Bilal Malik

Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt–PCP–JNK pathway

Although the mechanism of Aβ action in the pathogenesis of Alzheimer’s disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt–planar cell polarity (PCP)–c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt–PCP–JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt–PCP–JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.

Loss of neuronal cell cycle control as a mechanism of neurodegeneration in the presenilin-1 Alzheimer's disease brain

Presenilin-1 (PS1) is a component of the β-catenin degradation machinery, and PS1 mutations linked to familial Alzheimer’s disease (FAD) represent a loss of this function, leading, in non-neuronal cells, to accumulation of cyclin D1, aberrant cell cycle activation and hyperproliferation. In post-mitotic neurons, cell cycle activation is thought to be abortive and initiate apoptosis, thus contributing to AD pathogenesis. Consequently, we tested here the hypothesis that, in the PS1 FAD brain, cyclin D1 accumulation may occur and lead to neuronal apoptosis secondary to an abortive entry into the cell cycle.We show that cyclin D1 is indeed upregulated in cortical neurons of mice expressing the knock-in PS1 FAD mutation M146V, as well as in temporal cortex of FAD patients expressing different PS1 mutations. Cyclin D1 upregulation in mutant neurons leads to cell cycle-driven apoptosis, a phenotype reversed by blocking entry into the cell cycle by small interfering RNA (siRNA) cyclin D1 downregulation and by treatment with the cell cycle inhibitor quercetin, but not by γ-secretase inhibition. Furthermore, β-catenin accumulates in neurons and adult hippocampus of PS1 KIM146V mice, as well as in temporal cortex of PS1 FAD patients, strongly suggesting an initiating role for aberrant β-catenin signalling in cell cycle-driven neuronal apoptosis.The present work identifies a novel mechanism by which PS1 mutations may exacerbate neurodegeneration in the FAD brain beyond dysregulation of γ-secretase activity, and it lends support to the notion that Alzheimer’s disease may be, at least in part, a disease driven by loss of cell cycle control.

Cell cycle‐driven neuronal apoptosis specifically linked to amyloid peptide Aβ1–42 exposure is not exacerbated in a mouse model of presenilin‐1 familial Alzheimer’s disease

We have shown previously that β‐catenin and cyclin D1 are up‐regulated in cortical neurons from homozygous mice carrying the familial Alzheimer’s disease (FAD) presenilin‐1 M146V mutation in a knock‐in model (PS1 KIM146V mice), leading to cell cycle‐associated apoptosis. Here, we have aimed to determine (i) whether this phenotype is present in heterozygous PS1 KIM146V mice, which reflects more accurately the PS1 FAD condition in humans and (ii) whether Aβ1–42, which is invariably present in the PS1 FAD brain and is thought to affect neuronal cell cycle kinetics, may contribute to the abnormal cell cycle/cell death phenotype seen in PS1 KIM146V mice. We demonstrate that cell cycle‐linked apoptosis occurs in heterozygous PS1 KIM146V post‐mitotic neurons. In addition, there is a significant Aβ‐associated increase in cell cycle and cell death that is not further modified by the PS1 KIM146V mutation. Our results are consistent with a cell cycle‐associated neurodegeneration model in the PS1 FAD brain in which the loss of PS1‐dependent β‐catenin regulatory function is sufficient to commit susceptible neurons to an abortive cell cycle, and may act synergistically with the Aβ cytotoxic challenge present in the PS1 FAD brain to expand the neuronal population susceptible to cell cycle‐driven apoptosis.

Oligomeric amyloid-β peptide affects the expression of genes involved in steroid and lipid metabolism in primary neurons.

Amyloid-β peptide (Aβ) is the principal component of plaques in the brains of patients with Alzheimers disease (AD), and the most toxic form of Aβ may be as soluble oligomers. We report here the results of a microarray study of gene expression profiles in primary mouse cortical neurons in response to oligomeric Aβ(1-42). A major and unexpected finding was the down-regulation of genes involved in the biosynthesis of cholesterol and other steroids and lipids (such as Fdft1, Fdps, Idi1, Ldr, Mvd, Mvk, Nsdhl, Sc4mol), the expression of which was verified by quantitative real-time RT-PCR (qPCR). The ATP-binding cassette gene Abca1, which has a major role in cholesterol transport in brain and other tissues and has been genetically linked to AD, was notably up-regulated. The possible involvement of cholesterol and other lipids in Aβ synthesis and action in Alzheimers disease has been studied and debated extensively but remains unresolved. These new data suggest that Aβ may influence steroid and lipid metabolism in neurons via multiple gene-expression changes.

An Aβ transcription signature

levels of CaN and pCREB in their hippocampus. Results: We found that CaN was increased and pCREB was significantly reduced in the hippocampus and cortex of AD patients and, to a lesser extent in MCI subjects as compared to non-demented individuals. Indeed there was a significant inverse correlation between pCREB levels and MMSE scores among AD patients, MCI subjects and normal individuals. Furthermore, icv injection of Ab oligomers that results in memory dysfunction in mice increased CaN and decreased pCREB, establishing a causal link between Ab-driven cognitive deficits and CaN hyper-activation. Conclusions: Collectively these results reveal that increased CaN activity may be central to Ab oligomer-induced memory deficits in AD and suggest it as a potential pharmacological target.

AbstractPoster presentation: Tuesday posterP3-410: Analysis of the role of presenilin 1 on tau homeostasis

PS2 for regulating PLC signalling. Methods: To evaluate the dependence of PLC on PSs we measured PLC activity (as the hydrolysis of phosphoinositides), Protein kinase C (PKC) activation in mouse embryonic fibroblast (MEF) cells lacking PS1, PS2, or both. Results: PLC activity, and activation of the calcium-dependent PKC isoforms, PKC and PKC were lower in PS1 and PS2 double knockout MEFs after 2,4,6-trimethylN-(m/o-3-trifluoromethylphenyl) benzenesulfonamide; oxotremorine-M (3M3FBS) stimulation of PLC. Protein levels of PKC and PKC were significantly lower in PS1 and PS2 double knockout MEFs, as compared to the other cell types. In contrast, levels of the calcium-independent PKC isoform were significantly elevated in PS1 and PS2 double knockout as well as in PS1 knockout fibroblasts. Since ER calcium signalling can be regulated through APP dependent gene transcription, we investigated whether similar mechanisms regulate the levels of PKC isoforms. Using APP knockout MEFs and siRNA silencing of APP expression in HEK cells we showed that PKC expression is regulated in an APP dependent manner. Pharmacological inhibition of -secretase with L-685,458 did not affect protein levels of the different PKC isoforms. Conclusion: Together, our results show a PS dependence for the activation of PLC and PKC as well as for levels of different PKC isoforms. Levels of PKC were dependent on the presence of PS1 but not PS2. Furthermore, PKC levels may be APP dependent but -secretase independent.