Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are ...routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
J. Neurochem. (2011) 116, 240–247.
The β‐amyloid peptide that accumulate in Alzheimer’s disease (AD) brain derive from proteolytic processing of the amyloid precursor protein (APP). Recent evidence ...suggest that microRNAs (miRNAs) participate in the post‐transcriptional regulation of APP expression. Because gene dosage effects of the APP gene can cause genetic AD, dysregulation of the miRNA network could contribute significantly to disease. Here, we present evidence that, besides APP expression regulation, miRNAs are equally involved in the regulation of neuronal APP mRNA alternative splicing. Lack of miRNAs in post‐mitotic neurons in vivo is associated with APP exons 7 and 8 inclusion, while ectopic expression of miR‐124, an abundant neuronal‐specific miRNA, reversed these effects in cultured neurons. Similar results were obtained by depletion of endogenous polypyrimidine tract binding protein 1 (PTBP1) in cells, a recognized miR‐124 target gene. Furthermore, PTBP1 levels correlate with the presence of APP exons 7 and 8, while PTBP2 levels correlate with the skipping of these exons during neuronal differentiation. Finally, we show that miR‐124 is down‐regulated in AD brain. In sum, our results suggest that specific miRNAs are involved in the fine‐tuning of APP alternative splicing in neurons. Since abnormal neuronal splicing of APP affects β‐amyloid peptide production, these results could contribute to the understanding of the implication of miRNAs in brain health and disease.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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 the absence of Dicer in the adult forebrain is accompanied by a mixed neurodegenerative phenotype. Although 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. Finally, we show that miR-15a is specifically downregulated in Alzheimer's disease brain. In summary, these results support the hypothesis that changes in the miRNA network may contribute to a neurodegenerative phenotype by affecting tau phosphorylation.
Although the role of APP and PSEN genes in genetic Alzheimer's disease (AD) cases is well established, fairly little is known about the molecular mechanisms affecting Aβ generation in sporadic AD. ...Deficiency in Aβ clearance is certainly a possibility, but increased expression of proteins like APP or BACE1/β-secretase may also be associated with the disease. We therefore investigated changes in microRNA (miRNA) expression profiles of sporadic AD patients and found that several miRNAs potentially involved in the regulation of APP and BACE1 expression appeared to be decreased in diseased brain. We show here that miR-29a, -29b-1, and -9 can regulate BACE1 expression in vitro. The miR-29a/b-1 cluster was significantly (and AD-dementia-specific) decreased in AD patients displaying abnormally high BACE1 protein. Similar correlations between expression of this cluster and BACE1 were found during brain development and in primary neuronal cultures. Finally, we provide evidence for a potential causal relationship between miR-29a/b-1 expression and Aβ generation in a cell culture model. We propose that loss of specific miRNAs can contribute to increased BACE1 and Aβ levels in sporadic AD.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Alzheimer's disease (AD) and related tauopathies comprise a large group of neurodegenerative diseases associated with the pathological aggregation of tau protein. While much effort has focused on ...understanding the function of tau, little is known about the endogenous mechanisms regulating tau metabolism in vivo and how these contribute to disease. Previously, we have shown that the microRNA (miRNA) cluster miR-132/212 is downregulated in tauopathies such as AD. Here, we report that miR-132/212 deficiency in mice leads to increased tau expression, phosphorylation and aggregation. Using reporter assays and cell-based studies, we demonstrate that miR-132 directly targets tau mRNA to regulate its expression. We identified GSK-3β and PP2B as effectors of abnormal tau phosphorylation in vivo. Deletion of miR-132/212 induced tau aggregation in mice expressing endogenous or human mutant tau, an effect associated with autophagy dysfunction. Conversely, treatment of AD mice with miR-132 mimics restored in part memory function and tau metabolism. Finally, miR-132 and miR-212 levels correlated with insoluble tau and cognitive impairment in humans. These findings support a role for miR-132/212 in the regulation of tau pathology in mice and humans and provide new alternatives for therapeutic development.
Tauopathies represent a large class of neurological and movement disorders characterized by abnormal intracellular deposits of the microtubule-associated protein tau. It is now well established that ...mis-splicing of tau exon 10, causing an imbalance between three-repeat (3R) and four-repeat (4R) tau isoforms, can cause disease; however, the underlying mechanisms affecting tau splicing in neurons remain poorly understood. The small noncoding microRNAs (miRNAs), known for their critical role in posttranscriptional gene expression regulation, are increasingly acknowledged as important regulators of alternative splicing. Here, we identified a number of brain miRNAs, including miR-124, miR-9, miR-132 and miR-137, which regulate 4R:3R-tau ratios in neuronal cells. Analysis of miRNA expression profiles from sporadic progressive supranuclear palsy (PSP) patients, a major 4R-tau tauopathy, showed that miR-132 is specifically down-regulated in disease. We demonstrate that miR-132 directly targets the neuronal splicing factor polypyrimidine tract-binding protein 2 (PTBP2), which protein levels were increased in PSP patients. miR-132 overexpression or PTBP2 knockdown similarly affected endogenous 4R:3R-tau ratios in neuronal cells. Finally, we provide evidence that miR-132 is inversely correlated with PTBP2 during post-natal brain development at the time when 4R-tau becomes expressed. Taken together, these results suggest that changes in the miR-132/PTBP2 pathway could contribute to the abnormal splicing of tau exon 10 in the brain, and sheds light into the potential role played by miRNAs in a subset of tauopathies.
APP expression misregulation can cause genetic Alzheimer's disease (AD). Recent evidences support the hypothesis that polymorphisms located in microRNA (miRNA) target sites could influence the risk ...of developing neurodegenerative disorders such as Parkinson's disease (PD) and frontotemporal dementia. Recently, a number of single nucleotide polymorphisms (SNPs) located in the 3'UTR of APP have been found in AD patients with family history of dementia. Because miRNAs have previously been implicated in APP expression regulation, we set out to determine whether these polymorphisms could affect miRNA function and therefore APP levels.
Bioinformatics analysis identified twelve putative miRNA bindings sites located in or near the APP 3'UTR variants T117C, A454G and A833C. Among those candidates, seven miRNAs, including miR-20a, miR-17, miR-147, miR-655, miR-323-3p, miR-644, and miR-153 could regulate APP expression in vitro and under physiological conditions in cells. Using luciferase-based assays, we could show that the T117C variant inhibited miR-147 binding, whereas the A454G variant increased miR-20a binding, consequently having opposite effects on APP expression.
Taken together, our results provide proof-of-principle that APP 3'UTR polymorphisms could affect AD risk through modulation of APP expression regulation, and set the stage for further association studies in genetic and sporadic AD.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we ...generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly up-regulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly up-regulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNC3 expression may contribute to the ataxic phenotype.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates, resulting in alterations in autophagy. However, the consequences of impaired autophagy for ...neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation as well as post-mortem material from patients with Huntington’s disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNA-induced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together, these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation but also directly contributes to global alterations of intracellular post-transcriptional networks.
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•Mutant HTT overexpression causes impairment of autophagy, resulting in AGO2 accumulation•Activation of autophagy reverses AGO2 accumulation in neurons•AGO2 accumulation results in a global increase in miRNA levels•AGO2 re-localizes to stress granules, resulting in loss of miRNA activity
Pircs et al. report that aggregation of the mutant huntingtin protein, a hallmark of Huntington’s disease proteinopathy, impairs macroautophagy, leading to Argonaute-2 accumulation and global dysregulation of microRNAs. These results indicate that autophagy not only influences protein aggregation but also directly contributes to the global alterations of post-transcriptional networks in Huntington’s disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansions in the amino-terminal region of the huntingtin (Htt) protein. At the cellular level, ...neuronal death is accompanied by the proteolytic cleavage, misfolding and aggregation of huntingtin. Abnormal hyperphosphorylation of tau protein is a characteristic feature of a class of neurodegenerative diseases called tauopathies. As a number of studies have reported tau pathology in HD patients, we investigated whether HD pathology may promote tau hyperphosphorylation and if so tackle some of its underlying mechanisms. For that purpose, we used the R6/2 mouse, a well-characterized model of HD, and analyzed tau phosphorylation before and after the onset of HD-like symptoms. We found a significant increase in tau hyperphosphorylation at the PHF-1 epitope in pre-symptomatic R6/2 mice, whereas symptomatic mice displayed tau hyperphosphorylation at multiple tau phosphoepitopes (AT8, CP13, PT205 and PHF-1). There was no activation of major tau kinases that could explain this observation. However, when we examined tau phosphatases, we found that calcineurin/PP2B was downregulated by 30% in pre-symptomatic and 50% in symptomatic R6/2 mice, respectively. We observed similar changes in tau phosphorylation and calcineurin expression in Q175 mice, another HD model. Calcineurin was also reduced in Q111 compared with Q7 cells. Finally, pharmacological or genetic inhibition of endogenous calcineurin was sufficient to promote tau hyperphosphorylation in neuronal cells. Taken together, our data suggest that mutant huntingtin can induce abnormal tau hyperphosphorylation in vivo, via the deregulation of calcineurin.
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