Interneuron Accumulation of Phosphorylated tau Impairs Adult Hippocampal Neurogenesis by Suppressing GABAergic Transmission

Article

Interneuron Accumulation of Phosphorylated tau Impairs Adult Hippocampal Neurogenesis by Suppressing GABAergic Transmission Graphical Abstract

Authors Jie Zheng, Hong-Lian Li, Na Tian, ..., Longyu Ma, You Wan, Jian-Zhi Wang

Correspondence [email protected] (J.Z.), [email protected] (J.-Z.W.)

In Brief Impaired adult hippocampal neurogenesis contributes to the cognitive decline in Alzheimer’s disease. Zheng et al. report that phospho-tau accumulation in dentate gyrus GABAergic interneurons disrupts adult hippocampal neurogenesis and increased astrogliosis. Importantly, strengthening GABAergic signaling can rescue neurogenesis and improve cognitive functions in mouse models of Alzheimer’s disease.

Highlights d

Phospho-tau is accumulated in DG GABAergic interneurons of AD patients and mice

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Interneuron overexpressing human tau impairs adult hippocampal neurogenesis

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Tau accumulation reduces GABA, disinhibits local circuits, and promotes astrogliosis

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THIP, a d-GABAAR agonist, improves neurogenesis and cognition in AD mice

Zheng et al., 2020, Cell Stem Cell 26, 331–345 March 5, 2020 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.stem.2019.12.015

Cell Stem Cell

Article Interneuron Accumulation of Phosphorylated tau Impairs Adult Hippocampal Neurogenesis by Suppressing GABAergic Transmission Jie Zheng,1,7,* Hong-Lian Li,2,7 Na Tian,2 Fei Liu,1 Lu Wang,3 Yaling Yin,3 Lupeng Yue,4 Longyu Ma,5 You Wan,5 and Jian-Zhi Wang1,6,8,* 1Department of Pathophysiology, Key Laboratory of Ministry of Education for Neurological Disorders, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2Department of Histology and Embryology, Key Laboratory of Ministry of Education of China for Neurological Disorders, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 3Key Laboratory of Brain Research of Henan Province, Sino-UK Joint Laboratory of Brain Function and Injury of Henan Province, Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, China 4CAS Key Laboratory of Mental Health, Institute of Psychology, Department of Psychology, University of Chinese Academy of Sciences, Beijing 100101, China 5Neuroscience Research Institute and Department of Neurobiology, School of Basic Medical Sciences, Peking University, Beijing 100191, China 6Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226000, China 7These authors contributed equally 8Lead Contact *Correspondence: [email protected] (J.Z.), [email protected] (J.-Z.W.) https://doi.org/10.1016/j.stem.2019.12.015

SUMMARY

Phospho-tau accumulation and adult hippocampal neurogenesis (AHN) impairment both contribute importantly to the cognitive decline in Alzheimer’s disease (AD), but whether and how tau dysregulates AHN in AD remain poorly understood. Here, we found a prominent accumulation of phosphorylated tau in GABAergic interneurons in the dentate gyrus (DG) of AD patients and mice. Specific overexpression of human tau (hTau) in mice DG interneurons induced AHN deficits but increased neural stem cell-derived astrogliosis, associating with a downregulation of GABA and hyperactivation of neighboring excitatory neurons. Chemogenetic inhibition of excitatory neurons or pharmacologically strengthening GABAergic tempos rescued the tau-induced AHN deficits and improved contextual cognition. These findings evidenced that intracellular accumulation of tau in GABAergic interneurons impairs AHN by suppressing GABAergic transmission and disinhibiting neural circuits within the neurogenic niche, suggesting a potential of GABAergic potentiators for pro-neurogenic or cell therapies of AD.

INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia in the elderly. To date, therapeutic approaches targeting b-amyloid (Ab) and tau have been evidenced to exhibit limited effectiveness in halting AD progression in repeated clinical trials.

Repairing or reconstructing degenerated neural circuits in the AD brains, through either promoting innate neurogenesis (especially in the hippocampus) (Choi et al., 2018) or transplanting external stem cells or embryonic neurons (Hunsberger et al., 2016), brings new light in preventing cognitive decline in AD. However, how pathologic factors in the AD brains might dysregulate the fate of neurogenesis has not been fully elucidated. Adult hippocampal neurogenesis (AHN) derives from the proliferation of neural stem cells (NSCs), most of which remain quiescent under normal conditions due to both extensive GABAergic inputs from interneurons and interneuronal signaling mediated by direct cell-cell adhesion with glutamatergic neurons (Dong et al., 2019; Song et al., 2012). A remarkable decline of AHN was observed in AD patients and animals (Moreno-Jime´nez et al., 2019; Mu and Gage, 2011; Tobin et al., 2019), and this decline seems to occur even before the onset of AD (Dı´az-Moreno et al., 2018; Fu et al., 2019). Understanding how the fate of AHN is determined by AD pathological factors is cardinal for efficiently introducing neurogenesis, from either innate or grafted NSCs. Tau pathology is crucial in AD progression (Braak et al., 2006; Grundke-Iqbal et al., 1986; Lee et al., 1991). Recent studies also reveal a pivotal role of tau in experience-dependent regulation of AHN (Dioli et al., 2017; Pallas-Bazarra et al., 2016). Although AHN was found to be impaired in mice expressing full-length but not the anti-aggregated isoform of human tau (Joseph et al., 2017; Komuro et al., 2015), neurobiological mechanisms underlying how tau aggregation dysregulates AHN in AD are not known. We have previously reported that tau accumulation in mice hippocampus impaired local GABAergic transmission, presumably by dysregulating the GABA metabolism in interneurons (Li et al., 2017). Given the important role of GABAergic tempos in maintaining the NSC quiescence and shaping AHN (Bao et al., 2017), we hypothesized that tau accumulation, especially that in GABAergic interneurons, contributes to the AHN Cell Stem Cell 26, 331–345, March 5, 2020 ª 2019 Elsevier Inc. 331

Figure 1. Accumulation of Phosphorylated tau in GABAergic Interneurons in the Hippocampal SGZ and Hilus of AD Patients and 3xTg AD Mice (A) Representative images showing prominent accumulation of phosphorylated tau (pTau) in the SGZ and hilus of AD patients. Images were scanned and automatically spliced using the VS-ASW-S6 software (Olympus). Scale bars, 500 mm. (B and C) Most pTau-positive cells in the SGZ and hilus were identified as GABAergic interneurons, as indicated by co-labeling of pTau with GAD67 (B), and PV or SST (C). Arrows indicated co-labeled cells. Scale bars, 20 mm. (D–G) Prominent pTau accumulation in the SGZ and hilus of 6-month 3xTg AD mice (D, top panel). About 73.04% pTau-positive cells were identified as GABAergic interneurons (D, bottom panel, and F), and about 71.07% of PV-, or 82.12% of SST-positive interneurons showed pTau accumulation (E and G). Scale bars, 20 mm.

deficits in AD brains through dysregulating GABAergic transmissions and disrupting neural network dynamics in the hippocampal neurogenic niche. RESULTS

interneurons, the two major subpopulations of GABAergic interneuron, in the DG were found to have pTau accumulation (Figure 1G). These data together revealed a dominant pTau accumulation in the hippocampal DG GABAergic interneurons of AD patients and mice.

Prominent Accumulation of Phospho-tau in Hippocampal DG GABAergic Interneurons of AD Patients and Mice To investigate how tau aggregation dysregulates AHN in AD, we examined the distribution of phosphorylated tau (pTau) in the hippocampal neurogenic niche, i.e., the subgranular cell zone (SGZ) and hilus of dentate gyrus (DG) in AD patients and 3xTg AD mice. A remarkable accumulation of pTau was observed in the SGZ and hilus of AD patients (Figure 1A; Table S1), and the majority of those pTau-positive cells was identified as GABAergic interneurons by co-labeling with glutamate decarboxylase 67 (GAD67), parvalbumin (PV), and somatostatin (SST) (Figures 1B and 1C). Consistent with the pTau distribution in the DG of AD patients, prominent pTau accumulation was also observed in the SGZ and hilus of 6-month 3xTg AD mice, and most pTau-positive cells were also co-labeled with GAD67, PV, or SST (Figures 1D and 1E) but not CaMKII (a marker of projecting neurons, labeling glutamatergic neurons in the hippocampus) (data not shown). About 73.04% pTau+ (phospho-Thr181, Thr205, or Ser396) cells in the SGZ and hilus were identified as GABAergic interneurons (Figure 1F). Moreover, about 71.07% PV- and 82.12% SST-positive

Overexpressing hTau in DG GABAergic Interneurons Induced AHN Deficits To explore how human tau (hTau) accumulation in DG GABAergic interneurons affects AHN, we infused AAV-EF1a-DIO-hTaumCherry (the hTau group) or AAV-EF1a-DIO-mCherry (the mCherry group) into the DG subset of Dlx5/6-Cre-IRES-EGFP (termed as Dlx5/6-CIE), PV-Cre, and SST-Cre mice, respectively (Figures 2A–2D). Dlx5/6-CIE is a transgenic line in which Cre recombinase is specifically expressed in pan-GABAergic interneurons (Taniguchi et al., 2011) (Figures S1A–S1D; Table S2), and the Dlx5/6 promoter-driven expression of EGFP sharply drops to a nearly undetectable level in adult mice (Fu et al., 2012) (Figure S1E). We have previously evidenced that the AAV-mediated hTau overexpression is sufficient to mimic the AD-like pTau accumulation (Wei et al., 2018; Yin et al., 2016). One month after adeno-associated virus (AAV) injection, bromodeoxyuridine (BrdU, 50 mg/kg) was injected intraperitoneally for 5 consecutive days before the sacrifice of mice to evaluate cell proliferation. Doublecortin (DCX) and NeuroD1 were used as biomarkers to measure the number of immature granular cells (GCs). The Gfp gene-carried retrovirus (ROV-GFP) was co-injected with AAVs to examine the dendrite

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Figure 2. Interneuron-Specific Overexpression of hTau Induced AHN Deficits (A) Experimental scheme. AAV-EF1a-DIO-hTau-mCherry was stereotaxically infused into the DG of Dlx5/6-CIE, PV-Cre, or SST-Cre mice, respectively, to selectively overexpress human tau (hTau) in pan- or different subsets of GABAergic interneurons. (B) A diagram showing experimental strategies of measuring cell proliferation (BrdU), early survival and differentiation of immature neurons (DCX, neuroD1), and dendrite maturation of newborn neurons generated before hTau accumulation (ROV-GFP). (C and D) Representative images showing the expression pattern of mCherry (AAV) and GFP (ROV) in Dlx5/6-CIE mice (C), and PV-Cre or SST-Cre mice (D). Arrows indicate co-labeled cells. Scale bars were as indicated in each image. The image in (C, left) was scanned and automatically spliced using the VS-ASW-S6 software (Olympus). See also Figure S1. (E–H) hTau overexpression in pan-GABAergic interneurons decreased the number of BrdU-, DCX-, and NeruroD1-immunoreactive (IR) cells and (F) did not change the total number but decreased the dendrite length (G), complexity, and spine density (H) of ROV-GFP-labeled newborn neurons. Representative images are shown in (E). Scale bars are as indicated in each panel of image. The white circle indicates an astrocyte-like cell labeled by ROV-GFP. Unpaired t tests and two-way ANOVA, *p < 0.05, **p < 0.01. n = 6 mice (dots) or 13–18 neurons (rhombus). (legend continued on next page)

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maturation of pre-existing newborn GCs that generated before hTau was accumulated to a high level (Figure 2B). Selective overexpressing hTau in pan-GABAergic interneurons (Dlx5/6-positive) decreased the number of BrdU-, DCX-, and NeuroD1-labeled cells (Figures 2E and 2F), with reduced dendrite length, complexity, and spine density but unchanged total number of ROV-GFP-labeled newborn neurons (Figures 2G and 2H), compared with the mCherry group. These data suggest that specific overexpressing hTau in GABAergic interneurons induces multiple AHN deficits in mice. We further verified how PV- and SST-interneuron hTau overexpression independently contributes to the observed AHN impairments. Selective hTau overexpression in PV and SST interneurons resulted in comparable deficits of AHN: both reduced BrdU- and DCX-labeled cell number and deceased the dendrite length, complexity, and spine density without changing the number of ROV-GFP-labeled newborn neurons (Figures 2I and 2J). These data suggest that PV and SST interneurons may be of equal importance in regulating AHN. Overexpressing hTau in DG Excitatory or Immature GCs Had Limited Effects on AHN We also examined whether and how hTau accumulation in DG excitatory (including mature GCs and mossy cells) or immature GCs affects AHN, since accumulation of pTau (especially those phosphorylated at Thr205) was also detected in those cells in the current (data not shown) and previous studies (Bullmann et al., 2007). Specific overexpression of hTau in excitatory DG neurons, by AAV-CaMKIIa-hTau, reduced dendritic spine density of newborn neurons but exhibited only a limited effect on cell proliferation and the early survival/differentiation of immature neurons during AHN (Figures S2A–S2H). Unexpectedly, specific overexpression of hTau in immature GCs, by combinative infusion of ROVEF1a-Cre with AAV-EF1a-DIO-hTau, promoted the survival of newborn neurons but induced no significant changes in cell proliferation, differentiation, and dendrite maturation during AHN (Figures S2I–S2P). Therefore, we speculated that the tau accumulation in GABAergic, but not any of the other subsets of DG neurons, was the main player that contributed to the significantly impaired AHN in AD observed in the present and previous studies (Moreno-Jime´nez et al., 2019; Tobin et al., 2019). We then focused on investigating how the interneuron hTau accumulation induced AHN deficits. Interneuron-Specific Overexpression of hTau Increased NSC-Derived Astrogliosis We examined whether the observed AHN deficits, induced by GABAergic interneuron hTau overexpression, were derived from NSCs abnormality, using Dlx5/6-CIE:Nes-GFP mice obtained by crossbreeding Dlx5/6-CIE with Nestin-GFP transgenic mice. Typically, quiescent NSCs can be morphologically charac-

terized by smog-like and densely distributed branches at the very distal part of one or two main trunks originated from soma under 20–403 objective lens (Figure 3A). Interneuron-specific overexpression of hTau did not statistically change the total number of Nes-GFP-positive cells (Figures 3B and 3C) but remarkably increased the proportion of morphologically atypical cells, as characterized by decreased distal but increased proximal branches from soma (Figures 3D and 3E). We next measured the mRNA of several key genes that play pivotal roles in regulating NSC quiescence or determining cell fate during AHN in Nes-GFP-positive cells, by using fluorescence-activated cell sorting (FACS) and quantitative real-time PCR (Table S2). Interneuron-specific overexpression of hTau suppressed the gene expression of ID4 (a marker of cell quiescence) but upregulated the expression of MCM2 (a marker of cell activation) (Figures 3F and 3G), indicating that NSCs were exiting from quiescence (Song et al., 2012; Zhang et al., 2019). Moreover, hTau overexpression also downregulated the mRNAs of COUP-TF1 and Neurod1 but upregulated Aldh1l1, Gfap, and S100b in GFP-positive cells (Figure 3F), which together suggested a switch of NSC-derived neurogenesis toward astrogliosis (Bonzano et al., 2018; Boutin et al., 2010; Gonc¸alves et al., 2016). To further trace the final fate of NSC-derived daughter cells, newborn cells were labeled by stereotaxic infusing ROV-GFP into the DG of Dlx5/6-CIE mice 3 weeks after AAV-DIO-hTaumCherry injection, when hTau was empirically ensured to have accumulated to a high level. Most ROV-GFP-labeled cells differentiated into astrocyte-like cells, but not neurons, and expressed GFAP, a protein dominantly expressed in astrocytes (Figures 3H–3J), after another 3 weeks of ROV-GFP infusion. Interestingly, most GFP/GFAP double-labeled cells tended to migrate toward the molecular layer (ML) of DG (Figure 3I). Consistently, the increased number of GFAP-immunoreactive cells was observed in the DG ML of mice with interneuron hTau overexpression (Figure 3K). These data evidenced that overexpression of hTau in GABAergic interneurons increased NSC-derived astrogliosis and impaired neurogenesis. Overexpressing hTau in DG GABAergic Interneurons Induced Local Neural Network Hyperactivation To further explore neurobiological mechanisms underlying the AHN deficits and NSC-derived astrogliosis induced by interneuron hTau overexpression, we screened proteins expression and phosphorylation in the DG of mice. A total of 142/4,536 proteins and 576/6,414 phosphopeptides in 371/2,013 proteins were quantified to be statistically up- or downregulated (take 1.3 as the threshold of fold change) in proteomic and phosphoproteomic analyses (Figure 4A), including 29 proteins and 139 phosphopeptides (in 60 proteins) engaged in the process of neurogenesis or astrogliosis (Figures S3A and S3B). Importantly, the most dominant enrichments of changed proteins or

(I and J) Selective overexpression of hTau in PV- and SST-positive interneurons resulted in similar impairments of AHN: both reduced the number of BrdU- and DCX-labeled newborn cells and decreased the dendrite length, complexity, and spine density without changing the total number of ROV-GFP-labeled newborn neurons (J). Representative images are shown in (I). Scale bars are as indicated in each panel. White circles indicate astrocyte-like cells labeled by ROVGFP. Unpaired t tests and two-way ANOVA, *p < 0.05, **p < 0.01. n = 5–6 mice (dots) or 21–25 neurons (rhombus). See also Figure S4. Data are represented as mean ± SEM.

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Figure 3. Interneuron hTau Overexpression Increased NSC-Derived Astrogliosis (A) A representative image showing the typical morphology of quiescent NSCs. Scale bar, 20 mm. (B) Representative images showing that interneuron hTau-overexpression induced morphological alterations of NSCs in Dlx5/6-CIE:Nes-GFP mice. Scale bars, 50 mm. (C–E) Interneuron-specific overexpression of hTau did not change the total number of GFP-labeled cells (C) but significantly increased the portion of morphologically atypical NSCs (D and E). n = 5 (mCherry) or 7 (hTau) mice. An unpaired t test was used. (F) Interneuron hTau overexpression downregulated the mRNAs of ID4, COUP-TF1, and Neurod1 (black stars) but upregulated the mRNAs of MCM2, Aldh1l1, Gfap, and S100b (red stars) genes in Nes-GFP-labeled cells. Data were normalized by GAPDH and the mean value of each gene in mCherry group. Unpaired t tests, n = 6 mice in each group. *p < 0.05, **p < 0.01. (G) Interneuron hTau overexpression increased the co-labeling rate of Nes-GFP+ cells with MCM2 but decreased their co-labeling rate with ID4. Arrows indicate co-labeled cells. Unpaired t tests, n = 5 (mCherry) or 6 (hTau) mice. *p < 0.05, **p < 0.01. (H–J) ROV-GFP-labeled daughter cells of NSCs finally differentiated into astrocyte-like cells (H) (83.12%) and expressed GFAP (I and J). ML, molecular layer. GCL, granular cell layer. (K) Interneuron hTau overexpression increased the number of GFAP-immunoreactive cells in DG ML. Scale bars, 50 mm. Unpaired t tests, n = 4 (mCherry) or 8 (hTau) mice. *p < 0.05. Data are represented as mean ± SEM.

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Figure 4. GABAergic Interneuron hTau Overexpression Disrupted Synaptic Transmissions and Induced Neural Network Hyperactivation (A) A total of 142/4,536 proteins and 576/6,414 phosphopeptides in 371/2,013 proteins were statistically up- or downregulated in the DG of mice with interneuron hTau overexpression. n = 3 mice in each group. (B) Proteins exhibited significant changes in expression or phosphorylation, by interneuron hTau overexpression, were most dominantly enriched in biological processes involved in synaptic assembly, plasticity, and transmissions. Fisher’s exact tests were used. See also Figure S3. (C) hTau overexpression changed the expression or phosphorylation of many proteins involved in general, glutamatergic, or GABAergic synaptic transmissions. A cartoon illustrating interneuronal transmissions in the SGZ is shown in the left panel. NSCs respond to tonic GABAergic afferents from GABAergic interneurons (blue lines) and direct cell-cell adhesion with excitatory granular and mossy cells (purple but dashed lines). Numbers in histograms are mapping values, i.e., the sum of proteins and phosphopeptides. (D) Experimental procedures of AAV injection and in vivo electrophysiological recordings. The representative image (bottom) showed tetrodes location in the SGZ (by electric lesion). Scale bar, 200 mm. (E–G) Interneuron hTau overexpression upregulated overall firing rate of DG neurons (G). Representative raster plots of single-unit firing (E) and waveforms after median filtering (F) are shown. Scale bars, 1 s. n = 55 (mCherry) or 39 (hTau) single units. Mann-Whitney test, **p < 0.01. (H and I) hTau increased the LFP power of spectrum density (PSD) (H), with dominant increase of in d oscillation but minor decrease in low g oscillation on a minor order of magnitude (I). Unpaired t tests, n = 6 (mCherry) and 7 (hTau) mice. *p < 0.05. (J) Experimental procedures of AAVs injection and in vivo optic fiber recordings. The representative image (right) showed the pattern of virus infection. Scale bar, 50 mm. (K and L) Interneuron hTau overexpression enhanced calcium responses (5% DF/F as threshold) in DG excitatory neurons, as indicated by increased number of calcium response (L, left) and area under DF/F curves (AUC) (L, right). Unpaired t tests, n = 7 mice in each group, *p < 0.05. Representative DF/F signals are shown in (K). See also Figure S4. Data are represented as mean ± SEM.

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phosphopeptides were found in the biological processes involved in synaptic transmission, assembly, and plasticity (Figures 4B, S3C, and S3D), suggesting an important contribution of transsynaptic signaling dysregulation to the observed AHN deficits or NSC-derived astrogliosis in interneuron hTau overexpression. Indeed, disrupted GABAergic and glutamatergic tempos were both sufficient to result in rapid exit of NSCs from quiescent and finally derive astrogliosis instead of neurogenesis (Bao et al., 2017; Sierra et al., 2015). Here, we observed that interneuron hTau overexpression significantly changed the expression or phosphorylation of various proteins involved in general, glutamatergic, or GABAergic synaptic transmissions (Figures 4C, S3C, and S3D). To directly examine how the hTau overexpression in interneurons dysregulated local neural network dynamics, we recorded neural firing rate and oscillations in the SGZ of mice through in vivo electrophysiological recordings (Figure 4D). hTau overexpression in GABAergic interneurons increased the overall neuronal firing rate (Figures 4E–4G) and the power spectra density (PSD) local field potential (LFP), which exhibited a dominant increase in delta oscillation (1–4 Hz) but a minor decrease in slow gamma oscillation (30–45 Hz) (Figures 4H and 4I). In addition, neither a significant change in average speed nor a total traveling distance of mice was detected during electrophysiological recordings (data now shown). We next specifically examined how interneuron hTau overexpression affects the activity of neighboring excitatory neurons by infusing AAV-CaMKIIa-GCaMP6f into the DG of mice and measuring calcium responses (Figure 4J). Compared with mice infected with mCherry or GFP (AAVs), interneuron hTau overexpression significantly enhanced calcium responses (take 5% DF/F as threshold) in the DG (Figures 4K and 4L), indicating a hyperactivation of local excitatory neurons. Consistent with the effect of pan-GABAergic hTau-overexpression on the local neuronal excitability, selective hTau overexpression in the subset of PV-positive interneurons also resulted in a hyperactivation of neighboring excitatory neurons, while that in SST-positive interneurons tended to suppress neuronal activity of local excitatory neurons (Figure S4). The discrepancy potentially involves the tertiary synaptic connections among PV, SST, and excitatory neurons in the DG (Espinoza et al., 2018). Local Neural Network Hyperactivation Contributed to the Interneuron hTau-Induced AHN Deficits To verify the contribution of pan-interneuron hTau-overexpression-induced neural network hyperactivation to the observed AHN deficits and NSC-derived astrogliosis, we chronically inhibited DG excitatory neurons using designer receptors exclusively activated by designer drugs (DREADDs). AAV-EF1a-DIOhTau-mCherry and AAV-CaMKIIa-hM4Di-FLAG were co-infused into the DG of Dlx5/6-CIE:Nes-GFP or Dlx5/6-CIE mice. Clozapine N-oxide (CNO) was administrated through drinking water (1.25 mg/mL) for 6 consecutive weeks (Yeh et al., 2018) (Figures 5A and 5B). Chemogenetic inhibition of excitatory neurons (Figure 5C) significantly ameliorated the NSC abnormality and AHN deficits induced by interneuron hTau overexpression, as indicated by an increased portion of morphologically quiescent NSCs (Fig-

ures 5D and 5E), an increased number of BrdU- and DCX-labeled cells (Figure 5F), as well as an increased dendrite complexity and spine density of newborn neurons (Figures 5G and 5H), compared with the mice administrated with CNO but co-expressed with hTau+FLAG. It should be noted that the chemogenetic inhibition of excitatory neurons did not statistically alter the portion of neuron-like ROV-GFP-labeled newborn GCs (Figure 5I) and the number of GFAP-immunoreactive astrocytes in the ML (Figure 5J), possibly due to the still insufficiency of direct GABAergic inputs from local GABAergic interneurons onto NSCs, intermediate progenitor cells (IPCs), and immature GCs caused by hTau-overexpression. Taken together, these data confirmed a major contribution of interneuron hTau-accumulation-induced neural network hyperactivation to the AHN deficits, which can be partly rescued by chemogenic inhibition of local excitatory neurons (Figure 5K). Interneuron hTau Accumulation Impaired GABAergic Transmission We have previously reported that tau accumulation dysregulated GABA metabolism in hippocampus (Li et al., 2017). To test whether the observed hTau-induced neural network hyperactivation was a result of any impairment in GABAergic transmission, here, we directly measured GABAergic responses in vivo with iGABASnFR, a genetically engineered GABA sensor (Marvin et al., 2019) (Figures 6A–6C). Interneuron hTau overexpression significantly attenuated GABAergic responses (taken 5% DF/F as the threshold) in the DG compared with mice that expressed only mCherry (Figures 6D and 6E). Consistently, hTau also downregulated the local level of GABA in the DG (Figure 6F), especially that of intra-GABAergic interneurons (Figure 6G). To further explore molecular mechanisms underlying the impairments of GABAergic transmission by hTau overexpression, we examined the expression of several key proteins involved in GABA metabolism, including GABA synthesis (GAD65 and GAD67), transport (VGAT), release (synaptotagmin1, synapsin1, and VAMP2), reuptake (GAT1), and degradation (ABAT and SSADH). No statistical change was found in any proteins except a decrease of GAT1, a transporter that anchored on plasma membranes to reuptake GABA from synaptic cleft (GonzalezBurgos, 2010) in hTau-overexpressed mice (Figures 6H and 6I). However, the decrease of GAT1 seemed unlikely to be responsible for the attenuated GABA responses but might be just a compensatory result of the GABA reduction. Importantly, we found that interneuron-specific hTau overexpression significantly downregulated the phosphorylation of GAD67, but not GAD65 (Figure 6J), the two major enzymes responsible for GABA production (Fenalti et al., 2007), though no statistical change in total GAD67 and GAD65 was detected (Figures 6H and 6I). Consistent results were also obtained in the phosphoproteomic analysis (Figure S3D). Thus, the reduction of phospho-GAD67 (pGAD67) might dysregulate GABA synthesis and finally lead to the reduction of GABA. In addition, we also observed that overexpressing hTau(P301S), a mutant isoform of tau more likely to misfold than wild-type tau (Yoshiyama et al., 2007), in GABAergic interneurons induced only comparable reduction of pGAD67 and GAT1 as wild-type hTau (Figure S5), suggesting that the misfold of hTau may not aggravate its toxic effect on GABA metabolism. Cell Stem Cell 26, 331–345, March 5, 2020 337

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Figure 5. Chemogenetic Inhibition of Neural Network Hyperactivation Ameliorated AHN Deficits Induced by Interneuron hTau Overexpression (A) Experimental scheme of virus injection and CNO administration. Dlx5/6-CIE:Nes-GFP mice were used for evaluating the NSC morphology, and Dlx5/6-CIE mice were stereotaxically injected with ROV-GFP for evaluating the dendrite maturation of newborn neurons. CNO was administrated via drinking water. (B) A representative image showing the pattern of viruses infection in the SGZ of Dlx5/6-CIE:Nes-GFP mice. Scale bars, 50 mm. (C) hM4Di+CNO, administrated for a month, decreased overall neuronal firing rate in the DG of mice with interneuron hTau overexpression. n = 20 single units in each group. One-way ANOVA followed by Tukey’s multiple comparisons tests, *p < 0.05, **p < 0.01. (D–J) Chemogenetic inhibition partly rescued the AHN deficits induced by interneuron hTau overexpression, as indicated by increased portion of morphologically quiescent NSCs (E), increased number of BrdU- and DCX-labeled immature cells (F), increased dendrite complexity (G), and spine density (H) of newborn neurons, but no obvious change in the portion of ROV-GFP-labeled neuron-like cells (I), as well as GFAP-immunoreactive astrocytes in DG ML (J), compared with mice administrated with CNO but co-overexpressed hTau+FLAG. Representative images are shown in (D). One-way or repeated-measures ANOVA followed by Tukey’s multiple comparisons tests, n = 6 mice (dots), or 17–18 neurons (rhombus) in each group. *p < 0.05, **p < 0.01. Scale bars are as indicated in each panel. (K) A summary cartoon illustrating that intracellular accumulation of hTau in interneurons suppressed GABAergic transmissions within the neurogenic niche, while hM4Di+CNO suppressed the hyperactivation (disinhibition) of local excitatory neurons and partly rescued the hTau-induced AHN deficits. Data are represented as mean ± SEM.

338 Cell Stem Cell 26, 331–345, March 5, 2020

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Besides, intracellular overexpression of hTau in GABAergic interneurons also downregulated the expression of d, but not the a, b, or g2 subunits of GABAARs and gephyrin, a protein that regulates the postsynaptic clustering of GABAARs (Figures 6H and 6I). It seems unlikely for intracellular hTau to directly interact with postsynaptic GABAARs. Instead, the reduction of GABAAR d might be mainly attributed to the decrease of immature GCs, the subpopulation with abundant expression of d-containing GABAARs (Farrant and Nusser, 2005; Ge et al., 2007). Strengthening GABAergic Transmission by THIP Rescued the Interneuron hTau-Induced AHN Deficits To test whether strengthening GABAergic transmission can rescue the AHN deficits induced by interneuron hTau overexpression, mice were subcutaneously injected with 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP, also known as gaboxadol), once every other day for a month (3 mg/kg). THIP is an agonist of d-containing GABAARs, which was primarily used here to inhibit the hTau-induced hyperactivation of excitatory neurons and meanwhile enhance the tonic GABAergic inputs into NSCs, IPCs, and immature GCs (Figure S6). We observed that THIP efficiently rescued the interneuron hTau-overexpression-induced AHN deficits, as indicated by the increased number of morphologically quiescent NSCs, increased number of BrdU- and DCX-labeled cells, increased dendrite length and spine density of newborn GCs, and reduced GFAP-immunoreactive astrocytes in the ML of DG, compared with controls that overexpressed hTau but administrated only vehicle (Figures 6K and 6L). THIP Ameliorated AHN Deficits and Cognitive Impairment in 3xTg AD Mice Last, we tested whether prior administration of THIP can prevent AHN deficits and improve cognitive function in 6-month 3xTg AD mice, when prominent pTau accumulation was observed in DG GABAergic interneurons (Figures 1D–1G), and local GABAergic transmission was suppressed (Figures 7A and 7B). AHN was impaired in 6-month 3xTg mice, as indicated by the decreased number of BrdU- and DCX-labeled cells, decreased dendrite length and spine density of newborn neurons, but increased astrocytes in DG ML compared with age-matched wild-type mice, while most of these deficits were ameliorated by subcutaneous administration of THIP (3 mg/kg once every other day for 8 weeks) (Figures 7C and 7D). Moreover, since 3xTg mice were reported to exhibit mild cognitive impairments at 4–6 months (Billings et al., 2005), which might be at least partly attributed to the AHN deficits. Therefore,

we tested whether THIP could improve the AHN-associated spatial recognition in 3xTg AD mice. A poorer performance in object-place recognition task was observed in 6-month 3xTg mice, as indicated by lower bias scores toward the object removed to novel places in the test phase, while THIP ameliorated this impairment (Figure 7E). We also tested the effect of THIP on contextual pattern separation. In this paradigm, each mouse acquired a fear memory on the first day by a 65-mA foot shock delivered in context A and was tested the next day to discriminate a very distinct context (context B) (Figure 7F). Both 3xTg and wild-type mice performed well in distinguishing context A from B, with limited effect of THIP (Figure 7G). In the following days, when mice were trained to discriminate another pair of contexts that shared much more features (context A, paired with foot shock, and C, paired without foot shock), however, 3xTg mice learned much slower than wild-type mice to discriminate context C from A, suggesting an impairment of contextual pattern separation, while prior administration of THIP significantly attenuated this impairment in 3xTg mice (Figures 7H and 7I). Taking mice performance at the day 12 as an example, the subgroup of 3xTg mice administrated with THIP, but not vehicle, had successfully learned to distinguish the similar contexts but spent less time freezing in context C than A (Figure 7J). In addition, THIP also downregulated the phosphorylation and aggregation of tau in the DG of 3xTg mice but showed very limited effect on amyloid pathology, which was actually not yet evident in the DG of 6-month 3xTg AD mice (Figure S7). These data together suggested that strengthening GABAergic tone by prior administration of THIP was efficient in attenuating AHN deficits and improved AHN-dependent cognitive functions in 3xTg AD mice. DISCUSSION Tau Accumulation in GABAergic Interneurons Impaired AHN AHN declines but persists throughout aging (Boldrini et al., 2018; Sorrells et al., 2018). However, AD-like pTau accumulation, especially those in GABAergic interneurons, accelerated the age-dependent decline of AHN, by attenuating GABAergic tone and disinhibiting local neural circuits in the neurogenic niche. Indeed, GABAergic interneurons seem to be a subset of high vulnerability to AD factors, such as ApeoE4 and tauopathy (Andrews-Zwilling et al., 2010; Li et al., 2009; Wang et al., 2018a). There are two major subtypes of GABAergic interneurons, i.e., the PV- and SST-positive neurons. pTau accumulation in these

(F and G) hTau overexpression downregulated local level of GABA in DG detected by dot blotting (F). Unpaired t tests, n = 6 mice in each group, *p < 0.05. Representative immunofluorescence images showing that intracellular GABA was remarkably reduced in hTau-overexpressed interneurons (G). Scale bar, 5 mm. (H and I) Interneuron hTau overexpression downregulated total GAT1 and d-GABAAR but exhibited limited effects on many other proteins involved in either GABA synthesis, transport, release, degradation, and post-synaptic signaling. Representative blots are shown in (H). Data were normalized to b-actin and the mean value of mCherry group for each protein. Unpaired t tests, n = 4–8 mice, *p < 0.05. See also Figure S5. (J) Interneuron hTau overexpression downregulated the phosphorylation of GAD67 (highlighted by the red boxes) but not GAD65. (K and L) THIP, a d-GABAAR agonist, ameliorated the interneuron hTau-overexpression-induced AHN deficits, by increasing the portion of morphologically quiescent NSCs, the number of BrdU- and DCX-labeled cells, the dendrite length, and spine density of newborn neurons but decreasing the number of GFAPimmunoreactive astrocytes in ML, compared with mice with hTau overexpression but administrated with vehicle. Numbers inside histograms indicate mean percentages. Representative images are shown in (K). Two-way ANOVA followed by Tukey’s multiple comparisons tests, n = 6 mice (dots), or 9–16 neurons (rhombus) in each group, *p < 0.05, **p < 0.01. Scale bars are as indicated in each panel. See also Figure S6. Data are represented as mean ± SEM.

340 Cell Stem Cell 26, 331–345, March 5, 2020

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Figure 7. THIP Ameliorated AHN Deficits and Cognitive Impairments in 3xTg AD Mice (A and B) Attenuated GABAergic transmission in the DG of 6-month 3xTg AD mice (B). Unpaired t test, n = 7 in each group, *p < 0.05. The representative image in (A) shows the AAV expression and the location of optic fiber end. The image was scanned and automatically spliced using the ZEN software (Zeiss). (C) Experimental procedures of THIP administration, ROV injection, and behavioral tests. (D) THIP partly ameliorated AHN impairments in 3xTg mice, as indicated by no statistical change in BrdU-IR cells, but increased number of DCX-labeled cells, increased dendrite length and spine density of ROV-GFP labeled newborn neurons, and decreased number of astrocytes in DG ML, compared with 3xTg mice administrated with vehicle. Scale bars are as indicated in each panel. Two-way ANOVA followed by Tukey’s multiple comparisons tests, n = 6 in each group, *p < 0.05, **p < 0.01. (legend continued on next page)

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two subsets of interneurons contrarily dysregulated the excitability of local excitatory neurons, but both impaired AHN. GABA secreted from either PV or SST interneurons can both directly act on NSCs and immature neurons to shape AHN (Yang et al., 2019). On the other hand, either hyper- or hypoactivation of DG excitatory neurons, resulting from reduced GABAergic inputs onto PV or SST interneurons, respectively, can ultimately lead to AHN deficits, as observed in the current study. It is worth noting that the possible contribution of pTau accumulation in excitatory or immature neurons to the AD-associated AHN deficits should not be excluded, though selective hTau overexpression in those cells induced minor effects on AHN compared to those in GABAergic interneurons. Besides, although the role of extracellular Ab deposition in the dysregulation of AHN remains controversial (Yetman and Jankowsky, 2013; Ziegler-Waldkirch et al., 2018), whether and how soluble Ab affects AHN at the early stage of AD deserves future investigation. Interneuron Accumulation of hTau Dysregulated GABA Metabolism and Disinhibited Local Neural Circuits Interneuron overexpression of hTau downregulated the local level of GABA, presumably by dysregulating GABA synthesis through suppressing GAD67 phosphorylation (Fenalti et al., 2007; Wei et al., 2004). An intriguing question is that how tau, a cytosolic protein, leads to the downregulation of pGAD67 and GAT1. Proteomic and phosphoproteomic analyses in the present study revealed multiple changes in the expression or phosphorylation of various protein kinases, such as protein kinase C epsilon (PKCε), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) (data not shown). These kinases might somehow play important roles in the phosphorylation of GAD67 to dysregulate the synthesis of GABA (Blasio et al., 2018; Wei et al., 2004). Molecular mechanisms underlying how hTau induced the kinases/phosphatase imbalance and lead to GAD67 dephosphorylation remain to be elucidated. hTau accumulation impaired GABAergic transmission, thus disinhibited local excitatory neurons. Gamma oscillation was observed to be suppressed by hTau on a minor order of magnitude, possibly due to any potential functional deficits of PV interneurons, which primarily drives gamma oscillations and also shapes AHN (Buzsa´ki and Wang, 2012; Song et al., 2012). Under normal conditions, the majority of NSCs remains quiescent in response to both the tonic GABAergic inputs from local interneurons and the interneuronal signaling mediated by direct cell-cell adhesion with excitatory neurons (Dong et al., 2019; Song et al., 2012). Both GABAergic transmission impairments and glutamatergic neurons hyperactivation can force NSCs to exit from

quiescence to enhance its symmetrical self-renewal and finally derive astrogliosis instead of neurogenesis (Bao et al., 2017; Encinas et al., 2011; Sierra et al., 2015). Extracellular GABA can directly bind onto d-containing GABAARs on the surface of NSCs and IPCs, activating such as calcineurin/NFATc4 signaling, to regulate AHN (Quadrato et al., 2014). Insufficient GABAergic inputs could result in dendrite differentiation and integration impairments of immature neurons (Ge et al., 2006; Tozuka et al., 2005). Although it remains elusive whether NSCs or IPCs can directly react to extracellular glutamate secreted from neighboring excitatory neurons, recent studies suggested that disinhibition of GCs and mossy cells may also mislead the fate of AHN through dysregulating the transcellular EphB2 kinase-dependent signaling mediated by direct GC-NSC adhesion (Dong et al., 2019; Yeh et al., 2018) or though secreting proinflammatory cytokines or neuropeptides, which also play important roles in shaping neurogenesis (Borsini et al., 2015). Strengthening GABAergic Transmission Rescued hTau-Induced AHN Deficits and Improved Contextual Recognition GABAAR agonists, such as THIP and pentobarbital, were effective in preserving AHN in AD (Li et al., 2009). This is because, to date, there is no efficient drug for halting AD progression (Fan and Wang, 2019; Long and Holtzman, 2019). Promoting innate AHN, by exercise (Choi et al., 2018) or pro-neurogenic agents such as BDNF or P7C3 (Chen et al., 2018; Pieper et al., 2010; Wang et al., 2018b), might shed new light on preventing cognitive impairment at the early stage of AD. Alternatively, introducing neurogenesis by transplanting stem cells or embryonic neurons into AD brains, to repair or even reconstruct the degenerated neuronal network, holds potentials for the treatment of late-stage AD (Kim et al., 2015; MartinezLosa et al., 2018; Tong et al., 2014; Wray and Fox, 2016; Yue et al., 2015). However, it remains urgent to ensure that the innate NSCs or grafted stem cells can normally proliferate, differentiate, and subsequently integrate into the existed neuronal circuits in AD brains, since many pathologic factors in the AD brains might mislead the fate of neurogenesis. Our results strongly suggested a combinative application of pro-neurogenic agents or cell therapy with GABAergic enhancers for future trials. Furthermore, functional deficits or loss of GABAergic interneurons have been widely observed in AD brains (Andrews-Zwilling et al., 2010; Levenga et al., 2013; Palop and Mucke, 2016; Verret et al., 2012), and recent studies showed that transplanting GABAergic progenitors into the hippocampus of AD mice is efficient in ameliorating AD-like pathologies and improving cognitive functions (Martinez-Losa et al., 2018; Tong et al., 2014).

(E) THIP increased bias scores of 3xTg mice in the object-place recognition test. In this paradigm, mice were test to discriminate the object (object B) removed to a novel place (left panel). Representative heatmaps of mice traveling during the test phase are shown in the middle panel. (F) Training procedures of contextual acquisition and pattern separation. Each mouse acquired a fear memory by foot shock delivered in context A at day 1, and was tested to discriminate a very distinct context (context B) from A at the next day. In subsequent days, mice were trained to discriminate a pair of similar contexts (contexts A and C) sharing much more features (i.e., pattern separation). (G) At day 2, 3xTg mice performed normally in discriminating the context B from A, with limited effects of THIP. Paired t tests, n = 7–11 mice. *p < 0.05, **p < 0.01, ***p < 0.001, (H–J) At days 3–15, 3xTg mice administered with THIP learned more quickly than those administrated with vehicle, to discriminate the context C from A (H and I). At day 12, THIP significantly decreased the freezing time of 3xTg mice spent in context C (J). Repeated-measures ANOVA followed by Tukey’s multiple comparisons tests, or paired t tests. n = 7–11 mice. *p < 0.05, **p < 0.01, n.s. p > 0.05. Data are represented as mean ± SEM. See also Figure S7.

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Thus, a co-transplantation of NSCs/embryonic neurons with GABAergic progenitors might achieve better results in the cell therapies for AD. In conclusion, we found in the present study that hTau accumulation in GABAergic interneurons in the hippocampal neurogenic niche impaired AHN by suppressing GABAergic transmissions and disinhibiting local neuronal circuits. Strengthening GABAergic transmission by THIP is efficient for restoring AHN and improving spatial recognition in AD mice.

DECLARATION OF INTERESTS J.-Z.W. and J.Z. have just filed for a patent related to this work (CN201911140313.6). Received: June 28, 2019 Revised: October 27, 2019 Accepted: December 12, 2019 Published: January 23, 2020; corrected online: January 31, 2020 REFERENCES

STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Human subjects B Animals METHOD DETAILS B Stereotaxic injection B Immunostaining B AHN and astrogliosis evaluation B FACS and real time q-PCR B Proteomic and phosphoproteomic analysis B In vivo electrophysiology B In vivo optic fiber recording B Western blotting, dot blotting and co-immunoprecipitation B ELISA B Thioflavin T staining B Congo red staining B Behavioral tests B Contextual discrimination QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. stem.2019.12.015.

ACKNOWLEDGMENTS This work was supported in part by the National Key R&D Program of China (2016YFC1305800 to J.-Z.W.); the Natural Science Foundation of China (31730035, 91632305, 81721005, and 91949205 to J.-Z.W.; 81901107 to J.Z.); the China Postdoctoral Science Foundation (2018M632872 to J.Z.); and the Guangdong Provincial Key S&T Program (2018B030336001 to J.-Z.W.). The authors would like to thank Rui Guan in the Wan lab for her help with the housing and transport of the PV-Cre mice, and the authors thank all members in the Wang lab for helpful discussion and suggestions.

AUTHOR CONTRIBUTIONS Conceptualization, J.Z. and J.-Z.W.; Methodology, J.Z., L.M., and L.Y.; Investigation, J.Z., H.-L. L, N.T., F.L., L.M., L.Y., L.W., and Y.Y.; Manuscript – Writing, J.Z. and J.-Z.W.; Funding Acquisition, J.Z. and J.-Z.W.; Resources, Y.W. and J.-Z.W.; Supervision, H.-L.L., Y.W., and J.-Z.W.

Andrews-Zwilling, Y., Bien-Ly, N., Xu, Q., Li, G., Bernardo, A., Yoon, S.Y., Zwilling, D., Yan, T.X., Chen, L., and Huang, Y. (2010). Apolipoprotein E4 causes age- and Tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice. J. Neurosci. 30, 13707–13717. Bao, H., Asrican, B., Li, W., Gu, B., Wen, Z., Lim, S.A., Haniff, I., Ramakrishnan, C., Deisseroth, K., Philpot, B., and Song, J. (2017). Long-range GABAergic inputs regulate neural stem cell quiescence and control adult hippocampal neurogenesis. Cell Stem Cell 21, 604–617. Billings, L.M., Oddo, S., Green, K.N., McGaugh, J.L., and LaFerla, F.M. (2005). Intraneuronal Abeta causes the onset of early Alzheimer’s disease-related cognitive deficits in transgenic mice. Neuron 45, 675–688. Blasio, A., Wang, J., Wang, D., Varodayan, F.P., Pomrenze, M.B., Miller, J., Lee, A.M., McMahon, T., Gyawali, S., Wang, H.Y., et al. (2018). Novel SmallMolecule Inhibitors of Protein Kinase C Epsilon Reduce Ethanol Consumption in Mice. Biol. Psychiatry 84, 193–201. Boldrini, M., Fulmore, C.A., Tartt, A.N., Simeon, L.R., Pavlova, I., Poposka, V., Rosoklija, G.B., Stankov, A., Arango, V., Dwork, A.J., et al. (2018). Human Hippocampal Neurogenesis Persists throughout Aging. Cell Stem Cell 22, 589–599.e5. Bonzano, S., Crisci, I., Podlesny-Drabiniok, A., Rolando, C., Krezel, W., Studer, M., and De Marchis, S. (2018). Neuron-Astroglia Cell Fate Decision in the Adult Mouse Hippocampal Neurogenic Niche Is Cell-Intrinsically Controlled by COUP-TFI In Vivo. Cell Rep. 24, 329–341. Borsini, A., Zunszain, P.A., Thuret, S., and Pariante, C.M. (2015). The role of inflammatory cytokines as key modulators of neurogenesis. Trends Neurosci. 38, 145–157. Boutin, C., Hardt, O., de Chevigny, A., Core´, N., Goebbels, S., Seidenfaden, R., Bosio, A., and Cremer, H. (2010). NeuroD1 induces terminal neuronal differentiation in olfactory neurogenesis. Proc. Natl. Acad. Sci. USA 107, 1201–1206. Braak, H., Alafuzoff, I., Arzberger, T., Kretzschmar, H., and Del Tredici, K. (2006). Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 112, 389–404. Bragin, A., Jando´, G., Na´dasdy, Z., van Landeghem, M., and Buzsa´ki, G. (1995). Dentate EEG spikes and associated interneuronal population bursts in the hippocampal hilar region of the rat. J. Neurophysiol. 73, 1691–1705. Bullmann, T., de Silva, R., Holzer, M., Mori, H., and Arendt, T. (2007). Expression of embryonic tau protein isoforms persist during adult neurogenesis in the hippocampus. Hippocampus 17, 98–102. Buzsa´ki, G., and Wang, X.J. (2012). Mechanisms of gamma oscillations. Annu. Rev. Neurosci. 35, 203–225. Chen, C., Wang, Z., Zhang, Z., Liu, X., Kang, S.S., Zhang, Y., and Ye, K. (2018). The prodrug of 7,8-dihydroxyflavone development and therapeutic efficacy for treating Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 115, 578–583. Choi, S.H., Bylykbashi, E., Chatila, Z.K., Lee, S.W., Pulli, B., Clemenson, G.D., Kim, E., Rompala, A., Oram, M.K., Asselin, C., et al. (2018). Combined adult neurogenesis and BDNF mimic exercise effects on cognition in an Alzheimer’s mouse model. Science 361, eaan8821. €ela, R., Garcı´a-Corzo, L., Dı´az-Moreno, M., Armenteros, T., Gradari, S., Hortigu Fonta´n-Lozano, A´., Trejo, J.L., and Mira, H. (2018). Noggin rescues agerelated stem cell loss in the brain of senescent mice with neurodegenerative pathology. Proc. Natl. Acad. Sci. USA 115, 11625–11630.

Cell Stem Cell 26, 331–345, March 5, 2020 343

Dioli, C., Patrı´cio, P., Trindade, R., Pinto, L.G., Silva, J.M., Morais, M., Ferreiro, E., Borges, S., Mateus-Pinheiro, A., Rodrigues, A.J., et al. (2017). Tau-dependent suppression of adult neurogenesis in the stressed hippocampus. Mol. Psychiatry 22, 1110–1118.

Levenga, J., Krishnamurthy, P., Rajamohamedsait, H., Wong, H., Franke, T.F., Cain, P., Sigurdsson, E.M., and Hoeffer, C.A. (2013). Tau pathology induces loss of GABAergic interneurons leading to altered synaptic plasticity and behavioral impairments. Acta Neuropathol. Commun. 1, 34–48.

Dong, J., Pan, Y.B., Wu, X.R., He, L.N., Liu, X.D., Feng, D.F., Xu, T.L., Sun, S., and Xu, N.J. (2019). A neuronal molecular switch through cell-cell contact that regulates quiescent neural stem cells. Sci. Adv. 5, v4416.

Li, G., Bien-Ly, N., Andrews-Zwilling, Y., Xu, Q., Bernardo, A., Ring, K., Halabisky, B., Deng, C., Mahley, R.W., and Huang, Y. (2009). GABAergic interneuron dysfunction impairs hippocampal neurogenesis in adult apolipoprotein E4 knockin mice. Cell Stem Cell 5, 634–645.

Encinas, J.M., Michurina, T.V., Peunova, N., Park, J.H., Tordo, J., Peterson, D.A., Fishell, G., Koulakov, A., and Enikolopov, G. (2011). Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus. Cell Stem Cell 8, 566–579.

Li, X., Wang, Z., Tan, L., Wang, Y., Lu, C., Chen, R., Zhang, S., Gao, Y., Liu, Y., Yin, Y., et al. (2017). Correcting miR92a-vGAT-mediated GABAergic dysfunctions rescues human tau-induced anxiety in mice. Mol. Ther. 25, 140–152.

Espinoza, C., Guzman, S.J., Zhang, X., and Jonas, P. (2018). Parvalbumin+ interneurons obey unique connectivity rules and establish a powerful lateralinhibition microcircuit in dentate gyrus. Nat. Commun. 9, 4605.

Liu, E., Xie, A.J., Zhou, Q., Li, M., Zhang, S., Li, S., Wang, W., Wang, X., Wang, Q., and Wang, J.Z. (2017). GSK-3b deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory. Sci. Rep. 7, 5781.

Fan, D.Y., and Wang, Y.J. (2019). Early Intervention in Alzheimer’s Disease: How Early is Early Enough? Neurosci. Bull. Published online September 7, 2019. https://doi.org/10.1007/s12264-019-00429-x.

Long, J.M., and Holtzman, D.M. (2019). Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell 179, 312–339.

Farrant, M., and Nusser, Z. (2005). Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat. Rev. Neurosci. 6, 215–229. Fenalti, G., Law, R.H., Buckle, A.M., Langendorf, C., Tuck, K., Rosado, C.J., Faux, N.G., Mahmood, K., Hampe, C.S., Banga, J.P., et al. (2007). GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat. Struct. Mol. Biol. 14, 280–286. Fu, C., Cawthon, B., Clinkscales, W., Bruce, A., Winzenburger, P., and Ess, K.C. (2012). GABAergic interneuron development and function is modulated by the Tsc1 gene. Cereb. Cortex 22, 2111–2119. Fu, C.H., Iascone, D.M., Petrof, I., Hazra, A., Zhang, X., Pyfer, M.S., Tosi, U., Corbett, B.F., Cai, J., Lee, J., et al. (2019). Early Seizure Activity Accelerates Depletion of Hippocampal Neural Stem Cells and Impairs Spatial Discrimination in an Alzheimer’s Disease Model. Cell Rep. 27, 3741–3751.e4. Ge, S., Goh, E.L., Sailor, K.A., Kitabatake, Y., Ming, G.L., and Song, H. (2006). GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 439, 589–593. Ge, S., Pradhan, D.A., Ming, G.L., and Song, H. (2007). GABA sets the tempo for activity-dependent adult neurogenesis. Trends Neurosci. 30, 1–8. Gonc¸alves, J.T., Schafer, S.T., and Gage, F.H. (2016). Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell 167, 897–914. Gonzalez-Burgos, G. (2010). GABA transporter GAT1: a crucial determinant of GABAB receptor activation in cortical circuits? Adv. Pharmacol. 58, 175–204. Grundke-Iqbal, I., Iqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H.M., and Binder, L.I. (1986). Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. USA 83, 4913–4917. Hagihara, H., Toyama, K., Yamasaki, N., and Miyakawa, T. (2009). Dissection of hippocampal dentate gyrus from adult mouse. J. Vis. Exp. 33, 1543. Hunsberger, J.G., Rao, M., Kurtzberg, J., Bulte, J.W.M., Atala, A., LaFerla, F.M., Greely, H.T., Sawa, A., Gandy, S., Schneider, L.S., and Doraiswamy, P.M. (2016). Accelerating stem cell trials for Alzheimer’s disease. Lancet Neurol. 15, 219–230.

Longair, M.H., Baker, D.A., and Armstrong, J.D. (2011). Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27, 2453–2454. Martinez-Losa, M., Tracy, T.E., Ma, K., Verret, L., Clemente-Perez, A., Khan, A.S., Cobos, I., Ho, K., Gan, L., Mucke, L., et al. (2018). Nav1.1Overexpressing Interneuron Transplants Restore Brain Rhythms and Cognition in a Mouse Model of Alzheimer’s Disease. Neuron 98, 75–89.e5. Marvin, J.S., Shimoda, Y., Magloire, V., Leite, M., Kawashima, T., Jensen, T.P., Kolb, I., Knott, E.L., Novak, O., Podgorski, K., et al. (2019). A genetically encoded fluorescent sensor for in vivo imaging of GABA. Nat. Methods 16, 763–770. Moreno-Jime´nez, E.P., Flor-Garcı´a, M., Terreros-Roncal, J., Ra´bano, A., Cafini, F., Pallas-Bazarra, N., A´vila, J., and Llorens-Martı´n, M. (2019). Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat. Med. 25, 554–560. Mu, Y., and Gage, F.H. (2011). Adult hippocampal neurogenesis and its role in Alzheimer’s disease. Mol. Neurodegener. 6, 85. Nakashiba, T., Cushman, J.D., Pelkey, K.A., Renaudineau, S., Buhl, D.L., McHugh, T.J., Rodriguez Barrera, V., Chittajallu, R., Iwamoto, K.S., McBain, C.J., et al. (2012). Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion. Cell 149, 188–201. Pallas-Bazarra, N., Jurado-Arjona, J., Navarrete, M., Esteban, J.A., Herna´ndez, F., A´vila, J., and Llorens-Martı´n, M. (2016). Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis. EMBO J. 35, 1417–1436. Palop, J.J., and Mucke, L. (2016). Network abnormalities and interneuron dysfunction in Alzheimer disease. Nat. Rev. Neurosci. 17, 777–792. Pieper, A.A., Xie, S., Capota, E., Estill, S.J., Zhong, J., Long, J.M., Becker, G.L., Huntington, P., Goldman, S.E., Shen, C.H., et al. (2010). Discovery of a proneurogenic, neuroprotective chemical. Cell 142, 39–51. Quadrato, G., Elnaggar, M.Y., Duman, C., Sabino, A., Forsberg, K., and Di Giovanni, S. (2014). Modulation of GABAA receptor signaling increases neurogenesis and suppresses anxiety through NFATc4. J. Neurosci. 34, 8630–8645.

Joseph, M., Anglada-Huguet, M., Paesler, K., Mandelkow, E., and Mandelkow, E.M. (2017). Anti-aggregant tau mutant promotes neurogenesis. Mol. Neurodegener. 12, 88–108.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682.

Kim, H.J., Seo, S.W., Chang, J.W., Lee, J.I., Kim, C.H., Chin, J., Choi, S.J., Kwon, H., Yun, H.J., Lee, J.M., et al. (2015). Stereotactic brain injection of human umbilical cord blood mesenchymal stem cells in patients with Alzheimer’s disease dementia: A phase 1 clinical trial. Alzheimers Dement. (N. Y.) 1, 95–102.

Sierra, A., Martı´n-Sua´rez, S., Valca´rcel-Martı´n, R., Pascual-Brazo, J., Aelvoet, S.A., Abiega, O., Deudero, J.J., Brewster, A.L., Bernales, I., Anderson, A.E., et al. (2015). Neuronal hyperactivity accelerates depletion of neural stem cells and impairs hippocampal neurogenesis. Cell Stem Cell 16, 488–503.

Komuro, Y., Xu, G., Bhaskar, K., and Lamb, B.T. (2015). Human tau expression reduces adult neurogenesis in a mouse model of tauopathy. Neurobiol. Aging 36, 2034–2042. Lee, V.M., Balin, B.J., Otvos, L., Jr., and Trojanowski, J.Q. (1991). A68: a major subunit of paired helical filaments and derivatized forms of normal Tau. Science 251, 675–678.

344 Cell Stem Cell 26, 331–345, March 5, 2020

Song, J., Zhong, C., Bonaguidi, M.A., Sun, G.J., Hsu, D., Gu, Y., Meletis, K., Huang, Z.J., Ge, S., Enikolopov, G., et al. (2012). Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision. Nature 489, 150–154. Sorrells, S.F., Paredes, M.F., Cebrian-Silla, A., Sandoval, K., Qi, D., Kelley, K.W., James, D., Mayer, S., Chang, J., Auguste, K.I., et al. (2018). Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature 555, 377–381.

Taniguchi, H., He, M., Wu, P., Kim, S., Paik, R., Sugino, K., Kvitsiani, D., Fu, Y., Lu, J., Lin, Y., et al. (2011). A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013.

Yang, S.M., Groisman, A.I., and Schinder, A.F. (2019). Differential coupling of parvalbumin and somatostatin interneurons with adult-born granule cells. bioRxiv. https://doi.org/10.1101/598615.

Tobin, M.K., Musaraca, K., Disouky, A., Shetti, A., Bheri, A., Honer, W.G., Kim, N., Dawe, R.J., Bennett, D.A., Arfanakis, K., and Lazarov, O. (2019). Human Hippocampal Neurogenesis Persists in Aged Adults and Alzheimer’s Disease Patients. Cell Stem Cell 24, 974–982.e3.

Yeh, C.Y., Asrican, B., Moss, J., Quintanilla, L.J., He, T., Mao, X., Casse´, F., Gebara, E., Bao, H., Lu, W., et al. (2018). Mossy Cells Control Adult Neural Stem Cell Quiescence and Maintenance through a Dynamic Balance between Direct and Indirect Pathways. Neuron 99, 493–510.

Tong, L.M., Djukic, B., Arnold, C., Gillespie, A.K., Yoon, S.Y., Wang, M.M., Zhang, O., Knoferle, J., Rubenstein, J.L., Alvarez-Buylla, A., and Huang, Y. (2014). Inhibitory interneuron progenitor transplantation restores normal learning and memory in ApoE4 knock-in mice without or with Ab accumulation. J. Neurosci. 34, 9506–9515.

Yetman, M.J., and Jankowsky, J.L. (2013). Wild-type neural progenitors divide and differentiate normally in an amyloid-rich environment. J. Neurosci. 33, 17335–17341.

Tozuka, Y., Fukuda, S., Namba, T., Seki, T., and Hisatsune, T. (2005). GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron 47, 803–815.

Yin, Y., Gao, D., Wang, Y., Wang, Z.H., Wang, X., Ye, J., Wu, D., Fang, L., Pi, G., Yang, Y., et al. (2016). Tau accumulation induces synaptic impairment and memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling. Proc. Natl. Acad. Sci. USA 113, E3773–E3781.

Verret, L., Mann, E.O., Hang, G.B., Barth, A.M., Cobos, I., Ho, K., Devidze, N., Masliah, E., Kreitzer, A.C., Mody, I., et al. (2012). Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell 149, 708–721.

Yoshiyama, Y., Higuchi, M., Zhang, B., Huang, S.M., Iwata, N., Saido, T.C., Maeda, J., Suhara, T., Trojanowski, J.Q., and Lee, V.M. (2007). Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53, 337–351.

Wang, C., Najm, R., Xu, Q., Jeong, D.E., Walker, D., Balestra, M.E., Yoon, S.Y., Yuan, H., Li, G., Miller, Z.A., et al. (2018a). Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector. Nat. Med. 24, 647–657.

Yue, W., Li, Y., Zhang, T., Jiang, M., Qian, Y., Zhang, M., Sheng, N., Feng, S., Tang, K., Yu, X., et al. (2015). ESC-Derived Basal Forebrain Cholinergic Neurons Ameliorate the Cognitive Symptoms Associated with Alzheimer’s Disease in Mouse Models. Stem Cell Reports 5, 776–790.

Wang, Z.H., Wu, W., Kang, S.S., Liu, X., Wu, Z., Peng, J., Yu, S.P., Manfredsson, F.P., Sandoval, I.M., Liu, X., et al. (2018b). BDNF inhibits neurodegenerative disease-associated asparaginyl endopeptidase activity via phosphorylation by AKT. JCI Insight 3, 99007.

Zhang, R., Boareto, M., Engler, A., Louvi, A., Giachino, C., Iber, D., and Taylor, V. (2019). Id4 Downstream of Notch2 Maintains Neural Stem Cell Quiescence in the Adult Hippocampus. Cell Rep. 28, 1485–1498.e6.

Wei, J., Davis, K.M., Wu, H., and Wu, J.Y. (2004). Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications. Biochemistry 43, 6182–6189. Wei, Y.P., Ye, J.W., Wang, X., Zhu, L.P., Hu, Q.H., Wang, Q., Ke, D., Tian, Q., and Wang, J.Z. (2018). Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation. Neurosci. Bull. 34, 261–269. Wray, S., and Fox, N.C. (2016). Stem cell therapy for Alzheimer’s disease: hope or hype? Lancet Neurol. 15, 133–135.

Zheng, J., Jiang, Y.Y., Xu, L.C., Ma, L.Y., Liu, F.Y., Cui, S., Cai, J., Liao, F.F., Wan, Y., and Yi, M. (2017). Adult hippocampal neurogenesis along the dorsoventral axis contributes differentially to environmental enrichment combined with voluntary exercise in alleviating chronic inflammatory pain in mice. J. Neurosci. 37, 4145–4157. Ziegler-Waldkirch, S., d’Errico, P., Sauer, J.F., Erny, D., Savanthrapadian, S., Loreth, D., Katzmarski, N., Blank, T., Bartos, M., Prinz, M., and MeyerLuehmann, M. (2018). Seed-induced Ab deposition is modulated by microglia under environmental enrichment in a mouse model of Alzheimer’s disease. EMBO J. 37, 167–182.

Cell Stem Cell 26, 331–345, March 5, 2020 345

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Anti-ABAT

Abcam

Cat# ab216465; RRID: AB_2801302

Anti-AT8

Thermo Fisher

Cat# MN1020; RRID: AB_223647

Anti-b-actin

Abcam

Cat# ab6276; RRID: AB_2223210

Anti-BrdU

Bio-Rad

Cat# MCA2483; RRID: AB_808349

Anti-CaMKII

Abcam

Cat# ab22609; RRID: AB_447192

Anti-DCX

Santa Cruz

Cat# sc-8066; RRID: AB_2088494

Anti-DCX

Abcam

Cat# ab18723; RRID: AB_732011

Anti-Flag

Sigma

Cat# F7425; RRID: AB_439687

Anti-GABA

Sigma

Cat# A2052; RRID: AB_477652

Anti-GABAAR a1

Santa Cruz

Cat# sc-7348; RRID: AB_640758

Anti-GABAAR b1

Santa Cruz

Cat# sc-31424; RRID: AB_2109286

Anti-GABAAR g2

Abcam

Cat# ab87328; RRID: AB_10671894

Anti-GABAAR d

Abcam

Cat# ab110014; RRID: AB_10861508

Anti-GAD65

Abcam

Cat# ab26113; RRID: AB_448989

Anti-GAD67

Millipore

Cat# MAB5406; RRID: AB_2278725

Anti-GAT1

Abcam

Cat# ab426; RRID: AB_2189971

Anti-Gephyrin

Abcam

Cat# ab32206; RRID: AB_2112628

Anti-GFAP

Cell Signaling

Cat# 3670; RRID: AB_561049

Anti-Iba1

Abcam

Cat# ab5076; RRID: AB_2224402

Anti-ID4

Biocheck

Cat# BCH-9/82-12, RRID:AB_2814978

Anti-MCM2 (BM28)

BD Biosciences

Cat# 610700, RRID: AB_2141952

Anti-NeuN

Millipore

Cat# MABN140; RRID: AB_2571567

Anti-NeuroD1

Abcam

Cat# ab213725; RRID: AB_2801303

Anti-Parvalbumin

Abcam

Cat# ab11427, RRID: AB_298032

Anti-Phospho-Ser/Thr

Abcam

Cat# ab17464; RRID: AB_443891

Anti-Somatostatin

ImmunoStar

Cat# 20067, RRID: AB_572264

Anti-SSADH

Abcam

Cat# ab129017; RRID: AB_11154958

Anti-Synaptophysin

Abcam

Cat# ab32127; RRID: AB_2286949

Anti-Synaptotagmin

Abcam

Cat# ab13259; RRID: AB_299799

Anti-Tau (Phospho-Ser396)

Signalway

Cat# 11102; RRID: AB_896046

Anti-Tau (Phospho-Thr181)

Signalway

Cat# 11107; RRID: AB_896051

Anti-Tau (Phospho-Thr205)

Signalway

Cat# 11108-1, RRID: AB_896053

Anti-Tau (Phospho-Thr231) (PHF-6)

Thermo Fisher

Cat# 35-5200, RRID: AB_2533210

Anti-Tau (Phospho-Ser231) (PHF-13.6)

Thermo Fisher

Cat# 35-5300, RRID: AB_2533211

Anti-Tau (Tau5)

Abcam

Cat# ab80579, RRID: AB_1603723

Antibodies

Anti-Paired Helical Filaments (6E10)

Covance

Cat# SIG-39430-500, RRID: AB_663223

Anti-VAMP2

Abcam

Cat# ab3347; RRID: AB_2212462

Anti-VGAT

Santa Cruz

Cat# sc-393373; RRID: AB_2801273

Alex Fluor 488-conjugated donkey anti-mouse IgG

Jackson ImmunoResearch

Cat# 715-545-150; RRID: AB_2340846

Alex Fluor 647-conjugated donkey anti-mouse IgG

Jackson ImmunoResearch

Cat# 715-605-150; RRID: AB_2340862

Alex Fluor 594-conjugated donkey anti-rabbit IgG

Jackson ImmunoResearch

Cat# 711-585-152; RRID: AB_2340621

Alex Fluor 680-conjugated donkey anti-rabbit IgG

Jackson ImmunoResearch

Cat# 711-625-152; RRID: AB_2340627

HRP-conjugated donkey anti-goat IgG

ZSGB-BIO

Cat# PV-9003, RRID:AB_2814979

HRP-conjugated goat anti-mouse IgG

ZSGB-BIO

Cat# PV-9005 (Continued on next page)

e1 Cell Stem Cell 26, 331–345.e1–e6, March 5, 2020

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

HRP-conjugated goat anti-rabbit IgG

ZSGB-BIO

Cat# PV-6001

IRDye 800CW Goat anti-Mouse IgG (H + L)

LI-COR Biosciences

Cat# 926-32352; RRID: AB_2782999

IRDye 800CW Goat anti-Rabbit IgG (H + L)

LI-COR Biosciences

Cat# 925-32211; RRID: AB_2651127

IRDye 800CW Donkey anti-Goat IgG (H + L)

LI-COR Biosciences

Cat# 925-32214; RRID: AB_2687553

Bacterial and Virus Strains rAAV-EF1a-DIO-tau-mCherry-WPRE-pA

BrainVTA

Cat# PT-0527

rAAV-EF1a-DIO-mCherry-WPRE-pA

BrainVTA

Cat# PT-0013

rAAV-CaMKIIa-GCaMP6f-WPRE-pA

BrainVTA

Cat# PT-0119

rAAV-CaMKIIa-EGFP-WPRE-pA

BrainVTA

Cat# PT-0290

rAAV-hSyn-iGABASnFR-WPRE-SV40 pA

BrainVTA

Cat# PT-0249

pROV-U6-shRNA-EF1a(S)-EGFP

OBio

Cat# CN889

pROV-EF1a-3flag-2A-Cre

OBio

N/A

pAOV-CaMKIIa-tau-mCherry-WPRE-pA

OBio

N/A

pAOV-CaMKIIa-mCherry-WPRE-pA

OBio

N/A

pAOV-CaMKIIa-hM4D(Gi)-Flag-WPRE-pA

OBio

N/A

pAOV-CaMKIIa-3flag-WPRE-pA

OBio

N/A

pAOV-EF1a-DIO-tau(P301S)-mCherry

OBio

N/A

pAOV-EF1a-DIO-mCherry

OBio

N/A

Human Brain Bank of Chinese Academy of Medical Sciences & Peking Union Medical College

N/A

Biological Samples Human hippocampus sections, see also Table S1

Chemicals, Peptides, and Recombinant Proteins 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP)

Sigma

Cat# T101

Clozapine N-oxide (CNO)

Sigma

Cat# C0832

DAPI

Beyotime

Cat# C1002

Thioflavin T

Sigma

Cat# T3516

Congo red

Sigma

Cat# C6277

DAB-staining kit

ZSGB-BIO

Cat# ZLI9018

Human Ab1-40(Amyloid Beta 1-40) ELISA Kit

Elabscience

Cat# E-EL-H0542c

Human Ab1-42(Amyloid Beta 1-42) ELISA Kit

Elabscience

Cat# E-EL-H0543c

Single-cell sequence specific amplification kit

Vazyme

Cat# P621-01

TMTsixplex Isobaric Label Reagent Set

Thermo Fisher

Cat# 90061

This paper

ProteomeXchange Consortium: PXD014336

Mouse: Nestin-GFP

Cyagen Biotechnology

https://www.cyagen.com/cn/zh-cn/service/ transgenic-mouse-rat-gfp.html

Mouse: 129S4.Cg-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/LfaJ (3xTg)

The Jackson Laboratory

RRID:IMSR_JAX:031988

Mouse: 129S1/SvImJ

The Jackson Laboratory

RRID:IMSR_JAX:002448

Critical Commercial Assays

Deposited Data Mass spectrometry data in proteomic and phosphoproteomic analysis Experimental Models: Organisms/Strains

Mouse: Dlx5/6-Cre-ires-EGFP

Yang lab

RRID:IMSR_JAX:023724

Mouse: Parvalbumin-Cre

Wan lab

RRID:IMSR_JAX:017320

Mouse: Sst-Cre

The Jackson Laboratory

RRID:IMSR_JAX:013044

TsingKe

N/A

Oligonucleotides Primers for RT-qPCR, see also Table S2

(Continued on next page)

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SOURCE

IDENTIFIER

Fiji

https://imagej.net/Fiji

RRID:SCR_002285

KlustaKwik

http://klustakwik.sourceforge.net/

RRID:SCR_014480

GraphPad Prism

GraphPad Software

RRID:SCR_002798

MATLAB

MathWorks

RRID:SCR_001622

NeuroScope

http://neurosuite.sourceforge.net/

RRID:SCR_002455

Quantity One

Bio-Rad

RRID:SCR_014280

Simple Neurite Tracer

https://imagej.net/ Simple_Neurite_Tracer

RRID:SCR_016566

SPSS Statistics

IBM

RRID:SCR_002865

ZEN (blue edition)

Zeiss

RRID:SCR_013672

VS-ASW-S6

Olympus

N/A

Software and Algorithms

LEAD CONTACT AND MATERIALS AVAILABILITY For further information and requests for reagents and resource, please contact the leading correspondence, Jian-Zhi Wang (wangjz@ mail.hust.edu.cn). This study did not generate new unique reagents. EXPERIMENTAL MODEL AND SUBJECT DETAILS Human subjects The human brain sections were obtained from the Human Brain Bank of Chinese Academy of Medical Sciences & Peking Union Medical College, kindly provided by Prof. Chao Ma (Chinese Academy of Medical Sciences, Beijing, China). AD was diagnosed according to the criteria of the Consortium to Establish a Registry for AD and the National Institute on Aging. Subjects information, including dementia degree, age, sex, postmortem interval and comorbidities, were listed in Table S1. The study was approved by Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Animals Dlx5/6-Cre-ires-EGFP (Tg(mI56i-cre, EGFP)1Kc/J, termed as Dlx5/6-CIE) mice were generous gifts from Prof. Zhengang Yang (Fudan University, Shanghai, China). Parvalbumin-Cre (B6.129P2-Pvalbtm1(cre)Arbr/J) mice were obtained from Wan-lab. 3xTg (129S4.Cg-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/LfaJ) mice and wild-type 129 (129S1/SvImJ) mice were purchased from Jackson Laboratory. SST-Cre mice were purchased from Shanghai Model Organisms Center, Inc. (China). Nestin-GFP (Nes-GFP) mice were purchased from Cyagen (Suzhou, China). Dlx5/6-CIE:Nes-GFP mice were obtained by crossbreeding Dlx5/6-CIE with Nes-GFP mice. All mice were kept under standard laboratory conditions, with a 12 h alternating light/dark cycle, food and water available ad libitum. Only male mice weighing 20 – 30 g were used in all the experiments. The influence of sex was not evaluated in this study. Brain sections from 6-month 3xTg or age-matched wild-type 129 mice were used for phospho-tau immunostaining. 4-month 3xTg or age-matched wild-type mice were used for THIP administration. 8 – 12 weeks Dlx5/6-CIE, Dlx5/6-CIE:Nes-GFP, PV-Cre or SST-Cre mice were used for virus injection. The ages of mice used in each experiment were specified in the text. All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology. METHOD DETAILS Stereotaxic injection Mice weighing 20 – 30 g were anesthetized with 1% pentobarbital sodium (35 mg/kg), and then fixed in a stereotaxic instrument (RWD, Shenzhen, China). The scalp was sterilized with iodophors and 75% ethanol, in turn, and incised along the skull midline. Two holes were drilled bilaterally at posterior 1.9 mm, lateral ± 1.1 mm from begma. A total volume of: 1) 0.3 mL AAV-EF1a-DIOhTau-mCherry, AAV-EF1a-DIO-hTau(P301S)-mCherry, or AAV-EF1a-DIO-mCherry, 2) 1 mL ROV-EF1a-GFP, 3) 0.3 mL AAV-CaMKIIa-hTau-mCherry or AAV-CaMKIIa-mCherry, 4) 1 mL ROV-EF1a-Cre + 0.3 mL AAV-EF1a-DIO-hTau-mCherry or AAV-EF1a-DIOmCherry, 5) 0.3 mL AAV-CaMKIIa-GCaMP6f or AAV-CaMKIIa-GFP, 6) 0.3 mL AAV-CaMKIIa-hM4Di-flag or AAV-CaMKIIa-flag, 7) 0.3 mL AAV-hSyn-iGABASnFR or AAV-hSyn-GFP, were injected into each site of the dorsal DG (ventral 2.0 from the skull) (Liu et al., 2017), using an automatic microinjection system (World Precision Instruments, USA), at a rate of 50 nL/min. The titer of AAVs ranges from 2.27E+12 to 5.50E+12 vg/mL, the titer of ROV-GFP was 6.74E+08 vg/mL, and ROV-Cre 2.32E+07 vg/mL. In some experiments, different kinds of AAVs or ROVs were combinedly used, by sequentially injecting each virus, with an interval e3 Cell Stem Cell 26, 331–345.e1–e6, March 5, 2020

of about 20 minutes. The needle syringe was left in place for 5 minutes before withdrawal. The skin was sutured, and then sterilized with iodophors and 75% ethanol. The site of virus infection was confirmed after execution of mice by examining the virus-expressed mCherry, GFP or flag, respectively. Subjects with off-target virus infusion were excluded from analysis. Immunostaining For animal experiments, mice were anesthetized with 1% pentobarbital sodium and intracardially perfused with saline followed by 4% paraformaldehyde (PFA, in 0.1 M phosphate buffer, pH 7.4). Mice brains were removed, post-fixed in 4% PFA for 12 h, and then cryoprotected in 20% and 30% sucrose solutions in turn. Brain sections of 50 mm thickness were sliced in a cryostat microtome (CM1900, Leica). For immunofluorescence staining, free-floating sections were washed in PBS, blocked in a buffer containing 5% bull serum albumin and 0.3% Triton X-100 for 1 h, and then incubated with primary antibodies at 4 C for 24 h (1:200 dilution in 0.3% PBST for all antibodies). After washed in PBS, sections were incubated with secondary antibodies at 37 C for 1.5 h (1:500 dilution in 0.3% PBST for all antibodies), and finally mounted with a buffer (pH 9.0) containing NaHCO3 (220.2 mM), Na2CO3 (28.3 mM) and 50% glycerol. Specifically, for BrdU staining, sections were incubated in 2 N HCl at 37 C for 30 min to expose the epitopes in DNA, and then rinsed in 0.1 M sodium borate (pH 8.5) before blocking and antibodies incubation. For immunohistochemistry staining, endogenous peroxidase activity was eliminated by incubating brain slices in 0.3% H2O2 (in PBS) at 37 C for 30 min before serum blocking. Immunoreactions were developed using a DAB-staining kit (ZSGB-BIO). Sections were then dehydrated through graded ethanol series, and sealed with neutral balsam. For immunostaining on human brain sections, antigen-retrieval was performed in boiling sodium citrate buffer (10 mM), and sections were counterstained with DAPI (1:10,000 in PBS, 37 C, 1.5 h) in immunofluorescence or hematoxylin (10%, 37 C, 10 min) in immunohistochemistry. Images were taken by a virtual slide Microscope (SV120, Olympus) or two-photon laser-scanning confocal microscope (LSM710, Zeiss). AHN and astrogliosis evaluation Only AHN or astrogliosis in the dorsal DG (approximately from AP 1.7 to 2.5) was evaluated in the present study. Immunofluorescent images were obtained by scanning a z series stack at a 3 mm interval throughout the entire 50 mm-thickness. NSC morphology was evaluated using Nes-GFP or Dlx5/6-CIE:Nes-GFP mice. BrdU (B9285, Sigma-Aldrich) was dissolved in 0.01 M PBS (10 mg/ml) and injected (intraperitoneally, 50 mg/kg) for 5 consecutive days before execution for the evaluation of cell proliferation. The quantification of cell numbers was performed as described previously (Zheng et al., 2017). Briefly, every fifth and a total of 3 sections were stained and then counted by an experimenter blinded from animal groupings. The cell counts were multiplied by 5 and added to indicate the total number of cells in the dorsal DG. Cell numbers in left and right DG were counted separately but averaged, since no significant differences were observed (data not shown). The dendrite length and complexity of ROV-GFP-labeled newborn neurons were evaluated by Sholl-analysis using Simple Neurite Tracer plugin (Longair et al., 2011) and ImageJ (Fiji) software (Schindelin et al., 2012). Dendrite spines of ROV-GFP-labeled neurons were imaged under 100 3 oil immersion lens. Only secondary dendrites were analyzed in the present study. FACS and real time q-PCR Mice were executed by cervical dislocation and brains were acutely removed. Hippocampi tissues were isolated on ice, mechanically dissociated in DMEM/F12 media, and then incubated in 0.125% trypsin for 20 min at 37 C. Enzymatic activity was stopped by 10% FBS. Cell suspension was prepared by trituration, and then centrifuged at 1000 rpm at 4 C for 10 min. Each pellet was collected, resuspended and then filtered through a 0.45 mm membrane (Fisher Scientific) to obtain single-cell suspension in PBS. mCherryor GFP-positive cells were sorted through FACS (MOflo XDP, Beckman Coulter) and Summit software. Cell suspension from wildtype mice hippocampus was used as the non-fluorescent control. RNA extracted from collected samples were pre-amplified using a single-cell sequence specific amplification kit (P621-01, Vazyme). qPCR systems (10 mL each) included 2 3 ChamQ SYBR qPCR Master Mix (Q311-00, Vazyme) 5 mL, Forward Primer (2 mM) 1 mL, Reverse Primer (2 mM) 1 mL, 50 3 ROX Reference Dye 0.2 mL, RNase-free H2O 0.8 mL, and pre-amplified products 2 mL each. Samples were assayed on a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher). mRNA levels of the interested genes were normalized by GAPDH mRNA, a house-keeping gene not affected by the treatments. PCR primers employed in the present study were listed in Table S2. Proteomic and phosphoproteomic analysis Mice were executed by cervical dislocation, and brains were acutely removed. DG tissues were isolated on ice as described (Hagihara et al., 2009), grinded in liquid nitrogen, and then dissociated by sonication in a buffer containing 8 M urea and 1% protease inhibitor cocktail. Samples were collected after centrifugation at 4 C, 12000 g for 10 min. Protein concentrations were determined using BCA assays (Beyotime). For mass spectrometry, protein solutions were reduced with dithiothreitol (5 mM) for 30 min at 56 C, and alkylated with iodoacetamide (11 mM) for 15 min at room temperature in darkness. Samples were then diluted by Triethyl ammonium bicarbonate (make sure the final concentration of urea is less than 2M). Trypsin (Promega) was added (1:50, enzyme to protein) for the first digestion overnight, and 1:100 for a second digestion for 4 h. After the trypsin digestion, peptides were desalted by Strata X C18 SPE column Cell Stem Cell 26, 331–345.e1–e6, March 5, 2020 e4

(Phenomenex), vacuum-dried, reconstituted in 0.5 M TEAB, labeled using a TMT kit (Thermo Fisher), and then fractionated by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 mm particles, 4.6 mm ID, 250 mm length). Phosphopeptides were enriched using immobilized metal affinity chromatography (IMAC) microspheres. Mass spectrometry (MS) data were collected using a Q ExactiveTM Plus Hybrid Quadrupole-Orbitrap mass spectrometer coupled with EASY-nLC 1000 liquid chromatography pump (Thermo Fisher Scientific), and processed using Maxquant search engine (v.1.5.2.8). Mass spectra data were searched against SwissProt Mouse database concatenated with reverse decoy database. Expression level of each protein or phosphopeptide in all 6 samples were horizontally normalized, and converted into log2 values. The mean values were calculated as Mm (mCherry group) or Mt (hTau group). Two-tailed t tests were used to evaluate the differences between mCherry and hTau group. For each protein or phosphopeptide, p < 0.05 and Mt/Mm > 1.3 was considered as significantly upregulated, while p < 0.05 and Mt/Mm < 1/1.3 as significantly downregulated. Proteomic and phosphoproteomic data were combined in bioinformatics analysis. For Gene Ontology (GO) annotation, UniProt IDs (UniProt-GOA database) were converted to map GO IDs. If some identified proteins in MS were not annotated by UniProt-GOA database, the InterProScan software was used to annotate their GO functional based on a protein sequence alignment method. Then proteins were classified by GO annotation based on the biological process. Two-tailed Fisher’s exact test was employed to evaluate the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected p value < 0.05 is considered significant. In vivo electrophysiology Mice were anesthetized with 1% pentobarbital sodium, and fixed in a stereotaxic instrument. The skull was exposed, and three anchor screws were attached to the skull. A craniotomy (approximately 2 3 2 mm) was drilled on the left or right hemisphere. The dura was stripped using a needle. A custom designed recording drive, with 4 combined but movable tungsten tetrodes and two reference electrodes were implanted into the SGZ (AP 1.9, LM ± 1.1, DV 2.0), or cerebellum, respectively. The craniotomy was then sealed with paraffin wax, and the drive was secured in place with denture base resins (Medental, Beijing). Mice were housed individually on a 12 hours light/dark cycle with ad libitum access to water and food. Mice were handled for 5 min and adapted in a T-maze for 10 min per day for 3 consecutive days before the first time of recording, and allowed to freely travel in the T-maze during recording. Signals were recorded using a RHD2000 Recording System (Intan Technologies), amplified and digitized online at 20 kHz through a headstage (RHD2132, Intan) connected to the tetrodes through a connector (A79008-001, Omnetics). Data was stored for offline analysis with 16-bit format, visualized in NeuroScope, and then analyzed using MATLAB (MathWorks). Spike sorting was carried out in KlustaKwik using the principal component analysis. Tetrodes were slightly lowered through the drive over 3 - 5 days, until featured gamma activity and dentate spikes were detected in the local field potentials (LFP) according to previous studies (Bragin et al., 1995). Once putative DG units were detected, formal recordings were performed in the following days. Tetrodes location were confirmed by an electrical lesion (50 mA) delivered after the final recording. Subjects with off-target electrodes location were excluded from further analysis. In vivo optic fiber recording Optic fiber cannulas (NA = 0.37, Newdoon, China) were implanted into the SGZ of mice (AP 1.9, LM ± 1.1, DV 2.0), through similar procedures as tetrodes implantation described above. Each mouse was handled and adapted in a T-maze for 10 min per day for 3 consecutive days, before the first time of recording. GCaMP6f or iGABASnFR signals were recorded using a Fiber Photometry system (Thinker Tech, China), with the LED power of 65 mW. Mice were allowed to freely travel in a T-maze during recordings. Data were analyzed using MATLAB, DF/F was calculated as: DF/F = (F - F0) / (F0 - Foffset) 3 100%. A threshold of 5% DF/F was set to count calcium or GABA responses. Subjects with off-target fiber ends location were excluded from analysis. Western blotting, dot blotting and co-immunoprecipitation Western blotting and dot blotting were performed as previously described (Li et al., 2017). Briefly, dorsal hippocampal tissues were isolated and homogenized with RIPA lysis buffer (Beyotime). Proteins were separated in SDS-PAGE gels (in western blotting) and transferred onto nitrocellulose membranes (Merck Millipore). The membranes were then blocked with 5% BSA, incubated in turn with primary and IRDye-conjugated secondary antibodies (1:500 – 1:2000 dilutions), in turn. Blots or dots were visualized using an Odyssey Imaging System (LI-COR Biosciences), and quantified using Quantity One software (Bio-Rad). For co-immunoprecipitation, samples were incubated with protein G agarose and mouse IgG (Beyotime) at 4 C for 2 h, and then centrifuged at 4 C, 2,000 rpm for 5 min. Supernatants were subsequently incubated with specified antibodies and protein G agarose overnight at 4 C. Mixtures were then centrifuged at 4 C, 3,000 rpm for 3 min. Protein products were collected by washing the pellets with PBS for 3 times, mixed with loading buffer (2% SDS, 100 mM dithiothreitol, 10% glycerol, and 0.25% bromophenol blue), denatured at 95 C for 5 min, and subsequently analyzed through western blotting. ELISA Mice were executed by cervical dislocation. Dorsal hippocampal tissues were isolated on ice, homogenized with RIPA lysis buffer (Beyotime), and then centrifuged at 4 C, 14,000 rpm for 30 min. The supernatants were collected for the detection of soluble Ab, and the pellets were resuspended with 70% formic acid for the measurement of insoluble Ab. Protein concentration in each sample

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was measured through BCA assays (Thermo Fisher). Ab1-40 and Ab1-42 were measured using commercially available ELISA kits from Elabscience (Cat# E-EL-H0542c and E-EL-H0543c), respectively. All procedures followed the manufacturer’s instructions. Thioflavin T staining Free-floating brain sections were washed with Tris-buffered saline (TBS) for 3 3 5 min, and then incubated with 0.3% Thioflavin T (Sigma) (dissolved in 50% ethanol) at room temperature for 10 min. The sections were then decolorized in 50% ethanol for 3 3 5 min, washed in TBS, and subsequently co-stained with DAPI for 10 min. Congo red staining Free-floating brain sections were incubated in a sodium chloride solution (30% NaCl, 80% ethanol and 0.1& NaOH) at room temperature for 20 min, and 0.2% Congo red containing 0.1& NaOH for 40 min. The sections were then decolorized in ethanol, hydrated in PBS, and subsequently co-stained with Nissl staining solution (Beyotime). Behavioral tests Object-place recognition Mice were handled for 5 min per day for 3 consecutive days before test. In the probe phase, mice were placed in a box (50 3 50 3 50 cm, marked with a visual cue) with two identical objects (plastic cylinders) at two different corners, and allowed to freely explore for 5 min. Each mouse was then removed from the box for 2 min, during which the box and objects were cleaned with 75% ethanol, and one of the two objects (in pseudorandom, rendered as object B) was removed to a new corner while the other object (object A) remained unremoved. In the test phase, mice were allowed to freely explore in the box for another 5 min. Videos were recorded and analyzed online using OFT-100 system (Techman, China). Total area in the box was divided into 5 3 5 grids. Once mouse staying at the grid around each object was counted as exploring. The bias score toward object B was calculated as the exploring time (B - A) / (B + A). Contextual discrimination Contextual discrimination training began 5 days after the object-place recognition test, using a conditioning chamber (23 3 23 3 30 cm) with the floor made of 32 stainless steel rods wired to a shock generator in a sound-attenuating cubicle with 60 dB background noise from a fan. At day 1, each mouse was placed in the chamber (context A, paired with an additional white noise delivered through a small loudspeaker), and allowed to explore for 3 min. A 2 s footshock of 65 mA was then delivered, and the mouse was allowed to stay in the chamber for another 1 min. The chamber was cleaned with 75% ethanol between each animal. At the next day, mice were first placed in a very distinct context (context B, smooth plastic floor, no white noise, cylindrical wall), allowed to freely explore for 3 min, and subsequently place in the context A about 5 h later, allowed to freely explore for another 3 min. No footshock was delivered. Freezing behavior of mice were measured online using FCT-100 system (Techman, China). In the following days, mice were trained to discriminate a similar context (context C, floor of steel rods, no white noise, blue lighting, a plastic A-frame insert, and Lanyueliang cleaning the box between animals) from context A (Nakashiba et al., 2012). In context A, mice received a 2 s footshock of 65 mA after 3-min free-exploring, and allowed to stay in the chamber for another 1 min after the footshock. In context C, mice were placed in the chamber for an equivalent of time, but no footshock was delivered. Freezing time was measured during the first 3 min every day. The order of training followed a double alternation schedule, i.e., A-C, C-A, A-C, etc. Discriminative scores between context A and C were calculated as the freezing time (A - C) / (A + C). QUANTIFICATION AND STATISTICAL ANALYSIS Data were presented as means ± SEM, unless otherwise specified. All data were analyzed and plotted using SPSS Statistics (IBM), MATLAB (MathWorks) or GraphPad Prism (GraphPad Software). Unpaired or paired two-tailed t tests, ANOVA (one-way, two-way, or repeated-measures), two-tailed Fisher’s exact tests, Mann-Whitney tests, and post hoc Tukey’s multiple comparisons tests were used (as illustrated in figure legends), with p < 0.05 considered as statistically significant. DATA AND CODE AVAILABILITY The accession number for the mass spectrometry proteomics and phosphoproteomic data reported in this paper is ProteomeXchange Consortium: PXD014336. Other datasets and code supporting the current study have not been deposited in a public repository because they are presented in the main text and supplementary figures and tables, but are available from the corresponding authors on request.

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