Phase Separation of Disease-Associated SHP2 Mutants Underlies MAPK Hyperactivation.

The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.

Early blood immune molecular alterations in cynomolgus monkeys with a PSEN1 mutation causing familial Alzheimer's disease.

INTRODUCTION: More robust non-human primate models of Alzheimer's disease (AD) will provide new opportunities to better understand the pathogenesis and progression of AD. METHODS: We designed a CRISPR/Cas9 system to achieve precise genomic deletion of exon 9 in cynomolgus monkeys using two guide RNAs targeting the 3' and 5' intron sequences of PSEN1 exon 9. We performed biochemical, transcriptome, proteome, and biomarker analyses to characterize the cellular and molecular dysregulations of this non-human primate model. RESULTS: We observed early changes of AD-related pathological proteins (cerebrospinal fluid Aß42 and phosphorylated tau) in PSEN1 mutant (ie, PSEN1-ΔE9) monkeys. Blood transcriptome and proteome profiling revealed early changes in inflammatory and immune molecules in juvenile PSEN1-ΔE9 cynomolgus monkeys. DISCUSSION: PSEN1 mutant cynomolgus monkeys recapitulate AD-related pathological protein changes, and reveal early alterations in blood immune signaling. Thus, this model might mimic AD-associated pathogenesis and has potential utility for developing early diagnostic and therapeutic interventions. HIGHLIGHTS: A dual-guide CRISPR/Cas9 system successfully mimics AD PSEN1-ΔE9 mutation by genomic excision of exon 9. PSEN1 mutant cynomolgus monkey-derived fibroblasts exhibit disrupted PSEN1 endoproteolysis and increased Aß secretion. Blood transcriptome and proteome profiling implicate early inflammatory and immune molecular dysregulation in juvenile PSEN1 mutant cynomolgus monkeys. Cerebrospinal fluid from juvenile PSEN1 mutant monkeys recapitulates early changes of AD-related pathological proteins (increased Aß42 and phosphorylated tau).

A Simple and Efficient Method to Generate Dual Site-Specific Conjugation ADCs with Cysteine Residue and an Unnatural Amino Acid.

Antibody-drug conjugates (ADCs) are complex pharmaceutical molecules that combine monoclonal antibodies with biologically active drugs through chemical linkers. ADCs are designed to specifically kill disease cells by utilizing the target specificity of antibodies and the cytotoxicity of chemical drugs. However, the traditional ADCs were only applied to a few disease targets because of some limitations such as the huge molecular weight, the uncontrollable coupling reactions, and a single mechanism of action. Here we report a simple, one-pot, successive reaction method to produce dual payload conjugates with the site-specifically engineered cysteine and p-acetyl-phenylalanine using Herceptin (trastuzumab), an anti-HER2 antibody drug widely used for breast cancer treatment, as a tool molecule. This strategy enables antibodies to conjugate with two mechanistically distinct cytotoxic drugs through different functional groups sequentially, therefore, rendering the newly designed ADCs with functional diversity and the potential to overcome drug resistance and enhance the therapeutic efficacy.

Dihydroartemisinin suppresses ovalbumin-induced airway inflammation in a mouse allergic asthma model.

Asthma is a complex disease characterized by reversible airway obstruction, airway hyper-responsiveness (AHR) and chronic inflammation of the airways. Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, has been shown to possess antimalarial and antitumor activities, but whether it can be used in asthma treatment has not been investigated. In this study, we attempted to determine whether DHA regulates inflammatory mediators in the ovalbumin (OVA)-induced mouse asthma model. BALB/c mice were sensitized and challenged by OVA to induce chronic airway inflammation. The intragastrical administration of DHA at 30 mg/kg significantly decreased the number of infiltrating inflammatory cells, T-helper type 2 (Th2) cytokines, OVA-specific immunoglobulin E (IgE) and AHR. Treatment with DHA also attenuated OVA-induced mRNA expression of Muc5ac and chitinase 3-like protein 4 (Ym2) in lung tissues. In addition, lung histopathological studies revealed that DHA inhibited inflammatory cell infiltration and mucus hypersecretion. Then signal transduction studies showed that DHA significantly inhibited extracellular signal-regulated protein kinase (ERK), p38 mitogen-activated protein kinase phosphorylation. DHA also inhibited nuclear factor-κB (NF-κB) activation via the inhibition of phosphorylation of IκBα. These findings provide new insight into the immunopharmacological role of DHA in terms of its effects in a mouse model of asthma.

Attenuation of allergic airway inflammation in a murine model of asthma by Licochalcone A.

CONTEXT: Licochalcone A (Lico A) is a major and biogenetically characteristic chalcone isolated from the root of Xinjiang liquorice, Glycyrrhiza inflata. OBJECTIVE: We focused on investigating whether Lico A possesses distinct anti-inflammatory activity on a non-infectious mouse model of asthma, and we aimed to elucidate its involvement with the mitogen-activated protein kinases pathway. METHODS: BALB/c mice that were sensitized and challenged to ovalbumin (OVA) were treated with Lico A (50 mg/kg) 1 h before they were challenged with OVA. RESULTS: Our study demonstrated that Lico A may effectively inhibit the increase in T-helper type 2 cytokines, such as interleukin (IL)-4, IL-5 and IL-13 in bronchoalveolar lavage fluid, and reduced serum levels of OVA-specific IgE and IgG. Furthermore, Lico A substantially inhibited OVA-induced eosinophilia in lung tissue and mucus hyper-secretion by goblet cells in the airway. Meanwhile, pretreatment with Lico A resulted in a significant reduction in mRNA expression of acidic mammalian chitinase, chitinase 3-like protein 4 (Ym2), E-selectin, Muc5ac, CCL11 and CCR3 in lung tissues and airway hyper-responsiveness to methacholine. CONCLUSIONS: These findings suggest that Lico A may effectively delay the progression of airway inflammation and could be used as a therapy for patients with allergic airway inflammation.

Generation of a homozygous ABCA7-knockout human iPSC line using the CRISPR/Cas9 system.

ABCA7 encodes an ATP-binding cassette transporter, and its loss-of-function variants are associated with Alzheimer's disease. To investigate the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, we generated a homozygous ABCA7-knockout induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-mediated gene editing. This ABCA7-deficient iPSC line maintains a normal karyotype, expression of pluripotency markers, and trilineage differentiation capacity.

Differential and substrate-specific inhibition of γ-secretase by the C-terminal region of ApoE2, ApoE3, and ApoE4.

Aberrant low γ-secretase activity is associated with most of the presenilin mutations that underlie familial Alzheimer's disease (fAD). However, the role of γ-secretase in the more prevalent sporadic AD (sAD) remains unaddressed. Here, we report that human apolipoprotein E (ApoE), the most important genetic risk factor of sAD, interacts with γ-secretase and inhibits it with substrate specificity in cell-autonomous manners through its conserved C-terminal region (CT). This ApoE CT-mediated inhibitory activity is differentially compromised in different ApoE isoforms, resulting in an ApoE2 > ApoE3 > ApoE4 potency rank order inversely correlating to their associated AD risk. Interestingly, in an AD mouse model, neuronal ApoE CT migrates to amyloid plaques in the subiculum from other regions and alleviates the plaque burden. Together, our data reveal a hidden role of ApoE as a γ-secretase inhibitor with substrate specificity and suggest that this precision γ-inhibition by ApoE may protect against the risk of sAD.

Effects of a natural prolyl oligopeptidase inhibitor, rosmarinic acid, on lipopolysaccharide-induced acute lung injury in mice.

Rosmarinic acid (RA), a polyphenolic phytochemical, is a natural prolyl oligopeptidase inhibitor. In the present study, we found that RA exerted potent anti-inflammatory effects in in vivo models of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Mice were pretreated with RA one hour before challenge with a dose of 0.5 mg/kg LPS. Twenty-four hours after LPS was given, bronchoalveolar lavage fluid (BALF) was obtained to measure pro-inflammatory mediator and total cell counts. RA significantly decreased the production of LPS-induced TNF-a, IL-6, and IL-1ß compare with the LPS group. When pretreated with RA (5, 10, or 20 mg/kg) the lung wet-to-dry weight (W/D) ratio of the lung tissue and the number of total cells, neutrophils and macrophages in the BALF were decreased significantly. Furthermore, RA may enhance oxidase dimutase (SOD) activity during the inflammatory response to LPS-induced ALI. And we further demonstrated that RA exerts anti-inflammation effect in vivo models of ALI through suppresses ERK/MAPK signaling in a dose dependent manner. These studies have important implications for RA administration as a potential treatment for ALI.

Targeted gene correction and functional recovery in achondroplasia patient-derived iPSCs.

BACKGROUND: Achondroplasia (ACH) is the most common genetic form of dwarfism and belongs to dominant monogenic disorder caused by a gain-of-function point mutation in the transmembrane region of FGFR3. There are no effective treatments for ACH. Stem cells and gene-editing technology provide us with effective methods and ideas for ACH research and treatment. METHODS: We generated non-integrated iPSCs from an ACH girl's skin and an ACH boy's urine by Sendai virus. The mutation of ACH iPSCs was precisely corrected by CRISPR-Cas9. RESULTS: Chondrogenic differentiation ability of ACH iPSCs was confined compared with that of healthy iPSCs. Chondrogenic differentiation ability of corrected ACH iPSCs could be restored. These corrected iPSCs displayed pluripotency, maintained normal karyotype, and demonstrated none of off-target indels. CONCLUSIONS: This study may provide an important theoretical and experimental basis for the ACH research and treatment.

Long noncoding RNA BS-DRL1 modulates the DNA damage response and genome stability by interacting with HMGB1 in neurons.

Long noncoding RNAs (lncRNAs) are known to regulate DNA damage response (DDR) and genome stability in proliferative cells. However, it remains unknown whether lncRNAs are involved in these vital biological processes in post-mitotic neurons. Here, we report and characterize a lncRNA, termed Brain Specific DNA-damage Related lncRNA1 (BS-DRL1), in the central nervous system. BS-DRL1 is a brain-specific lncRNA and depletion of BS-DRL1 in neurons leads to impaired DDR upon etoposide treatment in vitro. Mechanistically, BS-DRL1 interacts with HMGB1, a chromatin protein that is important for genome stability, and is essential for the assembly of HMGB1 on chromatin. BS-DRL1 mediated DDR exhibits cell-type specificity in the cortex and cerebellum in gamma-irradiated mice and BS-DRL1 knockout mice show impaired motor function and concomitant purkinje cell degeneration. Our study extends the understanding of lncRNAs in DDR and genome stability and implies a protective role of lncRNA against neurodegeneration.

Protocatechuic acid suppresses ovalbumin-induced airway inflammation in a mouse allergic asthma model.

Protocatechuic acid (PCA) has been isolated from the leaves of ilex chinenses and has numerous pharmacologic effects, including anti-inflammatory and antitumoral activities. This study aims to evaluate the antiasthma activity of PCA and investigate its possible molecular mechanisms. BALB/c mice were sensitized and challenged to ovalbumin (OVA).Then mice were intraperitoneally (i.p.) injected with PCA 1h before OVA challenge. We found that PCA treatment at 15 or 30 mg/kg significantly decreased OVA-induced airway hyper-responsiveness (AHR) to inhaled methacholine. Type 2 helper T cell (Th2) cytokines in bronchoalveolar lavage (BAL) fluid, such as interleukin-4 (IL-4), interleukin 5 (IL-5) and interleukin-13 (IL-13), and serum OVA-specific immunoglobulin E (IgE) levels, were also reduced by PCA. Moreover treatment with PCA markedly decreased the number of inflammatory cells in BALF and attenuated OVA-induced mRNA expression of CCl11, CCR3, Muc5ac, acidic mammalian chitinase (AMCase), chitinase 3-like protein 4 (Ym2) and E-selectin in lung tissues, lung histopathological studies showed that PCA inhibited inflammatory cell infiltration and mucus hypersecretion compared with the OVA-induced mice group. We then investigated the possible molecular mechanisms which might be implicated in PCA activity. Our results suggested that the protective effect of PCA might be mediated by the inhibition of the extracellular signal-regulated protein kinase (ERK), p38 Mitogen-activated protein kinase (MAPK) phosphorylation and the nuclear factor-κB (NF-κB) activation.

Geniposide inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.

Our group recently reported the strong anti-inflammatory effects of geniposide (Gen), a bioactive iridoid glucoside derived from gardenia jasminoides, in a mouse acute lung injury model. Herein, we hypothesized that Gen might also have potential therapeutic benefits in treatment of asthma, which was tested in a mouse model of ovalbumin (Ova)-induced allergic airway inflammation. Ova-sensitized and -challenged BALB/c mice, as compared with control animals, displayed airway hyperresponsiveness (AHR), bronchoalveolar lavage eosinophilia, mucus hypersecretion, and increased T help 2 (Th2)-associated cytokine and chemokine amounts, as well as serum Ova-specific immunoglobulin E (IgE) level. Being compared with the Ova-induced hallmarks of asthma, intraperitoneal Gen treatment prevented eosinophilic pulmonary infiltration, attenuated the increases in interleukin (IL)-4, IL-5, and IL-13, and reduced eotaxin and vascular cell adhesion molecule 1 (VCAM-1) expression. Also, Gen significantly ameliorated the Ova-driven airway hyperresponsiveness, mucus hypersecretion, and allergen-specific IgE level, which are the cardinal pathophysiological symptoms in allergic airway diseases. In addition, the efficacy of Gen was comparable to that of dexamethasone (Dex), a currently available anti-asthmatic drug. Collectively, our findings reveal that the development of immunoregulatory strategies based on Gen may be considered as an effective adjuvant therapy for allergic asthma.

Licochalcone a inhibits lipopolysaccharide-induced inflammatory response in vitro and in vivo.

Licochalcone A (Lico A), a flavonoid found in licorice root (Glycyrrhiza glabra), is known for its antimicrobial activity and its reported ability to inhibit cancer cell proliferation. In the present study, we found that Lico A exerted potent anti-inflammatory effects in in vitro and in vivo models induced by lipopolysaccharide (LPS). The concentrations of TNF-α, interleukin (IL)-6, and IL-1ß in the culture supernatants of RAW 264.7 cells were determined at different time points following LPS administration. LPS (0.5 mg/kg) was instilled intranasally (i.n.) in phosphate-buffered saline to induce acute lung injury, and 24 h after LPS was given, bronchoalveolar lavage fluid was obtained to measure pro-inflammatory mediator and total cell counts. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) p65 protein was analyzed by Western blotting. Our results showed that Lico A significantly reduced the amount of inflammatory cells, the lung wet-to-dry weight (W/D) ratio, protein leakage, and myeloperoxidase activity and enhances oxidase dimutase activity in mice with LPS-induced acute lung injury (ALI). Enzyme-linked immunosorbent assay results indicated that Lico A can significantly down-regulate TNF-α, IL-6, and IL-1ß levels in vitro and in vivo, and treatment with Lico A significantly attenuated alveolar wall thickening, alveolar hemorrhage, interstitial edema, and inflammatory cells infiltration in mice with ALI. In addition, we further demonstrated that Lico A exerts an anti-inflammation effect in an in vivo model of acute lung injury through suppression of NF-κB activation and p38/ERK MAPK signaling in a dose-dependent manner.

Preventive effect of Imperatorin on acute lung injury induced by lipopolysaccharide in mice.

Imperatorin, a linear furanocoumarin, has many pharmacological effects such as antibacterial, anti-inflammatory and antiviral effects. The purpose of this study was to investigate the effect of Imperatorin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. BALB/c mice were pretreated with Imperatorin 1h before LPS challenge. We found that the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) were decreased significantly, and the level of interleukin-10 (IL-10) was up-regulated 8h after Imperatorin treatment. Pretreatment with Imperatorin (15 or 30 mg/kg) decreased lung wet-to-dry weight (W/D) ratio, the number of inflammatory cells and myeloperoxidase (MPO) activities. Additionally, Imperatorin attenuated lung histopathological changes and significantly inhibited the phosphorylation of IκB, JNK, ERK and p38/MAPK. These findings demonstrate that Imperatorin protects against LPS-induced ALI in mice.

Effects of an anthraquinone derivative from Rheum officinale Baill, emodin, on airway responses in a murine model of asthma.

Emodin is a component from traditional Chinese herbal medicines. We focused on investigating whether emodin possesses distinct anti-inflammatory activity on a non-infectious mouse model of asthma, and we aimed to elucidate its involvement with the NF-κB pathway. BALB/c mice that were sensitized and challenged to ovalbumin were treated with emodin (40 mg/kg) 1h before they were challenged with OVA. Our study demonstrated that emodin inhibited OVA-induced increases in eosinophil count; interleukin (IL)-4, IL-5, and IL-13 levels were recovered in bronchoalveolar lavage fluid and reduced serum levels of OVA-specific IgE, IgG, and IgG1. Histological studies demonstrated that emodin substantially inhibited OVA-induced eosinophilia in lung tissue and mucus hyper-secretion by goblet cells in the airway. Furthermore, pretreatment with emodin resulted in a significant reduction in mRNA expression of acidic mammalian chitinase (AMCase), chitinase 3-like protein 4 (Ym2) and Muc5ac in lung tissues and airway hyperresponsiveness to methacholine. These findings suggest that emodin may effectively delay the progression of airway inflammation and could be used as a therapy for patients with allergic airway inflammation.

Protocatechuic acid attenuates lipolysaccharide-induced acute lung injury.

Protocatechuic acid (PCA) is a major metabolite of anthocyanins. It has numerous pharmacological effects, including anti-inflammatory, antioxidant, and antitumoral activities. In the present study, we investigated the in vivo protective effect of PCA on acute lung injury (ALI) induced by lipolysaccharide (LPS) in mice. We treated mice with PCA 1 h before the intratracheal (i.n.) administration of LPS. The pulmonary injury severity was evaluated 6 h after LPS administration. We found that pretreatment with a 30 mg/kg of PCA markedly attenuated the LPS-induced histological alterations in the lung. In addition, PCA inhibited the production of several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6, at 6 h in the bronchoalveolar lavage fluid (BALF) after LPS challenge. Furthermore, PCA significantly reduced the number of total cells, neutrophils, and macrophages in the BALF, and it significantly decreased the wet/dry weight (W/D) ratio of lungs and the protein concentration in the BALF. Additionally, Western blotting showed that PCA efficiently blunted nuclear factor-kappa B (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα, as well as the translocation of p65 from cytoplasm to the nucleus. In conclusion, these results indicate that PCA was highly effective in inhibiting acute lung injury (ALI) and may be a promising potential therapeutic reagent for ALI treatment. PCA may utilize the NF-κB pathway to attenuate the nonspecific pulmonary inflammation induced by LPS administration.

Kaempferol regulates MAPKs and NF-κB signaling pathways to attenuate LPS-induced acute lung injury in mice.

Recent studies show that mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways are two pivotal roles contributing to the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI). The present study aimed to investigate the protective effect of kaempferol (Kae), a naturally occurring flavonoid compound, on ALI and explore its possible mechanisms. Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated or not with Kae (100 mg/kg, intragastrically) 1h prior to LPS exposure. Kae treatment attenuated pulmonary edema of mice with ALI after LPS challenge, as it markedly decreased the lung W/D ratio of lung samples, protein concentration and the amounts of inflammatory cells in BALF. Similarly, LPS mediated overproduction of proinflammatory cytokines in BALF, including TNF-α, IL-1ß and IL-6, was strongly reduced by Kae. Histological studies demonstrated that Kae substantially inhibited LPS-induced alveolar wall thickness, alveolar hemorrhage and leukocytes infiltration in lung tissue with evidence of reduced myeloperoxidase (MPO) activity. Kae also efficiently increased superoxide dismutase (SOD) activity of lung sample when compared with LPS group, which was obviously reduced by LPS administration. In addition, Western blot analysis indicated that the activation of MAPKs and NF-κB signaling pathways stimulated by LPS was significantly blocked by Kae. Taken together, our results suggest that Kae exhibits a protective effect on LPS-induced ALI via suppression of MAPKs and NF-κB signaling pathways, which may involve the inhibition of tissue oxidative injury and pulmonary inflammatory process.