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. 2024 Sep 24;7(12):3645–3657. doi: 10.1021/acsptsci.4c00400

Neprilysin-Mediated Amyloid Beta Clearance and Its Therapeutic Implications in Neurodegenerative Disorders

Shailendra K Saxena †,‡,*, Saniya Ansari †,‡, Vimal K Maurya †,‡, Swatantra Kumar †,‡, Deepak Sharma †, Hardeep S Malhotra §, Sneham Tiwari ∥, Chhitij Srivastava ⊥, Janusz T Paweska ‡,#, Ahmed S Abdel-Moneim ∇, Soniya Nityanand †
PMCID: PMC11651204  PMID: 39698259

Abstract

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Neprilysin (NEP) is a neutral endopeptidase, important for the degradation of amyloid beta (Aβ) peptides and other neuropeptides, including enkephalins, substance P, and bradykinin, in the brain, that influences various physiological processes such as blood pressure homeostasis, pain perception, and neuroinflammation. NEP breaks down Aβ peptides into smaller fragments, preventing the development of detrimental aggregates such as Aβ plaques. NEP clears Aβ plaques predominantly by enzymatic breakdown in the extracellular space. However, NEP activity may be regulated by a variety of factors, including its expression and activity levels as well as interactions with other proteins or substances present in the brain. The Aβ de novo synthesis results from the amyloidogenic and nonamyloidogenic processing of the amyloid precursor protein (APP). In addition to Aβ synthesis, enzymatic degradation and various clearance pathways also contribute to the degradation of the monomeric form of Aβ peptides in the brain. Higher production, dysfunction of degradation enzymes, defective clearance mechanisms, intracellular accumulation of phosphorylated tau proteins, and extracellular deposition of Aβ are hallmarks of neurodegenerative diseases. Strategies for promoting NEP levels or activity, such as pharmaceutical interventions or gene therapy procedures, are being studied as possible therapies for neurodegenerative diseases including Alzheimer’s disease. Therefore, in this perspective, we discuss the recent developments in NEP-mediated amyloidogenic and plausible mechanisms of nonamyloidogenic clearance of Aβ. We further highlight the current therapeutic interventions such as pharmaceutical agents, gene therapy, monoclonal antibodies, and stem-cell-based therapies targeting NEP for the management of neurodegenerative disorders.

Keywords: neurodegeneration, neuroprotection, neprilysin, amyloid beta, amyloid precursor protein

1. Introduction

Neurodegenerative disorders are primarily characterized by progressive degeneration of neurons in various brain regions, such as the cortex, hippocampus, and subcortical areas.1 This results in memory impairment and loss of cognitive abilities, along with physiological and behavioral abnormalities.2 The histopathological hallmarks of most neurodegenerative disorders include the formation of neurofibrillary tangles (extracellular β-amyloid plaques and hyperphosphorylated form of the microtubule-associated tau protein) in different regions of the brain.3 Worldwide, millions of individuals are affected by a wide range of neurodegenerative disorders, and these are characterized by a gradual loss of neurons in the peripheral or central nervous system (PNS or CNS).4 The prevalence of neurological disorders, including Alzheimer’s disease, Parkinson’s disease, stroke, and other forms of dementia, has significantly increased worldwide in the last three decades.5 According to the Global Burden of Disease (GBD) 2021, 3.4 billion people experienced a neurological condition in 2021.6

Globally, 55 million individuals were living with dementia in 2020, this figure is projected to nearly double every 20 years, to reach ∼78 million in 2030 and 139 million in 2050, and more than 60% of these cases occur in low- and middle-income countries.7 Approximately, 10 million new cases are reported annually, and the most frequent, 60–70% of cases, are Parkinson’s disease (PD) and Alzheimer’s disease (AD).8,9 Presently, there are already over 35 million cases of AD and 6–10 million cases of PD globally, and it is predicted that this figure will double every 20 years, accounting for over 115 million cases in 2050.10,11

The global prevalence of other neurological conditions includes amyotrophic lateral sclerosis (ALS), commonly referred to as Lou Gehrig’s disease, which is a rare neurological disorder that specifically degrades motor neurons. The global prevalence of ALS is approximately 2−5 cases per 100 000 individuals.12 Although the global prevalence of frontotemporal dementia (FTD) is estimated to be around 15–22 cases per 100 000 individuals under the age of 65, it is less prevalent than Alzheimer’s disease in older populations but rather more prevalent in younger individuals.13 Similarly, an estimated global prevalence of Huntington’s disease ranges from 2.7 to 13.7 per 100 000 individuals.14 Since neurological disorders are the primary cause of disease burden worldwide, they must be addressed through effective and long-term treatment, prevention, and rehabilitation strategies for neurological conditions.

The hallmarks that define neurodegenerative diseases include pathological protein aggregation, inflammation, cytoskeletal abnormalities, aberrant proteostasis, DNA and RNA defects, altered energy metabolism, neuronal cell death, etc.15 Dysregulation of pathological proteins (Aβ, α-syn, and p-tau), production, aggregation, and clearance are the characteristic features of neurological disorders, mainly Alzheimer’s disease and Parkinson’s disease.16,17 More specifically, the aggregation of intracellular neurofibrillary tangles (p-tau) and extracellular neuritic plaques (Aβ) are the hallmarks of AD and related dementias that have crucial roles in the neuropathogenesis and progression of AD.18,19 Clearance pathways of Aβ plaques involve various mechanisms such as enzymatic degradation, phagocytosis by microglia, transport across the blood–brain barrier (BBB), clearance via perivascular pathways, and transport via cerebrospinal fluid (CSF) that is aimed at removing excess Aβ from the brain.20,21 Among all of the clearance mechanisms, enzymatic degradation of Aβ provides selective and specific targeting of Aβ plaques into less toxic fragments and facilitates prompt clearance from the brain. Several enzymes, including neprilysin (NEP), IDE (insulin-degrading enzyme), ECE (endothelin-converting enzyme), and ACE (angiotensin-converting enzyme), play a role in the degradation of Aβ peptides.22,23 Among the enzymatic degradation of Aβ, NEP has gained more popularity nowadays due to its high specificity for Aβ peptides, minimal interaction with off-target molecules, and efficient cleavage of Aβ into less toxic fragments, making NEP the most promising avenue for the development of novel treatments targeting Aβ clearance mechanisms.24,25 Therefore, in this perspective, we discuss the NEP-mediated clearance of Aβ and therapeutic targets for NEP upregulation in defining neurodegenerative diseases, including AD, PD, ALS, FTD, MSA (multisystem atrophy), LBD (Lewy body dementia), and TBI (traumatic brain injury)26−28 (Figure 1).

Figure 1.

Figure 1

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Direct and indirect interactions of Aβ with tau and α-syn proteinopathies. Protein cross-talks that show the interactions between Aβ/α-syn, Aβ/tau, and α-syn/tau, as well as their potential convergence into a combined effect. In neurodegenerative diseases, the accumulation of Aβ, α-syn, and tau protein can be affected by both direct and indirect interactions between protein pairs. Direct interaction is represented by a continuous line, and indirect interaction is represented by a dotted line.

NEP is a zinc metalloprotease and is one of the key enzymes involved in Aβ degradation, breaking down insulin, and metabolizing neurotransmitters in the brain.29 NEP is referred to as neutral endopeptidase, also known as membrane metalloendopeptidase (MME), cluster of differentiation 10 (CD10), and common acute lymphoblastic leukemia antigen (CALLA).30,31 NEP also targets toxic neuropeptides, such as substance P (involved in inflammation and pain perception), bradykinin (controls inflammation and blood pressure), and enkephalins (opioid peptides for mood and pain regulation), in neurodegenerative diseases.32 Among all, Aβ is the preferred substrate for NEP.33 Aβ peptides are normally secreted into the intracellular and extracellular spaces of cells, which are the main target sites of NEP for the degradation of Aβ peptides.34 NEP is a type II membrane glycoprotein; the active site of this glycoprotein is located in intracellular and extracellular spaces into which toxic peptides of Aβ are secreted.35 In addition to having a protective effect of NEP against neurotoxicity, NEP overexpression prevented Aβ accumulation in neurons while NEP downregulation resulted in Aβ aggregation in neurons.36

Different mechanisms allow NEP to degrade Aβ from both the intracellular and extracellular spaces. Extracellular NEP degrades extracellular Aβ (Aβ 1–42), whereas in the secretory pathway intracellular NEP degrades intracellular Aβ (Aβ 1–40) before its secretion.37 In addition, NEP is also found in the endoplasmic reticulum (ER), early Golgi, and other subcellular spaces and can also be found in many different body organs and cells, such as the brain, kidney, intestine, adrenal gland, lung, gut, endothelium cells, fibroblasts, cardiac myocytes, smooth muscle cells, and hematopoietic cells.38 The soluble form of NEP is found in the blood, CSF, and urine. Age-related macular degeneration is also known to be facilitated by Aβ deposition in the retina. NEP activity and levels decrease with aging in both human and rodent brains, suggesting that a reduction in NEP levels may be a factor in the development of later-onset neurodegeneration.39 According to several studies in humans, Drosophila melanogaster, rats, and mice, NEP levels seem to be reduced during aging or mutations in the neprilysin gene (MME). Further, lower NEP activity is linked with increased Aβ peptide accumulation, which is thought to play a role in the development of neurodegenerative diseases like PD and AD.40,41

2. Structure and Function of NEP

NEP is a type II integral membrane protein which includes 742 amino acids (aa’s), with a molecular weight that ranges from 85 to 110 kDa.42 NEP appears in several tissues, including the brain, where it is mainly present in neurons as well as activated astrocytes and microglia. NEP is structurally composed of various domains that facilitate its function.43 A signal peptide directs its entry into the endoplasmic reticulum, which is followed by a transmembrane domain that attaches it to the cell membrane.44 The catalytic site responsible for peptide cleavage is located in the extracellular domain, which includes a substrate-binding region to aid in substrate identification. Along with having a wide range of substrate specificity, each substrate has multiple sites at which NEP can cleave it, but it prefers the amino side of hydrophobic residues.45

A cysteine-rich domain lies next to the catalytic region and may be important in enzyme stability or substrate binding. NEP is made up of three domains and is located at the plasma membrane.46 The larger extracellular catalytic domain has a length of 699 residues, while the short intracellular and transmembrane domains have lengths of 27 and 23 residues. The conserved zinc-binding motif HEXXH is found in the large central cavity of the extracellular catalytic domain, which is important for the proteolysis of various substrates. The zinc ion is coordinated by the two histidine residues, whereas catalysis is directly facilitated by glutamate.47

Neprilysin’s multidomain structure contributes to the breakdown of different peptides, including amyloid beta, which is implicated in AD pathogenesis. All known Aβ-degrading enzymes can break down monomeric Aβ, but their ability to break down oligomeric or fibrillar forms of the peptide is limited.48 On the other hand, NEP seems to be able to break down Aβ fibrils and oligomers. The monomeric Aβ 1–40 is more easily degraded by NEP than Aβ 1–42 is. Further, NEP cleaved only 27% of the monomeric Aβ 1–42 in an in vitro degradation assay, but it cleaved 73% of the monomers of Aβ 1–40.49 The ideal pH for NEP is 6.0 and is prevented by zinc-chelating substances.50

3. Peptide of Amyloid Beta Monomer

Aβ monomers combine to form amyloid fibrils, protofibrils, and oligomers. The soluble form of amyloid oligomers has the potential to migrate throughout the brain, while larger and insoluble forms of amyloid fibrils can aggregate in the form of amyloid plaques in different regions of the brain.51 X-ray crystallography, MD (molecular dynamics), and nuclear NMR (magnetic resonance spectroscopy) were used to determine the three-dimensional solution structures of various forms of the Aβ peptide. Most of the Aβ structural information is derived from MD and NMR.52 Different forms of Aβ peptide are found circulating in CSF, but predominantly 90% Aβ 1–40 is the major Aβ peptide found in CSF while Aβ 1–42 and other peptide lengths of Aβ are minor components. However, plaques mostly consist of Aβ 1–42 and contain smaller amounts of Aβ 1–40.53 It is known that Aβ 1–42 fragments such as Aβ 25–35 can form fibrils in vitro and are considered as highly toxic synthetic derivatives of Aβ peptides. The conformational property analysis of the Aβ 25–35 peptide reveals that, in a lipidic environment, the Aβ peptide forms fibrillar aggregates and behaves like a typical transmembrane helix, which indicates a direct mechanism of neurotoxicity54 (Table 1). Some studies suggested that the Aβ 1–28 peptide was not plaque-competent at any pH but may cause amyloid monomer formation in the early stages of AD.55 However, Aβ 10–35 and Aβ 1–40 have the potential to form plaques, but only in the pH range 5–9.56 Additionally, longer peptides like Aβ 1–42 can form fibrils over a wider pH range (pH >7.4), while shorter Aβ 1–39 peptides cannot form fibrils in this pH range (Figure 2). Some studies also showed a similar relationship between pH and fibril formation in an in vitro assay.57

Table 1. Molecular Docking Analysis of Neprilysin Protein with Aβ Peptides.

Amyloid beta monomer PDB Peptide Docking score Properties
Aβ 1–28 1AMB DAEFRHDSGYEVHHQKLVFFAEDVGSNK –220.55 The peptide folds to form a primarily α-helical structure with a bend centered at residue 12 in membranes like media.
Aβ 1–40 1BA4 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV –259.18 The C-terminal two-thirds of the peptide is an α-helix; without acidic amino acids in the helix, it promotes a helix–coil conformational transition.
Aβ 10–35 1HZ3 YEVHHQKLVFFAEDVGSNKGAIIGLM –218.96 In water Aβ is collapsed into a compact series of loops, strands, and turns and has no α-helical or β-sheet structure.
Aβ 1–42 1IYT DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA –214.14 The 3D structure of Aβ 1–42 shows two helical regions encompassing residues 8–25 and 28–38, connected by a regular type I β-turn.
Aβ 25–35 1QWP GSNKGAIIGLM –176.77 Aβ 25–35 is a highly toxic synthetic derivative of amyloid beta peptides (Aβ peptides) which forms fibrillary aggregates.
Aβ 16–21 2Y29 KLVFFA –180.88 These are microcrystal structures of fiber-forming segments of Aβ, self-complementing pairs of β-sheets termed steric zippers.
Aβ 35–42 2Y3L MVGGVVIA –203.62 Aβ molecules form β-sheet-containing structures that assemble into a variety of polymorphic oligomers, protofibers, and fibers that exhibit a range of lifetimes and cellular toxicities.
Aβ 17–36 5HOW LVFFAEDVGSNKGAIIGLMV –206.95 The X-ray crystallographic structure of Aβ 17–36 is stabilized as the β-hairpin.
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Figure 2.

Figure 2

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Protein–peptide docking analysis of neprilysin and amyloid beta binding. To study the molecular interaction between NEP and selected Aβ peptides, molecular docking was performed using the HDock web server (http://hdock.phys.hust.edu.cn/). Further, the 3D crystal structures of NEP protein and peptides of Aβ were downloaded from the RCSB PDB database (https://www.rcsb.org/) for NEP (PDB ID: 6GID), Aβ 1–28 (PDB ID: 1AMB), Aβ 1–40 (PDB ID: 1BA4), Aβ 10–35 (PDB ID: 1HZ3), Aβ 1–42 (PDB ID: 1IYT), Aβ 25–35 (PDB ID: 1QWP), Aβ 16–21 (PDB ID: 2Y29), Aβ 35–42 (PDB ID: 2Y3L), and Aβ 17–36 (PDB ID: 5HOW) in PDB format. Docking results demonstrated that Aβ 1–40 has a strong binding affinity for NEP proteins among all the Aβ peptides. The ribbon view of NEP shows the binding site for the monomers of Aβ peptide, which display H-bond interactions of target proteins with the metalloproteinase enzymes of NEP.

4. Biological Function of Amyloid Beta

The pathogenesis of neurodegenerative diseases is significantly determined by the expression of integral membrane protein, also termed APP, which is expressed in various tissues, most notably in the synapses of neurons. The monomeric forms of Aβ peptides cleaved from APP.58 The well-known function of APP is as the precursor molecule that is broken down by secretase enzymes (α-, β-, and γ-secretases) to form the 37–49 aa residue peptide of Aβ in amyloidogenic and nonamyloidogenic processing pathways. The amyloidogenic and nonamyloidogenic processing pathways are the two distinct cleavage pathways that play important roles in the proteolytic processing of APP.59 In the nonamyloidogenic processing pathway, an N-terminal fragment of sAPPα (soluble amyloid precursor protein alpha) is released from the cell surface by α-secretase cleavage and leaves an 83 amino acid α-C terminal fragment, CTFα (C83). Moreover, CTFα was digested by γ-secretase, which produced the extracellular nontoxic p3 (3 kDa) from the APP intracellular domain (AICD) and C83.60 In the amyloidogenic processing pathway of APP, β- and γ-secretases cleave the N- and C-terminals of Aβ. Further, the N-terminal sAPPβ and β-C-terminal fragments or 99 amino acids (CTFβ or C99) of APP are produced by β-secretase.61 Following this, CTFβ is processed by γ-secretase at several locations, resulting in fragments cleaved with 43, 45, 46, 48, 49, and 51 amino acids. Further, these fragments are cleaved in endocytic compartments and generate the ultimate forms of Aβ, which are 28 amino acids (Aβ 1–28), 40 amino acids (Aβ 1–40), and 42 amino acids (Aβ 1–42).62 The soluble forms of APP (sAPPα and sAPPβ) are cleaved by α- and β-secretases and released into the extracellular space.63 In both processing pathways, the AICD is released into the cytosol, where it can translocate to the nucleus and potentially regulate the expression of the NEP gene.

When Aβ is released into the extracellular space, it may migrate between several compartments via BBB by the P-glycoprotein efflux pump (Pgp/MDR1/ABCB1) and lipoprotein receptor-related protein (LRP), while RAGE (receptor for advanced glycation end products) primarily mediates the re-entry of circulating Aβ from blood to the brain.64 Aβ monomers aggregate to form protofibrils, oligomers, and amyloid fibrils, and the larger and insoluble forms of amyloid fibrils can further assemble into amyloid plaques. Oligomeric forms of Aβ are soluble and have the potential to spread throughout the brain.65 Ideally, an upregulation of the AICD cannot be determined as a positive inducer for Aβ clearance because it has been associated with the induction of other genes involved in both neuroprotection and neurotoxicity.66

5. Neprilysin-Mediated Clearance of Amyloid Beta

Protein aggregation and clearance are highly controlled parameters that represent both normal physiology and pathological conditions. Changes in the synthesis, accumulation, and clearance of neurotoxic proteins are characterized by specific disease states. In AD, Aβ accumulation and mutations in APP or in the enzymes that produce Aβ are signs of dysregulation in the production or clearance of Aβ.67 It has been demonstrated that the two distinct APP cleavage pathways (nonamyloidogenic and amyloidogenic) take place in different subcellular locations. The amyloidogenic APP processing is thought to primarily occur in endosomes, whereas the nonamyloidogenic pathway occurs at the plasma membrane.68 The nonamyloidogenic APP processing pathway generates the AICD. APP is produced in three major isoforms: APP695, APP770, and APP751.69 The APP751/770 isoforms are expressed at significantly lower levels than APP695. Some studies suggested that not all AICD pools are involved in nuclear signaling.70 Since APP695, the primary precursor of Aβ, sAPPβ, and AICD, occurs mainly in neurons, it suggests that β-secretase has a higher affinity or turnover of this isoform compared with the other isoforms. The main mechanism of APP751 and APP770 processing has been shown by α- and γ-secretase cleavage. Several studies showed that the formation of transcriptionally active AICD is primarily cleaved by β-secretase-mediated APP cleavage in endosomal compartments.71 In addition, APP695 is the predominant APP isoform in neuronal cells but not in non-neuronal cells; this could indicate an AICD-mediated gene regulatory mechanism that is specific to neurons.72

The AICD is produced by the nonamyloidogenic APP processing pathway and is quickly broken down into smaller fragments by various enzymes such as the proteasome, IDE, cathepsin B, and caspase-3.73,74 On the other hand, the AICD produced by processing of amyloidogenic APP, which requires the endocytic pathway, can translocate to the nucleus because it is shorter in distance and causes less peptide degradation. Further, the gene regulatory AFT (AICD, Fe65, Tip60) or AJT (AICD, JIP-1, Tip60) complexes are formed in the cytosol to stabilize the AICD.75 Fe65, Tip60, and MED12 can control the expression of the NEP gene. The AICD is transported to the nucleus and binds to the NEP promoter that expresses high levels of NEP in neuronal cells, while histone deacetylases (HDACs) repress the NEP promoter in neuronal cells that express low levels of NEP.76 These findings validate the role of the AICD in the regulation of NEP and its transport to the nucleus and suggest that the activation of the AICD is dependent on specific cell types and age-related factors (Figure 3). The effect of the AICD on NEP expression has been shown in multiple models by using different approaches that show the AICD and the NEP promoter region directly interact, whereas some suggest they do not. NEP expression both in vivo and in vitro enhances animal cognition and behavior, suggesting that the AICD-dependent regulation of NEP might be a promising therapeutic target.77

Figure 3.

Figure 3

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Possible mechanism of AICD-mediated NEP gene regulation. Two distinct APP cleavage pathways take place in different subcellular locations. (A) In the nonamyloidogenic pathway, α- and γ-secretases cleave at the plasma membrane and generate the AICD. These AICD fragments are then quickly broken down by the IDE, proteasome, and cathepsin-B. (B) The amyloidogenic APP processing pathway primarily takes place in endosomes, where β- and γ-secretase generate the AICD. This AICD can be stabilized by binding to the JIP-1 protein in cytosol and translocated to the nucleus, where the AICD, JIP-1, and Tip60 come together to form AJT complexes. Furthermore, the binding of the AICD to X11α in the cytosol results in an inhibition of AICD-induced transcription. (C) Cleavage sites of NEP within the released Aβ sequence. There are several cleavage sites for NEP, within the Aβ peptide. (D) Regulation of genes involved in neuroprotection and neurotoxicity by the AICD. The AICD increases tau phosphorylation, apoptosis, and Aβ accumulation by upregulating the expression and activity of APP, p53, and GSK3. The AICD also induces NEP gene expression resulting in increased Aβ degradation and reduced Aβ levels.

6. Neprilysin-Based Therapeutic Approach

Several pharmacological and epigenetic strategies have been used to enhance intracellular and extracellular NEP levels. Finding a pharmacological way to specifically increase brain NEP activity provides new therapeutic options for the treatment of neurodegenerative diseases. Therefore, we have discussed several therapeutic interventions such as pharmaceutical agents, gene therapy, monoclonal antibodies, and stem-cell-based therapies targeting NEP that could be used for the management of neurodegenerative diseases (Table 2).

Table 2. NEP-Targeting Drugs Used for the Treatment of Neurodegenerative Diseases.

6.

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6.1. Repurposed Pharmaceutical Agents

6.1.1. Somatostatin (SST)

The well-known neuropeptide SST is expressed throughout the brain and has been linked to several neurodegenerative disorders, such as major depressive disorder, PD, and AD. The reductions of SST and somatostatin receptor (SSTR) levels were found, and infusion of Aβ resulted in a decrease of NEP expression and impairment of somatostatin.78 Additionally, reduced expression of SST is also an early sign of many neurological conditions, such as depression, PD, and AD. Studies suggested that SST upregulates NEP activity through the concerted action with its SSTR2 and SSTR4 receptors.79

6.1.2. Minocycline

Minocycline is a semisynthetic second-generation derivative of tetracycline and has shown neuroprotective properties in cell culture as well as in animal model diseases by preventing microglial activation and neuronal damage. Studies using minocycline concurrently showed increased NEP expression, which reduces brain abnormalities and Aβ levels in AD. Minocycline has blood–brain barrier permeability and can activate the phospholipase-3-kinase (PI3-K)/AKT signaling in the brain leading to the prevention of Aβ aggregation.80

6.1.3. Imatinib

Imatinib (a tyrosine kinase inhibitor) is mainly used as an anticancer drug to treat various cancers, such as gastrointestinal stromal tumors (GISTs) and chronic myeloid leukemia (CML). Imatinib treatment has been shown to reduce secreted Aβ without affecting the cleavage of the intracellular domain of APP by γ-secretase in human neuroblastoma cells. Further, the activity of NEP and AICD levels increased and a decreased Aβ level was found after the imatinib treatment, suggesting it could be proven as a potential therapeutic agent for the management of neurodegenerative conditions like AD and PD.81

6.1.4. Valproic Acid

The epigenetic regulator valproic acid (VPA) has shown neuroprotective properties in neurodegenerative diseases. VPA treatment inhibits histone deacetylases (HDACs), delays neuronal death in degenerating neurons, and also increases NEP expression both in vivo and in vitro, suggesting it may be useful as a therapeutic agent for neurodegenerative disorders and improvement of cognitive functions of the aging brain.82 Recent studies suggest that VPA treatment can increase NEP levels and also its activity in human neuroblastoma cells (SH-SY5Y cells) and rat brains, especially in the hippocampus region.83

6.2. Natural Products

In addition to synthetic repurposing pharmaceutical agents, several natural compounds have also been shown to enhance NEP activity in cell culture and animal models including chrysin, amentoflavone, kaempferol, apigenin, resveratrol, epigallocatechin-3-gallate (EGCg), ginsenoside Rg3, etc.84 Apigenin, kaempferol, and chrysin all showed the same effects as resveratrol and EGCg, but amentoflavone was the most potent booster of NEP activity. Accordingly, it appeared that some flavonols, flavones, and flavonoids also had favorable effects on NEP upregulation. Therefore, several investigations have shown that resveratrol and EGCg have a wide range of health-promoting properties, such as neuroprotection, anti-inflammation, and antioxidant effects.85 More precisely, their therapeutic effects on neurodegenerative diseases seem to be related to the transcriptional and translational regulation of specific genes through the inhibition of histone deacetylase 1 (HDAC1) and activation of the extracellular CREB (cAMP-responsive element binding protein) signaling pathway.86 In previous investigations, NEP was shown to be downregulated in AD mouse models by HDAC1-dependent epigenetic inhibition, which implies that EGCg could increase the NEP level through the CREB signaling pathway.

While natural compounds are often recognized for upregulating NEP activity, some also have the ability to downregulate this enzyme. Researchers have reported that 3,5,3′-triiodothyronine isolated from Bos taurus domesticus acts as an inhibitor of neutral endopeptidase (NEP) activity.87 Additionally, several flavonoids isolated from Epilobium angustifolium, such as oenothein B and quercetin-3-O-glucuronide, have been identified as effective direct inhibitors of NEP.88

6.3. Neprilysin-Based Gene Delivery Therapy

Neprilysin-based gene delivery involves incorporating the NEP-encoding genetic materials into the target cells. Various preclinical studies show promising results in the clearance of Aβ, suggesting gene transfer strategies may be useful in the development of alternative therapies for neurodegenerative diseases. Further studies suggested that NEP-based gene therapy can improve cognitive function and potentially slowdown the progression of neurodegenerative diseases, mainly AD. Although NEP-based gene therapy has promising results in the preclinical model, currently it has several challenges such as target specificity, safety, efficacy, and lack of clinical trial data (Table 3).

Table 3. Role of NEP in Neurodegenerative Pathology.

No Author name Type of study Intervention Potential marker Outcome
PRECLINICAL STUDIES
1 Iwata et al., 2004 mice model gene therapy NEP, IDE, ECE Elevated NEP gene expression reduces Aβ levels in the hippocampus region of NEP-deficient mice.96
2 Poirier et al., 2006 transgenic mice model nucleic acid based therapy NEP Lower incidences of spatial memory deficit and the onset of AD are correlated with higher expression of NEP.97
3 Marr et al., 2003 transgenic mice model gene therapy NEP Neprilysin gene transfer decreases human amyloidopathy.98
4 Iijima-Ando et al., 2008 transgenic Drosophila model gene therapy NEP Human NEP significantly breaks down intraneuronal Aβ1−42 accumulation in transgenic Drosophila brains.99
5 Farris et al., 2007 transgenic mouse model loss of gene NEP, IDE Loss of human NEP function increases the amyloid plaque formation in different regions of the brain and also causes cerebral amyloid angiopathy.100
6 Kanemitsu et al., 2003 cell culture model gene therapy NEP, IDE, ECE Both the monomeric and pathological oligomeric forms of Aβ peptide can be broken down by human NEP.101
7 Huang et al., 2006 transgenic mice model gene therapy NEP Lack of NEP accelerates the formation of extracellular Aβ deposits, synaptic dysfunctions, Aβ angiopathy, and memory impairments.102
8 Leissring et al., 2003 transgenic mice model gene therapy NEP, IDE This study has demonstrated that overexpressing human NEP or IDE in the brains of APP transgenic mice lowers Aβ levels, reduces the amount of amyloid plaque, and delays premature death.103
9 Hemming et al., 2007 ex vivo study gene therapy NEP Extracellular Aβ deposits are reduced when NEP is administered to the brains of APP transgenic mice.104
10 Humpel, 2021 transgenic mice model gene therapy NEP Intranasal administration of collagen-loaded neprilysin reduces Aβ plaque formation.105
11 Spencer et al., 2008 transgenic mice model gene therapy NEP, IDE Long-term neprilysin gene transfer lowers the accumulation of intracellular Aβ.106
12 Iwata et al., 2013 mice model gene therapy NEP Neprilysin expression particularly in endosomes is thought to be useful for the efficient breakdown of Aβ oligomers.107
CLINICAL STUDIES
13 Yasojima et al., 2001 clinical study NA NEP Relationship between neprilysin and molecules which generate Aβ peptide in AD and healthy brains.108
14 de Almeida et al., 2018 clinical study NA NEP The level of NEP is reduced in the serum and CSF of HIV1-subtype patients with AD.109
15 Sorensen et al., 2013 clinical study NA NEP Neprilysin activity likely correlates with CSF-tau and phospho-tau in patients with AD.110
16 Brum et al., 2024 clinical trial sacubitril/valsartan NEP NEP inhibition has an effect on AD plasma biomarkers.111
17 Hellström-Lindahl et al., 2008 postmortem study NA NEP The occurrence and progression of AD are influenced by a decrease in NEP levels.112
18 Miners et al., 2009 postmortem study NA NEP, IDE The reduction in NEP and IDE activity is not the primary cause of Aβ accumulation in AD.113
19 Grimmer et al., 2019 clinical study NA NEP The NEP level is decreased in the patient’s CSF with cognitive impairments.114
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6.3.1. Sindbis Viral Vector

Sindbis viral vectors are derived from the Sindbis virus and are extensively used in gene therapy due to their target-specific delivery of genetic materials to target cells. Sindbis viral vectors have been used as a potential delivery system for the delivery of the NEP gene into neuronal cells, thereby increasing the NEP expression and activity in the brain. The increased NEP activity leads to the enhanced clearance of Aβ plaques in primary cortical neurons, demonstrating that increased NEP activity breaks down Aβ on the cell surface and possibly via a secretory pathway.89

6.3.2. Lentiviral Vector

A Lenti-Nep (lentiviral vector expressing human NEP) has been developed and examined in transgenic models of amyloidosis to investigate the function of NEP in Aβ deposition. This method was chosen over other viral vectors due to the significance that lentiviral vectors are noncytotoxic and have been demonstrated to transduce CNS cells effectively. According to the current study, Lenti-Nep decreased AD-like pathology in transgenic mice.90

6.3.3. Adeno-Associated Viral Vector

NEP was expressed via recombinant AAV vector mediated gene transfer after getting transported axonally to presynaptic sites via afferent projections of neuronal circuits.91 This gene transfer not only prevented the increased Aβ levels in the hippocampal formations of NEP deficient mice but also decreased the increase in young mutant APP transgenic mice.

6.3.4. Borna Disease Viral Vector

A known potentially effective gene therapy for AD is NEP gene delivery, which is a major Aβ-degrading enzyme in the brain. The BoDV vector allows foreign genes to be transduced into the CNS over an extended period. Therefore, the levels of Aβ 1–40 and Aβ 1–42 dramatically dropped when the proteolytic ability of NEP transduced by the BoDV vector was assessed, which suggests that NEP expressed from the BoDV vector is functional in that it breaks down toxic Aβ plaques in the brain.92

6.4. Stem Cell Therapy

Stem cell research has been shown to enhance the knowledge of progress and differentiation and subsequently facilitate the development of therapies for various diseases, including neurodegenerative disorders. Advancement in stem cell therapy could facilitate effective treatment of chronic conditions such as Alzheimer’s disease and Parkinson’s disease.93 Stem cells can also be used as vehicles to deliver substances into the CNS and to directly replace damaged cells. Recent findings suggest that the transplantation of adult mesenchymal stem cells resulted in a reduction of brain Aβ; the result was attributed to elevated levels of NEP mRNA and protein.94 Additionally, utilizing mesenchymal stem cells (hUCB-MSCs) from human umbilical cord blood, NEP expression was elevated in the transplanted cell into the hippocampal regions of APP AD mice, and Aβ plaques in other regions were reduced by the active migration of hUCB-MSCs toward Aβ deposits, However, the exact mechanism is still unclear.95

7. Conclusions

In neurodegenerative conditions, the monomeric form of Aβ peptides promptly aggregates and forms oligomers, protofibrils, and fibrils, which lead to the formation of amyloid plaques in different regions of the brain. Although there are an ample number of studies dealing with the signaling pathways of Aβ synthesis and associated enzymes, the investigation of molecules that regulate Aβ clearance and their mechanistic associations under neurodegenerative conditions is still unclear. Recent studies suggested that NEP is therapeutically significant in Aβ degradation, but there are still certain challenges that prevent its use as a targeted therapy for neurodegenerative disorders. Increased expression of NEP shows a potential treatment strategy for neurodegenerative diseases such as AD, ALS, and PD. Therefore, in the early stages of neurodegenerative diseases, upregulating NEP expression or activity may prevent or delay disease onset. Hence, increased NEP expression and activity can provide new prospects for therapeutic intervention, either by itself or in combination with different strategies.

8. Future Perspectives

Neprilysin’s role in the treatment of neurodegenerative diseases holds significant future potential, particularly in Alzheimer’s disease and Parkinson’s disease. It is important to understand the function of NEP in the Aβ metabolic pathway to design potential treatments for neurodegenerative disorders. The development of novel NEP modulators with selective targeting can minimize side effects and provide optimum therapeutic effects in terms of degrading toxic proteins such as amyloid beta.

Further, targeting multiple pathways involved in neurodegenerative processes like neuroinflammation, synaptic dysfunction, or aggregation of α-synuclein and p-tau with combination therapies involving existing treatment options with NEP modulators may provide synergistic benefits with better patient outcomes. However, small attempts have been made in the development of NEP activators. More emphasis should be given to the design and development of NEP activators, and their safety and efficacy should be evaluated in preclinical and clinical studies. Although a high peripheral level of NEP is associated with cardiac diseases, interdisciplinary coordination is needed between the cardiologist and neurologist. Developing NEP-based therapies for neurodegenerative disorders may face several limitations such as blood–brain barrier permeability, targeted delivery, broad substrate specificity of NEP, short half-life and stability, lack of comprehensive understanding of the mode of action, and lack of efficacy and safety data from clinical trials.

Acknowledgments

The authors are grateful to the Vice Chancellor, King George’s Medical University, Lucknow, India, for the encouragement for this work. S.K.S. was supported by the Indian Council of Medical Research; Department of Health Research, Ministry of Health and Family Welfare, Government of India; and CCRH, Ministry of Ayush, Government of India.

Glossary

Abbreviations

NEP

neprilysin

Aβ

amyloid beta

APP

amyloid precursor protein

CNS

central nervous system

CSF

cerebrospinal fluid

PNS

peripheral nervous system

BBB

blood–brain barrier

IDE

insulin-degrading enzyme

ECE

endothelin-converting enzyme

ACE

angiotensin-converting enzyme

AICD

APP intracellular domain

MME

membrane metalloendopeptidase

PD

Parkinson’s disease

AD

Alzheimer’s disease

ALS

amyotrophic lateral sclerosis

FTD

frontotemporal dementia

MSA

multisystem atrophy

LBD

Lewy body dementia

TBI

traumatic brain injury

SST

somatostatin

BoDV

Borna disease virus vector

HDAC

histone deacetylase

Author Contributions

S.K.S. conceived the idea and planned the study. S.K.S., S.A., V.K.M., and S.K. collected the data, devised the initial draft, reviewed the final draft, and contributed equally to this study as the first author. S.A., V.K.M., S.K., D.S., H.S.M., S.T.,C.S., J.T.P., A.S.A., S.N., and S.K.S. finalized the draft for submission. All authors read and approved the final version of the manuscript.

The authors declare no competing financial interest.

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