CHARACTERIZATION OF MOLECULAR ISOFORMS AND ROLE OF THE SURVIVAL MOTOR NEURON (SMN) IN MOTOR NEURONS DISEASES.

La sclerosi Laterale Amiotrofica (SLA) e l'Atrofia Muscolare Spinale (SMA) sono malattie neurodegenerative caratterizzate dalla perdita progressiva dei motoneuroni. La SMA è generalmente causata da delezione in omozigosi o mutazione del gene SMN, che codifica per una proteina ubiquitaria e multifunzionale, altamente espressa nel midollo spinale. La SLA è una malattia che può essere familiare o sporadica.Il 20% dei casi familiari è causato da una mutazione dominante nel gene SOD1. Inoltre ci sono altri geni coinvolti in questa malattia, tra cui FUS e TDP43. Lo scopo principale della tesi è quello di studiare il gene, le isoforme, la localizzazione subcellulare ed i partners molecolari di SMN. Inoltre, poiché sia FUS che TDP43 possiedono domini ricchi in glicina e questi sono necessari per l’interazione con SMN, sono state valutate le possibili interazioni, in modo da capire se le mutazioni dei rispettivi geni possano avere una ricaduta sulle loro interazioni e quindi sulla loro funzione. Dagli studi di RFLP-PCR, eseguiti su DNA estratto da campioni di sangue intero di pazienti SLA e di controlli neurologici, si evince che non c’è delezione a carico dell’esone 7 del gene SMN. Invece lo studio della proteina, attraverso Western blotting, ha rivelato la presenza di diverse isoforme, sia a livello nucleare che citoplasmatico di leucociti, di cellule HeLa e di cellule di neuroblastoma (SH-SY5Y). Anche le proteine TDP43 e FUS presentano diverse isoforme negli stessi campioni. Inoltre, studi di co-immunoprecipitazione SMN/FUS, fatti su cellule SH-SY5Y, hanno permesso di capire che le due proteine interagiscono a livello nucleare e nello specifico SMN interagisce con una specifica isoforma di FUS. Poi, attraverso immunofluorescenza, è stata valutata la localizzazione delle proteine studiate in cellule Hela, SH-SY5Y ed in fibroblasti umani; la distribuzione delle proteine rimane sempre la stessa nei 3 tipi cellulari : FUS in nucleoplasma, TDP43 in nucleoplasma e citoplasma, SMN in nucleoplasma, citoplasma e GEMS. Inoltre la localizzazione di FUS e di SMN e la loro interazione non cambia durante il differenziamento delle cellule di neuroblastoma in cellule neuroni-simili attraverso trattamento con acido retinoico e pretrattamento con polilisina/poliornitina. Invece la distribuzione di FUS cambia in fibroblasti umani provenienti da biopsia cutanea di un soggetto asintomatico con mutazione P525L nel gene FUS. In tali cellule la proteina FUS localizza sia nel nucleo che nel citoplasma ma anche in alcuni granuli citoplasmatici. Il fatto che FUS traslochi nel citoplasma in caso di mutazione era già stato visto in precedenza in pazienti affetti da SLA, noi qui dimostriamo per la prima volta che avviene lo stesso fenomeno in un caso pre-clinico.The Amyotrophic Lateral Sclerosis (ALS) and the Spinal Muscular Atrophy (SMA) are neurodegenerative disorders characterized by progressive loss of motor neurons. The SMA is generally caused by homozygous deletion or mutation of the SMN gene, which encodes for a protein that is ubiquitous and multifunctional and it is highly expressed in the spinal cord. The ALS is a familial or a sporadic disease. The 20% of the cases of the familial ALS is caused by a dominant mutation in the SOD1 gene. In addition FUS and TARDBP are two other genes involved in this disease. The purpose of my thesis is to study the gene, the isoforms, the subcellular localization and the molecular partners of SMN protein. We studied the SMN gene by RFLP-PCR and we discovered that there is not deletion in exon 7 and in exon 8 of this gene. Therefore, SMN is not implicated in the pathogenesis of ALS at genetic level, for this reason we analyzed the SMN protein. We chose also two other proteins, FUS and TDP-43 because they have the prerequisites for interacting with SMN protein; in fact they have a rich in glycine domains and this is fundamental for the interaction with the SMN protein. Our studies revealed that the proteins analyzed have different isoforms. In addition we found that SMN and TDP-43 proteins are both in the nucleus and in the cytoplasm, conversely the FUS protein is only in the nucleus. We subsequently evaluated the interaction of the SMN with the FUS protein by co-immunoprecipitation. It showed that only a specific isoform of FUS interacts with the SMN protein and this interaction occurs only in the nucleus. Then we understood that the localization of the FUS and the SMN proteins and their interaction does not change during differentiation of neuroblastoma cells (SH-SY5Y) into neuronal-like adult cells by retinoic acid treatment and pretreatment with poly-lysine/poly-ornithine. Conversely, the localization of the FUS protein changes in human fibroblasts, taken from skin biopsy of an asymptomatic subject with P525L FUS mutation. In these cells the FUS protein is found both in the nucleus and in the cytoplasm. The translocation of mutated FUS from the nucleus to the cytoplasm has already been discovered by other authors in patients with amyotrophic lateral sclerosis. Here we show, for the first time, that the same phenomenon is present in a subject with FUS mutation but asymptomatic

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Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms.

The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN

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 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

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

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