FIGURE 6.

Neuronatin is degraded by proteasome, and its overexpression leads to increased intracellular Ca²⁺ and apoptosis that can be suppressed by malin. A, Neuro 2a cells were plated onto 6-well tissue-cultured plates or two-well chamber slides and on the following day cells were treated with MG132 (10 μm for 6 h) and processed for immunofluorescence staining or subjected to preparation of soluble and insoluble fractions followed by immunoblot analysis using neuronatin antibody. Arrows indicate accumulation of neuronatin in perinuclear region. Scale bar, 20 μm. B, cells were seeded in a 6-well tissue culture plate and, on the following day, they were chased with 25 μg/ml cycloheximide for different time periods in the presence or absence of 10 μm MG132. Collected cells at each time point were then processed for immunoblotting using antibodies against neuronatin and GAPDH. Quantitation of band intensities was performed using NIH ImageJ analysis software, and values were normalized against GAPDH. Values represent the mean ± S.D. (error bars) of three independent experiments. MG132 treatment caused significant increase (a indicates p < 0.001) in half-life of neuronatin. C, Neuro 2a cells were plated onto 6-well tissue-cultured plates and 24 h later cells were transiently transfected with either neuronatin β plasmid (Nnat OE, 2 μg/well) or neuronatin and control siRNA (30 pmol/well) for 36 h. In some experiment, cells were treated with thapsigargin (1 μm for 12 h). The cells were then processed for either measurement of intracellular Ca²⁺ or immunoblot analysis using neuronatin, myc (to detect overexpressed neuronatin), and GAPDH antibodies. D, cells were transfected with plasmid encoding empty pcDNA3.1 (control) or neuronatin β plasmid alone or along with wild-type or C26S mutant of malin (2 μg of each plasmid/well of 6-well plate). Thirty-six hours after transfection, cells were subjected to the analysis of intracellular Ca²⁺ levels. In C and D, values are mean ± S.D. of three independent experiments each performed in triplicate. The a indicates p < 0.01 compared with control, and b indicates p < 0.01 compared with neuronatin-transfected group. E, neuronatin-induced cell death is protected by malin and aggravated by LD-associated C26S mutant of malin. Cells were transfected with LacZ or neuronatin β plasmid independently or along with malin plasmids as in D for 72 h. Cells were differentiated with dbcAMP (5 mm for 48 h) and then processed for immunofluorescence staining to identify neuronatin or neuronatin- and malin-transfected cells. Nuclei were stained with DAPI to identify the apoptotic cell (fragmented nuclei). Values are mean ± S.D. of three independent experiments with minimum of 200 transfected cells counted for each experiment. The a indicates p < 0.001 in comparison with LacZ transfected control, and b shows p < 0.01 compared with neuronatin-transfected group.