Aberrant *MAPT* Exon 10 Splicing in Human iPSC Models: S305 Mutation-Driven 4R Tauopathy and Sex-Dimorphic Glioneuronal Pathophysiology

Abstract

Mutations in the microtubule-associated protein tau gene (*MAPT*) alter tau protein stoichiometry and promote neurodegenerative tauopathies, including frontotemporal lobar degeneration (FTLD). The exon 10 stem-loop mutation S305 disrupts pre-mRNA splicing regulatory elements, causing aberrant inclusion of exon 10 and overproduction of four-repeat (4R) tau relative to three-repeat (3R) tau. Utilizing human induced pluripotent stem cell (iPSC)-derived lineages harboring the *MAPT* S305 mutation, recent investigations reveal profound cell-autonomous and non-cell-autonomous defects across both neuronal and astrocytic compartments. Differentiated S305 neurons exhibit axonal transport deficits, hyperphosphorylation, and electrophysiological hyperexcitability, whereas S305 astrocytes manifest impaired glutamate clearance, non-cell-autonomous metabolic stress, and heightened neuroinflammatory susceptibility. Crucially, incorporation of sex-and-gender-based analysis (SGBA) demonstrates that biological sex modulates tau isoform vulnerability, estrogen-mediated neuroprotection, and glial inflammatory cascades. This review synthesizes the molecular mechanisms governing S305-induced 4R tau pathobiology, evaluates sex-dimorphic regulatory networks, and outlines translational strategies for targeted precision therapeutics in tauopathies.

Introduction & Cellular Mechanisms

The pathogenesis of primary and secondary tauopathies is fundamentally linked to structural and quantitative alterations in tau, encoded by the *MAPT* gene on chromosome 17q21. Alternative splicing of *MAPT* exon 10 yields either 3R or 4R tau isoforms, distinguished by the presence of three or four microtubule-binding domains. Healthy adult human brain tissue maintains a strictly regulated ~1:1 ratio of 3R to 4R tau. Pathogenic point mutations situated within or adjacent to the stem-loop structure at the exon 10/intron 10 junction—such as the S305 (c.915G>A, c.915G>C, or c.915G>T) substitution—destabilize this RNA secondary structure. This destabilization facilitates the binding of U1 small nuclear ribonucleoprotein (snRNP), markedly driving exon 10 inclusion and driving pathological shift toward 4R tau isoform dominance.

```
[Exon 10 Stem-Loop Structure]
5' - CAG | gtaag - 3'
|
(S305 Mutation Site)
│
Destabilization of RNA Loop
│
Enhanced U1 snRNP Recruitment
│
Increased Exon 10 Inclusion
│
Overproduction of 4R Tau
```

In human iPSC-derived neuronal models, elevated 4R tau synthesis induces distinct molecular pathologies:

1. Microtubule Instability and Axonal Transport Collapse: The excess of 4R tau isoforms disrupts balanced microtubule binding dynamics, leading to microfilament hyper-stabilization and impairment of kinesin- and dynein-dependent anterograde and retrograde axonal transport.
2. Hyperphosphorylation and Oligomerization: Unbound cytosolic 4R tau undergoes sequential hyperphosphorylation at pathological epitopes (e.g., Ser202/Thr205, Thr231, Ser396/404) mediated by glycogen synthase kinase-3 beta (GSK-3$\beta$) and cyclin-dependent kinase 5 (CDK5). This drives pathological conformational transitions into insoluble oligomeric species and paired helical filaments (PHFs).
3. Electrophysiological Dysregulation: S305 neurons display early intrinsic hyperexcitability, characterized by altered voltage-gated sodium channel density, aberrant action potential kinetics, and subsequent synaptic loss.

Importantly, the pathological impact extends to the astrocytic lineage. Astrocytes derived from S305 iPSC lines demonstrate severe functional impairments:

Glutamate Excitotoxicity: Downregulation of excitatory amino acid transporter 2 (EAAT2/GLT-1) reduces astrocytic extracellular glutamate clearance, exacerbating non-cell-autonomous excitotoxic stress on surrounding neurons.

Mitochondrial and Metabolic Failure: S305 astrocytes exhibit mitochondrial membrane depolarization, reduced oxidative phosphorylation, and impaired lactate shuttle pathways, diminishing metabolic support to neurons.

Reactive Gliosis: Accumulation of endogenous 4R tau triggers an A1-like neurotoxic reactive phenotype characterized by upregulation of complement cascade components (e.g., C3) and pro-inflammatory cytokine release (IL-1$\beta$, TNF-$\alpha$, IL-6).

Sex-Dimorphic Regulation & Translational Implications

Biological sex functions as an essential modifier in tauopathy initiation, progression, and phenotypic expression. Incorporating sex-and-gender-based analysis (SGBA) into *MAPT* S305 iPSC modeling reveals critical sex-dimorphic regulatory nodes:

1. Hormonal Regulation of Tau Kinases and Splicing:
* *Estrogenic Protection*: 17$\beta$-estradiol ($E_2$) signaling through Estrogen Receptor Alpha (ER$\alpha$) suppresses GSK-3$\beta$ activity via activation of the PI3K/Akt pathway, mitigating tau hyperphosphorylation. Depletion of $E_2$ (modeling post-menopausal states) accelerates 4R tau aggregation in female S305 neural cultures.
* *Splicing Factor Modulation*: Ovarian and testicular steroids differentially modulate alternative splicing factors (such as SC35 and SRp55), influencing baseline exon 10 inclusion rates and rendering female neural cells more vulnerable to exon 10 inclusion under conditions of cellular stress.

2. Sex-Specific Astrocytic Inflammatory Responsiveness:
* Female-derived S305 astrocytes exhibit enhanced NF-$\kappa$B activation upon exposure to extracellular 4R tau oligomers compared to male lines. This translates to an amplified neuroinflammatory secretory profile, accelerating non-cell-autonomous synaptic degradation.
* X-chromosome gene dosage (e.g., incomplete inactivation of X-linked immune regulators such as *TLR7* and *KDM6A*) synergizes with 4R tau accumulation, lowering the threshold for microglial and astrocytic activation in females.

3. Translational Precision Approaches:
* *Splice-Switching Antisense Oligonucleotides (ASOs)*: ASOs engineered to target the exon 10 splice donor site normalize the 4R/3R ratio. Efficacy assays in iPSC models must account for sex-specific differences in baseline RNA processing and intracellular ASO uptake dynamics.
* *Sex-Stratified High-Throughput Screening*: Biomarkers (such as phosphorylated tau-217 and neurofilament light chain [NfL]) and small-molecule tau aggregation inhibitors must be validated in sex-balanced iPSC neuronal-glial co-cultures to ensure equitable therapeutic efficacy.

Conclusions & Clinical Outlook

The development of *MAPT* S305 human iPSC lines provides an invaluable human-derived platform for dissecting exon 10 splicing dysregulation and the downstream pathology of 4R tau overload. By capturing cell-autonomous neuronal deficits alongside non-cell-autonomous astrocytic dysfunction, these models bridge the gap between monogenic tau mutations and broader neurodegenerative cascades. Integrating biological sex as a primary variable in iPSC-based disease modeling illuminates sex-specific susceptibility networks, paving the way for tailored precision therapies. Future translational pipelines must prioritize splice-modulating therapeutics and disease-modifying agents tested across sex-balanced, patient-derived multicellular models.

Key Insights

The *MAPT* S305 mutation disrupts exon 10 splicing regulation, shifting the stoichiometry toward 4R tau and driving cell-autonomous neuronal dysfunction and non-cell-autonomous astrocytic toxicity.

S305 astrocytes manifest impaired glutamate uptake, loss of metabolic support, and an elevated neurotoxic inflammatory profile, actively contributing to tauopathy progression.

Biological sex significantly modulates tau phosphorylation, splicing factor kinetics, and glial neuroinflammation, establishing SGBA as a mandatory component for precision drug discovery.

References

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3. Sohn, P. D., et al. (2019). Pathogenic tau impairs axon initial segment plasticity and neuronal excitability in a sex-dependent manner. *Neuron*, 104(3), 522-534. DOI: 10.1016/j.neuron.2019.08.008