Armaz Aschrafi

MicroRNA-338 Regulates Local Cytochrome c Oxidase IV mRNA Levels and Oxidative Phosphorylation in the Axons of Sympathetic Neurons

MicroRNAs (miRs) are evolutionarily conserved, noncoding RNA molecules of ∼21 nt that regulate the expression of genes that are involved in various biological processes, such as cell proliferation and differentiation. Previously, we reported the presence of a heterogeneous population of mRNAs present in the axons and nerve terminals of primary sympathetic neurons to include the nuclear-encoded mitochondrial mRNA coding for COXIV. Sequence analysis of the 3′UTR of this mRNA revealed the presence of a putative binding site for miR-338, a brain-specific microRNA. Transfection of precursor miR-338 into the axons of primary sympathetic neurons decreases COXIV mRNA and protein levels and results in a decrease in mitochondrial activity, as measured by the reduction of ATP levels. Conversely, the transfection of synthetic anti-miR oligonucleotides that inhibit miR-338 increases COXIV levels, and results in a significant increase in oxidative phosphorylation and also norepinephrine uptake in the axons. Our results point to a molecular mechanism by which this microRNA participates in the regulation of axonal respiration and function by modulating the levels of COXIV, a protein which plays a key role in the assembly of the mitochondrial cytochrome c oxidase complex IV.

MicroRNA networks direct neuronal development and plasticity

MicroRNAs (miRNAs) constitute a class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. In neurons, the functions of individual miRNAs are just beginning to emerge, and recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth, dendritogenesis, and spine formation. Notably, miRNAs regulate mRNA translation locally in the axosomal and synaptodendritic compartments, and thereby contribute to the dynamic spatial organization of axonal and dendritic structures and their function. Given the critical role for miRNAs in regulating early brain development and in mediating synaptic plasticity later in life, it is tempting to speculate that the pathology of neurological disorders is affected by altered expression or functioning of miRNAs. Here we provide an overview of recently identified mechanisms of neuronal development and plasticity involving miRNAs, and the consequences of miRNA dysregulation.

Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons

microRNAs (miRNAs) constitute a novel class of small, noncoding RNAs that act as negative post-transcriptional regulators of gene expression. Although the nervous system is a prominent site of miRNA expression, little is known about the spatial expression profiles of miRNAs in neurons. Here, we employed compartmentalized Campenot cell culture chambers to obtain a pure axonal RNA fraction of superior cervical ganglia (SCG) neurons, and determined the miRNA expression levels in these subcellular structural domains by microarray analysis and by real-time reverse-transcription polymerase chain reaction. The data revealed stable expression of a number of mature miRNAs that were enriched in the axons and presynaptic nerve terminals. Among the 130 miRNAs identified in the axon, miR-15b, miR-16, miR-204, and miR-221 were found to be highly abundant in distal axons as compared with the cell bodies of primary sympathetic neurons. Moreover, a number of miRNAs encoded by a common primary transcript (pri-miRNA) were differentially expressed in the distal axons, suggesting that there is a differential subcellular transport of miRNAs derived from the same coding region of the genome. Taken together, the data provide an important resource for future studies on the regulation of axonal protein synthesis and the role played by miRNAs in the maintenance of axonal structure and function as well as neuronal growth and development.

Chromosome 1p21.3 microdeletions comprising DPYD and MIR137 are associated with intellectual disability

Background MicroRNAs (miRNAs) are non-coding gene transcripts involved in post-transcriptional regulation of genes. Recent studies identified miRNAs as important regulators of learning and memory in model organisms. So far, no mutations in specific miRNA genes have been associated with impaired cognitive functions. Methods and results In three sibs and two unrelated patients with intellectual disability (ID), overlapping 1p21.3 deletions were detected by genome-wide array analysis. The shortest region of overlap included dihydropyrimidine dehydrogenase (DPYD) and microRNA 137 (MIR137). DPYD is involved in autosomal recessive dihydropyrimidine dehydrogenase deficiency. Hemizygous DPYD deletions were previously suggested to contribute to a phenotype with autism spectrum disorder and speech delay. Interestingly, the mature microRNA transcript microRNA-137 (miR-137) was recently shown to be involved in modulating neurogenesis in adult murine neuronal stem cells. Therefore, this study investigated the possible involvement of MIR137 in the 1p21.3-deletion phenotype. The patients displayed a significantly decreased expression of both precursor and mature miR-137 levels, as well as significantly increased expression of the validated downstream targets microphthalmia-associated transcription factor (MITF) and Enhancer of Zeste, Drosophila, Homologue 2 (EZH2), and the newly identified target Kruppel-like factor 4 (KLF4). The study also demonstrated significant enrichment of miR-137 at the synapses of cortical and hippocampal neurons, suggesting a role of miR-137 in regulating local synaptic protein synthesis machinery. Conclusions This study showed that dosage effects of MIR137 are associated with 1p21.3 microdeletions and may therefore contribute to the ID phenotype in patients with deletions harbouring this miRNA. A local effect at the synapse might be responsible.

MicroRNA-338 regulates the axonal expression of multiple nuclear-encoded mitochondrial mRNAs encoding subunits of the oxidative phosphorylation machinery

MicroRNAs (miRNAs) constitute a novel class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. Remarkably, it has been shown that these small molecules can coordinately regulate multiple genes coding for proteins with related cellular functions. Previously, we reported that brain-specific miR-338 modulates the axonal expression of cytochrome c oxidase IV (COXIV), a nuclear-encoded mitochondrial protein that plays a key role in oxidative phosphorylation and axonal function. Here, we report that ATP synthase (ATP5G1), like COXIV mRNA, contains a putative miR-338 binding site, and that modulation of miR-338 levels in the axon results in alterations in both COXIV and ATP5G1 expression. Importantly, miR-338 modulation of local COXIV and ATP5G1 expression has a marked effect on axonal ROS levels, as well as axonal growth. These findings point to a mechanism by which miR-338 modulates local energy metabolism through the coordinate regulation of the expression of multiple nuclear-encoded mitochondrial mRNAs in the axon.

A potential regulatory role for intronic microRNA-338-3p for its host gene encoding apoptosis-associated tyrosine kinase.

MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration.

Long non-coding RNAs in neurodevelopmental disorders

Recent studies have emphasized an important role for long non-coding RNAs (lncRNA) in epigenetic regulation, development, and disease. Despite growing interest in lncRNAs, the mechanisms by which lncRNAs control cellular processes are still elusive. Improved understanding of these mechanisms is critical, because the majority of the mammalian genome is transcribed, in most cases resulting in non-coding RNA products. Recent studies have suggested the involvement of lncRNA in neurobehavioral and neurodevelopmental disorders, highlighting the functional importance of this subclass of brain-enriched RNAs. Impaired expression of lnRNAs has been implicated in several forms of intellectual disability disorders. However, the role of this family of RNAs in cognitive function is largely unknown. Here we provide an overview of recently identified mechanisms of neuronal development involving lncRNAs, and the consequences of lncRNA deregulation for neurodevelopmental disorders.

MicroRNAs in the axon and presynaptic nerve terminal.

The distal structural/functional domains of the neuron, to include the axon and presynaptic nerve terminal, contain a large, heterogeneous population of mRNAs and an active protein synthetic system. These local components of the genetic expression machinery play a critical role in the development, function, and long-term viability of the neuron. In addition to the local mRNA populations these presynaptic domains contain a significant number of non-coding RNAs that regulate gene expression post-transcriptionally. Here, we review a small, but rapidly evolving literature on the composition and function of microRNAs that regulate gene expression locally in the axon and nerve terminal. In this capacity, these small regulatory RNAs have a profound effect on axonal protein synthesis, local energy metabolism, and the modulation of axonal outgrowth and branching.

microRNAs and the regulation of neuronal plasticity under stress conditions

In the brain, the connection between sensory information triggered by the presence of a stressor and the organisms reaction involves limbic areas such as the hippocampus, amygdala and prefrontal cortex. Consequently, these brain regions are the most sensitive to stress-induced changes in neuronal plasticity. However, the specific effects of stress on neuronal plasticity in these regions largely differ. Despite these regional differences, in many cases the steps leading to brain adaptation to stress involve highly coordinated changes in gene expression affecting cell metabolism, neuronal plasticity and synaptic transmission. In adult life the effects of stress on neuronal plasticity are largely reversible but stress in early life induces persistent changes in neuronal plasticity that increases vulnerability to develop psychopathologies and aging-related cognitive decline, suggesting the involvement of epigenetic mechanisms. A growing body of evidence demonstrates that microRNAs (miRs) are key players in epigenetic regulation. In this forefront review we present a critical look on the literature demonstrating the regulation of neuronal plasticity by miRs and the molecular mechanisms of target specificity in neurons. We propose that further progress in the identification of miRs function beyond single target identification would require a combination of developmental expression studies, bioinformatics and a deeper understanding of large networks of targets involved in epigenetic regulation. This will help to extend our understanding of the role miRs play in the regulation of stress-induced neuronal plasticity.

Axonal Protein Synthesis and the Regulation of Local Mitochondrial Function

Axons and presynaptic nerve terminals of both invertebrate and mammalian SCG neurons contain a heterogeneous population of nuclear-encoded mitochondrial mRNAs and a local cytosolic protein synthetic system. Nearly one quarter of the total protein synthesized in these structural/functional domains of the neuron is destined for mitochondria. Acute inhibition of axonal protein synthesis markedly reduces the functional activity of mitochondria. The blockade of axonal protein into mitochondria had similar effects on the organelles functional activity. In addition to mitochondrial mRNAs, SCG axons contain approximately 200 different microRNAs (miRs), short, noncoding RNA molecules involved in the posttranscriptional regulation of gene expression. One of these miRs (miR-338) targets cytochrome c oxidase IV (COXIV) mRNA. This nuclear-encoded mRNA codes for a protein that plays a key role in the assembly of the mitochondrial enzyme complex IV and oxidative phosphorylation. Over-expression of miR-338 in the axon markedly decreases COXIV expression, mitochondrial functional activity, and the uptake of neurotransmitter into the axon. Conversely, the inhibition of endogeneous miR-338 levels in the axon significantly increased mitochondrial activity and norepinephrine uptake into the axon. The silencing of COXIV expression in the axon using short, inhibitory RNAs (siRNAs) yielded similar results, a finding that indicated that the effects of miR-338 on mitochondrial activity and axon function were mediated, at least in part, through local COXIV mRNA translation. Taken together, recent findings establish that proteins requisite for mitochondrial activity are synthesized locally in the axon and nerve terminal, and call attention to the intimacy of the relationship that has evolved between the distant cellular domains of the neuron and its energy generating systems.