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. 2004 Nov;165(5):1701-18.
doi: 10.1016/S0002-9440(10)63426-8.

Dysregulation of Stathmin, a Microtubule-Destabilizing Protein, and Up-Regulation of Hsp25, Hsp27, and the Antioxidant Peroxiredoxin 6 in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

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Free PMC article

Dysregulation of Stathmin, a Microtubule-Destabilizing Protein, and Up-Regulation of Hsp25, Hsp27, and the Antioxidant Peroxiredoxin 6 in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

Christoph W Strey et al. Am J Pathol. .
Free PMC article

Abstract

Gain-of-function mutations of the Cu/Zn superoxide dismutase (SOD1) gene cause dominantly inherited familial amyotrophic lateral sclerosis. The identification of differentially regulated proteins in spinal cords of paralyzed mice expressing SOD1(G93A) may contribute to understanding mechanisms of toxicity by mutant SOD1. Protein profiling showed dysregulation of Stathmin with a marked decrease of its most acidic and phosphorylated isoform, and up-regulation of heat shock proteins 25 and 27, peroxiredoxin 6, phosphatidylinositol transfer protein-alpha, apolipoprotein E, and ferritin heavy chain. Stathmin accumulated in the cytoplasm of 30% of spinal cord motor neurons with fragmented Golgi apparatus. Overexpression of Stathmin in HeLa cells was associated with collapse of microtubule networks and Golgi fragmentation. These results, together with the decrease of one Stathmin isoform, suggest a role of the protein in Golgi fragmentation. Mutant SOD1 co-precipitated and co-localized with Hsp25 in neurons and astrocytes. Mutant SOD1 may thus deprive cells of the anti-apoptotic and other protective activities of Hsp25. Astrocytes contained peroxiredoxin 6, a unique nonredundant antioxidant. The up-regulation of peroxiredoxin 6 probably constitutes a defense to oxidative stress induced by SOD1(G93A). Direct effects of SOD1(G93A) or sequential reactions triggered by the mutant may cause the protein changes.

Figures

Figure 1
Figure 1
Two-dimensional gel map of pooled spinal cord extracts from five WT animals containing the spots with significant abundance changes. Areas containing the Stathmin spot and the Hsp25 spot are compared in greater detail in Figure 2 (under A for Stathmin and B for Hsp25). The location of the spots with significant and greater than twofold abundance differences between the mutant and WT samples are labeled with their corresponding identification numbers. Note: in this and in the subsequent legends, WT is for mice that express the wild-type human SOD1.
Figure 2
Figure 2
Comparison of magnified gel areas (as highlighted in Figure 1) and spot abundance changes from WT and SOD1G93A mice. The left panel displays the gel areas of spot 586 (SOD1G93A) and spot 750 (Stathmin) (area A), and spot 522 (Hsp25) (area B) (compare also Figure 1) from a representative gel with protein extracts from five pooled spinal cords from SOD1G93A mice (top) and their controls (bottom). The bar plots on the right display the abundance differences of the corresponding spots. Hsp25 (spot 522) and SOD1G93A (spot 586) are more abundant in the SOD1G93A mice, whereas Stathmin (spot 750) is only present in the WT mice. Spot 586 in the WT samples, which is located in the same area as the SOD1G93A protein in the corresponding gels run with protein extracts from mutant mice, could not be identified using mass spectrometry, and is classified as unknown.
Figure 3
Figure 3
a: Hsp25 is up-regulated in pooled extracts from five spinal cords from paralyzed transgenic mice expressing SOD1G93A. Immunoblot of spinal cord extracts from age-matched mice expressing the WT and paralyzed transgenic mice expressing SOD1G93A probed with anti-Hsp25 antibody. The pellet obtained after centrifugation of the Triton X-100 extract was homogenized in 1% sodium dodecyl sulfate and clarified. Shown are the supernatants from the Triton (T) and sodium dodecyl sulfate (S) extracts. Fifteen μg of protein were loaded in each lane and transferred to nitrocellulose. b: WT, extract from five spinal cords of age matched mice expressing human SOD1; G93A, extract from five spinal cords of paralyzed mice expressing SOD1G93A. Unlike Hsp25, Stathmin is found only in the Triton X-100 soluble fraction. Stathmin plasmid: extract of CHO cells transfected with an expression vector for Stathmin (in Materials and Methods) and used in experiments in Figures 6 and 7. c:. Two-dimensional immunoblot of pooled spinal cord extracts using an antibody against Stathmin; CON, normal age-matched littermates not expressing mutant SOD1; WT, age-matched mice expressing the WT human SOD1; G93A, mice expressing the mutant. Asterisk marks the isoform that is down-regulated in the mutant.
Figure 4
Figure 4
Sections of spinal cord from symptom-free mice transgenic for WT human SOD1 (control, top row) or paralyzed mice expressing the mutant (G93A, bottom row). Immunostained for Stathmin, SCG10, or SCLIP. Note accumulation of Stathmin, SCG10, and SCLIP in neuronal perikarya and processes. Original magnifications, ×400.
Figure 5
Figure 5
Sections of spinal cord from paralyzed mice transgenic for the mutant G93A, double immunostained for Golgi apparatus (G) and Stathmin (ST). In the top pair, the cell without Stathmin has a normal Golgi network (g), whereas the cell with accumulated Stathmin has lost all Golgi staining (asterisk). The bottom pair shows a cell with fragmented Golgi (f) and accumulated Stathmin. Original magnifications, ×600.
Figure 6
Figure 6
HeLa cells transfected with a Stathmin expression vector. Stained for nuclei (DAPI), Golgi apparatus (GM130), and Stathmin. The two cells overexpressing Stathmin have fragmented Golgi apparatus. Original magnifications, ×500.
Figure 7
Figure 7
HeLa cells transfected with Stathmin were double immunostained for Stathmin (ST) and α-tubulin (TUB). Compare the intact microtubule network in an untransfected cell to the weak and collapsed network in a cell expressing Stathmin (asterisk). Original magnifications, ×600.
Figure 8
Figure 8
Sections of spinal cords immunostained for Hsp25. First panel: 90-day-old mice transgenic for WT human SOD1 (WT) or the mutant G93A (G93A). Note weak neuronal cytoplasmic stain. Second panel: note stain in astrocytes in paralyzed mouse (G93A-par). The nontransgenic control (non-Tgn) shows Hsp25 in neuronal perikarya. Third panel: 30-day-old mouse, transgenic for G93A (30 days G93A) and from control expressing the WT (30 days WT) showing no difference in Hsp25 immunostain. Fourth and bottom panel: double immunolabeling for Hsp25 (HSP, green) and GFAP (red) shows Hsp25 accumulation of Hsp in reactive astrocytes. Original magnifications, ×200.
Figure 9
Figure 9
Double-immunofluorescence labeling of Hsp25 (left) and SOD1 (right) in spinal cord sections from mice expressing SOD1G93A. a: Sixty-day-old mouse. Expression of both Hsp25 and SOD1 in neurons. b: Paralyzed mouse. Most reactive astrocytes (asterisk) express only Hsp25. c: Paralyzed mouse. Anterior horn area. Left, Hsp25; middle, SOD1; right, merge showing double labeling of probably dying neurons. Original magnifications: ×200 (a); 300 (b); 400 (c).
Figure 10
Figure 10
Immunoprecipitations: extracts from five pooled spinal cords from symptom-free mice expressing the WT, and paralyzed mice expressing the mutant (G93A), were immunoprecipitated with a: an anti-SOD1 antibody, followed by immunoblotting with an antibody against Hsp25; b: Immunoprecipitaions with an irrelevant antibody (MG160 IP) followed by immunoblotting with an anti-SOD1 antibody, or immunoprecipitations with an anti-Hsp25 antibody followed by immunoblotting with an anti-SOD1 antibody; SOD1 standard = bovine red blood cell SOD1.
Figure 11
Figure 11
Western blots from five pooled spinal cord extracts from age-matched transgenic mice expressing the WT, and paralyzed mice expressing the mutant (G93A), probed with an antibody against PRDX6.
Figure 12
Figure 12
Spinal cord sections immunostained with an anti-PRDX6 antibody. Top: 30-day-old mice expressing the WT or the mutant (G93A). Middle: Age-matched mouse expressing the WT (WT) and paralyzed mouse expressing the mutant (G93A). Note staining of astrocytes and processes in G93A. Bottom: Double labeling with an antibody against PRDX6 (red) and GFAP (green). Reactive astrocytes are labeled by PRDX6. Original magnifications, ×200.

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