Hexosamine pathway activation improves memory but does not extend lifespan in mice

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated

Bortezomib resistance mutations in PSMB5 determine response to second-generation proteasome inhibitors in multiple myeloma

Older patients with severe physical trauma are at high risk of developing neuropsychiatric syndromes with global impairment of cognition, attention, and consciousness. We employed a thoracic trauma (TxT) mouse model and thoroughly analyzed age-dependent spatial and temporal posttraumatic alterations in the central nervous system. Up to 5 days after trauma, we observed a transient 50% decrease in the number of excitatory synapses specifically in hippocampal pyramidal neurons accompanied by alterations in attention and motor activity and disruption of contextual memory consolidation. In parallel, hippocampal corticotropin-releasing hormone (CRH) expression was highly upregulated, and brain-derived neurotrophic factor (BDNF) levels were significantly reduced. In vitro experiments revealed that CRH application induced neuronal autophagy with rapid lysosomal degradation of BDNF via the NF-κB pathway. The subsequent synaptic loss was rescued by BDNF as well as by specific NF-κB and CRH receptor 1 (CRHR1) antagonists. In vivo, the chronic application of a CRHR1 antagonist after TxT resulted in reversal of the observed histological, molecular, and behavioral alterations. The data suggest that neuropsychiatric syndromes (i.e., delirium) after peripheral trauma might be at least in part due to the activation of the hippocampal CRH/NF-κB/BDNF pathway, which results in a dramatic loss of synaptic contacts. The successful rescue by stress hormone receptor antagonists should encourage clinical trials focusing on trauma-induced delirium and/or other posttraumatic syndromes

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia

Daniel Gündel,1 Mareli Allmeroth,2 Sarah Reime,1 Rudolf Zentel,2 Oliver Thews1 1Institute of Physiology, Martin Luther University Halle-Wittenberg, Halle (Saale), 2Institute of Organic Chemistry, Johannes Gutenberg-University, Mainz, Germany Background: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis.Materials and methods: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately.Results: The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis.Conclusion: The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO2) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient. Keywords: HPMA–LMA copolymers, endocytosis, tumor microenvironment, tumor lines, structure–property relationship&nbsp

Unbiased compound-protein interface mapping and prediction of chemoresistance loci through forward genetics in haploid stem cells

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis. Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca(2+) pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes. Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces