BCL2-associated Athanogene Research Articles

BAG2 and MAPK2 regulate differently on different periods of heat-induced programmed cell death in tomato

High temperature is a significant abiotic stress that affects growth and development of plants. BAG (Bcl-2 associated athanogene) protein family members act as co-chaperones and apoptosis inhibitors in multiple cellular processes. BAG, MAPK (Mitogen-activated protein kinase), and programmed cell death (PCD) play critical roles in plant growth and development, stress response, and disease resistance. In this study, we investigated the interaction of BAG, and MAPK as well as their putative role in PCD under either short or long-term heat stress. We constructed mutants of bag2 and mapk2 in tomato using CRISPR/Cas9. Our results revealed that tomato BAG2 and MAPK2 interacted positively both in vivo and in vitro. In addition, after 3 h of heat stress, the activities of Caspase 3 and antioxidant enzymes, expression levels of Caspase 3 and Caspase 9, and contents of H2O2 in bag2 and mapk2 mutant plants were lower than those in WT (wild type) plants. Moreover, under short-term (3 h) heat stress, the DNA fragmentation phenomena and trypan blue coloration in both mutants were less severe than in WT plants; however, DNA integrities were broken, and the number of dead cells was higher under long-term (24 h) heat stress. Additionally, the electrolyte leakage was increased, but the Fv/Fm (maximum photochemical efficiency) value was decreased in mutants following exposure to heat stress. These results suggested that tomato BAG2 and MAPK2 suppressed short-time heat-induced PCD while promoting long-time heat-induced PCD.

Generation of CRISPR-Cas9 edited human induced pluripotent stem cell line carrying BAG3 V468M mutation in its BAG domain

BCL2-Associated Athanogene 3 (BAG3) gene was identified mutated in patients with dilated cardiomyopathy (DCM), an important cause of heart failure and premature death. BAG3 is a cytoprotective co-chaperonne protein involved in many cellular process with a central role in the maintenance of protostasis. We generated two human induced pluripotent stem cell lines (hiPSc), one carrying the heterozygous, the other the homozygous p.V468M mutation identified in DCM familial cases. All lines expressed pluripotent markers, had normal karyotype, and differentiated into derivatives of the three germ layers. Sudies of hiPSc derived cardiomyocytes will help to understand the role of BAG3 in DCM.

Deciphering Immune Landscape Remodeling Unravels the Underlying Mechanism for Synchronized Muscle and Bone Aging.

Evidence from numerous studies has revealed the synchronous progression of aging in bone and muscle; however, little is known about the underlying mechanisms. To this end, human muscles and bones are harvested and the aging-associated transcriptional dynamics of two tissues in parallel using single-cell RNA sequencing are surveyed. A subset of lipid-associated macrophages (triggering receptor expressed on myeloid cells 2, TREM2+ Macs) is identified in both aged muscle and bone. Genes responsible for muscle dystrophy and bone loss, such as secreted phosphoprotein 1 (SPP1), are also highly expressed in TREM2+ Macs, suggesting its conserved role in aging-related features. A common transition toward pro-inflammatory phenotypes in aged CD4+ T cells across tissues is also observed, activated by the nuclear factor kappa B subunit 1 (NFKB1). CD4+ T cells in aged muscle experience Th1-like differentiation, whereas, in bone, a skewing toward Th17 cells is observed. Furthermore, these results highlight that degenerated myocytes produce BAG6-containing exosomes that can communicate with Th17 cells in the bone through its receptor natural cytotoxicity triggering receptor 3 (NCR3). This communication upregulates CD6 expression in Th17 cells, which then interact with TREM2+ Macs through CD6-ALCAM signaling, ultimately stimulating the transcription of SPP1in TREM2+ Macs. The negative correlation between serum exosomal BCL2-associated athanogene 6 (BAG6) levels and bone mineral density further supports its role in mediating muscle and bone synchronization with aging.

The oomycete-specific BAG subfamily maintains protein homeostasis and promotes pathogenicity in an atypical HSP70-independent manner

Protein homeostasis is vital for organisms and requires chaperones like the conserved Bcl-2-associated athanogene (BAG) co-chaperones that bind to the heat shock protein 70 (HSP70) through their C-terminal BAG domain (BD). Here, we show an unconventional BAG subfamily exclusively found in oomycetes. Oomycete BAGs feature an atypical N-terminal BD with a short and oomycete-specific α1 helix (α1'), plus a C-terminal small heat shock protein (sHSP) domain. In oomycete pathogen Phytophthora sojae, both BD-α1' and sHSP domains are required for P. sojae BAG (PsBAG) function in cyst germination, pathogenicity, and unfolded protein response assisting in 26S proteasome-mediated degradation of misfolded proteins. PsBAGs form homo- and heterodimers through their unique BD-α1' to function properly, with no recruitment of HSP70s to form the common BAG-HSP70 complex found in other eukaryotes. Our study highlights an oomycete-exclusive protein homeostasis mechanism mediated by atypical BAGs, which provides a potential target for oomycete disease control.

Why are aging and stress associated with dementia, cancer, and other diverse medical conditions? Role of pineal melatonin interactions with the HPA axis in mitochondrial regulation via BAG-1

Pineal melatonin and the cortisol awakening response (CAR) are integral aspects of the circadian rhythm. Pineal melatonin release during sleep is proposed to optimize mitochondrial function and dampen any residual oxidant and inflammatory activity. Little is known about CAR, which is generally thought to prepare the body for the coming day, primarily through the activation of the glucocorticoid receptor (GR). Melatonin, like the gut microbiome-derived butyrate, suppresses GR nuclear translocation, indicating that pineal melatonin and night-time butyrate may interact to modulate CAR effects via the GR, including CAR priming of immune and glia cells that underpin the pathogenesis of most medical conditions. Cutting edge research shows that the GR can be chaperoned by bcl2-associated athanogene (BAG)-1 to mitochondria, where GR can have significant and diverse impacts on mitochondrial function. A number of lines of evidence indicate that melatonin indirectly increases BAG-1, including via epigenetic mechanisms, such as derepressing miR-138 inhibition of BAG-1. The dramatic decrease in pineal melatonin production over aging will therefore have significant impacts on GR nuclear translocation, but also possibly the levels of BAG-1 mediated mitochondrial translocation of the GR. This may have dramatic consequences for how CAR ‘prepares the body for the coming day’, via the differential consequence of GR location in the cytoplasm, nucleus or mitochondria, with differential effects in different cell types. The interactions of melatonin/butyrate/BAG-1/GR are especially important in the hypothalamus, where a maintained heightened melatonin concentration occurs over the night due to the direct release of pineal melatonin, via the pineal recess, into the third ventricle. The interaction of melatonin/butyrate/BAG-1/GR will have differential effects in different cell types, thereby altering the intercellular homeostatic interactions in a given microenvironment that will contribute to the pathogenesis of many aging-associated conditions, including neurodegenerative conditions and cancer. This reframes the nature of the circadian rhythm as well as how stress-associated hypothalamus-pituitary-adrenal (HPA) axis may modulate both the pathogenesis and course of diverse medical presentations. This has a number of research and treatment implications across a host of current medical conditions.

Design, synthesis and biological evaluation of novel 2,4-thiazolidinedione derivatives able to target the human BAG3 protein

The Bcl-2-associated athanogene 3 (BAG3) protein plays multiple roles in controlling cellular homeostasis, and it has been reported to be deregulated in many cancers, leading tumor cell apoptosis escape. BAG3 protein is then an emerging target for its oncogenic activities in both leukemia and solid cancers, such as medulloblastoma. In this work a series of forty-four compounds were designed and successfully synthesized by the modification and optimization of a previously reported 2,4-thiazolidinedione derivative 28. Using an efficient cloning and transfection in human embryonic kidney HEK-293T cells, BAG3 was collected and purified by chromatographic techniques such as IMAC and SEC, respectively. Subsequently, through Surface Plasmon Resonance (SPR) all the compounds were evaluated for their binding ability to BAG3, highlighting the compound FB49 as the one having the greatest affinity for the protein (Kd = 45 ± 6 μM) also against the reference compound 28. Further analysis carried out by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) spectroscopy further confirmed the highest affinity of FB49 for the protein. In vitro biological investigation showed that compound FB49 is endowed with an antiproliferative activity in the micromolar range in three human tumoral cell lines and more importantly is devoid of toxicity in human peripheral mononuclear cell deriving from healthy donors. Moreover, FB49 was able to block cell cycle in G1 phase and to induce apoptosis as well as autophagy in medulloblastoma HD-MB03 treated cells. In addition, FB49 demonstrated a synergistic effect when combined with a chemotherapy cocktail of Vincristine, Etoposide, Cisplatin, Cyclophosphamide (VECC). In conclusion we have demonstrated that FB49 is a new derivative able to bind human BAG3 with high affinity and could be used as BAG3 modulator in cancers correlated with overexpression of this protein.

Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

Human adenovirus B7 (HAdV-B7) causes severe acute lower respiratory tract infections in children. However, neither the child-specific antivirals or vaccines are available, nor the pathogenesis is clear. Autophagy, as part of innate immunity, plays an important role in resistance to viral infection by degrading the virus and promoting the development of innate and adaptive immunity. This study provided evidence that HAdV-B7 infection induced complete autophagic flux, and the pharmacological induction of autophagy decreased HAdV-B7 replication. In this process, the host protein Bcl2-associated athanogene 3 (BAG3) mediated autophagy to inhibit the replication of HAdV-B7 by binding to the PPSY structural domain of viral protein pVI through its WW structural domain. These findings further our understanding of the host immune response during viral infection and will help to develop broad anti-HAdV therapies.

An Unbiased Screen Identified the Hsp70-BAG3 Complex as a Regulator of Myosin-Binding Protein C3

Variants in the gene myosin-binding protein C3 (MYBPC3) account for approximately 50% of familial hypertrophic cardiomyopathy (HCM), leading to reduced levels of myosin-binding protein C3 (MyBP-C), the protein product made by gene MYBPC3. Elucidation of the pathways that regulate MyBP-C protein homeostasis could uncover new therapeutic strategies. Toward this goal, we screened a library of 2,426 bioactive compounds and identified JG98, an allosteric modulator of heat shock protein 70 that inhibits interaction with Bcl-2-associated athanogene (BAG) domain co-chaperones. JG98 reduces MyBP-C protein levels. Furthermore, genetic reduction of BAG3 phenocopies treatment with JG-98 by reducing MYBP-C protein levels.. Thus, an unbiased compound screen identified the heat shock protein 70-BAG3 complex as a regulator of MyBP-C stability.

BAG3: An enticing therapeutic target for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a dreadful and fatal disease of unknown etiology, for which no cure exists. Autophagy, a lysosomal cellular surveillance pathway is insufficiently activated in both alveolar epithelial type II cells and fibroblasts of IPF patient lungs. Fine-tuning this pathway may result in the degradation of the accumulated cargo and influence cell fate. Based on our previous data, we here present our view on modulating autophagy via a unique co-chaperone, namely Bcl2-associated athanogene3 (BAG3) in IPF and discuss about how repurposing drugs that modulate this pathway may emerge as a promising novel therapeutic approach for IPF.

Optimal combination of immune checkpoint and senescence molecule predicts adverse outcomes in patients with acute myeloid leukemia

Background High expression of immune checkpoints (ICs) and senescence molecules (SMs) contributes to T cell dysfunction, tumor escape, and progression, but systematic evaluation of them in co-expression patterns and prognosis in acute myeloid leukemia (AML) was lacking. Methods Three publicly available datasets (TCGA, Beat-AML, and GSE71014) were first used to explore the effect of IC and SM combinations on prognosis and the immune microenvironment in AML, and bone marrow samples from 68 AML patients from our clinical center (GZFPH) was further used to validate the findings. Results High expression of CD276, Bcl2-associated athanogene 3 (BAG3), and SRC was associated with poor overall survival (OS) of AML patients. CD276/BAG3/SRC combination, standard European Leukemia Net (ELN) risk stratification, age, and French-American-British (FAB) subtype were used to construct a nomogram model. Interestingly, the new risk stratification derived from the nomogram was better than the standard ELN risk stratification in predicting the prognosis for AML. A weighted combination of CD276 and BAG3/SRC positively corrected with TP53 mutation, p53 pathway, CD8+ T cells, activated memory CD4+ T cells, T-cell senescence score, and Tumor Immune Dysfunction and Exclusion (TIDE) score estimated by T-cell dysfunction. Conclusion High expression of ICs and SMs was associated with poor OS of AML patients. The co-expression patterns of CD276 and BAG3/SRC might be potential biomarkers for risk stratification and designing combinational immuno-targeted therapy in AML. Key Messages High expression of CD276, BAG3, and SRC was associated with poor overall survival of AML patients. The co-expression patterns of CD276 and BAG3/SRC might be potential biomarkers for risk stratification and designing combinational immuno-targeted therapy in AML.

The Bcl-2-associated athanogene gene family in tobacco (Nicotiana tabacum) and the function of NtBAG5 in leaf senescence.

Leaf senescence in tobacco is closely related to leaf maturation and secondary metabolites. Bcl-2-associated athanogene (BAG) family members are highly conserved proteins and play key roles in senescence, growth and development, and resistance to biotic and abiotic stresses. Herein, the BAG family of tobacco was identified and characterized. In total, 19 tobacco BAG protein candidate genes were identified and divided into two classes, class I comprising NtBAG1a-e, NtBAG3a-b, and NtBAG4a-c and class II including NtBAG5a-e, NtBAG6a-b, and NtBAG7. Genes in the same subfamily or branch of the phylogenetic tree exhibited similarities in gene structure and the cis-element on promoters. RNA-seq and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that the expression of NtBAG5c-f and NtBAG6a-b was upregulated in senescent leaves, implying that they play a role in regulating leaf senescence. NtBAG5c was localized in the nucleus and cell wall as a homology of leaf senescence related gene AtBAG5. Further, the interaction of NtBAG5c with heat-shock protein 70 (HSP70) and sHSP20 was demonstrated using yeast two-hybrid experiment. Virus-induced gene silencing indicated that NtBAG5c reduced the lignin content and increased superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) accumulation. In NtBAG5c-silenced plants, the expression of multiple senescence-related genes cysteine proteinase (NtCP1), SENESCENCE 4 (SEN4) and SENESCENCE-ASSOCIATED GENE 12 (SAG12) was downregulated. In conclusion, tobacco BAG protein candidate genes were identified and characterized for the first time.

Regulation of BAG3 localization and sarcomeric protein turnover in the cardiac myocyte.

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone that we have previously shown localizes to the cardiac sarcomere and is critical for proteostasis and maintenance of normal sarcomeric function. Indeed, BAG3 loss in heart failure (HF) results in accumulation of ubiquitinated sarcomeric proteins, and depressed maximum force generating capacity (Fmax). However, how BAG3 is regulated in the cell and how it coordinates sarcomeric protein turnover is unknown. During our analysis of human heart tissue, BAG3 appears as a “doublet”, with one band at 74 kDa (BAG3-L) and a second at a higher 84 kDa (BAG3-H). Previous studies hypothesized the BAG3-H band was due to phosphorylation. To test this, we treated human myocardial tissue with 10 units of alkaline phosphatase for 1 hour, which decreased troponin I phosphorylation by ∼60%, but had no impact on the intensity of BAG3-H or BAG3-L bands, suggesting these two bands are not due to phosphorylation. When we ran purified human BAG3 protein, it ran near the same molecular weight as the BAG3-H band, suggesting the lighter BAG3-L band is the modified version. These results suggest the BAG3-L band has been cleaved at either the N- or C-termini, which would be around 90 amino acids to cause the observed mass shift. Mass spectrometry analysis is being performed to determine the cleavage site and the domains being interrupted by this cleavage. Importantly, the BAG3-H/BAG3-L is higher in the myofilament compared to the cytoplasm of cardiomyocytes, suggesting the heavier form of BAG3 binds with sarcomere with increased affinity. We are also using high resolution live-cell imaging reveal the kinetics of the sarcomeric protein quality control system orchestrated by BAG3. These findings reveal novel regulation of the critical BAG3 protein and provides potential for BAG3-based HF therapies.

Generation of two human iPSC lines with Exon 3 mutations in BCL2-Associated Athanogene 3 (BAG3) from dilated cardiomyopathy patients

Dilated cardiomyopathies (DCM) are one of the main causes of heart failure as one ages. BAG3 is a chaperone protein that is heavily implicated in the development of DCM and speed of progression toward heart failure. Here we generate two human iPSC lines from individuals with mutations in exon 3 of BAG3 and provide validation of their pluripotency and ability to differentiate toward the three primary germ layers. These two cell lines can help our understanding of BAG3 and its role in DCM by providing a good model for BAG3 inactivation and insufficiency.

BAG2 drives chemoresistance of breast cancer by exacerbating mutant p53 aggregate.

Rationale: Chemoresistance is a major challenge in the clinical management of patients with breast cancer. Mutant p53 proteins tend to form aggregates that promote tumorigenesis in cancers. We here aimed to explore the mechanism for the generation of mutant p53 aggregates in breast cancer and assess its role in inducing chemoresistance. Methods: Expression of BCL2-associated athanogene 2 (BAG2) was evaluated by qRT-PCR, western blotting, and immunohistochemistry in breast cancer patient specimens. The significance of BAG2 expression in prognosis was assessed by Kaplan-Meier survival analysis and the Cox regression model. The roles of BAG2 in facilitating the formation of mutant p53 aggregates were analyzed by co-immunoprecipitation, immunofluorescence, and semi-denaturing detergent-agarose gel electrophoresis assays. The effects of BAG2 on the chemoresistance of breast cancer were demonstrated by cell function assays and mice tumor models. Results: In the present study, we found that BAG2 was significantly upregulated in relapse breast cancer patient tissues and high BAG2 was associated with a worse prognosis. BAG2 localized in mutant p53 aggregates and interacted with misfolded p53 mutants. BAG2 exacerbated the formation of the aggregates and recruited HSP90 to promote the propagation and maintenance of the aggregates. Consequently, BAG2-mediated mutant p53 aggregation inhibited the mitochondrial apoptosis pathway, leading to chemoresistance in breast cancer. Importantly, silencing of BAG2 or pharmacological targeting of HSP90 substantially reduced the aggregates and increased the sensitivity of chemotherapy in breast cancer. Conclusion: These findings reveal a significant role of BAG2 in the chemoresistance of breast cancer via exacerbating mutant p53 aggregates and suggest that BAG2 may serve as a potential therapeutic target for breast cancer patients with drug resistance.

BAG3 attenuates tau hyperphosphorylation and gliosis induced by traumatic brain injury

AbstractBackgroundAlzheimer disease (AD) and traumatic brain injury (TBI) are two devastating brain disorders with complex relationships. Growing evidence supports that early or middle life of TBI may be a risk factor for developing late‐life AD and AD‐related dementias (ADRD). Tau hyperphosphorylation and gliosis may serve as a causative link between TBI and AD as well as ADRD. We have recently identified BCL2 associated athanogene 3 (BAG3) as a hub gene of regulating tau protein homeostasis. The protein level of BAG3 is significantly reduced in neurons, but it is increased in astrocytes of human AD compared to controls. We hypothesized that overexpression of BAG3 in neurons would attenuate tau hyperphosphorylation and gliosis induced by TBI.MethodWe injected 500 nL AAV9‐hSYN1‐eGFP‐2A‐hBAG3‐WPRE or AAV9‐hSYN1‐eGFP‐WPRE (control AAV9) into the hippocampal CA1 and DG regions of C57BL6/J and htau knock‐in mice. These mice were subjected to TBI surgery (controlled cortical impact, velocity: 3.00 m/s, depth: 0.8 mm, dwell time: 200 ms) three months post the injection. We then performed behavioral tests (open‐field test, Y‐maze, and Morris Water Maze) on these mice one month after the CCI surgery. Following the behavioral tests, we collected the brain tissues and performed the immunofluorescent staining of ptau (PHF1, pS396/404 tau), BAG3, IBA‐1 (microglia/macrophage marker) and GFAP (astrocyte marker) on fixed mouse brain floating sections.ResultTBI increased the immunoreactivity of PHF1, IBA‐1, GFAP and astrocytic BAG3, whereas reducing neuronal BAG3 compared to sham C57BL6/J and htau knock‐in mice. Furthermore, the immunoreactivity of BAG3 increased, while PHF1, IBA‐1 and GFAP decreased in the ipsilateral hippocampus after the injection of AAV9‐BAG3 compared to those TBI mice injected with control AAVs. Continued immunofluorescent staining and quantification for AAV9‐BAG3‐ and control AAV9‐injected mice is currently ongoing, as is behavior data quantification.ConclusionTBI induces tau hyperphosphorylation, microglia activation and reactive astrocytes, while reducing neuronal BAG3 in C57BL6/J and htau knock‐in mice. Neuronal overexpression of BAG3 can significantly attenuate tau hyperphosphorylation and gliosis induced by TBI. Our data suggests that targeting neuronal BAG3 may be a therapeutic strategy for preventing or reducing intra‐neuronal tau aggregates and gliosis found in TBI and TBI‐associated AD and ADRD.

Genetics of BAG3: A Paradigm for Developing Precision Therapies for Dilated Cardiomyopathies.

Nonischemic dilated cardiomyopathy is a common form of heart muscle disease in which genetic factors play a critical etiological role. In this regard, both rare disease-causing mutations and common disease-susceptible variants, in the Bcl-2-associated athanogene 3 (BAG3) gene have been reported, highlighting the critical role of BAG3 in cardiomyocytes and in the development of dilated cardiomyopathy. The phenotypic effects of the BAG3 mutations help investigators understand the structure and function of the BAG3 gene. Indeed, we report herein that all of the known pathogenic/likely pathogenic variants affect at least 1 of 3 protein functional domains, ie, the WW domain, the second IPV (Ile-Pro-Val) domain, or the BAG domain, whereas none of the missense nontruncating pathogenic/likely pathogenic variants affect the proline-rich repeat (PXXP) domain. A common variant, p.Cys151Arg, associated with reduced susceptibility to dilated cardiomyopathy demonstrated a significant difference in allele frequencies among diverse human populations, suggesting evolutionary selective pressure. As BAG3-related therapies for heart failure move from the laboratory to the clinic, the ability to provide precision medicine will depend in large part on having a thorough understanding of the potential effects of both common and uncommon genetic variants on these target proteins. The current review article provides a roadmap that investigators can utilize to determine the potential interactions between a patient's genotype, their phenotype, and their response to therapeutic interventions with both gene delivery and small molecules.

Abstract 12697: Pharmacological Inhibition of BAG3-HSP70 With the Proposed Breast Cancer Therapeutic JG-98: In-vivo Mice Study

Introduction: The co-chaperone Bcl2-associated athanogene-3 (BAG3), along with its cofactor heat shock protein 70 (HSP70), is fundamental for protein quality control and cell survival in a healthy heart. However, elevated expression of BAG3 is also associated with metastasis in breast and other cancers, and small molecules such as JG-98 that disrupt BAG3-HSP70 binding have been shown to reduce cancer cell proliferation. We previously showed JG-98 is cardiotoxic in neonatal rat ventricular myocytes. Hypothesis: As BAG3-HSP70 is fundamental for autophagic protein turnover, JG-98 will reduce tumor size but have cardiotoxic effects. Methods: BT474 Resistant breast cancer cells were injected bilaterally into mammary fat pads of female athymic nude mice. When tumors reached 25 mm 2 , mice received a baseline echocardiography and were randomized to intraperitoneal injections of either PBS/DMSO vehicle or 3 mg/kg JG-98 twice weekly for 3 weeks (n = 4/group) or 6 weeks (n=10/group). Tumor growth was monitored using calipers and echocardiography was performed prior to sacrifice. Results: After 3 weeks, tumor volume increased by ~44% in Vehicle treated mice but decreased by 17% in JG-98 treated mice, effectively halting tumor growth. However, cardiac echocardiography did not reveal any functional or structural differences between the vehicle and JG-98 treated mice after either 3 or 6 weeks. JG-98 did result in dysregulation of BAG3 and its interactome in the LV. JG-98 slightly increased BAG3 levels in the LV at 3 weeks, but slightly decreased levels at 6 weeks. This agrees with data showing acute stress upregulates BAG3, but chronic stress downregulates it. Similarly, we found altered protein expression of the BAG3 binding partners HSBP8, HSPB5, and HSP70. Conclusions: JG-98 had no overt cardiotoxic effects, suggesting the adult heart may be resistant to disrupted BAG3-HSP70. However, we also observed dysregulated protein levels in the BAG3-mediated autophagy pathways that could precede dysfunction with more chronic treatment. Furthermore, mutations in BAG3 or altered expression levels could sensitize individuals to JG-98 treatment. Further work is necessary to understand the cardiac impact of cancer therapeutics that target BAG3.

Abstract 11262: Effect of Active Cancer/Cachexia on the Development of Cardiac Dysfunction in Mice

Introduction: Cancer is independently associated with the alteration of cardiac function prior to cardiotoxic chemotherapy (CCT) exposure. Similar to cancer associated cachexia (CAC), the elevation and the deleterious role of IL-6 in plasma was associated with a reduced cardiac function in heart failure (HF) patients subpopulation. Cancer cells manipulate BCL-2-associated athanogene 3 (BAG3)-HSP70-regulated pathways in tumor cells, which is a key regulator of protein turnover and contractility in cardiomyocyte. Hypothesis: Here, we aimed to characterize the progression of cardiac dysfunction and the expression of BAG3 and HSP70 in tumor-bearing mice. Methods: Colon-26 adenocarcinoma cells (C26; n=22) with/without shIL-6 (C26 shIL-6; n=22) were injected subcutaneously adult male BALB/c mice. Control mice were injected with PBS (n=13). Echocardiographic examinations and invasive hemodynamic measurements ( in vivo and ex vivo using isolated working hearts system) were performed at 10 (early) and 20 (late) days post injection, respectively. The expression of BAG3 and Hsp70 were determined by Western blot. Results: The tumor size was comparable between the cancer groups. However, only C26 group showed a significant loss of subcutaneous fat and skeletal muscle (p<0.05, respectively), confirming cachectic condition. Echocardiography results show a tendency to decline of ejection fraction at the early phase (p~0.08 vs Control), and turned significance lower at late stage (p<0.05 vs Control) in tumor-bearing mice. In line with that, invasive hemodynamic and isolated working heart measurements confirmed LV systolic and diastolic dysfunction (late stage, p<0.05 vs Control, respectively). Interestingly, heart rate and aortic flow were predominantly declined in cachectic animals (p<0.05 vs Control). Importantly, cardiac dysfunction was associated with a significant reduction in both BAG3 and Hsp70 in the myocardium independently of cachexia. Conclusions: Cancer rather than CAC is a main driver for the development of cardiac contractile dysfunction prior to CCT exposure. In addition, our data suggest that targeting BAG3-Hsp70 complex in the cardiomyocytes may provide a novel strategy to improve the cancer associated cardiac dysfunction.

BAG3 promotes autophagy and suppresses NLRP3 inflammasome activation in Parkinson’s disease

Neuroinflammation mediated by microglia plays a key role in the pathogenesis of Parkinson's disease (PD), and our previous studies showed this was significantly inhibited by enhanced autophagy. In the autophagy pathway, Bcl2-associated athanogene (BAG)3 is a prominent co-chaperone, and we have shown BAG3 can regulate autophagy to clear the PD pathogenic protein α-synuclein. However, the connection between BAG3 and microglia mediated neuroinflammation is not clear. In this study, we explored whether BAG3 regulated related neuroinflammation and its original mechanism in PD. An inflammatory model of PD was established by injecting adeno-associated virus (AAV)-BAG3 into the bilateral striatum of C57BL/6 male mice to induce overexpression of BAG3, followed by injection of lipopolysaccharide (LPS). The striatum was extracted at 3 days after injection of LPS for Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and immunohistochemical staining was performed at 21 days after injection. At the same time, LPS was used to induce activation of BV2 cells to verify the effect of BAG3 in vitro. Overexpression of BAG3 reduced LPS-induced pyroptosis by reducing activation of caspase-1, the NOD-like receptor family, and the pyrin domain-containing 3 (NLRP3) inflammasome, and by release of interleukin (IL)-1β and tumor necrosis factor (TNF)-α. The LPS-induced inflammatory environment inhibits autophagy, and overexpression of BAG3 can restore autophagy, which may be the mechanism by which BAG3 reduces neuronal inflammation in PD. Our results demonstrate BAG3 promotes autophagy and suppresses NLRP3 inflammasome formation in PD.

LncRNA TUG1 Promoted Stabilization of BAG5 by Binding DDX3X to Exacerbate Ketamine-Induced Neurotoxicity.

As a clinically widely used anesthetic, ketamine (KET) has been reported to cause neurotoxicity in patients. Our work aimed to probe the function of long-chain non-coding RNA taurine-upregulated gene 1 (lncRNA TUG1) in KET-induced neurotoxicity. HT22 cells were subjected to KET to build the cell model. 3-(4, 5-Dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay was employed to determine cell viability. Additionally, cell apoptosis was evaluated by flow cytometry. The binding relationships among TUG1, DEAD-box RNA helicase 3X (DDX3X), and Bcl-2-associated athanogene 5 (BAG5) were verified by RIP and RNA pull-down assays. Cell viability was impaired and cell apoptosis was increased in KET-treated HT22 cells accompanied by increased TUG1, DDX3X, and BAG5 expressions. TUG1 knockdown dramatically enhanced cell viability and repressed the of KET-induced apoptosis in HT22 cells, while TUG1 overexpression presented the opposite effects. In addition, we found that TUG1 promoted DDX3X expression via directly binding with DDX3X. As expected, DDX3X overexpression abolished the palliative effect of TUG1 knockdown on KET-induced neurotoxicity. Further research proved that TUG1 increased the stability of BAG5 through interacting with DDX3X. Finally, as expected, the moderating effect of TUG1 knockdown on KET-induced neuron injury was abolished by BAG5 overexpression. Taken together, TUG1 promoted BAG5 expression by binding DDX3X to exacerbate KET-induced neurotoxicity.