Pascal Y. Smith

Genetic ablation of Dicer in adult forebrain neurons results in abnormal tau hyperphosphorylation and neurodegeneration

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 Alzheimers 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.

In vivo regulation of amyloid precursor protein neuronal splicing by microRNAs.

J. Neurochem. (2011) 116, 240–247.

miR-132/212 deficiency impairs tau metabolism and promotes pathological aggregation in vivo

Alzheimers 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.

Memory formation and retention are affected in adult miR-132/212 knockout mice.

The miR-132/212 family is thought to play an important role in neural function and plasticity, while its misregulation has been observed in various neurodegenerative disorders. In this study, we analyzed 6-month-old miR-132/212 knockout mice in a battery of cognitive and non-cognitive behavioral tests. No significant changes were observed in reflexes and basic sensorimotor functions as determined by the SHIRPA primary screen. Accordingly, miR-132/212 knockout mice did not differ from wild-type controls in general locomotor activity in an open-field test. Furthermore, no significant changes of anxiety were measured in an elevated plus maze task. However, the mutant mice showed retention phase defects in a novel object recognition test and in the T-water maze. Moreover, the learning and probe phases in the Barnes maze were clearly altered in knockout mice when compared to controls. Finally, changes in BDNF, CREB, and MeCP2 were identified in the miR-132/212-deficient mice, providing a potential mechanism for promoting memory loss. Taken together, these results further strengthen the role of miR-132/212 in memory formation and retention, and shed light on the potential consequences of its deregulation in neurodegenerative diseases.

Preclinical Evaluation of miR-15/107 Family Members as Multifactorial Drug Targets for Alzheimer's Disease

Alzheimers disease (AD) is a multifactorial, fatal neurodegenerative disorder characterized by the abnormal accumulation of Aβ and Tau deposits in the brain. There is no cure for AD, and failure at different clinical trials emphasizes the need for new treatments. In recent years, significant progress has been made toward the development of miRNA-based therapeutics for human disorders. This study was designed to evaluate the efficiency and potential safety of miRNA replacement therapy in AD, using miR-15/107 paralogues as candidate drug targets. We identified miR-16 as a potent inhibitor of amyloid precursor protein (APP) and BACE1 expression, Aβ peptide production, and Tau phosphorylation in cells. Brain delivery of miR-16 mimics in mice resulted in a reduction of AD-related genes APP, BACE1, and Tau in a region-dependent manner. We further identified Nicastrin, a γ-secretase component involved in Aβ generation, as a target of miR-16. Proteomics analysis identified a number of additional putative miR-16 targets in vivo, including α-Synuclein and Transferrin receptor 1. Top-ranking biological networks associated with miR-16 delivery included AD and oxidative stress. Collectively, our data suggest that miR-16 is a good candidate for future drug development by targeting simultaneously endogenous regulators of AD biomarkers (i.e., Aβ and Tau), inflammation, and oxidative stress.

microRNA-132/212 deficiency enhances Aβ production and senile plaque deposition in Alzheimer’s disease triple transgenic mice

The abnormal regulation of amyloid-β (Aβ) metabolism (e.g., production, cleavage, clearance) plays a central role in Alzheimer’s disease (AD). Among endogenous factors believed to participate in AD progression are the small regulatory non-coding microRNAs (miRs). In particular, the miR-132/212 cluster is severely reduced in the AD brain. In previous studies we have shown that miR-132/212 deficiency in mice leads to impaired memory and enhanced Tau pathology as seen in AD patients. Here we demonstrate that the genetic deletion of miR-132/212 promotes Aβ production and amyloid (senile) plaque formation in triple transgenic AD (3xTg-AD) mice. Using RNA-Seq and bioinformatics, we identified genes of the miR-132/212 network with documented roles in the regulation of Aβ metabolism, including Tau, Mapk, and Sirt1. Consistent with these findings, we show that the modulation of miR-132, or its target Sirt1, can directly regulate Aβ production in cells. Finally, both miR-132 and Sirt1 levels correlated with Aβ load in humans. Overall, our results support the hypothesis that the miR-132/212 network, including Sirt1 and likely other target genes, contributes to abnormal Aβ metabolism and senile plaque deposition in AD. This study strengthens the importance of miR-dependent networks in neurodegenerative disorders, and opens the door to multifactorial drug targets of AD by targeting Aβ and Tau.

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

Background 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 in vivo, particularly in the adult mammalian brain. Methodology/Principal Findings We analyzed whole genome microarrays from mice lacking Dicer, 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 Dicer 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 in vitro. 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 Dicer 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. Conclusions/Significance 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.

Extracellular Na(+) levels regulate formation and activity of the NaX/alpha1-Na(+)/K(+)-ATPase complex in neuronal cells.

MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na+]out). The mechanism underlying Na+-sensing involves Na+-flow through the NaX channel, directly regulated by the Na+/K+-ATPase α1-isoform which controls Na+-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na+]in). Here we aim to determine whether environmental changes in Na+ could actively modulate the NaX/Na+/K+-ATPase complex activity. We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na+] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na+] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na+/K+-ATPase remained unaltered. This unbalance between NaX and Na+/K+-ATPase pump proportion would induce a bigger portion of Na+/K+-ATPase-control-free NaX channel. Thus, we suggest that hypernatremic environment increases NaX/Na+/K+-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.