Tau depletion in human neurons mitigates Aβ-driven toxicity

Abstract

Alzheimer's disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-β (Aβ) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aβ-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aβ were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aβ-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aβ-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

Fig. 1. Generation of MAPT−/− iPSCs.

A Illustrations of gene editing strategies showing the positions of gRNA(s) in Exon 1 and 4 DNA loci. The intended double-stranded break in Exon 1 is indicated by an arrow, whereas the intended 25-bp deletion is indicated between the gRNA pair used to target Exon 4. B Sequencing results of edited MAPT loci, including all combinations of alleles in the Exon 1 isogenic panel. For the Exon 4 isogenic panel, both MAPT+/− and MAPT−/− lines harbour the 25-bp deletion at the same site. C Schematic of the cortical neuron differentiation protocol used throughout this study – iPSC lines were first differentiated concurrently to NPCs, before they were subject to lentiviral transduction for Ngn2 expression (“NPC-Ngn2 protocol”) for maturation either in co-culture with primary rat cortical astrocytes or in monoculture on coated surface.

Fig. 2. Validation of MAPT−/− iPSCs.

A qRT-PCR results using cDNA samples from Day 35 NPCs of both isogenic panels. Each bar graph title indicates the primer pairs used for MAPT transcript detection. Exon 4(del) describes the the CRISPR targeted and excised locus. The data points were normalised to the respective MAPT+/+ NPCs for each differentiation. Mean ± SEM and n = three independent cortical neuron differentiation repeats. Kruskal–Wallis with Dunn’s multiple comparison test was used for statistical analysis. B ICC of Day 50 iPSC-derived cortical neurons from both isogenic panels using Tau-12 antibody targeting to probe for total tau, showing representative images (top) and quantifications (bottom). The parameters are Tau-12+ cytoplasm normalised to the total number of nuclei (top row) and Tau-12+ area relative to the total beta-3 tubulin (B3T)+ and MAP2+ areas (bottom row). Scale bar = 50 μm. Mean ± SEM. N = two wells of neurons for the IgG control and four wells of neurons for the positive Tau-12 staining from one differentiation. Two-way ANOVA with Sidak’s multiple comparison test was performed for statistical analysis. C Western blots probing for tau using three different antibodies (Tau-1 – mid-region, Tau-5 – mid-region and Tau-46 – C-terminus) either on Day 30 (NPC) or Day 50 (neuron) of neuronal differentiation for both MAPT−/− isogenic panels. 6 ng of recombinant tau ladder was used and 5 μg of lysate was added per lane (except for the Tau-46 blot where 10 μg of lysate was added). Anti-β-actin blots were used as the expression control housekeeping protein for the lysates. Full blots are shown in Supplementary Fig. 3. D Nanopore long-read sequencing of MAPT transcripts in the Exon 4 isogenic panel Day 35 NPCs. Bar graph of MAPT transcript long-read sequencing depth (normalised across genotypes) focusing on Exon 4 showing lower abundance of MAPT transcripts in the MAPT+/− and MAPT−/− lines as compared to the MAPT+/+ line. A truncated form of Exon 4 is included in a minority of reads in the MAPT+/− and MAPT−/− lines indicated by the red arrows. E Sashimi plot illustrating Exon 4 inclusion in the MAPT+/+ line and skipping in the MAPT+/− and MAPT−/− lines. Splicing patterns supported by fewer than 10% of total reads per sample were filtered for clarity.

Fig. 3. MAPT−/− iPSC-derived cortical neurons demonstrate reductions in neuronal activity and protection from Aβ-driven hyperactivity.

A Representative raster plots showing individual neuronal activity spikes for each of the sixteen electrodes (row) in each MEA well over 2 min for both Day 90 MAPT+/+ and MAPT−/− neurons (MAPT−/− #1 from the Exon 1 panel) from each isogenic panel at baseline; Quantification of baseline neuronal activity parameters measured by MEA assays on Day 90–100 iPSC-derived cortical neurons from both MAPT−/− isogenic panels. Mean ± SEM. n = 53–55 (Exon 1 MAPT+/+) or 51–53 (Exon 1 MAPT−/−) wells across three independent neuronal differentiation repeats; 101–105 (Exon 4 MAPT+/+) or 103–114 (Exon 4 MAPT−/−) wells across six independent neuronal differentiation repeats. Some wells did not achieve the threshold needed to register network activities. Two-tailed Mann-Whitney test was used for statistical analysis. B Representative raster plots showing individual neuronal activity spikes for each of the sixteen electrodes (row) in each MEA well over 2 min for both Day 90 Exon 4 MAPT+/+ and MAPT−/− neurons treated with either AD brain homogenate or Aβ-immunodepleted (ID) AD brain homogenate at 25% v/v in the neuronal media for 5 days; Quantification of neuronal activity parameters measured by MEA assays over 5 days on Day 90–93 Exon 4 MAPT+/+ and MAPT−/− iPSC-derived cortical neurons treated with either AD brain homogenate or Aβ-ID homogenate at 25% v/v in the neuronal media. All datapoints were normalised to the baseline recording pre-treatment, and for each time point relative to the wells subject to aCSF (vehicle) control treatment. Mean ± SEM. n = 7–14 (MAPT+/+ID), 11–14 (MAPT+/+AD) or 5–16 (MAPT−/− ID and AD) wells across three independent neuronal differentiation repeats. Two-way ANOVA with Dunnett’s multiple comparison correction was used for statistical analysis compared against the MAPT+/+AD wells 5 days post-treatment.

Fig. 4. Aβ-driven retrograde impairment of axonal transport of mitochondria is absent in MAPT−/− iPSC-derived cortical neurons.

A Schematic of the experiments designed to measure axonal transport of mitochondria using microfluidic chambers. B Quantification of ratio of motile mitochondria (motile to stationary) in Day 70–95 iPSC-derived cortical neurons from the Exon 4 isogenic panel with or without directionality over 150 s of live imaging. The neurons were treated with either 2 μM scrambled Aβ_1–42 or Aβ_1–42 oligomers for 1 h before imaging. Mean ± SEM. n = 8 (MAPT + /+ scrambled Aβ_1–42), 13 (MAPT + / + Aβ_1–42), 6–9 (MAPT−/− scrambled Aβ_1–42) and 12–14 (MAPT−/− Aβ_1–42) microfluidic chambers measured across five independent neuronal differentiation repeats. Two-tailed Mann-Whitney test was used for statistical analysis.

Fig. 5. Aβ-driven neurodegeneration is absent in MAPT−/− iPSC-derived cortical neurons.

A Representative immunofluorescence images of Day 79–83 (Exon 1 isogenic panel) and Day 79–86 (Exon 4 isogenic panel) iPSC-derived cortical neurons treated with either 10 μM scrambled Aβ_1–42 or Aβ_1–42 oligomers for 5 days. The neurons were probed with antibodies against human nuclei (green) and cleaved caspase 3 (CC3; yellow) which was used as the marker for cell death. Scale bar = 100 μm. B Quantification of relative CC3+ neuron count post-treatment with either 10 μM scrambled Aβ_1–42 or Aβ_1–42 oligomers for 5 days. Mean ± SEM. n = three (Exon 1) or four (Exon 4) independent neuronal differentiation repeats. Two-way ANOVA with Šídák’s multiple comparison correction was used for statistical analysis compared against the scrambled Aβ_1–42 treatment.