Amyloid precursor protein induces reactive astrogliosis.

AIM: Astrocytes respond to stressors by acquiring a reactive state characterized by changes in their morphology and function. Molecules underlying reactive astrogliosis, however, remain largely unknown. Given that several studies observed increase in the Amyloid Precursor Protein (APP) in reactive astrocytes, we here test whether APP plays a role in reactive astrogliosis. METHODS: We investigated whether APP instigates reactive astroglios by examining in vitro and in vivo the morphology and function of naive and APP-deficient astrocytes in response to APP and well-established stressors. RESULTS: Overexpression of APP in cultured astrocytes led to remodeling of the intermediate filament network, enhancement of cytokine production, and activation of cellular programs centered around the interferon (IFN) pathway, all signs of reactive astrogliosis. Conversely, APP deletion abrogated remodeling of the intermediate filament network and blunted expression of IFN-stimulated gene products in response to lipopolysaccharide. Following traumatic brain injury (TBI), mouse reactive astrocytes also exhibited an association between APP and IFN, while APP deletion curbed the increase in glial fibrillary acidic protein observed canonically in astrocytes in response to TBI. CONCLUSIONS: The APP thus represents a candidate molecular inducer and regulator of reactive astrogliosis. This finding has implications for understanding pathophysiology of neurodegenerative and other diseases of the nervous system characterized by reactive astrogliosis and opens potential new therapeutic avenues targeting APP and its pathways to modulate reactive astrogliosis.

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.

The mechanical regulation of RNA binding protein hnRNPC in the failing heart.

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by reexpressing fetal contractile proteins via transcriptional and posttranscriptional processes, such as alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is up-regulated and relocates to the sarcomeric Z-disc upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates with the translation machinery. Alterations in hnRNPC expression, phosphorylation, and localization can be mechanically determined and affect the AS of mRNAs involved in mechanotransduction and cardiovascular diseases, including Hippo pathway effector Yes-associated protein 1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by affecting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNA homeostatic mechanoregulation in pathological conditions.

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.

ADAR1 entraps sinister cellular dsRNAs, thresholding antiviral responses.

ADAR1 edits adenosines to inosines in cellular double-stranded (ds)RNA, thereby preventing aberrant activation of antiviral dsRNA sensors, as well as interferon (IFN) induction in Aicardi-Goutières syndrome (AGS) encephalopathy. Recently, Nakahama et al., Tang et al., Maurano et al., and de Reuver et al. demonstrated that Adar1 Zα domain-mutant mice show aberrant MDA5 and PKR activation, developing encephalopathies; short Z-RNA patches within cellular dsRNA are unexpectedly crucial in causing aberrant antiviral responses.

Interplays of different types of epitranscriptomic mRNA modifications.

Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences.

ADAR RNA Modifications, the Epitranscriptome and Innate Immunity.

Modified bases act as marks on cellular RNAs so that they can be distinguished from foreign RNAs, reducing innate immune responses to endogenous RNA. In humans, mutations giving reduced levels of one base modification, adenosine-to-inosine deamination, cause a viral infection mimic syndrome, a congenital encephalitis with aberrant interferon induction. These Aicardi-Goutières syndrome 6 mutations affect adenosine deaminase acting on RNA 1 (ADAR1), which generates inosines in endogenous double-stranded (ds)RNA. The inosine base alters dsRNA structure to prevent aberrant activation of antiviral cytosolic helicase RIG-I-like receptors. We review how effects of inosines, ADARs, and other modified bases have been shown to be important in innate immunity and cancer.

Biallelic variants in ADARB1, encoding a dsRNA-specific adenosine deaminase, cause a severe developmental and epileptic encephalopathy.

BACKGROUND: Adenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects. METHODS: We studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing. RESULTS: All patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity. CONCLUSION: In conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.

Bi-allelic ADARB1 Variants Associated with Microcephaly, Intellectual Disability, and Seizures.

The RNA editing enzyme ADAR2 is essential for the recoding of brain transcripts. Impaired ADAR2 editing leads to early-onset epilepsy and premature death in a mouse model. Here, we report bi-allelic variants in ADARB1, the gene encoding ADAR2, in four unrelated individuals with microcephaly, intellectual disability, and epilepsy. In one individual, a homozygous variant in one of the double-stranded RNA-binding domains (dsRBDs) was identified. In the others, variants were situated in or around the deaminase domain. To evaluate the effects of these variants on ADAR2 enzymatic activity, we performed in vitro assays with recombinant proteins in HEK293T cells and ex vivo assays with fibroblasts derived from one of the individuals. We demonstrate that these ADAR2 variants lead to reduced editing activity on a known ADAR2 substrate. We also demonstrate that one variant leads to changes in splicing of ADARB1 transcript isoforms. These findings reinforce the importance of RNA editing in brain development and introduce ADARB1 as a genetic etiology in individuals with intellectual disability, microcephaly, and epilepsy.

Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila.

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.

Membrane and synaptic defects leading to neurodegeneration in Adar mutant Drosophila are rescued by increased autophagy.

BACKGROUND: In fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration. RESULTS: A genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested. CONCLUSIONS: These findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage.

Long-term tracking demonstrates effectiveness of a partnership-led training program to advance the careers of biomedical researchers from underrepresented groups.

The demographic profile of the biomedical workforce in the U.S. does not reflect the population at large, raising concerns that there will be insufficient trained researchers in the future, and the scope of research interests will not be sufficiently broad. To diversify and expand the pool of researchers trained to conduct research on cancer and cancer health disparities, a series of training activities to recruit and train primarily Hispanic students at both the undergraduate and graduate level were developed. The strengths of both a Hispanic Serving Institution and an NIH-designated Comprehensive Cancer Center were leveraged to develop appropriate research training and professional development activities. The career progression of the participants and degree completion rates was tracked, along with persistent interest in biomedical research in general and cancer and cancer health disparities research in particular for these underrepresented individuals. Finally, this report demonstrates that these training activities increased general knowledge about cancer among participants.

Activation of innate immunity by mitochondrial dsRNA in mouse cells lacking p53 protein.

Viral and cellular double-stranded RNA (dsRNA) is recognized by cytosolic innate immune sensors, including RIG-I-like receptors. Some cytoplasmic dsRNA is commonly present in cells, and one source is mitochondrial dsRNA, which results from bidirectional transcription of mitochondrial DNA (mtDNA). Here we demonstrate that Trp53 mutant mouse embryonic fibroblasts contain immune-stimulating endogenous dsRNA of mitochondrial origin. We show that the immune response induced by this dsRNA is mediated via RIG-I-like receptors and leads to the expression of type I interferon and proinflammatory cytokine genes. The mitochondrial dsRNA is cleaved by RNase L, which cleaves all cellular RNA including mitochondrial mRNAs, increasing activation of RIG-I-like receptors. When mitochondrial transcription is interrupted there is a subsequent decrease in this immune-stimulatory dsRNA. Our results reveal that the role of p53 in innate immunity is even more versatile and complex than previously anticipated. Our study, therefore, sheds new light on the role of endogenous RNA in diseases featuring aberrant immune responses.

ADAR RNA editing in innate immune response phasing, in circadian clocks and in sleep.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in dsRNA. ADAR editing in pre-mRNAs recodes open reading frames and alters splicing, mRNA structure and interactions with miRNAs. Here, we review ADAR gene expression, splice forms, posttranslational modifications, subcellular localizations and functions of ADAR protein isoforms. ADAR1 edits cellular dsRNA to prevent aberrant activation of cytoplasmic antiviral dsRNA sensors; ADAR1 mutations lead to aberrant expression of interferon in Aicardi Goutières syndrome (AGS), a human congenital encephalopathy. We review related studies on mouse Adar1 mutant phenotypes, their rescues by preventing signaling from the antiviral RIG-I-like Sensors (RLRs), as well as Adar1 mechanisms in innate immune suppression and other roles of Adar1, including editing-independent effects. ADAR2, expressed primarily in CNS, edits glutamate receptor transcripts; regulation of ADAR2 activity in response to neuronal activity mediates homeostatic synaptic plasticity of vertebrate AMPA and kainite receptors. In Drosophila, synapses and synaptic proteins show dramatic decreases at night during sleep; Drosophila Adar, an orthologue of ADAR2, edits hundreds of mRNAs; the most conserved editing events occur in transcripts encoding synapse-associated proteins. Adar mutant flies exhibit locomotion defects associated with very increased sleep pressure resulting from a failure of homeostatic synaptic processes. A study on Adar2 mutant mice identifies a new role in circadian rhythms, acting indirectly through miRNAs such as let-7 to modulate levels of let-7 target mRNAs; ADAR1 also regulates let-7 miRNA processing. Drosophila ADAR, an orthologue of vertebrate ADAR2, also regulates let-7 miRNA levels and Adar mutant flies have a circadian mutant phenotype.

Accumulation of bioactive metabolites in cultivated medical Cannabis.

There has been an increased use of medical Cannabis in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers. As phytochemists in a state with legal medical Cannabis we sought to characterize the accumulation of phytochemicals in material grown by licensed commercial producers. We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids. Through analysis of developing flowers on plants, we can identify sources of variability of floral metabolites due to flower maturity and position on the plant. The terpenoid composition varied by accession and was used to cluster cannabis strains into specific types. Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.

Knowledge and social engagement change in intention to be screened for colorectal cancer.

OBJECTIVE: Innovative technologies have been used to promote colorectal cancer (CRC) screening among the underserved. However, the impact of these innovative technologies on knowledge and social engagement likelihood as they relate to subsequent intention to be screened across different populations has not been fully explored. DESIGN: Using a pre-post-test design with an inflatable walk-through colon, we assessed changes in knowledge and social engagement likelihood across populations and their associations with intention to be screened in two community settings. One was a community setting in Washington State (WA); the other, a college campus in New Mexico (NM). Differential effects on knowledge and social engagement likelihood were examined across demographic groups (race/ethnicity, gender, age, education, insurance status, and geographic region). Finally, we assessed if changes in knowledge and social engagement likelihood were associated with CRC screening intention. RESULTS: NM males had greater gains in CRC knowledge than NM females; in WA, Hispanics, younger, less educated, and uninsured participants had greater gains in knowledge. NM females and younger WA participants were more likely to discuss CRC with their social networks than NM males and older WA participants. In WA, Hispanics and older adults reported greater intention to be screened for CRC. Change in social engagement likelihood, but not knowledge, was associated with intention to be screened. CONCLUSIONS: The effectiveness of health promotion technologies on knowledge and social engagement may vary across different demographic characteristics. Further, the importance of social engagement likelihood in interacting with intention to be screened was substantiated.

Dynamic landscape and regulation of RNA editing in mammals.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

ADAR RNA editing in human disease; more to it than meets the I.

We review the structures and functions of ADARs and their involvements in human diseases. ADAR1 is widely expressed, particularly in the myeloid component of the blood system, and plays a prominent role in promiscuous editing of long dsRNA. Missense mutations that change ADAR1 residues and reduce RNA editing activity cause Aicardi-Goutières Syndrome, a childhood encephalitis and interferonopathy that mimics viral infection and resembles an extreme form of Systemic Lupus Erythmatosus (SLE). In Adar1 mouse mutant models aberrant interferon expression is prevented by eliminating interferon activation signaling from cytoplasmic dsRNA sensors, indicating that unedited cytoplasmic dsRNA drives the immune induction. On the other hand, upregulation of ADAR1 with widespread promiscuous RNA editing is a prominent feature of many cancers and particular site-specific RNA editing events are also affected. ADAR2 is most highly expressed in brain and is primarily required for site-specific editing of CNS transcripts; recent findings indicate that ADAR2 editing is regulated by neuronal excitation for synaptic scaling of glutamate receptors. ADAR2 is also linked to the circadian clock and to sleep. Mutations in ADAR2 could contribute to excitability syndromes such as epilepsy, to seizures, to diseases involving neuronal plasticity defects, such as autism and Fragile-X Syndrome, to neurodegenerations such as ALS, or to astrocytomas or glioblastomas in which reduced ADAR2 activity is required for oncogenic cell behavior. The range of human disease associated with ADAR1 mutations may extend further to include other inflammatory conditions while ADAR2 mutations may affect psychiatric conditions.

Major transcriptional changes observed in the Fulani, an ethnic group less susceptible to malaria.

The Fulani ethnic group has relatively better protection from Plasmodium falciparum malaria, as reflected by fewer symptomatic cases of malaria, lower infection rates, and lower parasite densities compared to sympatric ethnic groups. However, the basis for this lower susceptibility to malaria by the Fulani is unknown. The incidence of classic malaria resistance genes are lower in the Fulani than in other sympatric ethnic populations, and targeted SNP analyses of other candidate genes involved in the immune response to malaria have not been able to account for the observed difference in the Fulani susceptibility to P.falciparum. Therefore, we have performed a pilot study to examine global transcription and DNA methylation patterns in specific immune cell populations in the Fulani to elucidate the mechanisms that confer the lower susceptibility to P.falciparum malaria. When we compared uninfected and infected Fulani individuals, in contrast to uninfected and infected individuals from the sympatric ethnic group Mossi, we observed a key difference: a strong transcriptional response was only detected in the monocyte fraction of the Fulani, where over 1000 genes were significantly differentially expressed upon P.falciparum infection.