Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors

BACKGROUND: The mutation in Huntington\u27s disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. RESULTS: Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. CONCLUSION: We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified

Micafungin prophylaxis for acute leukemia patients undergoing induction chemotherapy

Background Micafungin is a well-tolerated and effective prophylactic antifungal agent used in hematologic diseases. In this prospective trial, we evaluated the efficacy and safety of prophylactic micafungin during first induction chemotherapy in patients with acute leukemia. We also compared outcomes of prophylactic micafungin with those of prophylactic posaconazole in acute myeloid leukemia (AML). Methods Medically fit patients with newly diagnosed acute leukemia received 50 mg micafungin intravenously once daily from the initiation of first induction chemotherapy to recovery of neutrophil count, suspected fungal infection, or unacceptable drug-related toxicity (Clinicaltrials.gov number, NCT02440178). The primary end point was incidence of invasive fungal infection, and the secondary end points were adverse events of prophylactic micafungin and mortality during induction therapy. Results The 65 patients (median age = 51 years, male:female = 34:31) enrolled in this study had diagnoses of AML (33, 50.8%), acute lymphoblastic leukemia (31, 47.7%), and acute biphenotypic leukemia (1, 1.5%). Median duration of micafungin treatment was 24 days (range 1–68), with proven invasive fungal disease in one patient (1.5%) and possible fungal infection in two patients (3.1%). Three of the patients (4.6%) experienced the following adverse events, but all events were tolerable: liver function abnormality (Grade 2, n = 1; Grade 3, n = 1) and allergic reaction (Grade 2, n = 1). Three patients died during induction therapy, and invasive aspergillosis pneumonia was the cause of death for one of those patients. Overall, 19 patients (29.2%) discontinued prophylactic micafungin, and 18 (27.7%) patients switched to another antifungal agent. We observed no fungal infections caused by amphotericin B-resistant organisms. In AML patients, outcomes of prophylactic micafungin during induction chemotherapy did not differ significantly with those of prophylactic posaconazole with regard to incidence of fungal infections, rate of discontinuation, or safety. Conclusions Our study demonstrates that prophylactic micafungin is safe and effective in patients with acute leukemia undergoing induction chemotherapy. Outcomes in patients with AML were similar to those of prophylactic posaconazole, indicating the usefulness of micafungin as a prophylactic antifungal agent during induction chemotherapy for AML. Trial registration Clinicaltrials.gov NCT02440178, registered May 12th 2015.This study was funded by Astellas Pharma Korea, Inc. Funding source had no role in the study design, data collection, data analysis or data interpretation

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders

Ginsenoside Rh2 epigenetically regulates cell-mediated immune pathway to inhibit proliferation of MCF-7 breast cancer cells

Background: Ginsenoside Rh2 has been known to enhance the activity of immune cells, as well as to inhibit the growth of tumor cells. Although the repertoire of genes regulated by Rh2 is well-known in many cancer cells, the epigenetic regulation has yet to be determined, especially for comprehensive approaches to detect methylation changes. Methods: The effect of Rh2 on genome-wide DNA methylation changes in breast cancer cells was examined by treating cultured MCF-7 with Rh2. Pyrosequencing analysis was carried out to measure the methylation level of a global methylation marker, LINE1. Genome-wide methylation analysis was carried out to identify epigenetically regulated genes and to elucidate the most prominent signaling pathway affected by Rh2. Apoptosis and proliferation were monitored to examine the cellular effect of Rh2. Results: LINE1 showed induction of hypomethylation at specific CpGs by 1.6–9.1% (p < 0.05). Genome-wide methylation analysis identified the “cell-mediated immune response”-related pathway as the top network. Cell proliferation of MCF-7 was retarded by Rh2 in a dose-dependent manner. Hypermethylated genes such as CASP1, INSL5, and OR52A1 showed downregulation in the Rh2-treated MCF-7, while hypomethylated genes such as CLINT1, ST3GAL4, and C1orf198 showed upregulation. Notably, a higher survival rate was associated with lower expression of INSL5 and OR52A1 in breast cancer patients, while with higher expression of CLINT1. Conclusion: The results indicate that Rh2 induces epigenetic methylation changes in genes involved in immune response and tumorigenesis, thereby contributing to enhanced immunogenicity and inhibiting the growth of cancer cells. Keywords: Breast cancer, CpG methylation, epigenetics, ginsenoside, Rh

Survival of Verwey transition in gadolinium-doped ultrasmall magnetite nanoparticles

We have demonstrated that the Verwey transition, which is highly sensitive to impurities, survives in anisotropic Gd-doped magnetite nanoparticles. Transmission electron microscopy analysis shows that the nanoparticles are uniformly distributed. X-ray photoelectron spectroscopy and EDS mapping analysis confirm Gd-doping on the nanoparticles. The Verwey transition of the Gd-doped magnetite nanoparticles is robust and the temperature dependence of the magnetic moment (zero field cooling and field cooling) shows the same behaviour as that of the Verwey transition in bulk magnetite, at a lower transition temperature (similar to 110 K). In addition, irregularly shaped nanoparticles do not show the Verwey transition whereas square-shaped nanoparticles show the transition. Mossbauer spectral analysis shows that the slope of the magnetic hyperfine field and the electric quadrupole splitting change at the same temperature, meaning that the Verwey transition occurs at similar to 110 K. These results would provide new insights into understanding the Verwey transition in nano-sized materials.113sciescopu

ID1-Mediated BMP Signaling Pathway Potentiates Glucagon-Like Peptide-1 Secretion in Response to Nutrient Replenishment

Glucagon-like peptide-1 (GLP-1) is a well-known incretin hormone secreted from enteroendocrinal L cells in response to nutrients, such as glucose and dietary fat, and controls glycemic homeostasis. However, the detailed intracellular mechanisms of how L cells control GLP-1 secretion in response to nutrients still remain unclear. Here, we report that bone morphogenetic protein (BMP) signaling pathway plays a pivotal role to control GLP-1 secretion in response to nutrient replenishment in well-established mouse enteroendocrinal L cells (GLUTag cells). Nutrient starvation dramatically reduced cellular respiration and GLP-1 secretion in GLUTag cells. Transcriptome analysis revealed that nutrient starvation remarkably reduced gene expressions involved in BMP signaling pathway, whereas nutrient replenishment rescued BMP signaling to potentiate GLP-1 secretion. Transient knockdown of inhibitor of DNA binding (ID)1, a well-known target gene of BMP signaling, remarkably reduced GLP-1 secretion. Consistently, LDN193189, an inhibitor of BMP signaling, markedly reduced GLP-1 secretion in L cells. In contrast, BMP4 treatment activated BMP signaling pathway and potentiated GLP-1 secretion in response to nutrient replenishment. Altogether, we demonstrated that BMP signaling pathway is a novel molecular mechanism to control GLP-1 secretion in response to cellular nutrient status. Selective activation of BMP signaling would be a potent therapeutic strategy to stimulate GLP-1 secretion in order to restore glycemic homeostasis

Platelet storage induces accelerated desialylation of platelets and increases hepatic thrombopoietin production

Abstract Background Stored platelets undergo deleterious changes, referred to as platelet storage lesions (PSLs), which accelerate the desialylation of platelets and result in their phagocytosis and clearance by hepatic macrophages. Recent studies have reported that Ashwell–Morell receptor binds to desialylated platelets, thereby inducing hepatic thrombopoietin (TPO) production in a mouse model. Therefore, this study aimed to demonstrate these relationships between PSL and hepatic TPO production in human study. Methods Platelet concentrates (PCs) were obtained from 5 healthy volunteers and the remaining were discarded samples from the blood bank. PCs were divided into two halves, and stored either at 22 or 4 °C. Experiments were conducted using serial samples. Desialylation was assessed using flow cytometry, and structural changes were visualized using electron microscopy. Following co-culture of HepG2 cells (HB-8065, ATCC) with isolated platelets, hepatic TPO production was determined using real-time quantitative polymerase chain reaction and the supernatant TPO level was measured using a Luminex kit. Results For 5 days of storage duration, platelet counts were not influenced by the storage conditions, but the degree of desialylation was proportional to the storage duration. Significant changes in the platelet surface and structure according to storage conditions were noted in electron microscopy. HepG2 cells incubated with aged platelets expressed more TPO mRNA, and supernatant TPO levels were proportional to the storage duration. Refrigeration also influenced on the results of this study, but they were not statistically significant. Conclusions This is the first study to demonstrate that, in vitro, aging and refrigeration affect the integrity of human platelets, resulting in induction of hepatic TPO mRNA and protein expression

AMPK–SKP2–CARM1 signalling cascade in transcriptional regulation of autophagy

Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation. Although the components of autophagy in the cytoplasm have been well studied, the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARM1-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK-SKP2-CARM1 in the regulation of autophagy induction after nutrient starvation