Small Maf compound mutants display central nervous system neuronal degeneration, aberrant transcription, and Bach protein mislocalization coincident with myoclonus and abnormal startle response

Abstract

The small Maf proteins form heterodimers with CNC and Bach family proteins to elicit transcriptional responses from Maf recognition elements (MAREs). We previously reported germ line-targeted deficiencies in mafG plus mafK compound mutant mice. The most prominent mutant phenotype was a progressive maf dosage-dependent neuromuscular dysfunction. However, there has been no previous report regarding the effects of altered small-maf gene expression on neurological dysfunction. We show here that MafG and MafK are expressed in discrete central nervous system (CNS) neurons and that mafG::mafK compound mutants display neuronal degeneration coincident with surprisingly selective MARE-dependent transcriptional abnormalities. The CNS morphological changes are concurrent with the onset of a neurological disorder in the mutants, and the behavioral changes are accompanied by reduced glycine receptor subunit accumulation. Bach/small Maf heterodimers, which normally generate transcriptional repressors, were significantly underrepresented in nuclear extracts prepared from maf mutant brains, and Bach proteins fail to accumulate normally in nuclei. Thus compound mafG::mafK mutants develop age- and maf gene dosage-dependent cell-autonomous neuronal deficiencies that lead to profound neurological defects.

FIG. 1.

Behavioral analysis of mafG::mafK compound mutant mice. (A) A mafG^−/−::mafK^+/− mutant mouse (6 weeks old) displaying characteristic hind leg clasping. (B) A mafG^−/−::mafK^+/− mutant mouse (10 weeks) with long-lasting myoclonus. (C) The penetrance of the motor disorder was examined at 3, 6, and 9 weeks of age. Bars indicate frequencies of mice displaying each phenotype. G1K1 and G0K1, mafG^+/−::mafK^+/− control and mafG^−/−::mafK^+/− mutant, respectively. (D) Representative pattern of locomotor activities of mafG^−/−::mafK^+/− (mutant) and mafG^+/−::mafK^+/− (control) mice. Each bar indicates the frequency of ambulation per minute. Arrows, times of acoustic stimuli. (E) Comparison of the influences of acoustic stimuli on the locomotor activities of mutant and control mice. The mean ambulation count for 2 min after stimulus (red bars in panel D) was divided by that for 5 min before stimulus. This value was averaged for five independent animals and depicted as a bar. Error bars, standard deviations.

FIG. 2.

mafG gene expression in the CNS. Tissue sections from a mafG^+/− mouse were prepared and stained for β-galactosidase activity. LacZ activity (blue) was observed exclusively in neurons (not in glial cells) in the spinal cord (A and B), pons (C), medulla (D), and cerebellar nuclei (E). Solid arrowheads, neurons; open arrowheads, glia; arrows beside V, ventral side. Scale bar, 450 (A and E), 90 (B), 110 (C), or 150 μm (D). cc, central canal; ah, anterior horn; ph, posterior horn; pn, pontine nuclei; rn, reticular nuclei; cn, cerebellar nuclei; ce, cerebellum.

FIG. 3.

Histopathological changes in CNS neurons of maf mutant mice. (A to D) Nissl staining of spinal cord sections from 10-week-old mafG^+/−::mafK^+/− control mice (A and C) and mafG^−/−::mafK^+/− mutant mice (B and D) displaying myoclonus. Solid arrowheads (D), swollen nuclei with minimal cytoplasm in the affected mice. (E to G) Immunostaining for ubiquitin in spinal cord sections from control mafG^+/−::mafK^+/− mice (E) and mafG^−/−::mafK^+/− mutant mice (F and G). The mutants display far more intense staining, primarily in neuronal nuclei (G). (H) Double staining for β-galactosidase activity and ubiquitin immunohistological activity on brain stem sections prepared from 4-week-old mafG^−/−::mafK^+/− mutant mice. Note that all the cells in which ubiquitin has accumulated (intense brown signal) are also LacZ positive (open arrowheads). Scale bar, 400 (A, B, E, and F), 40 (C and D), 120 (G), or 60 μm (H). Abbreviations are as defined in the legend to Fig. 2.

FIG. 4.

Expression of glycine receptor α1 and β subunits in the CNS of small maf gene compound mutant mice. (A) RNA samples prepared from individual mafG^+/−::mafK^+/− control mice (lanes 1 to 3) or mafG^−/−::mafK^+/− mutant mice (lanes 4 to 6) were hybridized to probes corresponding to the Glra1, Glrb, or NMDAR1 genes; these were normalized to β-actin expression as the internal control. (B) The data shown in panel A were quantified on a phosphorimager and then normalized to the internal control. ∗, P < 0.05. (C) The abundance of a Glra1 receptor subunit was examined by immunoblotting using an anti-α1 subunit antibody. Protein samples were prepared from individual mafG^+/−::mafK^+/− control mice (lanes 1 to 3) and mafG^−/−::mafK^+/− mutant mice (lanes 4 to 6). Protein staining was used to confirm equivalent protein loading. G0K1 and G1K1 are as defined for Fig. 1. (D) Glra1 reduction was examined by RNA blot analysis usingRNA samples prepared from the CNS of individual newborn mice (lanes 1 to 4) or animals 3 (lanes 5 to 8), 10 (lanes 9 to 12), or 30 weeks of age (lanes 13 to 16). RNA samples from mafG^+/−::mafK^+/− control (lanes 1, 2, 5, 6, 9, 10, 13, and 14) and mafG^−/−::mafK^+/− mutant littermates (lanes 3, 4, 7, 8, 11, 12, 15, and 16) were examined.

FIG. 5.

HO-1 is induced in the mafG^−/−::mafK^+/− mutant CNS. (A) RNA blot analysis was performed to examine the expression of HO-1 and GST-Pi by using RNA samples prepared from the cerebrums (lanes 1 to 4), cerebellums (lanes 5 to 8), brain stems (lanes 9 to 12), and spinal cords (lanes 13 to 16) of individual 10-week-old mafG^+/−::mafK^+/− control mice (lanes 1, 2, 5, 6, 9, 10, 13, and 14) and mafG^−/−::mafK^+/− mutant mice (lanes 3, 4, 7, 8, 11, 12, 15, and 16). G0K1 and G1K1 are as defined for Fig. 1. (B and C) In situ hybridization was performed to examine the distribution of HO-1-expressing cells in the mafG^−/−::mafK^+/− mutant brain stems at 5 weeks of age. Arrowheads, positively stained neurons. Scale bar (C), 60 μm. (D) HO-1 induction was examined by RNA blot analysis using RNA samples prepared from the CNS of individual newborn mice (lanes 1 to 4) and animals 3 (lanes 5 to 8), 10 (lanes 9 to 12), or 30 weeks of age (lanes 13 to 16). RNA samples from mafG^+/−::mafK^+/− controls (lanes 1, 2, 5, 6, 9, 10, 13, and 14) and mafG^−/−::mafK^+/− mutant littermates (lanes 3, 4, 7, 8, 11, 12, 15, and 16) were examined.

FIG. 6.

MARE-binding activity of the Bach/small Maf heterodimer is reduced due to inhibition of Bach nuclear localization in the maf mutant mouse brain. (A) EMSA was performed with a probe containing a consensus MARE (see Materials and Methods). The probe was incubated without added nuclear extract (lanes 1 and 8) or with nuclear extract prepared from mafG^+/−::mafK^+/− control mouse brains (lanes 2 to 4 and 9 to 15) and mafG^−/−::mafK^+/− mutant mouse brains (lanes 5 to 7). The intensity of the lowest-mobility complex (arrow) was reduced in the mutant brains (lanes 5 to 7). Formation of this complex was inhibited by preincubation of extracts with an anti-small Maf antibody (lane 9) or an anti-Bach antibody (lane 12) but not with anti-p45 NF-E2, anti-Nrf1, anti-Fos, anti-Jun, or preimmune antibodies (lanes 10, 11, 13, 14, and 15, respectively). G0K1 and G1K1 are as defined for Fig. 1. (B and C) Nuclear extracts (lanes 1 to 4) and whole-cell extracts (lanes 7 to 10) were prepared from control mafG^+/−::mafK^+/− mouse brains (lanes 1, 2, 7, and 8) or mafG^−/−::mafK^+/− mutant mouse brains (lanes 3, 4, 9, and 10). Recombinant Bach1 and Bach2 proteins served as the positive control (rBach; lanes 5, 6, 11, and 12). Anti-Bach (B) and anti-Nrf1 (C) antibodies were used for immunoblot analysis. Arrows 1 and 2, mobilities of Bach1 and Bach2, respectively.

FIG. 7.

Small Maf proteins promote nuclear localization of Bach2. NIH 3T3 cells were examined for subcellular localization of transfected Bach2 protein (A to C) or location of nuclei in these cells using DAPI (D to F). The Bach2 expression plasmid was either transfected alone (A and D) or cotransfected with a vector directing expression of mafK (B and E) or a mafK mutant in two positions that disrupts dimerization (MafKL2PM4P; C and F). (G) Quantitative analysis of subcellular localization of Bach2. A total of 100 transfected cells were examined for Bach2 localization and classified into three different categories: C>N, predominantly cytoplasmic; C=N, equally distributed; C<N, predominantly nuclear.