Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 colocalize 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 Ca 2 þ -dependent cysteine protease, by upregulation of Ca 2 þ -permeable AMPA receptors generates carboxyterminal-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 Ca 2 þ -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.

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.

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and the OPDM_LRP12 Study Group IMPORTANCE Repeat expansion of CGG in LRP12 has been identified as the causative variation of oculopharyngodistal myopathy (OPDM). However, to our knowledge, the clinicopathologic features of OPDM with CGG repeat expansion in LRP12 (hereafter referred to as OPDM_LRP12) remain unknown.OBJECTIVE To identify and characterize the clinicopathologic features of patients with OPDM_LRP12. DESIGN, SETTING, AND PARTICIPANTSThis case series included 208 patients with a clinical or clinicopathologic diagnosis of oculopharyngeal muscular dystrophy (OPDM) from January 1, 1978, to December 31, 2020. Patients with GCN repeat expansions in PABPN1 were excluded from the study. Repeat expansions of CGG in LRP12 were screened by repeat primed polymerase chain reaction and/or Southern blot.MAIN OUTCOMES AND MEASURES Clinical information, muscle imaging data obtained by either computed tomography or magnetic resonance imaging, and muscle pathologic characteristics.RESULTS Sixty-five Japanese patients with OPDM (40 men [62%]; mean [SD] age at onset, 41.0 [10.1] years) from 59 families with CGG repeat expansions in LRP12 were identified. This represents the most common OPDM subtype among all patients in Japan with genetically diagnosed OPDM. The expansions ranged from 85 to 289 repeats. A negative correlation was observed between the repeat size and the age at onset (r 2 = 0.188, P = .001). The most common initial symptoms were ptosis and muscle weakness, present in 24 patients (37%). Limb muscle weakness was predominantly distal in 53 of 64 patients (83%), but 2 of 64 patients (3%) had predominantly proximal muscle weakness. Ptosis was observed in 62 of 64 patients (97%), and dysphagia or dysarthria was observed in 63 of 64 patients (98%). A total of 21 of 64 patients (33%) had asymmetric muscle weakness. Aspiration pneumonia was seen in 11 of 64 patients (17%), and 5 of 64 patients (8%) required mechanical ventilation. Seven of 64 patients (11%) developed cardiac abnormalities, and 5 of 64 patients (8%) developed neurologic abnormalities. Asymmetric muscle involvement was detected on computed tomography scans in 6 of 27 patients (22%) and on magnetic resonance imaging scans in 4 of 15 patients (27%), with the soleus and the medial head of the gastrocnemius being the worst affected. All 42 muscle biopsy samples showed rimmed vacuoles. Intranuclear tubulofilamentous inclusions were observed in only 1 of 5 patients. CONCLUSIONS AND RELEVANCEThis study suggests that OPDM_LRP12 is the most frequent OPDM subtype in Japan and is characterized by oculopharyngeal weakness, distal myopathy that especially affects the soleus and gastrocnemius muscles, and rimmed vacuoles in muscle biopsy.

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.

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alpha-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 Ca(2+)-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.

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