CRISPRa-mediated NEAT1 lncRNA upregulation induces formation of intact paraspeckles

Long noncoding RNAs (lncRNAs) are fundamental genomic regulatory factors under various physiological and pathological conditions. A class of lncRNAs termed architectural RNAs (arcRNAs) plays an essential scaffolding role in building nuclear bodies. NEAT1 arcRNA is an abundant, nuclear-retained lncRNA that constructs paraspeckle nuclear bodies. NEAT1 is upregulated in various developmental and disease conditions including cancer and virus infection. However, it remains unclear how elevated expression of NEAT1 influences such conditions. Here, we set up an experimental method to selectively increase NEAT1 expression. We applied the synergistic activation mediator (SAM) system using catalytically dead Cas9 (dCas9) proteins to activate transcription of the NEAT1 gene. We examined 10 pre-designed and 15 originally designed single-guide RNAs (sgRNAs) in the NEAT1 promoter region for CRISPR activation (CRISPRa). We validated several sgRNAs that we designed for the SAM system to strongly activate NEAT1 expression in two human cell lines and induced formation of paraspeckles with intact core-shell structures. Thus, this selective NEAT1 upregulation method using the SAM system would be useful for further functional analyses of NEAT1 lncRNA in both basic and applied research

Regulatory mechanism of CCN2 production by serotonin (5-HT) via 5-HT2A and 5-HT2B receptors in chondrocytes.

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones

Early Phase Increase in Serum TIMP-1 in Patients with Acute Encephalopathy with Biphasic Seizures and Late Reduced Diffusion

Background: Acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) is the most frequent subtype of acute encephalopathy syndrome among Japanese children. Exanthem subitum is the most common causative infectious disease of AESD. We herein retrospectively analyzed serum and cerebrospinal fluid (CSF) concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor matrix metalloproteinase-1 (TIMP-1), and seven cytokines in patients with AESD or prolonged febrile seizure (FS) to assess the pathophysiology of AESD and detect biomarkers for diagnosing AESD in the early phase. Methods: Serum and CSF samples were obtained from 17 patients with AESD (1st seizure phase group, n = 7; 2nd seizure phase group, n = 10) and 8 with FS. The concentrations of MMP-9, TIMP-1, and seven cytokines were measured by enzyme-linked immunosorbent assays or cytometric bead arrays. Results: Serum concentrations of TIMP-1 were significantly higher in the 1st seizure phase group than in the 2nd seizure phase group. No significant differences were observed in serum concentrations of MMP-9 or the MMP-9/TIMP-1 ratio. Conclusions: The MMP-9-independent increase in circulating TIMP-1 concentrations observed in the present study may be associated with the pathophysiology of AESD in the 1st seizure phase

Childhood-Onset Progressive Dystonia With Mitochondrial DNA G14459A Mutation

This article reports the case of an 11-year-old boy with progressive dystonia caused by the homoplasmic G14459A mitochondrial DNA mutation. The patient presented with focal dystonia in the right upper limb at 3 years of age, which progressed over 4 years to exhibit dystonia in both the upper and lower limbs. At 7 years of age, high signal intensity lesions in the bilateral striata and the midbrain were observed on fluid-attenuated inversion recovery images. It was observed on diffusion-weighted images that with time, these high signal intensity lesions migrated from the putamen to the caudate nuclei, which closely correlated with disease progression. Because his symptoms and abnormal magnetic resonance imaging findings progressed despite treatment with coenzyme Q10 and l -carnitine, at 7 years of age he was then started on sodium succinate, hoping to improve his complex I deficiency. After treatment, progression of MRI abnormalities appeared to have been suppressed for 4 years, although no improvement was observed in dystonia

Protein production of CCN2 regulated by 5-HT signaling via 5-HT_2AR and 5-HT_2BR.

<p>HCS-2/8 cells were grown until they had reached confluence. (A) The cells were treated with vehicle control (PBS and/or DMSO), 5-HT, ritanserin alone, the combination of 5-HT and ritanserin, SB204741 alone, or the combination of 5-HT and SB204741 at the indicated dose. Cell lysates were prepared 24 h later, and Western blot analysis was performed with anti-CCN2 and β-actin antibodies. The graph indicates relative densitometry to untreated controls (ratio = 1.0; dotted line) from 5 independent cultures and analyzed by Bonferroni’s test, and <i>p</i> < 0.05 (*) was considered significant. (B) HCS-2/8 cells were treated with 5-HT alone (10 μM), NBOH-2C-CN (10 μM or 100 μM) or BW723C86 (1 μM or 10 μM) for 24 h. Then cell lysates were prepared, and Western blot analysis was performed. Relative densitometry (untreated control = 1.0; dotted line) from 5 independent cultures are presented and analyzed by Bonferroni’s test, and <i>p</i> < 0.05 (*) was considered significant. (C) HCS-2/8 cells were treated with 5-HT at the concentration of 10 μM for 24 h, and the cell culture supernatant and cell layer fraction were harvested. Quantification of 5-HT was performed by using an ELISA system. The concentration of 5-HT produced by HCS-2/8 cells was determined by subtracting the 5-HT concentration in fresh media from that in conditioned media. Results are presented as the mean and standard deviations of 3 independent cultures and analyzed by Bonferroni’s test, and <i>p</i> < 0.05 (*) was considered significant. (D) HCS-2/8 cells were treated with 5-HT at the concentration of 10 and 50 μM for 24 h, and Western blot analysis was performed. The graph indicates relative densitometry to untreated controls (ratio = 1.0; dotted line) from 3 measurements and analyzed by Dunnett’s test, and there was no significant difference.</p

Real-time RT-PCR analysis of <i>COL2A1</i>, <i>ACAN</i>, and <i>CCN2</i> mRNAs in HCS-2/8 cells treated with the combination of 5-HT and SB204741, an antagonist of 5-HT_2BR or retanserin, an antagonist of 5-HT_2AR.

<p>(A) HCS-2/8 cells were grown until they had reached confluence. Then, the cells were treated with 5-HT (10 μM), ritanserin, an antagonist of 5-HT_2AR (100 μM), SB204741, an antagonist of 5-HT_2BR (30 μM) or the combination of 5-HT and SB206553 or ritanserin. After 12 h, total RNA was collected; and real-time RT-PCR analysis was then performed. The amounts of these mRNAs were normalized to that amount of <i>GAPDH</i> mRNA. The graphs show the expression levels of (a) <i>COL2a1</i>, (b) <i>ACAN</i>, (c) <i>CCN2</i> mRNAs in HCS-2/8 cells treated with vehicle control (PBS and/or DMSO, n = 4), 5-HT (n = 4), ritanserin alone (n = 3), the combination of 5-HT and ritanserin (n = 3), SB204741 alone (n = 4), or the combination of 5-HT and SB204741 (n = 4). (B) HCS-2/8 cells were treated with 5-HT, ritanserin, SB204741, or the combination of 5-HT and SB206553 or ritanserin for 24 h. The graphs show the expression levels of (a) <i>COL2a1</i>, (b) <i>ACAN</i>, (c) <i>CCN2</i> in HCS-2/8 cells treated with vehicle control (PBS and/or DMSO, n = 4), 5-HT (n = 4), ritanserin alone (n = 4), the combination of 5-HT and ritanserin (n = 4), SB204741 alone (n = 4), or the combination of 5-HT and SB204741 (n = 4). In all graphs, the ordinate indicates fold induction with respect to control sample (ratio = 1.0), and bars represent mean and standard deviation. The data were analyzed by Tukey’s test for multiple comparisons, and <i>p</i> < 0.05 (*), <i>p</i> < 0.01 (**) was considered significant.</p

Activation of MAPKs in HCS-2/8 cells treated with 5-HT or agonist of each 5-HT₂ receptor and rescue effect of inhibitors of ERK1/2 and JNK on the BW723C86-decreased CCN2 production.

<p>(A) HCS-2/8 cells were grown until they had reached confluence. Then, the cells were treated with 5-HT, NBOH-2C-CN or BW723C86 at the indicated concentrations. After 15 min, cell lysates were prepared; and Western blot analysis was performed with the antibodies against the indicated proteins. The levels of phosphorylated ERK1/2 and JNK were increased by the treatment with BW723C86 at a concentration of 10 μM. In contrast, the levels of phosphorylated p38 MAPK were increased by the treatment with NBOH-2C-CN at the concentrations of 10 μM and 100 μM. (B) HCS-2/8 cells were grown until they had reached confluence. Then, when the cells were treated with 5-HT (10 μM) or BW723C86 (10 μM), PD98059 (MEK1 inhibitor; 50 μM) or SP600125 (JNK inhibitor; 50 μM) was applied to the cultures simultaneously. After 24 h, the cell lysates were prepared; and Western blot analysis was performed with anti-human CCN2 rabbit serum and β-actin antibody. When HCS-2/8 cells were treated with 5-HT, PD98059 or SP600125 had no effects. In contrast, when the cells were treated with BW723C86, CCN2 production was rescued by either PD98059 or SP600125. The graphs give the results of Western blotting using anti-CCN2 antibody and quantified by densitometric analysis, with normalization by the levels of β-actin. The ordinate indicates the fold change relative to untreated controls (ratio = 1.0; dotted line). The graph indicates relative densitometry (untreated control = 1.0; dotted line) from 6 independent cultures and analyzed by Bonferroni's test, and <i>p</i> < 0.05 (*) was considered significant.</p

Immunohistochemical analysis of whole knee joints from 60 day-old male mice by use of anti-5-HT_2AR and 5-HT_2BR antibodies.

<p>(A-C) Sections of the frontal knee joints were stained with toluidine blue, and cartilage tissues showed metachromatic staining. The areas surrounded by the boxes are enlarged in “B” (articular cartilage tissues) and “C” (growth plate). (D-H) In the low-power magnification view of the knee joint stained with anti-5-HT_2AR (D) the areas surrounded by the boxes are enlarged (E, H). Images of “E” and “F” represent articular cartilage tissues (E) and the growth plate (F). A serial section was stained with a non-immune antibody as a negative control, and images of the same areas as seen in “E” and “F” are shown in “G” and “M”, respectively. The immunoreactivity for 5-HT_2AR was detected in cells from the proliferating to prehypertrophic regions of the growth plate. (I-M) The knee joint stained with anti-5-HT_2BR. In the low-power-magnification view (I), the areas indicated by the boxes are enlarged in “J” and “K”. Images in “J” and “K” represent articular cartilage tissues (J) and the growth plate (K). Images in “L” and “M” represent the same areas as seen in “J” and “K,” respectively, in a serial section stained with a non-immune antibody as a negative control. The immunoreactivity for 5-HT_2BR was detected in the surface layer of articular cartilage tissues. The sizes of scale bars are indicated.</p