Donepezil, an anti-Alzheimer's disease drug, promotes differentiation and regeneration in injured skeletal muscle through the elevation of the expression of myogenic regulatory factors.

We previously demonstrated that donepezil, an anti-Alzheimer's disease drug, improved skeletal muscle atrophy by enhancing the angiogenesis of endothelial cells and activating the proliferation of satellite cells in a mouse model of peripheral arterial disease. However, the effect of donepezil on muscle differentiation during regeneration remains unclear. Therefore, we measured the expressions of myogenic regulatory factors and late muscle differentiation markers in donepezil-treated C2C12 myoblast cells before and after the induction of cell differentiation. The results indicate that the expressions of myogenin, troponin T (TnT) and myosin heavy chain (MyHC) were significantly increased and myotube formation was accelerated in donepezil-treated cells under the differentiation condition. However, the promotive effect of donepezil on muscle differentiation could not be reproduced by the addition of acetylcholine (ACh) and was not disrupted after treatment with ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to promote muscle differentiation in C2C12 cells. These results indicate that the specific characteristics of donepezil in the promotion of muscle differentiation are independent of its acetylcholinesterase-inhibitory action. We further found that donepezil induced an incremental shift of the cross-sectional area of myofibers and elevated the expressions of myogenin, TnT and MyHC in a mouse model of cardiotoxin injury. These results suggest that donepezil promotes the differentiation of muscle regeneration upon injury via the elevation of the expressions of myogenic regulatory factors and late muscle differentiation markers. Our findings suggest that donepezil can be a useful therapeutic agent for injured skeletal muscle treatment.

Burden of migraine among Japanese patients: a cross-sectional National Health and Wellness Survey.

BACKGROUND: Limited studies have measured the burden of migraine in Japan. This study aimed at estimating the disease burden of migraine in Japan and identifying factors associated with the burden using the 2017 National Health and Wellness Survey. METHODS: Migraine patients were defined by ICHD-3 like criteria with ≥4 monthly headache days (MHDs), and non-migraine respondents were selected using 1:4 propensity score matching. Multivariate analyses were conducted to compare Health-related Quality of Life (HRQoL), work productivity and activity impairment (WPAI), healthcare resource utilization (HRU) and costs between the two groups, and to identify factors associated with these outcomes in migraine patients. RESULTS: In 30,001 respondents, 378 migraine patients were identified. Compared to matched controls (N = 1512), migraine patients had lower physical (45.17 vs. 49.89), mental (42.28 vs. 47.71) and role/social (37.91 vs. 44.19) component summary scores (p < 0.001). Migraine patients had higher absenteeism (6.4% vs. 2.2%), presenteeism (40.2% vs. 22.5%), total work productivity impairment (44.3% vs. 24.5%), total activity impairment (45.0% vs. 23.9%), indirect costs (1,492,520 JPY vs. 808,320 JPY) and more visits to healthcare providers in the past 6 months (7.23 vs. 3.96) (p < 0.001). More MHDs was associated with worse HRQoL, and higher HRU and indirect costs. CONCLUSIONS: Japanese migraine patients experience an incremental burden. This demonstrates the unmet needs among Japanese migraine patients.

Relationship between the fall in blood pressure in the standing position and diaphragmatic muscle thickness: proof of concept study.

BACKGROUND: The diaphragm is an important muscle of respiration, and regulates the intrathoracic pressure. Blood pressure is regulated by the baroreceptor reflex system, and is also affected by intrathoracic pressure. We examined the relationship between the diaphragmatic muscle thickness and the degree of drop in blood pressure in the standing position. METHODS: We prospectively studied 15 healthy subjects. The diaphragmatic muscle thickness was measured using a B-mode ultrasonic imaging device. The blood pressure before and after standing was measured by a head-up tilt test. RESULTS: The diastolic blood pressure difference during expiration and inspiration showed a significant correlation with the diaphragmatic muscle thickness (r = 0.578, P = 0.024 and r = 0.518, P = 0.048, respectively). CONCLUSION: The diaphragmatic muscle thickness was related to the fall in diastolic blood pressure in the standing position. This indicates that adequate diaphragmatic muscle thickness helps to maintain intrathoracic pressure and prevents excessive drop in blood pressure in the standing position.

Candidate plasticity gene 16 mediates suppression of insulin gene expression in rat insulinoma INS-1 cells under glucotoxic conditions.

Chronic hyperglycemia causes pancreatic ß-cell dysfunction, impaired insulin secretion and suppression of insulin gene expression, referred to as glucotoxicity. Insulin gene expression is regulated by several protein kinases and protein phosphatases. However, the molecular mechanisms of the suppressed insulin gene expression in glucotoxicity are not fully understood. In this study, we employed rat insulinoma INS-1 cells as a model of pancreatic glucotoxicity. In INS-1 cells, insulin gene expression is up-regulated by incubation with 11.2 mM glucose for 7 days and down-regulated by incubation with 22.4 mM glucose for the same period. To identify the protein kinases and protein phosphatases involved in the suppression of insulin gene expression, we analyzed gene expression in INS-1 cells cultured with 11.2 mM or 22.4 mM glucose for 7 days using microarray analysis and real-time PCR. The expression levels of nine protein kinases were affected by glucotoxic conditions. In particular, CPG16 expression level was increased in INS-1 cells under these conditions. Transfection of CPG16 decreased insulin promoter activity, whereas kinase-dead mutant of CPG16 did not affect this. These results suggest that CPG16 plays a role in the suppression of insulin gene expression in pancreatic ß-cells under glucotoxic conditions.

A negative feedback loop between nuclear factor 90 (NF90) and an anti-oncogenic microRNA, miR-7.

Alterations in microRNAs (miRNAs) levels deeply correlate with tumorigenesis. However, the molecular mechanism for the regulation of the miRNA production in tumors is not fully understood. We previously reported that downregulation of miR-7, which is an anti-oncogenic miRNA, was caused by overexpression of the nuclear factor 90 (NF90)-nuclear factor 45 (NF45) complex through the binding of double-stranded (ds) RNA-binding proteins to primary miR-7, resulting in promotion of tumorigenesis (Higuchi et al 2016). During this study, we found that the level of NF90 protein was dramatically decreased by overexpression of miR-7. Interestingly, the miR-7-mediated reduction in NF90 family proteins was only observed in NF90 protein, but not in NF110 protein, which is a longer form of the NF90 gene. Luciferase reporter analysis indicated that the overexpression of miR-7 significantly repressed the luciferase activity in the coding region of NF90 mRNA harboring a predicted target sequence of miR-7. The luciferase activity of the reporter vector, which has a mutated miR-7 target site in the coding region, was the same in the control and miR-7 overexpressed cells. Furthermore, the translation of TARGET-tagged NF90 mRNA without the 3'UTR of the NF90 mRNA was inhibited by the overexpression of miR-7. These results imply that miR-7 suppresses NF90 at the protein level through the binding of miR-7 to the complementary site of the seed sequence in the coding region of the NF90 mRNA. We further confirmed increased endogenous NF90 protein levels in SK-N-SH cells transfected with antisense oligonucleotides targeting miR-7, indicating that miR-7-mediated translational repression of NF90 is a physiological event. Taken together with our previous findings (Higuchi et al 2016), it suggests that the level of NF90 is increased by a negative feedback loop between NF90 and miR-7 in tumor tissues under physiological conditions.

Suppression of MicroRNA-7 (miR-7) Biogenesis by Nuclear Factor 90-Nuclear Factor 45 Complex (NF90-NF45) Controls Cell Proliferation in Hepatocellular Carcinoma.

MicroRNA-7 (miR-7)has been characterized as an anti-oncogenic microRNA (miRNA) in several cancers, including hepatocellular carcinoma (HCC). However, the mechanism for the regulation of miR-7 production in tumors remains unclear. Here, we identified nuclear factor 90 (NF90) and NF45 complex (NF90-NF45) as negative regulators of miR-7 processing in HCC. Expression of NF90 and NF45 was significantly elevated in primary HCC tissues compared with adjacent non-tumor tissues. To examine which miRNAs are controlled by NF90-NF45, we performed an miRNA microarray and quantitative RT-PCR analyses of HCC cell lines. Depletion of NF90 resulted in elevated levels of mature miR-7, whereas the expression of primary miR-7-1 (pri-miR-7-1) was decreased in cells following knockdown of NF90. Conversely, the levels of mature miR-7 were reduced in cells overexpressing NF90 and NF45, although pri-miR-7-1 was accumulated in the same cells. Furthermore, NF90-NF45 was found to bind pri-miR-7-1 in vitro These results suggest that NF90-NF45 inhibits the pri-miR-7-1 processing step through the binding of NF90-NF45 to pri-miR-7-1. We also found that levels of the EGF receptor, an oncogenic factor that is a direct target of miR-7, and phosphorylation of AKT were significantly decreased in HCC cell lines depleted of NF90 or NF45. Of note, knockdown of NF90 or NF45 caused a reduction in the proliferation rate of HCC cells. Taken together, NF90-NF45 stimulates an elevation of EGF receptor levels via the suppression of miR-7 biogenesis, resulting in the promotion of cell proliferation in HCC.

Donepezil, an acetylcholinesterase inhibitor, attenuates LPS-induced inflammatory response in murine macrophage cell line RAW 264.7 through inhibition of nuclear factor kappa B translocation.

We have previously demonstrated that the pharmacotherapy with donepezil, an acetylcholinesterase inhibitor, suppresses cardiac remodeling in a mouse model of ischemic heart failure after myocardial infarction (MI). However, the precise mechanisms of the cardioprotective effect of donepezil have not been completely delineated. Because post-ischemic inflammation is a pathological key event in the cardiac remodeling process following MI, we investigated the hypothesis that donepezil acts as an inhibitor of inflammatory mediators. RAW 264.7 murine macrophage cells were pretreated with donepezil (100µM) prior to a pro-inflammatory stimulation by administration of lipopolysaccharide (LPS, 10ng/ml). Donepezil significantly reduced intra- and extracellular levels of various kinds of inflammatory mediators such as TNF-α, IL-1ß, IL-2, IL-6 and IL-18 after the LPS stimulation, and attenuated LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB). These results indicate that donepezil possesses an anti-inflammatory property. However, the inhibitory effect of donepezil on the macrophage inflammatory responses was never reproduced by ACh, nor was disrupted by ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to inhibit the inflammatory responses in LPS-stimulated macrophage cells. These results suggest that a cholinergic anti-inflammatory pathway would not be involved in the anti-inflammatory effect of donepezil and that the specific characteristics of donepezil in suppressing the LPS-induced cytokine release and the NF-κB activation would be independent of its acetylcholinesterase inhibition. The present study showed that donepezil exerts an anti-inflammatory effect independently of acetylcholinesterase inhibitory action, thereby donepezil may contribute to cardioprotection during cardiac remodeling process in an ischemic heart failure after MI.

Overexpression of NF90-NF45 Represses Myogenic MicroRNA Biogenesis, Resulting in Development of Skeletal Muscle Atrophy and Centronuclear Muscle Fibers.

MicroRNAs (miRNAs) are involved in the progression and suppression of various diseases through translational inhibition of target mRNAs. Therefore, the alteration of miRNA biogenesis induces several diseases. The nuclear factor 90 (NF90)-NF45 complex is known as a negative regulator in miRNA biogenesis. Here, we showed that NF90-NF45 double-transgenic (dbTg) mice develop skeletal muscle atrophy and centronuclear muscle fibers in adulthood. Subsequently, we found that the levels of myogenic miRNAs, including miRNA 133a (miR-133a), which promote muscle maturation, were significantly decreased in the skeletal muscle of NF90-NF45 dbTg mice compared with those in wild-type mice. However, levels of primary transcripts of the miRNAs (pri-miRNAs) were clearly elevated in NF90-NF45 dbTg mice. This result indicated that the NF90-NF45 complex suppressed miRNA production through inhibition of pri-miRNA processing. This finding was supported by the fact that processing of pri-miRNA 133a-1 (pri-miR-133a-1) was inhibited via binding of NF90-NF45 to the pri-miRNA. Finally, the level of dynamin 2, a causative gene of centronuclear myopathy and concomitantly a target of miR-133a, was elevated in the skeletal muscle of NF90-NF45 dbTg mice. Taken together, we conclude that the NF90-NF45 complex induces centronuclear myopathy through increased dynamin 2 expression by an NF90-NF45-induced reduction of miR-133a expression in vivo.

Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE.

Protein kinase expression and activity play important roles in diverse cellular functions through regulation of phosphorylation signaling. The most commonly used tools for detecting the protein kinase are protein kinase-specific antibodies, and phosphorylation site-specific antibodies were used for detecting activated protein kinase. Using these antibodies, only one kinase was analyzed at a time, however, a method for analyzing the expression and activation of a panel of protein kinases in cells is not established. Therefore, we developed a combined method using Multi-PK antibody and Phos-tag SDS-PAGE for profiling the expression and phosphorylation state of intracellular protein kinases. Using the new method, changes in the expression and phosphorylation state of various protein kinases were detected in cells treated with anticancer agent which inhibit multiple tyrosine kinase activities. Therefore, the new method is a useful technique for analysis of intracellular protein kinases.•Multi-PK antibody recognizes a wide variety of protein kinases in various species.•Using Phos-tag SDS-PAGE, phosphorylated proteins are visualized as slower migration bands compared with corresponding non-phosphorylated proteins.•This combined method can be used for detecting changes in the expression and phosphorylation state of various intracellular protein kinases.

Heterotrimeric guanosine triphosphate-binding protein-coupled modulatory actions of motilin on K+ channels and postsynaptic γ-aminobutyric acid receptors in mouse medial vestibular nuclear neurons.

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice-patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti-motilin receptor antibody. Bath application of motilin (0.1 µm) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose-dependent manner (K(d) , 0.03 µm). The motilin action on spontaneous action potentials was blocked by apamin (100 nm), a blocker of small-conductance Ca(2+) -activated K(+) channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 µg/µL) or guanosine triphosphate-γ-S (1 mm) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild-type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ-aminobutyric acid transporter-Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small-conductance Ca(2+) -activated K(+) channels and postsynaptic γ-aminobutyric acid receptors through heterotrimeric guanosine triphosphate-binding protein-coupled receptor in a group of glutamatergic MVNs.

High expression of nuclear factor 90 (NF90) leads to mitochondrial degradation in skeletal and cardiac muscles.

While NF90 has been known to participate in transcription, translation and microRNA biogenesis, physiological functions of this protein still remain unclear. To uncover this, we generated transgenic (Tg) mice using NF90 cDNA under the control of ß-actin promoter. The NF90 Tg mice exhibited a reduction in body weight compared with wild-type mice, and a robust expression of NF90 was detected in skeletal muscle, heart and eye of the Tg mice. To evaluate the NF90 overexpression-induced physiological changes in the tissues, we performed a number of analyses including CT-analysis and hemodynamic test, revealing that the NF90 Tg mice developed skeletal muscular atrophy and heart failure. To explore causes of the abnormalities in the NF90 Tg mice, we performed histological and biochemical analyses for the skeletal and cardiac muscles of the Tg mice. Surprisingly, these analyses demonstrated that mitochondria in those muscular tissues of the Tg mice were degenerated by autophagy. To gain further insight into the cause for the mitochondrial degeneration, we identified NF90-associated factors by peptide mass fingerprinting. Of note, approximately half of the NF90-associated complexes were ribosome-related proteins. Interestingly, protein synthesis rate was significantly suppressed by high-expression of NF90. These observations suggest that NF90 would negatively regulate the function of ribosome via its interaction with the factors involved in the ribosome function. Furthermore, we found that the translations or protein stabilities of PGC-1 and NRF-1, which are critical transcription factors for expression of mitochondrial genes, were significantly depressed in the skeletal muscles of the NF90 Tg mice. Taken together, these findings suggest that the mitochondrial degeneration engaged in the skeletal muscle atrophy and the heart failure in the NF90 Tg mice may be caused by NF90-induced posttranscriptional repression of transcription factors such as PGC-1 and NRF-1 for regulating nuclear-encoded genes relevant to mitochondrial function.

The NF90-NF45 complex functions as a negative regulator in the microRNA processing pathway.

The positive regulatory machinery in the microRNA (miRNA) processing pathway is relatively well characterized, but negative regulation of the pathway is largely unknown. Here we show that a complex of nuclear factor 90 (NF90) and NF45 proteins functions as a negative regulator in miRNA biogenesis. Primary miRNA (pri-miRNA) processing into precursor miRNA (pre-miRNA) was inhibited by overexpression of the NF90 and NF45 proteins, and considerable amounts of pri-miRNAs accumulated in cells coexpressing NF90 and NF45. Treatment of cells overexpressing NF90 and NF45 with an RNA polymerase II inhibitor, alpha-amanitin, did not reduce the amounts of pri-miRNAs, suggesting that the accumulation of pri-miRNAs is not due to transcriptional activation. In addition, the NF90 and NF45 complex was not found to interact with the Microprocessor complex, which is a processing factor of pri-miRNAs, but was found to bind endogenous pri-miRNAs. NF90-NF45 exhibited higher binding activity for pri-let-7a than pri-miR-21. Of note, depletion of NF90 caused a reduction of pri-let-7a and an increase of mature let-7a miRNA, which has a potent antiproliferative activity, and caused growth suppression of transformed cells. These findings suggest that the association of the NF90-NF45 complex with pri-miRNAs impairs access of the Microprocessor complex to the pri-miRNAs, resulting in a reduction of mature miRNA production.

Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia.

Animals sense various ranges of temperatures by cutaneous thermal stimuli. Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated at a warm temperature (over 30 degrees C) in exogenously expressed cells. We found in the present study that TRPV4 is essential in thermal hyperalgesia at a warm temperature in vivo. TRPV4-/- and TRPV4+/+ mice exhibited the same latency of escape from 35-50 degrees C hotplates. Neuronal activity in the femoral nerve, however, revealed that the number and activity level of neurons decreased in response to a warm temperature in TRPV4-/- mice. TRPV4-/- mice displayed a significantly longer latency to escape from the plates at 35- 45 degrees C when hyperalgesia was induced by carrageenan without changes in foot volumes. TRPV4 therefore determines the sensitivity rather than the threshold of painful heat detection and plays an essential role in thermal hyperalgesia.