NPT088 reduces both amyloid-β and tau pathologies in transgenic mice

Abstract

Introduction: Alzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-β (Aβ) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein ("NPT088") consisting of the active fragment of g3p and human-IgG₁-Fc.

Methods: Aged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically.

Results: NPT088-lowered Aβ plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice.

Discussion: These observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aβ and tau, the hallmark pathologies of AD.

Keywords: Brain weight; Cerebrospinal fluid; GAIM; General amyloid interaction motif; Limb clasping; Novel object recognition; Phospho-tau; Spontaneous alternation; Thioflavin S.

Fig. 1

Generation and in vitro characterization of NPT088, an immunoglobulin-GAIM fusion protein. (A) Native bacteriophage M13 potently and broadly binds to and disrupts a variety of misfolded protein assemblies, including Aβ, tau, α-synuclein, and yeast prion Sup35. M13, owing to its large size (17 MDa), represents a significant hurdle for effective CNS drug delivery. Subsequent characterization of amyloid fiber binding and remodeling of M13 revealed that the minor capsid protein, gene 3 protein (g3p), is critical for this activity and, furthermore, that the two N-terminal domains of g3p facilitate binding and disruption of amyloids as a general amyloid interaction motif (GAIM). The engineered fusion protein, NPT088 (NPT088), consists of the active fragment of g3p and human-IgG₁-Fc. (B) Incubation of differentiated N2a cells with 2 μM (9 μg/mL) Aβ₄₂ ADDLs for 24-hour-induced robust cytotoxicity. Preincubation of ADDLs with NPT088 for 3 hours significantly inhibited cytotoxicity. Co-application of ADDL and NPT088 (Time 0 control) had no effect on induction of cytotoxicity. Asterisks indicate significant difference from ADDL alone (Dunnett's test, ^∗∗∗P < .001, ^∗∗∗∗P < .0001). (C) NPT088 was used to precipitate Aβ from formic acid lysates of aged Tg2576 brain. Precipitates were resolved on SDS-PAGE and western blots probed with a monoclonal anti-Aβ antibody (6E10). NPT088 precipitated Aβ from formic acid extracts of brain prepared from two different Tg2576 mice (Tg). No Aβ was extracted from lysates prepared from WT mice. NS indicates nonspecific band that is present in formic acid extracts from WT brains and is recognized by 6E10. hAPP represents human amyloid precursor protein. Note the enrichment of all species of Aβ in the immunoprecipitated lanes relative to the Input material lane. (D) Transmission electron microscopy images of Aβ₄₂ fiber preparations incubated for 7 days and stained with 1% uranyl acetate. (a–b) Examples of Aβ fiber structure after incubation for 7 days in buffer alone. (c–d) Examples of Aβ₄₂ fiber structure after incubation with NPT088 (0.25 μM) for 7 days. Note the dramatic loss of fiber structure.

Fig. 2

Systemically administered NPT088 improves cognition in aged Tg2576 mice. Behavior was assessed in aged Tg2576 mice (16–19 months at the start of dosing) after repeated, weekly IP administration of NPT088. (A) Spontaneous alternation in a Y-maze was significantly increased after 10 weeks of weekly dosing with NPT088 at 20 mg/kg (q₄₇ = 2.8, P = .01). N = 13 (PBS), 13 (0.2 mg/kg), 12 (2 mg/kg), 13 (20 mg/kg). (B) Performance of novel object recognition was significantly increased after 14 weeks of weekly dosing with NPT088 at 20 mg/kg (q₄₀ = 2.1, P = .05). N = 11 (PBS), 11 (0.2 mg/kg), 12 (2 mg/kg), 10 (20 mg/kg). (C) Systemic NPT088 significantly reduced levels of Aβ₄₂ from the soluble fraction of hippocampus at all doses tested (0.2 mg/kg: q₄₄ = 3.6, P = .003; 2 mg/kg: q₄₄ = 3.3, P = .005; 20 mg/kg: q₄₄ = 2.4, P = .05). N = 12 (PBS), 11 (0.2 mg/kg), 12 (2 mg/kg), 13 (20 mg/kg). (D) Systemic NPT088 significantly reduced levels of Aβ₄₂ from the insoluble fraction of hippocampus when administered at 2 mg/kg (q₄₆ = 2.7, P = .02). N = 13 (PBS), 12 (0.2 mg/kg), 12 (2 mg/kg), 13 (20 mg/kg). (E) Systemic NPT088 significantly reduced levels of Aβ₄₂ from the soluble fraction of cortex at all doses tested (0.2 mg/kg: q₄₇ = 3.1, P = .01; 2 mg/kg: q₄₇ = 3.1, P = .01; 20 mg/kg: q₄₇ = 2.4, P = .05). N = 13 (PBS), 13 (0.2 mg/kg), 13 (2 mg/kg), 12 (20 mg/kg). (F) Systemic NPT088 significantly reduced levels of Aβ₄₂ in the insoluble fraction of cortex at 2 and 20 mg/kg (2 mg/kg: q₄₇ = 3.6, P = .002; 20 mg/kg: q₄₇ = 2.1, P = .05). N = 13 (PBS), 12 (0.2 mg/kg), 13 (2 mg/kg), 13 (20 mg/kg). In all panels, asterisks indicate significant difference versus Tg-PBS.

Fig. 3

Systemically administered NPT088 reduces Aβ plaque and Aβ42 in aged Tg2576 mice. Aged Tg2576 mice (16–19 months at the start of dosing) received 14 weeks of weekly, IP injections of NPT088 at ascending doses (0.2, 2, 20 mg/kg). Tissue was harvested 24 hours after the last injection. (A) Representative images of 82E1-immunostained Aβ in the hippocampus showing reduction after treatment with NPT088. Scale bars = 500 μm. (B) Summary quantification of 82E1 immunostaining from three coronal sections at 240-μm intervals indicating significant reduction of total Aβ plaque in the hippocampus at all doses tested. N = 11 (PBS), 10 (0.2 mg/kg), 12 (2 mg/kg), 10 (20 mg/kg). Asterisks indicate significant difference versus Tg-PBS. (C) Representative images of 82E1 immunostaining in the cortex showing reduction of Aβ after treatment with NPT088. Scale bars = 500 μm. (D) Summary quantification of 82E1 immunostaining from three sections indicating significant reduction of Aβ in the hippocampus at all doses tested. N = 11 (PBS), 10 (0.2 mg/kg), 11 (2 mg/kg), 9 (20 mg/kg). Asterisks indicate significant difference vs. Tg-PBS. (E) Representative images of thioflavin-S staining in the cortex showing reduction in the number of fibrillar Aβ plaques after treatment with NPT088. Scale bars = 500 μm. (F) Summary quantification from two sections of thioflavin-S staining indicating significant reduction of fibrillar Aβ plaque number in the hippocampus at 2 and 20 mg/kg (2 mg/kg: t₄₁ = 1.9, P = .04; 20 mg/kg: t₄₁ = 2.0, P = .03). N = 10 (PBS), 11 (0.2 mg/kg), 13 (2 mg/kg), 11 (20 mg/kg). Asterisks indicate significant difference versus Tg-PBS.

Fig. 4

Systemically administered NPT088 reduces Aβ in CSF of aged Tg2576 mice. CSF was collected from aged Tg2576 mice (20–21 months at experiment start) before dosing or after administration of 13 weeks of weekly, IP injections of NPT088 (2 mg/kg). (A) The concentration of Aβ_1–38 significantly increased in CSF from aged Tg2576 mice over 13 weeks (q₅₄ = 2.3, P = .05). Weekly IP dosing with NPT088 significantly reduced Aβ₁₋₃₈ in CSF (q₅₄ = 2.4, P = .04). N = 19 (Tg-Time 0), N = 21 (Tg-PBS), N = 17 (Tg-NPT088). (B) The concentration of Aβ_1–40 significantly increased in CSF from aged Tg2576 mice over 13 weeks (q₅₃ = 2.5, P = .03). Weekly IP dosing with NPT088 significantly reduced Aβ_1–40 in CSF (q₅₃ = 2.4, P = .04). N = 19 (Tg-Time 0), 20 (Tg-PBS), N = 17 (Tg-NPT088). One significant outlier (Grubb test) in the Tg-PBS group (77.7 ng/mL) was excluded from Aβ_1–40 analysis. (C) The concentration of Aβ_1–42 did not significantly change in CSF from aged Tg2576 mice over 13 weeks of dosing (q₅₂ = 1.1, P = .4). Weekly IP dosing with NPT088 did not affect Aβ_1–42 in CSF (q₅₂ = 1.8, P = .15). N = 19 (Tg-Time 0), 20 (Tg-PBS), N = 17 (Tg-NPT088). One significant outlier (Grubb test) in the Tg-PBS group (6.2 ng/mL) was excluded from Aβ_1–42 analysis. Asterisks indicate significant difference versus Tg-PBS.

Fig. 5

Systemically administered NPT088 improves cognitive and motor phenotypes and reverses tau pathology in rTg4510 mice. Behavior and levels of p-tau were assessed in rTg4510 mice (3.5 mo) that received 14 weeks of weekly IP treatment with NPT088 (20 mg/kg) or PBS. (A) rTg4510 mice treated with NPT088 exhibited significantly better performance in the novel object recognition task relative to rTg4510 mice receiving PBS (t = 2.3, df = 7, P = .02). N = 4 (WT-PBS), 4 (Tg-PBS), 5 (Tg-NPT088). Asterisk indicates significant difference versus rTg4510-PBS. (B) rTg4510 mice exhibited significant limb clasping relative to WT mice when suspended by the tail (q₉ = 2.8, P = .02). Treatment with NPT088 significantly reversed limb splay abnormality (q₉ = 2.8, P = .02). N = 4 (WT-PBS), 4 (Tg-PBS), 5 (Tg-NPT088). (C) Treatment with NPT088 significantly decreased somatodendritic AT8 (pSer202/pThr205) immunostaining of aggregated p-tau in pyramidal cells of the hippocampus (U_5,5 = 3, P = .03). Representative images of AT8 immunostaining shown to the left of summary quantification. AT8 immunostaining was quantified in three sections at 240-μm intervals per animal. Scale bars = 100 μm. N = 5 (Tg-PBS), 5 (Tg-NPT088). (D) Treatment with NPT088 significantly decreased levels of neuronal thioS in cortex from mice treated with NPT088 (U_3,5 = 0, P = .02). Representative images of thioS staining shown to the left of summary quantification. ThioS levels were quantified from three sections per animal. Scale bars = 100 μm. N = 3 (Tg-PBS), 5 (Tg-NPT088). In all panels, asterisk indicates significant difference from Tg-PBS.

Fig. 6

Systemically administered NPT088 reduced neurodegeneration in rTg4510 mice. rTg4510 mice in this experiment (5.7 mo at start) received 13 weekly IP doses of NPT088 (2 mg/kg) or PBS. Tissue from a group of 13 rTg4510 mice (5.7 mo) was harvested at the beginning of the study to serve as a Time 0 control, and tissue from group of 3 WT mice (8.8 months) was harvested at the end of the study. (A) Representative images of brains from WT mice and rTg4510 mice that received either NPT088 or PBS. Scale bar indicates 5 mm. (B) Wet weight of brains, measured at harvest, from rTg4510 mice receiving NPT088 was significantly more than brain weights measured from rTg4510 mice receiving PBS (t = 1.7, df = 30, P = .049). Brain weights measured from WT mice (N = 3) shown for comparative purposes. N = 3 (WT), 19 (Tg-PBS), 18 (Tg-NPT088). (C) Representative Nissl-stained sagittal sections of hippocampus from rTg4510 mice harvested at the beginning of the study (Time 0), rTg4510 mice dosed with either PBS (Tg-PBS) or NPT088 (Tg-NPT088), or littermate non-Tg mice (WT). Scale bars are 200 μm. (D) Weight of left hippocampus of rTg4510 mice treated with NPT088 (8.8 mo at harvest) was significantly greater than the weights measured from rTg4510 mice dosed with PBS (q₄₇ = 2.3, P = .046). Hippocampus weight measured from rTg4510 mice harvested at the beginning of the study (5.7 mo) was also significantly greater than weights from rTg4510 mice dosed with PBS (q₄₇ = 3.3, P = .004). Weight of hippocampus from non-Tg littermate mice (WT) is shown for comparison. N = 3 (WT), 19 (Tg-PBS), 18 (Tg-NPT088), 13 (Tg-Time 0). (E) Thickness of stratum pyramidale in area CA1 of hippocampus was significantly greater in rTg4510 mice dosed with NPT088 relative to rTg4510 mice that received PBS (q₄₀ = 2.8, P = .02). Stratum pyramidale of area CA1 from tissue harvested at the beginning of the study exhibited a trend for increased thickness relative to mice that were dosed with PBS (q₄₀ = 1.8, P = .15). Data from WT mice are shown for comparison. N = 2 (WT), 16 (Tg-PBS), 14 (Tg-NPT088), 13 (Tg-Time 0). One significant outlier (Grubb's test) was excluded from analyses (Tg-PBS). (F) The area of thioS-positive deposits was quantified from 2 sections per mouse in a defined region of interest with a fixed area (∼0.19 mm²) in sagittal sections of the parietal cortex, dorsal to hippocampus. A significant increase in thioS staining was observed over the course of the study (q₄₇ = 4.9, P < .0001). Treatment with NPT088 significantly reduced thioS staining in parietal cortex (q₄₇ = 2.3, P = .045). N = 19 (Tg-PBS), 18 (Tg-NPT088), 13 (Tg-Time 0). (G) Limb clasping was assessed during week 4 of dosing. rTg4510 mice receiving PBS exhibited significantly more severe limb clasping relative to rTg4510 mice assessed at the beginning of the study (q₅₀ = 2.7, P = .02). Mice treated with NPT088 exhibited a trend for reduced limb clasping (q₅₀ = 1.8, P = .13). N = 20 (Tg-PBS), 20 (Tg-NPT088), 13 (Tg-Time 0). (H) rTg4510 mice that were treated with NPT088 exhibited significantly better performance in the novel object recognition test compared to mice that received PBS (t = 2, df = 21, P = .03). N = 14 (Tg-PBS), 12 (Tg-NPT088). In all panels, asterisks indicate significant difference from Tg-PBS.