Genetic ablation of Dicer in adult forebrain neurons results in abnormal tau hyperphosphorylation and neurodegeneration
ABSTRACT: The type III RNase Dicer is responsible for the maturation and function of microRNA (miRNA) molecules in the cell. It is now well documented that Dicer and the fine-tuning of the miRNA gene network are important for neuronal integrity. However, the underlying mechanisms involved in neuronal death, particularly in the adult brain, remain poorly defined. Here, we show that absence of Dicer in the adult forebrain is accompanied by a mixed neurodegenerative phenotype. While neuronal loss is observed in the hippocampus, cellular shrinkage is predominant in the cortex. Interestingly, neuronal degeneration coincides with the hyperphosphorylation of endogenous tau at several epitopes previously associated with neurofibrillary pathology. Transcriptome analysis of enzymes involved in tau phosphorylation identified ERK1 as one of the candidate kinases responsible for this event in vivo. We further demonstrate that miRNAs belonging to the miR-15 family are potent regulators of ERK1 expression in mouse neuronal cells and co-expressed with ERK1/2 in vivo. Last, we show that miR-15a is specifically downregulated in Alzheimer’s disease brain. In sum, these results support the hypothesis that changes in the miRNA network may contribute to a neurodegenerative phenotype by affecting tau phosphorylation. Dicer KO vs. control
Project description:The type III RNase Dicer is responsible for the maturation and function of microRNA (miRNA) molecules in the cell. It is now well documented that Dicer and the fine-tuning of the miRNA gene network are important for neuronal integrity. However, the underlying mechanisms involved in neuronal death, particularly in the adult brain, remain poorly defined. Here, we show that absence of Dicer in the adult forebrain is accompanied by a mixed neurodegenerative phenotype. While neuronal loss is observed in the hippocampus, cellular shrinkage is predominant in the cortex. Interestingly, neuronal degeneration coincides with the hyperphosphorylation of endogenous tau at several epitopes previously associated with neurofibrillary pathology. Transcriptome analysis of enzymes involved in tau phosphorylation identified ERK1 as one of the candidate kinases responsible for this event in vivo. We further demonstrate that miRNAs belonging to the miR-15 family are potent regulators of ERK1 expression in mouse neuronal cells and co-expressed with ERK1/2 in vivo. Last, we show that miR-15a is specifically downregulated in Alzheimer’s disease brain. In sum, these results support the hypothesis that changes in the miRNA network may contribute to a neurodegenerative phenotype by affecting tau phosphorylation. Overall design: Dicer KO vs. control
Project description:Early-onset Alzheimer’s disease-like pathology in Down syndrome (DS, trisomy 21) is commonly attributed to an increased dosage of the amyloid precursor protein (APP) gene. To test this central tenet of the amyloid-cascade hypothesis we deleted the supernumerary copy of the APP gene in trisomic DS iPSC, or upregulated APP expression in euploid human pluripotent stem cell lines with dCas9-VP64, and subjected these lines to prolonged cortical neural differentiation. Our data reveal that increased APP gene dosage and expression is necessary and sufficient for increased beta-amyloid production and pyroglutamate(E3)-containing plaque deposition, but is neither sufficient nor required for tau hyperphosphorylation, neurofibrillary tangle formation, or increased oxidative stress-induced apoptosis in neurons. Transcriptome comparisons of the isogenic neurons demonstrates that the supernumerary APP gene copy has profound temporally-modulated genome-wide effects on gene expression during differentiation and maturation of DS neuronal cultures that link APP function to regulation of genes involved in neuronal synaptic function and outgrowth of neuronal processes. Collectively, our data reveal that APP plays an important role in the amyloidogenic aspects of Alzheimer’s disease, but challenge the hypothesis that increased APP levels are solely responsible for hyperphosphorylation of tau or enhanced oxidative stress-induced neuronal cell death in Down syndrome associated AD-pathogenesis. Overall design: 34 samples
Project description:TRBP has two known functions as Dicer co-factor and PKR inhibitor. However, the role of TRBP in miRNA biogenesis is controversial and its regulation of PKR in mitosis remains unexplored. Here, we generate TRBP KO HeLa cells and find that TRBP depletion alters Dicer processing sites of a subset of miRNAs, but does not affect Dicer stability, miRNA abundance, or Argonaute loading. By generating PACT, another Dicer interactor, and TRBP/PACT double-KO cells, we further show that TRBP and PACT do not functionally compensate each other and that only TRBP contributes to Dicer processing. We also report that TRBP is hyperphosphorylated by JNK in M phase when PKR is activated by cellular dsRNAs. Hyperphosphorylation potentiates the inhibitory activity of TRBP on PKR, suppressing PKR in M-G1 transition. By generating the first human TRBP KO, our study clarifies the role of TRBP and unveils negative feedback regulation of PKR through TRBP phosphorylation. small RNAs of wild type, TRBP knockout, PACT knockout and TRBP/PACT double knockout cells were sequenced by Illumina Miseq.
Project description:Transcriptional profiling of C. elegans strains grown in the presence and absence of ethosuximide. Two mutant alleles of dnj-14 (tm3223 and ok237) and two wild type control strains (N2 and CZ1200) were analysed. dnj-14 is the worm orthologue of the human DNAJC5 gene, mutations in which cause the neurodegenerative disease, adult onset neuronal ceroid lipofucinosis. Ethosuximide ameliorates neurodegenerative phenotypes in the dnj-14 model and also in C. elegans neurodegenerative disease models based on expression of human mutant Tau and TDP-43 proteins. It is hoped that transcriptional profiling of the effect of ethosuximide on gene expression in both wild type and mutant strains might help to identify the mechanism by which ethosuximide exerts its generally neuroprotective mechanism of action. The untreated control samples of this study are also used in the experiment with accession E-MTAB-3147.
Project description:Background: Activation of microglia, the resident immune cells of the central nervous system, is a prominent pathological hallmark of Alzheimer’s disease (AD). However, the gene expression changes underlying microglia activation in response to tau pathology remain elusive. Furthermore, it is not clear how murine gene expression changes relate to human gene expression networks. Methods: Microglia cells were isolated from rTg4510 tau transgenic mice and gene expression was profiled using RNA sequencing. Four age groups of mice (2-, 4-, 6-, and 8-months) were analyzed to capture longitudinal gene expression changes that correspond to varying levels of pathology, from minimal tau accumulation to massive neuronal loss. Statistical and system biology approaches were used to analyze the genes and pathways that underlie microglia activation. Differentially expressed genes were compared to human brain co-expression networks. Results:Statistical analysis of RNAseq data indicated that more than 4000 genes were differentially expressed in rTg4510 microglia compared to wild type microglia, with the majority of gene expression changes occurring between 2- and 4-months of age. These genes belong to four major clusters based on their temporal expression pattern. Genes involved in innate immunity were continuously up-regulated, whereas genes involved in the glutamatergic synapse were down-regulated. Up-regulated innate inflammatory pathways included NF-κB signaling, cytokine-cytokine receptor interaction, lysosome, oxidative phosphorylation, and phagosome. NF-κB and cytokine signaling were among the earliest pathways activated, likely driven by the RELA, STAT1 and STAT6 transcription factors. The expression of many AD associated genes such as APOE and TREM2 was also altered in rTg4510 microglia cells. Differentially expressed genes in rTg4510 microglia were enriched in human neurodegenerative disease associated pathways, including Alzheimer’s, Parkinson’s, and Huntington’s diseases, and highly overlapped with the microglia and endothelial modules of human brain transcriptional co-expression networks. Conclusion: This study revealed temporal transcriptome alterations in microglia cells in response to pathological tau perturbation and provides insights into the molecular changes underlying microglia activation during tau mediated neurodegeneration. Overall design: Compare the microglial cell gene expression changes in rTg4510 tau transgenic mice and wild type at four age groups (2-, 4-, 6-, and 8-months)
Project description:Alzheimer’s disease (AD) is a neurodegenerative disease displaying plaques formed by the neurotoxic amyloid β-peptide (Aβ) and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relates to the massive neuronal death that occurs in AD brains remain elusive. To identify proteins, the levels of which are affected by increased Aβ levels, we performed a proteomic analysis of the AD mouse model APPsw and compared it to that of APPsw mice lacking the major Aβ degrading enzyme neprilysin. This was achieved by establishing an LC-MS/MS method to analyze brain homogenate, using an 18O-labeled internal standard to accurately quantify the protein levels. By this approach we identified approximately 600 proteins and could quantify the levels of 300 of these. Interestingly, several proteins, including some not previously reported to be associated with AD, were identified. The relevance of these proteins for AD warrants further research.
Project description:DICER has a well-characterized role in the processing of microRNAs (miRNAs) and small interfering RNAs (siRNA) that are important for post-transcriptional gene regulation. Emerging evidence suggests that DICER also has several non-canonical functions beyond miRNA/siRNA biogenesis, for example in transcriptional gene silencing at the chromatin level, as well as in RNA degradation and maintenance of genomic integrity. We have shown that the function of DICER in germ cells is essential for normal spermatogenesis; male mice lacking DICER in postnatal male germ cells are infertile due to severe defects in haploid differentiation. To better understand the function of DICER in male germ cells, we immunoprecipitated DICER from juvenile mouse testes and performed mass spectrometric analysis to identify DICER-interacting proteins.
Project description:The activity of the endoribonuclease Dicer is crucial to produce the mature form of most microRNAs (miRNAs). Recent studies indicate that lack of miRNAs in different neuronal types results in a range of anatomical and behavioural phenotypes. In the present study we aimed to investigate the developmental and metabolic consequences of miRNA ablation in hypothalamic POMC neurons studying mice with a conditional deletion of Dicer in this population of neurons (POMCDicerKO). These mice exhibited a progressive obese phenotype characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that was complete by 6 weeks of age. Furthermore, immunohistochemistry and gene expression studies in control C57Bl/6 adult mice showed that Dicer was expressed in relevant hypothalamic areas and neurons implicated in energy balance, and that its expression was regulated by nutrient availability. Collectively, our results highlight a crucial role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity. Total RNA was extracted from hypothalamic microdissections of 2-week old control and POMCDicerKO mice using the RNeasy micro spin columns (Qiagen, Venlo, The Netherlands). Ten micrograms of total RNA were converted into cRNA, biotinylated, fragmented, and hybridized to GeneChip Mouse Genome 430 2.0 (Affymetrix, Santa Clara, CA). Six microarrays were hybridized with three independent samples from control and POMCDicerKO mice.
Project description:microRNA (miRNA)-mediated gene silencing is commonly deregulated in a wide variety of diseases. Therefore, a better understanding of how miRNAs govern the translation and stability of mRNAs is of paramount importance. We recently demonstrated that the cap-binding protein 4EHP is recruited by the miRNA machinery to effect translational repression. However, the impact of 4EHP on regulation of endogenous mRNAs and its role in cell biology is debated. Herein, using the ribosome profiling assay, we identify a subset of endogenous mRNAs that are sensitive to translational repression by 4EHP. We show that expression of DUSP6, a phosphatase that reverses ERK1/2 phosphorylation, is regulated by 4EHP. This regulation, which occurs exclusively at the level of mRNA translation, without affecting the stability of Dusp6 mRNA, is engendered through 4EHP- and miRNA-dependent elements in Dusp6 3´UTR. Consequently, 4EHP protein controls ERK1/2 phosphorylation, promotes cell growth and inhibits apoptosis. Our data reveal a crucial role for translational control mechanisms in the regulation of the ERK pathway. Overall design: Ribosome profiling and mRNA-seq in 4EHP KO and WT MEFs.