Julie A. Saugstad

Identification and characterization of human cytomegalovirus-encoded microRNAs

ABSTRACT MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.

MicroRNAs as effectors of brain function with roles in ischemia and injury, neuroprotection, and neurodegeneration

MicroRNAs are small RNAs that function as regulators of posttranscriptional gene expression. MicroRNAs are encoded by genes, and processed to form ribonucleoprotein complexes that bind to messenger RNA (mRNA) targets to repress translation or degrade mRNA transcripts. The microRNAs are particularly abundant in the brain where they serve as effectors of neuronal development and maintenance of the neuronal phenotype. They are also expressed in dendrites where they regulate spine structure and function as effectors in synaptic plasticity. MicroRNAs have been evaluated for their roles in brain ischemia, traumatic brain injury, and spinal cord injury, and in functional recovery after ischemia. They also serve as mediators in the brains response to ischemic preconditioning that leads to endogenous neuroprotection. In addition, microRNAs are implicated in neurodegenerative disorders, including Alzheimers, Huntington, Parkinson, and Prion disease. The discovery of microRNAs has expanded the potential for human diseases to arise from genetic mutations in microRNA genes or sequences within their target mRNAs. This review discusses microRNA discovery, biogenesis, mechanisms of gene regulation, their expression and function in the brain, and their roles in brain ischemia and injury, neuroprotection, and neurodegeneration.

Subunit-Dependent High-Affinity Zinc Inhibition of Acid-Sensing Ion Channels

Acid-sensing ion channels (ASICs), a novel class of ligand-gated cation channels activated by protons, are highly expressed in peripheral sensory and central neurons. Activation of ASICs may play an important role in physiological processes such as nociception, mechanosensation, and learning-memory, and in the pathology of neurological conditions such as brain ischemia. Modulation of the activities of ASICs is expected to have a significant influence on the roles that these channels can play in both physiological and/or pathological processes. Here we show that the divalent cation Zn2+, an endogenous trace element, dose-dependently inhibits ASIC currents in cultured mouse cortical neurons at nanomolar concentrations. With ASICs expressed in Chinese hamster ovary cells, Zn2+ inhibits currents mediated by homomeric ASIC1a and heteromeric ASIC1a-ASIC2a channels, without affecting currents mediated by homomeric ASIC1β, ASIC2a, or ASIC3. Consistent with ASIC1a-specific modulation, high-affinity Zn2+ inhibition is absent in neurons from ASIC1a knock-out mice. Current-clamp recordings and Ca2+-imaging experiments demonstrated that Zn2+ inhibits acid-induced membrane depolarization and the increase of intracellular Ca2+. Mutation of lysine-133 in the extracellular domain of the ASIC1a subunit abolishes the high-affinity Zn2+ inhibition. Our studies suggest that Zn2+ may play an important role in a negative feedback system for preventing overexcitation of neurons during normal synaptic transmission and ASIC1a-mediated excitotoxicity in pathological conditions.

Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain

The large number of metabotropic glutamate receptor subtypes suggests diverse roles in brain function, although specific distribution patterns can give clues to subtype-specific functions [Hayashi Y. et al. (1993) Nature 366, 687-690; Nakajima Y. et al. (1993) J. biol. Chem. 268, 11868-11873; Nomura A. et al. (1994) Cell 77, 361-369; Ohishi H. et al. (1993), 1009-1018]. The metabotropic glutamate receptor mGluR7 is sensitive to the agonist L-2-amino-4-phosphonobutyric acid, a presynaptic inhibitor of neurotransmitter release. We examined the anatomic distribution of mGluR7 messenger RNA expression by in situ hybridization in the developing and adult rat central nervous systems. Our results demonstrate that mGluR7 messenger RNA is among the most widely distributed of metabotropic glutamate receptors in both the developing and adult rat nervous system and that mGluR7 messenger RNA is expressed in most neuronal groups known to respond to L-2-amino-4-phosphonobutyric acid, including mitral cells of the olfactory bulb, granule cells of the dentate gyrus and neurons of the entorhinal cortex and dorsal root ganglion. mGluR7 exhibits preferential expression in sensory afferent pathways and is highly represented in the periventricular zone of the hypothalamus, the latter implying a modulatory role for mGluR7 in neuroendocrine pathways. Most strikingly, the majority of neurons at all levels of olfactory circuitry are among the areas of highest mGluR7 messenger RNA content. The anatomic distribution of mGluyR7 messenger RNA suggests that mGluR7 activation may participate in the processing of hippocampal, sensory and olfactory information.

Ischemic preconditioning regulates expression of microRNAs and a predicted target, MeCP2, in mouse cortex

Preconditioning describes the ischemic stimulus that triggers an endogenous, neuroprotective response that protects the brain during a subsequent severe ischemic injury, a phenomenon known as ‘tolerance’. Ischemic tolerance requires new protein synthesis, leads to genomic reprogramming of the brains response to subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate posttranscriptional gene expression by exerting direct effects on messenger RNA (mRNA) translation. We examined miRNA expression in mouse cortex in response to preconditioning, ischemic injury, and tolerance. The results of our microarray analysis revealed that miRNA expression is consistently altered within each group, but that preconditioning was the foremost regulator of miRNAs. Our bioinformatic analysis results predicted that preconditioning-regulated miRNAs most prominently target mRNAs that encode transcriptional regulators; methyl-CpG binding protein 2 (MeCP2) was the most prominent target. No studies have linked MeCP2 to preconditioning or tolerance, yet miR-132, which regulates MeCP2 expression, is decreased in preconditioned cortex. Downregulation of miR-132 is consistent with our finding that preconditioning ischemia induces a rapid increase in MeCP2 protein, but not mRNA, in mouse cortex. These studies reveal that ischemic preconditioning regulates expression of miRNAs and their predicted targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could serve as effectors of ischemic preconditioning-induced tolerance.

miRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.

Rapid degradation of bim by the ubiquitin-proteasome pathway mediates short-term ischemic tolerance in cultured neurons

A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke.

ASIC1a-Specific Modulation of Acid-Sensing Ion Channels in Mouse Cortical Neurons by Redox Reagents

Acid-sensing ion channel (ASIC)-1a, the major ASIC subunit with Ca2+ permeability, is highly expressed in the neurons of CNS. Activation of these channels with resultant intracellular Ca2+ accumulation plays a critical role in normal synaptic plasticity, learning/memory, and in acidosis-mediated glutamate receptor-independent neuronal injury. Here we demonstrate that the activities of ASICs in CNS neurons are tightly regulated by the redox state of the channels and that the modulation is ASIC1a subunit dependent. In cultured mouse cortical neurons, application of the reducing agents dramatically potentiated, whereas the oxidizing agents inhibited the ASIC currents. However, in neurons from the ASIC1 knock-out mice, neither oxidizing agents nor reducing reagents had any effect on the acid-activated current. In Chinese Hamster Ovary cells, redox-modifying agents only affected the current mediated by homomeric ASIC1a, but not homomeric ASIC1b, ASIC2a, or ASIC3. In current-clamp recordings and Ca2+-imaging experiments, the reducing agents increased but the oxidizing agents decreased acid-induced membrane depolarization and the intracellular Ca2+ accumulation. Site-directed mutagenesis studies identified involvement of cysteine 61 and lysine 133, located in the extracellular domain of the ASIC1a subunit, in the modulation of ASICs by oxidizing and reducing agents, respectively. Our results suggest that redox state of the ASIC1a subunit is an important factor in determining the overall physiological function and the pathological role of ASICs in the CNS.

Ubiquitin proteasome-mediated synaptic reorganization: a novel mechanism underlying rapid ischemic tolerance.

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin–proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), MG115 (carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal), and clasto-lactacystin-β-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin–proteasome system, rendering neurons resistant to excitotoxicity.

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin.

Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor.