Crystal structure of Bak bound to the BH3 domain of Bnip5, a noncanonical BH3 domain-containing protein.

The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.

Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1.

Bak is a critical executor of apoptosis belonging to the Bcl-2 protein family. Bak contains a hydrophobic groove where the BH3 domain of proapoptotic Bcl-2 family members can be accommodated, which initiates its activation. Once activated, Bak undergoes a conformational change to oligomerize, which leads to mitochondrial destabilization and the release of cytochrome c into the cytosol and eventual apoptotic cell death. In this study, we investigated the molecular aspects and functional consequences of the interaction between Bak and peroxisomal testis-specific 1 (Pxt1), a noncanonical BH3-only protein exclusively expressed in the testis. Together with various biochemical approaches, this interaction was verified and analyzed at the atomic level by determining the crystal structure of the Bak-Pxt1 BH3 complex. In-depth biochemical and cellular analyses demonstrated that Pxt1 functions as a Bak-activating proapoptotic factor, and its BH3 domain, which mediates direct intermolecular interaction with Bak, plays a critical role in triggering apoptosis. Therefore, this study provides a molecular basis for the Pxt1-mediated novel pathway for the activation of apoptosis and expands our understanding of the cell death signaling coordinated by diverse BH3 domain-containing proteins.

The Bcl-2 family protein bid interacts with the ER stress sensor IRE1 to differentially modulate its RNase activity.

IRE1 is a transmembrane signalling protein that activates the unfolded protein response under endoplasmic reticulum stress. IRE1 is endowed with kinase and endoribonuclease activities. The ribonuclease activity of IRE1 can switch substrate specificities to carry out atypical splicing of Xbp1 mRNA or trigger the degradation of specific mRNAs. The mechanisms regulating the distinct ribonuclease activities of IRE1 have yet to be fully understood. Here, we report the Bcl-2 family protein Bid as a novel recruit of the IRE1 complex, which directly interacts with the cytoplasmic domain of IRE1. Bid binding to IRE1 leads to a decrease in IRE1 phosphorylation in a way that it can only perform Xbp1 splicing while mRNA degradation activity is repressed. The RNase outputs of IRE1 have been found to regulate the homeostatic-apoptotic switch. This study, thus, provides insight into IRE1-mediated cell survival.

Apoptotic mitochondrial poration by a growing list of pore-forming BCL-2 family proteins.

The pore-forming BCL-2 family proteins are effectors of mitochondrial poration in apoptosis initiation. Two atypical effectors-BOK and truncated BID (tBID)-join the canonical effectors BAK and BAX. Gene knockout revealed developmental phenotypes in the absence the effectors, supporting their roles in vivo. During apoptosis effectors are activated and change shape from dormant monomers to dynamic oligomers that associate with and permeabilize mitochondria. BID is activated by proteolysis, BOK accumulates on inhibition of its degradation by the E3 ligase gp78, while BAK and BAX undergo direct activation by BH3-only initiators, autoactivation, and crossactivation. Except tBID, effector oligomers on the mitochondria appear as arcs and rings in super-resolution microscopy images. The BH3-in-groove dimers of BAK and BAX, the tBID monomers, and uncharacterized BOK species are the putative building blocks of apoptotic pores. Effectors interact with lipids and bilayers but the mechanism of membrane poration remains elusive. I discuss effector-mediated mitochondrial poration.

Effect of UV Stress on the Structure and Function of Pro-apoptotic Bid and Anti-apoptotic Bcl-xl proteins.

Ultraviolet C (UV-C) radiation induces apoptosis in mammalian cells via the mitochondrion-mediated pathway. The Bcl-2 family of proteins are the regulators of the mitochondrial pathway of apoptosis and appears responsive to UV-C radiation. It is unknown how the structure and, effectively, the function of these proteins are directly impacted by UV-C exposure. Here, we present the effect of UV-C irradiation on the structure and function of pro-apoptotic Bid-FL and anti-apoptotic Bcl-xlΔC proteins. Using a variety of biophysical tools, we show that, following UV-C irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV stress than Bid-FL Interestingly, UV-C exposure shows dramatic chemical shift perturbations in consequence of dramatic structural perturbations (α-helix to ß-sheet) in the BH3- binding region, a crucial segment of Bcl-xlΔC. Furter it has been shown that UV-exposed Bcl-xlΔC has reduced efficacy of its interactions with pro-apoptotic tBid.

Design, synthesis and anti-tumor activity studies of novel pyrido[3, 4-d]pyrimidine derivatives.

In order to find high-efficiency and low-toxic anti-tumor drugs, 29 pyrido[3,4-d]pyrimidine compounds were designed, synthesized and evaluated by MTT assay in vitro. The results presented that most of the compounds had good antitumor activities, among which compound 30 had the best anti-tumor activity on MGC803 cells (IC50 = 0.59 µM). Mechanistic studies exhibited that compound 30 inhibited migration of MGC803 and induced apoptosis. It was proved that compound 30 up-regulated expression of Bid and PARP, down-regulated expression of CycD1 by western blot experiments. This study indicated that compound 30 might be served as a lead agent for the treatment of human gastric cancers.

TRAF3/p38-JNK Signalling Crosstalk with Intracellular-TRAIL/Caspase-10-Induced Apoptosis Accelerates ROS-Driven Cancer Cell-Specific Death by CD40.

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.

Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor-mediated extrinsic apoptosis, specifically in cancer cells, and Bid (BH3-interacting domain death agonist) plays an important role in TRAIL-induced apoptosis. Ferroptosis is a newly defined form of regulated cell death known to be distinct from other forms of cell death. However, our previous studies have shown that ferroptosis shares common pathways with other types of programmed cell death such as apoptosis. In this study, we investigated the role of Bid in the crosstalk between the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and TRAIL-induced apoptosis. When human colorectal carcinoma HCT116 cells were treated with the ferroptosis-inducing agents artesunate and erastin in combination with TRAIL, TRAIL-induced activation of caspase-8 was enhanced, and subsequently, the truncation of Bid was increased. Similar results were observed when ovarian adenocarcinoma OVCAR-3 cells were treated with the ferroptotic agents in combination with TRAIL. Results from studies with Bid mutants reveal that the truncation of Bid and the presence of intact BH3 domains are critical for synergistic apoptosis. Nonfunctional Bid mutants were not able to activate the mitochondria-dependent apoptosis pathway, which is required for the conversion of p19 to p17, the active form of caspase-3. These results indicate that Bid plays a critical role in the crosstalk between the ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

BID- and BAX-mediated mitochondrial pathway dominates A-1331852-induced apoptosis in senescent A549 cells.

Recovered senescent tumor cells harbor higher migration and invasion potential, owing to which they play a crucial role in tumor recurrence and drug resistance. The aim of this study was to explore the ability of BH3 mimetics in clearing senescent A549 cells and elucidate their underlying killing mechanism. Doxorubicin-induced cell senescence was determined using augmented senescence-associated beta-galactosidase (SA-ß-Gal) staining and increased P16 expression. CCK-8 and crystal violet staining demonstrated that A-1331852, BH3 mimetic, could kill senescent tumor cells without affecting the proliferating cells. A-1331852 induced caspase-dependent senescent cell death accompanied by nuclear concentration, decreased mitochondrial membrane potential, and cleavage of poly (ADP-ribose) polymerase. Most importantly, A-1331852 upregulated the expression of BID and BAX indicating their role in mediating A-1331852-induced apoptosis in senescent A549 cells. The results of fluorescence resonance energy transfer showed that A-1331852 loosened or even released the binding between BCL-xL and tBID, releasing tBID. In addition, A-1331852 also dissociated the binding between BCL-xL and BAX, eventually leading to BAX oligomerization in the mitochondria, and resulting in apoptosis via the mitochondrial pathway. In conclusion, our data demonstrate for the first time that A-1331852 promotes apoptosis of senescent A549 cells by influencing the interaction between BCL-xL and tBID and that between BCL-xL and BAX.

The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK.

The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX, BAK and BOK, we have previously defined the processes during development that require apoptosis. Rare Bok-/- Bax-/- Bak-/- triple knockout (TKO) mice developed to adulthood and several tissues that were thought to require apoptosis during development appeared normal. This raises the question if all apoptosis had been abolished in the TKO mice or if other BCL-2 family members could act as effectors of apoptosis. Here, we investigated the role of BID, generally considered to link the extrinsic and intrinsic apoptosis pathways, acting as a BH3-only protein initiating apoptosis upstream of BAX and BAK. We found that Bok-/- Bax-/- Bak-/- Bid-/- quadruple knockout (QKO) mice have additional developmental anomalies compared to TKO mice, consistent with a role of BID, not only upstream but also in parallel to BAX, BAK and BOK. Mitochondrial experiments identified a small cytochrome c-releasing activity of full-length BID. Collectively, these findings suggest a new effector role for BID in the intrinsic apoptosis pathway.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.

Structural basis of BAK activation in mitochondrial apoptosis initiation.

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Curcumin, a potential initiator of apoptosis via direct interactions with Bcl-xL and Bid.

Apoptosis is a naturally occurring process during the growth and development of multicellular organisms and is increasingly active during times of cellular stress such as in response to intracellular DNA damage when removal of the host cell is paramount to prevent cancer. Unfortunately, once formed, cancer cells become impervious to apoptosis, creating a desperate need to identify an approach to induce apoptosis in these cells. An attractive option is to focus efforts on developing and locating compounds which activate apoptosis using natural compounds. Curcumin is a natural component in turmeric and is well-known for its pharmacological effects in preventing and combating many ailments and has been shown to decrease the rapid proliferation of a wide variety of tumor cells. However, to date, the apoptotic intermediates and interactions through which curcumin exerts its cytotoxic effects are unknown. Motivated by reports linking the intracellular modulation of the concentrations of Bid and Bcl-xL, following curcumin administration to cancer cells, we set out to probe for potential intermolecular interactions of these proteins with curcumin. Using several biophysical techniques, most notably, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy, we reveal binding interactions of curcumin with both Bcl-xLΔC and full-length Bid (Bid-FL) and prove that this binding is hydrophobically driven and localized to well-known functional regions of each protein. Specifically, our NMR studies show that while Bid-FL interacts with curcumin through its hydrophobic and pore forming helices (α6-α7), Bcl-xLΔC interacts with curcumin via its BH3 binding pocket (α2-α3-α4-α5), a critical region for mediating apoptosis.

BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane.

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6µm-2 to ≃0.1µm-2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

Influence of thyroid hormone in the expression of the marker pro-apoptosis BID, in spite of the predominance of anti-apoptosis activation in intratiroidal lymphocytic infiltration in Hashimoto's thyroiditis.

Cell destruction in Hashimoto's thyroiditis (HT) involves autoantibodies and cytotoxic T lymphocytes. Thyrocytes maintenance occurs by pro-apoptotic, anti-apoptotic and cell proliferation balance. OBJECTIVES: To characterize factors related to the mechanisms of apoptosis and cell proliferation in thyroid cells and intrathyroid lymphocytic infiltrate in HT. METHODS: We assessed lymphocytic infiltrate and thyroid cells from HT and normal thyroid by immunohistochemical analysis of cell proliferation (Ki-67), antiproliferation (p27Kip1), pro-apoptosis (Fas, Fas-ligand, BID) and anti-apoptosis (MCL-1, BCL2) markers. RESULTS: Lymphocytic infiltrate presented BCL2 and MCL-1 higher expression, Ki-67 and p27kip1 balance. Thyrocytes exhibited Fas and FasL balance, higher BID expression; MCL-1, BCL-2, Ki-67 similar to the normal thyroid. T4 and higher lymphocytes BID expression were associated. CONCLUSIONS: In lymphocytic infiltrate predominated anti-apoptosis in relation to pro-apoptosis except for BID. Thyrocytes presented pro-apoptosis and anti-apoptosis balance and cell proliferation similar to normal thyroid. T4-associated BID expression in HT lymphocytes suggests the influence of thyroid hormone as a signal to up-regulate the BID pro-apoptotic protein and thus increase lymphocytic apoptosis rates.

Methylation of Promoters of Apoptosis-Related Genes in Blood Lymphocytes of Workers Exposed to Occupational External Irradiation.

We studied the effect of technogenic radiation on the degree of promoter methylation in genes involved in apoptosis in blood lymphocytes of workers exposed to long-term γ-radiation during their professional activities. Blood samples for the analysis were obtained from 11 conventionally healthy men aged from 54 to 71 years (mean 66 years), workers of the Siberian Group of Chemical Enterprises working experience from 27 to 40 years (mean 30 years); the external exposure dose was 175.88 mSv (158.20-207.81 mSv). In all examined subjects, the degree of methylation of the promoters of apoptosis-related genes ranged from 0.22 to 50.00%. A correlation was found between the degree of methylation of BCLAF1 promoters (p=0.035) with the age of workers, BAX promoters (p=0.0289) with high content of aberrant cells, and APAF1 promoters (p=0.0152) with increased number of dicentric chromosomes. A relationship was found between the dose of external irradiation and the degree of methylation of gene promoters of BAD (p=0.0388), BID (р=0.0426), and HRK (р=0.0101) genes.

The effect of bee bread (Perga) with chemotherapy on MDA-MB-231 cells.

Bee bread (BB) is a bee product like propolis and honey. It is the main food for larvae and bees producing royal jelly in the hive. It also known as Perga. As with other bee products, it is increasingly popular due to its antioxidant properties. The aim of this study was to examine the effects of BB on MDA-MB-231 breast cancer cells and the effects on these cells when administered together with Doxorubicin (DOX) and Cisplatin (CDDP), used in cancer treatment. The proliferation of the cells was determined by applying 5 mg/mL BB together with different concentrations of DOX and CDDP. In addition to these studies, the effect of DOX+BB and CDDP+BB combinations on the migration of MDA-MB-231 cells was determined by the wound healing method. The expression levels of Bid and Bcl-2 were determined by RtqPCR. According to these studies, as expected, BB did not show a significant toxic effect on MDA-MB-231 cells at different concentrations. BB significantly suppressed the effect of DOX and CDDP on the proliferation of MDA-MB-231 cells. BB with DOX and CDDP suppressed the proapoptotic Bid gene while overexpressing the anti-apoptotic Bcl-2 gene, separately. Interestingly, BB blocked the migration of MDA-MB-231 cells by 50% even after 72 h. As a result, BB significantly reduced the toxicity of DOX and CDDP on MDA-MB-231 cells. The most interesting result of the study is that BB prevented the migration of cancer cells.

Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity.

Abnormalities of the tumor vasculature result in insufficient blood supply and development of a tumor microenvironment that is characterized by low glucose concentrations, low extracellular pH, and low oxygen tensions. We previously reported that glucose-deprived conditions induce metabolic stress and promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. In this study, we examined whether the metabolic stress-associated endoplasmic reticulum (ER) stress response pathway plays a pivotal role in the enhancement of TRAIL cytotoxicity. We observed no significant cytotoxicity when human colorectal cancer SW48 cells were treated with various doses of TRAIL (2-100 ng/ml) for 4 h or glucose (0-25 mM) for 24 h. However, a combination of TRAIL and low glucose-induced dose-dependent apoptosis through activation of caspases (-8, -9, and -3). Studies with activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), or death receptor 5 (DR5)-deficient mouse embryonic fibroblasts or HCT116 cells suggest that the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis are involved in the combined treatment-induced apoptosis. Moreover, the combined treatment-induced apoptosis was completely suppressed in BH3 interacting-domain death agonist (Bid)- or Bcl-2-associated X protein (Bax)-deficient HCT116 cells, but not Bak-deficient HCT116 cells. Interestingly, the combined treatment-induced Bax oligomerization was suppressed in PUMA-deficient HCT116 cells. These results suggest that glucose deprivation enhances TRAIL-induced apoptosis by integrating the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis, consequently amplifying the Bid-Bax-associated mitochondria-dependent pathway.

Syringin Prevents Aß25-35-Induced Neurotoxicity in SK-N-SH and SK-N-BE Cells by Modulating miR-124-3p/BID Pathway.

Alzheimer's disease (AD) is a neurodegenerative disorder disease, disturbing people's normal life. Syringin was mentioned to antagonize Amyloid-ß (Aß)-induced neurotoxicity. However, the action mechanism is still not fully elucidated. This study aimed to explore a molecular mechanism of syringin in defending Aß-induced neurotoxicity. SK-N-SH and SK-N-BE cells were treated with amyloid ß-protein fragment 25-35 (Aß25-35) to induce cell neurotoxicity. The injury effects were distinguished by assessing cell viability and cell apoptosis using cell counting kit-8 (CCK-8) assay and flow cytometry assay, respectively. The expression of Cleaved-caspase3 (Cleaved-casp3), B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and BH3 interacting domain death agonist (BID) at the protein level was determined by western blot. The expression of miR-124-3p and BID was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-124-3p and BID was predicted by the online database starBase and confirmed by dual-luciferase reporter assay plus RNA pull-down assay. Aß25-35 treatment inhibited cell viability and induced cell apoptosis, while the addition of syringin recovered cell viability and suppressed cell apoptosis. MiR-124-3p was significantly downregulated in Aß25-35-treated SK-N-SH and SK-N-BE cells, and BID was upregulated. Nevertheless, the addition of syringin reversed their expression. BID was a target of miR-124-3p, and its downregulation partly prevented Aß25-35-induced injuries. Syringin protected against Aß25-35-induced neurotoxicity by enhancing miR-124-3p expression and weakening BID expression, and syringin strengthened the expression of miR-124-3p to diminish BID level. Syringin ameliorated Aß25-35-induced neurotoxicity in SK-N-SH and SK-N-BE cells by regulating miR-124-3p/BID pathway, which could be a novel theoretical basis for syringin to treat AD.