Neprilysin Protein Research Articles

Increased Expression of Neprilysin Is Associated with Inflammation in Preeclampsia

Preeclampsia (PE) is associated with a finely tuned equilibrium between trophoblast cell invasion and fetal-maternal immunological tolerance. An imbalance between proinflammatory (IL-6) and anti-inflammatory (IL-10) cytokines is a hallmark of PE. Neprilysin (NEP), a membrane-bound metalloprotease, is vulnerable to the inflammatory environment and plays a significant role in modulating vascular tone. The aim of this study was to determine the correlation between NEP (mRNA and protein) levels and the inflammatory status in PE patients compared to healthy pregnant women and to identify the role of NEP in evaluating the severity of preeclampsia. The study group comprised 52 pregnant women with PE while the control group comprised 47 normotensive pregnant women. After a caesarean section, placental tissue samples from patients and controls were collected to measure the expression levels of IL-6, TGF-β, IL-10, and NEP mRNA. In addition, an enzyme-linked immunosorbent assay was used to assess the quantity of NEP protein in blood samples. Our results revealed a significant positive correlation between NEP (mRNA and protein) and proinflammatory markers IL-6 and TGF-β levels in patients compared to controls and a significant inverse correlation between NEP and anti-inflammatory cytokine IL-10. Moreover, this is the first study to find a strong positive correlation between NEP level and PE severity. In conclusion, in PE patients, there is a substantial relationship between NEP, the degree of inflammation, and PE severity. NEP could act as a potential biomarker for diagnosis and prognosis of PE.

Neprilysin activity is increased in metabolic dysfunction-associated steatotic liver disease and normalizes after bariatric surgery or GLP-1 therapy

Inhibitors of neprilysin improve glycemia in patients with heart failure and type 2 diabetes (T2D). The effect of weight loss by diet, surgery, or pharmacotherapy on neprilysin activity (NEPa) is unknown. We investigated circulating NEPa and neprilysin protein concentrations in obesity, T2D, metabolic dysfunction-associated steatotic liver disease (MASLD), and following bariatric surgery, or GLP-1-receptor-agonist therapy. NEPa, but not neprilysin protein, was enhanced in obesity, T2D, and MASLD. Notably, MASLD associated with NEPa independently of BMI and HbA1c. NEPa decreased after bariatric surgery with a concurrent increase in OGTT-stimulated GLP-1. Diet-induced weight loss did not affect NEPa, but individuals randomized to 52-week weight maintenance with liraglutide (1.2mg/day) decreased NEPa, consistent with another study following 6-week liraglutide (3mg/day). A 90-min GLP-1 infusion did not alter NEPa. Thus, MASLD may drive exaggerated NEPa, and lowered NEPa following bariatric surgery or liraglutide therapy may contribute to the reported improved cardiometabolic effects.

SAT032 Circulating Neprilysin Activity Is Increased In Type 2 Diabetes And Fatty Liver Disease, And Reduces Following Bariatric Surgery And Liraglutide Therapy

Abstract Disclosure: S.A. Kjeldsen: None. L. Gluud: None. M. Werge: None. J. Pedersen: None. F. Bendtsen: None. K. Alexiadou: None. T. Tan: None. S.S. Torekov: None. E.W. Iepsen: None. N.J. Jensen: None. M. Richter: None. B. Hartmann: None. J.J. Holst: None. B. Holst: None. J. Holt: None. S. Madsbad: None. M.S. Svane: None. K.N. Bojsen-Møller: None. N.J. Wewer Albrechtsen: None. The neutral endopeptidase neprilysin (NEP) cleaves peptides important for metabolism, including GLP-1. Plasma levels of GLP-1 are reduced in patients with T2D, but the reason is unknown. Long-term treatment with the NEP inhibitor sacubitril (in combination with valsartan) improves glycemic control in individuals with T2D and heart failure. Here, we investigate plasma activity and protein levels of NEP in patients with obesity, T2D, and/or NAFLD diagnosed using biopsy or ultrasound, following bariatric surgery (RYGB and sleeve gastrectomy), and after treatment with GLP-1. We hypothesized that NEP activity (NEPa) is increased in patients with T2D, and that GLP-1 agonism reduces NEPa in a weight loss-independent manner. Here we show that plasma levels of NEPa are increased by 51% in patients with T2D (n=33) compared to individuals with normal glucose tolerance (NGT, n=160) (mean ± SD; 2734 ± 3646 vs. 1809 ± 2039 pmol/ml/min, P=0.02). Plasma NEPa was not increased in obesity (n=188) compared to lean controls (n=15) (1944 ± 2392 vs. 1373 ± 1918, P=0.4), but was increased by 76% in NAFLD (n=51) compared to non-NAFLD controls (n=43) (2629 ± 3219 vs. 1497 ± 1404, P=0.02). There were no differences in plasma NEP protein between any of the comparator groups, suggesting that changes in NEPa in metabolic disease are post-translational modifications of the enzyme or other mechanisms influencing NEPa. Bariatric surgery (RYGB, n=66 and SG, n=20) reduced plasma NEPa by 60% (P<0.001), while plasma NEP protein levels were unaltered or increased in individuals with and without preoperative T2D. Six weeks GLP-1 (liraglutide) treatment (3 mg/day) in individuals with overweight and obesity (n=14) reduced NEPa by 28% (P=0.04; apparent after 4 weeks), and at follow-up (3 weeks after discontinuation) NEPa levels returned to baseline, whereas NEP protein levels were unchanged throughout the study. In a study of individuals with obesity randomized to complete a 52-week weight maintenance intervention by diet or liraglutide (1.2 mg/day) following an 8-week diet-induced weight loss period, plasma NEPa was not affected by the initial 12% weight loss (n=48, P=0.3). However, plasma NEPa reduced in response to 52-week liraglutide therapy (n=15, P=0.05). A 90-minute low dose infusion of native GLP-1 (7-36 amide,1 pmol/kg/min) in individuals with overweight and obesity (n=19) did not alter plasma NEPa, which suggests that the attenuating effects on NEPa with GLP-1R agonism is not acute. In conclusion, our data support that plasma NEPa is increased in T2D and NAFLD independent of obesity but may be normalized by bariatric surgery, and that chronic treatment with GLP-1 reduces NEPa. Altered NEPa may therefore contribute to altered metabolism of GLP-1 in diabetes and following bariatric surgery. Whether liraglutide-induced reduction in NEPa contributes to its metabolic effects demands further investigation. Presentation: Saturday, June 17, 2023

Changes in Aβ42, Neprilysin, and γ-Secretase in the Hippocampus of Male Rats Alzheimer’s model: The Effects of Aerobic Training and Omega-3 Intake

Background: Alzheimer›s disease (AD) is characterized by excessive deposition of the amyloid-β peptide (Aβ) in the central nervous system and reducing its level is the goal of many medications. This study aimed to investigate the effect of aerobic training and omega-3 intake on Aβ42, neprilysin, and γ-secretase levels in the hippocampus of male rats Alzheimer›s model. Methods: Fifty male Wistar rats (age: 12 weeks-old and weight: 222.31±11.91 g), were divided into the five groups including control Alzheimer’s (AC), Alzheimer’s with omega-3 intake (AO), Alzheimer’s training (AT), Alzheimer’s with omega-3 intake and training (AOT) and Healthy Control (HC). AD was induced by the injection of homocysteine (60mM) into the rat brain ventricle. Training on the treadmill with a speed of 20 m/min (60 minutes and 5 days/week) was applied. The supplement group received omega-3 supplement 800 mg/kg of body weight, daily for eight weeks. Levels of Aβ42, γ-secretase, and neprilysin protein were measured using ELISA method. In data analysis, one-way ANOVA and Tukey test as post hoc were used (P<0.05). Results: The obtained results showed that the level of Aβ42 in the hippocampus of AC group was significantly higher than that of the HC group (P=0.001). Also, the level of Aβ42 in the hippocampus of AC group was significantly higher as compared to AO, AT, and AOT groups (P values 0.001, 0.007, and 0.003 respectively). The γ-Secretase level in the hippocampus of AC group was significantly higher than that in the HC group (P=0.001). Moreover, the γ-secretase levels in the hippocampus of the AC group were significantly higher compared to AO, AT, and AOT groups (P values: 0.002, 0.001, and 0.001 respectively). There was no significant difference in neprilysin levels of the hippocampus among the research groups (P=0.534). Conclusion: It appears that exercise training and omega-3 consumption, can affect amyloidogenic pathways through reducing the level of γ-secretase, and lead to reduced level of hippocampus Aβ in AD subjects. Therefore, aerobic exercise training and omega-3 intake can be studied as a complementary therapy in Alzheimer’s patients.

Blood–brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease

BackgroundAggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood–brain barrier (BBB).MethodsTo provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples.ResultsWhen tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain.ConclusionA one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced.

Poly(I:C) promotes neurotoxic amyloid β accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid β (Aβ) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aβ-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aβ degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aβ toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aβ was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aβ1-42. Altogether, these results suggest that viral infections induce Aβ neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aβ degradation.

Abstract 9449: Increased Neprilysin Expression Links Atrial Fibrotic Remodeling in Cardiovascular Surgery Patients

Introduction: Deposition of extracellular matrix (ECM) such as collagen leads to fibrosis, which plays essential roles in structural remodeling in atrial fibrillation (AF). Neprilysin (NEP) is an integral membrane-bound metallopeptidase with a wide spectrum of substrates and physiological functions. Hypothesis: We investigated the association of NEP and the atrial fibrosis-related genes in cardiovascular surgery patients including heart valve disease. Methods: Atrial mRNA expression including collagen and NEP were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (sinus rhythm (SR) 24 patients, AF 35 patients) in which LA tissues were removed from the appendage during surgery. NEP mRNA expression was also analyzed in adipose tissues (AT) obtained from both epicardial and subcutaneous adipose tissues. The NEP protein expression was measured by western blotting analysis and immunohistochemistry staining, and serum level of NEP was measured by ELISA. Results: The immunohistochemical staining showed the expression of NEP in both AT and LA. Higher expression of NEP mRNA and protein in adipose tissues was observed, compared with LA. Serum NEP concentration did not significantly correlate with NEP mRNA and protein expression of AT and LA. The significant correlations were observed among ECM (COL1A1, COL3A1, fibronectin) and the five selected fibrosis-related genes (TGFβ1, CTGF, miR21, NOX2 and BAX) attributed to the fibrosis of myocardium in LA. NEP mRNA expression of LA also positively correlated with the ECM and the fibrosis-related gene expression level. The multiple regression analysis revealed TGFβ1, NOX2, and NEP as independent predictors of atrial COL1A1 mRNA expression level. In addition, it revealed NEP as an independent predictor of COL1A1 mRNA expression level, even adjusted by age, sex, and BMI. Serum NEP concentration did not correlate with the ECM and the fibrosis-gene expression level. Patients with AF had higher atrial expression of NEP protein, compared to those with SR. Conclusions: Increased atrial expression of NEP links atrial fibrotic remodeling and developing AF in cardiovascular surgery patients. However, serum NEP did not reflect atrial NEP expression in LA.

Abstract 11451: Myocardial Neprilysin Expression in Patients With Obstructive Hypertrophic Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and fibrosis, the underlying mechanisms of which remain poorly understood. Neprilysin (NEP) inhibition has been shown to attenuate hypertrophy and fibrosis in animal models of heart failure, and NEP inhibition is an established therapeutic target in dilated and ischemic cardiomyopathy. However, the myocardial expression of NEP in HCM has not been previously reported. Hypothesis: The expression of NEP is increased in patients with obstructive HCM (oHCM). Methods/Results: We analyzed publicly available gene expression data from two independent cohorts that used myectomy samples from patients with oHCM, and healthy unused donor hearts served as controls. Cohort #1 (NCBI GEO accession number GSE36961) included 107 patients with oHCM and 39 controls and used microarray to assess gene expression. Cohort #2 (NCBI GEO accession number GSE160997) included 18 patients with oHCM and 5 controls and used RNA-Seq to assess gene expression. Both cohorts showed NEP gene expression was significantly increased in oHCM compared to controls (P<0.0001 and P<0.001, respectively). We then examined NEP expression in our surgical cohort (#3), which included 40 patients with oHCM and 8 controls. Using qPCR we showed NEP gene expression was significantly elevated in oHCM compared to controls (P<0.001, see Figure 1). Conclusions: Myocardial NEP gene expression is significantly increased in patients with oHCM undergoing septal myectomy. Future studies are needed to determine if increased myocardial NEP gene expression is accompanied by elevated NEP protein expression and/or activity. Furthermore, future studies are required to determine the mechanism of increased NEP, and whether these findings extend to HCM patients who have not progressed to symptomatic obstruction.

Analyzing Corin-BNP-NEP Protein Pathway Revealing Differential Mechanisms in AF-Related Ischemic Stroke and No AF-Related Ischemic Stroke.

BackgroundThe incidence of atrial fibrillation (AF)-related stroke increases with aging. Natriuretic peptides (NPs) family, including Corin-B type natriuretic peptide (BNP)-neprilysin (NEP) protein levels increased with age and are risk markers of cardiovascular and cerebrovascular diseases, such as AF and cardioembolic stroke. Aging is also linked to epigenetics, specifically DNA methylation. However, only a few studies have investigated the effect of DNA methylation on the NP system. Thus, the present study aimed to investigate whether the Corin-BNP-NEP protein pathway is involved in the pathogenesis of AF-stroke and CpG methylation in the promoter region of the Corin protein gene has an effect on AF-related ischemic stroke.MethodsA total of 82 patients hospitalized with acute ischemic strokes were enrolled in this study. The differences in clinical information were compared between the AF-stroke (n = 37) and no AF-stroke groups (n = 45). Plasma-soluble Corin and NEP were detected using an ELISA kit. CpG methylation in the promoter region of the gene was assessed by a next-generation sequencing-based bisulfite sequencing polymerase chain reaction (BSP).Results(1) Patients in AF-stroke were older, had higher initial NIHSS score, 90-day mRs, higher D2-dimer, INR, and APTT, and low TG, TC, and HbA1c (all p < 0.05). (2) Serum levels of Corin and BNP in the AF-stroke group were significantly higher than that in the no AF-stroke group (p < 0.05). No significant difference was detected in the serum levels of NEP between the two groups. (3) The levels of CpG methylation in the promoter region of the Corin protein gene in the AF-stroke group was significantly lower than that in the no AF-stroke group (p < 0.05). The CpG sites with maximal methylation differences between the two groups were CORIN:678, CORIN:682, CORIN:694, and CORIN:700.ConclusionThe current findings raise the possibility that the Corin–BNP–NEP protein pathway may be involved in the pathogenesis of AF-related ischemic stroke. Deficient CpG methylation in the promoter region of the Corin protein gene is associated with AF-related ischemic stroke.

A nonsense mutation in MME gene associates with autosomal recessive late-onset Charcot-Marie-Tooth disease.

BackgroundThe genetic cause for the majority of patients with late‐onset axonal form of neuropathies have remained unknown. In this study we aimed to identify the causal mutation in a family with multiple affected individuals manifesting a range of phenotypic features consistent with late‐onset sensorimotor axonal polyneuropathy.MethodsWhole exome sequencing (WES) followed by targeted variant screening and prioritization was performed to identify the candidate mutation. The co‐segregation of the mutation with the phenotype was confirmed by Sanger sequencing.ResultsWe identified a nonsense mutation (c.1564C>T; p.Q522*) in membrane metalloendopeptidase (MME) gene as the cause of the disease condition. The mutation has a combined annotation‐ dependent depletion (CADD) score 45 and predicted to be deleterious based on various algorithms. The mutation was inherited in an autosomal recessive mode and further confirmed to co‐segregate with the disease phenotype in the family and showed to has the required criteria including rarity and deleteriousness to be considered as pathogenic.ConclusionThe MME gene encodes for the membrane bound endopeptidase neprilysin (NEP) which is involved in processing of various peptide substrates. The identified mutation causes a complete loss of carboxy‐terminal region of the NEP protein which contains the zinc binding site and the catalytic domain and thus considered to be a loss‐of‐function mutation. The loss of NEP activity is likely associated with impaired myelination and axonal injury which is hallmark of CMT diseases.

Renin-angiotensin system (RAS) enzymes and placental trophoblast syncytialisation

Placental renin-angiotensin system (RAS) components; prorenin, angiotensinogen, and angiotensin (Ang) II type 1 receptor (AT1R) are upregulated during syncytialisation. This study examined whether angiotensin-converting enzyme (ACE), ACE2 and neprilysin (NEP) are also altered during syncytialisation. Two in vitro models of syncytialisation were used: forskolin-treated BeWo cells and spontaneously syncytialising primary human trophoblast cells. Term placentae and primary trophoblasts had the highest levels of ACE, ACE2 and NEP mRNA. In primary trophoblasts, ACE mRNA levels significantly increased with syncytialisation, ACE2 and NEP mRNA levels decreased. ACE, ACE2 and NEP protein levels and ACE2 activity did not change. Syncytialisation of primary trophoblasts decreased soluble (s)ACE and sNEP but not sACE2 levels. In primary trophoblasts, the balance between the enzymes controlling the two opposing pathways of the RAS was maintained. These findings were unable to be reproduced in BeWo cells. Future studies exploring placental levels of these enzymes in pregnancies complicated by placental insufficiency are warranted.

Ginsenoside F1 Protects the Brain against Amyloid Beta-Induced Toxicity by Regulating IDE and NEP.

Ginsenoside F1, the metabolite of Rg1, is one of the most important constituents of Panax ginseng. Although the effects of ginsenosides on amyloid beta (Aβ) aggregation in the brain are known, the role of ginsenoside F1 remains unclear. Here, we investigated the protective effect of ginsenoside F1 against Aβ aggregation in vivo and in vitro. Treatment with 2.5 μM ginsenoside F1 reduced Aβ-induced cytotoxicity by decreasing Aβ aggregation in mouse neuroblastoma neuro-2a (N2a) and human neuroblastoma SH-SY5Y neuronal cell lines. Western blotting, real-time PCR, and siRNA analysis revealed an increased level of insulin-degrading enzyme (IDE) and neprilysin (NEP). Furthermore, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis confirmed that ginsenoside F1 could pass the blood–brain barrier within 2 h after administration. Immunostaining results indicate that ginsenoside F1 reduces Aβ plaques in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice. Consistently, increased levels of IDE and NEP protein and mRNA were observed after the 8-week administration of 10 mg/kg/d ginsenoside F1. These data indicate that ginsenoside F1 is a promising therapeutic candidate for AD.

Molecular Profiling of Neprilysin Expression and its Interactions with SARS-CoV-2 Spike Proteins to Develop Evidence Base Pharmacological Approaches for Therapeutic Intervention

Introduction: Neprilysin due to its peptidase activity is involved in several physiological and pathological processes. Recently our group has reported the association of neprilysin with angiotensin-converting enzyme 2 (ACE2) network proteins which facilitate the entry of SARS-CoV-2 virus. The potential role of neprilysin beyond its peptidase activity is not known. Materials and Methods: Using the established sequence analysis and molecular docking tools, this study evaluated the molecular profile of neprilysin interaction with SARSCoV- 2 virus proteins. Results: Human neprilysin protein showed a significant sequence similarity with SARS-CoV-2 spike protein, which was further confirmed by observation of considerable interaction in the molecular docking. Human neprilysin protein was also found to additionally interact with SARS-CoV-2 proteins facilitating virus replication. The potential of neprilysin inhibitors (Sacubitril and Sacubitrilat) to interfere with neprilysin and SARS-CoV-2 proteins interactions was assessed. The neprilysin inhibitors showed binding efficacy within therapeutically feasible concentration range (1 to 150 uM). Conclusion: This study while reporting a novel role of neprilysin as potential receptor for SARS-CoV-2 virus, highlights the merit in assessing clinical efficacy of neprilysin inhibitors for the management of SARS-CoV-2 infection.

Mutations in MME gene causing distal hereditary motor neuropathy and MCI-AD

MME gene encodes neprilysin protein(NEP). It is expressed in a wide variety of tissues and it is known to cleave and degrade beta-amyloid. It may have a role in Alzheimer's disease (AD), but it is not yet well understood. Distal hereditary motor neuropathy(dHMN) is a rare cause of neuropathy. MME homozygous mutations cause dHMN. To our knowledge, there are not previous reports of MME dHMN and AD. AIM:Report a patient with dHMN caused by MME homozygous mutation and MCI-AD.

Impact of age, hypercholesterolemia, and the vitamin D receptor on brain endogenous β-amyloid peptide accumulation in mice.

Age, hypercholesterolemia, and vitamin D deficiency are risk factors that increase the brain accumulation of pathogenic β-amyloid peptides (40 and 42), precursors leading to Alzheimer's disease (AD) in humans. The relative changes accompanying aging, high cholesterol, and/or treatment of calcitriol, active vitamin D receptor (VDR) ligand, under normal physiology are unknown. We examined these relative changes in C57BL/6 mice of ages 2, 4-8, and more than 10 months old, which were fed a normal or high fat / high cholesterol diet and treated with calcitriol, active ligand of the vitamin D receptor (0 or 2.5μg/kg ×4, intraperitoneally, every other day to elicit cholesterol lowering in liver). Aβ40 but not Aβ42 accumulation in brain and lower P-glycoprotein (P-gp) and neprilysin protein expressions for Aβ efflux and degradation, respectively, were found to be associated with aging. But there was no trend for BACE1 (β-secretase 1, a cholesterol-sensitive enzyme) toward Aβ synthesis with age. In response to calcitriol treatment, P-gp was elevated, mitigating partially the age-related changes. Although age-dependent decreasing trends in mRNA expression levels existed for Cyp46a1, the brain cholesterol processing enzyme, whose inhibition increases BACE1 and ApoE to facilitate microglia Aβ degradation, mRNA changes for other cholesterol transporters: Acat1 and Abca1, and brain cholesterol levels remained unchanged. There was no observable change in the mRNA expression of amyloid precursor protein (APP) and the influx (RAGE) and efflux (LRP1) transporters with respect to age, diet, or calcitriol treatment. Overall, aging poses as a risk factor contributing to Aβ accumulation in brain, and VDR-mediated P-gp activation partially alleviates the outcome.

Aluminum chloride-induced amyloid β accumulation and endoplasmic reticulum stress in rat brain are averted by melatonin

Accumulation of aluminium (Al) in the brain is known to be a toxic insult that result in neurodegenerative diseases and melatonin is known to have neuroprotective role. The present study was designed to investigate the neuroprotective effects of melatonin for aluminium chloride (AlCl3)-induced neurotoxicity in rats. Twelve-week old male Wistar rats were orally received 175 mg/kg AlCl3 with or without 5 mg/kg melatonin intraperitoneal pretreatment. Group 3 intraperitoneally recieved 5 mg/kg melatonin and group 4 rats were orally treated with saline solution for 8 weeks. A series of behavioral tests, biochemical analysis and expression of AD-associated proteins in the brain were determined after 7 weeks of all treatments. Our results indicated that AlCl3 treatment tends to induce memory and cognitive impairment. However, melatonin treatment attenuated amyloid beta (Aβ) (1–42) level by decreasing β-secretase, augmented low-density lipoprotein receptor-related protein 1, and neprilysin protein expression. Moreover, AlCl3 -induced endoplasmic reticulum (ER) stress and oxidative stress was attenuated by melatonin supplementation. In conclusion, these findings demonstrate a protective role of melatonin against Aβ peptide accumulation, ER stress and oxidative stress in the AlCl3 -treated AD model. Hence, the melatonin supplement might be an alternative way to alleviate the development of AD.

Sex-Specific Modulation of Blood Pressure and the Renin-Angiotensin System by ACE (Angiotensin-Converting Enzyme) 2.

We showed ACE (angiotensin-converting enzyme) 2 is higher in the kidney of male compared with female mice. To further investigate this sex difference, we examined the role of ACE2 in Ang-[1-8] (angiotensin [1-8])-induced hypertension and regulation of the renin-angiotensin system in the kidney of WT (wild type) and Ace2 KO (knockout) mice. Mean arterial pressure rose faster in WT male than WT female mice after Ang-[1-8] infusion. This sex difference was attenuated in ACE2 KO mice. Ang-[1-8] infusion reduced glomerular AT1R (angiotensin type 1 receptor) binding in WT female mice by 30%, and deletion of Ace2 abolished this effect. In contrast, Ang-[1-8] infusion increased glomerular AT1R binding in WT male mice by 1.2-fold, and this effect of Ang-[1-8] persisted in Ace2 KO male mice (1.3-fold). ACE2 also had an effect on renal protein expression of the neutral endopeptidase NEP (neprilysin), the enzyme that catabolizes Ang-[1-10] (angiotensin [1-10]), the precursor of Ang-[1-8]. Ang-[1-8] infusion downregulated NEP protein expression by 20% in WT male, whereas there was a slight increase in NEP expression in WT female mice. Deletion of Ace2 resulted in lowered NEP expression after Ang-[1-8] infusion in both sexes. These findings suggest sex-specific ACE2 regulation of the renin-angiotensin system contributes to female protection from Ang-[1-8]-induced hypertension. These findings have ramifications for the current coronavirus disease 2019 (COVID-19) pandemic, especially in hypertension since ACE2 is the SARS-CoV-2 receptor and hypertension is a major risk factor for poor outcomes.

Protein kinases A and C regulate amyloid-β degradation by modulating protein levels of neprilysin and insulin-degrading enzyme in astrocytes

The pathology of sporadic Alzheimer’s disease is hallmarked by altered signal transduction via the neurotransmitter receptor-G-protein-mediated protein kinase A (PKA) and protein kinase C (PKC) pathways. Because the accumulation of amyloid-β (Aβ) depends on its rates of synthesis and clearance, the metabolic pathway of Aβ in the brain and the entire body warrants exploration. The two major enzymes involved in Aβ degradation in the brain are believed to be the neprilysin and insulin-degrading enzyme (IDE). This study investigated whether PKA and PKC regulate the degradation of Aβ by modulating the protein levels of neprilysin and IDE in astrocytes. Activation of PKA induced a significant decrease in neprilysin protein levels in cultured astrocytes, whereas activation of PKC induced a significant decrease in the protein level of neprilysin and an increase in the protein level of IDE. Following activation of PKC, the reduction of neprilysin was achieved by its secretion into the culture media. Moreover, PKA-activated astrocytes significantly delayed the degradation of exogenous Aβ, whereas PKC-activated astrocytes significantly facilitated its degradation. These results suggest that PKA and PKC regulate Aβ degradation in astrocytes through a decrease in the protein level of neprilysin and an increase in neprilysin secretion and protein levels of IDE, respectively.

Effect of hyperglycemia and rosiglitazone on renal and urinary neprilysin in db/db diabetic mice.

Alteration in renin‐angiotensin system (RAS) has been implicated in the pathophysiology of diabetic kidney disease (DKD). The deleterious actions of angiotensin II (Ang II) could be antagonized by the formation of Ang‐(1–7), generated by the actions of angiotensin‐converting enzyme 2 (ACE2) and neprilysin (NEP). NEP degrades several peptides, including natriuretic peptides, bradykinin, amyloid beta, and Ang I. Although combination of Ang II receptor and NEP inhibitor treatment benefits patients with heart failure, the role of NEP in renal pathophysiology is a matter of active research. NEP pathway is a potent enzyme in Ang I to Ang‐(1–7) conversion in the kidney of ACE2‐deficient mice, suggesting a renoprotective role of NEP. The aim of the study is to test the hypothesis that chronic hyperglycemia downregulates renal NEP protein expression and activity in db/db diabetic mice and treatment with rosiglitazone normalizes hyperglycemia, renal NEP expression, and attenuates albuminuria. Mice received rosiglitazone (20 mg kg−1 day−1) for 10 weeks. Western blot analysis, immunohistochemistry, and enzyme activity revealed a significant decrease in renal and urinary NEP expression and activity in 16‐wk db/db mice compared with lean control (p < .0001). Rosiglitazone also attenuated albuminuria and increased renal and urinary NEP expressions (p < .0001). In conclusion, data support the hypothesis that diabetes decreases intrarenal NEP, which could have a pivotal role in the pathogenesis of DKD. Urinary NEP may be used as an index of intrarenal NEP status. The renoprotective effects of rosiglitazone could be mediated by upregulation of renal NEP expression and activity in db/db diabetic mice.

Retrograde transport of masseter muscle-derived neprilysin to hippocampus

Although the effects of neprilysin (NEP), also called CD10, on the clearance of Alzheimer’s disease (AD)-associated amyloid-β (Aβ) have been reported, NEP is not made in the brain, and the mechanism for the transport of NEP to the brain has not been investigated. Our hypothesis is that muscle packages NEP in exosomes in response to a neuromuscular signal and sends it to the brain via retrograde axonal transport. The masseter muscle (MM) and the trigeminal nerve (TGN) are good candidates for this mechanism by virtue of their proximity to the brain. The aim of this study was to trace the NEP protein from the MM, through the TGN, and to the hippocampus (HPC) in muscle contraction models in vitro and in vivo. NEP expression in mouse tissue lysates was analyzed by RT-PCR and Western blot. Four-week-old mice were perfused to remove blood NEP contamination. The MM expressed substantial levels of NEP protein and mRNA. On the other hand, a remarkably high level of NEP protein was measured in the TGN in the absence of mRNA. NEP protein, without the corresponding mRNA, was also detected in the HPC. These results suggested that the MM derived NEP was taken up by the TGN, which in turn permitted NEP access to the central nervous system and within it the HPC. When the MM was induced to contract by electric stimulation in freshly euthanized mice, NEP protein decreased in the MM in a stimulus time-dependent manner, while that in the TGN and the HPC increased sequentially. Furthermore, NIR-labeled exosomes tracked along the same route. Finally, carbachol induced secretion of exosomal NEP in C2C12-derived myotube cells. These results support our hypothesis that MM-derived NEP is transported along the TGN to reach the HPC following electrical or cholinergic stimulation.