Takenari Yamashita

Induced Loss of ADAR2 Engenders Slow Death of Motor Neurons from Q/R Site-Unedited GluR2

GluR2 is a subunit of the AMPA receptor, and the adenosine for the Q/R site of its pre-mRNA is converted to inosine (A-to-I conversion) by the enzyme called adenosine deaminase acting on RNA 2 (ADAR2). Failure of A-to-I conversion at this site affects multiple AMPA receptor properties, including the Ca2+ permeability of the receptor-coupled ion channel, thereby inducing fatal epilepsy in mice (Brusa et al., 1995; Feldmeyer et al., 1999). In addition, inefficient GluR2 Q/R site editing is a disease-specific molecular dysfunction found in the motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients (Kawahara et al., 2004). Here, we generated genetically modified mice (designated as AR2) in which the ADAR2 gene was conditionally targeted in motor neurons using the Cre/loxP system. These AR2 mice showed a decline in motor function commensurate with the slow death of ADAR2-deficient motor neurons in the spinal cord and cranial motor nerve nuclei. Notably, neurons in nuclei of oculomotor nerves, which often escape degeneration in ALS, were not decreased in number despite a significant decrease in GluR2 Q/R site editing. All cellular and phenotypic changes in AR2 mice were prevented when the mice carried endogenous GluR2 alleles engineered to express edited GluR2 without ADAR2 activity (Higuchi et al., 2000). Thus, loss of ADAR2 activity causes AMPA receptor-mediated death of motor neurons.

Profound downregulation of the RNA editing enzyme ADAR2 in ALS spinal motor neurons.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset fatal motor neuron disease. In spinal motor neurons of patients with sporadic ALS, normal RNA editing of GluA2, a subunit of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, is inefficient. Adenosine deaminase acting on RNA 2 (ADAR2) specifically mediates RNA editing at the glutamine/arginine (Q/R) site of GluA2 and motor neurons expressing Q/R site-unedited GluA2 undergo slow death in conditional ADAR2 knockout mice. Therefore, investigation into whether inefficient ADAR2-mediated GluA2 Q/R site-editing occurs universally in motor neurons of patients with ALS would provide insight into the pathogenesis of ALS. We analyzed the extents of GluA2 Q/R site-editing in an individual laser-captured motor neuron of 29 ALS patients compared with those of normal and disease control subjects. In addition, we analyzed the enzymatic activity of three members of the ADAR family (ADAR1, ADAR2 and ADAR3) in ALS motor neurons expressing unedited GluA2 mRNA and those expressing only edited GluA2 mRNA. Q/R site-unedited GluA2 mRNA was expressed in a significant proportion of motor neurons from all of the ALS cases examined. Conversely, motor neurons of the normal and disease control subjects expressed only edited GluA2 mRNA. ADAR2, but not ADAR1 or ADAR3, was significantly downregulated in all the motor neurons of ALS patients, more extensively in those expressing Q/R site-unedited GluA2 mRNA than those expressing only Q/R site-edited GluA2 mRNA. These results indicate that ADAR2 downregulation is a profound pathological change relevant to death of motor neurons in ALS.

TDP-43 pathology in sporadic ALS occurs in motor neurons lacking the RNA editing enzyme ADAR2

Both the appearance of cytoplasmic inclusions containing phosphorylated TAR DNA-binding protein (TDP-43) and inefficient RNA editing at the GluR2 Q/R site are molecular abnormalities observed specifically in motor neurons of patients with sporadic amyotrophic lateral sclerosis (ALS). The purpose of this study is to determine whether a link exists between these two specific molecular changes in ALS spinal motor neurons. We immunohistochemically examined the expression of adenosine deaminase acting on RNA 2 (ADAR2), the enzyme that specifically catalyzes GluR2 Q/R site-editing, and the expression of phosphorylated and non-phosphorylated TDP-43 in the spinal motor neurons of patients with sporadic ALS. We found that all motor neurons were ADAR2-positive in the control cases, whereas more than half of them were ADAR2-negative in the ALS cases. All ADAR2-negative neurons had cytoplasmic inclusions that were immunoreactive to phosphorylated TDP-43, but lacked non-phosphorylated TDP-43 in the nucleus. Our results suggest a molecular link between reduced ADAR2 activity and TDP-43 pathology.

A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology

Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 co-localize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca2+-dependent cysteine protease, by upregulation of Ca2+-permeable AMPA receptors generates carboxy-terminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca2+-impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.

AMPA receptor-mediated neuronal death in sporadic ALS

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor‐mediated excitotoxicity has been proposed to play a role in death of motor neurons in amyotrophic lateral sclerosis (ALS). We demonstrated that RNA editing of GluR2 mRNA at the glutamine/arginine (Q/R) site was decreased in autopsy‐obtained spinal motor neurons, but not in cerebellar Purkinje cells, of patients with sporadic ALS. This molecular change occurs in motor neurons of sporadic ALS cases with various phenotypes, but not in degenerating neurons of patients with other neurodegenerative diseases, including SOD1‐associated familial ALS. Because GluR2 Q/R site‐editing is specifically catalyzed by adenosine deaminase acting on RNA 2 (ADAR2), it is likely that regulatory mechanism of ADAR2 activity does not work well in the motor neurons of sporadic ALS. Indeed, ADAR2 expression level was significantly decreased in the spinal ventral gray matter of sporadic ALS as compared to normal control subjects. It is likely that ADAR2 underactivity selective in motor neurons induced deficient GluR2 Q/R site‐editing, which results in the neuronal death of sporadic ALS. Thus, among multiple different molecular mechanisms underlying death of motor neurons, it is likely that an increase of the proportion of Q/R site‐unedited GluR2‐containing Ca2+‐permeable AMPA receptors initiates the death of motor neurons in sporadic ALS. To this end, normalization of ADAR2 activity in motor neurons may become a therapeutic strategy for sporadic ALS.

Determination of editors at the novel A-to-I editing positions

A-to-I RNA editing modifies a variety of biologically important mRNAs, and is specifically catalyzed by either adenosine deaminase acting on RNA type 1 (ADAR1) or type 2 (ADAR2) in mammals including human. Recently several novel A-to-I editing sites were identified in mRNAs abundantly expressed in mammalian organs by means of computational genomic analysis, but which enzyme catalyzes these editing sites has not been determined. Using RNA interference (RNAi) knockdowns, we found that cytoplasmic fragile X mental retardation protein interacting protein 2 (CYFIP2) mRNA had an ADAR2-mediated editing position and bladder cancer associated protein (BLCAP) mRNA had an ADAR1-mediated editing position. In addition, we found that ADAR2 forms a complex with mRNAs with ADAR2-mediated editing positions including GluR2, kv1.1 and CYFIP2 mRNAs, particularly when the editing sites were edited in human cerebellum by means of immunoprecipitation (IP) method. CYFIP2 mRNA was ubiquitously expressed in human tissues with variable extents of K/E site editing. Because ADAR2 underactivity may be a causative molecular change of death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), this newly identified ADAR2-mediated editing position may become a useful tool for ALS research.

Rescue of amyotrophic lateral sclerosis phenotype in a mouse model by intravenous AAV9-ADAR2 delivery to motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult‐onset motor neuron disease, and the lack of effective therapy results in inevitable death within a few years of onset. Failure of GluA2 RNA editing resulting from downregulation of the RNA‐editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) occurs in the majority of ALS cases and causes the death of motor neurons via a Ca2+‐permeable AMPA receptor‐mediated mechanism. Here, we explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno‐associated virus serotype 9 (AAV9) vector that provides gene delivery to a wide array of central neurons after peripheral administration. A single intravenous injection of AAV9‐ADAR2 in conditional ADAR2 knockout mice (AR2), which comprise a mechanistic mouse model of sporadic ALS, caused expression of exogenous ADAR2 in the central neurons and effectively prevented progressive motor dysfunction. Notably, AAV9‐ADAR2 rescued the motor neurons of AR2 mice from death by normalizing TDP‐43 expression. This AAV9‐mediated ADAR2 gene delivery may therefore enable the development of a gene therapy for ALS.

The molecular link between inefficient GluA2 Q/R site-RNA editing and TDP-43 pathology in motor neurons of sporadic amyotrophic lateral sclerosis patients

TAR DNA-binding protein (TDP-43) pathology and reduced expression of adenosine deaminase acting on RNA 2 (ADAR2), which is the RNA editing enzyme responsible for adenosine-to-inosine conversion at the GluA2 glutamine/arginine (Q/R) site, concomitantly occur in the same motor neurons of amyotrophic lateral sclerosis (ALS) patients; this finding suggests a link between these two ALS-specific molecular abnormalities. AMPA receptors containing Q/R site-unedited GluA2 in their subunit assembly are Ca(2+)-permeable, and motor neurons lacking ADAR2 undergo slow death in conditional ADAR2 knockout (AR2) mice, which is a mechanistic ALS model in which the ADAR2 gene is targeted in cholinergic neurons. Moreover, deficient ADAR2 induced mislocalization of TDP-43 similar to TDP-43 pathology seen in the sporadic ALS patients in the motor neurons of AR2 mice. The abnormal mislocalization of TDP-43 specifically resulted from activation of the Ca(2+)-dependent serine protease calpain that specifically cleaved TDP-43 at the C-terminal region, and generated aggregation-prone N-terminal fragments. Notably, the N-terminal fragments of TDP-43 lacking the C-terminus were demonstrated in the brains and spinal cords of ALS patients. Because normalization of either the Ca(2+)-permeability of AMPA receptors or the calpain activity in the motor neurons normalized the subcellular localization of TDP-43 in AR2 mice, it is likely that exaggerated calpain-dependent TDP-43 fragments played a role at least in the initiation of TDP-43 pathology. Elucidation of the molecular cascade of neuronal death induced by ADAR2 downregulation could provide a new specific therapy for sporadic ALS. In this review, we summarized the work from our group on the role of inefficient GluA2 Q/R site-RNA editing and TDP-43 pathology in sporadic ALS, and discussed possible effects of inefficient ADAR2-mediated RNA editing in general.

Newly identified ADAR-mediated A-to-I editing positions as a tool for ALS research

Among the extensively occurring adenosine to inosine (A-to-I) conversions in RNA, RNA editing at the GluR2 Q/R site is crucial for the survival of mammalian organisms. Editing at this site is incomplete in the motor neurons of patients with sporadic amyotrophic lateral sclerosis (ALS). Adenosine deaminase acting on RNA type 2 (ADAR2) specifically mediates GluR2 Q/R site-editing, hence, it is likely a molecule relevant to the pathogenesis of sporadic ALS. Since no other transcript with ADAR2-mediated A-to-I positions is abundantly expressed in most neurons, the editors at the newly identified A-to-I positions were investigated. CYFIP2 and FLNA mRNAs were identified together with mRNAs having known ADAR2-mediated editing positions in ADAR2-immunoprecipitates of the human cerebellum, indicating that these mRNAs probably possessed ADAR2-mediated positions. Furthermore, an in vitro RNAi knockdown system demonstrated that the CYFIP2 mRNA K/E site and the BLCAP mRNA Y/C site were edited predominantly by ADAR2 and ADAR1, respectively. CYFIP2 mRNA was ubiquitously expressed and particularly abundant in the central nervous system. The extent of CYFIP2 K/E site-editing was between 30% and 80% in the central nervous system. Therefore, the extent of CYFIP2 K/E site-editing may be an additional marker for ADAR2 activity in neuronal and other types of cells in vivo, as well as in vitro, and thus is considered to be a good tool for sporadic ALS research.

Co-Occurrence of TDP-43 Mislocalization with Reduced Activity of an RNA Editing Enzyme, ADAR2, in Aged Mouse Motor Neurons

TDP-43 pathology in spinal motor neurons is a neuropathological hallmark of sporadic amyotrophic lateral sclerosis (ALS) and has recently been shown to be closely associated with the downregulation of an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) in the motor neurons of sporadic ALS patients. Because TDP-43 pathology is found more frequently in the brains of elderly patients, we investigated the age-related changes in the TDP-43 localization and ADAR2 activity in mouse motor neurons. We found that ADAR2 was developmentally upregulated, and its mRNA expression level was progressively decreased in the spinal cords of aged mice. Motor neurons normally exhibit nuclear ADAR2 and TDP-43 immunoreactivity, whereas fast fatigable motor neurons in aged mice demonstrated a loss of ADAR2 and abnormal TDP-43 localization. Importantly, these motor neurons expressed significant amounts of the Q/R site-unedited AMPA receptor subunit 2 (GluA2) mRNA. Because expression of unedited GluA2 has been demonstrated as a lethality-causing molecular abnormality observed in the motor neurons, these results suggest that age-related decreases in ADAR2 activity play a mechanistic role in aging and serve as one of risk factors for ALS.