SummaryAlzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased β-amyloid production, altered cytokine release, and dysregulated Ca2+ homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca2+ transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.

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Seladin-1 is a neuroprotective protein selectively downregulated in brain regions affected in Alzheimer disease (AD).Seladin-1 protects cells against ␤-amyloid (A␤) peptide 42-and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the ␤-secretase (BACE1) function and ␤-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that ϳ60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or A␤ secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized ␥-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced ␤-amyloidogenic processing of APP and ultimately increased A␤ production. These adverse changes associated with decreased cell viability in seladin-1 siRNAtransfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased ␤-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis. Alzheimer disease (AD)2 is the most common neurodegenerative disorder leading to dementia. It is neuropathologically characterized by extracellular amyloid plaques as well as intraneuronal neurofibrillary tangles, composed of ␤-amyloid peptide (A␤) and hyper-phosphorylated Tau, respectively. A␤ is generated from the ␤-amyloid precursor protein (APP) after sequential cleavages by ␤-and ␥-secretases (1). Importantly, mutations in APP and PSEN1 and -2 genes have been shown to increase the production of the 42-amino acid-long A␤42 and to cause the familial autosomal dominant form of AD. Based on this notion, it has been proposed that aberrant metabolism of APP is the initiating event in AD pathogenesis, which is followed by other adverse events, such as inflammation, oxidative stress, and neurofibrillary tangle formation (2). Recently, this model was challenged by a dual pathway hypothesis, which proposed that A␤ and Tau can actually be linked by separate mechanisms driven by common upstream initiators, such as apolipoprotein E (apoE) or glycogen synthase kinase-3 (3). Therefore, considering th...

AD risk loci polygenically contribute to Aβ pathology in the CSF and temporal cortex, and this effect is potentially associated with increased γ-secretase activity.

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Agonist-induced activation of the ␦-opioid receptor (␦OR) was recently shown to augment ␤-and ␥-secretase activities, which increased the production of ␤-amyloid peptide (A␤), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the ␦OR variant with a phenylalanine at position 27 (␦OR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (␦OR-Cys27) variant. For this study, we expressed these variants in human SH-SY5Y and HEK293 cells expressing exogenous or endogenous amyloid precursor protein (APP) and assessed the effects on APP processing. Expression of ␦OR-Cys27, but not ␦OR-Phe27, resulted in a robust accumulation of the APP C83 C-terminal fragment and the APP intracellular domain, while the total soluble APP and, particularly, the ␤-amyloid 40 levels were decreased. These changes upon ␦OR-Cys27 expression coincided with decreased localization of APP C-terminal fragments in late endosomes and lysosomes. Importantly, a long-term treatment with a subset of ␦OR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the ␦OR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the ␦OR-Cys27 variant affects APP processing through altered endocytic trafficking of APP.Alzheimer's disease (AD) is the most common neurodegenerative disorder in the aging population. It is neuropathologically characterized by well-known hallmarks, such as extracellular amyloid plaques and intraneuronal neurofibrillary tangles, composed of ␤-amyloid peptide (A␤) and hyperphosphorylated tau, respectively. A␤ is generated from the amyloid precursor protein (APP) after sequential cleavages by ␤ (BACE1)-and ␥-secretases. It is a well-established fact that the molecular mechanisms underlying AD pathogenesis involve alterations in APP processing which lead to increased A␤ production or, alternatively, decreased enzymatic degradation and clearance of A␤ (39). To facilitate the design of novel intervention approaches for AD, it is important to identify and functionally characterize genetic alterations which play a role in AD pathogenesis. A plausible candidate in this context is the OPRD1 gene, encoding the ␦-opioid receptor (␦OR), which was recently shown to form a complex with ␤-and ␥-secretases (28, 40). Following agonist-induced activation, ␦OR mediates coendocytic sorting of this complex to late endosomes and lysosomes (LEL) (28,40), in which compartments A␤ production primarily takes place. Conversely, ␤-and ␥-secretase activities as well as A␤ levels were found to be significantly reduced in transgenic APP/PS1⌬E9 mice (overexpressing human APP with the Swedish mutation together with human presenilin-1 harboring the exon 9 deletion) treated with a selective nonpeptide antagonist for ␦OR (40). These results suggest that the amyloidogenic processing of APP is enhanced upon ␦OR activation and that the selective antagonist-mediated modulation of ␦OR ma...

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