Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of ␤-amyloid (A␤) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The A␤ peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases. The endocytic system has been implicated in the cleavages leading to the formation of A␤. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated A␤ is released into the extracellular milieu are not clear. Here, we show that ␤-cleavage occurs in early endosomes followed by routing of A␤ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of A␤ peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD. multivesicular bodies ͉ rafts ͉ amyloid precursor protein ͉ ␤-secretase ͉ endocytosis A lzheimer's disease (AD) is a late-onset neurological disorder with progressive loss of memory and cognitive abilities as a result of excessive neurodegeneration (1). AD is characterized by extracellular aggregates of ␤-amyloid (A␤) peptides known as amyloid plaques (2). The A␤ peptide is derived from the sequential processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases. ␤-secretase [(␤-APP cleaving enzyme (BACE)] is a type-1 transmembrane aspartyl protease and is mainly localized to endosomes, lysosomes and the transGolgi network (3). ␥-Secretase is a multicomponent complex that is composed of presenilin-1͞presenilin-2, nicastrin, Aph-1, and PEN-2 (4) and is localized to the early secretory (5, 6) and the endocytic compartments (7,8). Nonamyloidogenic processing of APP involves ␣-secretase that cleaves APP inside the A␤ region, giving rise to the ␣-cleaved ectodomain, thus precluding the formation of A␤ (9). Hence, the availability of APP to either ␣-or ␤-secretase determines whether A␤ peptide will be generated. Lateral organization of membranes (10) and subcellular localization (11, 12) of the substrate and the secretases have been documented to regulate A␤ generation. Recent work suggests that ␤-secretase associates with lipid rafts, liquid-ordered domains in the membrane (13,14), and that integrity of raft domains is required for ␤-cleavage of APP to occur (ref. 10; see, however, ref. 15). ␣-Cleavage, in contrast, occurs outside raft domains (10). The ␥-secretase complex is also raft-associated (16); hence, amyloidogenic processing of APP could occur in clustered raft domains to generate A␤ (10). Inhibition of endocytosis reduces ␤-cleavage but not ␣-cleavage, suggesting that ␤-cleavage mainly occurs in endosomes (10,11,(17)(18)(19). Accumulation of A␤ peptides in extracellular...

Background: Brown adipose tissue (BAT) is abundant in small mammals and in newborns and helps them to survive cold temperatures. In adults, it had long been considered to be absent or at least of no relevance. Recent investigations, however, have fuelled interest in adult BAT. Objective: We aimed at (1) summarizing structural and physiological characteristics of BAT versus white adipose tissue (WAT); (2) discussing the development of the two adipose tissue types; (3) reviewing the data available from human studies on BAT, and (4) discussing the impact of aging. Methods: We summarize recent descriptions of BAT and WAT based on the original literature and reviews in the field, with emphasis on human BAT. Results: WAT and BAT have essentially antagonistic functions: WAT stores excess energy as triglycerides and BAT is specialized in the dissipation of energy through the production of heat. Considerable amounts of BAT are present in a substantial proportion of adult humans and relatively high quantities of BAT are associated with lower body weight. With increasing age, BAT decreases and body weight increases. Conclusions: Although the available cross-sectional data do not allow definite conclusions to be drawn concerning a causal relationship between loss of BAT and increasing body weight with advancing age or obesity-related metabolic disorders of older age, stimulation of BAT appears to be an attractive novel candidate target for the treatment of age-related obesity.

Protein kinase-B (PKB) and its target, the forkhead transcription factor like 1 (FKHRL1)/FoxO3a, have been suggested as regulators of neurotrophin-mediated cell survival in neuronal cells. We analyzed human neuroblastoma cells and found that FKHRL1 was phosphorylated, suggesting its inactivation. To study FKHRL1 function, we infected SH-EP and NB15 cells with a 4OH-tamoxifen-regulated FKHRL1(A3)ER tm transgene. Activation of FKHRL1 promoted cytochrome-c release and caspasedependent apoptosis. FKHRL1 induced TRAIL and the BH3-only proteins Noxa and Bim, implicating both extrinsic and intrinsic death pathways. However, expression of dnFADD did not inhibit FKHRL1-induced cell death, whereas Bcl2 protected against apoptosis. This excluded the death-receptor pathway and suggested that cell death decision is regulated by Bcl2-rheostat. Importantly, RNAi knockdown of Noxa or Bim decreased apoptosis, indicating that Noxa and Bim cooperate to mediate FKHRL1-induced cell death. We conclude that Noxa and Bim establish a connection between FKHRL1 and mitochondria, and that both BH3-only proteins are critically involved in FKHRL1-induced apoptosis in neuroblastoma.

Glioblastomas belong to the most aggressive human cancers with short survival times. Due to the blood-brain barrier, they are mostly inaccessible to traditional chemotherapy. We have recently shown that doxorubicin bound to polysorbate-coated nanoparticles crossed the intact blood-brain barrier, thus reaching therapeutic concentrations in the brain. Here, we investigated the therapeutic potential of this formulation of doxorubicin in vivo using an animal model created by implantation of 101/8 glioblastoma tumor in rat brains. Groups of 5-8 glioblastoma-bearing rats (total n ‫؍‬ 151) were subjected to 3 cycles of 1.5-2.5 mg/kg body weight of doxorubicin in different formulations, including doxorubicin bound to polysorbate-coated nanoparticles. The animals were analyzed for survival (% median increase of survival time, Kaplan-Meier). Preliminary histology including immunocytochemistry (glial fibrillary acidic protein, ezrin, proliferation and apoptosis) was also performed. Rats treated with doxorubicin bound to polysorbate-coated nanoparticles had significantly higher survival times compared with all other groups. Over 20% of the animals in this group showed a long-term remission. Preliminary histology confirmed lower tumor sizes and lower values for proliferation and apoptosis in this group. All groups of animals treated with polysorbatecontaining formulations also had a slight inflammatory reaction to the tumor. There was no indication of neurotoxicity. Additionally, binding to nanoparticles may reduce the systemic toxicity of doxorubicin. This study showed that therapy with doxorubicin bound to nanoparticles offers a therapeutic potential for the treatment of human glioblastoma.

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2␣. In contrast, HIF-1␣ served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1␣ resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2␣-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2␣-dependent erythrocytosis, whereas HIF-1␣ protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia. (Blood.

As effective stroke treatment by thrombolysis is bound to a narrow time window excluding most patients, numerous experimental treatment strategies have been developed to gain new options for stroke treatment. However, all approaches using neuroprotective agents that have been successfully evaluated in rodents have subsequently failed in clinical trials. Existing large animal models are of significant scientific value, but sometimes limited by ethical drawbacks and mostly do not allow for long-term observation. In this study, we are introducing a simple, but reliable stroke model using permanent middle cerebral artery occlusion in sheep. This model allows for control of ischemic lesion size and subsequent neurofunctional impact, and it is monitored by behavioral phenotyping, magnetic resonance imaging, and positron emission tomography. Neuropathologic and (immuno)-histologic investigations showed typical ischemic lesion patterns whereas commercially available antibodies against vascular, neuronal, astroglial, and microglial antigens were feasible for ovine brain specimens. Based on absent mortality in this study and uncomplicated species-appropriate housing, long-term studies can be realized with comparatively low expenditures. This model could be used as an alternative to existing large animal models, especially for longitudinal analyses of the safety and therapeutic impact of novel therapies in the field of translational stroke research.

Mutations in the oncogenes KRAS and BRAF have been identified as prognostic factors in patients with colorectal diseases and as predictors of negative outcome in epidermal growth factor receptor-targeted therapies. Therefore, accurate mutation detection in both genes, KRAS and BRAF, is of increasing clinical relevance. We aimed at optimizing allele-specific real-time PCR assays for the detection of common mutations in KRAS and the BRAF Val600Glu mutation using allele-specific PCR primers for allelic discrimination and probes (TaqMan) for quantification. Each reaction mix contains a co-amplified internal control to exclude false-negative results. Allele-specific real-time PCR assays were evaluated on plasmid model systems providing a mutation detection limit of 10 copies of mutant DNA in proportions as low as 1% of the total DNA. Furthermore, we analyzed 125 DNA samples prepared from archived, formalin-fixed, paraffin-embedded colorectal carcinomas and compared results with those obtained from direct-sequence analysis. All mutations determined by sequence analysis could be recovered by allele-specific PCR assays. In addition, allele-specific PCR assays clearly identified three additional samples affected by a mutation. We propose these allele-specific real-time PCR assays as a low-cost and fast diagnostic tool for accurate detection of KRAS and BRAF mutations that can be applied to clinical samples.

BackgroundFOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.MethodsThe effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.ResultsWe report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.ConclusionThe combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-224) contains supplementary material, which is available to authorized users.