Adenosine Deaminase Acting on RNA (ADAR) Enzymes: A Journey from Weird to Wondrous.

ConspectusThe adenosine deaminase acting on RNA (ADAR) enzymes that catalyze the conversion of adenosine to inosine in double-stranded (ds)RNA are evolutionarily conserved and are essential for many biological functions including nervous system function, hematopoiesis, and innate immunity. Initially it was assumed that the wide-ranging biological roles of ADARs are due to inosine in mRNA being read as guanosine by the translational machinery, allowing incomplete RNA editing in a target codon to generate two different proteins from the same primary transcript. In humans, there are approximately seventy-six positions that undergo site-specific editing in tissues at greater than 20% efficiency that result in recoding. Many of these transcripts are expressed in the central nervous system (CNS) and edited by ADAR2. Exploiting mouse genetic models revealed that transgenic mice lacking the gene encoding Adar2 die within 3 weeks of birth. Therefore, the role of ADAR2 in generating protein diversity in the nervous system is clear, but why is ADAR RNA editing activity essential in other biological processes, particularly editing mainly involving ADAR1? ADAR1 edits human transcripts having embedded Alu element inverted repeats (AluIRs), but the link from this activity to innate immunity activation was elusive. Mice lacking the gene encoding Adar1 are embryonically lethal, and a major breakthrough was the discovery that the role of Adar1 in innate immunity is due to its ability to edit such repetitive element inverted repeats which have the ability to form dsRNA in transcripts. The presence of inosine prevents activation of the dsRNA sensor melanoma differentiation-associated protein 5 (Mda5). Thus, inosine helps the cell discriminate self from non-self RNA, acting like a barcode on mRNA. As innate immunity is key to many different biological processes, the basis for this widespread biological role of the ADAR1 enzyme became evident.Our group has been studying ADARs from the outset of research on these enzymes. In this Account, we give a historical perspective, moving from the initial purification of ADAR1 and ADAR2 and cloning of their encoding genes up to the current research focus in the field and what questions still remain to be addressed. We discuss the characterizations of the proteins, their localizations, posttranslational modifications, and dimerization, and how all of these affect their biological activities. Another aspect we explore is the use of mouse and Drosophila genetic models to study ADAR functions and how these were crucial in determining the biological functions of the ADAR proteins. Finally, we describe the severe consequences of rare mutations found in the human genes encoding ADAR1 and ADAR2.

ADAR2 enzymes: efficient site-specific RNA editors with gene therapy aspirations.

The adenosine deaminase acting on RNA (ADAR) enzymes are essential for neuronal function and innate immune control. ADAR1 RNA editing prevents aberrant activation of antiviral dsRNA sensors through editing of long, double-stranded RNAs (dsRNAs). In this review, we focus on the ADAR2 proteins involved in the efficient, highly site-specific RNA editing to recode open reading frames first discovered in the GRIA2 transcript encoding the key GLUA2 subunit of AMPA receptors; ADAR1 proteins also edit many of these sites. We summarize the history of ADAR2 protein research and give an up-to-date review of ADAR2 structural studies, human ADARBI (ADAR2) mutants causing severe infant seizures, and mouse disease models. Structural studies on ADARs and their RNA substrates facilitate current efforts to develop ADAR RNA editing gene therapy to edit disease-causing single nucleotide polymorphisms (SNPs). Artificial ADAR guide RNAs are being developed to retarget ADAR RNA editing to new target transcripts in order to correct SNP mutations in them at the RNA level. Site-specific RNA editing has been expanded to recode hundreds of sites in CNS transcripts in Drosophila and cephalopods. In Drosophila and C. elegans, ADAR RNA editing also suppresses responses to self dsRNA.

7-Ketocholesterol and 7ß-hydroxycholesterol: In vitro and animal models used to characterize their activities and to identify molecules preventing their toxicity.

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7ß-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7ß-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

Neuroprotective effects of PACAP against paraquat-induced oxidative stress in the Drosophila central nervous system.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that can arise after long-term exposure to environmental oxidative stressors, such as the herbicide paraquat (PQ). Here we investigated the potential neuroprotective action of vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) against PQ in Drosophila. We found that pre-treatment with this neuropeptide applied to the ventral nerve cord (VNC) at low doses markedly extended the survival of wild-type decapitated flies exposed to neurotoxic levels of PQ or dopamine (DA). In contrast and interestingly, application of a PACAP receptor antagonist, PACAP-6-38, had opposite effects, significantly decreasing the resistance of flies to PQ. PACAP also reduced PQ-induced caspase activation and reactive oxygen species (ROS) accumulation in the VNC. We then searched for the endogenous neuropeptide receptor potentially involved in PACAP-mediated neuroprotection in Drosophila. Knocking down the gene encoding the receptor Han/PDFR of the neuropeptide pigment-dispersing factor (PDF) in all neurons conferred to flies higher resistance to PQ, whereas PDFR downregulation restricted to PDF or DA neurons did not increase PQ resistance, but remarkably suppressed the neuroprotective action of PACAP. Further experiments performed with Pdf and Pdfr-deficient mutant strains confirmed that PDF and its receptor are required for PACAP-mediated neuroprotection in flies. We also provide evidence using split-green fluorescent protein (split-GFP) reconstitution that PDF neurons make synaptic contacts onto DA neurons in the abdominal VNC. Our results therefore suggest that the protective action of PACAP against PQ-induced defects in the Drosophila nervous system involves the modulation of PDFR signaling in a small number of interconnected neurons.