Testing of the therapeutic efficacy and safety of AMPA receptor RNA aptamers in an ALS mouse model.

In motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients, the RNA editing at the glutamine/arginine site of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors is defective or incomplete. As a result, AMPA receptors containing the abnormally expressed, unedited isoform of GluA2 are highly Ca2+-permeable, and are responsible for mediating abnormal Ca2+ influx, thereby triggering motor neuron degeneration and cell death. Thus, blocking the AMPA receptor-mediated, abnormal Ca2+ influx is a potential therapeutic strategy for treatment of sporadic ALS. Here, we report a study of the efficacy and safety of two RNA aptamers targeting AMPA receptors on the ALS phenotype of AR2 mice. A 12-wk continuous, intracerebroventricular infusion of aptamers to AR2 mice reduced the progression of motor dysfunction, normalized TDP-43 mislocalization, and prevented death of motor neurons. Our results demonstrate that the use of AMPA receptor aptamers as a novel class of AMPA receptor antagonists is a promising strategy for developing an ALS treatment approach.

Altered Intracellular Milieu of ADAR2-Deficient Motor Neurons in Amyotrophic Lateral Sclerosis.

Transactive response DNA-binding protein (TDP-43) pathology, and failure of A-to-I conversion (RNA editing) at the glutamine/arginine (Q/R) site of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of most patients with amyotrophic lateral sclerosis (ALS). Adenosine deaminase acting on RNA 2 (ADAR2) specifically catalyzes GluA2 Q/R site-RNA editing. Furthermore, conditional ADAR2 knockout mice (AR2) exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons, which is the most reliable pathological marker of ALS. Therefore, the evidence indicates that ADAR2 downregulation is a causative factor in ALS, and AR2 mice exhibit causative molecular changes that occur in ALS. We discuss the contributors to ADAR2 downregulation and TDP-43 pathology in AR2 mouse motor neurons. We describe mechanisms of exaggerated Ca2+ influx amelioration via AMPA receptors, which is neuroprotective in ADAR2-deficient motor neurons with normalization of TDP-43 pathology in AR2 mice. Development of drugs to treat diseases requires appropriate animal models and a sensitive method of evaluating efficacy. Therefore, normalization of disrupted intracellular environments resulting from ADAR2 downregulation may be a therapeutic target for ALS. We discuss the development of targeted therapy for ALS using the AR2 mouse model.

Calpain-dependent disruption of nucleo-cytoplasmic transport in ALS motor neurons.

Nuclear dysfunction in motor neurons has been hypothesized to be a principal cause of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we investigated the mechanism by which the nuclear pore complex (NPC) is disrupted in dying motor neurons in a mechanistic ALS mouse model (adenosine deaminase acting on RNA 2 (ADAR2) conditional knockout (AR2) mice) and in ALS patients. We showed that nucleoporins (Nups) that constituted the NPC were cleaved by activated calpain via a Ca2+-permeable AMPA receptor-mediated mechanism in dying motor neurons lacking ADAR2 expression in AR2 mice. In these neurons, nucleo-cytoplasmic transport was disrupted, and the level of the transcript elongation enzyme RNA polymerase II phosphorylated at Ser2 was significantly decreased. Analogous changes were observed in motor neurons lacking ADAR2 immunoreactivity in sporadic ALS patients. Therefore, calpain-dependent NPC disruption may participate in ALS pathogenesis, and inhibiting Ca2+-mediated cell death signals may be a therapeutic strategy for ALS.

The AMPA receptor antagonist perampanel robustly rescues amyotrophic lateral sclerosis (ALS) pathology in sporadic ALS model mice.

Both TDP-43 pathology and failure of RNA editing of AMPA receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of the majority of patients with amyotrophic lateral sclerosis (ALS). AR2 mice, in which an RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is conditionally knocked out in the motor neurons, exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons through a Ca(2+)-permeable AMPA receptor-mediated mechanism. Therefore, amelioration of the increased Ca(2+) influx by AMPA receptor antagonists may be a potential ALS therapy. Here, we showed that orally administered perampanel, a selective, non-competitive AMPA receptor antagonist significantly prevented the progression of the ALS phenotype and normalized the TDP-43 pathology-associated death of motor neurons in the AR2 mice. Given that perampanel is an approved anti-epileptic drug, perampanel is a potential candidate ALS drug worthy of a clinical trial.

General malaise and physical symptoms in young women with untouched toe.

Untouched toe is a condition in which a toe does not touch the ground while standing. It is frequently observed in women even under physiological conditions. Deformities or symptoms of the toes are not observed in these women. The clinical significance of untouched toe has not been fully elucidated. Two hundred young healthy women were recruited into the present study after informed consent. We evaluated the prevalence of untouched toe by measuring various indexes of the toe using a foot-sole-measuring equipment. We also conducted a self-administered questionnaire regarding general malaise. Untouched toe was observed in 114 of these 200 women (57.0%). The fifth toe was more frequently affected than the other toes. There were no significant differences in size of foot except the area and proportion touching the ground between women with untouched toe and those without untouched toe. The prevalence of general malaise was significantly higher in women with at least one untouched toe (57.0%) compared with those without untouched toe (43.0%) (p<0.05). Twelve symptoms-irritability, headache, tired eyes, hazy vision, congested or runny nose, irregular menstruation or menstrual pain, shoulder stiffness, neck stiffness, low back pain, cold hands, swollen feet, and cold feet-were more frequently observed in women with at least one untouched toe compared with those without untouched toes. Untouched toe was associated with various symptoms of general malaise. However, the pathological mechanism by which untouched toe causes these symptoms has not been determined. Further analysis of gait and exercise habits in women with untouched toe is necessary.

A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation.

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely-not only in the nucleus, but also in the cytoplasm-and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well.

[Hereditary diffuse leukoencephalopathy with neuroaxonal spheroids (HDLS) in early-onset dementia].

By reviewing and collating data in a 2-step postal survey sent to all of the institutions for individuals with dementia in Ibaraki prefecture requesting information on early-onset dementia (EOD) cases, 617 subjects with EOD were identified. The estimated prevalence of EOD in the target population was 42.3 per 100,000. Of the illness causing EOD, vascular dementia was the most frequent followed by Alzheimer's disease, head trauma, dementia with Lewy body/Parkinson's disease with dementia, frontotemporal lobar degeneration, and other causes. On the other hand, hereditary diffuse leucoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white matter disease with variable phenotypes. The onset of symptoms is usually in the fourth or fifth decade, progressing to dementia with death within 6 years. Recently, several mutations of the colony stimulating factor 1 receptor encoded by CSF1R segregating HDLS were identified. Since clinical presentations varied substantially within and across families with HDLS, CSF1R mutation carriers may be present in clinical series of Alzheimer's disease, frontotemporal lobar degeneration, corticobasal syndrome, multiple sclerosis, CADASIL, Parkinson's disease and ischemic stroke with additional white matter changes, all causing EOD. In the differential diagnosis of EOD, we should always consider HDLS and if necessary perform CSF1R gene analysis.

Adsorption characteristics of various proteins to a titanium surface.

Adsorption characteristics of 18 proteins, with different sizes and isoelectric points, to a titanium oxide surface were studied. The adsorption isotherms were categorized based on protein type and pH: type 1, irreversible adsorption; type 2, Langmuir-type reversible adsorption; and type 3, reversible and irreversible adsorption. Most of the proteins tested were irreversibly adsorbed in the pH range of 3-8, whereas most adsorbed reversibly at pH 8.5-9.4. Protamine, with a pI value of 12, adsorbed reversibly in the pH range of 3-9. pH values that gave maximal sums of irreversibly and reversibly adsorbed proteins were in the pH range of 3-8 and tended to increase slightly with the pI value of the corresponding protein. pH values that gave maximal quantities of irreversibly adsorbed protein ranged between 4-6 and were nearly independent of pI.