Small-Molecule DEPTAC TP2 Selectively Reverses Tau Hyperphosphorylation and Restores Cognition in Tauopathy Mouse Models

The researchers identified a pyrrolo[2,3-c]pyridine derivative (compound 11-5, derived from US patent US10022461) from the BindingDB database as the Tau-binding moiety. For the PP2A-recruiting moiety, they selected a phenothiazine-based scaffold (structurally related to perphenazine) previously validated to activate PP2A. The two moieties were linked via a PEG linker matching the length used in the earlier peptide-based DEPTAC. Through a sequence of reactions — substitution, nitro reduction, Ullmann coupling, and amide condensation — the team synthesized the final bifunctional molecule, TP2, with purity confirmed by HPLC to exceed 98%.

Figure 1. Design and synthesis of TP2

The researchers confirmed this mechanistic hypothesis using three independent methods:

• Co-IPCo-immunoprecipitation: In primary neurons, immunoprecipitating endogenous Tau with a Tau5 antibody after TP2 treatment revealed a marked increase in co-precipitated PP2A regulatory subunit.
  

  Figure 2. Assessing the interaction between endogenous Tau and PP2A-Bα by Co-IP
• FLIM-FRETLive-cell imaging: In cells co-expressing GFP-Tau and mCherry-PP2A-Bα, TP2 treatment significantly shortened the donor fluorescence lifetime and increased FRET efficiency, indicating that the two proteins were brought within 10 nanometers of each other.
  

  Figure 3. FLIM-FRET experiments performed in live HEK293 cells to detect TP2-induced proximity between Tau and PP2A-Bα
• SPRSurface plasmon resonance: The dissociation constant (K_D) of TP2 for GST-Tau was 7.36 μM, compared with 92.5 μM for His-PP2A-Bα — indicating substantially higher affinity for Tau. When 1 μM PP2A-Bα was added to the system, TP2's apparent affinity for Tau increased roughly threefold (K_D dropped to 2.44 μM), demonstrating that the three components can form a cooperatively stabilized ternary complex.
  

  Figure 4. Surface plasmon resonance (SPR) characterization of TP2's direct binding to its targets and TP2-induced ternary complex formation

In vitro microtubule polymerization assays showed that TP2 alone neither promotes microtubule assembly nor alters the kinetics of Tau-induced assembly, indicating that TP2 binding does not interfere with Tau's normal physiological function.

In primary rat hippocampal neurons, TP2 reduced levels of multiple phosphorylation sites (pT181, pS199, pS396, pS404, AT8) in a dose-dependent manner, with total Tau (Tau5) also showing a modest decline. CCK-8 viability assays confirmed that TP2 did not affect neuronal survival within the effective concentration range. The researchers then used K18 fibrils (an aggregation-prone fragment of the Tau microtubule-binding repeat domain) to induce Tau pathological toxicity: neurons exposed to K18 fibrils developed pronounced somatic aggregation and neurite dystrophy, while TP2 treatment ameliorated this phenotype and preserved a more intact dendritic network.

Figure 5. TP2 effectively reduces Tau phosphorylation and rescues K18-induced neurotoxicity in primary neurons

Compared with the previously reported peptide-based DEPTAC D16, TP2 achieved comparable dephosphorylation at a lower effective concentration and sustained its effect for longer after drug washout, demonstrating greater intracellular potency and a more durable duration of action.

Pharmacokinetic data showed that TP2 was still detectable in thoroughly perfused brain tissue 30 minutes after a single intravenous injection (2.5 mg/kg), confirming that systemically administered TP2 reaches the brain. Building on this, the researchers administered seven intravenous doses to two model systems:

P301L mice (a mature-tangle model) and AAV-hTau-N368 mice (a fragment-toxicity model): TP2 significantly reduced pathological phosphorylated Tau at multiple sites in both models, while total Tau levels showed no substantial decline. This reflects the core feature of the DEPTAC strategy — correcting the phosphorylation imbalance rather than indiscriminately clearing Tau. TP2 was equally effective against already-aggregated insoluble Tau, oligomeric Tau, and neurofibrillary tangles, indicating that its effect is not limited to early-stage disease.

Figure 6. TP2 effectively reduces hyperphosphorylated, oligomeric, and aggregated Tau, improves outcomes in tauopathy models, and leaves global PP2A activity unchanged

Beyond clearing pathological Tau, TP2 also repaired the neuron's structural "hardware." In P301L mice, TP2 reversed neuronal loss in the hippocampal CA3 region and restored dendritic branching complexity and spine density in CA1 pyramidal neurons, with MAP2 staining confirming the integrity of dendritic structure. Under finer-resolution electron microscopy, TP2 also reversed the decline in synaptic density, and key pre- and post-synaptic proteins (SYP, PSD95, GluA2, GluN2B) showed a trend toward recovery. Microtubule fragmentation was likewise improved, indicating that once Tau is dephosphorylated, its native microtubule-stabilizing function is restored in parallel.

Figure 7. TP2 treatment rescues neuronal morphology, synaptic integrity, and cytoskeletal structure in P301L mice

Tau pathology is commonly accompanied by neuroinflammation. Microglia in the dentate gyrus of P301L mice showed clear signs of activation (increased numbers, altered morphology); TP2 treatment significantly reduced microglial numbers and restored a resting-state morphology. Astrocytes told a different story: rather than the proliferative response typically seen, these 12-month-old mice showed astrocyte atrophy — reduced numbers and simplified processes — consistent with observations in human AD brain tissue. TP2 treatment reversed this atrophy, restoring both cell density and morphological complexity. qPCR results supported these findings: TP2 significantly reduced expression of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, while the anti-inflammatory cytokine IL-10 trended upward without reaching statistical significance.

Figure 8. Effect of TP2 treatment on microglial activation and astrocyte atrophy in P301L mice

Having validated TP2's effects at the molecular, cellular, and structural levels, the researchers assessed functional outcomes using three behavioral tests:

• NORNovel object recognition: TP2-treated mice recovered discrimination indices close to wild-type levels;
• MWMMorris water maze: TP2 significantly shortened escape latency and increased target-quadrant platform crossings in both P301L and N368 mice;
• CFCContextual fear conditioning: TP2 significantly increased freezing time, indicating restored consolidation of fear memory.

Consistent cognitive improvement across two mechanistically distinct models — the mature-tangle P301L model and the fragment-induced AAV-N368 model — provides strong support for TP2's therapeutic potential.

Figure 9. TP2 administration improves cognitive deficits in P301L and AAV-hTau-N368 mouse models