Resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-infected T-cell lines

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-kappaB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-kappaB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-lLnorleucinal (LLnL), an NF-kappaB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-kappaB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-kappaB may be a useful adjunct in clinical use of Apo2L against ATL.     

Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease.

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Abeta42 or anti-Abeta40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Abeta40 or anti-Abeta42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 micro m were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Abeta40 was localized to core-like structures, mainly in layers I-III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I-VI, were labeled with anti-Abeta42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Abeta deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.     

Deletion of vitamin E enhances phenotype of Alzheimer disease model mouse

Increased oxidative damage is a prominent and early feature in Alzheimer disease (AD). However, whether it is a primary cause or merely a downstream consequence in AD pathology is still unknown. We previously generated alpha-tocopherol transfer protein knockout (Ttpa-/-) mice, in which lipid peroxidation in the brain was significantly increased by complete depletion of alpha-tocopherol (alpha-Toc). Here we crossed AD transgenic (APPsw) model mice (Tg2576) with Ttpa-/- mice. The resulting double-mutant (Ttpa-/- APPsw) mice showed earlier and more severe cognitive dysfunction in the Morris water maze, novel-object recognition, and contextual fear conditioning tests. They also showed increased amyloid beta-peptide (Abeta) deposits in the brain by immunohistochemical analysis, which was ameliorated with alpha-Toc supplementation. In this report we provide clear evidence indicating that chronic lipid peroxidation due to alpha-Toc depletion enhances AD phenotype in a mouse model.     

Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles

Amplification with catalyzed reporter deposition (CARD) greatly enhances peroxidase signals, which has been utilized to amplify immunohistochemical labelings including fluorochromes. Here we describe a strategy to amplify each of two immunofluorescent signals without crosstalk on double-stained histological sections from human autopsied brains with Alzheimer's disease (AD). One of the two primary antibodies (anti-Abeta or anti-PHF-tau) was probed by a species-specific secondary antibody conjugated with horseradish peroxidase (HRP), which was visualized by FITC-labeled tyramide. After inactivation of HRP, the other primary antibody was probed by another species-specific secondary antibody conjugated with HRP. Amplification with biotinylated tyramide was followed by streptavidin-conjugated Cy-5, which specifically labeled the latter epitope. It was found that Abeta and PHF-tau were localized to senile plaques and neurofibrillary tangles (NFTs), respectively, which verified lack of crosstalk on the double-stained section. Localization of ubiquitin and PHF-tau was looked for at higher magnification in NFT-bearing neurons. Although these two epitopes were colocalized in some neurons, ubiquitin was not always present in PHF-tau positive NFTs. Discrepancy between PFH-tau and ubiquitin, verified inter- and intracellularly, may represent different stages of NFT formation. This is the first report of successful CARD amplification of two different fluorescent signals on double-labeling immunohistochemistry, which is now proved to be powerful in detecting epitopes in relation to AD-related lesions. Improved intensity over tenfold of the two fluorescent signals without crosstalk will expand the application of the multilabeling method with fluorochromes.     

Local cortical circuit model inferred from power-law distributed neuronal avalanches

How cortical neurons process information crucially depends on how their local circuits are organized. Spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called “neuronal avalanches”. They obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local circuits developed in slice cultures. However, the explicit network structure underlying the power-law statistics remains unclear. Here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. We demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. The resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. Interestingly, the recurrent synaptic connections formed by this wiring rule limit the number of cell assemblies embeddable in a neuron pool of given size. We investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. Our model suggests that local cortical circuits may have a more complex topological design than has previously been thought. Competing financial interests: The authors declare that they have no competing financial interests. Action Editor: Peter Latham     

Genetic vitamin E deficiency does not affect MPTP susceptibility in the mouse brain

Oxidative stress is involved in the degeneration of the nigrostriatal dopaminergic system in Parkinson's disease (PD). Vitamin E (alpha-tocopherol) is a potent antioxidant in the cell membrane that can trap free radicals and prohibit lipid peroxidation. The retention and secretion of vitamin E are regulated by alpha-tocopherol transfer protein (TTP) in the brain and liver. Dysfunction of TTP results in systemic deficiency of vitamin E in humans and mice, and increased oxidative stress in mouse brain. In this study, we investigated the effect of vitamin E deficiency in PD development by generating an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD using TTP knockout (TTP-/-) mice. Vitamin E concentration in the brains of TTP+/- mice was half that in TTP+/+ mice, and in TTP-/- mice, was undetectable. MPTP treatment tended to decrease striatal dopamine, but the effect was comparable and not significant in any of the three genotypes. Furthermore, the extent of loss of dopaminergic cell bodies in the substantia nigra did not differ among the groups. One the other hand, oral administration of vitamin E resulted in the partial protection of striatal dopaminergic terminals against MPTP toxicity. Our results suggest that vitamin E does not play a major protective role in MPTP-induced nigrostriatal dopaminergic neurodegeneration in the brain.     

Utility of tricalcium phosphate and osteogenic matrix cell sheet constructs for bone defect reconstruction

AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate (TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells (BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds (1) seeded with BMSCs; (2) wrapped with osteogenic matrix cell sheets; or (3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk.RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone non-union. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness (mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.     

Complex evolution of spike patterns during burst propagation through feed-forward networks

Stable signal transmission is crucial for information processing by the brain. Synfire-chains, defined as feed-forward networks of spiking neurons, are a well-studied class of circuit structure that can propagate a packet of single spikes while maintaining a fixed packet profile. Here, we studied the stable propagation of spike bursts, rather than single spike activities, in a feed-forward network of a general class of excitable bursting neurons. In contrast to single spikes, bursts can propagate stably without converging to any fixed profiles. Spike timings of bursts continue to change cyclically or irregularly during propagation depending on intrinsic properties of the neurons and the coupling strength of the network. To find the conditions under which bursts lose fixed profiles, we propose an analysis based on timing shifts of burst spikes similar to the phase response analysis of limit-cycle oscillators.