Negative features of sporadic amyotrophic lateral sclerosis: Motor neurons of Onuf's nucleus survive in ADAR2-conditional knockout mice.

Patients with amyotrophic lateral sclerosis (ALS) do not develop oculomotor disturbances and vesicorectal dysfunction until end-stage disease owing to the survival of certain motor neurons (MNs), including oculomotor neurons and MNs within Onuf's nucleus. In sporadic ALS, adenosine deaminase acting on RNA 2 (ADAR2)-mediated editing of GluA2 mRNA at the Q/R site is compromised in lower MNs. We previously developed genetically modified mice with a conditional knockout of ADAR2 in cholinergic neurons (ADAR2flox/flox/VAChT-Cre, Fast; AR2). These mice displayed slow and progressive lower motor neuron death with TAR DNA-binding protein 43 (TDP-43) pathology, attributable to insufficient editing at the GluA2 Q/R site due to ADAR2 deficiency. MN death was more common in fast-fatigable MNs owing to differential vulnerability under conditions of ADAR2 deficiency. Although facial and hypoglossal nerves were impaired in AR2 mice, cell death did not occur within the oculomotor nerve nucleus, as observed in patients with sporadic ALS. Since the basis for avoiding cystorectal damage in ALS is unknown, we compared the features of Onuf's nucleus MNs in 12-month-old AR2 mice with those in age-matched wild-type mice. Although the number of MNs was not significantly lower in AR2 mice, the neurons exhibited a shrunken morphology and TDP-43 pathology. Onuf's nucleus MNs could survive in an ADAR2-deficient state and mainly included fast fatigue-resistant (FR) and slow (S) MNs. In summary, FR and S MNs show increased resilience to ADAR2 deficiency, potentially participating in an important neuronal death avoidance mechanism in ALS.

Enhanced Marine Biodegradation of Polycaprolactone through Incorporation of Mucus Bubble Powder from Violet Sea Snail as Protein Fillers.

Microplastics' spreading in the ocean is currently causing significant damage to organisms and ecosystems around the world. To address this oceanic issue, there is a current focus on marine degradable plastics. Polycaprolactone (PCL) is a marine degradable plastic that is attracting attention. To further improve the biodegradability of PCL, we selected a completely new protein that has not been used before as a functional filler to incorporate it into PCL, aiming to develop an environmentally friendly biocomposite material. This novel protein is derived from the mucus bubbles of the violet sea snail (VSS, Janthina globosa), which is a strong bio-derived material that is 100% degradable in the sea environment by microorganisms. Two types of PCL/bubble composites, PCL/b1 and PCL/b5, were prepared with mass ratios of PCL to bubble powder of 99:1 and 95:5, respectively. We investigated the thermal properties, mechanical properties, biodegradability, surface structure, and crystal structure of the developed PCL/bubble composites. The maximum biochemical oxygen demand (BOD) degradation for PCL/b5 reached 96%, 1.74 times that of pure PCL (≈55%), clearly indicating that the addition of protein fillers significantly enhanced the biodegradability of PCL. The surface morphology observation results through scanning electron microscopy (SEM) definitely confirmed the occurrence of degradation, and it was found that PCL/b5 underwent more significant degradation compared to pure PCL. The water contact angle measurement results exhibited that all sheets were hydrophobic (water contact angle > 90°) before the BOD test and showed the changes in surface structure after the BOD test due to the newly generated indentations on the surface, which led to an increase in surface toughness and, consequently, an increase in surface hydrophobility. A crystal structure analysis by wide-angle X-ray scattering (WAXS) discovered that the amorphous regions were decomposed first during the BOD test, and more amorphous regions were decomposed in PCL/b5 than in PCL, owing to the addition of the bubble protein fillers from the VSS. The differential scanning calorimeter (DSC) and thermal gravimetric analysis (TGA) results suggested that the addition of mucus bubble protein fillers had only a slight impact on the thermal properties of PCL. In terms of mechanical properties, compared to pure PCL, the mucus-bubble-filler-added composites PCL/b1 and PCL/b5 exhibited slightly decreased values. Although the biodegradability of PCL was significantly improved by adding the protein fillers from mucus bubbles of the VSS, enhancing the mechanical properties at the same time poses the next challenging issue.

Pathological features of glial cells and motor neurons in the anterior horn of the spinal cord in sporadic ALS using ADAR2 conditional knockout mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective degeneration of motor neurons (MNs). In the MNs of patients with ALS, adenosine deaminase acting on RNA 2 (ADAR2)-mediated RNA editing of GluA2 mRNA at the Q/R site is profoundly deficient. In genetically modified mice (ADAR2flox/flox/VAChT-Cre.Fast; AR2), the selective knockout of ADAR2 in cholinergic neurons induced progressive loss of lower MNs. MNs exhibiting an age-related increase in abnormal TDP-43 localization and reduced ADAR2 immunoreactivity are localized in the lateral areas of the anterior horns (AHs) in aged wild-type mice. However, the patterns in the AHs of AR2 mice remain unknown. In this study, we investigated whether similar degeneration is observed in AR2 mice. We compared the number of astrocytes and MNs in the lateral and medial AHs of the lumbar spinal cord of 12-month-old AR2 mice with age-matched wild-type mice. The number of MNs significantly decreased in both the lateral and medial areas in AR2 mice AHs, particularly in the former. The number of reactive astrocytes increased significantly in the lateral areas of the AHs of AR2 mice. In conclusion, stronger activation of astrocytes with reduction of MNs in the ADAR2 deficiency-related lateral area increases in AR2 mice AHs. Fast fatigable MNs are expected to be present in the lateral area of the AHs. We found that MN death is more common in the lateral area of AHs associated with FF MNs due to differences in vulnerability to MN under ADAR2 deficiency.

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.

ADAR2-dependent A-to-I RNA editing in the extracellular linear and circular RNAs.

Currently, no reliable biomarkers of amyotrophic lateral sclerosis (ALS) exist. In sporadic ALS, RNA editing at the glutamine/arginine site of GluA2 mRNA is specifically reduced in the motor neurons due to the downregulation of adenosine deaminase acting on RNA 2 (ADAR2). Furthermore, TDP-43 pathology, the pathological hallmark of ALS, is observed in the ADAR2-lacking motor neurons in ALS patients and conditional ADAR2 knockout mice, suggesting a pivotal role of ADAR2 downregulation in the ALS pathogenesis. Extracellular RNAs were shown to represent potential disease biomarkers and the editing efficiencies at their ADAR2-dependent sites may reflect cellular ADAR2 activity, suggesting that these RNAs isolated from the body fluids may represent the biomarkers of ALS. We searched for ADAR2-dependent sites in the mouse motor neurons and human-derived cultured cells and found 10 sites in five host RNAs expressed in SH-SY5Y cells and their culture medium. Of these, the arginine/glycine site of SON mRNA was newly identified as an ADAR2-dependent site. Furthermore, we detected a circular RNA with an ADAR2-dependent site in the SH-SY5Y cells and their culture medium. Therefore, the changes in the editing efficiencies at the identified host RNA sites isolated from the body fluids may represent potential biomarkers of ALS.

Ca2+ -permeable AMPA receptors associated with epileptogenesis of hypothalamic hamartoma.

Hypothalamic hamartoma (HH), composed of neurons and glia without apparent cytologic abnormalities, is a rare developmental malformation in humans. Patients with HH often have characteristic medically refractory gelastic seizures, and intrinsic epileptogenesis within the lesions has been speculated. Herein we provide evidence to suggest that in HH neurons, Ca2+ permeability through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is aberrantly elevated. In needle biopsy specimens of HH tissue, field potential recordings demonstrated spontaneous epileptiform activities similar to those observed in other etiologically distinct epileptogenic tissues. In HH, however, these activities were clearly abolished by application of Joro Spider Toxin (JSTX), a specific inhibitor of the Ca2+ -permeable AMPA receptor. Consistent with these physiologic findings, the neuronal nuclei showed disappearance of adenosine deaminase acting on RNA 2 (ADAR2) immunoreactivity. Furthermore, examination of glutamate receptor 2 (GluA2) messenger RNA (mRNA) revealed that editing efficiency at the glutamine/arginine site was significantly low. These results suggest that neurons in HH may bear Ca2+ -permeable AMPA receptors due to dislocation of ADAR2.

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.

Phosphorylated TDP-43 becomes resistant to cleavage by calpain: A regulatory role for phosphorylation in TDP-43 pathology of ALS/FTLD.

TAR DNA-binding protein-43 (TDP-43) pathology, which includes the presence of abnormal TDP-43-containing inclusions with a loss of nuclear TDP-43 in affected neurons, is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobar degeneration (FTLD). TDP-43 in the pathological brains and spinal cords of ALS/FTLD patients is abnormally fragmented and phosphorylated. It is believed that the generation of aggregation-prone TDP-43 fragments initiates TDP-43 pathology, and we previously reported that calpain has an important role in the generation of such aggregation-prone TDP-43 fragments. However, the role of phosphorylation in TDP-43 pathology has not been largely elucidated, despite previous observations that several kinases and their kinases are involved in TDP-43 phosphorylation. Here, we investigated the role of TDP-43 phosphorylation in the calpain-dependent cleavage of TDP-43 and found that phosphorylated, full-length TDP-43 and calpain-dependent TDP-43 fragments were more resistant to cleavage by calpain than endogenous full-length TDP-43 was. These results suggest that both phosphorylated and calpain-cleaved TDP-43 fragments persist intracellularly for a length of time that is sufficient for self-aggregation, thereby serving as seeds for inclusions.

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 Ca(2+) -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.

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.

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.

The abnormal processing of TDP-43 is not an upstream event of reduced ADAR2 activity in ALS motor neurons.

TDP-43 pathology in motor neurons is a hallmark of ALS. In addition, the reduced expression of an RNA editing enzyme, adenosine deaminase acting on RNA 2 (ADAR2), increases the expression of GluA2 with an unedited Q/R site in the motor neurons of patients with sporadic ALS. As the occurrence of these two disease-specific abnormalities in the same motor neurons suggests a molecular link between them, we examined the effects of altered TDP-43 processing on ADAR2 activity in TetHeLaG2m and Neuro2a cells. We found that ADAR2 activity did not consistently change due to the overexpression or knockdown of TDP-43 or the expression of abnormal TDP-43, including caspase-3-cleaved fragments, truncated TDP-43 lacking either nuclear localization or export signals and ALS-linked TDP-43 mutants. These results suggest that the abnormal processing of TDP-43 is not an upstream event of inefficient GluA2 Q/R site editing in the motor neurons of sporadic ALS patients.

Classification of neural differentiation-associated genes in P19 embryonal carcinoma cells by their expression patterns induced after cell aggregation and/or retinoic acid treatment.

Expression of neural differentiation-associated genes was examined by RT-PCR and macroarray analyses during neural differentiation of P19 embryonal carcinoma cells induced by cell aggregation and/or retinoic acid (RA) treatment. Results revealed that the neural genes examined could be classified into 4 groups based on their expression patterns. The 1st group included the Wnt-1, Id-1, Id-3 and cdc42 genes, expression of which was altered by cell aggregation alone, but not by RA treatment alone. The 2nd group included the alphaN-catenin, Neuro D and GDNFRbeta genes, expression of which was altered by RA treatment alone, but not by cell aggregation. The 3rd group consisted of the Brn-2, TrkA, bcl-X, N-cadherin, E-cadherin and Otx-2 genes, expression of which was altered by either treatment. The 4th group included the ACTH, D4DR, NGC and Oct-3 genes, the expression of which changed only when both treatments were applied simultaneously. Expression of the Ets-1 and Fli-1 transcription factor genes was up-regulated by either treatment alone at initial stages of neural differentiation of P19 cells, although overexpression of these genes alone could not induce cell differentiation. Our results suggest that although both treatments are required for complete neural differentiation of P19 cells, cell aggregation or RA treatment alone drive differentiation to a certain extent at the gene expression level.

Effects of overexpression of the Ets family transcription factor TEL on cell growth and differentiation of K562 cells.

Functional roles of several Ets transcription factors in megakaryocytic and erythroid differentiation of the human chronic myelogenous leukemia cell line K562 was investigated. When K562 cells were induced to differentiate into the megakaryocyte lineage by treatment with TPA, the expression of the c-ets-1, Fli-1 and TEL2 genes was increased, and that of the TEL gene was decreased at the onset of differentiation. When the cells were induced to differentiate into the erythroid lineage by treatment with Hemin, expression of the c-ets-1 gene was increased, and that of the Fli-1 and TEL genes was decreased. To investigate the role of TEL in the growth and differentiation of K562 cells, we introduced an expression plasmid containing the TEL gene into the cells. Overexpression of TEL in K562 cells leads to inhibition of the endogenous expression of megakaryocyte-specific GPIIb and GPIbalpha genes, and inhibition of cell growth. DNA array analysis revealed that expression of genes such as the RhoG and D4-GDI genes was down-regulated in TEL-overexpressing cells, while that of the representative growth-related genes such as the c-myc, c-fos and c-jun genes was not remarkably changed. These results suggest that several Ets family transcription factors are implicated in megakaryocytic and erythroid differentiation of K562 cells and TEL inhibits the expression of some megakaryocyte-specific genes and growth in K562 cells.