ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers

<div><p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motoneurons degenerate leading to muscle wasting, paralysis and eventually death from respiratory failure. Several studies indicate that skeletal muscle contributes to disease progression; however the molecular mechanisms remain elusive. Fibrosis is a common feature in skeletal muscle under chronic damage conditions such as those caused by muscular dystrophies or denervation. However, the exact mechanisms of fibrosis induction and the cellular bases of this pathological response are unknown. We show that extracellular matrix (ECM) components are augmented in skeletal muscles of symptomatic hSOD1^G93A mice, a widely used murine model of ALS. These mice also show increased TGF-β1 mRNA levels, total Smad3 protein levels and p-Smad3 positive nuclei. Furthermore, platelet-derived growth factor receptor-α (PDGFRα), Tcf4 and α-smooth muscle actin (α-SMA) levels are augmented in the skeletal muscle of symptomatic hSOD1^G93A mice. Additionally, the fibro/adipogenic progenitors (FAPs), which are the main producers of ECM constituents, are also increased in these pathogenic conditions. Therefore, FAPs and ECM components are more abundant in symptomatic stages of the disease than in pre-symptomatic stages. We present evidence that fibrosis observed in skeletal muscle of symptomatic hSOD1^G93A mice is accompanied with an induction of TGF-β signaling, and also that FAPs might be involved in triggering a fibrotic response. Co-localization of p-Smad3 positive cells together with PDGFRα was observed in the interstitial cells of skeletal muscles from symptomatic hSOD1^G93A mice. Finally, the targeting of pro-fibrotic factors such as TGF-β, CTGF/CCN2 and platelet-derived growth factor (PDGF) signaling pathway might be a suitable therapeutic approach to improve muscle function in several degenerative diseases.</p></div

Muscle architecture is altered in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A-D)</b> Sirius red staining of gastrocnemius muscle from wild-type (120 days old) and symptomatic (120 days old) hSOD1^G93A age-matched mice seen under polarized light in cross-sections. Bar corresponds to 100 μm. <b>(E)</b> Fiber diameter in wild-type (120 days old; black circles) and symptomatic hSOD1^G93A (120 days old; black squares) mice, values correspond to the mean ± SEM of three animals for each experimental condition. Two-way ANOVA, *** p<0.001, n.s: not significant.</p

FAPs markers are increased in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A)</b> PDGFRα, Tcf4 and α-SMA were detected by western-blot in protein extracts from wild-type (60 days old), pre-symptomatic (60 days old) hSOD1^G93A age-matched mice, wild-type (120 days old), and symptomatic (120 days old) hSOD1^G93A age-matched mice. GAPDH protein levels are shown as loading control. <b>(B-I)</b> Tcf4 (white and green) and laminin (white and red) were detected by indirect immunofluorescence in cross-sections of gastrocnemius muscle from wild-type (120 days old) and hSOD1^G93A symptomatic (120 days old) age-matched mice, bar corresponds to 50 μm, nuclei were stained with Hoechst. Arrows show Tcf4-positive nuclei. <b>(J)</b> Tcf4-positive cells were quantified in gastrocnemius muscle from wild-type (120 days old) and symptomatic (120 days old) hSOD1^G93A age-matched mice, values correspond to the mean ± SEM of three animals for each experimental condition. One-way ANOVA, ** p<0.005. <b>(K-L)</b> PDGFRα (green) and WGA (red) <b>(M-N)</b> PDGFRα and p-Smad3 (red) were detected by indirect immunofluorescence in cross-sections of gastrocnemius muscle from wild-type (120 days old) and hSOD1^G93A symptomatic (120 days old) age-matched mice, bar corresponds to 50 μm, nuclei were stained with Hoechst. Arrows and asterisk show PDGFRα/p-Smad3 co-localization and PDGFRα^-/p-Smad3⁺ cells, respectively.</p

Extracellular matrix (ECM) components deposition in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A-D)</b> H&E staining. <b>(E-H)</b> Sirius red staining. <b>(I-L)</b> fibronectin (red) and <b>(M-P)</b> collagen-I (green) were detected by indirect immunofluorescence in cross-sections of gastrocnemius muscle from wild-type (60 days old), hSOD1^G93A pre-symptomatic (60 days old), wild-type (120 days old), and hSOD1^G93A symptomatic (120 days old) age-matched mice. Bar corresponds to 50 μm. Representative images of three mice per condition.</p

Extracellular matrix (ECM) components are augmented in gastrocnemius muscle from symptomatic hSOD1^G93A mice.

<p><b>(A)</b> Fibronectin and collagen-III were detected by western-blot in protein extracts from wild-type mice (60 days old), pre-symptomatic (60 days old) hSOD1^G93A age-matched mice, wild-type (120 days old), and symptomatic (120 days old) hSOD1^G93A age matched mice. GAPDH protein levels are shown as loading control. <b>(B-C)</b> Protein levels of fibronectin and collagen-III were quantified using densitometric analysis. Values correspond to the mean ± SEM of four animals for each experimental condition. One-way ANOVA, ** p<0.05; *** p<0.001, n.s: not significant.</p

Changes in dendritic architecture during hippocampal development <i>in vitro</i>.

<p><b><i>A</i></b>, Cultured hippocampal neurons were transfected with GFP at 0 DIV with Amaxa nucleofection and fixed at 2, 5, 7, 12, 15 and 20 DIV. Images of representative GFP-expressing hippocampal neurons at the different stages are shown. Scale bar is 25 μm. Inset: Magnified views of boxed areas showing examples of dendritic branches. Note that dendritic arborization increases until 7 DIV and then gradually, but profoundly, drops during the next 2 weeks. Also note that whereas immature neurons display filopodia-like structures, mature neurons are predominantly decorated by dendritic spines. <b><i>B</i></b>, Quantification of the average number of primary, secondary or tertiary dendritic branches of neurons expressing GFP at the developmental stages indicated in the graph. <b><i>C</i></b>, Sholl analysis of neurons at different stages of development documents that cells at 7 DIV display an increased number of intersections close to the soma (25–50 μm). <b><i>D–E</i></b>, Averaged length of primary, secondary and tertiary branches (<b><i>D</i></b>) and total outgrowth (<b><i>E</i></b>) of hippocampal neurons at different developmental stages. At least 20 neurons, obtained from 3 independent experiments, were analyzed for each stage. Figures show Mean ± SEM.</p

Expression of NMDAR subunits NR2A and NR2B, and MAGUKs PSD95 and SAP102, at different stages of development.

<p><b><i>A</i></b>, Total protein extracts were obtained from hippocampal neurons at 2, 5, 7, 12, 15, 18, and 20 DIV and immunoblotted with antibodies against NR2B, NR2A, PSD95, SAP102, or N-cadherin as loading control. <b><i>B</i></b>, Results of qRT-PCR run with primers designed to determine mRNA levels for NR2B, NR2A, PSD95, or SAP102 at the same developmental stages; results were normalized against levels of GAPDH mRNA. Note that NR2B expression decreases with development, while NR2A and PSD95 increases. Expression of SAP102 remains relatively stable at these developmental stages. <b><i>C</i></b>, Immunodetection of endogenous NR2B, NR2A, PSD95, or SAP102 (green) and co-localization with presynaptic marker Bassoon or Piccolo (red) at 5 and 20 DIV. Insets show magnified views of boxed areas showing examples of synaptic clusters located in close apposition to presynaptic clusters.</p

Over-expression of NR2B in hippocampal neurons promotes dendritic branching only in immature cells.

<p>Cultured hippocampal neurons were transfected with GFP alone (control) or with NR2B (NR2B) and fixed at 7, 12, 15 and 20 DIV. To express GFP and/or NR2B in neurons for 3 days, neurons were transfected with either CaPO₄ (<12 DIV) or magnetofection-based methods (>12 DIV). <b><i>A–B</i></b>, Images of representative control hippocampal neurons or expressing NR2B at 7 DIV (<b><i>A</i></b>) or 20 DIV (<b><i>B</i></b>). Scale bar is 25 μm. Note that NR2B expression induces branching only at 7 DIV, while in hippocampal neurons at 20 DIV NR2B expression results in a dendritic architecture that is similar to that of controls. <b><i>C–E</i></b>, Averaged length of secondary (<b><i>C</i></b>) and tertiary (<b><i>D</i></b>) branches, and total outgrowth (<b><i>E</i></b>) of hippocampal neurons expressing GFP alone or GFP plus NR2B at different developmental stages. <b><i>F</i></b>, Sholl analysis of control neurons or NR2B-expressing neurons at 7 DIV and 20 DIV. For each developmental stage and condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. *** p<0.001 (ANOVA).</p

Simultaneous over-expression of NR2B and knockdown of PSD95 induce dendritic branching in mature hippocampal neurons.

<p><b><i>A</i></b>, Cultured hippocampal neurons were transfected with a magnetofection-based method at 15 DIV with GFP alone (control; left image), GFP plus shRNA-PSD95 (shRNA-PSD95; middle image), or GFP plus NR2B and shRNA-PSD95 (NR2B+shRNA-PSD95; right image). At 20 DIV, cultures were fixed and images taken. Scale bar is 25 μm. Inset: Magnified views of boxed areas showing examples of dendritic branches. Note that neurons that express NR2B+shRNA-PSD95 display a more complex dendritic architecture compared to control or shRNA-PSD95-expressing cells. <b><i>B–C</i></b>, Quantification of the average number of secondary (<b><i>B</i></b>) and tertiary (<b><i>C</i></b><b>)</b> processes: neurons expressing NR2B+shRNA-PSD95 have more branches relative to control neurons and to cells transfected with shRNA-PSD95. <b><i>D–E</i></b>, Total outgrowth (<b><i>D</i></b>) and Sholl analysis (<b><i>E</i></b>) of hippocampal neurons expressing the different constructs as indicated. For each condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. *** p<0.001 (ANOVA).</p

The C-terminal domain of NR2B is required to promote dendritic branching.

<p><b><i>A</i></b>, Schematic representation of a wild-type NR2B subunit (NR2B), the chimera NR2A_headB_tail (NR2A_hB_t), a wild-type NR2A subunit (NR2A), and the chimera NR2B_headA_tail (NR2B_hA_t). <b><i>B–C</i></b>, Cultured hippocampal neurons were transfected with a magnetofection-based method at 15 DIV with GFP and the different wild-type and chimeric NR2 constructs, as indicated. As controls, cells were transfected with GFP alone (control; red continuous lines) or GFP plus shRNA-PSD95 (shPSD95; red dotted lines). For transfection with constructs that contain the NR2B tail (NR2B and NR2A_hB_t), PSD95 was knocked-down to allow clustering of these NR subunits. At 20 DIV, cultures were fixed, images taken and average number of secondary (<b>B</b>) and tertiary (<b>C)</b> dendritic branches was quantified. Note that in hippocampal neurons, the expression of a NR2 construct containing the C-terminal of the NR2B is a prerequisite for an increase in the number secondary and tertiary dendritic branches relative to control and shPSD95 conditions. For each condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. * p<0.05, ** p<0.01, and *** p<0.001 (ANOVA) for branches relative to control conditions (control or shRNA-PSD95); and ### p<0.001 (t-test) for branches between the indicated conditions. N.S.  =  not significantly different.</p