MicroRNAs and Alzheimer's Disease Mouse Models: Current Insights and Future Research Avenues

Evidence from clinical trials as well as from studies performed in animal models suggest that both amyloid and tau pathologies function in concert with other factors to cause the severe neurodegeneration and dementia in Alzheimer's disease (AD) patients. Accumulating data in the literature suggest that microRNAs (miRNAs) could be such factors. These conserved, small nonprotein-coding RNAs are essential for neuronal function and survival and have been implicated in the regulation of key genes involved in genetic and sporadic AD. The study of miRNA changes in AD mouse models provides an appealing approach to address the cause-consequence relationship between miRNA dysfunction and AD pathology in humans. Mouse models also provide attractive tools to validate miRNA targets in vivo and provide unique platforms to study the role of specific miRNA-dependent gene pathways in disease. Finally, mouse models may be exploited for miRNA diagnostics in the fight against AD

miRNA-dependent target regulation: functional characterization of single-nucleotide polymorphisms identified in genome-wide association studies of Alzheimer’s disease

Other miRNA targeting sites identified by less stringent analysis near rs7143400-C/G, rs2847655-T/C, rs610923-C/A and rs9909-G/C. A summary of the genes, PolymiRTSs, effects of minor alleles, targeting miRNAs and miRNA expression alterations observed in AD (when available; refer to the cited references). The grayed miRNAs were also found in the stringent screening described in Fig. 2a in the main text. (XLS 23 kb

Synphilin-1 Enhances α-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner

© 2010 Büttner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Parkinson’s disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive. Methodology/Principal Findings: We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation. Conclusions/Significance: Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.This work was supported by grants from IWT-Vlaanderen (SBO NEURO-TARGET), the K.U.Leuven Research Fund (K.U.Leuven BOF-IOF) and K.U.Leuven R&D to JW, a Tournesol grant from Egide (Partenariat Hubert Curien) in France in collaboration with the Flemish Ministry of Education and the Fund of Scientific Research of Flanders (FWO) in Belgium to JW, MCG and LB, a shared PhD fellowship of the EU-Marie Curie PhD Graduate School NEURAD to JW, MCG and LB, grants of the Austrian Science Fund FWF (Austria) to FM and DR (S-9304-B05), to FM and SB (LIPOTOX), and to SB (T-414-B09; Hertha-Firnberg Fellowship) and an EMBO Installation Grant, a Marie Curie IRG, and a grant of the Fundação para a Ciência e Tecnologia (PTDC/SAU-NEU/105215/2008) to TFO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson

Parkinson's disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated α-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of α-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced α-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for α-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the α-synuclein membrane interaction. In addition, α-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of α-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes

Gene Network and Pathway Analysis of Mice with Conditional Ablation of Dicer in Post-Mitotic Neurons

<div><h3>Background</h3><p>The small non-protein-coding microRNAs (miRNAs) have emerged as critical regulators of neuronal differentiation, identity and survival. To date, however, little is known about the genes and molecular networks regulated by neuronal miRNAs <em>in vivo</em>, particularly in the adult mammalian brain.</p> <h3>Methodology/Principal Findings</h3><p>We analyzed whole genome microarrays from mice lacking <em>Dicer</em>, the enzyme responsible for miRNA production, specifically in postnatal forebrain neurons. A total of 755 mRNA transcripts were significantly (P<0.05, FDR<0.25) misregulated in the conditional <em>Dicer</em> knockout mice. Ten genes, including Tnrc6c, Dnmt3a, and Limk1, were validated by real time quantitative RT-PCR. Upregulated transcripts were enriched in nonneuronal genes, which is consistent with previous studies <em>in vitro</em>. Microarray data mining showed that upregulated genes were enriched in biological processes related to gene expression regulation, while downregulated genes were associated with neuronal functions. Molecular pathways associated with neurological disorders, cellular organization and cellular maintenance were altered in the <em>Dicer</em> mutant mice. Numerous miRNA target sites were enriched in the 3′untranslated region (3′UTR) of upregulated genes, the most significant corresponding to the miR-124 seed sequence. Interestingly, our results suggest that, in addition to miR-124, a large fraction of the neuronal miRNome participates, by order of abundance, in coordinated gene expression regulation and neuronal maintenance.</p> <h3>Conclusions/Significance</h3><p>Taken together, these results provide new clues into the role of specific miRNA pathways in the regulation of brain identity and maintenance in adult mice.</p> </div

Multiple microRNA regulation of lipoprotein lipase gene abolished by 3'UTR polymorphisms in a triglyceride-lowering haplotype harboring p.Ser474Ter

International audienceBACKGROUND: Lipoprotein lipase (LPL) is a key enzyme in triglyceride (TG) metabolism. LPL gene single nucleotide polymorphisms (SNPs) are associated with TG concentrations however the functionality of many of these SNPs remains poorly understood. MicroRNAs (miR) exert post-transcriptional down-regulation and their target sequence on the 3'UTR may be altered by SNPs. We therefore investigated whether LPL 3'UTR SNPs could modulate plasma TG concentration through the alteration of miR binding-sites. METHODS AND RESULTS: We performed genetic association studies of LPL 3'UTR SNPs with TG concentrations in 271 type 2 diabetic patients and in general population samples (2997 individuals). A specific LPL haplotype (Hap4) was associated with lower plasma TG concentration (TG-0.18, IC95% [-0.30, -0.07] mmol/L or logTG-0.13, IC95% [-0.18, -0.08], p = 4.77.10(-8)) in the meta-analysis. Hap4 comprises seven 3'UTR SNP minor alleles and p.Ser474Ter (rs328) a well-documented nonsense mutation associated with low TG concentration although by an unknown mechanism so far. Bio-informatic studies identified several putative miRNA binding-sites on the wild-type Hap1 haplotype, lost on Hap4. Functional validation performed in HEK-293T cells using luciferase expression constructs with various LPL 3'UTR allele combinations demonstrated a binding of miR-29, miR-1277 and miR-410 on Hap1, lost on Hap4. This loss of specific miR binding-site in presence of Hap4 was independent of the allelic variation of p.Ser474Ter (rs328). CONCLUSIONS: We report the regulation of LPL by the miR-29, miR-1277 and miR-410 that is lost in presence of Hap4, a specific LPL TG-lowering haplotype. Consequently p.Ser474Ter association with TG concentration could be at least partially explained by its strong linkage disequilibrium with these functional 3'UTR SNPs

Validation of neurofilament changes in the <i>Dicer</i> cKO mice.

<p>(<b>A</b>) Immunohistochemistry of Nefl in the cortex (a–f) and the dentate gyrus (g–l) of control (a–c, g–i) and <i>Dicer</i> cKO (d–f, j–l) mice. Note the reduction in Nefl signal (in green) in the mutant mice (highlighted in white square). Of mention, changes in the cortex were more pronounced in 13 week-old <i>Dicer</i> cKO mice (shown here) when compared to age-matched controls. Dentate gyrus stainings gave similar results in both 9–10.5 and 13 week-old mice (9.5 week-old mouse shown here). DAPI (nuclei) stainings are shown in blue. Scale bars 20 µm (a–l). (<b>B</b>) Representative (n = 5) western blot analysis of Nefl and Nefh in cortex samples of 9–10.5 week-old control and <i>Dicer</i> cKO mice. Note that only Nefl was downregulated in the <i>Dicer</i> cKO mice. Gapdh was used as internal loading control.</p