A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis

Abstract

Alzheimer disease (AD) is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of amyloid-β peptide (Aβ) in the neuropil and around the blood vessels, and formation of neurofibrillary tangles. The endopeptidase neprilysin has been successfully used to reduce the accumulation of Aβ following intracranial viral vector delivery or ex vivo manipulated intracranial delivery. These therapies have relied on direct injections into the brain, whereas a clinically desirable therapy would involve i.v. infusion of a recombinant enzyme. We previously characterized a recombinant neprilysin that contained a 38-amino acid brain-targeting domain. Recombinant cell lines have been generated expressing this brain-targeted enzyme (ASN12). In this report, we characterize the ASN12 recombinant protein for pharmacology in a mouse as well as efficacy in two APPtg mouse models of AD. The recombinant ASN12 transited to the brain with a t½ of 24 h and accumulated to 1.7% of injected dose at 24 h following i.v. delivery. We examined pharmacodynamics in the tg2576 APPtg mouse with the prion promoter APP695 SWE mutation and in the Line41 mThy1 APP751 mutation mouse. Treatment of either APPtg mouse resulted in reduced Aβ, increased neuronal synapses, and improved learning and memory. In addition, the Line41 APPtg mice showed increased levels of C-terminal neuropeptide Y fragments and increased neurogenesis. These results suggest that the recombinant brain-targeted neprilysin, ASN12, may be an effective treatment for AD and warrant further investigation in clinical trials.

Keywords: Alzheimer Disease; Aβ; Blood Brain Barrier; Brain Penetrating Peptides; Lipoprotein Receptor; NPY; Neprilysin; Neurogenesis; Protein Targeting; Transcytosis.

FIGURE 1.

Purified ASN12 is taken up and transported by the LDL receptor. A, ASN12 and SN5 were affinity column-purified. Recombinant protein was analyzed by Coomassie-stained polyacrylamide gel. M, marker; RFU, relative fluorescence units. B, recombinant protein was assayed for enzymatic activity with the DAGNP substrate. C, dose response of ASN12 and SN5 protein uptake by serum-starved HepG2 cells. D, diagram of the blood-brain barrier, and E, in vitro system used to examine the rate of protein transport at the blood-brain barrier. F, rates of blood-brain barrier transcytosis of ASN12 and SN5 were calculated with the in vitro blood-brain barrier system. Competition for transport was determined with the LDLR binding GCmB6 recombinant protein.

FIGURE 2.

Pharmacokinetics of ASN12 and SN5 in C57/Bl6 mice. C57/Bl6 mice received 1 mg/kg recombinant ASN12 or SN5 in a 100-μl volume or 100 μl of saline as a control by intravenous delivery to the tail vein. A, at various times after injection, mice were sacrificed and blood (A), liver (B), and brain (C) were sampled for recombinant human neprilysin with an ELISA. n = 3 for each group. D, fixed brains (frontal cortex) were sectioned and stained for recombinant human neprilysin followed by anti-mouse Alexa488 (green) and counterstained with DAPI (nuclei, blue). Scale bars = 25 μm.

FIGURE 3.

Distribution of ASN12 and SN5 following repeat i.v. administration to tg2576 APPtg mice. tg2576 APPtg and non-tg mice received i.v. injections of ASN12 or SN5 (1 mg/kg) or saline every 3 days for 3 weeks. A, 3 days after the last injection, whole blood was sampled and assayed for human neprilysin with an ELISA. Mice were sacrificed, and whole brains were removed half-frozen for protein analysis and half-fixed for tissue section immunohistochemistry. B, brain homogenates were assayed for human neprilysin with an ELISA. C, brain sections were stained for human neprilysin (green), the neuronal marker MAP2 (red), and counterstained for nuclei (DAPI, blue). * indicates ANOVA p < 0.05 compared with mice that received SN5. Arrows indicate co-localization of neprilysin with the neuronal marker MAP2. Scale bars = 7 μm. Arrows highlight cells staining for neprilysin. n = 5 for each group.

FIGURE 4.

ASN12 reduces intraneuronal Aβ and ameliorates Aβ-induced neuropathology in the tg2576 APPtg mice. tg2576 APPtg and non-tg mice received ASN12, SN5 (1 mg/kg), or saline i.v. every 3 days for 3 weeks. Following protein delivery, mice were sacrificed, and brains were removed and divided sagitally with one-half fixed in paraformaldehyde. A, representative brain sections were stained for Aβ (4G8, 82E1, or D129), neuronal dendritic marker (MAP2), post-synaptic marker (synaptophysin), or astrocytic marker (GFAP). B, image analysis of the % area of the neuropil covered or corrected optical density for images analyzed. * indicates statistical significance p < 0.05 when compared with non-tg controls. # indicates statistical significance p < 0.05 when ASN12 or SN5 are compared with saline-treated APPtg mice. Scale bars = 7 μm. n = 5 for each group.

FIGURE 5.

Neither recombinant protein, ASN12 or SN5, appears to show toxicity in APP tg2576 or non-tg mice. A, mice were weighed prior to treatment and weekly afterward during treatment. B, survival curve of mice during the course of the experiment. C, representative Western blot of autoantibodies to human neprilysin or the human 38-amino acid LDLR domain from apoB detected at the conclusion of the 3-week experiment. Recombinant ASN12 was run on a gel, blotted to PVDF, and probed with whole blood from mice following the conclusion of the 3-week dosing. A positive control consisted of probing with an anti-human neprilysin antibody. n = 5 for each group.

FIGURE 6.

ASN12 transits to the CNS following intraperitoneal delivery to the Line41 APPtg mouse. Line41 APPtg and non-tg mice received intraperitoneal injections of ASN12 or SN5 (1 mg/kg) or saline twice weekly for 3 weeks. A, 10 days after the last injection, mice were sacrificed, and whole brains were removed half-frozen for protein analysis and half-fixed for tissue section immunohistochemistry. B, brain sections were stained for human neprilysin (green), the neuronal marker MAP2 (red), and counterstained for nuclei (DAPI, blue). * indicates ANOVA p < 0.05 compared with mice that received SN5. # indicates ANOVA p < 0.05 compared with non-tg mice that received ASN12. Arrows indicate co-localization of neprilysin with the neuronal marker MAP2. Scale bars = 7 μm. Arrows highlight cells staining for neprilysin. n = 5 for each group.

FIGURE 7.

ASN12 is taken up by neurons in the CNS of the Line41 APPtg mouse. Line41 APPtg and non-tg mice received intraperitoneal injections of ASN12 or SN5 (1 mg/kg) or saline twice weekly for 3 weeks. A, brain sections were stained for human neprilysin (red), the neuronal marker NeuN (green), and counterstained for nuclei (DAPI, blue). B, quantitation of % co-localization of neprilysin and NeuN staining. C, brain sections stained for neprilysin (red), the astrocytic marker GFAP (green), and counterstained for nuclei (DAPI, blue). D, quantitation of % co-localization of neprilysin and GFAP staining. * indicates ANOVA p < 0.05 compared with mice that received SN5. Arrows highlight cells staining for neprilysin. Scale bars = 7 μm. n = 5 for each group.

FIGURE 8.

ASN12 reduces intraneuronal Aβ and ameliorates Aβ-induced neuropathology in the Line41 APPtg mouse. Line41 APPtg and non-tg mice received ASN12, SN5 (1 mg/kg), or saline intraperitoneally twice weekly for 3 weeks. Following protein delivery, mice were sacrificed, brains removed, divided sagitally with one-half fixed in paraformaldehyde. A, representative brain sections were stained for Aβ (4G8, 82E1, or D129), neuronal dendritic marker (MAP2), post-synaptic marker (synaptophysin), or astrocytic marker (GFAP). B, image analysis of the % area of the neuropil covered or corrected optical density for images analyzed. * indicates statistical significance p < 0.05 when compared with non-tg controls. # indicates statistical significance p < 0.05 when ASN12 or SN5 are compared with saline-treated APPtg mice. Scale bars = 7 μm. n = 5 for each group.

FIGURE 9.

ASN12 restores levels of C-terminal NPY fragment in the Line41 APPtg mouse. Line41 APPtg and non-tg mice received ASN12, SN5 (1 mg/kg) or saline intraperitoneally twice weekly for 3 weeks. A, brains were fixed in paraformaldehyde and representative sections were stained for full-length NPY (FL-NPY), C-terminal NPY (CT-NPY), or NPY Y2 receptor (Y2-R). B, analysis of tissue staining was completed by examining cell counts by stereology or total optical density. ML, medial layer; GC, granular cells; SG, subgranular layer. * indicates statistical significance p < 0.05 compared with non-tg controls. # indicates statistical significance p < 0.05 compared with vehicle or SN5-treated Line41 APPtg mice. Scale bars = 20 μm. n = 5 per group.

FIGURE 10.

ASN12 restores neurogenesis in the Line41 APPtg mouse. Line41 APPtg and non-tg mice received ASN12, SN5 (1 mg/kg), or saline intraperitoneally twice weekly for 3 weeks. A, brains were fixed in paraformaldehyde, and representative sections were stained for DCX or PCNA. B, analysis of tissue staining was completed by examining cell counts by stereology. ML, medial layer; GC, granular cells; SG- subgranular layer. * indicates statistical significance p < 0.05 compared with Non-tg controls. # indicates statistical significance p < 0.05 compared with vehicle or SN5-treated Line41 APPtg mice. Arrows indicate cells positive for CT-NPY or Y2-R staining. Arrows indicate cells positive for PCNA staining. Scale bars = 20 μm. n = 5 per group.

FIGURE 11.

ASN12 improves water maze performance in Line41 APPtg mice. A, mice were trained on the cued platform on days 1–3 and then tested for spatial learning on days 4–7. B, probe test was performed at day 8, and the number of entrances of the mouse in the target quadrant containing the hidden platform was quantified. * indicates statistical significance p < 0.05 when compared with non-tg controls. One-way ANOVA with post hoc Dunnett's. n = 5 per group.