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Mutant SOD1 causes up-regulation of Stathmin 1 and 2 gene expression

To identify the upstream triggers of microtubule loss and tubular/vesicular Golgi fragmentation in mutant SOD1 motor neurons, we performed data mining and bioinformatic analyses. We first analyzed five studies comparing the gene expression profiles of SOD1^G93A and control motor neurons isolated by laser capture microdissection from adult spinal cord [19, 28, 33, 40, 42]. Two studies showed that cytoskeleton-related genes annotated to the Gene Ontology (GO) terms “cytoskeletal part/GO:0044430” and “cytoskeleton/GO:0005856” are significantly dys-regulated: 14 genes at presymptomatic stage [33]), 76 genes at early disease stage [33]) and 42 genes at end stage [42]). Among those, genes annotated to the microtubule cytoskeleton (GO:0015630) display significant dys-regulation [33].

To identify individual dys-regulated genes linked to microtubules, we then compared by bioinformatics analysis the publically available gene expression profiles from mutant SOD1 G93A and control motor neurons deposited in the Gene Omnibus database by Ferraiuolo et al. as GSE10953 [19] and by Nardo et al. as GSE46298 [40]. Given the similarities between pmn and SOD1, we first focused on tubulin binding co-factors but found that the six genes Tbca, Tbcb, Tbcc, Tbcd, Tbce and Tbcel (Tbce-like) are not significantly dys-regulated in mutant SOD1 motor neurons at any time point (fold change??1.5, p??0.05, Table 1).

Gene expression profiles of tubulin-binding cofactors in mutant SOD1 motor neurons

Ferraiuolo et al. 2007

Presymptomatic G93A vs. Ctrl (P60)

Symptomatic G93A vs. Ctrl (P90)

Endstage G93A vs. Ctrl (P120)

Gene Symbol

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Tbca

up

1,74

1,61 E-01

up

1,13

6,11E-01

down

1,63

9,62E-02

Tbcb

up

1,03

9,24E-01

down

1,08

7,49E-01

down

1,33

6,17E-02

Tbcc

down

1,09

8,24E-01

down

1,25

4,57E-01

up

1,03

9,71E-01

Tbcd

down

1,14

8,10E-01

up

1,07

7,65E-01

down

1,03

9,32E-01

Tbce

up

1,29

6,22E-01

up

1,01

9,87E-01

down

1,07

9,18E-01

Tbcel

down

1,07

9,21 E-01

down

1,12

7,91 E-01

up

1,10

8,09E-01

Nardo et al. 2013

Presymptomatic C57 G93A vs. Ctrl

Onset C57 G93A vs. Ctrl

Symptomatic C57 G93A vs. Ctrl

Gene

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Tbca

up

1,23

1,81 E-01

down

1,03

8,53E-01

up

1,04

3,83E-01

Tbcb

up

1,06

7,17E-01

down

1,15

5,21 E-01

down

1,04

8,46E-01

Tbcc

up

1,16

2,81 E-01

down

1,10

2,12E-01

up

1,07

6,83E-01

Tbcd

up

1,09

6,15E-01

up

1,01

9,47E-01

up

1,10

5,20E-01

Tbce

up

1,11

6,02E-01

up

1,17

2,70E-01

up

1,10

1,05E-02

Tbcel

up

1,06

6,01 E-01

up

1,10

4,47E-01

down

1,09

6,51E-01

Nardo et al. 2013

Presymptomatic 129sv G93A vs. Ctrl

Onset 129sv G93A vs. Ctrl

Symptomatic 129sv G93A vs. Ctrl

Gene

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Tbca

up

1,11

5,19E-01

down

1,11

2,51 E-01

down

1,10

4,67E-01

Tbcb

up

1,22

2,89E-01

down

1,45

3,00E-03

down

1,45

1,61E-02

Tbcc

up

1,20

1,50E-01

down

1,23

4,28E-02

down

1,06

3,85E-01

Tbcd

up

1,28

1,37E-01

down

1,11

4,26E-01

down

1,06

5,97E-01

Tbce

up

1,17

2,44E-01

down

1,20

8,76E-03

down

1,05

5,69E-01

Tbcel

up

1,10

6,18E-01

up

1,01

9,11E-01

down

1,02

8,61E-01

By contrast, we found three Stathmin genes whose expression was significantly modulated in presymptomatic mutant SOD1^G93A motor neurons when compared to control. Stathmin 1 and Stathmin 2 were up-regulated by 1.87 and 1.63 fold respectively, whereas Stathmin 3 was down-regulated by 1.71 fold at P60 (p??0.05, Table 2). Stathmin 1 up-regulation (by 1.68 fold) also tended to occur at end stage in an independent dataset of Perrin et al. [42], although not reaching statistical significance (p?=?0.06, Table 2).

Gene expression profiles of Stathmins in mutant SOD1 motor neurons

Ferraiuolo et al. 2007

Presymptomatic G93A vs. Ctrl (P60)

Symptomatic G93A vs. Ctrl (P90)

Endstage G93A vs. Ctrl (P120)

Gene

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Stmnl

up

1,87

3,80E-02

up

1,21

4,54E-01

down

1,40

3,32E-01

Stmn2

down

1,03

9,47E-01

down

1,39

2,65E-01

up

1,91

3,96E-01

Stmn3

down

1,71

9,50E-03

down

1,44

2,73E-01

down

1,21

2,92E-01

Stmn4

down

1,02

9,61 E-01

up

1,39

5,15E-01

up

1,09

8,50E-01

Nardo et al. 2013

Presymptomatic C57 G93A vs. Ctrl

Onset C57 G93A vs. Ctrl

Symptomatic C57 G93A vs. Ctrl

Gene

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Stmnl

down

1,35

9,31 E-02

up

1,17

1,49E-01

up

1,16

3,48E-01

Stmn2

down

1,05

8.98E-01

down

1,52

2,38E-01

up

1,36

4,89E-01

Stmn3

up

1,07

7.28E-01

down

1,24

1,41 E-02

down

1,14

1,50E-02

Stmn4

down

1,06

6,69E-01

up

1,43

1,63E-01

up

1,36

3,15E-02

Nardo et al. 2013

Presymptomatic 129v G93A vs. Ctr

Onset 129sv G93A vs. Ctrl

Symptomatic 129sv G93A vs. Ctrl

Gene

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Stmnl

up

1,05

5,50E-01

up

1,06

3,40E-01

down

1,04

7,57E-01

Stmn2

up

1,63

1.98E-02

down

1,46

1,76E-01

down

1,71

9,08E-02

Stmn3

up

1,08

6,24E-01

down

1,25

6,38E-03

down

1,30

1,18E-02

Stmn4

down

1,13

3,75E-01

up

1,15

3,11E-01

up

1,23

2,98E-03

Perrin et al. 2006

Presymptomatic G93A vs. Ctrl (P60)

Symptomatic G93A vs. Ctrl (P90)

Endstage G93A vs. Ctrl (P120)

Gene

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Regulation

Fold-Change

P-Value

Stmnl

down

1,38

c

down

1,38

c

up

1,68

6,00E-02

Stmn2

up

1,20

c

up

1,30

1,36E-01

up

1,12

6,44E-01

Stmn3

down

1,03

c

up

1,07

5,63E-01

down

1,02

8,36E-01

Stmn4

up

1,75

c

up

1,30

6,49E-01

up

1,85

c

Stathmins represent a family of four proteins (Stathmins 1 to 4), which destabilize microtubules either by sequestering tubulin dimers or by increasing the frequency of microtubule catastrophes, i.e. their transition from steady growth to rapid depolymerization [3, 10]. All Stathmins are highly expressed in neurons, several of them localize to the Golgi [10] and Stathmin 1 protein levels were found increased in spinal cords of paralyzed (late stage) SOD1^G93A mice [55]. We therefore considered Stathmins as good candidates for mutant SOD1-linked microtubule alterations and Golgi fragmentation.

We analyzed whether one of the four Stathmins is dysregulated at the protein level in the spinal cord of mutant SOD1 mice. Using Western blot, we found that levels of Stathmin 1 (19 kD) and Stathmin 2 (also called SCG10, ~22 kD) are both increased by more than 3 fold in mutant SOD1^G85R and SOD1^G93A spinal cords at age 240 days (Fig. 4a) whereas they are not in SOD1^wt mice (not shown). Of note, this up-regulation is not observed for Stathmin 3 (RB3, ~26 kD) (not shown) and Stathmin 4 (RB3’, ~24 kD) is undetectable. We therefore validate that expression of mutant SOD1 triggers up-regulation of Stathmins 1 and 2 in motor neurons and conclude that this could cause the observed microtubule defects and Golgi fragmentation.

Early and rapidly progressive co-accumulation of Stathmin 1, Stathmin 2 and GS28 in motor neurons of mutant SOD1 mice. a. Western blots show expression of Stathmin 1, Stathmin 2 and GS28 in lumbar spinal cords of SOD1^G85R mice (upper panels), SOD1^G93A mice (lower panels) and corresponding litter mates. Analyses were performed at ages P8, P30, P180 and P240. Loading control ?-actin. Each blot was performed in duplicate and is representative of four animals per genotype. b. Diagrams showing kinetics of Stathmin 1, Stathmin 2 and GS28 protein levels in lumbar spinal cords of mutant SOD1^G85R mice and SOD1^G93A mice. Stathmin 1 and 2 levels are already significantly increased at P8. Fold changes (mean?±?sd) are determined from four spinal cords per genotype and time point and expressed relative to the levels in non-transgenic littermate controls (set to 1). Differences between mutant SOD1 and control are statistically significant as measured by Mann Whitney test (*, p??0.01). c–d. Confocal images of lumbar spinal cord cross sections from non-transgenic, SOD1^G85R and SOD1^G93A mice aged 240 days showing accumulation of Stathmin 1 (C, upper panels), Stathmin 2 (D, upper panels) and GS28 (C-D, lower panels) in motor neurons, which sometimes appear as degenerating. Note motor neurons with either low expression (arrowheads) or high expression (arrows) of Stathmins and Golgi SNAREs. Scale bar 20 ?m. e. Diagram showing immunoreactivities (IR) of Stathmin 1 (x-axis) and GS28 (y-axis) in motor neurons of control non-transgenic mice (in blue) and mutant SOD1^G85R mice (in red). Pearson analysis demonstrates significant correlation between both parameters: r?=?0.28, p??0.0066 (ctrl) and r?=?0.47, p??0.0001 (SOD1^G85R). The slopes of the regression curves are 0.49?±?0.18 (ctrl) and 0.68?±?0.13 (SOD1^G85R), n?=?91 cells (ctrl) and n?=?99 cells (SOD1^G85R) analyzed from n?=?3 mice per genotype. f. Immunoreactivities of Stathmin 2 and GS28 also show significant correlation by Pearson analysis: r?=?0.40, p??0.0047 (ctrl), r?=?0.69, p??0.0001 (SOD1^G85R). The slopes of the regression curves are 0.29?±?0.09 (ctrl) and 0.60?±?0.09 (SOD1^G85R) by linear regression analysis, n?=?47 cells and n?=?48 cells analyzed respectively from n?=?3 mice per genotype