The Antiproteinopathy, Antioxidant, and Antiapoptotic Effects of Methylene Blue and 4-Phenylbutyric Acid Alone, and in Combination on Familial Alzheimer's Disease PSEN1 I416T Cholinergic-Like Neurons.

Familial Alzheimer's disease (FAD) is a chronic neurological condition that progresses over time. Currently, lacking a viable treatment, the use of multitarget medication combinations has generated interest as a potential FAD therapy approach. In this study, we examined the effects of 4-phenylbutyric acid (4-PBA) and methylene blue (MB) either separately or in combination on PSEN1 I416T cholinergic-like neuron cells (ChLNs), which serve as a model for FAD. We found that MB was significantly efficient at reducing the accumulation of intracellular Aß, phosphorylation of TAU Ser202/Thr205, and increasing Δψm, whereas 4-PBA was significantly efficient at diminishing oxidation of DJ-1Cys106-SH, expression of TP53, and increasing ACh-induced Ca2+ influx. Both agents were equally effective at blunting phosphorylated c-JUN at Ser63/Ser73 and activating caspase 3 (CASP3) into cleaved caspase 3 (CC3) on mutant cells. Combination of MB and 4-PBA at middle (0.1, 1) concentration significantly reduced iAß, p-TAU, and oxDJ-1 and augmented the ACh-induced Ca2+ influx compared to combined agents at low (0.05, 0.5) or high (0.5, 5) concentration. However, combined MB and 4-PBA were efficient only at dropping DJ-1Cys106-SO3 and increasing ACh-induced Ca2+ inward in mutant ChLNs. Our data show that the reagents MB and 4-PBA alone possess more than one action (e.g., antiamyloid, antioxidant, anti-TAU, antiapoptotic, and ACh-induced Ca2+ influx enhancers), that in combination might cancel or diminish each other. Together, these results strongly argue that MB and 4-PBA might protect PSEN1 I416T ChLNs from Aß-induced toxicity by working intracellularly as anti-Aß and anti-Tau agents, improving Δψm and cell survival, and extracellularly, by increasing ACh-induced Ca2+ ion influx. MB and 4-PBA are promising drugs with potential for repurposing in familial AD.

4-Phenylbutyric acid suppresses psoralen-induced hepatotoxicity by inhibiting ERS and reestablishing mitochondrial fusion-fission balance in mice.

Psoralen is a main active molecule of the traditional Chinese herb medicine Fructus Psoraleae. Our previous studies have shown that psoralen induced liver injury through the endoplasmic reticulum stress (ERS) signaling pathways. In this article, we studied whether the ERS inhibitor, 4-phenylbutyrate acid (4-PBA) could inhibit the liver toxicity caused by psoralen, and explored the underlying mechanisms. Mice were given the solvent, 20mg/kg, 40mg/kg, 80mg/kg of psoralen, or 80mg/kg of psoralen plus 4-PBA for 14 days. We found that 4-PBA significantly reduced the serum LDH and liver tissue MDA level, increased the activities of SOD and CAT, reduced liver weight and coefficient, repaired histopathological damage, and inhibited hepatocytes apoptosis induced by psoralen. RNA-seq transcriptomics found that except for the endoplasmic reticulum, the mitochondria was severely affected by psoralen. And genes involved in mitochondrial fusion, apoptosis, protein folding, and autophagy were found differently expressed in the psoralen group. Further studies found that 4-PBA inhibited the overexpression of GRP78 and CHOP, increased the Bcl-2/Bax ratio, and reduced the expression of Caspase-3. Moreover, 4-PBA reduced the overexpression of mitochondrial fission protein DRP1, increased the expression of fusion proteins Mfn-2 and OPA1, but has no inhibitory effects on autophagy proteins Atg5 or LC3A/B. In conclusion, 4-PBA inhibited ERS and reestablished mitochondrial fusion-fission balance, thereby blocking cell apoptosis, oxidative stress, and mitochondrial dysfunction, thus prevented against psoralen-induced hepatotoxicity.

Role of histone deacetylases and their inhibitors in neurological diseases.

Histone deacetylases (HDACs) are zinc-dependent deacetylases that remove acetyl groups from lysine residues of histones or form protein complexes with other proteins for transcriptional repression, changing chromatin structure tightness, and inhibiting gene expression. Recent in vivo and in vitro studies have amply demonstrated the critical role of HDACs in the cell biology of the nervous system during both physiological and pathological processes and have provided new insights into the conduct of research on neurological disease targets. In addition, in vitro and in vivo studies on HDAC inhibitors show promise for the treatment of various diseases. This review summarizes the regulatory mechanisms of HDAC and the important role of its downstream targets in nervous system diseases, and summarizes the therapeutic mechanisms and efficacy of HDAC inhibitors in various nervous system diseases. Additionally, the current pharmacological situation, problems, and developmental prospects of HDAC inhibitors are described. A better understanding of the pathogenic mechanisms of HDACs in the nervous system may reveal new targets for therapeutic interventions in diseases and help to relieve healthcare pressure through preventive measures.

The Derlin-1-Stat5b axis maintains homeostasis of adult hippocampal neurogenesis.

Adult neural stem cells (NSCs) in the hippocampal dentate gyrus continuously proliferate and generate new neurons throughout life. Although various functions of organelles are closely related to the regulation of adult neurogenesis, the role of endoplasmic reticulum (ER)-related molecules in this process remains largely unexplored. Here we show that Derlin-1, an ER-associated degradation component, spatiotemporally maintains adult hippocampal neurogenesis through a mechanism distinct from its established role as an ER quality controller. Derlin-1 deficiency in the mouse central nervous system leads to the ectopic localization of newborn neurons and impairs NSC transition from active to quiescent states, resulting in early depletion of hippocampal NSCs. As a result, Derlin-1-deficient mice exhibit phenotypes of increased seizure susceptibility and cognitive dysfunction. Reduced Stat5b expression is responsible for adult neurogenesis defects in Derlin-1-deficient NSCs. Inhibition of histone deacetylase activity effectively induces Stat5b expression and restores abnormal adult neurogenesis, resulting in improved seizure susceptibility and cognitive dysfunction in Derlin-1-deficient mice. Our findings indicate that the Derlin-1-Stat5b axis is indispensable for the homeostasis of adult hippocampal neurogenesis.

Sustained feeding of a diet high in fat resulted in a decline in the liver's insulin-degrading enzyme levels in association with the induction of oxidative and endoplasmic reticulum stress in adult male rats: Evaluation of 4-phenylbutyric acid.

The current study explored the impact of high fat diet (HFD) on hepatic oxidative and endoplasmic reticulum (ER) stress and its insulin degrading enzyme (IDE) content with the injection of 4-phenyl butyric acid (4-PBA) in adult male rats. Following the weaning period, male offspring were distributed among six distinct groups. The corresponding diet was used for 20 weeks, subsequently 4-PBA was administered for three consecutive days. Plasma glucose and insulin levels, HOMA-ß (homeostasis model assessment of ß-cell), hepatic ER and oxidative stress biomarkers and IDE protein content were assessed. Long-term ingestion of HFD (31 % cow butter) induced oxidative and ER stress in the liver tissue. Accordingly, a rise in the malondialdehyde (MDA) content and catalase enzyme activity and a decrease in the glutathione (GSH) content were detected within the liver of the HFD and HFD + DMSO groups. Consumption of this diet elevated the liver expression of binding immunoglobulin protein (BIP) and C/enhancer-binding protein homologous protein (CHOP) levels while reduced its IDE content. The HOMA-ß decreased significantly. The injection of the 4-PBA moderated all the induced changes. Findings from this study indicated that prolonged HFD consumption led to a reduction in plasma insulin levels, likely attributed to pancreatic ß cell malfunction, as evidenced by a decline in the HOMA-ß index. Also, the HFD appears to have triggered oxidative and ER stress in the liver, along with a decrease in its IDE content.

Protective Effects and Mechanisms of Inhibiting Endoplasmic Reticulum Stress on Cold Seawater Immersion Combined with Hemorrhagic Shock.

Purpose: Cold seawater immersion aggravates hemorrhagic shock-induced homeostasis imbalance and organ dysfunction, leading to increased mortality. Previous studies have shown that treatments targeting oxidative stress and mitochondrial dysfunction have limited efficacy for cold seawater immersion combined with hemorrhagic shock (SIHS). Thus, the mechanisms responsible for SIHS need further investigation. Methods and Results: Data from the hemorrhagic shock transcriptome and cold seawater immersion targets used for bioinformatics analysis revealed the involvement of endoplasmic reticulum stress (ERS) in SIHS occurrence and progression. Based on these findings, the effects and possible mechanism of inhibiting ERS in SIHS rats were investigated. SIHS causes a lethal triad and impairment of vital organ function, leading to death. Compared to lactated Ringer's solution, the ERS inhibitor 4-phenylbutyric acid (PBA)significantly ameliorated acidosis and coagulopathy and protected vital organ function while prolonging survival and the golden treatment time. Through target screening and validation, 7 targets were identified for the ERS inhibitor PBA for the treatment of SIHS, among which S1PR1, MMP8 and CFTR may play more important roles. Conclusion: ERS plays a crucial role in the progression of SIHS. Inhibition of ERS caused by SIHS alleviates the lethal triad, protects organ function, and prolongs survival and the golden treatment time. The ERS inhibitor PBA may be an effective therapeutic measure for treating SIHS.

Development of 4-phenylbutyric acid microsponge gel formulations for the treatment of lewisite-mediated skin injury.

Lewisite, a chemical warfare agent, causes skin blisters, erythema, edema, and inflammation, requiring mitigation strategies in case of accidental or deliberate exposure. 4-phenyl butyric acid (4-PBA), a chemical chaperone, reduces endoplasmic reticulum stress and skin inflammation. The study aimed to encapsulate 4-PBA in microsponges for effective, sustained delivery against lewisite injury. Porous microsponges in a topical gel would potentially sustain delivery and improve residence time on the skin. Microsponges were developed using the quasi-emulsion solvent diffusion method with Eudragit RS100. Optimized formulation showed 10.58%w/w drug loading was incorporated in a carboxymethylcellulose (CMC) and Carbopol gel for in vitro release and permeation testing using dermatomed human skin. A sustained release was obtained from all vehicles in the release study, and IVPT results showed that compared to the control (41.52 ± 2.54 µg/sq.cm), a sustained permeation profile with a reduced delivery was observed for microsponges in PBS (14.16 ± 1.23 µg/sq.cm) along with Carbopol 980 gel (12.55 ± 1.41 µg/sq.cm), and CMC gel (10.09 ± 1.23 µg/sq.cm) at 24 h. Optimized formulation showed significant protection against lewisite surrogate phenyl arsine oxide (PAO) challenged skin injury in Ptch1+/-/SKH-1 hairless mice at gross and molecular levels. A reduction in Draize score by 29%, a reduction in skin bifold thickness by 8%, a significant reduction in levels of IL-1ß, IL6, and GM-CSF by 54%, 30%, and 55%, respectively, and a reduction in apoptosis by 31% was observed. Thus, the translational feasibility of 4-PBA microsponges for effective, sustained delivery against lewisite skin injury is demonstrated.

Implementation of a Multi-Functional-Group Strategy for Enhanced Performance of Perovskite Solar Cells through the Incorporation of 3-Amino-4-Phenylbutyric Acid Hydrochloride.

Reducing the defect density of perovskite films during the crystallization process is critical in preparing high-performance perovskite solar cells (PSCs). Here, a multi-functional molecule, 3-phenyl-4-aminobutyric acid hydrochloride (APH), with three functional groups including a benzene ring, ─NH3 + and ─COOH, is added into the perovskite precursor solution to improve perovskite crystallization and device performance. The benzene ring increases the hydrophobicity of perovskites, while ─NH3 + and ─COOH passivate defects related to I- and Pb2+, respectively. Consequently, the power conversion efficiency (PCE) of the optimal device increased to 24.65%. Additionally, an effective area of 1 cm2 with a PCE of 22.45% is also prepared using APH as an additive. Furthermore, PSCs prepared with APH exhibit excellent stability by 87% initial PCE without encapsulation after exposure at room temperature under 25% humidity for 5000 h and retaining 70% of initial PCE after aging at 85 °C in an N2 environment for 864 h.

4-phenylbutyric acid improves sepsis-induced cardiac dysfunction by modulating amino acid metabolism and lipid metabolism via Comt/Ptgs2/Ppara.

INTRODUCTION: Cardiac dysfunction after sepsis the most common and severe sepsis-related organ failure. The severity of cardiac damage in sepsis patients was positively associated to mortality. It is important to look for drugs targeting sepsis-induced cardiac damage. Our previous studies found that 4-phenylbutyric acid (PBA) was beneficial to septic shock by improving cardiovascular function and survival, while the specific mechanism is unclear. OBJECTIVES: We aimed to explore the specific mechanism and PBA for protecting cardiac function in sepsis. METHODS: The cecal ligation and puncture-induced septic shock models were used to observe the therapeutic effects of PBA on myocardial contractility and the serum levels of cardiac troponin-T. The mechanisms of PBA against sepsis were explored by metabolomics and network pharmacology. RESULTS: The results showed that PBA alleviated the sepsis-induced cardiac damage. The metabolomics results showed that there were 28 metabolites involving in the therapeutic effects of PBA against sepsis. According to network pharmacology, 11 hub genes were found that were involved in lipid metabolism and amino acid transport following PBA treatment. The further integrated analysis focused on 7 key targets, including Comt, Slc6a4, Maoa, Ppara, Pparg, Ptgs2 and Trpv1, as well as their core metabolites and pathways. In an in vitro assay, PBA effectively inhibited sepsis-induced reductions in Comt, Ptgs2 and Ppara after sepsis. CONCLUSIONS: PBA protects sepsis-induced cardiac injury by targeting Comt/Ptgs2/Ppara, which regulates amino acid metabolism and lipid metabolism. The study reveals the complicated mechanisms of PBA against sepsis.

PBA alleviates cadmium-induced mouse spermatogonia apoptosis by suppressing endoplasmic reticulum stress.

OBJECTIVE: Endoplasmic reticulum (ER) stress mediates Cd-caused germ cell apoptosis in testis. The effects of 4-phenylbutyric acid (PBA), a classical chaperone, were investigated on Cd-induced apoptosis in mouse GC-1 spermatogonia cells. METHODS: The cells were pretreated with PBA before Cd exposure. TUNEL and flow cytometry assays were applied to determine apoptosis. Some key biomarkers of ER stress were analyzed using RT-PCR and western blot. RESULTS: as expected, the apoptotic cells exposed to Cd apparently increased. The mRNA and protein expression levels of GRP78 and ATF6α, were elevated in the Cd groups. Additional experiments displayed that Cd notably increased IRE1α and JNK phosphorylation, and upregulated XBP-1 mRNA and protein expression. Moreover, p-eIF2α and CHOP expressions were clearly elevated in the Cd groups. Interestingly, PBA almost completely inhibited ER stress and protected spermatogonia against apoptosis induced by Cd. CONCLUSION: PBA alleviated Cd-induced ER stress and spermatogonia apoptosis, and may have the therapeutic role in Cd-induced male reproductive toxicity.

Tri-ortho-cresyl phosphate induces hepatic steatosis by mTOR activation and ER stress induction.

Tri-ortho-cresyl phosphate (TOCP), an organophosphorus compound (OP), which is widely used as plasticizer, flame retardant and other industrial products, has been reported to cause multiple toxicities including neurotoxicity and reproductive toxicity. However, it remains to be elusive whether TOCP induces hepatotoxicity. The purpose of this study was to investigate the effect of TOCP on hepatocytes and the lipid metabolism in particular. The adult mice were given a single dose of TOCP (800 mg/kg, p.o.) and the histological changes in liver tissue and lipid content in serum were determined. The results showed that more vacuoles and lipid droplets were observed in the liver of the mice exposed to TOCP. And triglyceride concentrations in serum and liver tissue significantly increased. However, the histopathological changes of the liver and the elevated triglyceride levels in the exposed mice can be reversed by endoplasmic reticulum (ER) stress inhibitor 4-phenylbutyric acid and mTOR signal inhibitor rapamycin. It was also found that the changes of expression levels of the biomarkers of ER stress and mTOR signaling pathway, such as GRP78, CHOP, and p-mTOR, in the exposed mice were consistent with those observed in the cultured primary hepatocytes treated with the same chemicals. These results showed that TOCP activated mTOR signal and ER stress to induce de novo lipid synthesis, which led to the hepatic steatosis in mouse.

Chemical Chaperone 4-PBA Mitigates Tumor Necrosis Factor Alpha-Induced Endoplasmic Reticulum Stress in Human Airway Smooth Muscle.

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells' phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 µM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.

4-Phenylbutyric acid may prevent mouse ovarian and uterine damage due to procymidone-induced alteration of circRNA Scar and circZc3h4 levels by controlling excessive unfolded protein response.

Procymidone (PCM) below the no-observed-adverse-effect-level (NOAEL) has previously been proven to induce ovarian and uterine damage in adolescent mice due to its raised circRNA Scar, decreased circZc3h4, and overactivated unfolded protein response (UPR). Also, 4-phenylbutyric acid (4-PBA) inhibits histone deacetylase and endoplasmic reticulum stress, reduces UPR, improves metabolism, and ensures homeostasis within the endoplasmic reticulum. In this study, 20, 40 and 80 mM of 4-PBA were utilized respectively to intervene the damage caused by 1.0 × 10-5 M PCM to ovaries and uterus in vitro culture. Besides, 100 mg/kg /d 4-PBA was intraperitoneally injected to female adolescent mice before, during and after oral administration of 100 mg/kg /d PCM for prevention and cure to observe tissue changes in the ovaries and uteri, and levels of circRNA Scar, circZc3h4 and UPR members. Our findings demonstrated that in vitro experiments, all doses of 4-PBA could inhibit ovarian and uterine damage caused by PCM, and the effect of 80 mM was especially noticeable. In the in vivo experiments, the best results were obtained when PCM was given with simultaneous 4-PBA intervention, i.e., minimal ovarian and uterine damage. Both in vivo and in vitro, 4-PBA in the ovary and uterus resulted in decreased circRNA Scar levels, increased circZc3h4 abundance, and moderately elevated levels of UPR members. So, it is suggested that 4-PBA moderately activates UPR, partially or completely antagonizing the elevated circRNA Scar and decreased circZc3h4 and consequently preventing PCM-induced ovarian and uterine damage effectively in adolescent mice.

Endoplasmic reticulum stress and alterations of peroxiredoxins in aged hearts.

Aging-related cardiovascular disease is influenced by multiple factors, with oxidative stress being a key contributor. Aging-induced endoplasmic reticulum (ER) stress exacerbates oxidative stress by impairing mitochondrial function. Furthermore, a decline in antioxidants, including peroxiredoxins (PRDXs), augments the oxidative stress during aging. To explore if ER stress leads to PRDX degradation during aging, young adult (3 mo.) and aged (24 mo.) male mice were studied. Treatment with 4-phenylbutyrate (4-PBA) was used to alleviate ER stress in young adult and aged mice. Aged hearts showed elevated oxidative stress levels compared to young hearts. However, treatment with 4-PBA to attenuate ER stress reduced oxidative stress in aged hearts, indicating that ER stress contributes to increased oxidative stress in aging. Moreover, aging resulted in reduced levels of peroxiredoxin 3 (PRDX3) in mitochondria and peroxiredoxin 4 (PRDX4) in myocardium. While 4-PBA treatment improved PRDX3 content in aged hearts, it did not restore PRDX4 content in aged mice. These findings suggest that ER stress not only leads to mitochondrial dysfunction and increased oxidant stress but also impairs a vital antioxidant defense through decreased PRDX3 content. Additionally, the results suggest that PRDX4 may contribute an upstream role in inducing ER stress during aging.

Deoxynivalenol induces endoplasmic reticulum stress-associated apoptosis via the IRE1/JNK/CHOP pathway in porcine alveolar macrophage 3D4/21 cells.

The interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention. Nevertheless, the precise involvement of the unfolded protein response (UPR) signaling in the apoptosis of porcine macrophage cells induced by Deoxynivalenol (DON) remains enigmatic. In this study, we revealed that exposure to 2 µM DON resulted in a substantial decline in cell viability, concomitant with the initiation of cell apoptosis and the halting of the G1 phase cell cycle in the porcine alveolar macrophage line 3D4/21. Transcriptomic analysis of DON-exposed cells showed distinct expression patterns in 3104 genes, with notable upregulation of ER stress-related genes, including IRE1, CHOP, XBP1 and JNK. Our subsequent validation via qPCR and Western blot analyses confirmed the attenuation of GRP78 and BCL-2, coupled with the upregulation of IRE1, CHOP, JNK, p-JNK, and Bax in DON-induced cells, indicating the instigation of ER stress-associated apoptosis by DON. The addition of 5 mM 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, decreased levels of CHOP, IRE1, JNK, p-JNK, and Bax, while increasing levels of GRP78 and Bcl-2, suggesting that 4-PBA alleviated DON-induced ER stress and apoptosis. Overall, our findings provide new insights into DON-induced ER stress via the IRE1/JNK/CHOP pathway, leading to subsequent cellular apoptosis.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

4-Phenylbutyric acid attenuates amyloid-ß proteotoxicity through activation of HSF-1 in an Alzheimer's disease model of the nematode Caenorhabditiselegans.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common form of dementia. The pathogenesis is a complex process, in which the proteotoxicity of amyloid-ß (Aß) was identified as a major factor. 4-Phenylbutyric acid (4-PBA) is an aromatic short-chain fatty acid that may attenuate Aß proteotoxicity through its already shown properties as a chemical chaperone or by inhibition of histone deacetylases (HDACs). In the present study, we investigated the molecular effects of 4-PBA on Aß proteotoxicity using the nematode Caenorhabditis elegans as a model. Computer-based analysis of motility was used as a measure of Aß proteotoxicity in the transgenic strain GMC101, expressing human Aß1-42 in body wall muscle cells. Aß aggregation was quantified using the fluorescent probe NIAD-4 to correlate the effects of 4-PBA on motility with the amount of the proteotoxic protein. Furthermore, these approaches were supplemented by gene regulation via RNA interference (RNAi) to identify molecular targets of 4-PBA. 4-PBA improved the motility of GMC101 nematodes and reduced Aß aggregation significantly. Knockdown of hsf-1, encoding an ortholog essential for the cytosolic heat shock response, prevented the increase in motility and decrease in Aß aggregation by 4-PBA incubation. RNAi for hda-1, encoding an ortholog of histone deacetylase 2, also increased motility. Double RNAi for hsf-1 and hda-1 revealed a dominant effect of hsf-1 RNAi. Moreover, 4-PBA failed to further increase motility under hda-1 RNAi. Accordingly, the results suggest that 4-PBA attenuates Aß proteotoxicity in an AD-model of C. elegans through activation of HSF-1 via inhibition of HDA-1.

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer.

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco's Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38-24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer's disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines.

Phenylbutyric acid robustly increases Npy mRNA expression in hypothalamic neurons by increasing H3K9/14 acetylation at the Npy promoter.

Phenylbutyric acid (PBA) is a commonly used inhibitor of endoplasmic reticulum stress, as well as a histone deacetylase (HDAC) inhibitor, that increases hypothalamic expression of orexigenic neuropeptide Y (Npy). Elucidation of the dose-response relationship and mechanism of action of PBA may position this compound as a potential therapeutic for eating disorders where Npy is dysregulated, such as anorexia nervosa. The hypothalamic neuronal model mHypoE-41 was exposed to PBA (5 µM-5 mM) to assess the maximal Npy upregulation. Transcription factors and histone acetylation-related genes were assessed by qRT-PCR, as well as the involvement estrogen receptors (ER) using siRNA knockdown. Changes in global and Npy promoter-specific H3K9/14 acetylation were detected using western analysis and chromatin immunoprecipitation. Treatment with 5 mM PBA led to a 10-fold and 206-fold increase in Npy mRNA at 4 and 16 h, respectively, as well as increased NPY secretion. This induction was not observed with another orexigenic neuropeptide Agrp. PBA significantly increased the expression of Foxo1, Socs3 and Atf3 and the ERs Esr1 and Esr2 mRNA, but the PBA-mediated induction of Npy was not dependent on ERα or ERß. PBA induced histone H3K9/14 acetylation at 3 distinct Npy promoter regions, suggesting increased Npy transcriptional activation due to a more open chromatin structure. We also report changes in Hdac mRNAs by PBA and the fatty acid palmitate, highlighting the importance of epigenetic regulation in Npy transcription. Overall, we conclude that PBA has strong orexigenic potential and can robustly and specifically induce Npy in hypothalamic neurons through a mechanism likely involving histone H3 acetylation.

Immortalized Alzheimer's Disease Astrocytes: Characterization of Their Proteolytic Systems.

Alzheimer's disease (AD) is a progressive neurodegeneration with dysfunctions in both the ubiquitin-proteasome system (UPS) and autophagy. Astroglia participation in AD is an attractive topic of research, but molecular patterns are partially defined and available in vitro models have technical limitations. Immortalized astrocytes from the hippocampus of 3xTg-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively) have been obtained as an attempt to overcome primary cell line limitations and this study aims at characterizing their proteolytic systems, focusing on UPS and autophagy. Both 26S and 20S proteasomal activities were downregulated in 3Tg-iAstro, in which a shift in catalytic subunits from constitutive 20S proteasome to immunoproteasome occurred, with consequences on immune functions. In fact, immunoproteasome is the specific complex in charge of clearing damaged proteins under inflammatory conditions. Parallelly, augmented expression and activity of the lysosomal cathepsin B, enhanced levels of lysosomal-associated membrane protein 1, beclin1, and LC3-II, together with an increased uptake of monodansylcadaverine in autophagic vacuoles, suggested autophagy activation in 3Tg-iAstro. The two proteolytic pathways were linked by p62 that accumulated in 3Tg-iAstro due to both increased synthesis and decreased degradation in the UPS defective astrocytes. Treatment with 4-phenylbutyric acid, a neuroprotective small chemical chaperone, partially restored proteasome and autophagy-mediated proteolysis in 3Tg-iAstro. Our data shed light on the impaired proteostasis in 3Tg-iAstro with proteasome inhibition and autophagic compensatory activation, providing additional validation of this AD in vitro model, and propose a new mechanism of action of 4-phenylbutyric acid in neurodegenerative disorders.