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

Abstract

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 Ca 2ϩ 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.

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

“…To test whether ADAR2 downregulation causes TDP-43 pathology, we examined the subcellular localization of TDP-43 in motor neurons of ADAR2 flox/flox /VAChT-Cre.Fast (AR2) mice in which the gene encoding ADAR2 is conditionally targeted in motor neurons 18 . Approximately half of the motor neurons were devoid of ADAR2 activity and expressed only Q/R site-unedited GluA2, AR2 mice exhibited motor dysfunctions as a result of the slow death of ADAR2-lacking motor neurons 18 . Spinal cord motor neurons lacking ADAR2 were devoid of TDP-43 immunoreactivity in both the nucleus and the cytoplasm, whereas neurons that expressed ADAR2 had TDP-43-positive nuclei, similar to the motor neurons in control mice ( Fig.…”

“…To test whether the mislocalization of TDP-43 in AR2 mice was induced by activation of calpain in response to amplified Ca 2 þ influx through AMPA receptors, we investigated changes in TDP-43 and calpains in AR2res (AR2/GluR-B R/R ) mice 18 . In these mice, the endogenous GluA2 alleles have been replaced with the GluR-B R allele, which encodes a Q/R site-edited GluA2.…”

“…The mutant animals used in this study were homozygous (ADAR2 flox/flox / VAChT-Cre.Fast; AR2) 18 and heterozygous (ADAR2 flox/ þ /VAChT-Cre.Fast; AR2H) conditional ADAR2 knockout mice 25 , AR2res (ADAR2 flox/flox /VAChTCre.Fast/GluR-B R/R ) mice 18 , CAST Tg 28 and knockout (CAST KO) mice 27 . AR2res (ADAR2 flox/flox /VAChT-Cre.Fast/GluR-B R/R ) mice were generated by crossbreeding AR2 mice with GluR-B R/R mice in which the endogenous GluA2 gene (Glia2) were engineered to encode directly edited GluA2, and ADAR2-lacking motor neurons did not undergo neuronal death by 6 months of age 18,25 . CAST Tg and CAST KO mice were bred as described previously 27,28 .…”

“…Immunohistochemistry was performed as previously described by Hideyama et al 18 Immunofluorescent staining of the sections was performed using rabbit anti-TDP-43 (ProteinTech Group Inc., 1:100) and sheep anti-rat RED1 (ADAR2; Exalpha Biologicals, Inc., 1:100) for the primary antibodies. Sections were then incubated with Alexa Fluor 488 donkey anti-sheep IgG (Invitrogen, 1:100) and Alexa Fluor 647 chicken anti-rabbit IgG (Invitrogen, 1:100), respectively, as the secondary antibodies.…”

“…17). ADAR2 activity at the GluA2 Q/R site is indispensable for motor neuron survival, because motor neurons lacking ADAR2 undergo slow death specifically via a failure of GluA2 RNA editing, which results in the upregulation of Ca 2 þ -permeable AMPA receptors 18 .…”