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. 2012 Dec 14;287(51):42984-94.
doi: 10.1074/jbc.M112.417600. Epub 2012 Oct 24.

Motor neuron-specific disruption of proteasomes, but not autophagy, replicates amyotrophic lateral sclerosis

Affiliations

Motor neuron-specific disruption of proteasomes, but not autophagy, replicates amyotrophic lateral sclerosis

Yoshitaka Tashiro et al. J Biol Chem. .

Abstract

Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.

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Figures

FIGURE 1.
FIGURE 1.
Generation of Rpt3 conditional knock-out mice, Rpt3 delta/flox; VAChT-Cre.Fast+ (Rpt3-CKO), and influence on expression of proteasome subunits. A, schematic diagram of the Rpt3flox construct. Cre removes the region of the Rpt3 gene that encodes exons 7–10 (black boxes), which are flanked by loxP (triangles). The neomycin resistance gene (white box) between FRT (closed half-circles) was already removed by crossingFLP66 mice with Rpt3 floxed mice. F1, Rpt3 fl-for primer; R1, Rpt3 fl-rev primer; R2, Rpt3 del-rev primer; A, AatI; AL, ApaLI; H, HindIII. B, genotyping of floxed Rpt3 mice and Rpt3 delta mice with PCR. Rpt3+/+ (WT) mice (1), Rpt3 delta/+ mice (2), Rpt3+/flox mice (3), and Rpt3 delta/flox mice (4) are detected with PCR using tail DNA. Left, flox detection PCR; right, delta detection PCR. C, graphic representation of quantitative analysis of serial sections immunostained for Rpt3 and ChAT from control and Rpt3-CKO mice at 6 weeks of age. The number of ChAT-positive motor neurons with decreased Rpt3 immunoreactivity is significantly higher in Rpt3-CKO mice than in controls (n = 3, §, p < 0.01). NS indicates not significant. D, immunohistochemistry for ChAT, Rpt3, and ubiquitin in serial sections from the spinal cord of Rpt3-CKO mice (d–f) and control littermates (a–c). Representative image of ChAT-positive spinal motor neurons in Rpt3-CKO mice, showing that Rpt3-negative neurons show ubiquitin accumulation in the cytoplasm (arrows). Scale bars, 10 μm. E, immunohistochemistry for Rpt6 (a and d), Rpn2 (b and e), and α6 (c and f) on spinal cord sections from control and Rpt3-CKO mice. The motor neurons from Rpt3-CKO mice show no immunoreactivity for Rpt6 or Rpn2 in either the cytoplasm or the nuclei, or for α6 in the cytoplasm (arrows). Scale bars, 10 μm.
FIGURE 2.
FIGURE 2.
Motor dysfunction in Rpt3-CKO mice. Motor performance of Rpt3-CKO mice was evaluated by measuring rotarod retention time (A), body weight (B), grip strength (C), and tail suspension (D). A, rotarod analysis indicates that the mean retention time on the moving rod progressively decreases after 16 weeks of age in Rpt3-CKO mice. The differences in retention time between Rpt3-CKO mice and control littermates are statistically significant at 26 weeks of age and later (n = 8, *, p < 0.05, §, p < 0.01). B, progressive reduction in body weight of Rpt3-CKO mice. The mean body weight of Rpt3-CKO mice is significantly lower than that of control mice after 36 weeks of age (n = 8, *, p < 0.05, §, p < 0.01). C, grip strengths of both forelimbs and hindlimbs are significantly weaker in Rpt3-CKO mice than those in control mice after 12 weeks of age (n = 8, *, p < 0.05, §, p < 0.01). D, representative photographs of a control and an Rpt3-CKO mouse at 35 weeks of age displaying an abnormal limb-clasping reflex during tail hanging in the Rpt3-CKO mouse (b) and normal clasping in the control littermate (a). Representative photo of a Rpt3-CKO mouse at the advanced stage, showing severe kyphosis caused by weakness of the paraspinal muscles (c).
FIGURE 3.
FIGURE 3.
Neuronal loss and cytopathology of the spinal motor neurons in Rpt3-CKO mice. A, ChAT immunohistochemistry of spinal anterior horn cells from Rpt3-CKO mice (d–f) and control mice (a–c) at 6, 12, and 40 weeks of age. The number of motor neurons, indicated by ChAT-positive immunoreactivity, in Rpt3-CKO mice is significantly lower at 12 and 40 weeks of age than that of control mice (g)(n = 3, §, p < 0.01). NS indicates not significant. B, H&E staining of anterior horns from control (a–c) and Rpt3-CKO mice (d–f). Rpt3-CKO mice (12 weeks) have abnormal motor neurons with eosinophilic substances in the cytosol (arrows). C, spinal motor neurons from Rpt3-CKO mice show chromatolysis and basophilic inclusions, resembling those in ALS patients. a and c, Nissl staining; b and d, H&E staining. Scale bars, 30 μm (A), 20 μm (B), and 10 μm (C).
FIGURE 4.
FIGURE 4.
Aberrant staining patterns of TDP-43, FUS, ubiquilin 2, and optineurin in the spinal motor neurons of Rpt3-CKO mice. A, immunohistochemistry for TDP-43 (a–c), FUS (e–g), optineurin (i–k), and ubiquilin 2 (m–o) in spinal cords from Rpt3-CKO mice at 6 (b, f, j, n) and 12 (c, g, k, o) weeks of age and control mice at 6 weeks of age (a, e, i, m). Cytoplasmic mislocalization of TDP-43, FUS, and ubiquilin 2 and increased optineurin are observed in motor neurons from Rpt3-CKO mice as early as 6 weeks of age. Noticeable intracytoplasmic inclusions of TDP-43, FUS, and ubiquilin 2 (arrows) are recognizable at 12 weeks of age. Note that these inclusions resemble those in human ALS patients (d, h, arrowheads). B, double immunofluorescence investigations demonstrating co-localization of TDP-43 (a, d, green) with FUS (b, red) or ubiquilin 2 (e, red) in the intracytoplasmic inclusions of spinal motor neurons (arrows). Merged images are shown in c and d. Nuclei are stained with DAPI (blue). Scale bars, 10 mm. C, immunoreactivity to TDP-43 in the nucleus and the cytosol was compared, and the number of positive cells was counted under the microscope. A few motor neurons with nuclear exclusions were found in control mice (a). In contrast, the number of motor neurons in which the cytoplasm was more intensely stained with anti-TDP-43 antibody than the nucleus was significantly increased in 12-week-old Rpt3-CKO mice (b) (n = 3, §, p < 0.01).
FIGURE 5.
FIGURE 5.
Glial activation in spinal anterior horns of Rpt3-CKO mice. A, immunohistochemistry for GFAP in spinal anterior horns of control (a–f) and Rpt3-CKO mice (g–l), showing an increase in size and number of reactive astrocytes in anterior horns at 6 weeks of age, which further progressed along with the disease progression (a–f). Control mice demonstrate apparently low levels of astrogliosis (g–l). Scale bars, 10 μm. B, immunohistochemistry for MAC-2 in the spinal anterior horns of control (a–f) and Rpt3-CKO mice (g–l). MAC-2-positive reactive microglia with amoeboid morphology proliferate in the anterior horn at 12 weeks of age, but not at 6 weeks of age. Note that MAC-2-positive microglia have regressed by 40 weeks of age in Rpt3-CKO mice (k and l). Scale bars, 10 μm. C, quantitative evaluation of astrocytosis (a) and microgliosis (b). Number of GFAP-positive reactive astrocytes increases significantly in the spinal anterior horns of Rpt3-CKO mice from 6 weeks (upper), and remains high until 40 weeks (a). MAC-2-positive microglia showed a later increase, which is statistically significant at 12 weeks of age (b) (n = 3, §, p < 0.01).
FIGURE 6.
FIGURE 6.
Histochemical analysis of mice with defective autophagy in motor neurons (Atg7-CKO). A, genotyping of transgenic mice for floxed and delta Atg7 with PCR. Atg7 flox allele (1) and Atg7 delta allele (2) were detected agarose gel electrophoresis showing Cre-dependent deletion of loxP-franked region for Atg7 in Atg7-CKO mice (2) compared with Atg+ control (1). B, Nissl staining of anterior horns from control (a) and Atg7-CKO mice (b). Atg7-CKO mice have no motor neurons loss. Immunohistochemistry for Atg7 on spinal cord sections of control and Atg7-CKO mice (c and d). Atg7-negative neurons (arrowhead) showed inclusions (asterisks) instead of Atg7-positive neurons (arrow). Scale bars, 40 μm (a and b), 10 μm (c and d), C, H&E staining (a and e) and immunohistochemistry for ubiquitin (b, f), p62 (c, g), Nbr1 (d, h), TDP-43 (i, m), FUS (j, o), optineurin (k, p), and ubiquilin 2 (l, q) in spinal anterior horns from Atg7+control (a–d, i–l) and Atg7-CKO (e–h, m–p) mice. Motor neurons in Atg7-CKO mice showed marked accumulation of p62, ubiquitin, Nbr1, and ubiquilin 2 without cytoplasmic mislocalization or aggregate formation of TDP-43, FUS, or optineurin (asterisks). Scale bars, 20 μm (low magnification), 10 μm (high magnification). D, electron microscopy imaging of large inclusions (asterisks) in which amorphous structures accumulated in an Atg7-CKO mouse at 2 years of age. Scale bars, 5 μm (left), 1 μm (right).
FIGURE 7.
FIGURE 7.
Temporal profiles demonstrating the ALS-like phenotypes with proteasome inhibition and TDP-43 mislocalization. Rpt3 down-regulation is induced by excision of the Rpt3 gene by Cre until 6 weeks of age. The population of motor neurons with TDP-43 mislocalization gradually increased after Rpt3 down-regulation. Astrogliosis precedes paralytic symptoms, motor neuron loss, and microgliosis.

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