Shin Kwak

Glutamate receptors: RNA editing and death of motor neurons

The aetiology of sporadic amyotrophic lateral sclerosis (ALS), a fatal paralytic disease, is largely unknown. Here we show that there is a defect in the editing of the messenger RNA encoding the GluR2 subunit of glutamate AMPA receptors in the spinal motor neurons of individuals affected by ALS. This failure to swap an arginine for a glutamine residue at a crucial site in the subunit, which occurs normally in the affected brain areas of patients with other neurodegenerative diseases, will interfere with the correct functioning of the glutamate receptors and may be a contributory cause of neuronal death in ALS patients.

Deficient RNA editing of GluR2 and neuronal death in amyotropic lateral sclerosis

One plausible hypothesis for selective neuronal death in sporadic amyotropic lateral sclerosis (ALS) is excitotoxicity mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, which are a subtype of ionotropic glutamate receptors. The Ca2+ conductance of AMPA receptors differs markedly depending on whether the GluR2 (or GluR-B) subunit is a component of the receptor. The properties of GluR2 are generated posttranscriptionally by RNA editing at the Q/R site in the putative second membrane domain (M2), during which the glutamine (Q) codon is substituted by an arginine (R) codon. AMPA receptors containing the unedited form of GluR2Q have high Ca2+ permeability in contrast to the low Ca2+ conductance of those containing the edited form of GluR2R. The role of Ca2+-permeable AMPA receptors, particularly GluR2 Q/R site RNA editing status, in neuronal death has been clearly demonstrated both in mice deficient in editing at the GluR2 Q/R site and in mice transgenic for an artificial Ca2+-permeable GluR2 subunit. We analyzed the expression level of mRNA of each AMPA receptor subunit in individual motor neurons, as well as the editing efficiency of GluR2 mRNA at the Q/R site in the single neuron level in control subjects and ALS cases. There was no significant difference as to the expression profile of AMPA receptor subunits or the proportion of GluR2 mRNA to total GluRs mRNA between normal subjects and ALS cases. By contrast, the editing efficiency varied greatly, from 0% to 100%, among the motor neurons of each individual with ALS, and was not complete in 44 of them (56%), whereas it remained 100% in normal controls. In addition, GluR2 editing efficiency was more than 99% in the cerebellar Purkinje cells of ALS, spinocerebellar degeneration and normal control groups. Thus, GluR2 underediting occurs in a disease specific and region selective manner. GluR2 modification by RNA editing is a biologically crucial event for neuronal survival, and its deficiency is a direct cause of neuronal death. Therefore, marked reduction of RNA editing in ALS motor neurons may be a direct cause of the selective motor neuron death seen in ALS. It is likely that the molecular mechanism underlying the deficiency in RNA editing is a reduction in the activity of ADAR2, a double- strand RNA specific deaminase. The restoration of this enzyme activity in ALS motor neurons may open the novel strategy for specific ALS therapy.

Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis

Enhancement of calcium influx through the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptor is a plausible mechanism underlying selective neuronal death in amyotrophic lateral sclerosis (ALS). The calcium conductance of the AMPA receptor is regulated by the GluR2 subunit that is edited at the glutamine/arginine residue site in the subunit assembly. We investigated the molecular changes of GluR2 mRNA in the spinal cord of ALS cases, those of cases with other neurological diseases, and those of normal cases using reverse transcription–polymerase chain reaction combined with restriction enzyme cleavage. We found that the editing efficiency was significantly lower only in the ventral gray of ALS cases (virtually 0% in 2 cases) than in any spinal region of the disease controls and normal controls. In addition, expression of GluR2 mRNA is lower in the ventral gray of the ALS cases and disease controls than in that of the normal controls. The above molecular changes of GluR2 mRNA in the ventral gray of ALS cases may enhance calcium influx through AMPA receptors, thereby promoting neuronal vulnerability. The decrement of GluR2 mRNA editing efficiency is unique to the ventral gray of ALS cases and may be closely linked to the etiology of ALS.

Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS

AMPA receptor‐mediated neurotoxicity is currently the most plausible hypothesis for the etiology of amyotrophic lateral sclerosis (ALS). The mechanism initiating this type of neuronal death is believed to be exaggerated Ca2+‐influx through AMPA receptors, which is critically determined by the presence or absence of the glutamate receptor subunit 2 (GluR2) in the assembly. We have provided the first quantitative measurements of the expression profile of AMPA receptor subunits mRNAs in human single neurons by means of quantitative RT–PCR with a laser microdissector. Among the AMPA subunits, GluR2 shared the vast majority throughout the neuronal subsets and tissues examined. Furthermore, both the expression level and the proportion of GluR2 mRNA in motoneurons were the lowest among all neuronal subsets examined, whereas those in motoneurons of ALS did not differ from the control group, implying that selective reduction of the GluR2 subunit cannot be a mechanism of AMPA receptor‐mediated neurotoxicity in ALS. However, the low relative abundance of GluR2 might provide spinal motoneurons with conditions that are easily affected by changes of AMPA receptor properties including deficient GluR2 mRNA editing in ALS.

Evaluation of corticospinal tracts in ALS with diffusion tensor MRI and brainstem stimulation.

Objective: To assess corticospinal tract involvement in patients with amyotrophic lateral sclerosis (ALS) by correlating diffusion tensor imaging (DTI) measures with intra- and extracranial central motor conduction time (CMCT) and clinical features of the patients. Methods: We investigated 31 patients with ALS and 31 normal volunteers by DTI and measured fractional anisotropy (FA) within the corticospinal tracts and in the extramotor white matter. We measured CMCT for the first dorsal interosseous muscle and segmented it into cortical-brainstem (CTX-BS CT) and brainstem-cervical root (BS-CV CT) conduction times by magnetic brainstem stimulation at the foramen magnum level. Clinical status of each patient was evaluated with the ALS Functional Rating Scale–Revised (ALSFRS-R) and upper motor neuron (UMN) score devised for this study. Results: We found a significant decrease of mean FA in all regions of the corticospinal tracts in patients with ALS as compared with controls. We found that FA along the corticospinal tract decreased significantly with higher UMN scores. There was no significant correlation between FA and ALSFRS-R, to which both upper and lower motoneuron involvements contribute. FA showed a significant correlation with the intracranial part of the central motor conduction (CTX-BS CT) but not with the extracranial conduction time. Conclusions: Fractional anisotropy reflects functional abnormality of intracranial corticospinal tracts and can be used for objective evaluation of upper motor neuron impairment in amyotrophic lateral sclerosis. GLOSSARY: ALS = amyotrophic lateral sclerosis; ALSFRS-R = ALS Functional Rating Scale–Revised; BS-CV CT = brainstem-cervical root conduction time; CMCT = central motor conduction time; CTX-BS CT = cortical-brainstem conduction time; FA = fractional anisotropy; FDI = first dorsal interosseous; LMN = lower motor neuron; ROI = region of interest; UMN = upper motor neuron.

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.

Underediting of GluR2 mRNA, a neuronal death inducing molecular change in sporadic ALS, does not occur in motor neurons in ALS1 or SBMA.

Deficient RNA editing of the AMPA receptor subunit GluR2 at the Q/R site is a primary cause of neuronal death and recently has been reported to be a tightly linked etiological cause of motor neuron death in sporadic amyotrophic lateral sclerosis (ALS). We quantified the RNA editing efficiency of the GluR2 Q/R site in single motor neurons of rats transgenic for mutant human Cu/Zn-superoxide dismutase (SOD1) as well as patients with spinal and bulbar muscular atrophy (SBMA), and found that GluR2 mRNA was completely edited in all the motor neurons examined. It seems likely that the death cascade is different among the dying motor neurons in sporadic ALS, familial ALS with mutant SOD1 and SBMA.