Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.     

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

Background

Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer''s β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.

Methodology

Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.

Conclusions

The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer''s disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.     

Proteomic Profiling of γ-Secretase Substrates and Mapping of Substrate Requirements

The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes.     

γ-Secretase Processing and Effects of γ-Secretase Inhibitors and Modulators on Long Aβ Peptides in Cells

Understanding how different species of Aβ are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aβx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aβ fusion protein expression constructs designed to generate peptides from Aβ1–38 to Aβ1–55 and C99 (CTFβ) were transfected into cells, and Aβ production was assessed. Secreted and cell-associated Aβ were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aβ peptides from 1–38 to 1–55 were readily detected in the cells and as soluble full-length Aβ proteins in the media. Aβ peptides longer than Aβ1–48 were efficiently cleaved by γ-secretase and produced varying ratios of Aβ1–40:Aβ1–42. γ-Secretase cleavage of Aβ1–51 resulted in much higher levels of Aβ1–42 than any other long Aβ peptides, but the processing of Aβ1–51 was heterogeneous with significant amounts of shorter Aβs, including Aβ1–40, produced. Two PSEN1 variants altered Aβ1–42 production from Aβ1–51 but not Aβ1–49. Unexpectedly, long Aβ peptide substrates such as Aβ1–49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aβ substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aβ, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.     

Glu-333 of Nicastrin Directly Participates in ��-Secretase Activity

γ-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for γ-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in γ-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in γ-secretase activity. By normalizing to the active pool of γ-secretase with two separate methods, we establish that γ-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by γ-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because γ-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in γ-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.The brains of Alzheimer disease patients are characterized by dense neuritic plaques that consist of the insoluble β-amyloid peptide (Aβ)² and neurons containing neurofibrillary tangles of the Tau protein (1, 2). The Aβ peptide is produced via the sequential proteolysis of APP by β- and γ-secretase (3). γ-secretase is a multisubunit complex consisting of at least four proteins: presenilin (PS), NCT, APH-1, and PEN-2, all of which are necessary and sufficient for activity (4–9). The formation of the γ-secretase complex is tightly controlled, with an ordered assembly of subunits coupled to spatial restriction (10). It is believed that the last step of the complicated γ-secretase maturation and activation process involves in cis endoproteolysis of the PS holoprotein (11–13). It is this form of γ-secretase with PS in its N- and C-terminal fragments (NTF and CTF, respectively) that represents the fully mature, proteolytically active enzyme.γ-Secretase is a unique protease that cleaves within the lipid bilayer a large number of Type 1 single transmembrane-spanning proteins that vary widely in their sequence and size (14–16). In a previous report, we demonstrated that NCT functions as a substrate receptor for γ-secretase (4). In that report, we showed that NCT recruits substrates that have had their large extracellular domains first removed by an upstream protease in a process termed “ectodomain shedding.” This process generates a new, short extracellular stub with a free N terminus, which is required for proteolysis by γ-secretase. We also established that Glu-333 of NCT participates in activity within the larger context of the DYIGS and peptidase-like (DAP) domain, which shares distant homology to amino- and carboxypeptidases. A recent study by Chávez-Gutiérrez et al. (17) confirmed that mutations at the equivalent rodent residue impair γ-secretase. However, the authors attributed the reduction in activity to a role for Glu-333 in γ-secretase maturation but not directly in activity per se. Although a role for NCT and Glu-333 in γ-secretase assembly and maturation is consistent with our early work (4, 18, 19), the authors'' conclusion that mature γ-secretase complexes containing the Glu-333 mutant NCT are fully active presents a challenge to the model that NCT is a receptor for γ-secretase substrates in mature, active enzyme. Although PS-NTF or -CTF alone is an adequate measure of active γ-secretase complexes, Chávez-Gutiérrez et al. (17) measured specific activity by normalizing γ-secretase products to the sum of PS1-CTF and PEN-2 presumably due to the levels of PS-NTF/CTF by themselves being at the detection limit of Western blotting with electrochemiluminescence (ECL). Such an approach has caveats, as normalizing to the sum of PS1 and PEN-2 does not represent a measurement of the intrinsic activity per single, active enzyme; rather, this mode of normalization instead skews the data to minimize the effects of the mutations, especially when compounded with the unreliability of ECL measurement at the detection limit of Western blotting. Indeed, normalizing to the amount of mature, active γ-secretase in a rigorous, quantitative manner would be necessary to accurately compare the intrinsic activities of wild-type and mutant enzymes.In this study we used two γ-secretase reconstitution methods, including one that bypasses endoproteolysis and two separate normalization approaches to demonstrate that γ-secretase complexes containing NCT-E333A are indeed intrinsically less active than wild-type NCT. We show that this mutant is deficient in its ability to directly bind to γ-secretase substrates. Moreover, we confirm our observations with a second γ-secretase substrate, C83, which is itself the physiological product of α-secretase cleavage of APP. We also examine a series of substrate truncation mutants and find that γ-secretase can cleave substrates that lack the entire extracellular domain, provided that such substrates also contain a free N-terminal amine. Taken together, we conclude that Glu-333 participates directly in activity after γ-secretase complex maturation. Finally, we put forth a model wherein the dual role of Glu-333 in γ-secretase maturation and substrate recognition could be explained in the context of NCT being a substrate receptor. In this model Glu-333 partakes in the recruitment of not only the ectodomain-shed Type 1 membrane proteins but also of the intracellular loop of PS for its endoproteolysis, a hallmark event of γ-secretase maturation and activation.     

The mechanism of γ-Secretase dysfunction in familial Alzheimer disease

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain‐of‐toxic‐function, mechanism. However, many PSEN mutations paradoxically impair γ‐secretase and ‘loss‐of‐function’ mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ε‐cleavage function is not generally observed among FAD mutants. On the other hand, γ‐secretase inhibitors used in the clinic appear to block the initial ε‐cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase‐like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.     

β-Secretase (BACE-1) inhibitory effect of biflavonoids

Here, we describe amentoflavone-type biflavonoids, which were isolated from natural sources and were found to inhibit β-secretase (BACE-1). The structure–activity relationship was studied, and compounds 1–8, 10, 17, and 18 showed BACE-1 inhibitory activity. Among these compounds, 2,3-dihydroamentoflavone 17 and 2,3-dihydro-6-methylginkgetin 18 exhibited potent inhibitory effects with IC₅₀ values of 0.75 and 0.35 μM, respectively.     

Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

Background

A key player in the development of Alzheimer''s disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active γ-secretase in the affected organ of AD, namely the brain.

Principal Findings

We show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy.

Significance

The information about the subcellular localization of γ-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active γ-secretase.     

S-Palmitoylation of ��-Secretase Subunits Nicastrin and APH-1

Proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases generates β-amyloid (Aβ) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Aβ production. Previously, we and others reported that the four integral subunits of the γ-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of γ-secretase subunits. We report that in cultured cells and in brain the γ-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 is S-palmitoylated at Cys¹⁸² and Cys²⁴⁵. S-Palmitoylation-defective nicastrin and APH-1 form stable γ-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for γ-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Aβ40, Aβ42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate γ-secretase processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that cause dementia. This debilitating disorder is pathologically characterized by the cerebral deposition of 39–42 amino acid peptides termed Aβ, which are generated by proteolytic processing of amyloid precursor protein (APP)² by β- and γ-secretases (1, 2). The β-site APP cleavage enzyme 1 cleaves full-length APP within its luminal domain to generate a secreted ectodomain leaving behind a C-terminal fragment (β-CTF). γ-Secretase cleaves β-CTF within the transmembrane domain to release Aβ and APP intracellular C-terminal domain (AICD). γ-Secretase is a multiprotein complex, comprising at least four subunits: presenilins (PS1 and PS2), nicastrin, APH-1, and PEN-2 for its activity (3). PS1 is synthesized as a 42–43-kDa polypeptide and undergoes highly regulated endoproteolytic processing within the large cytoplasmic loop domain connecting putative transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and C-terminal fragments (CTF) by an uncharacterized proteolytic activity (4). This endoproteolytic event has been identified as the activation step in the process of PS1 maturation as it assembles with other γ-secretase subunits (3). Nicastrin is a heavily glycosylated type I membrane protein with a large ectodomain that has been proposed to function in substrate recognition and binding (5), but this putative function has not been confirmed by others (6). APH-1 is a seven-transmembrane protein encoded by two human or three rodent genes that are alternatively spliced (7). Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for γ-secretase processing of APP, a type I membrane protein, termed p23 (also referred toTMP21), was recently identified as a γ-secretase component that modulates γ-secretase activity and regulates secretory trafficking of APP (8, 9).A growing number of type I integral membrane proteins has been identified as γ-secretase substrates within the last few years, including Notch1 homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin receptor DCC, and others (10). Mounting evidence suggests that APP processing occurs within cholesterol- and sphingolipid-enriched lipid rafts, which are biochemically defined as detergentresistant membrane microdomains (DRM) (11, 12). Previously we reported that each of the γ-secretase subunits localizes in lipid rafts in post-Golgi and endosome membranes enriched in syntaxin 6 (13). Moreover, loss of γ-secretase activity by gene deletion or exposure to γ-secretase inhibitors results in the accumulation of APP CTFs in lipid rafts indicating that cleavage of APP CTFs likely occurs in raft microdomains (14). In contrast, CTFs derived from Notch1, Jagged2, N-cadherin, and DCC are processed by γ-secretase in non-raft membranes (14). The mechanisms underlying association of γ-secretase subunits with lipid rafts need further clarification to elucidate spatial segregation of amyloidogenic processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as a potential mechanism for regulating raft association, stability, intracellular trafficking, and function of several cytosolic and transmembrane proteins (15–17). S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety to certain cysteine residues through thioester linkage. Cysteines close to transmembrane domains or membrane-associated domains in non-integral membrane proteins are preferred S-palmitoylation sites, although no conserved motif has been identified (18). Palmitoylation modifies numerous neuronal proteins, including postsynaptic density protein PSD-95 (19), a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors (20), nicotinic α7 receptors (21), neuronal t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin (22, 23), neuronal growth-associated protein GAP-43 (24), protein kinase CLICK-III (CL3)/CaMKIγ (25), β-secretase (26), and Huntingtin (27). Although palmitoylation can occur in vitro without the involvement of an enzyme, a family of palmitoyltransferases that specifically catalyze S-palmitoylation has been identified (28, 29).In this study, we have identified S-palmitoylation of γ-secretase subunits nicastrin and APH-1, and characterized its role on DRM association, protein stability, and γ-secretase enzyme activities. We show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 at Cys¹⁸² and Cys²⁴⁵. Mutagenesis of palmitoylation sites results in increased degradation of nascent nicastrin and APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured cells overexpression of S-palmitoylation-deficient nicastrin and APH-1 does not modulate γ-secretase processing of APP or other substrates.     

γ-Secretase inhibitors and modulators

γ-Secretase is a fascinating, multi-subunit, intramembrane cleaving protease that is now being considered as a therapeutic target for a number of diseases. Potent, orally bioavailable γ-secretase inhibitors (GSIs) have been developed and tested in humans with Alzheimer's disease (AD) and cancer. Preclinical studies also suggest the therapeutic potential for GSIs in other disease conditions. However, due to inherent mechanism based-toxicity of non-selective inhibition of γ-secretase, clinical development of GSIs will require empirical testing with careful evaluation of benefit versus risk. In addition to GSIs, compounds referred to as γ-secretase modulators (GSMs) remain in development as AD therapeutics. GSMs do not inhibit γ-secretase, but modulate γ-secretase processivity and thereby shift the profile of the secreted amyloid β peptides (Aβ) peptides produced. Although GSMs are thought to have an inherently safe mechanism of action, their effects on substrates other than the amyloid β protein precursor (APP) have not been extensively investigated. Herein, we will review the current state of development of GSIs and GSMs and explore pertinent biological and pharmacological questions pertaining to the use of these agents for select indications. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Modulators of γ-Secretase Activity Can Facilitate the Toxic Side-Effects and Pathogenesis of Alzheimer's Disease

Background

Selective modulation of different Aβ products of an intramembrane protease γ-secretase, could be the most promising strategy for development of effective therapies for Alzheimer''s disease. We describe how different drug-candidates can modulate γ-secretase activity in cells, by studying how DAPT affects changes in γ-secretase activity caused by gradual increase in Aβ metabolism.

Results

Aβ 1–40 secretion in the presence of DAPT shows biphasic activation-inhibition dose-response curves. The biphasic mechanism is a result of modulation of γ-secretase activity by multiple substrate and inhibitor molecules that can bind to the enzyme simultaneously. The activation is due to an increase in γ-secretase''s kinetic affinity for its substrate, which can make the enzyme increasingly more saturated with otherwise sub-saturating substrate. The noncompetitive inhibition that prevails at the saturating substrate can decrease the maximal activity. The synergistic activation-inhibition effects can drastically reduce γ-secretase''s capacity to process its physiological substrates. This reduction makes the biphasic inhibitors exceptionally prone to the toxic side-effects and potentially pathogenic. Without the modulation, γ-secretase activity on it physiological substrate in cells is only 14% of its maximal activity, and far below the saturation.

Significance

Presented mechanism can explain why moderate inhibition of γ-secretase cannot lead to effective therapies, the pharmacodynamics of Aβ-rebound phenomenon, and recent failures of the major drug-candidates such as semagacestat. Novel improved drug-candidates can be prepared from competitive inhibitors that can bind to different sites on γ-secretase simultaneously. Our quantitative analysis of the catalytic capacity can facilitate the future studies of the therapeutic potential of γ-secretase and the pathogenic changes in Aβ metabolism.     

Discovery of a novel sulfonamide-pyrazolopiperidine series as potent and Efficacious γ-Secretase Inhibitors

Discovery of a series of pyrazolopiperidine sulfonamide based γ-secretase inhibitors and its SAR evolution is described. Significant increases in APP potency on the pyrazolopiperidine scaffold over the original N-bicyclic sulfonamide scaffold were achieved and this potency increase translated in an improved in vivo efficacy.     

Inhibition of Notch Signaling by a γ-Secretase Inhibitor Attenuates Hepatic Fibrosis in Rats

Notch signaling is essential to the regulation of cell differentiation, and aberrant activation of this pathway is implicated in human fibrotic diseases, such as pulmonary, renal, and peritoneal fibrosis. However, the role of Notch signaling in hepatic fibrosis has not been fully investigated. In the present study, we show Notch signaling to be highly activated in a rat model of liver fibrosis induced by carbon tetrachloride (CCl₄), as indicated by increased expression of Jagged1, Notch3, and Hes1. Blocking Notch signaling activation by a γ-secretase inhibitor, DAPT, significantly attenuated liver fibrosis and decreased the expression of snail, vimentin, and TGF-β1 in association with the enhanced expression of E-cadherin. The study in vitro revealed that DAPT treatment could suppress the EMT process of rat hepatic stellate cell line (HSC-T6). Interestingly, DAPT treatment was found not to affect hepatocyte proliferation in vivo. In contrast, DAPT can inhibit hepatocyte apoptosis to some degree. Our study provides the first evidence that Notch signaling is implicated in hepatic fibrogenesis and DAPT treatment has a protective effect on hepatocytes and ameliorates liver fibrosis. These findings suggest that the inhibition of Notch signaling might present a novel therapeutic approach for hepatic fibrosis.