Ischemia-responsive protein 94 is a key mediator of ischemic neuronal injury-induced microglial activation.

Neuroinflammation, especially activation of microglia, the key immune cells in the brain, has been proposed to contribute to the pathogenesis of ischemic stroke. However, the dynamics and the potential mediators of microglial activation following ischemic neuronal injury are not well understood. In this study, using oxygen/glucose deprivation and reoxygenation with neuronal and microglial cell cultures as an in vitro model of ischemic neuronal injury, we set out to identify neuronal factors released from injured neurons that are capable of inducing microglial activation. Conditioned media (CM) from hippocampal and cortical neurons exposed to oxygen/glucose deprivation and reoxygenation induced significant activation of microglial cells as well as primary microglia, evidenced by up-regulation of inducible nitric oxide synthase, increased production of nitrite and reactive oxygen species, and increased expression of microglial markers. Mechanistically, neuronal ischemia-responsive protein 94 (Irp94) was a key contributor to microglial activation since significant increase in Irp94 was detected in the neuronal CM following ischemic insult and immunodepletion of Irp94 rendered ischemic neuronal CM ineffective in inducing microglial activation. Ischemic insult-augmented oxidative stress was a major facilitator of neuronal Irp94 release, and pharmacological inhibition of NADPH oxidase significantly reduced the ischemic injury-induced neuronal reactive oxygen species production and Irp94 release. Taken together, these results indicate that neuronal Irp94 may play a pivotal role in the propagation of ischemic neuronal damage. Continued studies may help identify Irp94 and/or related proteins as potential therapeutic targets and/or diagnostic/prognostic biomarkers for managing ischemia-associated brain disorders.

Alpha-1 antitrypsin inhibits RANKL-induced osteoclast formation and functions.

Osteoporosis is a global public health problem affecting more than 200 million people worldwide. We previously showed that treatment with alpha-1 antitrypsin (AAT), a multifunctional protein with anti-inflammatory properties, mitigated bone loss in an ovariectomized mouse model. However, the underlying mechanisms of the protective effect of AAT on bone tissue are largely unknown. In this study, we investigated the effect of AAT on osteoclast formation and function in vitro. Our results showed that AAT dose-dependently inhibited the formation of RANKL (receptor activator of nuclear factor κB ligand) induced osteoclasts derived from mouse bone marrow macrophages/monocyte (BMM) lineage cells and the murine macrophage cell line, RAW 264.7 cells. In order to elucidate the possible mechanisms underlying this inhibition, we tested the effect of AAT on the gene expression of cell surface molecules, transcription factors, and cytokines associated with osteoclast formation. We showed that AAT inhibited M-CSF (macrophage colony-stimulating factor) induced cell surface RANK expression in osteoclast precursor cells. In addition, AAT inhibited RANKL-induced TNF-α production, cell surface CD9 expression, and dendritic cell-specific transmembrane protein (DC-STAMP) gene expression. Importantly, AAT treatment significantly inhibited osteoclast-associated mineral resorption. Together, these results uncovered new mechanisms for the protective effects of AAT and strongly support the notion that AAT has therapeutic potential for the treatment of osteoporosis.

Alpha 1-antitrypsin therapy mitigated ischemic stroke damage in rats.

Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin (AAT), a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic, antimicrobial, and cytoprotective activities, could be beneficial in stroke. The goal of this study is to test whether AAT can improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial (i.c.) microinjection of endothelin-1. Five to 10 minutes after stroke induction, rats received either i.c. or intravenous delivery of human AAT. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours, the infarct volumes of the human AAT treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (P < .0001 and P < .05, respectively) compared with control rats. Human AAT significantly limited sensory motor system deficits. Human AAT could be a potential novel therapeutic drug for the protection against neurodegeneration after ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models.

The under-appreciated world of the serpin family of serine proteinase inhibitors.

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.

Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors.

Recently developed serotypes of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the use of rAAV vectors for gene therapy. However, host immune responses to the transgene products from different serotypes remain uncharacterized. In the present study, we evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors. In non-obese diabetic (NOD) mice, which have a hypersensitive immunity, rAAV serotype 1 vector (rAAV1-hAAT) induced high levels of both humoral and cellular responses, while rAAV8-hAAT did not. In vitro studies showed that rAAV1, but not rAAV8 vector transduced dendritic cells (DCs) efficiently. In vivo studies indicated that vector transduction of DCs was essential for the immune responses; while the presence of a transgene product (or foreign gene product produced by host cells) was not immunogenic. Intriguingly, preimmunization with rAAV8-hAAT vector or with serum of hAAT transgenic NOD mouse induced immune tolerance to rAAV1-hAAT injection. These results demonstrate the immunogenic differences of rAAV1 and rAAV8 and imply tremendous potential for these vectors in different applications, where an immune response to transgene is to be either elicited or avoided.

Human Alpha 1 Antitrypsin Suppresses NF-κB Activity and Extends Lifespan in Adult Drosophila.

Human alpha 1 antitrypsin (hAAT) is a multifunctional protein that has been shown to have anti-inflammatory and cellular protective properties. While previous studies demonstrated the antiaging potential of hAAT, the mechanism(s) underlying the antiaging effect remain elusive. In this study, we performed a detailed analysis of transcriptomic data that indicated that NF-κB-targeted genes and NF-κB-regulated pathways were selectively inhibited by hAAT treatment. We further showed that the first detectable impact of hAAT treatment was the inhibition of the nuclear activity of NF-κB. Subsequently, hAAT treatment suppressed the mRNA levels of NF-κB-targeted genes, as well as NF-κB itself (P65 and P50), in human senescent cells. Using Drosophila models, we further examined the impact of hAAT on locomotor activity and endurance. Finally, using an adult-specific promotor, we demonstrated that overexpression of hAAT in the late stage of life significantly extended the lifespan of transgenic flies. These results extend the current understanding of the anti-inflammatory function of hAAT.

Human Alpha-1 Antitrypsin Attenuates ENaC and MARCKS and Lowers Blood Pressure in Hypertensive Diabetic db/db Mice.

Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells.

Adipose tissue-derived mesenchymal stem cell-based liver gene delivery.

BACKGROUND & AIMS: The adipose tissue represents an accessible, abundant, and replenishable source of adult stem cells for potential applications in regenerative medicine. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs) with respect to morphology, immune-phenotype, and multiple differentiation capability. In the present study, we investigated the feasibility of AT-MSC-based liver gene delivery for the treatment of alpha 1-antitrypsin deficiency. METHODS: Mouse AT-MSCs were transduced by rAAV vectors and transplanted into the mouse liver. RESULTS: We showed that AT-MSCs can be transduced by recombinant adeno-associated viral vector serotype 1 (rAAV1-CB-hAAT). After transplanting to the mouse liver, ex vivo transduced AT-MSCs expressed the transgene product, human alpha 1-antitrypsin (hAAT). Importantly, serum levels of hAAT were sustained and no anti-hAAT antibody was detected in any recipients. CONCLUSIONS: These results demonstrated that AT-MSCs can be transduced by rAAV vectors, engrafted into recipient livers, contribute to liver regeneration, and serve as a platform for transgene expression without eliciting an immune response. AT-MSC-based gene therapy presents a novel approach for the treatment of liver diseases, such as AAT deficiency.

Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model.

BACKGROUND: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). METHODS: DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA. RESULTS: Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially. CONCLUSION: These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.

Ex vivo transduction and transplantation of bone marrow cells for liver gene delivery of alpha1-antitrypsin.

Adult stem cell-based gene therapy holds several unique advantages including avoidance of germline or other undesirable cell transductions. We have previously shown that liver progenitor (oval) cells can be used as a platform for liver gene delivery of human alpha1-antitrypsin (hAAT). However, this cell source cannot be used in humans for autologous transplantation. In the present study, we tested the feasibility of bone marrow (BM) cell-based liver gene delivery of hAAT. In vitro studies showed that BM cells can be transduced by lentiviral vector (Lenti-CB-hAAT) and recombinant adeno-associated viral vectors (rAAV1-CB-hAAT, and rAAV8-CB-hAAT). Transplantation studies showed that transplanted BM cells homed into liver, differentiated into hepatocytes and expressed hAAT in the liver. Importantly, we showed that transplantation of rAAV8-CB-hAAT vector-transduced BM cells resulted in sustained levels of hAAT in the systemic circulation of recipient mice. These results demonstrated that rAAV vector-mediated BM cell-based liver gene therapy is feasible for the treatment of AAT deficiency and implies a novel therapy for the treatment of liver diseases.

Transgenic Mice Overexpressing Human Alpha-1 Antitrypsin Exhibit Low Blood Pressure and Altered Epithelial Transport Mechanisms in the Inactive and Active Cycles.

Human alpha-1 antitrypsin (hAAT) is a versatile protease inhibitor, but little is known about its targets in the aldosterone-sensitive distal nephron and its role in electrolyte balance and blood pressure control. We analyzed urinary electrolytes, osmolality, and blood pressure from hAAT transgenic (hAAT-Tg) mice and C57B/6 wild-type control mice maintained on either a normal salt or high salt diet. Urinary sodium, potassium, and chloride concentrations as well as urinary osmolality were lower in hAAT-Tg mice maintained on a high salt diet during both the active and inactive cycles. hAAT-Tg mice showed a lower systolic blood pressure compared to C57B6 mice when maintained on a normal salt diet but this was not observed when they were maintained on a high salt diet. Cathepsin B protein activity was less in hAAT-Tg mice compared to wild-type controls. Protein expression of the alpha subunit of the sodium epithelial channel (ENaC) alpha was also reduced in the hAAT-Tg mice. Natriuretic peptide receptor C (NPRC) protein expression in membrane fractions of the kidney cortex was reduced while circulating levels of atrial natriuretic peptide (ANP) were greater in hAAT-Tg mice compared to wild-type controls. This study characterizes the electrolyte and blood pressure phenotype of hAAT-Tg mice during the inactive and active cycles and investigates the mechanism by which ENaC activation is inhibited in part by a mechanism involving decreased cathepsin B activity and increased ANP levels in the systemic circulation.

Combination of alpha-1 antitrypsin and doxycycline suppresses collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT). METHODS: DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control of a tetracycline-dependent promoter. Control groups received doxycycline alone or saline. Macroscopic arthritis development as well as histopathological changes in the joint were evaluated. In addition, the effects of hAAT and doxycycline on lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-induced interleukin (IL)-6 production from mouse fibroblast cells were also determined. RESULTS: Combination therapy significantly reduced arthritis development and progression compared to the control group in respect to macroscopic as well as histopathological changes. Doxycycline and hAAT in combination also inhibited IL-6 expression from LPS-stimulated NIH/3T3 mouse fibroblast cells, indicating a contributing mechanism of arthritis inhibition. CONCLUSIONS: The results obtained in the present study indicate that a combination therapy using AAT and doxycycline holds promising potential as a new therapy for RA.

Regulated hAAT Expression from a Novel rAAV Vector and Its Application in the Prevention of Type 1 Diabetes.

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

Alpha-1-Antitrypsin Ameliorates Pristane Induced Diffuse Alveolar Hemorrhage in Mice.

Diffuse alveolar hemorrhage (DAH) is a fatal complication in patients with lupus. DAH can be induced in B6 mice by an intraperitoneal injection of pristane. Since human alpha-1-antitrypsin (hAAT) is an anti-inflammatory and immuno-regulatory protein, we investigated the protective effect of hAAT against pristane-induced DAH in B6 mice and hAAT transgenic (hAAT-Tg) mice. We first showed that hAAT Tg expression lowers TNF-α production in B cells, as well as CD4+ T cells in untreated mice. Conversely, the frequency of regulatory CD4+CD25+ and CD4+CD25-IL-10+ cells was significantly higher in hAAT-Tg than in B6 mice. This confirmed the anti-inflammatory effect of hAAT that was observed even at steady state. One week after a pristane injection, the frequency of peritoneal Ly6Chi inflammatory monocytes and neutrophils in hAAT-Tg mice was significantly lower than that in B6 mice. Importantly, pristane-induced DAH was completely prevented in hAAT-Tg mice and this was associated with a modulation of anti- to pro-inflammatory myeloid cell ratio/balance. We also showed that treatment with hAAT decreased the severity of DAH in B6 mice. These results showed for the first time that hAAT has a therapeutic potential for the treatment of DAH.

Alpha-1 Antitrypsin Therapy for Autoimmune Disorders.

Autoimmune diseases are conditions caused by an over reactive immune system that attacks self-tissues and organs. Although the pathogenesis of autoimmune disease is complex and multi-factorial, inflammation is commonly involved. Therefore, anti-inflammatory therapies hold potential for the treatment of autoimmune diseases. However, long-term control of inflammation is challenging and most of the currently used drugs have side effects. Alpha-1 antitrypsin (AAT) is an anti-inflammatory protein with a well-known safety profile. The therapeutic potential of AAT has been tested in several autoimmune disease models. The first study using a recombinant adeno-associated viral (rAAV) vector showed that AAT gene transfer prevented the development of type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Subsequent studies showed that treatment with AAT protein prevented and reversed type 1 diabetes. The beneficial effects of AAT treatment have also been observed in other autoimmune disease models such as rheumatoid arthritis and systemic lupus erythematosus. This paper reviews the therapeutic application of AAT and discusses possible mechanisms of action in various autoimmune diseases.

Gene Delivery of Alpha-1-Antitrypsin Using Recombinant Adeno-Associated Virus (rAAV).

The challenge for alpha-1-antitrypsin (AAT also known as SERPINA1) gene therapy is to achieve long term and high levels of AAT production. Recombinant adeno-associated virus (rAAV) vector has several advantages for AAT gene delivery including no viral genes in the vector, no requirement of integration for long-term transgene expression, low immunogenicity, and wide tropism. AAV-mediated AAT gene therapy has been developed and tested in animal models for AAT deficiency, type 1 diabetes, rheumatoid arthritis, and osteoporosis. AAV-mediated AAT gene therapy has also been tested in clinical studies and has shown promising results. Here we describe the methods of rAAV-AAT vector construction and production as well as AAT gene delivery through (1) liver-directed, (2) muscle-directed, and (3) mesenchymal stem cell (MSC)-mediated routes. We will also describe methods for the evaluation of AAT expression for each delivery approach.

In Vivo Analysis of Alpha-1-Antitrypsin Functions in Autoimmune Disease Models.

Alpha-1-antitrypsin (AAT) is a circulating protein, a serpin, with multiple protective functions. Beside the well-known proteinase inhibitory function, which protects the lungs from chronic obstructive pulmonary disease (COPD), many studies have shown that AAT inhibits pro-inflammatory cytokine gene expression and functions. These anti-inflammatory and immune-regulatory properties have led to studies testing the therapeutic effect of AAT in autoimmune disease models. Initially, a study using recombinant adeno-associated viral (rAAV) vector showed that AAT gene therapy prevented type 1 diabetes (T1D) development in a nonobese diabetic (NOD) mouse model. Consequently, several studies confirmed that AAT therapy prevented and reversed T1D. AAT therapy has also been tested and has demonstrated protective effects in a collagen-induced arthritis model and a systemic lupus erythematosus (SLE) mouse model. This chapter describes methods that evaluate AAT functions in autoimmune mouse models.

Alpha 1 Antitrypsin Gene Therapy Extends the Lifespan of Lupus-Prone Mice.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by high levels of pathogenic autoantibodies and tissue damage. Multiple studies showed that dendritic cell (DC) activation plays a critical role in SLE pathogenesis. Human alpha 1 antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory and cytoprotective properties. In this study, we first examined the effects of hAAT on the functions of DCs from lupus-prone mice, and we showed that hAAT treatment efficiently inhibited CpG- (TLR9 agonist) induced activation of bone marrow-derived conventional and plasmacytoid DCs as well as the production of pro-inflammatory cytokines. The hAAT treatment also attenuated DC help for B cell proliferation and immunoglobulin M (IgM) production. We next tested the protective effect of hAAT protein and gene therapy using recombinant adeno-associated virus 8 (rAAV8-CB-hAAT) in a spontaneous lupus mouse model, and we showed that both treatments decreased autoantibody levels. Importantly, rAAV8-CB-hAAT did not induce an immune response to its transgene product (hAAT), but it showed more pronounced therapeutic effects in reducing urine protein levels and extending the lifespan of these mice. These results indicate that AAT has therapeutic potential in the treatment of SLE in humans.

Anti-inflammaging effects of human alpha-1 antitrypsin.

Inflammaging plays an important role in most age-related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha-1 antitrypsin (hAAT) has immune-regulatory, anti-inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti-inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT-expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti-aging effect of hAAT, we monitored the expression of aging-associated genes and found that aging-induced expressions of Relish (NF-ĸB orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA-seq analysis revealed that innate immunity genes regulated by NF-kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti-inflammaging effect in human cells, we treated X-ray-induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL-6 and IL-8, two major factors of senescence-associated secretory phenotype. Consistent with results from Drosophila,RNA-seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA-approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging-related diseases.

Electroacupuncture relieves depression-like symptoms in rats exposed to chronic unpredictable mild stress by activating ERK signaling pathway.

Electroacupuncture (EA) has been shown to alleviate the symptoms associated with major depressive disorder; however, the underlying mechanisms remain unclear. While the mainstay treatment for depression are pharmacological agents that modulate serotonergic and/or noradrenergic activity of the brain, recent data suggest that, neurotrophins may play a larger role in the pathogenesis of depression and may offer better therapeutic potential in alleviating symptoms associated with depression. One downstream target of neurotrophins is the extracellular signal-regulated kinase (ERK)/Mitogen-activated protein kinase (MAPK) cascade, a major mediator of cellular stress often associated with clinical depression. In this study, we assessed whether the efficacy of EA is due to regulation of these novel pathways using an animal model of depression induced by chronic unpredictable mild stress (CUMS). We found that EA stimulation at specific locations, Baihui (GV20), and Yintang (GV29) ameliorated the behavioral responses of CUMS, which included reduced locomotion, decreased sucrose intake and weight loss. Furthermore, EA increased the activation of ERK and ribosomal s6 kinase (RSK) levels under stress. Both the behavioral and biochemical responses to EA were attenuated with administration of ERK inhibitor, suggesting that EA improves depression-like symptoms in stressed rats, in part, by activation of ERK signaling.