The amyloid ␤-peptide (A␤) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by A␤ and accumulation of A␤ has been detected in mitochondria. Because A␤ is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial A␤ uptake. Our results from rat mitochondria show that A␤ is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed A␤ association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of A␤ to mitochondrial cristae. A similar distribution pattern of A␤ in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for A␤ in mitochondria and demonstrate both in vitro and in vivo that A␤ is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied A␤ can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of A␤, a peptide considered to be of major significance in Alzheimer's disease.Alzheimer disease ͉ protein import ͉ human brain biopsies T he amyloid-␤ peptide (A␤) is produced by regulated intramembrane proteolysis of the A␤ precursor protein (APP) by the sequential cleavage by ␤-and ␥-secretases (1-2). Plaques consisting mainly of aggregated A␤ are detected in the neuropil in aged subjects and in particular in subjects with Alzheimer's disease (AD) (3-5). Recently, it has been argued that it is A␤ oligomers and fibrils that cause toxicity, loss of synapses, and ultimately neuronal death (6-9). The exact mechanisms of how A␤ damages the neurons are still unknown; however, several lines of evidence implicate that A␤ exerts its toxicity intracellularly (10, 11) and point toward a role of mitochondria in this process (12). It has been reported that mitochondrial A␤ accumulation impairs neuronal function and, thus, contributes to cellular dysfunction in a transgenic APP mouse model (13). It is noteworthy that in AD at an early stage there is already a reduction in the number of mitochondria (14), the brain glucose metabolism is decreased (15), and the activities of both tricarboxylic acid cycle enzymes (16) and cytochrome c oxidase (COX) are reduced (17)(18)(19)(20). In vitro studies with isolated mitochondria suggest that A␤ 1-42 inhibits COX activity in a copper-dependent manner (21). Furthermore, mitochondrial A␤-binding alcohol dehydrogenase (ABAD) has been found to be up-regulated in neurons from AD patients (22), and A␤ has been shown to interact with ABAD, resulting in free radical production and neuronal apoptosis. Recently, we have shown that preseque...

The ribosomal S6 protein kinase p70 S6 kinase is known for its role in modulating cell-cycle progression, cell size, and cell survival. In response to mitogen stimulation, p70 S6 kinase activation up-regulates ribosomal biosynthesis and enhances the translational capacity of the cell. In Alzheimer's disease (AD), there is a marked increase in total tau protein in the form of abnormally hyperphosphorylated tau (PHF-tau) in neurons with neurofibrillary tangles (NFTs). In the present study, we investigated whether p70 S6 kinase activation is associated with PHF-tau accumulation in AD. By immunohistochemistry, we found that the levels of phosphorylated p70 S6 kinase (at Thr389 or at Thr421/Ser424) were increased in accordance with the progressive sequence of neurofibrillary changes according to Braak's criteria. Confocal microscopy showed that in AD brain, phosphorylated p70 S6 kinase appeared especially in neurons that are known to later develop NFTs. This pattern of neurons showed dot-like structures of phosphorylated p70 S6 kinase and hyperphosphorylated tau, which partially correlated with rab5 (endosome marker), lamp-1 (lysosome marker), and ubiquitin (ubiquitin-proteasomal system marker). By indirect enzyme-linked immunosorbent assay, phosphorylated p70 S6 kinase (Thr389 or Thr421/Ser424), total tau, and PHF-tau were found to be significantly increased in AD brain as compared to control cases. The levels of total p70 S6 kinase and p70 S6 kinase phosphorylated at Thr421/Ser424 showed significant correlations with the levels of both total tau and PHF-tau. Regression analyses revealed a significant dependence of total tau or PHF-tau on p70 S6 kinase phosphorylated at Thr421/Ser424 rather than at Thr389. The levels of ribosomal protein S6 as well as the levels of markers for the proteolytic system were also significantly increased in AD as compared to control brain. Using a SH-SY5Y neuroblastoma cell model, we found that 100 micro mol/L zinc sulfate could induce p70 S6 kinase phosphorylation and activation, in particular at Thr421/Ser424. This up-regulation of the activated kinase resulted in an increased expression and phosphorylation of tau. Pretreatment of cells with rapamycin (an inhibitor of FRAP/mTOR which is the immediate upstream kinase of the p70 S6 kinase) attenuated the effects induced by zinc. In primary cultured neurons of rat cortical cortex, zinc sulfate treatment could repeat p70 S6 kinase phosphorylation and activation at Thr421/Ser424, followed by increased expression and phosphorylation of tau. Taken together, these data suggest that activated p70 S6 kinase could mediate an up-regulation of tau translation. The partial co-localization of phosphorylated p70 S6 kinase with rab5, lamp-1 and ubiquitin, or PHF-tau with ubiquitin suggests that the activated proteolytic system might not be sufficient to degrade the over-produced and over-phosphorylated tau protein. A p70 S6 kinase modulated up-regulation of tau translation might contribute to PHF-tau accumulation in neurons with neurofibrillary changes.

Rabbit monoclonal antibodies are reliable markers of SSTRs in somatotroph adenomas. SSTR2a expression correlated with the response to octreotide and was reduced after octreotide treatment, indicating the need for adjustment when SSTR2a expression is correlated with baseline characteristics. Evaluation of SSTR subtypes may be an important aspect of improving the medical treatment for acromegaly.

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BackgroundDiffusion kurtosis imaging (DKI) allows for assessment of diffusion influenced by microcellular structures. We analyzed DKI in suspected low-grade gliomas prior to histopathological diagnosis. The aim was to investigate if diffusion parameters in the perilesional normal-appearing white matter (NAWM) differed from contralesional white matter, and to investigate differences between glioma malignancy grades II and III and glioma subtypes (astrocytomas and oligodendrogliomas).Patients and methodsForty-eight patients with suspected low-grade glioma were prospectively recruited to this institutional review board-approved study and investigated with preoperative DKI at 3T after written informed consent. Patients with histologically proven glioma grades II or III were further analyzed (n=35). Regions of interest (ROIs) were delineated on T2FLAIR images and co-registered to diffusion MRI parameter maps. Mean DKI data were compared between perilesional and contralesional NAWM (student’s t-test for dependent samples, Wilcoxon matched pairs test). Histogram DKI data were compared between glioma types and glioma grades (multiple comparisons of mean ranks for all groups). The discriminating potential for DKI in assessing glioma type and grade was assessed with receiver operating characteristics (ROC) curves.ResultsThere were significant differences in all mean DKI variables between perilesional and contralesional NAWM (p=<0.000), except for axial kurtosis (p=0.099). Forty-four histogram variables differed significantly between glioma grades II (n=23) and III (n=12) (p=0.003−0.048) and 10 variables differed significantly between ACs (n=18) and ODs (n=17) (p=0.011−0.050). ROC curves of the best discriminating variables had an area under the curve (AUC) of 0.657−0.815.ConclusionsMean DKI variables in perilesional NAWM differ significantly from contralesional NAWM, suggesting altered microstructure by tumor infiltration not depicted on morphological MRI. Histogram analysis of DKI data identifies differences between glioma grades and subtypes.