Zfra restores memory deficits in Alzheimer's disease triple-transgenic mice by blocking aggregation of TRAPPC6AΔ, SH3GLB2, tau, and amyloid β, and inflammatory NF-κB activation

Abstract

Introduction: Zinc finger-like protein that regulates apoptosis (Zfra) is a naturally occurring 31-amino-acid protein. Synthetic peptides Zfra1-31 and Zfra4-10 are known to effectively block the growth of many types of cancer cells.

Methods: Ten-month-old triple-transgenic (3×Tg) mice for Alzheimer's disease (AD) received synthetic Zfra peptides via tail vein injections, followed by examining restoration of memory deficits.

Results: Zfra significantly downregulated TRAPPC6AΔ, SH3GLB2, tau, and amyloid β (Αβ) aggregates in the brains of 3×Tg mice and effectively restored their memory capabilities. Zfra inhibited melanoma-induced neuronal death in the hippocampus and plaque formation in the cortex. Mechanistically, Zfra blocked the aggregation of amyloid β 42 and many serine-containing peptides in vitro, suppressed tumor necrosis factor-mediated NF-κB activation, and bound cytosolic proteins for accelerating their degradation in ubiquitin/proteasome-independent manner.

Discussion: Zfra peptides exhibit a strong efficacy in blocking tau aggregation and amyloid Αβ formation and restore memory deficits in 3×Tg mice, suggesting its potential for treatment of AD.

Keywords: Neurodegeneration; Peptide polymerization; Protein degradation; SH3GLB2; TRAPPC6A; WWOX; Zfra.

Fig. 1

Effect of Zfra peptide on learning and memory of 3×Tg mice. (A) The nonspatial learning and memory were determined by novel object recognition task. The abilities of nonspatial memory are expressed as the percentages of novel object exploring time (time spent on novel object/time spent on both objects) during acquisition, short-term (2 hours) delay, and long-term (24 hours) delay (PBS group, n = 6; Zfra group, n = 5). (B) The spatial learning and memory were determined by Morris Water Maze: latency of training sessions; probe test in water maze; swimming speeds. Statistics: Zfra versus respective PBS group, two-tailed Mann–Whitney test (PBS group, n = 12; Zfra group, n = 10). Abbreviations: PBS, phosphate buffered saline; Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 2

Effect of Zfra peptide on Aβ deposition in 3×Tg mice. Representative micrographs of Aβ-positive cells are shown on the left panels, the boxed regions are enlarged and shown on the middle, and the quantitative results are shown on the right. (A) Dorsal hippocampus. (B) Ventral hippocampus. *P < .05 versus respective PBS group, two-tailed unpaired t-test (PBS group, n = 3; Zfra group, n = 5). Also, representative micrographs of cortical Aβ plaques (white arrows) are shown for the PBS controls and Zfra-treated mice on the left panel (C), and the data quantified on the right. Statistics: Zfra versus respective PBS group, two-tailed unpaired t-test (PBS group, n = 3; Zfra group, n = 5). Abbreviations: Aβ, amyloid β; Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 3

Effect of Zfra on tau phosphorylation of the dorsal part hippocampi in 3×Tg mice. Representative micrographs of pS412-tau immunostaining are shown on the left panels, the boxed regions are enlarged and shown on the right, and the quantitative results are shown on the bottom. (A) Dorsal hippocampus. (B) Ventral hippocampus. *P < .05 versus respective PBS group, two-tailed Mann–Whitney test. (C) Zfra inhibition of pT181-tau aggregation is shown (scale bar = 200 μm for top panel and 100 μm for bottom panel). (D) Zfra suppressed intracellular pS35-TPC6AΔ expression (40 neurons counted) and extracellular pS35-TPC6AΔ plaque formation (see the blue asterisk). (E) Zfra blocked the aggregation of self-polymerizing SH3GLB2 (∼70% suppression). The quality of homemade antibody is shown. Magnification: top panel 20× and bottom panel 100×. Abbreviations: Alv., alveus; S.L., stratum lacunosum; S.O., stratum oriens; S.P., stratum pyramidale; S.R., stratum radiatum; Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 4

Zfra suppresses melanoma B16F10-mediated neurodegeneration in the hippocampus. Nude mice were preinjected with 100 μL of sterile Milli-Q water or Zfra4–10 (1 mM in sterile water) in 3 consecutive weeks. After treatment for a week, these mice were inoculated with melanoma B16F10 cells on both flanks (2 × 10⁵ cells in 100-μL PBS). Mice were sacrificed when the tumor sizes grew up to 2000–3000 mm³ in about a month. (A) Zfra blocked neurodegeneration in the hippocampus (top panel) and prevented the formation of TPC6AΔ plaques in the cortex (bottom panel), which negatively correlates with pS14-WWOX expression. Note the presence of condensed apoptotic nuclei in the hippocampal neurons in the control mice. (B) Metastasis of B16F10 cells to the lung was blocked in Zfra-treated mice. Compared to controls, Zfra suppressed p-S14WWOX expression in the lung by ∼72% suppression (three sections). The WWOX protein levels were not suppressed. Abbreviation: Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 5

Zfra suppresses polymerization or aggregation of serine-containing TPC6AΔ and other peptides in PBS. Zfra4–10 was incubated with an indicated serine-containing peptide in PBS at room temperature for 24 hours (final 200 μM each peptide). The mixtures were subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (A) Full-length Zfra or red-fluorescent TMR-Zfra blocked aggregation of Aβ42 (lanes 3, 5, and 7). In controls, Aβ40 did not undergo aggregation. The membrane was stained with antibody against Aβ. (B) Zfra4–10 and one of the TPC6AΔ peptides were resuspended in Milli-Q water or PBS and incubated for 24 hours at room temperature. Zfra4–10 suppressed the polymerization of serine-containing peptides. % Reduction in intensity = [1 − (Z.P)/(Z + P)] × 100%, where Z = Zfra4–10, P = an indicated peptide, and Z.P = peptide mixture. (C) Under similar conditions, serine-containing peptides derived from WWOX and ANKRD40 were synthesized, possessing with or without phosphorylation at a specific serine or tyrosine residue. Zfra4–10 suppressed polymerization of these peptides in PBS. Abbreviation: Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 6

Zfra covalently binds and accelerates protein degradation. (A) On dissolving in PBS for 30–120 minutes at 37°C, Zfra peptide (3.5 kDa) underwent self-polymerization and formed large-size complexes (78 and 80 kDa), as determined by reducing SDS-PAGE and Western blotting (see the vertical arrow at lane 10, and lanes 1, 4, and 7). Aliquots of Zfra-deficient breast MCF7 cell lysates (100 μg) were incubated with Zfra (100 μM) for various durations in the presence or absence of proteasome inhibitor MG-132 (50 μM). Zfra formed complexes with cytosolic proteins (or zfrated; see arrows), and disappeared with time even in the presence of MG-132. (B and C) Zfra-negative prostate DU145 cells were exposed to UV (160–720 mJ/cm²), and aliquots of whole cell lysates (100 μg) were incubated with Zfra peptide (100 μM) for various durations (in PBS) at 37°C, in the presence of a cocktail of protease inhibitors (1:10 dilution). Zfrated proteins (70, 72, 200 kDa and minor band ladders) and exogenous Zfra peptide were degraded with time (>99% in 120 minutes; reducing SDS-PAGE). Synthetic Zfra was in PBS at the first lane at the left, and in Milli-Q water at the last lane at the right. Zfra-free control lysates had only less than 10% degradation of total proteins after incubation for 120 minutes, in the presence of protease inhibitors (data not shown). (D) L929 cell lysates (100 μg) were incubated with Zfra peptide (100 μM) for 30 minutes at 37°C (in PBS). A 30-kDa protein was zfrated in 30 minutes, and 22- and 80-kDa protein was zfrated rapidly and then degraded in 30 minutes (reducing SDS-PAGE). *A nonspecific immunoreactive or a zfrated protein in L929 cells. (E) Zfra peptide (100 μM) bound recombinant His-Tau (2 μg protein) in PBS for 24 hours at room temperature, and the resulting complex resisted dissociation by reducing SDS-PAGE. Abbreviation: Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 7

Zfra is upregulated in the AD hippocampi and interacts with tau and Aβ and blocks NF-κB promoter activation. (A and B) By antibody FRET microscopy, Zfra bound Aβ with a high affinity (FRETc greater than 45) in the hippocampal sections of postmortem AD patients (see yellow punctates in B). However, Zfra may colocalize with Aβ but without binding (FRETc less than 5). (C) Confocal microscopy analysis revealed the colocalization of Zfra with Aβ. (D) Zfra binds PHF-tau with a similar pattern. (E) A bar graph of Zfra binding with Aβ or PHF-tau is shown (n = 20). (F) By filter retardation assay using insoluble human hippocampal extracts, aggregation of Zfra and pSer8-Zfra was increased by 45 and 85%, respectively, in the older AD patients (81 ± 9.7 years old; n = 70), compared to younger nondemented controls (60 ± 13.3 years old; n = 46). Aβ was increased by 170% in the AD patients. (G) Zfra significantly blocked TNF-α (50 ng/mL)-mediated activation of NF-κB promoter in COS7 cells. Abbreviations: AD, Alzheimer's disease; Neg, negative control; PHF, paired helical filament; Pos, positive control; Zfra, zinc finger-like protein that regulates apoptosis.

Fig. 8

Zfra induces spleen Z cells to relocate out of the spleen, but the cells do not appear to migrate to the brain. (A) Spleen sections from Zfra-treated and control 3×Tg mice were stained with TMR-Zfra, followed by determining the presence of Z cells by fluorescence microscopy. Z-cell migration out of the spleen is shown in the Zfra-treated 3×Tg mice. Scale bar is 100 μm in length (100× magnification). (B) Similar experiments were carried out using brain tissue sections of 3×Tg mice. Brain sections were examined. (C and D) Similarly, spleens were harvested from Zfra-treated and control nude mice, which were inoculated with melanoma B16F10. Zfra induced Z-cell relocation out of the spleen, and the cells were found in the skin cancer lesions. Abbreviations: TMR-Zfra, tetramethylrhodamine-labeled Zfra; Zfra, zinc finger-like protein that regulates apoptosis.