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Breakthrough: Successfully established an in vivo neuronal model of Alzheimer’s disease derived from iPSCs that spontaneously forms neurofibrillary tangles.

　　Introduction

　　Tau protein primarily functions to stabilize the microtubule network within neurons. During transcription of the human MAPT (tau protein) gene, alternative splicing of exon 10 gives rise to multiple isoforms: 3R tau, which is produced from transcripts lacking exon 10 and contains three microtubule-binding repeats; and 4R tau, which results from inclusion of this exon and contains four repeats.

　　Researchers have long been puzzled by a fundamental issue: although we can generate human neurons in the laboratory using induced pluripotent stem cells (iPSCs), these neurons invariably remain in a “fetal‑like” state. This means they predominantly express the 3R isoform of tau protein, failing to faithfully recapitulate the complex pathology observed in the adult Alzheimer’s disease (AD) brain, where both 3R and 4R tau proteins are co‑tangled.

　　The latest research from the laboratory of Dominik Paquet at Ludwig Maximilian University (LMU) in Munich, published in Science Translational Medicine (highlighted by Alzforum), has successfully addressed this challenge using a scarless CRISPR‑Cas9 gene-editing approach.

　　01 Core Breakthrough: Enabling Neurons to “Mature Overnight”

　　In the brains of healthy adults, tau protein maintains a balance between its 3R and 4R isoforms. However, conventional iPSC-derived neurons cultured in the laboratory remain perpetually in a “fetal state,” predominantly expressing the 3R isoform, which prevents them from accurately recapitulating the hallmark co‑existing 3R–4R tangles observed in adult Alzheimer’s disease (AD).

　　Researcher Angelika Dannert successfully achieved precise regulation of the tau gene in cortical neurons derived from human induced pluripotent stem cells (iPSCs) by combining CRISPR-Cas9 with the PiggyBac transposon system.

Figure 1.3 R to 4R. The combination of CRISPR editing with the PiggyBac selection transposon ensures enrichment of iPSC‑derived neurons expressing 3R:4R tau. Using gene-editing techniques, researchers have induced iPSC‑derived neurons to bypass developmental stages and directly express adult‑specific Tau protein isoforms.

　　First‑step excision and insertion (positive selection): First, CRISPR‑Cas9 is used to precisely delete the entire intron between exons 10 and 11—including the splicing regulatory elements—thereby forcing this allele to continuously produce 4R tau. Subsequently, a PiggyBac transposon containing two essential selection markers is inserted at the cleavage site. Positive selection is achieved using the puromycin resistance gene: upon addition of puromycin to the culture medium, cells that failed to excise the intron and integrate the transposon are killed, thereby eliminating unedited cells.

　　Step 2: Transposon excision and purification (negative selection): The TK enzyme substrate FIAU is introduced into the culture system; its cytotoxic metabolites, generated selectively in cells harboring residual transposons, specifically eliminate the negative cell population that has not undergone excision. Leveraging the PiggyBac transposon system’s unique “precise integration and seamless excision” mechanism, the surviving resistant clones achieve truly “trace‑free” editing—leaving no CRISPR components, exogenous transposon sequences, or recombination scars in the host genome, with editing precision at the single‑base pair level.

　　Step 3: Genotype Identification and Molecular Phenotype Validation: Two rounds of amplification and PCR-based genotyping were performed using primers specific for the 3R and 4R splicing isoforms, ultimately confirming the successful construction of the target allele and demonstrating that this cell line achieves the intended molecular phenotype of balanced expression of both 3R and 4R Tau proteins.

　　02 “PLSF” neurons: spontaneously formed “ghost entanglements”

　　To induce tau protein pathological tangles resembling those seen in Alzheimer’s disease (AD) in cultured neurons, researchers further introduced the pathogenic P301L and S320F mutations into exons 10 and 11, respectively, of the 4R isoform of the tau gene—both mutations are associated with frontotemporal dementia accompanied by tau pathology—and designated the mutated cells as PLSFs. This approach enabled neurons to spontaneously form pathological tau aggregates without the need for overexpression.

　　Over time, 4R PLSF heterozygous cells exhibit a remarkably strong capacity to induce tau protein aggregation and display pathological features highly consistent with those observed in the brains of human Alzheimer’s disease (AD) patients, including misfolding, oligomerization, hyperphosphorylation, and the accumulation of insoluble tau. Comprehensive analyses using electron microscopy and multiple specific antibodies and dyes have confirmed that these cells harbor, for the first time, large aggregated fibrils that fill the entire cell body, thereby overcoming the longstanding challenge of poorly pronounced phenotypes in previous models.

Figure 2. Over time, 4R PLSF‑het accumulates hyperphosphorylated (AT8), fibrillar (PHF‑1), and insoluble (K9JA) tau. Under fluorescence microscopy, PLSF neurons spontaneously generate highly phosphorylated tau protein fibrils during culture.

　　03 The model’s translational value in target discovery and CRO pipeline development

　　Upon its publication, this system immediately attracted significant attention from the industry, primarily because it offers a highly standardized in vitro platform for evaluating the pharmacological efficacy of live cells.

　　High consistency of pathological biomarkers: The spontaneously generated Tau fibrils in the model exhibit strong binding to classical conformation-specific, oligomeric, and hyperphosphorylated antibodies—such as MC‑1, ALZ50, PHF‑1, and AT8—found in human Alzheimer’s disease brain tissue.

　　Recreating the full cytotoxic cascade: cells not only exhibited a reduction in synaptic density and nuclear envelope rupture, but also, following apoptosis, left behind the classic “ghost tangles” observed in clinical pathology sections, thereby confirming the capacity of Tau aggregates to propagate across neuronal networks.

　　Pharmacological validation loop: The fibrils within this model can bind to a clinical‑grade Tau PET imaging tracer (PI‑2620). Upon addition of a small‑molecule aggregation inhibitor (Anle138b) to the culture system, fibril growth and seeding capacity were successfully inhibited, demonstrating the model’s full practical feasibility for high‑throughput drug screening and target validation.

　　Figure 3. Amyloid-like fibrils. Electron microscopy reveals that in 4R PLSF homozygous cells, the fibrils form connections between AT8 (top) and PHF‑1 (bottom).

Breakthrough: Successfully established an in vivo neuronal model of Alzheimer’s disease derived from iPSCs that spontaneously forms neurofibrillary tangles.

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