Alzheimer's risk variants in the clusterin gene are associated with alternative splicing

Genetic variation in CLU encoding clusterin has been associated with Alzheimer's disease (AD) through replicated genome-wide studies, but the underlying mechanisms remain unknown. Following earlier reports that tightly regulated CLU alternative transcripts have different functions, we tested CLU single-nucleotide polymorphisms (SNPs), including those associated with AD for quantitative effects on individual alternative transcripts. In 190 temporal lobe samples without pathology, we found that the risk allele of the AD-associated SNP rs9331888 increases the relative abundance of transcript NM_203339 (P=4.3 × 10−12). Using an independent set of 115 AD and control samples, we replicated this result (P=0.0014) and further observed that multiple CLU transcripts are at higher levels in AD compared with controls. The AD SNP rs9331888 is located in the first exon of NM_203339 and therefore, it is a functional candidate for the observed effects. We tested this hypothesis by in vitro dual luciferase assays using SK-N-SH cells and mouse primary cortical neurons and found allelic effects on enhancer function, consistent with our results on post-mortem human brain. These results suggest a biological mechanism for the genetic association of CLU with AD risk and indicate that rs9331888 is one of the functional DNA variants underlying this association

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-β (Aβ) deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.Boryana S Stamova, Michelle Apperson, Wynn L Walker, Yingfang Tian, Huichun Xu, Peter Adamczy, Xinhua Zhan, Da-Zhi Liu, Bradley P Ander, Isaac H Liao, Jeffrey P Gregg, Renee J Turner, Glen Jickling, Lisa Lit and Frank R Shar

Modes of Aβ toxicity in Alzheimer’s disease

Alzheimer’s disease (AD) is reaching epidemic proportions, yet a cure is not yet available. While the genetic causes of the rare familial inherited forms of AD are understood, the causes of the sporadic forms of the disease are not. Histopathologically, these two forms of AD are indistinguishable: they are characterized by amyloid-β (Aβ) peptide-containing amyloid plaques and tau-containing neurofibrillary tangles. In this review we compare AD to frontotemporal dementia (FTD), a subset of which is characterized by tau deposition in the absence of overt plaques. A host of transgenic animal AD models have been established through the expression of human proteins with pathogenic mutations previously identified in familial AD and FTD. Determining how these mutant proteins cause disease in vivo should contribute to an understanding of the causes of the more frequent sporadic forms. We discuss the insight transgenic animal models have provided into Aβ and tau toxicity, also with regards to mitochondrial function and the crucial role tau plays in mediating Aβ toxicity. We also discuss the role of miRNAs in mediating the toxic effects of the Aβ peptide

Functional genomics dissects pathomechanisms in tauopathies: mitosis failure and unfolded protein response

Background: Alzheimer's disease (AD) is characterized by β-amyloid (Aβ) peptide-containing plaques and tau-containing neurofibrillary tangles. By intracerebral injection of Aβ, both pathologies have been combined in P301L tau mutant mice. Furthermore, in cell culture, Aβ induces tau aggregation. While both Aβ and mutant tau cause neuronal dysfunction, their modes of action are only vaguely understood. Methods: To determine which processes are disrupted by Aβ and/or P301L mutant tau, we used transcriptomic and proteomic techniques followed by functional validation and analysis of human AD tissue. Results: Our transcriptomic study in the SH-SY5Y cell culture system revealed that Aβ and P301L tau expression independently affect genes controlling the cell cycle and cell proliferation. Proteomics applied to Aβ-treated P301L tau-expressing SH-SY5Y cells and the amygdala of Aβ-injected P301L transgenic mice revealed that a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. Among the proteins identified was valosin-containing protein (VCP), a component of the quality control system during endoplasmic reticulum stress. Mutations in VCP have recently been linked to frontotemporal dementia. Conclusion: Our data support the mitosis failure hypothesis that claims that aberrant cell cycle reentry of postmitotic neurons induces apoptosis. Furthermore, our data underline a role of Aβ in the stress response associated with protein folding

Target gene repression mediated by miRNAs miR-181c and miR-9 both of which are down-regulated by amyloid-β.

MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that are essential for normal brain development and function. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer's disease (AD) that is characterized by amyloid-β (Aβ) and tau deposition in brain. How deregulated miRNAs contribute to AD is not understood, as their dysfunction could be both a cause and a consequence of disease. To address this question we had previously profiled miRNAs in models of AD. This identified miR-9 and -181c as being down-regulated by Aβ in hippocampal cultures. Interestingly, there was a remarkable overlap with those miRNAs that are deregulated in Aβ-depositing APP23 transgenic mice and in human AD tissue. While the Aβ precursor protein APP itself is a target of miRNA regulation, the challenge resides in identifying further targets. Here, we expand the repertoire of miRNA target genes by identifying the 3' untranslated regions (3' UTRs) of TGFBI, TRIM2, SIRT1 and BTBD3 as being repressed by miR-9 and -181c, either alone or in combination. Taken together, our study identifies putative target genes of miRNAs miR-9 and 181c, which may function in brain homeostasis and disease pathogenesis

Oligomeric and fibrillar species of β-amyloid (Aβ42) both impair mitochondrial function in P301L tau transgenic mice

We recently provided evidence for a mitochondrial dysfunction in P301L tau transgenic mice, a strain modeling the tau pathology of Alzheimer's disease (AD) and frontotemporal dementia (FTD). In addition to tau aggregates, the AD brain is further characterized by Aβ peptide-containing plaques. When we addressed the role of Aβ, this indicated a synergistic action of tau and Aβ pathology on the mitochondria. In the present study, we compared the toxicity of different Aβ42 conformations in light of recent studies suggesting that oligomeric rather than fibrillar Aβ might be the actual toxic species. Interestingly, both oligomeric and fibrillar, but not disaggregated (mainly monomeric) Aβ42 caused a decreased mitochondrial membrane potential in cortical brain cells obtained from FTD P301L tau transgenic mice. This was not observed with cerebellar preparations indicating selective vulnerability of cortical neurons. Furthermore, we found reductions in state 3 respiration, the respiratory control ratio, and uncoupled respiration when incubating P301L tau mitochondria either with oligomeric or fibrillar preparations of Aβ42. Finally, we found that aging specifically increased the sensitivity of mitochondria to oligomeric Aβ42 damage indicating that oligomeric and fibrillar Aβ42 are both toxic, but exert different degrees of toxicity