RanBP9 Overexpression Accelerates Loss of Pre and Postsynaptic Proteins in the APΔE9 Transgenic Mouse Brain

There is now compelling evidence that the neurodegenerative process in Alzheimer’s disease (AD) begins in synapses. Loss of synaptic proteins and functional synapses in the amyloid precursor protein (APP) transgenic mouse models of AD is well established. However, what is the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to robustly increased amyloid β peptide (Aβ) generation leading to enhanced amyloid plaque burden in a mouse model of AD. In this study we compared synaptic protein levels among four genotypes of mice, i.e., RanBP9 single transgenic (Ran), APΔE9 double transgenic (Dbl), APΔE9/RanBP9 triple transgenic (Tpl) and wild-type (WT) controls. We found significant reductions in the levels of synaptic proteins in both cortex and hippocampus of 5- and 6-months-old but not 3- or 4-months-old mice. Specifically, at 5-months of age, rab3A was reduced in the triple transgenic mice only in the cortex by 25% (p<0.05) and gap43 levels were reduced only in the hippocampus by 44% (p<0.01) compared to wild-type (WT) controls. Interestingly, RanBP9 overexpression in the Tpl mice reduced gap43 levels by a further 31% (p<0.05) compared to APΔE9 mice. RanBP9 also further decreased the levels of drebrin in the hippocampus by 32% (p<0.01) and chromogranin in the cortex by 24% (p<0.05) compared to APΔE9 mice. At 6-months of age, RanBP9 expression in the cortex led to further reduction of rab3A by 30% (p<0.05) and drebrin by 38% (p<0.01) compared to APΔE9 mice. RanBP9 also increased Aβ oligomers in the cortex at 6 months. Similarly, in the hippocampus, RanBP9 expression further reduced rab3A levels by 36% (p<0.01) and drebrin levels by 33% (p<0.01). Taken together these data suggest that RanBP9 overexpression accelerates loss of synaptic proteins in the mouse brain.     

TNF-alpha opens a paracellular route for HIV-1 invasion across the blood-brain barrier.

BACKGROUND: HIV-1 invades the central nervous system early after infection when macrophage infiltration of the brain is low but myelin pallor is suggestive of blood-brain-barrier damage. High-level plasma viremia is a likely source of brain infection. To understand the invasion route, we investigated virus penetration across in vitro models with contrasting paracellular permeability subjected to TNF-alpha. MATERIALS AND METHODS: Blood-brain-barrier models constructed with human brain microvascular endothelial cells, fetal astrocytes, and collagen I or fibronectin matrix responded in a dose-related fashion to cytokines and ligands modulating paracellular permeability and cell migration. Virus penetration was measured by infectious and quantitative HIV-1 RNA assays. Barrier permeability was determined using inulin or dextran. RESULTS: Cell-free HIV-1 was retained by the blood-brain barrier with close to 100% efficiency. TNF-alpha increased virus penetration by a paracellular route in a dose-dependent manner proportionately to basal permeability. Brain endothelial cells were the main barrier to HIV-1. HIV-1 with monocytes attracted monocyte migration into the brain chamber. CONCLUSIONS: Early after the infection, the blood-brain barrier protects the brain from HIV-1. Immune mediators, such as TNF-alpha, open a paracellular route for the virus into the brain. The virus and viral proteins stimulate brain microglia and macrophages to attract monocytes into the brain. Infiltrating macrophages cause progression of HIV-1 encephalitis.     

Associations between common variants in <Emphasis Type="Italic">GC</Emphasis> and <Emphasis Type="Italic">DHCR7</Emphasis>/<Emphasis Type="Italic">NADSYN1</Emphasis> and vitamin D concentration in Chinese Hans

Recent studies have identified common variants in or near GC, CYP2R1 and NADSYN1/DHCR7 to be associated with 25-hydroxyvitamin D [25(OH)D] levels in European populations. We aimed to examine whether these variants also influence 25(OH)D levels in Chinese. Seven common variants were successfully genotyped and tested for associations with plasma 25(OH)D levels in a population-based cohort of 3,210 Chinese Hans from Beijing and Shanghai. Six common variants at GC (rs4588, rs7041, rs2282679 and rs1155563) and NADSYN1/DHCR7 (rs3829251 and rs1790349) loci were all significantly associated with lower plasma 25(OH)D levels (−0.036 ≤ β ≤ −0.076 per risk-allele, P ≤ 5.7 × 10⁻⁵), while CYP2R1-rs2060793 showed a trend toward association with 25(OH)D levels in the Shanghai subpopulation (P = 0.08), but not in the Beijing subpopulation (P = 0.82). Haplotype-based association analyses of the four GC variants showed that only the haplotype that contained all risk-alleles (TACC) was significantly associated with lower plasma 25(OH)D levels (β = −0.085, P = 2.3 × 10⁻⁹), while the haplotype containing the risk-alleles of rs4588 and rs2282679 (TATC) was marginally associated with lower 25(OH)D levels (β = −0.054, P = 0.0562) when compared with GCTA haplotype carrying the four protective alleles. Most notably, conditional analyses showed that only GC-rs4588 and GC-rs2282679 (r ² = 0.97) remained significantly associated with 25(OH)D concentrations (P ≤ 1.9 × 10⁻⁵) after adjusting for the other two SNPs in GC. In conclusion, GC and NADSYN1/DHCR7 loci individually and collectively contribute to variation in plasma vitamin D levels in Chinese Hans.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Yeast thioredoxin genes

Based on the conserved protein sequence of thioredoxins from yeast and other organisms, two primers were synthesized for polymerase chain reaction of yeast genomic DNA. A 34-base pair (bp) sequence around the active site of yeast thioredoxin was obtained from the polymerase chain reaction product. This specific sequence was used as a probe in Southern blot analysis of total yeast genomic DNA digested with various restriction enzymes. Under conditions of high stringency, more than one DNA species hybridized with the probe, suggesting that more than one gene encodes yeast genomic library. Two Sau3A1 fragments, 825 and 2045 bp, respectively, from two different clones were cloned into pUC13. Sequence analysis of these fragments gave two different open reading frames without introns. The 825-bp Sau3A1 fragment encodes a 103-amino acid residue protein named thioredoxin I. The 2045-bp Sau3A1 fragment contains a sequence encoding thioredoxin II which has 102 amino acid residues. This is the first report of the cloning and sequencing of eukaryotic thioredoxin genes from any source. Both yeast thioredoxins contain a dithiol active site sequence, Cys-Gly-Pro-Cys. Thioredoxins I and II show 78% amino acid sequence identity. They display more amino acid sequence similarity with mammalian thioredoxin than with Escherichia coli and plant chloroplast thioredoxins.     

Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1(+) hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1(+) cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation.     

Dual effects of histone deacetylase inhibition by trichostatin A on endothelial nitric oxide synthase expression in endothelial cells

Inhibition of histone deacetylases by trichostatin A (TSA) has pleiotropic effects on gene expression. We demonstrated that at low dose (0.1 microg) TSA increased the eNOS mRNA levels, which was followed by a time- and dose-dependent down-regulation. Cycloheximide, a protein synthesis inhibitor, completely abolished TSA-induced decrease in eNOS expression, indicating that new protein synthesis is required for the inhibiting effect. Mevastatin--an inhibitor HMG-CoA reductase and geranylgeranylation reaction dose-dependently antagonized TSA-induced reduction. This mevastatin-mediated antagonism was completely abolished by geranylgeranylpyrophosphate, suggesting that geranylgeranyl modification is needed to activate the eNOS mRNA destabilizing factor--a mechanism responsible for statin-mediated eNOS upregulation.