Hyperphosphorylated tau targeting human serum albumin Fusion protein as therapeutics for Alzheimer's diseases

Abstract

Introduction: Neurofibrillary tangles (NFTs) are composed of hyperphosphorylated forms of microtubule-associated protein tau (Tau), which is responsible for neurodegeneration in Alzheimer's disease (AD). The hippocampal region has been a major focus of AD research because the deposits of phosphorylated tau protein in these regions are correlated with early memory deficits. Despite extensive studies, therapeutic strategies to reduce tau hyperphosphorylation and NFTs deposition remain unclear. AL04, a recently developed recombinant fusion protein comprising Cystatin C, human serum albumin, and a novel blood brain barrier (BBB) penetrating peptide, is currently under investigation. Previous studies have demonstrated its effectiveness in reducing amyloid beta plaques in AD mouse model.

Methods: In this study, we investigated the effects of AL04 on lowering hyperphosphorylated tau and NFTs in JNPL3 mouse model harboring human tau-P301L mutation. 3-month-old female mice intraperitoneally received AL04 (5 mg/kg) or PBS treatment every other week for 24 weeks. We used confocal microscopy and western blot to visualize and analyze changes in hyperphosphorylated tau Ser202/Thr205 labeled with AT8 antibody in the brain.

Results: We found that the AL04 treatment decreases hyperphosphorylated tau at PP2A-sensitive epitope Ser202/Thr205 in the hippocampus of the brain. In the brain lysates of AL04 treated mice, we observed the reduction of I2PP2A, inhibitor of PP2A, and the induction of autophagy receptor proteins such as SQSTM-1/p62 and OPTN.

Conclusion: Our data suggests that AL04 can be used as an AD prophylactic/therapeutic agent as it lowers the hyperphosphorylated tau by downregulating I2PP2A. We also propose that AL04 can induce the degradation of hyperphosphorylated tau aggregates through the upregulation of the autophagy pathway.

Keywords: Alzheimer’s disease; Anti-Tau Therapeutics; Hyperphosphorylated Tau; Neurodegeneration; Phosphatase PP2A; Protein-degradation.

Fig. 1

Schematic illustration of AL04 (83 kDa) and roles of each fusion component. Cystatin C (CysC) as an action moiety protein, human serum albumin (HSA), modified TAT peptide as cell permeable peptide (CPP), GS linker (GGSAS), and cleavable linker (GFLG): The numbers on the boxes denote the amino acid numbers from the N-terminal of AL04.

Fig. 2

AL04 attenuates the accumulation of hyperphosphorylated tau in the hippocampal CA3 and DG of JNPL3 Mice. (A and B) Representative immunohistochemistry (IHC) images with antibody against AT8 (p-Tau, Ser202/Thr205) in hippocampal CA3 and DG in PBS-treated animal (A), AL04-treated animals (B). AT8-staining is brown, and nuclei counter staining are in light blue. The boxed areas in CA3 and DG are shown in the upper panels on the right side, respectively (10x magnification). Higher magnification (25x magnification) views of the corresponding dashed black squares are shown in the lower panel. Red arrowheads indicated AT8 in the DG. Scale bars = 100 μm (A and B).

Fig. 3

Treatment with AL04 lowers phospho-tau at PP2A-sensitive Ser202/Thr205 in the brain of JNPL3 mouse. (A) Representative blots of AT8 and total P301L tau (HT-7) upon the treatment of PBS or AL04 for 24 weeks. GAPDH was used as loading control. (B) Quantitative analysis of the ratio of phosphorylation level of tau at Ser202/Thr205 normalized against total tau in brain homogenates from PBS-treated (black, n=1) or AL04 (white, n=3) for 24 weeks. The results are expressed as mean ± SD of samples. *, P < 0.005, Student's t-test.

Fig. 4

AL04-treated inhibition of Akt-GSK3β signaling in JNPL3 mice brain is dependent on PP2A. (A and C) Mice were treated with 5 mg/kg of AL04 every other week for 24 weeks. Brain lysates were subjected to SDS-PAGE and immunoblotting for 20 kDa active I2PP2A, phospho-GSK3β (P-GSK3β, Ser9) and its upstream kinase phospho-AKT (P-AKT, Ser473). GAPDH was used as a loading control. (B and D) Relative quantifications of active I2PP2A, P-GSK3β, and P-AKT levels are shown. Values are expressed as means ± SE of 3 determinations. *, P < 0.05; **, P < 0.005, Student's t test.

Fig. 5

AL04 enhances the expressions of proteins involved in protein-degradation pathways. (A) Brain lysates obtained from PBS-treated or AL04-treated mice were immunoblotted with a P-ULK1 (Ser757), ULK1, LC3-II, p62, OPTN, Nrf2, and GAPDH antibody. GAPDH was used as a loading control. (B) The quantified ratio of P-ULK1 (Ser 757), p62, OPTN, Nrf2, or LC3-II are shown. Values are expressed as means ± SE of 3 determinations. *, P < 0.05; **, P < 0.005, Student's t test.