Application of integrative physiological approach to evaluate human physiological responses to the inhalation of essential oils of Japanese citrus fruits iyokan (Citrus iyo) and yuzu (Citrus junos).

This study investigated the effects of essential oil odors from Japanese citrus fruits, iyokan (Citrus iyo) and yuzu (Citrus junos), on human psychology and both the autonomic and central nervous systems. The inhalation of both essential oils significantly increased miosis rate and fingertip temperature and could induce parasympathetic dominance by suppressing sympathetic nerve activity. Oxyhemoglobin concentration in the prefrontal cortex increased after the inhalation of yuzu essential oil and decreased after the inhalation of iyokan essential oil. Subjectively, the inhalation of both essential oils reduced the feelings of fatigue and improved the feelings of refreshment, suggesting that the effect of autonomic nervous activity might involve in these psychological changes directly. Moreover, we observed that task performance improved after inhaling yuzu essential oil, which may be due to the increase in oxyhemoglobin concentration in the prefrontal cortex.

Effect of Tongue-Hold Swallow on Pharyngeal Contractile Properties in Healthy Individuals.

Tongue-hold swallow (THS) is a swallow exercise in which an individual swallows saliva while holding the anterior portion of the tongue between the front teeth. The effect of THS on pharyngeal contractile vigor is still unclear. The purpose of this study was to quantify THS using high-resolution manometry with a contractile integral analysis. Twenty-two healthy participants performed three different saliva swallow tasks: normal swallow, weak THS (in which the tongue was protruded 1 cm outside the upper incisors), and strong THS (in which the tongue was protruded 2 cm outside the upper incisors). The participants repeated each task twice randomly. Pharyngeal and upper esophageal sphincter metrics, including the pharyngeal contractile integral, were analyzed. Both weak and strong THS enhanced the velopharyngeal contractile integral and peak pressure compared with normal swallow (P < 0.01). THS also prolonged mesopharyngeal contraction (P < 0.01). Holding the tongue anteriorly during swallow requires significant biomechanical changes to pharyngeal contractile properties at the superior and middle pharyngeal constrictor levels; thus, it may serve as a resistance exercise for the muscles that are involved in bolus propulsion.

Survival motor neuron protein regulates oxidative stress and inflammatory response in microglia of the spinal cord in spinal muscular atrophy.

The deficiency of survival motor neuron (SMN) protein can result in the onset of spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by a progressive loss of motor neurons and skeletal muscle atrophy. The mechanism underlying SMA pathology remains unclear. Here, we demonstrate that SMN protein regulates oxidative stress and inflammatory response in microglia. Antisense oligonucleotide, which increases SMN protein expression (SMN-ASO), attenuated SMA model mice phenotypes and suppressed the activation of microglia in the spinal cord. The expression of oxidative stress marker in microglia was decreased by SMN-ASO injection in SMA model mice. Increased reactive oxygen species production and subsequent antioxidative stress reaction was observed in SMN protein-depleted RAW264.7. Furthermore, nuclear factor kappa B (NFκB) and c-Jun amino terminal kinase (JNK) signaling, which mainly mediate the inflammatory response, are activated in SMN protein-depleted RAW264.7. Tumor necrosis factor-α (TNF-α) production is also increased in SMN protein-depleted RAW264.7. These findings suggest that SMN protein regulates oxidative stress and inflammatory response in microglia, supporting current claims that microglia can be an effective target for SMA therapy.

The Protective Effects of Levetiracetam on a Human iPSCs-Derived Spinal Muscular Atrophy Model.

Spinal muscular atrophy (SMA) is an inherited disease characterized by progressive motor neuron death and subsequent muscle weakness and is caused by deletion or mutation of survival motor neuron (SMN) 1 gene. Protecting spinal motor neuron is an effective clinical strategy for SMA. The purpose of this study was to investigate the potential effect of an anti-epileptic drug levetiracetam on SMA. In the present study, we used differentiated spinal motor neurons (MNs) from SMA patient-derived induced pluripotent stem cells (SMA-iPSCs) to investigate the effect of levetiracetam. Levetiracetam promoted neurite elongation in SMA-iPSCs-MNs. TUNEL-positive spinal motor neurons were significantly reduced by levetiracetam in SMA-iPSCs-MNs. In addition, the expression level of cleaved-caspase 3 was decreased by levetiracetam in SMA-iPSCs-MNs. Furthermore, levetiracetam improved impaired mitochondrial function in SMA-iPSCs-MNs. On the other hand, levetiracetam did not affect the expression level of SMN protein in SMA-iPSCs-MNs. These findings indicate that levetiracetam has a neuroprotective effect for SMA.

SMN Protein Contributes to Skeletal Muscle Cell Maturation Via Caspase-3 and Akt Activation.

BACKGROUND/AIM: In spinal muscular atrophy (SMA), systemic deficiency of survival motor neurons (SMN) caused by loss or mutation of SMN1 leads to SMA symptoms. SMA was, for a long time, considered as a selective motor-neuron disease. However, accumulated evidence suggests that skeletal muscle cells are affected by low levels of SMN protein. The purpose of this study was to elucidate the function of SMN protein in skeletal cell differentiation and maturation. MATERIALS AND METHODS: In SMNΔ7 mice, which exhibit a systemic reduction of SMN protein, muscle atrophy was evaluated. To direct the effect of SMN against muscle cells, SMN functions were examined by knockdown of SMN in mouse myoblasts cell line C2C12 using siRNA. RESULTS: SMNΔ7 mice showed muscle atrophy accompanied by decreased both expression of a myogenesis marker and a proliferating marker. In SMN-knockdown myoblasts, early expression of myosin heavy chain and reduced multinuclear myotube formation were found. Decreased caspase-3 activity and reduced phosphorylation of Akt were observed at an early stage of differentiation in SMN-knockdown myoblasts. CONCLUSION: A critical role of SMN protein in muscle cell differentiation via caspase-3 and Akt activation was shown.

Discovery of a CNS penetrant small molecule SMN2 splicing modulator with improved tolerability for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor neuron disease, typically resulting from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Nusinersen/SPINRAZA, a splice-switching oligonucleotide that modulates SMN2 (a paralog of SMN1) splicing and consequently increases SMN protein levels, has a therapeutic effect for SMA. Previously reported small-molecule SMN2 splicing modulators such as risdiplam/EVRYSDI and its analog SMN-C3 modulate not only the splicing of SMN2 but also that of secondary splice targets, including forkhead box protein M1 (FOXM1). Through screening SMA patient-derived fibroblasts, a novel small molecule, designated TEC-1, was identified that selectively modulates SMN2 splicing over three secondary splice targets. TEC-1 did not strongly affect the splicing of FOXM1, and unlike risdiplam, did not induce micronucleus formation. In addition, TEC-1 showed higher selectively on galactosylceramidase and huntingtin gene expression compared to previously reported compounds (e.g., SMN-C3) due to off-target effects on cryptic exon inclusion and nonsense-mediated mRNA decay. Moreover, TEC-1 significantly ameliorated the disease phenotype in an SMA murine model in vivo. Thus, TEC-1 may have promising therapeutic potential for SMA, and our study demonstrates the feasibility of RNA-targeting small-molecule drug development with an improved tolerability profile.

Reversible Redox Property of Co(III) in Amorphous Co-doped SiO2/γ-Al2O3 Layered Composites.

This paper reports on a unique reversible reducing and oxidizing (redox) property of Co(III) in Co-doped amorphous SiO2/γ-Al2O3 composites. The Fenton reaction during the H2O2-catalyzed sol-gel synthesis utilized in this study lead to the partial formation of Co(III) in addition to Co(II) within the composites. High-resolution transmission electron microscopy (HRTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analyses for the composite powder sample with a composition of Al:Si:Co = 85:10:5 showed the amorphous state of the Co-doped SiO2 that modified γ-Al2O3 nanocrystalline surfaces. In situ X-ray absorption fine structure (XAFS) spectroscopic analysis suggested reversible redox reactions of Co species in the composite powder sample during heat-treatment under H2 at 500 °C followed by subsequent cooling to RT under Ar. Further analyses by in situ IR spectroscopy combined with cyclic temperature programmed reduction/desorption (TPR/TPD) measurements and X-ray photoelectron spectroscopic (XPS) analysis revealed that the alternating Co(III)/(II) redox reactions were associated with OH formation (hydrogenation)-deformation (dehydrogenation) of the amorphous aluminosilicate matrix formed in situ at the SiO2/γ-Al2O3 hetero interface, and the redox reactions were governed by the H2 partial pressure at 250-500 °C. As a result, a supported mesoporous γ-Al2O3/Co-doped amorphous SiO2/mesoporous γ-Al2O3 three-layered composite membrane exhibited an H2-triggered chemical valve property: mesopores under H2 flow (open) and micropores under He flow (closure) at 300-500 °C.

Notch Signaling Mediates Astrocyte Abnormality in Spinal Muscular Atrophy Model Systems.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of spinal motor neurons and muscle atrophy. The disease is mainly caused by low level of the survival motor neuron (SMN) protein, which is coded by two genes, namely SMN1 and SMN2, but leads to selective spinal motor neuron degeneration when SMN1 gene is deleted or mutated. Previous reports have shown that SMN-protein-deficient astrocytes are abnormally abundant in the spinal cords of SMA model mice. However, the mechanism of the SMN- deficient astrocyte abnormality remains unclear. The purpose of this study is to identify the cellular signaling pathways associated with the SMN-deficient astrocyte abnormality and propose a candidate therapy tool that modulates signaling. In the present study, we found that the astrocyte density was increased around the central canal of the spinal cord in a mouse SMA model and we identified the dysregulation of Notch signaling which is a known mechanism that regulates astrocyte differentiation and proliferation, in the spinal cord in both early and late stages of SMA pathogenesis. Moreover, pharmacological inhibition of Notch signaling improved the motor functional deficits in SMA model mice. These findings indicate that dysregulated Notch signaling may be an underlying cause of SMA pathology.

Impairment of oligodendrocyte lineages in spinal muscular atrophy model systems.

Survival motor neuron (SMN) deficiency indicates that various cellular processes are impaired in spinal muscular atrophy (SMA). Previous reports have shown that SMN deficiency causes motor neuron degeneration, whereas the numbers of astrocytes and microglia are significantly increased or activated in SMA model systems. Only a few groups have studied the role of oligodendrocyte (OL) lineages such as OL precursor cell and nerve/glial antigen 2 (NG2)-glia in SMA pathology. Our aim in this study was to investigate whether OL lineages are impaired in SMA model systems. We investigated the expression of myelin basic protein (MBP) and NG2, which are OL lineage markers, using SMNΔ7 mice (mSmn, SMN2, SMNΔ7) and cell cultures derived from induced pluripotent stem cells generated from SMA patients. We showed for the first time that the OL lineages, including NG2-positive OL precursor cells and MBP-positive myelinating OLs were impaired in SMNΔ7 mice and induced pluripotent stem cells derived from SMA patients. Notch was involved in the decline of NG2 expression in the spinal cord of SMNΔ7 mice. In addition, pharmacological Notch inhibition promoted MBP-positive OL differentiation in SMNΔ7 mice. These findings indicate that OL differentiation was impaired in SMA, which might be involved in the Notch dysregulation.

Efficacy of Prednisolone in Generated Myotubes Derived From Fibroblasts of Duchenne Muscular Dystrophy Patients.

Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy characterized by progressive muscle degeneration. This disease is caused by the mutation or deletion of the dystrophin gene. Currently, there are no effective treatments and glucocorticoid administration is a standard care for DMD. However, the mechanism underlying prednisolone effects, which leads to increased walking, as well as decreased muscle wastage, is poorly understood. Our purpose in this study is to investigate the mechanisms of the efficacy of prednisolone for this disease. We converted fibroblasts of normal human cell line and a DMD patient sample to myotubes by MyoD transduction using a retroviral vector. In myotubes from the MyoD-transduced fibroblasts of the DMD patient, the myotube area was decreased and its apoptosis was increased. Furthermore, we confirmed that prednisolone could rescue these pathologies. Prednisolone increased the expression of not utrophin but laminin by down-regulation of MMP-2 mRNA. These results suggest that the up-regulation of laminin may be one of the mechanisms of the efficacy of prednisolone for DMD.

Effect of Timolol on Optineurin Aggregation in Transformed Induced Pluripotent Stem Cells Derived From Patient With Familial Glaucoma.

Purpose: To determine a chemical agent that can reduce the aggregation of optineurin (OPTN) in cells differentiated from induced pluripotent stem cells obtained from a patient with normal-tension glaucoma (NTG) caused by an E50K mutation in the OPTN gene (OPTNE50K-NTG). Methods: Retinal ganglion cells (RGCs) were created from induced pluripotent stem cells derived from a healthy individual (wild-type [WT]-iPSCs) and from a patient with NTG due to OPTNE50K (E50K-iPSCs) mutation. The death of the induced RGCs was evaluated by counting the number of TUNEL- and ATH5-positive cells. Axonal growth was determined by measuring the axonal length of TUJ1-positive cells. OPTN aggregation was assessed by measuring the OPTN-positive area by immunofluorescence and by Western blotting. Autophagic flux assay was investigated by determining the light chain 3 (LC3)B-II/LC3B-I ratio and p62 expression by Western blotting. Results: The results showed OPTNE50K aggregation, activation of astrocytes, reduction in the number of RGCs, and enhancement of apoptotic cell death in the in vitro OPTNE50K model of NTG. Timolol was found to reduce the OPTNE50K-positive area and decreased the insoluble OPTNE50K, suggesting that it has the potential of reducing the OPTNE50K aggregation. Timolol also increased the ATH5-positive cells, decreased TUNEL-positive cells, increased the LC3B-II/LC3B-I ratio, and decreased the expression of p62. These findings suggest that timolol might enhance autophagic flux, leading to reduced OPTNE50K aggregation. Conclusions: Timolol should be considered a potential therapeutic agent specific to OPTNE50K-NTG because it can reduce the OPTNE50K aggregation in E50K-iPSCs-RGCs by enhancing autophagic flux and neuroprotective effects.

Edaravone is a candidate agent for spinal muscular atrophy: In vitro analysis using a human induced pluripotent stem cells-derived disease model.

Spinal muscular atrophy (SMA) is an intractable disease characterized by a progressive loss of spinal motor neurons, which leads to skeletal muscle weakness and atrophy. Currently, there are no curative agents for SMA, although it is understood to be caused by reduced levels of survival motor neuron (SMN) protein. Additionally, why reduced SMN protein level results in selective apoptosis in spinal motor neurons is still not understood. Our purpose in this study was to evaluate the therapeutic potential of edaravone, a free radical scavenger, by using induced pluripotent stem cells from an SMA patient (SMA-iPSCs) and to address oxidative stress-induced apoptosis in spinal motor neurons. We first found that edaravone could improve impaired neural development of SMA-iPSCs-derived spinal motor neurons with limited effect on nuclear SMN protein expression. Furthermore, edaravone inhibited the generation of reactive oxygen species and mitochondrial reactive oxygen species upregulated in SMA-iPSCs-derived spinal motor neurons, and reversed oxidative-stress induced apoptosis. In this study, we suggest that oxidative stress might be partly the reason for selective apoptosis in spinal motor neurons in SMA pathology, and that oxidative stress-induced apoptosis might be the therapeutic target of SMA.

Established Stem Cell Model of Spinal Muscular Atrophy Is Applicable in the Evaluation of the Efficacy of Thyrotropin-Releasing Hormone Analog.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of spinal motor neurons. This disease is mainly caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Currently, no effective treatment is available, and only symptomatic treatment can be provided. Our purpose in the present study was to establish a human SMA-derived induced pluripotent stem cell (SMA-iPSC) disease model and assay a therapeutic drug in preparation for the development of a novel treatment of SMA. We generated iPSCs from the skin fibroblasts of a patient with SMA and confirmed that they were pluripotent and undifferentiated. The neural differentiation of SMA-iPSCs shortened the dendrite and axon length and increased the apoptosis of the spinal motor neurons. In addition, we found activated astrocytes in differentiated SMA-iPSCs. Using this model, we confirmed that treatment with the thyrotropin-releasing hormone (TRH) analog, 5-oxo-l-prolyl-l-histidyl-l-prolinamide, which had marginal effects in clinical trials, increases the SMN protein level. This increase was mediated through the transcriptional activation of the SMN2 gene and inhibition of glycogen synthase kinase-3ß activity. Finally, the TRH analog treatment resulted in dendrite and axon development of spinal motor neurons in differentiated SMA-iPSCs. These results suggest that this human in vitro disease model stimulates SMA pathology and reveal the potential efficacy of TRH analog treatment for SMA. Therefore, we can screen novel therapeutic drugs such as TRH for SMA easily and effectively using the human SMA-iPSC model. Significance: Platelet-derived growth factor (PDGF) has recently been reported to produce the greatest increase in survival motor neuron protein levels by inhibiting glycogen synthase kinase (GSK)-3ß; however, motor neurons lack PDGF receptors. A human in vitro spinal muscular atrophy-derived induced pluripotent stem cell model was established, which showed that the thyrotropin releasing hormone (TRH) analog promoted transcriptional activation of the SMN2 gene and inhibition of GSK-3ß activity, resulting in the increase and stabilization of the SMN protein and axon elongation of spinal motor neurons. These results reveal the potential efficacy of TRH analog treatment for SMA.

[An outbreak of encephalopathy after eating autumn mushroom (Sugihiratake; Pleurocybella porrigens) in patients with renal failure: a clinical analysis of ten cases in Yamagata, Japan].

In September and October, 2004, an outbreak of encephalopathy of unknown etiology occurred in certain areas of Japan including Yamagata, Akita, and Niigata prefectures. These patients had a history of chronic renal failure, most of them had undergone hemodialysis, and also had a history of eating Sugihiratake (Pleurocybella porrigens), an autumn mushroom without known toxicity. Since clinical details of this type of encephalopathy remain unknown, we analyzed the clinical, radiological and electroencephalographic (EEG) features of ten cases of this encephalopathy in Yamagata prefecture. The summary of the present study is as follows: 1. Ten patients had chronic renal failure, and seven underwent hemodialysis. 2. Each patient had a history of eating Sugihiratake within 2-3 weeks of the onset of neurological symptoms. 3. The onset was subacute; the initial symptoms were tremor, dysarthria, and/or weakness of the extremities, which lasted an average of 4.5 days (ranging from 2 to 11 days), followed by severe consciousness disturbance and intractable seizures, resulting in status epilepticus in 5 patients. Myoclonus was also seen in 4 patients and Babinski reflex in 3. 4. Brain CT and MRI examinations were unremarkable in the early stages of the disease. Three to eight days after onset, however, conspicuous lesions appeared in the areas of the insula and basal ganglia in 6 patients. On MRI, these brain lesions were hyperintense on T2-weighted and FLAIR images, and hypointense on T1-weighted images. 5. EEG examination was performed in 6 patients, all of whom showed abnormal EEG findings. Periodic synchronous discharge (PSD) was seen in 2 patients, spike and wave complex in one patient, and non-specific slow waves in 3. 6. Prognosis was different from case to case. Three patients died at 13, 14, and 29 days after onset. Two patients still showed persistent disturbance of consciousness one month after onset. One patient showed parkinsonism after recovering from consciousness disturbance. Four patients recovered nearly completely around one month after onset In 3 of the 4 recovered patients, renal failure was not severe and they did not need to undergo hemodialysis. This suggests that the degree of renal failure is a key for the prognosis of this type of encephalopathy. The present study suggests that this endemic disease is a newly recognized clinical entity of encephalopathy.