Modeling MAPT S305-Driven Tauopathy in Human iPSC-Derived Neuronal and Astrocytic Lineages: Mechanistic Insights and Sex-Dimorphic Implications

Abstract

Pathological hyperphosphorylation and aggregation of the microtubule-associated protein tau (MAPT) define a broad spectrum of neurodegenerative diseases known as tauopathies. The *MAPT* S305 mutation, localized within the exon 10 splice donor site, shifts pre-mRNA splicing to favor the inclusion of exon 10, resulting in an overproduction of four-repeat (4R) tau isoforms relative to three-repeat (3R) isoforms. Utilizing human induced pluripotent stem cell (hiPSC) models harboring the *MAPT* S305 mutation provides unprecedented fidelity in capturing human-specific pathogenic mechanisms. Elevated 4R tau stoichiometry alters microtubule dynamics, triggers axonal transport deficits, induces mitochondrial dysfunction, and precipitates cell-autonomous neuronal hyperexcitability followed by synaptic decay. Crucially, non-cell-autonomous pathological cascades in *MAPT* S305 astrocytic lineages disrupt glutamate homeostasis, exacerbate neuroinflammatory cytokine cascades, and impair neurovascular coupling. Incorporating sex-and-gender-based analysis (SGBA) reveals that sexual dimorphisms—mediated by estrogen receptor signaling networks and X-chromosome gene dosage (e.g., *USP9X*, *KDM6A*)—significantly modulate 4R tau accumulation, astrocytic reactivity, and neurodegenerative susceptibility. This review synthesizes the molecular mechanics of *MAPT* S305 hiPSC models, highlighting translational pathways for sex-stratified precision therapeutics.

Introduction & Cellular Mechanisms

The human *MAPT* gene undergoes alternative splicing at exon 10 to yield tau isoforms containing either three or four microtubule-binding repeats (3R and 4R tau, respectively). Under physiological conditions in the adult human brain, the ratio of 3R to 4R tau isoforms is maintained near unity. The *MAPT* S305 point mutation, situated at the junction of exon 10 and intron 10, destabilizes the secondary stem-loop structure that naturally limits exon 10 inclusion. This structural destabilization enhances the binding affinity of the U1 small nuclear ribonucleoprotein (snRNP) complex, driving constitutive inclusion of exon 10 and perturbing the baseline 3R:4R stoichiometric balance in favor of 4R tau.

Human iPSC-derived neurons harboring the *MAPT* S305 mutation demonstrate that an excess of 4R tau isoforms impairs microtubule dynamics due to the higher affinity of 4R tau for tubulin dimers. Paradoxically, this heightened affinity leads to localized hyperphosphorylation at pathological epitopes (e.g., Thr181, Ser202/Thr205, Ser396/404) mediated by aberrant activation of glycogen synthase kinase-3 beta (GSK-3β) and cyclin-dependent kinase 5 (CDK5). Hyperphosphorylated 4R tau dissociates from the microtubule lattice, forming toxic soluble oligomers and neurofibrillary tangles (NFTs).

At the organelle level, 4R tau oligomerization severely compromises anterograde and retrograde axonal transport by occluding kinesin and dynein motor protein trafficking. Consequently, localized mitochondrial distribution is disrupted, leading to:

Inner mitochondrial membrane potential hyperpolarization followed by collapse,

Elevated production of reactive oxygen species (ROS),

Impaired oxidative phosphorylation and ATP depletion, and

Perturbation of store-operated calcium entry (SOCE) and endoplasmic reticulum (ER) stress (activation of the PERK-eIF2α unfolded protein response axis).

In *MAPT* S305 hiPSC-derived astrocytes, toxic 4R tau aggregates accumulate via liquid-liquid phase separation (LLPS) and endocytic internalization from the extracellular matrix. Astrocytic tau accumulation triggers a transition toward a pro-inflammatory reactive state. Key biochemical alterations in *MAPT* S305 astrocytes include:
1. Downregulation of the primary excitatory amino acid transporter 2 (EAAT2/GLT-1), leading to impaired extracellular glutamate clearance and subsequent non-cell-autonomous excitotoxicity in adjacent neurons.
2. Dysregulated calcium ($Ca^{2+}$) wave propagation, causing aberrant release of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6).
3. Metabolic decoupling from neurons, characterized by diminished lactate transport across the astrocyte-neuron lactate shuttle (ANLS).

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[ MAPT Exon 10 S305 Mutation ]
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Disruption of Pre-mRNA Stem-Loop
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U1 snRNP Recruitment & Exon 10 Inclusion
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Elevated 4R:3R Tau Stoichiometry
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┌────────┴────────────────────────┐
▼ ▼
[ Neuronal Compartment ] [ Astrocytic Compartment ]
- Microtubule destabilization - EAAT2/GLT-1 downregulation
- Axonal transport blockade - Impaired glutamate clearance
- Mitochondrial ROS & PERK UPR - Pro-inflammatory cytokine secretion
- Excitotoxicity & synaptic loss - Disruptions in ANLS metabolic coupling
```

Sex-Dimorphic Regulation & Translational Implications

Translational research in neurodegenerative tauopathies demonstrates substantial sex-dimorphic variance in pathological severity, progression rate, and glial reactivity. Incorporating Sex-and-Gender-Based Analysis (SGBA) into *MAPT* S305 hiPSC models is critical for unraveling the biological underpinnings of these clinical differences.

1. Hormonal Regulation of Tau Kinases and Microtubule Dynamics:
Estrogen signaling via Estrogen Receptor Alpha (ERα) and Beta (ERβ) directly modulates the phosphorylation state of tau through the Akt/GSK-3β signaling axis. 17β-estradiol activates Akt via phosphoinositide 3-kinase (PI3K), inducing inhibitory phosphorylation of GSK-3β at Serine 9. In female hiPSC-derived neurons post-menopause or under estrogen-depleted conditions, the attenuation of ERα activation leads to disinhibition of GSK-3β, driving hyperphosphorylation of the elevated 4R tau species induced by the S305 mutation. Conversely, androgen receptor (AR) signaling in male neuronal networks modulates CDK5 p35/p25 cleavage, exhibiting distinct kinetic profiles of tau phosphorylation.

2. X-Chromosome Inactivation Escape and Glial Neuroinflammation:
Females possess two X chromosomes, and incomplete X-chromosome inactivation (XCI escape) leads to the over-expression of X-linked genes in female neural cells. Key escapees include *USP9X* (an X-linked deubiquitinase) and *KDM6A* (a histone demethylase):
* *USP9X* directly deubiquitinates tau, preventing its proteasomal degradation and promoting 4R tau stabilization in female hiPSC lines.
* *KDM6A* regulates epigenomic landscapes in astrocytes and microglia; elevated *KDM6A* expression in female *MAPT* S305 astrocytes accelerates the transition into a pro-inflammatory state upon exposure to oligomeric 4R tau.

3. Translational Implications for Precision Medicine:
The intersection of the *MAPT* S305 mutation and biological sex dictates target responsiveness to emerging tau-targeted therapeutics. Antisense oligonucleotides (ASOs) designed to target the splice donor site of exon 10 to correct the 4R:3R tau ratio display differential efficacy depending on sex-specific baseline RNA processing machinery. Furthermore, small-molecule tau aggregation inhibitors and anti-tau monoclonal antibodies exhibit sex-dependent pharmacokinetics and microglial clearance rates, driven by differential Fc receptor expression and phagocytic capacity between male and female glia.

Conclusions & Clinical Outlook

The development of *MAPT* S305 mutant human iPSC lines represents a decisive advancement in modeling 4R tauopathies within human neuronal and astrocytic compartments. These models demonstrate that alternative splicing mutations do not act in isolation; rather, they initiate a cascade of cell-autonomous neuronal pathology and non-cell-autonomous astrocytic toxicity.

To translate these cellular insights into clinical breakthroughs, future research must prioritize sex-stratified high-throughput screening platforms. Co-culture systems comprising patient-derived neurons, astrocytes, and microglia must be evaluated under rigorous SGBA frameworks to capture the full spectrum of hormonal, genetic, and epigenetic modifiers. Advancements in splice-switching ASOs, CRISPR-Cas-mediated epigenome editing of the *MAPT* locus, and cell-type-specific tau degradation technologies (e.g., Tau-PROTACs) offer promising therapeutic avenues when tailored to the sex-specific physiological landscape of the patient.

Key Insights

The *MAPT* S305 mutation alters pre-mRNA secondary structure to enforce exon 10 inclusion, driving an overproduction of 4R tau that induces microtubule dysfunction, mitochondrial failure, and synaptic loss in hiPSC-derived neurons.

Non-cell-autonomous astrocytic pathology—characterized by EAAT2 downregulation, glutamate excitotoxicity, impaired metabolic coupling, and pro-inflammatory signaling—is an integral driver of *MAPT* S305-mediated neurodegeneration.

Biological sex significantly modifies tauopathy pathogenesis: X-chromosome inactivation escapees (e.g., *USP9X*, *KDM6A*) and sex-steroid receptor signaling differentially regulate 4R tau degradation, hyperphosphorylation kinetics, and glial neuroinflammatory phenotypes.

References

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