We previously demonstrated that TSC22D4, a protein encoded by the TGF-β1-activated gene Tsc22d4 (Thg-1pit) and highly expressed in postnatal and adult mouse cerebellum with multiple post-translationally modified protein forms, moves to nucleus when in vitro differentiated cerebellum granule neurons (CGNs) are committed to apoptosis by hyperpolarizing KCl concentrations in the culture medium. We have now studied TSC22D4 cytoplasmic/nuclear localization in CGNs and Purkinje cells: (1) during CGN differentiation/maturation in vivo, (2) during CGN differentiation in vitro, and (3) by in vitro culturing ex vivo cerebellum slices under conditions favoring/inhibiting CGN/Purkinje cell differentiation. We show that TSC22D4 displays both nuclear and cytoplasmic localizations in undifferentiated, early postnatal cerebellum CGNs, irrespectively of CGN proliferation/migration from external to internal granule cell layer, and that it specifically accumulates in the somatodendritic and synaptic compartments when CGNs mature, as indicated by TSC22D4 abundance at the level of adult cerebellum glomeruli and apparent lack in CGN nuclei. These features were also observed in cerebellum slices cultured in vitro under conditions favoring/inhibiting CGN/Purkinje cell differentiation. In vitro TSC22D4 silencing with siRNAs blocked CGN differentiation and inhibited neurite elongation in N1E-115 neuroblastoma cells, pinpointing the relevance of this protein to CGN differentiation.
A representative confocal immunofluorescence analysis of TSC22D4 expression and localization in PN9 cerebellum histological sections. The specificity of TSC22D4 immunostaining was assessed by omitting the anti-TSC22D4 antibody (CTRL). Note the somatodendritic, but not nuclear, TSC22D4 localization in Purkinje cells (asterisks). EGL, external granular layer; IGL, internal granule layer. Scale bar indicates 30 μm (JPEG 332 kb)
Double TSC22D4 and Synaptophysin (Syp) immunostaining of PN40 cerebellum, analyzed by confocal microscopy. A representative histological section is shown in the panels. Inserts represent higher magnification (100×) of glomeruli. Asterisks and arrows indicate Purkinje cells and glomeruli, respectively. IGL, internal granular layer. Scale bars indicate 30 μm (panels) and 10 μm (inserts) (JPEG 2567 kb)
Tsc22d4 mRNA expression in differentiating N1E-115 cells. DIV0 cells were cultured in vitro under conditions promoting either cell proliferation (medium containing 10% FBS) or differentiation (medium containing 2% FBS) and then processed for semiquantitative real-time RT-PCR analysis of Tsc22d4 mRNA at increasing days of in vitro culture (DIV1-DIV6), taking the amount of GAPDH message as internal reference. Histograms represent the mean ± SEM of Tsc22d4/GAPDH ratios obtained in three independent experiments. (JPEG 211 kb)
a Analysis of CGN and Purkinje cell differentiation and viability in in vitro cultured cerebellum slices. Ex vivo PN12 cerebellum slices were cultured in vitro for 10 days in a medium containing 5 or 25 mM KCl, then incubated with 5 μM SYTOX orange fluorescent dye in PBS for 5 min and analyzed by DIC and fluorescence microscopy. Dead cells were identified by their SYTOX orange positivity staining. Brackets indicate Purkinje cells. Scale bars indicate 40 μm. b Analysis of DNA fragmentation. DNA was extracted from either freshly made PN12 cerebellum slices (CTRL) or slices that had been in vitro cultured for 10 days in the presence of either 25 mM or 5 mM KCl (JPEG 1369 kb)