α7nAChR activation protects against oxidative stress, neuroinflammation and central insulin resistance in ICV-STZ induced sporadic Alzheimer's disease.

Central insulin resistance is considered as one of the pathological hallmarks of Alzheimer's disease (AD), similar to formation of amyloid plaques and neurofibrillary tangles (NFT). Activation of α7nAChR by GTS-21 has been indicated to reverse peripheral insulin resistance and exert neuroprotection. Therefore, the aim of the present study was to determine the effect of α7nAChR agonist (GTS-21) on intracerebroventricular administration of streptozotocin (ICV-STZ)-induced oxidative stress, neuroinflammation, cholinergic dysfunction, central insulin resistance and cognitive deficits. GTS-21 (1, 4 and 8 mg/kg; i.p.) was administered for 21 days following bilateral ICV-STZ administration (3 mg/kg) in C57BL/6 mice. Neurobehavioral assessments were performed using Morris water maze (MWM) and novel object recognition (NOR). Inflammatory markers (TNF-α, IL-6 and IL-1ß) were determined using ELISA. Oxido-nitrosative stress (GSH, MDA and nitrite) and cholinergic activity (acetylcholine esterase and choline acetyltransferase) were estimated in the cortex and hippocampus through biochemical methods. Gene expression of insulin receptor (IR), IRS1, IRS2, BACE1, APP, PI3-K, AKT and GSK3ß were determined by q-RT-PCR. ICV-STZ administration induced memory impairment, increased oxidative stress and neuroinflammation, and caused cholinergic dysfunction. Our results demonstrated that activation of α7nAChR by GTS-21 treatment improved memory in MWM and NOR test. Moreover, GTS-21 treatment significantly decreased oxido-nitrosative stress, inflammatory markers and cholinergic dysfunction in cortex and hippocampus. Finally, GTS-21 treatment restored ICV-STZ induced downregulation of IR, IRS1, IRS2, PI3-k, Akt and attenuated GSK3ß, APP and BACE-1 indicating improved insulin signalling. Therefore, activation of α7nAChR through GTS-21 might be the potential target for the amelioration of central insulin resistance induced AD.

Vitamin D3 attenuates cognitive deficits and neuroinflammatory responses in ICV-STZ induced sporadic Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by intracellular neurofibrillary tangles and extracellular Aß deposition. Growing experimental evidence indicate diverse biological effects of vitamin D3 including antioxidant, neuroprotective, anti-inflammatory and cardiovascular benefits. However, the underlying neuroprotective mechanism of vitamin D3 is still largely elusive. Therefore, the present study was aimed to investigate the neuroprotective effects of vitamin D3 on ICV-STZ induced sporadic AD. Our study demonstrated that vitamin D3 pretreatment significantly improved spatial learning and memory functions and effectively mitigated ICV-STZ mediated neuronal oxidative stress, mitochondrial aberrations and improved cholinergic functions. Moreover, vitamin D3 attenuated hippocampal neuroinflammatory response and reduced neuronal death in cortex and hippocampus. Our findings indicated that prophylactic vitamin D3 supplementation ameliorated ICV-STZ mediated neurobehavioral alterations, oxidative stress and neuroinflammation thereby improving cholinergic functions and reversed degenerative changes in brain. Thus, our study further provides evidence for its therapeutic supplementation for various neurodegenerative disorders including AD.

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aß1-16 peptide.

The binding of metal ions to Aß peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu(2+) on redox properties and cytotoxicity of Aß peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aß peptide has higher propensity of H2O2 generation. The oxidation of Aß1-16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aß1-16-Cu(2+) (1:2) complex.

Mass spectral studies reveal the structure of Aß1-16-Cu2+ complex resembling ATCUN motif.

In Alzheimer's disease, copper binds to amyloid beta (Aß) peptide and generates oxidative stress. The coordination of histidine (His) residues to Cu(2+) is still uncertain. We studied Cu(2+) binding to Aß1-16 peptide using the diethyl pyrocarbonate (DEPC) assay and mass spectrometry. Our results show that only one His is involved in Cu(2+) coordination, which is identified as His6 using mass spectral studies. Novel nickel displacement studies have further supported the proposal that the Cu(2+) binding site of Aß1-16 peptide resembles the ATCUN motif of human serum albumin.

Solution structure of physiological Cu(His)2: novel considerations into imidazole coordination.

A disagreement on the mode of histidine binding to copper and the structure of [Cu(2+)(His)(2)] in solution still exists. Spectroscopic data in solution support a six-coordinate species with N4O2 donor atoms, while X-ray crystallography reveals five-coordinate N(3)O(2) donor atoms. We modified [Cu(2+)(His)(2)] in solution using diethyl pyrocarbonate (DEPC) and monitored the products spectrophotometrically and by mass spectrometry. Our spectrophotometric study indicates the presence of a free imidazole in the [Cu(2+)(His)(2)] complex in solution. Mass spectral characterization of a DEPC-modified [Cu(2+)(His)(2)] complex yielded a peak at 587.8 amu corresponding to three DEPC adducts. Taken together, our data indicate that the [Cu(2+)(His)(2)] complex in solution exists as a neutral five-coordinate structure with N3O2 donor atoms.

Spectrum of ATM gene mutations in a hospital-based series of unselected breast cancer patients.

Blood relatives of patients with the inherited disease ataxia telangiectasia (A-T) have an increased susceptibility for breast cancer. We therefore looked for sequence alterations of the ATM gene in a large hospital-based series of unselected breast cancer patients. The whole ATM coding sequence was analyzed in genomic DNA samples from a core group of 192 consecutive breast cancer cases to define the spectrum of ATM gene mutations. Common sequence alterations were then screened in the whole series of 1000 breast cancer patients and in 500 random individuals. In the core group, 21 distinct sequence alterations were identified throughout the ATM coding region, and 1 common splicing mutation was uncovered in intron 10. Almost half of the breast cancer patients (46%) were heterozygotes for 1 of 16 different amino acid substitutions, and three patients (1.6%) carried a truncating mutation. These data indicate that approximately 1 in 50 German breast cancer patients is heterozygous for an A-T-causing mutation. In our extended series, the most common A-T mutation 1066-6T-->G was disclosed in 7 of 1000 (0.7%) breast cancer patients. Transcript analyses indicated that the loss of exon 11 in the ATM mRNA was the pathogenic consequence of this splicing mutation, which produced a <10% of full-length ATM mRNA and ATM protein in a homozygous A-T patient. We also found an excess of rare missense substitutions in the breast cancer cohort compared with random individuals (7.9% versus 5.3% of alleles; odds ratio = 1.6; P < 0.01). One missense substitution, S707P in exon 15, was two times more frequent in breast cancer patients (odds ratio = 2.4; 95% confidence interval, 1.0-5.8) and five times more frequent in patients with bilateral disease than in random individuals (P < 0.001). We conclude that a large variety of distinct ATM mutations and variants exist among breast cancer patients, some of which can contribute to the etiology and progression of the malignancy. Screening for frequent A-T mutations such as the 1066-6-->G splice site substitution can be effective to prospectively identify A-T heterozygotes in an unselected cancer patient population.

Marek's Disease Herpesviruses I. Production and Preliminary Characterization of Marek's Disease Herpesvirus A Antigen.

A method was developed for the large-scale production of Marek's disease herpesvirus A antigen in duck embryo fibroblast roller bottle cultures in quantities sufficient to permit its purification and characterization. Maximum yield was obtained in serum-free culture medium harvested daily. The Marek's disease herpesvirus A antigen was stable at pH 2.0 and was a glycoprotein based on its sensitivity to trypsin, specific immune co-precipitation of radioactive amino acids and glucosamine, and detection of radioactive glucosamine by immunodiffusion and autoradiography. The antigen aggregated and lost titer upon storage but dissociated readily and regained titer in 1 or 2 M urea and 0.05% Brij 35. Fresh unaggregated antigen or antigen dissociated with urea and Brij 35 sedimented at 3.7S on sucrose gradients. The apparent molecular weight of the glycoprotein antigen was estimated to be 44,800 by gel filtration on Sephadex G-200 in the presence of 2 M urea and 0.05% Brij 35.