Gm14230 controls Tbc1d24 cytoophidia and neuronal cellular juvenescence

Abstract

It is not fully understood how enzymes are regulated in the tiny reaction field of a cell. Several enzymatic proteins form cytoophidia, a cellular macrostructure to titrate enzymatic activities. Here, we show that the epileptic encephalopathy-associated protein Tbc1d24 forms cytoophidia in neuronal cells both in vitro and in vivo. The Tbc1d24 cytoophidia are distinct from previously reported cytoophidia consisting of inosine monophosphate dehydrogenase (Impdh) or cytidine-5'-triphosphate synthase (Ctps). Tbc1d24 cytoophidia is induced by loss of cellular juvenescence caused by depletion of Gm14230, a juvenility-associated lncRNA (JALNC) and zeocin treatment. Cytoophidia formation is associated with impaired enzymatic activity of Tbc1d24. Thus, our findings reveal the property of Tbc1d24 to form cytoophidia to maintain neuronal cellular juvenescence.

Fig 1. Tbc1d24 is expressed predominantly in juvenile in the cerebral cortex and forms cytoophidia.

(A) IGV view of the transcriptome analysis in the mouse cerebral cortex at postnatal day (P) 1 and P56. Numbers next to the read patterns (0 and 220) indicate fragments per kilobase of exon per million reads mapped (FPKM) values. (B) qPCR analysis of mouse Tbc1d24 in the postnatal mouse cerebral cortex at P1, P14 and P56. Data were normalized to Polr2a (n = 3). (C) Immunofluorescence analysis of Tbc1d24 in Neuro2a cells. Cells were also stained with the acetylated tubulin antibody and DAPI. Scale bar = 25 μm. (D) Super-resolution microscopy of Tbc1d24 cytoophidia in Neuro2a cells. Scale bar = 25 μm. (E) Immunofluorescence analysis of Tbc1d24 and Vimentin with a different antibody against Tbc1d24 (LSBio, LS-C679739) in Neuro2a cells. Scale bar = 25 μm. (F) Immunofluorescence analysis of Tbc1d24 in the mouse cortical tissue. The arrows indicate Tbc1d24 cytoophidia. Scale bar = 25 μm. (G) Immunofluorescence analysis of TBC1D24 in SH-SY5Y cells. The arrows indicate TBC1D24 cytoophidia. Scale bar = 10 μm. **p<0.01; Student’s t-test. The data were presented as the means ± standard error of the mean (SEM).

Fig 2. Tbc1d24 cytoophidia are distinct from Impdh and Ctps cytoophidia.

(A) Costaining of Impdh and Tbc1d24 in Neuro2a cells. Scale bar = 25 μm. (B) Costaining of Ctps and Tbc1d24 in Neuro2a cells. Scale bar = 25 μm. (C) Immunofluorescence analysis of Tbc1d24 in Neuro2a cells treated with control distilled water or 2 mM 6-diazo-5-oxo-L-norleucine (DON) for 24 hours (hrs). Scale bar = 25 μm. (D) Frequency of Tbc1d24 cytoophidium-positive Neuro2a cells treated with 2 mM DON for 24 hrs. (E) Immunofluorescence analysis of Tbc1d24 in Neuro2a cells treated with control dimethyl sulfoxide (DMSO) or 2 μM mycophenolate (MPA) for 24 hrs. Scale bar = 25 μm. (F) Frequency of Tbc1d24 cytoophidium-positive Neuro2a cells treated with 2 μM MPA for 24 hrs. (G) Immunofluorescence analysis of Tbc1d24 in Neuro2a cells treated with control distilled water or 2 mM Acivicin for 24 hrs. Scale bar = 25 μm. (H) Frequency of Tbc1d24 cytoophidium-positive Neuro2a cells treated with 2 mM Acivicin for 24 hrs. **p<0.01; Student’s t-test. The data were presented as the means ± SEM.

Fig 3. Gm14230 controls cellular juvenescence and Tbc1d24 cytoophidia.

(A) qPCR analysis of Gm14230 in Neuro2a cells transfected with control siRNA (siCtrl) or Gm14230 siRNA. Data were normalized to the expression of Tubb5 (n = 3). (B) The appearance of Neuro2a cells 72 hrs after transfection of control siRNA or Gm14230 siRNA. Scale bar = 100 μm. (C) Number of cells per field 72 hrs after transfection with control siRNA or Gm14230 siRNA. (D) Senescence-associated (SA) beta-galactosidase activity staining in Neuro2a cells 72 hrs after transfection with control siRNA or Gm14230 siRNA. Scale bar = 100 μm. (E) Number of SA beta-gal-positive cells per field 72 hrs after transfection with control siRNA or Gm14230 siRNA. (F) Western blot analysis of Neuro2a cells 72 hrs after transfection with control siRNA or Gm14230 siRNA. β-actin (Actb) was used as a loading control. MW, molecular weight. (G) The densitometric analysis for western blot analyses from which the representative images were shown as Fig 3F. The intensity of the bands was quantified and normalized to those of Actb. The ratios to Actb were further normalized to siCtrl. (H) Immunofluorescence analysis of Tbc1d24 and Vimentin in Neuro2a cells transfected with control siRNA or Gm14230 siRNA. Scale bar = 25 μm. (I) Frequency of Tbc1d24 cytoophidia in Neuro2a cells transfected with control siRNA or Gm14230 siRNA. *p<0.05 and **p<0.01; Student’s t-test. The data were presented as the means ± SEM.

Fig 4

Influence of Gm14230 expression on Tbc1d24 cytoophidia formation. (A) qPCR analysis of Gm14230 in Neuro2a cells transfected with the empty control or the Gm14230 plasmid. Data were normalized to Tubb5 (n = 3). (B) The appearance of Neuro2a cells 72 hrs after transfection of the empty control or the Gm14230 plasmid. Scale bar = 100 μm. (C) Number of cells per field 72 hrs after transfection of the empty control or the Gm14230 plasmid. (D) Immunofluorescence analysis of Tbc1d24 and Vimentin in Neuro2a cells transfected with the empty control or the Gm14230 plasmid. Scale bar = 100 μm. (E) Frequency of Tbc1d24 cytoophidia in Neuro2a cells transfected with the empty control or the Gm14230 plasmid. *p < 0.05 and **p < 0.01; Student’s t-test. The data were presented as the means ± SEM.

Fig 5. Tbc1d24 cytoophidia formation is enhanced in zeocin-induced loss of cellular juvenescence.

(A) The appearance of Neuro2a cells 72 hrs after treatment with zeocin. Scale bar = 100 μm. (B) Number of cells per field 72 hrs after treatment with or without zeocin. (C) Immunofluorescence analysis of Tbc1d24 and Vimentin treated with or without zeocin for 72 hrs. Scale bar = 25 μm. (D) Frequency of cytoophidia in Neuro2a cells with or without zeocin treatment for 72 hrs. (E) qPCR analysis of Tbc1d24 in Neuro2a cells transfected with control siRNA or Tbc1d24 siRNA. Data were normalized to Tubb5 (n = 3). (F) The appearance of Neuro2a cells treated with or without zeocin for 96 hrs and simultaneously transfected with control siRNA or Tbc1d24 siRNA. Scale bar = 100 μm. (G) Number of cells per field 96 hrs after treatment with or without zeocin. (H) The appearance of cells with Sytox blue staining in the same fields as in (F). The gray color dots indicate dead cells stained with Sytox blue. (I) Frequency of Sytox blue-positive cells. **p < 0.01; Student’s t-test. The data were presented as the means ± SEM.

Fig 6. Lower GAP activity of Tbc1d24 is associated with cytoophidia formation.

(A) GTP-bound Arf6 pulldown assay in Neuro2a cells transfected with control siRNA or Gm14230 siRNA. Total Arf6 levels were examined to evaluate the equal expression of Arf6 protein after Gm14230-depletion. (B) The densitometric analysis for western blot analyses from GTP bound Arf6 pulldown assay. The intensity of the bands was quantified and normalized to those of total Arf6. The ratios to total Arf6 were further normalized to siCtrl. **p < 0.01; Student’s t-test. Data were represented as the means ± SEM. (C) Schematics for the role of Tbc1d24 cytoophidia to control GAP activity in a cellular juvenescence-dependent manner.