spinal muscular atrophy (SMA)

Niko Hensel^a,b, Hella Brinkmann^a and Peter Claus^a,b,c,*

aInstitute of Neuroanatomy, Hannover Medical School, 30625 Hannover, Germany, ^bCenter for Systems Neuroscience (ZSN), 30559 Hannover, Germany, ^cNiedersachsen Research Network on Neuroinfection

(N-RENNT), Germany

Manuscript in submission

Niko Hensel contributed by: Conceiving and designing experiments Performing experiments

Analyzing data

Writing the manuscript

Neurotrophic pathway activation leads to a transient mis-location of extracellular-signal-regulated-kinase (ERK) in

Niko Hensel^a,b, Hella Brinkmann^a and Peter Claus^a,b,c,*

aInstitute of Neuroanatomy, Hannover Medical School, 30625 Hannover, Germany, ^bCenter for Systems Neuroscience (ZSN), 30559 Hannover, Germany, ^cNiedersachsen Research Network on Neuroinfection

(N-RENNT), Germany

Introduction

Spinal Muscular Atrophy (SMA) is characterized by a degeneration of lower motoneurons within spinal cord leading to a progressive proximal muscle weakness (Hoffmann, 1900; Werdnig, 1894). The disease is caused by mutations or deletions within the Survival of motoneuron 1 (Smn1) gene (Lefebvre et al., 1995).

However, humans possess two or more copies of the Smn2 gene. Smn2 only differs from Smn1 in one translational silent basepair exchange within an exonic splice enhancer region leading to only low levels of functional full length SMN-protein (Lorson and Androphy, 2000; Lorson et al., 1999; Monani et al., 1999).

Thus, Smn2 only partially rescues for Smn1 deficiencies and the disease phenotype correlates with the Smn2 copy number (Dubowitz, 1991; Taylor et al., 1998). Although originally described as a lower motoneuron disease other neuronal populations came into focus recently. SMA patients also display impairments in sympathetic nerve function (Hachiya et al., 2005). Moreover, SMA-mice exhibit defects in sympathetic heart innervation, indicating neuronal outgrowth defects for this specific cell type (Heier et al., 2010). However, outgrowth defects have not been described for SMA-mice motoneurons in vivo so far (McGovern et al., 2008); the motoneuron phenotype was preferentially linked to defects in neuromuscular junction (NMJ) maintenance as well as defects in central synapses at the motoneuron perikaryon (Martinez-Hernandez et al., 2013; Mentis et al., 2011). Interestingly, we could previously show outgrowth defects in sympathetic-like PC12 cells with SMN knock-down, while a SMA model of the motoneuron-like NSC34 cell line displayed enhanced neurite outgrowth (Hensel et al., 2013; Nölle et al., 2011). We could further link the outgrowth deficits in PC12 cells with a unique dysregulation pattern of the ROCK pathway controlling actin dynamics at the neuronal growth cone. Moreover, neuronal profilin2a, a direct interactor of both ROCK- as well as SMN-proteins, served as a molecular bridge causing ROCK dysregulations in SMA (Nölle et al., 2011). In motoneuron-like NSC34 cells, however, we identified extracellular regulated kinase (ERK) acting upstream of enhanced neurite outgrowth in SMA. ERK was upregulated by an enhanced expression of its upstream activator fibroblast growth factor receptor 1 (FGFR-1), a receptor for neurotrophic signalling. Although we additionally reported an enhanced ROCK activity, this had no consequences regarding neurite outgrowth, indicating that ERK overcompensates for enhanced ROCK activity (Hensel et al., 2012; Hensel et al., 2013).

Therefore, we further elucidate the dysregulations in ROCK- and ERK-pathways in motoneuron-like NSC34 cells. Here, we report that ROCK pathway dysregulations in NSC34 cells are not caused by growth conditions but are specific for this cell type. Moreover, neuronal profilin2a was excluded as a molecular bridge leading to ERK dependent outgrowth defects in motoneuron-like NSC34 cells. However, concerning neurotrophic factor signalling, we found SMN dependent-alterations of short distance signalling controlling neuronal differentiation. On the subcellular level, activated ERK became mis-located in SMN knock-down cells, a phenotype that is linked with cellular degeneration. Finally, enhanced ERK-activation was reported in SMA-mice hearts indicating a mechanism not restricted to neuronal cells and highlighting the exclusion of neuronal profilin2a as a molecular link for ERK-pathway dysregulations in SMA.

Results and Discussion

Dysregulation of ROCK-pathway and neurite outgrowth in SMN deprived NSC34 cells is cell type specific

We could previously show that both PC12 cells as well as NSC34 cells display SMN dependent alterations of the ROCK pathway. In PC12 cells, SMN reduction leads to a release of profilin2a (prof2a) from SMN-prof2a-complex and an enhanced activity of ROCK towards the released prof2a, thereby inducing its subsequent hyper-phosphorylation. As a consequence, ROCK is sequestered leading to hypo-phosphorylation of several other downstream-targets such as Cofilin (Cof). This mechanism could be linked to a reduced neurite outgrowth (Hensel et al., 2013; Nölle et al., 2011). In NSC34 cells, however, ROCK-activity is generally upregulated leading to enhanced cofilin-phosphorylation (P-Cof) and an inhibition of the ROCK-pathway by ERK is impaired under SMN knock-down. On the morphological level NSC34-cells displayed an enhanced neurite outgrowth which was dependent upon ERK-activity. Thus, phospho-ERK might be compensationally upregulated as a reaction for enhanced ROCK-activity and impaired ERK to ROCK crosstalk. However, in contrast to PC12 cells ROCK inhibition in SMN knock-down NSC34 cells had no morphological consequences (Hensel et al., 2013; Nölle et al., 2011), indicating different mechanisms.

To check if the different behaviour between NSC34- and PC12-cells is cell intrinsic or dependent upon media conditions, we applied PC12-cell differentiation conditions to NSC34 cell cultures (Fig. 1). Next we evaluated the ROCK activity by phosphorylation of its downstream target Cofilin (Fig. 1, A, B) and measured the neurite outgrowth (Fig. 1, C).

Figure 1: Cell specifity of ROCK-pathway and neurite outgrowth dysregulations under SMN knock-down. NSC34 cells were grown under standard differentiation conditions (NSC34-medium) or in PC12-medium with or without NGF. (A) Cell lysates were blotted with phospho-Cofilin (P-Cof), SMN and α-tubulin (α-Tub) antibodies. (B) Densitometrical analysis of phospho-Cofilin blots.

(C) Sister cultures were grown under same conditions as described in (A), except for a transfection of EGFP to trace and measure neurite lengths. n=3, Two Way ANOVA, ## (p<0.01) and ### (p<0.001) with Bonferroni post-test, * (p<0.05), ** (p<0.01) and *** (p<0.001).

Application of PC12-cell medium induced P-Cof levels selectively for SMN knock-down cells, amplifying the SMN-dependent increase in ROCK-activity (Fig. 1, B). However, this had no influence on the neurite length; magnitudes of enhanced neurite length upon SMN knock-down did not change (Fig. 1, C).

Interestingly, SMN knock-down had no influence on ROCK-pathway activation as well as neurite outgrowth when cells were grown in PC12-medium containing nerve growth factor (NGF), reflecting the standard PC12-cell differentiation conditions (Fig. 1, B, C). Among others, the ERK pathway is a common downstream target of NGF signalling (Xiao and Liu, 2003). Thus, NGF treatment might mask SMN-dependent ERK activation leading to equal neurite lengths between knock-down and control cells.

Supporting this hypothesis, control cells displayed a tendency for enhanced neurite outgrowth when treated with NGF (Fig. 1, C). However, growing NSC34 cells under PC12-cell conditions did not induce a PC12-cell like behaviour with reduced neurite lengths and phospho-Cofilin levels. Although modulated by growth conditions, the observed differences are therefore cell-type specific.

SMN-interacting protein profilin2a does not link ROCK- to ERK-dysregulations

A compensational mechanism of enhanced ERK activity as a reaction to enhanced ROCK activity suggests ROCK to act upstream of ERK regarding ERK induced neurite outgrowth in SMN knock-down cells.

However, ROCK inhibition did not modulate ERK dependent alterations of neurite outgrowth, indicating a mechanism independent of kinase activity of ROCK (Hensel et al., 2013). Such a mechanism could be an alteration of freely available ROCK dependent upon SMN. As profilin2a SMN dependently controls for availability of ROCK for Cofilin in PC12 cells (Nölle et al., 2011), a similar mechanism might induce ERK dependent changes in NSC34 cells. We therefore evaluated whether the enhanced neurite lengths in SMN knock-down NSC34-cells, could be regulated by over-expression of profilin2a (Fig. 2).

Figure 2: Influence of profilin2a and its phosphosite Serine 137 on neurite outgrowth. NSC34-cells were co-transfected with plasmids coding for profilin-2a wild type (Prof2a), profilin-2a non-phospho mutant S137A and phospho-mimic mutant S137D with either SMN-siRNA (si) and scrambled control siRNA (scr) (A) or SMN-Flag or Flag-control vector (B). Neurites were traced by EGFP-coexpression from IRES-vectors coding for the Profilin-2a-constructs. N=5, Two-Way ANOVA, ### p<0.001, # p<0.05 with Bonferroni posttest * p<0.05.

As ROCK downstream target dysregulations in PC12 cells were coupled to hyper-phosphorylation of profilin2a (Nölle et al., 2011) we additionally measured non-phospho-mutant S137A as well as profilin2a phospho-mutant S137D (Fig. 2, A). However, no modifying changes in behaviour of NSC34 cells under SMN-knockdown could be measured. This emphasizes that the effects measured in NSC34 cells are distinct from those seen in PC12-cells. As profilin2a and SMN directly interact on the protein level, it is though possible that an effect can only be seen when co-expressed in similar concentrations (Fig. 2, B). Indeed, under SMN-overexpression profilin2a negatively regulates neurite outgrowth. As this effect could be rescued by non-phospho mutant S137A, it seems to be dependent upon S137-phosphorylation. Although profilin2a phosphorylation thus is relevant for neurite outgrowth in NSC34-cells, we could only show a modulative role when co-overexpressed with SMN. In our NSC34-cell disease model profilin2a does not have any effects on neurite outgrowth, which therefore can be excluded as a possible relevant mechanism upstream of ERK induced neurite outgrowth.

SMN knock-down leads to enhanced susceptibility for cell density dependent neuronal differentiation

Regarding the molecular mechanism for compensational upregulation of ERK-activity, we could previously show that NSC34-cells display an enhanced FGFR-1 expression leading to sustained ERK-activation under treatment with SMN-siRNA (Hensel et al., 2012). However, these results were obtained by a classical signalling approach, leaving the cells in serum free starvation medium 2 h in advance of analysis.On the functional level, with regard to neurite outgrowth as well as the number of differentiated cells, we could not show any influence of FGFR-signalling. Incubation of the cells for three days with FGFR-inhibitor PD173074 had no influence on the enhanced number of differentiated cells under SMN-knock-down (Hensel et al., 2013). In principle, receptors for neurotrophic factors display a redundancy in signalling and function (Xu et al., 2013). Thus, it is possible that during a three day period of FGFR-inhibitor incubation, another receptor, not targeted by PD173074, became upregulated instead. This mechanism would be in agreement with the susceptibility of SMN-knock-down induced enhanced number of differentiated cells towards ERK-inhibitor treatment (Hensel et al., 2013) and suggests a more general compensatory mechanism of ERK activity upregulation as a response to SMN knock-down. However, to verify if paracrine signalling might be responsible for enhanced number of differentiated cells, we transfected NSC34 cells with SMN siRNA and scrambled control siRNA, respectively. Then, we counted the number of nuclei and the number of differentiated cells per frame and performed a correlation analysis between both parameters (Fig.3). Although there were no detectable differences between scrambled control and knock-down cells in cell density (Fig. 3, A), SMN knock down induced differentiation as shown by the enhanced number of differentiated cells normalized by the total number of cells (Fig. 3, B), recapitulating our results reported previously (Hensel et al., 2013). However, variations in cell density between experimental repetitions and unequal spatial distributions of cells on each coverslip within each experiment provide for a continuum of cell density affecting microenvironments (Fig. 3, D-G). This might in turn lead to a continuum of local growth factor levels influencing the differentiation. Interestingly, a correlation analysis revealed a positive correlation of the number of differentiated cells with the number of nuclei only for cells with a SMN knock down (Fig. 3, C). To test, if this correlation behaviour differs between knock-down and control cells, we performed a correlation analysis for each biological independent experiment (Fig. 3, H), and statistically tested the slopes against each other: Whereas SMN knock down cells displayed a reproducible positive correlation between cell density and number of differentiated cells, this could not be observed in control cells (Fig. 3, K).

Figure 3: Correlation between the number of differentiated cells (#diff’ cells) with the number of nuclei (# nuclei). NSC34 cells were transfected with siRNAs against SMN (si) and scrambled control siRNA (scr), as well as with an EGFP-plasmid for visualization of cells. After immunofluorescent staining with an anti-SMN antibody, microscopical images of the cells were obtained and the number of nuclei per picture (A) and the number of differentiated EGFP-positive cells normalized by the number of nuclei (B) was measured for each frame. Thus, two values, the number of differentiated cells as well as the number of nuclei could be assigned to one frame and depicted as one point in a two-dimensional graph (C). Examples of such frames are given in (D-G) and allocated to their respective individual points depicted in the graph (C, D’-G’). For each biological replicate (n) 10 datapoints were measured (k) and analyzed for correlation. One representative example is given (H) with the respective slopes derived by linear regression. Those slopes were averaged over all biological replicates (K). Correlation analysis was applied to cells treated with ROCK-inhibitor Y27632, ERK inhibitor FR180204 and FGFR inhibitor PD173074 against DMSO control treated cells. Student’s t-test for A, B, K with * (p<0.05) and ns (non significant), n=3. Correlation analysis and linear regression with F-test for C, H with *** (p<0.0001) and ns (non significant), n=3. Two way ANOVA with * (p<0.01) and Bonferroni post tests with ns (non significant) for L, n=5.

Therefore, NSC34 cells are more susceptible in induction of neuronal differentiation by surrounding cells under SMN knock-down, most probably caused by either cell-cell contacts or short distance paracrine signalling between neighbouring cells. This is in accordance with previous results demonstrating enhanced FGFR-1 expression (Hensel et al., 2013) as this might lead to the observed enhanced susceptibility in neuronal differentiation towards cell density: More cells producing secreted FGFs might encounter a cell with an enhanced FGFR-expression leading to enforced ERK-activity and ultimately to enhanced neuronal differentiation. Although we could not show any influence of FGFR inhibition on the total number of differentiated cells (Hensel et al., 2013), the correlation analysis provides a tool to evaluate inter-cellular effects such as paracrine signalling or cell-cell contacts. To test if FGFR-1 upregulation is the molecular basis for the enhanced susceptibility of SMN knock-down cells for induction of neuronal differentiation by surrounding cells, we performed a correlation analysis of already published data (Hensel et al., 2013, Fig. 5) where we applied different pathway specific inhibitors to our cells (Fig. 3, K). Importantly, this data set recapitulates an enhanced susceptibility for neuronal differentiation dependent upon cell density under SMN knock-down. Generally the measured slopes are significantly larger for SMN siRNA transfected cells (Fig 3, K). However, due to more restrictive Bonferroni-correction of posttests, compared to a simple t-test in figure 1, A, no significant differences between knock-down and control cells within inhibitor treatment groups could be observed. Moreover, inhibitors did not influence the enhanced dependency of neuronal differentiation upon cell density observed under SMN knock-down. Interestingly, slopes of siRNA transfected cells could not be normalized to control levels by either FGFR inhibitor treatment PD173074 or ERK inhibitor treatment FR180204 indicating that none of those pathways influenced the enhanced susceptibility of SMN knock-down cells for cell density effects on neuronal differentiation (Fig. 3, K).

Previously, we could show that ERK pathway is responsible for enhanced neurite outgrowth as well as enhanced number of differentiated cells (Hensel et al., 2013). Thus, underlying mechanisms must be fundamentally different. Whereas changes in neurite outgrowth and neuronal differentiation rely on ERK-activity this is not the case for changes in cell density dependent induction of neuronal differentiation.

Subcellular localization of activated ERK by quantitative Immunofluorescence

To further study the consequences of ERK activation in SMA we aimed to analyze its subcelluar localization.

Therefore, we developed a protocol to stain for phospho-ERK (P-ERK) (Fig. 4). Fixation can have a significant influence on signal to noise ratio in quantitative Immunofluorescence (qIF). Interestingly, phospho-ERK signal for suspended cells measured in flow cytometry is most intense when fixed with 1.5 %

paraformaldehyde (PFA) in medium followed by a methanol fixation step for 10 minutes at room temperature (Chow et al., 2005). We therefore tested different fixation protocols like 4% PFA on PBS-washed cells (Fig. 4, A1-A4), methanol fixation for 15 min at -20°C (Fig. 4, B1-B4) as well as the already described protocol using a combination of PFA-in medium fixation followed by a methanol fixation step (Fig. 4, C1-C4). To check for specificity of the staining, we included a first antibody negative control (Fig. 4, A3, B3, C3). Moreover, cells were induced with FGF-2 for 10 min before fixation (Fig 4, A2, B2, C2). To assure the specificity of the antibody towards phospho-site, FGF-2 induced cells were additionally treated with shrimp alkaline phosphatase (SAP) after fixation (Fig. 4, A4, B4, C4). In all conditions, the first antibody-negative control almost showed no fluorescence independent of fixation protocol indicating a low background due to secondary antibody incubation (Fig. 4, A3, B3, C3).

Figure 4: Establishment of a protocol for quantitative phospho-ERK immunofluorescence. NSC34 cells were fixed employing three different protocols. 4 % paraformaldehyde (PFA) in PBS for 15 min at room temperature (A1-A4), methanol for 15 min at -20°C (B1-B4). And a combination of PFA in medium-fixation with a final concentration of 1.5 % for 10 min at room temperature followed by a methanol fixation step for 10 min at 4°C (C1-C4). Before fixation, cells were incubated with 50 ng/ml FGF-2 for 10 min (A2, A4, B2, B4, C2, C4). After fixation some of those cells were incubated with 10 U shrimp alkaline phosphatase (SAP) for 2 h at 37°C (A4, B4, C4). Cells were stained by anti-phospho-ERK antibody and an Alexa488-conjugated secondary antibody except for A3, B3 and C3 where first antibody incubation was omitted. (D, E) Confocal image of NSC34 cells stained with DAPI in blue and for phospho-ERK in green.

Whole cell regions of interest (D) and nuclear regions of interest (E) automatically determined by ImageJ are shown in turquoise.

Including an anti-phopho-ERK first antibody, only the combinatorial PFA/methanol-fixation protocol displayed a mentionable signal (Fig. 4, C1 compared with A1, B1). When induced with FGF-2, cells fixed by the PFA/methanol combination, displayed a very prominent signal (Fig. 4, C2 compared with A2, B2), which was almost downregulated to control levels by 2 h incubation with SAP (Fig. 4, C4 compared to C1) confirming the phospho-site specificity of the signal. Thus, in-medium-fixation of cells by PFA followed by a methanol fixation step at room temperature ensures the best signal to noise ratio of phospho-ERK staining. In epi-fluorescence microscopy, fluorescent signals from cytosolic cell layers located above nuclei, disturb an accurate quantification of nuclear signals. Therefore, we applied confocal microscopy to our stained cells (Fig. 4, D, E). To measure the signal intensity in specific regions, we used ImageJ software for automatic detection of regions of interest (ROIs) for whole cells (Fig. 4, D turquoise labels) and nuclei (Fig. 4, E, turquoise labels). By measuring the sum of all grey values within these ROIs (integrated density) the relative amount of P-ERK can be measured for nuclei and –by subtracting values for nuclei from those for whole cells– the cytosolic compartment. Normalization by the area of DAPI-positive staining on a frame equivalent to the number of cells leads to the relative intensity.

Activated ERK transiently accumulates in cytosol of SMA model cells

The function of activated ERK is dependent upon its subcellular distribution. P-ERK is rapidly transported to the nucleus where it promotes cellular survival and proliferation (Sano et al., 1995). In contrast, activated ERK triggers non-apoptotic modes of cell death, when sequestered in cytosolic compartements of some cell types (Mebratu and Tesfaigzi, 2009; Subramaniam and Unsicker, 2010). In neuronal PC12-cell differentiation, NGF treatment of PC12 cells triggers ERK-phosphorylation, which is subsequently transported to the nucleus. In consequence the majority of activated ERK locates within nuclei (Sano et al., 1995). Previously, Parker and collegues reported an enhanced apoptosis of NSC34 cells upon SMN knock-down (Parker et al., 2008). Thus, it is possible that a SMN induced enhanced ERK activation is not only involved in neuronal differentiation processes (Hensel et al., 2013). Besides that, enhanced P-ERK-levels might be dyslocated within SMN deprived cells leading to the reported apoptosis phenotype.

To test for SMN-dependency of P-ERK localization, we transfected siRNA against SMN (si) as well as a scrambled control siRNA (scr) (Fig. 5). Cells were incubated with FGF-2 for either 10 min (Fig 5, C, D) or 1 h (Fig. 5, E, F), fixed and stained for SMN and P-ERK. Relative intensities for nuclear and cytosolic phospho-ERK were calculated from confocal images for the different conditions (Fig. 5, G).

Figure 5: Subcellular distribution of activated ERK and its SMN dependency: NSC34 cells were transfected with siRNA against SMN (si), scrambled control siRNA (scr) and differentiated for three days. 10 min (C, D) or 60 min (E,F) before staining and confocal analysis, cells were induced with 50 ng/ml FGF-2. Relative intensities of phospho-ERK were measured in nuclear (nucl.) or cytosolic compartments (cyt.) and statistically analyzed (G). For that purpose, we performed Two-Way ANOVAs with treatment condition (Ctrl., 10 min FGF-2 and 60 min FGF-2) vs. localization (nucl., cyt.) or with treatment conditions vs. knock-down (scr, si). n=3, ## p<0.01 and ### p<0.0001 for ANOVA statistic and * p<0.05 and

Im Dokument Analysis and inhibition of dysregulated pathways in a mouse model of the motoneuron disease spinal muscular atrophy (Seite 33-70)