 ± 6.56 Hz, p = 0.99, df = 28). d Summary data of AP properties. L2 LEC excitatory cells after hAPP injection display an unchanged Membrane Capacitance (p = 0.83, t = 0.27, Ctrl: minimum: 77.13, 25% percentile: 189.5, median: 208.5, 75% percentile: 261.9, maximum: 377.5, range: 300.3; hAPP: minimum: 103.0, 25% percentile: 189.9, median: 226.4, 75% percentile: 281.1, maximum: 324.4, range: 221.3) as well as an unchanged input resistance (p = 0.15, t = 1.50, df = 28; Ctrl: minimum: 30.98, 25% percentile: 60.00, median: 85.43, 75% percentile: 122.9, maximum: 259.8, range: 228.9; hAPP: minimum: 40.70, 25% percentile: 41.29, median: 59.46, 75% percentile: 106.4, maximum: 146.2, range: 105.5), data points represent biological replicates; Ctrl n = 19, hAPP n = 11. e Graphical summary of AAV.EF1a.hAPP (or for Ctrl, saline) stereotactic injection in the Somatosensory Cortex. Excitatory neurons in L5 were targeted for whole-cell current clamp recordings. f AP firing elicited by square pulse current injections of varying magnitude normalized to cellular capacitance during recording in excitatory cells from L5 SS Ctx at 12 pA/pF. g Group data summary of AP firing frequency in L5 SS Ctx from Ctrl (black) and hAPP injected mice (magenta). SS Ctx excitatory neurons from hAPP injected mice show no significant change in AP Frequency (Hz) when compared to Ctrl (Ctrl: Max: 46.35 ± 5.38 Hz, hAPP: Max: 61.43 ± 6.78 Hz, p > 0.05, df = 40). h Summary data of AP properties. SS Ctx interneurons after hAPP injection display an unchanged Membrane Capacitance (Ctrl: 176.9 ± 11.58, hAPP: 140.5 ± 14.31, p = 0.06, t = 1.98, df = 40, two-tailed unpaired t-test; Ctrl: minimum: 63.66, 25% percentile: 141.4, median: 180.5, 75% percentile: 229.4, maximum: 248.3, range: 184.7; hAPP: minimum: 47.71, 25% percentile: 88.57, median: 127.2, 75% percentile: 191.3, maximum: 285.2, range: 237.5) and input resistance (Ctrl: 117.8 ± 8.93, hAPP: 140.5 ± 20.43, p = 0.23, t = 1.05, df=40, two-tailed unpaired t-test; Ctrl: minimum: 58.62, 25% percentile: 91.03, median: 117.1, 75% percentile: 145.8, maximum: 202.1, range: 143.5; hAPP: minimum: 39.12, 25% percentile: 54.81, median: 111.9, 75% percentile: 215.4, maximum: 326.2, range: 287.5) data points represent biological replicates; Ctrl n = 21, hAPP n = 21. i RNAscope representative images at 40x magnification for Ctrl injected (top) and hAPP injected mice (bottom(: mAPP mRNA (cyan), CaMKIIa mRNA (green), human APP mRNA (magenta), and a final merged image. j RNAscope quantification for hAPP copies per CaMKIIa+ cell comparing control to hAPP injected. hAPP injected show a significant increase in hAPP copies per CaMKIIa+ cell (p = 0.0007, t = 7.42, df = 5; two-tailed paired t-test). For all summary graphs, data are expressed as mean ( ± SEM). For c, g Statistical significance is denoted as *p < 0.05, as determined by Two-way ANOVA with Sidak’s multiple comparison test. For d, h Individual data points and box plots are displayed. Statistical significance is denoted as *p < 0.05, as determined by two-tailed unpaired t-test. Source data are provided as a Source Data File.

Fig. 5. Human APP expression induces hyperexcitability in the LEC but not SS Ctx.

a Graphical summary of AAV.EF1a.hAPP (or for Ctrl, saline) stereotactic injection in the Lateral Entorhinal Cortex. Excitatory cells were targeted for whole-cell voltage-clamp recordings. b Spontaneous events obtained by holding cell voltage at 0 mV (inhibitory post-synaptic currents, IPSCs [top]) and −70 mV (excitatory post-synaptic currents, EPSCs [bottom]), interleaved. c Top: Cumulative distribution curve for spontaneous EPSCs in the LEC showing the relationship of relative frequency of events to the inter-event interval (IEI) (left). Quantified averages of IEIs are displayed for each cell as individual data points and compared between Ctrl (black) and hAPP injected (magenta) conditions (right). L2 LEC sEPSCs show a significant reduction in the IEIs (231.7 ± 12.25 ms, 272.7 ± 12.24 ms, hAPP and Ctrl respectively, p = 0.029, t = 2.361, df = 19, two-tailed unpaired t-test). Ctrl (top) box plot: minimum: 214.5, 25% percentile: 237.7, median: 277.7, 75% percentile: 301.8, maximum: 334.4, range: 120.0; hAPP: minimum: 147.3, 25% percentile: 196.8, median: 247.2, 75% percentile: 264.9, maximum: 276.1, range: 128.8. See Supplementary Fig. 13 for mEPSC data. Bottom: Cumulative distribution curve for spontaneous IPSCs in the LEC showing the relationship of relative frequency of events to the inter-event interval (left). Quantified averages of IEIs are displayed for each cell as individual data points and compared between Ctrl (black) and hAPP injected (magenta) conditions (right). L2 LEC sIPSCs show a significant increase in the IEIs (219.9 ± 15.84 ms, 177.3 ± 12.02 ms, hAPP and Ctrl respectively, p = 0.047, t = 2.097, df = 19, two-tailed unpaired t-test). Box plot (bottom) Ctrl: minimum: 123.8, 25% percentile: 142.7, median: 174.2, 75% percentile: 218.5, maximum: 223.2, range: 99.44; hAPP: minimum: 152.4, 25% percentile: 182.5, median: 199.5, 75% percentile: 249.3, maximum: 320.9, range: 168.6. See Supplementary Fig. 13 for mIPSC data. d Graphical summary of AAV.EF1a.hAPP (or for Ctrl, saline) stereotactic injection in the Somatosensory Cortex. Excitatory cells were targeted for whole-cell voltage-clamp recordings. e Spontaneous events obtained by holding cell voltage at 0 mV (IPSCs [top]) and −70 mV (EPSCs [bottom]), interleaved. f Top: Cumulative distribution curve for spontaneous EPSCs in the SS Ctx showing the relationship of relative frequency of events to the IEIs (left). Quantified averages of IEI are displayed for each cell as individual data points and compared between Ctrl (black) and hAPP injected (magenta) conditions (right). L5 SS Ctx sEPSCs show no change in the IEIs (p = 0.7372, t = 0.3450, df = 15; two-tailed unpaired t-test). Ctrl (top) box plot: minimum: 196.8, 25% percentile: 207.9, median: 243.5, 75% percentile: 260.9, maximum: 285.8, range: 89.01; hAPP: minimum: 146.2, 25% percentile: 227.8, median: 257.5, 75% percentile: 277.8, maximum: 289.2, range: 143.0. See Supplementary Fig. 13 for mEPSC data. Bottom: Cumulative distribution curve for spontaneous IPSCs in the SS Ctx showing the relationship of relative frequency of events to the inter-event interval (left). Quantified averages of IEIs are displayed for each cell as individual data points and compared between Ctrl (black) and hAPP injected (magenta) conditions (right). L5 SS Ctx sIPSCs show no change in the IEIs (p = 0.0812, t = 1.890, df = 15; two-tailed unpaired t-test). Box plot (bottom) Ctrl: minimum: 66.86, 25% percentile: 99.89, median: 138.2, 75% percentile: 191.3, maximum: 195.5, range: 128.7; hAPP: minimum: 86.38, 25% percentile: 133.9, median: 209.0, 75% percentile: 254.7, maximum: 277.0, range: 190.6. See Supplementary Fig. 13 for mIPSC data. Source data are provided as a Source Data File.

Fig. 6. hAPP-induced hyperexcitability observed in vivo can be attenuated by enhancement of PV interneuron excitability.

a Graphical summary of AAV.EF1a.hAPP (or for Ctrl, E2.tdTom) stereotactic injection in the Lateral Entorhinal Cortex. b 3 weeks post-injection, mice were anesthetized for in vivo glass electrode LFP recordings. b is a representative slice at 10x magnification from the LEC where DiI coated electrode recorded from a patch of E2.tdTom+ expressing cells. c Raw LFP signal from in vivo glass electrode recordings. d Power Spectral Density [PSD] analysis (dark lines: mean, light lines: ±SEM) comparing Ctrl (E2.tdTom+saline injected) to hAPP (E2.tdTom+hAPP injected). e Power derived from PSD analysis averaged for each mouse in the Delta (1–4 Hz), Theta (4–12 Hz), Gamma (30–80 Hz), and High Gamma (80–160 Hz) frequency ranges and compared between Ctrl and hAPP. Delta (Ctrl: 31.11 ± 0.94, Min. 28.86, Max. 33.43, Range. 4.57; hAPP: 37.02 ± 1.15, Min. 33.62, Max. 38.43, Range. 4.81; p = 0.0073, t = 3.98, df = 6); Theta (Ctrl: 26.20 ± 0.88, Min. 24.57, Max. 28.22, Range. 3.65; hAPP: 31.86 ± 1.34, Min. 27.97, Max. 33.98, Range. 6.01; p = 0.012, t = 3.54, df = 6), Gamma (Ctrl: 4.56 ± 1.59, Min. 0.24, Max. 7.13, Range. 6.89; hAPP: 11.17 ± 1.20, Min. 8.01, Max. 13.15, Range. 5.14; p = 0.016, t = 3.32, df=6), High Gamma (Ctrl: −1.22 ± 1.63, Min. −5.76, Max. 1.35, Range. 7.11; hAPP: 1.49 ± 1.65, Min. −2.98, Max. 4.97, Range. 7.96; p = 0.2864, t = 1.170, df = 6). Individual data points (representing biological replicates, n = 4 for each group) and box plots are displayed. Statistical significance is denoted as *p < 0.05, as determined by two-tailed unpaired t-test. f Graphical summary of AAV.E2.Gq.DREADD with AAV.EF1a.hAPP (or for Ctrl DREADD, E2.Gq.DREADD + E2.tdTom) stereotactic injection in the Lateral Entorhinal Cortex. 3 weeks post-injection, mice were anesthetized for in vivo glass electrode LFP recordings with administration of saline or 0.5 mg/kg administration of Clozapine-n-oxide [CNO] during the recording with a wait time after administration of 10–15 min prior to recording. g Spectrogram heatmap of LFP power (normalized) from the recording of one mouse after intraperitoneal saline injection (left, 15 min.) and intraperitoneal CNO injection (right, 15 min.). h Power Spectral Density analysis (dark lines: mean, light lines: ±SEM) comparing Ctrl DREADD (E2.Gq.DREADD + E2.tdTom + saline) to hAPP DREADD (E2.Gq.DREADD + E2.tdTom + hAPP). i [Left] Power Spectral Density analysis only for the Delta frequency range (1-4 Hz) (dark lines: mean, light lines: ±SEM) comparing Ctrl DREADD (black), Ctrl DREADD after CNO (Gray), hAPP DREADD (maroon), and hAPP DREADD after CNO (green). [Right] Power [derived from PSD analysis] averaged for each mouse in the Delta frequency range. (hAPP to Ctrl DREADD + CNO: p = 0.0063, hAPP DREADD to hAPP DREADD + CNO: p = 0.0193, Ctrl DREADD + CNO to hAPP DREADD + CNO: p = 0.5758, df = 7, One-Way ANOVA with Multiple Comparisons) Data represent mean ± SEM, individual data points displayed represent biological replicates, n = 3 for each group. j Power Spectral Density analysis (dark lines: mean, light lines: ±SEM) comparing Ctrl DREADD after 0.5 mg/kg Clozapine-n-oxide [CNO] injection (E2.Gq.DREADD + E2.tdTom + saline) to hAPP DREADD after 0.5 mg/kg CNO injection (E2.Gq.DREADD + E2.tdTom + hAPP). k [Left] Power Spectral Density analysis only for the Theta frequency range (4–10 Hz) (dark lines: mean, light lines: ±SEM) comparing Ctrl DREADD (black), Ctrl DREADD after CNO (Gray), hAPP DREADD (maroon), and hAPP DREADD after CNO (green). [Right] Power [derived from PSD analysis] averaged for each mouse in the Theta frequency range. (hAPP to Ctrl DREADD + CNO: p = 0.0493, hAPP DREADD to hAPP DREADD + CNO: p = 0.0261, Ctrl DREADD + CNO to hAPP DREADD + CNO: p = 0.8593, df = 7, One-Way ANOVA with Multiple Comparisons). Data represent mean ± SEM, individual data points displayed represent biological replicates, n = 3 for each group. Also see Supplementary Fig. 14 for related datasets. Source data are provided as a Source Data File.

Fig. 7. hTau co-expression with hAPP quells hyperexcitability but increases pathological tau.

a Graphical summary of AAV.EF1a.hAPP, AAV.EF1a.MAPT (hTau), or co-injected AAV.EF1a.hAPP with AAV.EF1a.MAPT stereotactic injection in the Lateral Entorhinal Cortex. Excitatory cells were targeted for whole-cell voltage-clamp recordings. b Spontaneous events obtained by holding cell voltage at −70 mV (excitatory post-synaptic currents, EPSCs [top]) and 0 mV (inhibitory post-synaptic currents, IPSCs [bottom]), interleaved. Quantified averages of event frequency are displayed for each cell normalized to Ctrl values as a ratio of EPSC Frequency to IPSC frequency and compared between hAPP injected (magenta), hTau injected (gray) and hAPP + hTau co-injected (pink) conditions. L2 LEC injected with hAPP showed a significantly elevated E:I ratio compared to hTau injected (p = 0.0136, df=20). hAPP and hTau co-injected E:I ratio was not significantly different from hAPP injected (p = 0.3323, df = 20) or hTau injected (p = 0.2175, df = 20). For all summary graphs, data are expressed as mean ( ± SEM), data points represent technical replicates (cells). Statistical significance is denoted as *p < 0.05, as determined by an Ordinary one-way ANOVA with Multiple comparisons. Box plot data: hAPP: minimum: 1.292, 25% percentile: 1.315, median: 1.539, 75% percentile: 2.317, maximum: 2.765, range: 1.473; hTau: minimum: 0.6859, 25% percentile: 0.7222, median: 0.8825, 75% percentile: 1.318, maximum: 1.600, range: 0.9138; hAPP+ hTau: minimum: 0.8832, 25% percentile: 1.062, median: 1.410, 75% percentile: 1.913, maximum: 1.989, range: 1.105. IHC representative images at 60x magnification for hTau (top) or hAPP+hTau (bottom) injected mice (for Ctrl or hAPP injected, see Supplementary Fig. 15) with staining for either AH36 (c) or T22 (e). d hAPP, hTau, and hAPP+hTau were analyzed for AH36 brightness in the first 100 um of every slice. AH36 brightness was normalized to CaMKII.eYFP brightness to control for any potential variability in viral expression. All groups were then normalized to the Ctrl injected condition. hAPP+hTau showed the highest level of AH36 brightness, although it was not significant over hAPP (p = 0.1267) or hTau (p = 0.4900) (df = 8, One-Way ANOVA with Multiple Comparisons). hAPP and hTau were also not significantly different (p = 0.5328). Box plot data: hAPP: minimum: 0.3056, 25% percentile: 0.3258, median: 0.9949, 75% percentile: 1.986, maximum: 2.113, range: 1.807; hTau: minimum: 0.9517, 25% percentile: 1.112, median: 1.673, 75% percentile: 2.038, maximum: 2.133, range: 1.182; hAPP+hTau: minimum: 1.916, 25% percentile: 1.916, median: 2.193, 75% percentile: 2.469, maximum: 2.469, range: 0.5531. f hAPP, hTau, and hAPP+hTau were analyzed for T22 brightness in the first 100 um of every slice. T22 brightness was normalized to CaMKII.eYFP brightness to control for any potential variability in viral expression. All groups were then normalized to the Ctrl injected condition. hAPP+hTau showed a significantly higher level of T22 brightness, above both hAPP (p = 0.0350) and hTau (p = 0.0.0389) (df = 8, One-Way ANOVA with Multiple Comparisons). hAPP and hTau were not significantly different (p = 0.9526). Box plot data: hAPP: minimum: 0.5981, 25% percentile: 0.5981, median: 1.048, 75% percentile: 1.206, maximum: 1.206, range: 0.6079; hTau: minimum: 0.4814, 25% percentile: 0.5575, median: 0.9968, 75% percentile: 1.699, maximum: 1.863, range: 1.382; hAPP+hTau: minimum: 1.776, 25% percentile: 1.782, median: 2.164, 75% percentile: 3.071, maximum: 3.252, range: 1.476. Source data are provided as a Source Data File.