Neuritic processes of multi-neuritic neurons display mixed axonal/dendritic phenotype.

<p>(A-C) Confocal images of control (A₁-C₁), a-SMN- (A₂-C₂) and FL-SMN- (A₃-C₃) silenced hippocampal neurons fixed at 8 DIV and co-labeled with the dendritic marker MAP2 (blue, C₁-C₃) and the axonal marker ankG (red, B₁-B₃), specifically staining the axon initial segment (AIS). (D₁-D₃) Merged images. Neurons treated with the control non-targeting siRNA displayed a unique AIS strongly labeled by ankG and different dendrites selectively labeled by MAP2. Staining selectivity was lost in multi-neuritic neurons silenced with a-SMN or FL-SMN, variably displaying axon and dendritic labeling in the same neuritic process. Arrowheads evidence ankG staining. (E) Stacked histograms showing the percent ratio of multi-neuritic neurons with normal vs. abnormal ankG and MAP2 distribution after control, a-SMN or FL-SMN silencing. Data are presented as mean ± SEM of three different experiments. Statistical analysis was performed by chi-square test (** p<0.01, ***p<0.001) among the three experimental groups (control, a-SMN siRNA and FL-SMN siRNA). Scale bar: 25 μm.</p

FL-SMN and a-SMN knockdown: Multi-neurite neurons.

<p>(A₁-B₃) Confocal images of hippocampal neurons co-transfected with control or a-SMN or FL-SMN-specific siRNAs together with Venus plasmid (green, A₁-A₃), fixed after 3DIV and labeled with the anti-a-SMN #553 (red, B₁-B₂) or anti-FL-SMN antibodies (red, B₃). After selective a-SMN or FL-SMN silencing, a significant fraction of hippocampal neurons showed several processes with similar length and no clear evidence of axonal polarization. (C) Percentages of Venus⁺ hippocampal neurons displaying multi-neuritc morphology after transfection with control (white bars), FL-SMN SiRNA (grey bars) or a-SMN-specific siRNAs (red bars). Data are presented as mean ± SEM of three different experiments of each group (* p < 0.05; ** p < 0.01; n.s.: not significant). Statistical analysis were performed by means of one-way ANOVA followed by Tukey HSD as post hoc comparison test. Scale bar: 10 μm.</p

Quantification of a-SMN neuritic labeling during early neuronal differentiation.

<p>Stage 2–3 rat primary hippocampal neurons were co-labeled with the rat-specific anti-a-SMN antibody #553 (A₁-C₁, red) and the pan-cellular marker 5-DTAF (A₂-C₂, green). (A₃-C₃) Pseudocolor images showing heat maps of fluorescence intensity ratio between a-SMN and DTAF, with warm colors denoting higher signal (0–255). (A₄-C₄) Intensity quantification expressed as a-SMN/DTAF fluorescence ratio. The a-SMN/DTAF ratio was similar in primary neurites at stage 2 (A₁-A₄) and in primary (arrowheads) vs. minor neurites (arrows) at stage 2⁺ (B₁-B₄). By contrast, a-SMN staining was significantly more intense in axons <i>vs</i>. dendrites at stage 3 (C₁-C₄: axons 1.79 ± 0.14 and dendrites 1.00 ± 0.09). Data are presented as mean ± SEM of 30 random sampled cells for every stage (2, 2+ and 3) from three different experiments. Statistical analysis was performed by Student’s t-test (** p<0.01). Scale bars: 10 μm.</p

Effect of FL-SMN and a-SMN knockdown on axon growth.

<p>Confocal images of stage 3 hippocampal neurons co-transfected with the Venus plasmid (green) and control (A₁-C₁) or FL-SMN (A₂-C₂) or a-SMN specific siRNAs (A₃-C₃), labeled with anti-a-SMN (red, B₁ and B₃), anti-SMN (red, B₂), and III-ß-tubulin (blue, C₁-C₃) antibodies. Note that, if compared with neurons treated with control siRNA, both FL-SMN- and a-SMN silenced hippocampal neurons showed after 3 DIV shorter and less extensively branched axons. (D) Morphometric analysis revealed that FL-SMN or a-SMN knock-down were equally effective in reducing axon elongation (FL-SMN and a-SMN <i>vs</i>. control siRNA: * p < 0.05, ** p < 0.01: D₁), total axon length (FL-SMN and a-SMN <i>vs</i>. control siRNA: * p < 0.05: D₂) and axonal branching (FL-SMN and a-SMN <i>vs</i>. control siRNA: * p < 0.05, ** p < 0.01: D₃). By contrast, dendrite length (D₄) and number (D₅) were unaffected by either FL-SMN or a-SMN knock-down. Data are presented as mean ± SEM of three independent experiments (axon elongation n > 250 cells, total axon length and axonal branching n > 140 cells, dendrite length and number n > 240 cells). Statistical analysis were performed by means of one-way ANOVA followed by Tukey HSD as post hoc comparison test. Scale bar: 10 μm.</p

Axon markers SMI31 and tau in multi-neuritic neurons.

<p>(A-D) Confocal images of hippocampal neurons co-transfected with control (A₁-D₁), a-SMN (A₂-D₃) or FL-SMN (A₄-D₅) specific siRNAs and the Venus plasmid (green, A₁-A₅ and C₁-C₅), fixed at 3DIV and labeled with the axonal markers SMI31 (blue, B₁-B₅) or tau (red, D₁-D₅). While neurons transfected with control siRNA had a single axon labeled by SMI31(A₁-B₁), in a-SMN and FL-SMN-silenced neurons displaying multi-neuritic morphology, SMI31or tau axonal staining was mainly in soma (B₅, D₂, D₄) or variably localized in two or more neuritic processes (B₂-B₄, D₃, D₅). (E) Quantification of axon labeling in individual multi-neuritic neurons. Stacked histograms showing SMI31 distribution in a-SMN and FL-SMN silenced neurons with multi-neuritic morphology. Data are presented as mean ± SEM. At least 70 cells from three different experiments were analyzed. Percent ratio of axonal markers distribution (multineurites/ only soma/axonal labeling) was compared by means of chi-square test (** p<0.01) between the two experimental conditions (a-SMN or FL-SMN siRNA). Scale bar: 10 μm.</p

a-SMN subcellular localization during neuronal differentiation.

<p>Confocal images of primary hippocampal neurons from embryonic rats co-labeled with DTAF; (A₁-A₄, green), rat-specific anti-a-SMN antibody #553 (B₁-B_4, red) and axonal marker anti-tau antibody (C₁-C_4, blue). Note the early a-SMN staining within cell bodies in stage 1 (B₁) and newly formed primary neurites in stage 2/2⁺ (B₂), and the more selective staining of the forming primary axon in stages 2⁺-4 (B_3-4), with a distribution similar to tau axonal staining (C₂-C₄). Scale bar: 25 μm.</p

Quantification of FL-SMN and a-SMN silencing in NSC34 motor neurons.

<p>(A) Schematic representation of the binding sites of FL-SMN (blue lines) and a-SMN (red line) specific siRNAs along each respective mRNA sequence. Note that the FL-SMN siRNA against the exon 3/exon 4 junction can make a partial annealing to the end of exon 3 and beginning of exon 4 sequence on the a-SMN mRNA (lower blue lines). (B) Western blot analysis of siRNA silencing in NSC34 cells performed with BD Bioscience anti-SMN antibody against the N-terminal region. (B₁) NSC34 motor neurons were co-transfected with rat FL-SMN tagged construct and control or FL-SMN or a-SMN siRNAs as indicated at the bottom of the blot. Labels on the left indicate the native FL-SMN and the transfected tagged protein as a slightly higher band. Tubulin was reported as loading marker (upper red band). Molecular weights are reported on the right (MW). (B₂) NSC34 motor neurons were co-transfected with rat a-SMN tagged construct and control or FL-SMN or a-SMN siRNAs as indicated at the bottom of the blot. Transfection of NSC34 with tag-a-SMN led to the expression of three SMN bands, as indicated in the label on the left. Molecular weights are reported on the right (MW). (C) Histograms showing the quantification of immunoreactive ratio of FL-SMN or a-SMN/tubulin in all experimental groups. (C₁) The 42 KDa tagged-FL-SMN protein expression was significantly down-regulated by FL-SMN siRNA to 12% (***p < 0.001; B₁, blue bar) compared to control non-target siRNA (white bar). a-SMN siRNA did not modify FL-SMN protein levels (red bar). (C₂) Native FL-SMN was significantly down-regulated by FL-SMN siRNA to 47% (*p < 0.05; blue bar), compared to control (white bar). No significant difference was obtained with a-SMN siRNA (red bar) vs. control (white bar). (C₃) The expression of all the transfected tag-a-SMN proteins were down-regulated by a-SMN specific a-SMN siRNA to 12% (***p < 0.001; C₁, red bar), compared to control non-target siRNA (white bar). Note that the exogenous a-SMN was significantly reduced also by the FL-SMN siRNA (to 74%, **p < 0.01; C₁, blue bar) probably due to the partial annealing of the FL-SMN siRNA to the exon 3 and 4 sequence of the a-SMN mRNA. Data were normalized versus α-tubulin protein levels and were presented as mean ± SEM of three different experiments. Statistical analysis was performed by Student’s t-test.</p

a-SMN subcellular localization in stage 4 neurons.

<p>Confocal images of primary hippocampal neurons from E18 embryonic rats co-labeled with #553 anti-rat-a-SMN (A₁–D₁, red) and anti-MAP2 antibody (A₂, green) or anti-tau antibody (B₂-D_2, green). Merged images are shown in column 3 (A₃-D₃). Note how a-SMN fluorescence shows a preferential localization in cell soma and axon, similar to tau (B₁-B₃) while it is less intense in the dendritic compartment, highlighted by dendritic marker MAP2 (A₁-A₃). Higher magnification images show how a-SMN staining is less intense at the growth cone tip (C₁-C₃). In the cell soma a-SMN fluorescence presents a punctuate pattern (D₁-D₃) with localization both cytoplasmic and nuclear, while tau staining is very low in the cytoplasm and nearly absent from the nucleus (D₂). Scale bars: 25μm in A and B; 7,5μm in C and D.</p

Stereological analysis of ChAT⁺ motor neurons.

<p>(<b>A</b>) Low-power images of ChAT immunoreacted spinal cord sections at ventral C3-C5 cervical levels from WT (upper) and SMA (lower) mice at pre- (P4, left) and late-symptomatic (P13, right) disease stages. Note that the C5 area of ChAT⁺ immunoreactivity was smaller in SMA than in WT mice at both P4 and P13. (<b>B</b>) Stereological analysis revealed that total motor neuron number was reduced at P4 (left) and significantly decreased at P13 (right) in SMA compared to WT mice (^*p<0.05, t-test). (<b>C</b>) Western-blot analysis revealed increased ChAT expression between P4 and P13 in WT mice. Values are expressed as mean ± SEM. Scale bar: 100 µm.</p

Glial activation in Δ7 SMA mice.

<p>Coronal sections from cervical spinal cord (<b>A</b>) and brain (<b>C</b>) of WT (left) and SMA mice (right) stained with Neurotrace (red) and GFAP-immunoreacted (green). Note glial activation in the spinal cord and cortical layer V at P13 (bottom right in A and C) but not at P4. (<b>B</b>, <b>D</b>) Quantification of fluorescence intensity (expressed in arbitrary units, AU) revealed a significantly increased glial activation in both ventral (VH) and dorsal horns (DH) of the spinal cord (<b>B</b>) and in cortical layer V but not layers II-III (D) of SMA mice at P13 (^***p<0.001, t-test). Values are expressed as mean ± SEM. Scale bar: 100 µm.</p