Neuronal RNA Research Articles

Adventiously-bound redox active iron and copper are at the center of oxidative damage in Alzheimer disease.

Central to oxidative damage in Alzheimer disease is the production of metal-catalyzed hydroxyl radicals that damage every category of macromolecule. Studies on redox-competent copper and iron indicate that redox activity in Alzheimer disease resides exclusively within the cytosol of vulnerable neurons and that chelation with deferoxamine or DTPA removes this activity. We have also found that while proteins that accumulate in Alzheimer disease such as tau, amyloid beta, and apolipoprotein E possess metal-binding sites, metal-associated cellular redox activity is more dependent on metal-nucleic acid binding. Consistent with this finding is the large amount of cytoplasmic RNA in pyramidal neurons. Still, the source of metal-catalyzed redox activity is controversial. Heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in Alzheimer disease suggesting increased heme turnover as a source of redox-active iron. Additionally, the role of mitochondria as a potential source of redox-active metals and oxygen radical production is assuming more prominence. In recent studies, we have found that while mitochondrial DNA and cytochrome C oxidase activity are increased in Alzheimer disease, the number of mitochondria is decreased, indicating accelerated mitochondria turnover. This finding, as well as preliminary studies demonstrating a reduction in microtubule density in neurons in Alzheimer disease suggests mitochondrial dysfunction as a potentially inseparable component of the initiation and progression of Alzheimer disease.

Delaying the mitochondrial decay of aging in the brain

Oxidative mitochondrial decay is a major contributor to aging and neural degeneration. In old rats (vs. young rats) mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O 2 uptake are lower; oxidants/O 2, neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher. Ambulatory activity and cognition declines with age. Feeding old rats for a few weeks with acetyl- l-carnitine (ALCAR), a mitochondrial metabolite, plus R-lipoic acid (LA), a mitochondrial coenzyme and antioxidant, restores mitochondrial function; lowers oxidants, neuronal RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (Skinner box/Morris water maze). The mechanism appears to be that with age increased oxidative damage to protein causes a deformation of structure of key enzymes, with a consequent lessening of affinity ( K m) for the substrate. The loss with age of carnitine acetyl transferase binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product which increases with age). Feeding the substrate ALCAR with LA restores the velocity of the reaction, K m for ALCAR and CoA, and mitochondrial function. Heme biosynthesis is predominantly in the mitochondria. Interfering with heme synthesis causes specific loss of complex IV with consequent release of oxidants and neuronal degeneration. Iron deficiency (25% of menstruating women in the US ingest <50% of the RDA) also causes release of oxidants and mitochondrial decay, presumably through lack of heme, accelerating brain aging. Vitamin B6 or zinc inadequacy (10% of Americans ingest <50% of each RDA) should also cause a heme deficiency.

The cytoplasmic polyadenylation element binding protein and polyadenylation of messenger RNA in Aplysia neurons

Translation of some mRNAs in nerve terminals has been shown to be regulated by polyadenylation in an experience-dependent manner. The transcripts whose translation is controlled by regulated polyadenylation contain the cytoplasmic polyadenylation element (CPE), which binds to the highly conserved CPE-binding protein (CPEB). In Aplysia, neuron-specific actin mRNA, which has a CPE in its 3′ UTR, is located both in cell bodies and at nerve endings (synaptosomes). We found that actin mRNA from pleural ganglion sensory neurons becomes polyadenylated during long-term facilitation produced by treatment with serotonin or 8-bromo cAMP. We cloned two isoforms of CPEB (ApCPEB77 and ApCEPB49) from Aplysia nervous tissue. The larger form, which is predominant in nervous tissue, is similar to p82, the clam binding protein, as well as to vertebrate CPEBs. Moreover, synaptosomal actin mRNAs are polyadenylated following the treatment with 5-HT. Since both CPEB and polyadenylated actin mRNA are present in synaptosomes and synaptosomal actin protein increases during long-term facilitation, we suggest that the translation of actin message in nerve endings is up-regulated by polyadenylation to grow new synapses.

Neuronal RNA oxidation is a prominent feature of dementia with Lewy bodies.

An approach was used to identify the oxidized nucleoside, 8-hydroxyguanosine in brains of dementia with Lewy bodies. Neurons with marked immunoreaction of 8-hydroxyguanosine in the cytoplasm were widely distributed in the hippocampal region and temporal neocortex. Relative intensity measurements of neuronal 8-hydroxyguanosine immunoreactivity showed that there was a significant increase in nucleic acid oxidation in dementia with Lewy bodies compared with controls. Treatment with nuclease (DNase or RNase) before the immunostaining demonstrated that RNA was a major site of nucleic acid oxidation. Together with the previously reported RNA oxidation in vulnerable neurons in Alzheimer and Parkinson diseases, neuronal RNA oxidation in dementia with Lewy bodies might represent one of the fundamental abnormalities in age-associated neurodegenerative diseases.

SODIUM CHANNEL BLOCKING AND ANTIARRHYTHMIC ACTIONS OF THE NOVEL ARYLPIPERAZINE RSD992

Bolus doses (4–128 μmol kg −1) and infusions (2–32 μmol kg −1 min −1) of the novel arylpiperazine drug RSD992 produced bradycardia in rats and guinea pigs but had minimal effect on ECG variables. RSD992 (2–32 μmol kg −1 min −1) increased threshold current ( I T) for induction of extra-systoles and induction of sustained ventricular fibrillation (VF T) and also increased the effective refractory period (ERP) and decreased the maximum following frequency (MFF) in rat and guinea pig hearts. RSD992 (32–512 μM) significantly increased PR and QRS intervals in isolated rat hearts subjected to conditions that mimic ischaemia (pH 6.4, K + 11 mM) but not in isolated hearts under normal perfusion conditions (pH 7.4, K + 3 mM). RSD992 (0.1–3.0 mM) reduced peak sodium current in rat cardiac (rNa v1.5) sodium channels more potently than neuronal (rNa v1.2a) sodium channels expressed in Xenopus oocytes. The voltage-dependence of sodium channel activation was unaffected whereas inactivation was shifted in a hyperpolarized direction thus suggesting RSD992 may preferentially interact with the inactive state of the sodium channel, a state usually associated with myocardial cell depolarization in ischaemic myocardium. RSD992 (2–24 μmol kg −1 min −1) decreased the incidence of ventricular arrhythmias and mortality in rats subject to coronary artery ligation. RSD992 exhibits frequency- and ischaemia-selective actions on myocardial sodium currents and antiarrhythmic actions in ischaemic rat myocardium.

Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

Transport of Neuronal BC1 RNA in Mauthner Axons

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons <i>in vivo</i>. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 μm/sec. Whereas a BC1 5′ segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3′ segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

Ectopic expression of Drosophila ELAV and human HuD in Drosophila wing disc cells reveals functional distinctions and similarities.

Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements (AREs) in their 3' untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes, which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame (ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3) with the intron nASI whose splicing is regulated in neurons by ELAV, inserted into the ORF. The two sets differed from each other only in their 3'UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that: (1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3'UTR, but not of transgenes with Act5C 3'UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm, ELAV is localized to both the cytoplasm and the nucleus.

The message and the messenger: delivering RNA in neurons.

Synaptic plasticity results in enduring changes in synaptic function. Localized protein synthesis is part of this process. Kosik and Krichevsky describe how a dynamic macromolecular structure, the RNA granule, may be a key element contributing to changes in protein production leading to synaptic plasticity.

The Message and the Messenger: Delivering RNA in Neurons

The Message and the Messenger: Delivering RNA in Neurons

Neuronal RNA granules: get ready for translation

Local de novo protein synthesis in response to synaptic activity is thought to be important for neuronal plasticity. In neurons, mRNA translation can be induced rapidly at sites that are very distant from the cell body, where the mRNAs are produced. This suggests that local stimulation-dependent translation involves pre-existing local mRNAs. RNA granules are motile structures that can translocate mRNAs to specific loci. In addition, they contain ribosomal components. However, it is not known whether RNA granules are translationally competent, nor whether they are involved in activity-regulated protein synthesis.

Neuronal RNA granules: get ready for translation

Local de novo protein synthesis in response to synaptic activity is thought to be important for neuronal plasticity. In neurons, mRNA translation can be induced rapidly at sites that are very distant from the site at which these mRNAs are produced (i.e. the cell body). This suggests that local, stimulation-dependent translation involves pre-existing local mRNAs rather than the initiation of mRNA transport from the cell body. RNA granules are motile structures that can translocate mRNAs to specific loci; they also contain ribosomal components. However, it is not known whether RNA granules are translationally competent, nor whether they are involved in activity-regulated protein synthesis.

Hyperphosphorylation of RNA polymerase II and reduced neuronal RNA levels precede neurofibrillary tangles in Alzheimer disease.

Affected neurons of Alzheimer disease (AD) brain are distinguished by the presence of the cell cycle cdc2 kinase and mitotic phosphoepitopes. A significant body of previous data has documented a decrease in neuronal RNA levels and nucleolar volume in AD brain. Here we present evidence that integrates these seemingly distinct findings and offers an explanation for the degenerative outcome of the disease. During mitosis cdc2 phosphorylates and inhibits the major transcriptional regulator RNA polymerase II (RNAP II). We therefore investigated cdc2 phosphorylation of RNAP II in AD brain. Using the H5 and H14 monoclonal antibodies specific for the cdc2-phosphorylated sites in RNAP II, we found that the polymerase is highly phosphorylated in AD. Moreover, RNAP II in AD translocates from its normally nuclear compartment to the cytoplasm of affected neurons, where it colocalizes with cdc2. These M phase-like changes in RNAP II correlate with decreased levels of poly-A RNA in affected neurons. Significantly, they precede tau phosphorylation and neurofibrillary tangle formation. Our data support the hypothesis that inappropriate activation of the cell cycle cdc2 kinase in differentiated neurons contributes to neuronal dysfunction and degeneration in part by inhibiting RNAP II and cellular processes dependent on transcription.

Poised for Action

Local protein synthesis allows neurons to respond rapidly to stimuli and is one mechanism that underlies long-lasting synaptic changes. Krichevsky and Kosik investigated how RNA granules in neurons were affected by depolarization. The RNA granules became more accessible to antibody labeling and were ultrastructurally more dispersed, less compact structures after depolarization. RNA granules did not incorporate radioactive amino acids, nor could various translation factors be detected in them, indicating that they are not translationally competent. Analysis of the distribution of mRNAs in response to depolarization showed that certain somatodendritic RNAs, such as those for calcium-calmodulin-dependent kinase II and the neurotrophin receptor TrkB, redistributed from the granules to the polysome pool for translation. The authors suggest that the RNA granules are a storage site to hold inactive mRNAs, from which they can be rapidly released for translation in response to stimulation. A. M. Krichevsky, K. S. Kosik, Neuronal RNA granules: A link between RNA localization and stimulation-dependent translation. Neuron 32 , 683-696 (2001). [Online Journal]

Poised for Action

Local protein synthesis allows neurons to respond rapidly to stimuli and is one mechanism that underlies long-lasting synaptic changes. Krichevsky and Kosik investigated how RNA granules in neurons were affected by depolarization. The RNA granules became more accessible to antibody labeling and were ultrastructurally more dispersed, less compact structures after depolarization. RNA granules did not incorporate radioactive amino acids, nor could various translation factors be detected in them, indicating that they are not translationally competent. Analysis of the distribution of mRNAs in response to depolarization showed that certain somatodendritic RNAs, such as those for calcium-calmodulin-dependent kinase II and the neurotrophin receptor TrkB, redistributed from the granules to the polysome pool for translation. The authors suggest that the RNA granules are a storage site to hold inactive mRNAs, from which they can be rapidly released for translation in response to stimulation. A. M. Krichevsky, K. S. Kosik, Neuronal RNA granules: A link between RNA localization and stimulation-dependent translation. Neuron 32, 683-696 (2001).[Online Journal]

Neuronal RNA Granules: A Link between RNA Localization and Stimulation-Dependent Translation

RNA granules are a macromolecular structure observed in neurons, where they serve as motile units that translocate mRNAs. Isolated RNA granules are highly enriched in Staufen protein and ultrastructurally contain densely packed clusters of ribosomes. With depolarization, many mRNAs, including those involved in plasticity, rapidly shift from the RNA granule fraction to polysomes. Depolarization reorganizes granules and induces a less compact organization of their ribosomes. RNA granules are not translationally competent, as indicated by the failure to incorporate radioactive amino acids and the absence of eIF4E, 4G, and tRNAs. We concluded that RNA granules are a local storage compartment for mRNAs under translational arrest but are poised for release to actively translated pools. Local release of mRNAs and ribosomes from granules may serve as a macromolecular mechanism linking RNA localization to translation and synaptic plasticity.

Hypothalamic Pituitary Adrenal Axis and Hypothalamic–Neurohypophyseal Responsiveness in Water-Deprived Rats

The differential effects of osmotic stimulation on magnocellular and parvocellular hypothalamic neurons were studied by analysis of corticotropin-releasing hormone (CRH) and vasopressin (VP) expression in controls and 48-h water-deprived rats subjected to either restraint for 1 h or a single lipopolysaccharide injection (250 μg/100 g). Water deprivation reduced basal CRH mRNA levels but the increments following 4 h of restraint or 6 h lipopolysaccharide (LPS) injection were similar to those in controls. In contrast, water deprivation had no effect on basal VP heteronuclear RNA (hnRNA) and mRNA levels in parvocellular neurons, but responses to restraint or LPS injection were reduced. VP expression in magnocellular paraventricular and supraoptic nuclei, and plasma sodium and vasopressin were higher in water-deprived rats, changes which were unaffected by restraint. LPS injection reduced VP mRNA but not hnRNA levels in magnocellular neurons and increased plasma vasopressin levels only in water-deprived rats independently of changes in plasma sodium. This was accompanied by an increase in vasopressin mRNA content in the posterior pituitary. The data show that the blunted ACTH responses to acute stress during chronic osmotic stimulation are correlated with the inability of parvocellular neurons to increase VP rather than CRH expression. In addition, LPS-induced endotoxemia causes disturbances of the magnocellular vasopressinergic system with an unexpected potentiation of osmotic simulated VP secretion. The lack of increase in VP transcription after LPS and changes in VP mRNA distribution suggest that endotoxemia affect the secretory process at the levels of the neurohypophyseal axon terminal.

Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

Corrigendum to “Alteration of expression levels of neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA in the hippocampi and cortices of young adult and aged cognitively unimpaired and impaired Long–Evans rats”

Corrigendum to “Alteration of expression levels of neuronal nitric oxide synthase and haem oxygenase-2 messenger RNA in the hippocampi and cortices of young adult and aged cognitively unimpaired and impaired Long–Evans rats”

Neuronal BC1 RNA: Co-expression with growth-associated protein-43 messenger RNA

Brain-specific cytoplasmic RNA 1 (BC1-RNA), a non-coding RNA polymerase III transcript, is a neuronal RNA that is specifically targeted to dendritic domains. It is co-localized with components of the dendritic protein synthetic machinery, and it has been suggested to operate in the regulation of local translation-related processes in postsynaptic microdomains, thus subserving long-term synaptic plasticity in neurons. To probe the relevance of BC1 expression in neuronal plasticity, we have analyzed the expression pattern of BC1 RNA in the rat nervous system. We found that BC1 RNA is expressed by a specific subset of neurons (but not by non-neuronal cells) in the central and peripheral nervous system of the adult rat. The BC1 labeling pattern indicates that the subcellular location of the RNA is typically postsynaptic which, depending on cell type, manifests itself in a predominantly somatic, somatodendritic, or dendritic location. Our results further show that BC1-expressing neurons typically co-express the messenger RNA for growth-associated protein-43 (GAP-43). Such co-expression was observed in diverse brain areas, including the olfactory bulb, neocortex, and hippocampus, among others. While BC1 RNA was in many neuronal cell types detectable in distal dendritic domains, GAP-43 messenger RNA was typically more restricted to neuronal perikarya. In the mature nervous system, expression of GAP-43 has been described as an intrinsic determinant of predominantly presynaptic plasticity, while BC1 RNA has been implicated in postsynaptic plasticity. Co-expression of both RNAs, as reported here, thus identifies a distinct subset of neurons in the rat nervous system that exhibits both types of plasticity.