Silvia Carolina Lenzken

Valentina Romeo

Francesca Zolezzi

Francesca Cordero

Giuseppe Lamorte

Davide Bonanno

Donatella Biancolini

Mauro Cozzolino

Maria Grazia Pesaresi

Alessia Maracchioni

John Wiley

Sons

Remo Sanges

Tilmann Achsel

Maria Teresa Carrì

Raffaele A Calogero

Silvia M L Barabino
email: silvia.barabino@unimib.it

Mutant SOD1 and mitochondrial damage alter expression and splicing of genes controlling neuritogenesis in models of neurodegeneration

Keywords: ALS, axon guidance, microarray, mitochondria, neurodegeneration, RNA splicing, SOD1 ALS, axon guidance, microarray, mitochondria, neurodegeneration, RNA splicing, SOD1

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Introduction

Mitochondrial dysfunction has been directly or indirectly implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer and Huntington (Gibson, et al., 2009). In recent years, mitochondrial damage has also emerged as an early factor contributing to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS, [START_REF] Magrane | Mitochondrial function, morphology, and axonal transport in amyotrophic lateral sclerosis[END_REF].

ALS is a progressive, invariably fatal, neurodegenerative disease caused by the degeneration of motor neurons [START_REF] Boillee | ALS: a disease of motor neurons and their nonneuronal neighbors[END_REF]. Sporadic ALS (sALS) is a genetically and clinically very heterogeneous disease [START_REF] Chio | A two-stage genome-wide association study of sporadic amyotrophic lateral sclerosis[END_REF]. The aetiology of sALS is largely unknown but is likely linked to both environmental and genetic factors inducing mechanisms of motor neuron damage. These mechanisms include oxidative damage, accumulation of intracellular aggregates, mitochondrial failure, defects in axonal transport, growth factor deficiency, aberrant RNA metabolism, glial cell pathology, and glutamate excitotoxicity [START_REF] Cozzolino | Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications[END_REF][START_REF] Rothstein | Current hypotheses for the underlying biology of amyotrophic lateral sclerosis[END_REF]. Familial ALS (fALS) accounts for approximately 5%-10% of all ALS cases and is caused by genetic factors. Of these, approximately 1 in 5 are linked to a mutation in copper/zinc superoxide dismutase 1 (SOD1,#MIM 147450), an enzyme responsible for scavenging the free O 2 -radicals.

There is a substantial body of evidence indicating that mitochondrial dysfunction is a feature of motor neuron degeneration in ALS. Mitochondrial damage has been reported both in sporadic and familial ALS patients and in cellular and animal models that express mutant SOD1 [START_REF] Shi | Mitochondrial dysfunction in amyotrophic lateral sclerosis[END_REF]. Mitochondrial dysfunction may lead to the degeneration of motor neurons because of energy deprivation following impairment of the respiratory chain, alteration of intracellular calcium handling and activation of the apoptotic pathway. We have recently reported that mitochondrial another, quite unexpected effect, i.e. it modulates alternative splicing of a set of mRNAs in cells of neuronal origin [START_REF] Maracchioni | Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration[END_REF]. The importance of alternative splicing (AS) in regulating gene expression is illustrated by a growing number of diseases associated with abnormal mRNA patterns (for review see [START_REF] Cooper | RNA and disease[END_REF]. Alterations of the splicing pattern of several mRNAs have been reported in ALS patients and in murine models [START_REF] Aerbajinai | Increased expression level of the splicing variant of SIP1 in motor neuron diseases[END_REF][START_REF] Munch | Alternative splicing of the 5'sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis[END_REF][START_REF] Pantelidou | Differential expression of molecular motors in the motor cortex of sporadic ALS[END_REF][START_REF] Robertson | A neurotoxic peripherin splice variant in a mouse model of ALS[END_REF][START_REF] Tomiyama | Flip and flop splice variants of AMPA receptor subunits in the spinal cord of amyotrophic lateral sclerosis[END_REF].

Recently, the identification in fALS patients of mutations in TARDBP (MIM# 605068) and FUS (MIM# 13070) that encode DNA/RNA-binding proteins has further strengthened the idea that splicing abnormalities may contribute to neurodegeneration [START_REF] Lagier-Tourenne | Rethinking ALS: the FUS about TDP-43[END_REF].

Based on these reports, we hypothesized that not only gene expression but also alternative pre-mRNA splicing might be altered in neurodegenerative conditions involving mitochondrial damage such as ALS, or Parkinson's disease. We thus performed a whole-genome, exon level analysis of the cellular response to mitochondrial failure. Using Affimetrix Exon 1.0 ST GeneChips® microarrays we profiled human neuroblastoma SH-SY5Y cells treated with PQ, and SH-SY5Y cells stably expressing the mutant G93A-SOD1protein, which is one of the genetic causes of ALS. We then combined the data sets from the two experiments to identify common molecular pathways underlying the response to mitochondrial insufficiency.

The results revealed alterations in the transcriptional and post-transcriptional expression of several genes involved in neuritogenesis and support a role for the

Materials and Methods

Cell culture

Human neuroblastoma SH-SY5Y untransfected or stably transfected with cDNAs coding for wild type SOD1 or the mutant G93A-SOD1 [START_REF] Carri | Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells[END_REF], were cultured in D-MEM/F-12 media with GlutaMAX™ (Gibco, Invitrogen, UK), 10%

FBS, 100 Units/ml penicillin G, 100µg/ml streptomycin/ penicillin (Euroclone, Milano, Italy). Stably transfected cells were also maintained in the presence of 400µg/ml Geneticin (G418 sulphate, Euroclone). Cells were fed every 2-3 days and passed once a week. For the microarray experiment, after an initial amplification of each cell type, cells were aliquoted and stored in liquid nitrogen. For every experiment an aliquot of each cell line was thawed and seeded. After having reached confluence, cells were reseeded at 3x10 6 cells in 100mm dish. Paraquat (N,N'dimethyl-4,4'-bipyridinium dichloride, Sigma-Aldrich) treatment was carried out essentially as described in [START_REF] Maracchioni | Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration[END_REF] but for 18 h at 0,75 mM concentration.

RNA preparation

Total RNA was from cultured cells extracted using TRIzol® Reagent (Invitrogen), and subsequently purified using silica membrane spin columns from RNeasy Mini kit (Qiagen). RNA quantity and purity were assessed using a NanoDrop® instrument (Thermo Fisher Scientific Inc.). Total RNA integrity was assessed by using a 2100

Bioanalyzer (Agilent Technologies) and the RNA Integrity Number (RIN) was Reagent (Invitrogen) from eleven healthy female mice (controls) and fourteen female G93A-SOD1mice sacrificed between 93 and 170 days.

Microarray hybridization and data analysis

Each condition was replicated 5 times. After extraction and quality check 1.5 µg of total RNA was subjected to removal of ribosomal RNA following the procedure suggested by the manufacturer (Affymetrix). The resulting total RNA was then used to created the biotin-labelled library to be hybridized on GeneChip® Exon 1.0 ST human microarrays following the procedure described by the manufacturer (Affymetrix). The CEL files resulting from the hybridization were analyzed using oneChannelGUI 1.6.5 [START_REF] Sanges | oneChannelGUI: a graphical interface to Bioconductor tools, designed for life scientists who are not familiar with R language[END_REF]. Gene-level calculation was performed by RMA (Robust Multichip Average, (Irizarry, et al., 2003) and normalization by quantile sketch [START_REF] Bolstad | A comparison of normalization methods for high density oligonucleotide array data based on variance and bias[END_REF]. To assess differential expression at gene-level, we used an empirical Bayes method [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF] together with a false discovery rate (FDR) correction of the p-value [START_REF] Westfall | Resampling-based multiple testing: examples and Materials and Methods for p-value adjustment[END_REF]. Thus, the list of differentially expressed genes was generated using an FDR ≤ 0.05 together with an absolute log 2 (fold-change) threshold of 1. Differential expression was detected by linear model statistics. This method is based on the fitting of a linear model to estimate the variability in the data. In case of one-channel microarray data this approach is the same as the analysis of variance except that a model is fitted for every gene. For the detection of the differential expression an empirical Bayes method is used to moderate the standard errors. The use of moderated statistics for the detection of differential expression is especially useful in cases of experiments with a small number of replicates. Alternative splicing events (ASEs) were detected as described in [START_REF] Beffa | Dissecting an alternative splicing analysis workflow for GeneChip Exon 1.0 ST Affymetrix arrays[END_REF]. Briefly, an intensity filter was applied at gene-level to remove not expressed and low expressed genes, i.e. genes were retained for exon-level analysis if in all biological replication gene-level signal was greater than 5. Subsequently, only genes characterized to have at least two RNA isoforms annotated in Ensembl database (Ensembl 56 release, September 2009, [START_REF] Flicek | Ensembl 2008[END_REF]) were retained for further analysis. The Splicing Index value was calculated by taking the log 2 ratio of the normalized exon intensity (NI) in Sample 1 and the NI in Sample 2. The normalized exon intensity (NI) is the ratio of the probe set intensity to the gene intensity.

The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] and are accessible through GEO Series accession number GSE21450 embedding dataset SOD1 (GSE21298) and dataset PQ (GSE21305).

Quantitative RT-PCR assays

For the experiments performed on cultured cells, the relative mRNA levels of selected transcripts were determined by qPCR on three biological replicates and a pool of the

Immunoblotting

Cell monolayers were washed twice with ice cold PBS and lysed on the tissue culture dish by addition of ice-cold lysis buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, and 1% NP-40, and complete protease inhibitor cocktail (Roche Diagnostics). The samples were then centrifuged at 15,000 rcf for 15 min at 4°C, and the supernatants were collected. An aliquot of the cell lysate was used for protein analysis with the Bio-Rad Bradford kit for protein quantification. Proteins were subjected to SDS-PAGE and then transferred onto nitrocellulose membrane (Thermo Fisher Scientific).

The membranes was blocked for 1 h with 5% non-fat dry milk in PBS-buffered saline containing 1% Tween 20 (PBS-T). Membranes were immunoblotted with antibodies of interest for 1 h. Anti-cyclin A , and anti-cyclin B, were from BD Biosciences, (Pharmingen). The anti-NTRK1 (TrkA) antibody was from Abcam. The detection was carried out by incubation with horseradish peroxidase conjugated sheep anti-mouse IgG (Amersham) for 1 h. The blots were then washed extensively and the proteins visualized using an enhanced chemiluminescent detection system (Amersham).

Animals

Transgenic mice expressing the human SOD1 gene with the G93A mutation (strain B6.Cg-Tg(Sod1-G93A)1Gur=J, [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF] were purchased from the when the mice showed the first impairment in grip strength. The symptomatic phase stage of disease was considered when the mice showed a 10% weight loss that was usually accompanied with the first impairment in Rotarod performance. All animal studies were conducted in accordance with standard ethical guidelines

Results

The fact that neurons are highly dependent on the oxidative energy metabolism has suggested a common pathogenic mechanism for neurodegeneration based on an underlying dysfunction in mitochondrial activity. To investigate the cellular response to mitochondrial insufficiency both at the level of gene expression and at the pre-mRNA splicing level we performed a whole-genome, splicing-sensitive microarray analysis of two experimental paradigms. First, we profiled untreated human neuroblastoma SH-SY5Y cells vs. cells treated with PQ. PQ is a neurotoxic herbicide that belongs to the class of redox cycling compounds capable of inducing mitochondrial damage, increasing reactive oxygen species (ROS) production and oxidative stress (Birney, et al., 2007;Castello, et al., 2007). For this experiment, the most effective PQ concentration was determined by concentration-response time course studies using an MTT assay to monitor mitochondrial activity, and RT-PCR to verify alternative splicing changes of endogenous transcripts (see Supporting Results).

Second, we analyzed SH-SY5Y cells stably expressing wild type SOD1 (WT-SOD1)

vs. cells expressing a pathogenic mutation (G93A-SOD1, [START_REF] Carri | Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells[END_REF]. We have previously demonstrated that this mutant SOD1 induces mitochondrial defects such as depolarization, impaired respiratory activity and ATP production, altered 13 calcium buffering and cytochrome c release in this and other neuronal cell lines [START_REF] Cozzolino | Oligomerization of mutant SOD1 in mitochondria of motoneuronal cells drives mitochondrial damage and cell toxicity[END_REF][START_REF] Ferri | Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials[END_REF][START_REF] Jaiswal | Cu/Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of mitochondria and perturbs Ca2+ homeostasis in SOD1G93A mice[END_REF]. Mitochondrial damage has been repeatedly reported also in transgenic mice overexpressing Sod1G93A [START_REF] Damiano | Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice[END_REF][START_REF] Jaiswal | Cu/Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of mitochondria and perturbs Ca2+ homeostasis in SOD1G93A mice[END_REF][START_REF] Mattiazzi | Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice[END_REF]Nguyen, et al., 2009). In this experiment, WT-SOD1 cells were used as a control to rule out unspecific effects due to the transfection and/or to an increased level of SOD1 activity.

After having assessed their quality (see Supporting Material), biologic quintuplicate RNA samples prepared from each experimental paradigm were hybridized to Human Exon 1.0 ST Arrays (Affymetrix), which allow the definition of both transcription patterns (gene-level analysis) and alternative pre-mRNA maturation events (exonlevel analysis). Probesets on the Exon Array are classified as Core, Extended, or Full according to the reliability of the annotation used to define the putative genomic regions of interest. All analyses reported here utilized only the highest confidence "core" probesets, which are based on RefSeq and GenBank full-length mRNAs.

Probesets are grouped into "transcript clusters" corresponding to all possible isoforms transcribed from a single locus or gene; therefore, for simplicity we refer to transcript clusters as genes throughout these results. As illustrated in Figure 1A, gene-level and exon-level data sets were generated for each experiment. Hierarchical clustering showed good segregation of treated samples from control samples (PQ vs. untreated and G93A-SOD1 vs. WT-SOD1, respectively (Figure 1B). Sample group homogeneity was also confirmed by Principal Component Analysis (not shown)

Differential expression was detected by linear model statistics (see Materials and Methods, [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF]). Exon-level calculation was done by RMA (Irizarry, et al., 2003) and normalization by quantile sketch [START_REF] Bolstad | A comparison of normalization methods for high density oligonucleotide array data based on variance and bias[END_REF]. Also in this case F o r P e e r R e v i e w 14 hierarchical cluster analysis showed a good correlation between the two experimental groups (Figure 1C).

Gene-level expression profiling reveals extensive transcriptional changes upon mitochondrial dysfunction

We first analyzed gene expression changes in PQ-treated cells. To identify differentially expressed genes we applied, as cut off an absolute log 2 -fold change ≥ ±1 and a p-value ≤ 0.05. Among the 790 genes that were differentially expressed in the treated samples compared with the controls, 403 were upregulated and 387 were downregulated (Supporting Material, Supp. Table S1 "Paraquat gene-level analysis").

The selected genes were analyzed for their molecular and cellular functions and involved pathways using the Ingenuity Pathways Analysis software (IPA7.0, Ingenuity Systems®). The analysis identified Cellular Growth and Proliferation, Cell Death, and Cell Cycle, as the top three categories among the known affected biological functions (Table 1). Consistent with the idea that oxidative stress is a critical mechanism in PQ-induced neurotoxicity (Castello, et al., 2007), IPA 7.0 analysis identified 29 genes that are associated with oxidative stress (Table 2).

However, the dominant pathway affected by PQ treatment was p53 signalling. The activation of the p53-mediated response is indicated by the upregulation of ATF3, BTG2, CDKN1A, GADD45A/B/G, MDM2, PPM1D, SESN1/2, TP53INP1, and by POLH (for gene name abbreviations see Supp. Table S8).

Next, we carried out whole-genome profiling of SH-SY5Y cells stably transfected with cDNAs coding for WT-SOD1 or the mutant G93A-SOD1 protein. Among the 192 genes that were found to be differentially expressed in the G93A-SOD1 samples, 81 were upregulated and 111 were down-regulated (Supp. 3).

In order to identify common deregulated pathways, we then combined those genes from the two data sets, which were characterized by an absolute log 2 -fold change ≥ 0.5 and by an uncorrected p-value ≤ 0.05. This intersection resulted in the detection of 156 genes that were differentially expressed in both conditions (Supp. Table S5 "Common gene-level analysis", and Figure 1D). These genes were used to query the Ingenuity IPA 7.0 knowledge database. The analysis identified Nervous System Development and Function as the top affected biological function (Table 4).

Moreover, 28 genes were found to be associated to neurological disorders, 8 to axonal guidance signalling, 2 to glycosphingolipid biosynthesis, and 3 to the glycerolipid metabolism (Supp. Table S6). Additional genes involved in neuritogenesis were differentially expressed in only one of the two data sets (Supp. Table S6).

Exon-level profiling reveals the expression of alternative mRNA isoforms

Array data were next examined for evidence of differential exon expression, i. e.

alternative pre-mRNA splicing. Differentially expressed exon probe sets, i.e. putative alternative splicing events (ASEs), were detected according to Della [START_REF] Beffa | Dissecting an alternative splicing analysis workflow for GeneChip Exon 1.0 ST Affymetrix arrays[END_REF]. The integration of the results of MiDAS [START_REF] Gardina | Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array[END_REF] and Rank Product [START_REF] Breitling | Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments[END_REF] characterized by a p ≤ 0.05 lead to the identification of 816 putative ASEs, associated to 521 genes (Supp. Table S3 "PQ-ASEs"). Analysis with IPA 7.0 identified Cellular Growth and Proliferation, Cell Death, and Cell Cycle as the most affected cellular and molecular functions (Table 5).

A similar analysis was performed for the G93A-SOD1 experiment. A total of 406 putative ASEs, associated to 242 genes were eventually identified (Supporting Material, Table S4 "SOD1 ASEs"). In this case, pathway analysis detected Calcium Signaling, cAMP-mediated Signaling, and G-Protein Coupled Receptor Signaling as the most affected top canonical pathways (Table 6). Therefore from the in silico analysis of the alternative splicing data, the two experimental paradigms appear to differentially affect pre-mRNA splicing. Indeed, the integration of the two exon-level datasets led to the identification of only 48 common ASEs associated to 40 genes (Supp. Table S7, "Common ASEs"). Interestingly, 10 of these common genes function in Cellular Movement. Finally, we examined the position of the alternatively spliced exons with respect to their position in RefSeq transcripts. We classified exons as initial, internal or last. According to this classification 14 events affected initial exons, 3 occurred in terminal exons, and 24 fell in internal exons. Seven events could either not be assigned or occurred in intronic positions. Interestingly, 9 events (18,7%) reflected the possible use of alternative promoters. This observation is consistent with the result of the analyses of the ENCODE regions (Birney, et al., 2007), which identified >20% of genes having functional alternative promoters [START_REF] Cooper | Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome[END_REF]. Finally, 18 out of 40 common AS genes are also differentially This result is consistent with previous reports that suggest that regulation of AS of cassette exons and regulation of transcription appear to act on separate genes [START_REF] Pan | Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform[END_REF].

Experimental validation of the gene-level analysis of the microarray data confirmed altered expression of axon guidance genes

We first performed the experimental validation of the gene expression data obtained from the PQ experiment. Different cellular and biochemical approaches confirmed the soundness of the microarray data and the biological significance of the pathways identified with IPA 7.0 (see Supporting Material "Results" and Supp. Figure S1). For the validation of the gene-level data obtained from the intersection of the two experimental paradigms we chose twelve of the 156 differentially expressed genes that showed a common trend upon PQ treatment and in G93A-SOD1 cells. Genes were prioritized by fold change variation and functional classification. Specifically, we focused on genes encoding molecules involved in the Nervous System Development and Function category. Expression of these genes was analyzed by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR). As shown in Figure 2, qRT-PCR data were in good agreement to the microarray results for all genes except for VSNL1 and ADAMTS1.

As mentioned above, pathway analysis of the genes that showed a common trend in the two experimental paradigms identified eight genes involved in axon guidance signalling (Supp. 18 signalling, etc.) were also found differentially expressed in both data sets. To identify molecular interactions among these genes we analyzed them further by IPA 7.0. The Ingenuity program can convert data sets into networks containing direct and indirect relationships between genes based on known interactions in the literature. The software is capable not only of constructing associations of genes identified by microarray (including relative expression levels), but also of predicting involvement of additional molecules not associated with significant transcriptional changes. We thus compiled a list of genes related to neuritogenesis from which the Ingenuity software created a network, on which our transcriptomics data were overlaid. In this network the 24 genes that were differentially expressed in our data sets were connected to 16 additional genes (Figure 3A). The network contained several transcription factors that are interconnected and involved in neuronal commitment and differentiation (ASCL1, ERBB3/4, NEUROG3, NKX2.2, and PAX6). Of these genes, NKX2.2, and PAX6 have been shown to play crucial roles in controlling the timing of neuritogenesis and gliogenesis in the developing ventral spinal cord modulating the activities of the proneural factors NEUROG1/2/3, and ASCL1 [START_REF] Sugimori | Combinatorial actions of patterning and HLH transcription factors in the spinal cord[END_REF].

Interestingly, ASCL1 is strongly downregulated both in PQ-treated and in G93A-SOD1 cells. In addition, the network connected genes encoding cytoplasmic proteins that may regulate actin dynamics such as WASF3 and EVL.

Hierarchical cluster analyses of the expression data of the subset of genes involved in neuritogenesis identified the majority of them as being downregulated (Figure 3B).

qPCR analysis of a subset of these genes confirmed the array data (Figure 3C). We also tested by Western blotting the level of SEMA3A in the supernatant of PQ-treated and G93A-SOD1 cells as well as the expression of NTRK1. As shown in Figure 3D and 3E, these experiments confirmed the increased expression of the repulsive

Altered expression of genes involved in neuritogenesis in G93A-Sod1 mice

To determine whether changes in the expression of genes involved in axon growth and guidance could be observed in an animal model of neurodegeneration, we investigated the expression of a subset of the identified genes in the G93A-Sod1 mouse model of ALS. Disease onset in our strain is around 120 days [START_REF] Pizzasegola | Treatment with lithium carbonate does not improve disease progression in two different strains of SOD1 mutant mice[END_REF]. Thirteen of the differentially expressed genes involved in neuritogenesis were analyzed by RT-qPCR on RNA extracted from spinal cord and brain of four transgenic mice carrying the G93A-Sod1 mutation and three normal control

Experimental validation of alternative splicing events

Next, we went on to confirm the putative ASEs identified by the exon-level analysis.

We chose ASEs affecting 15 transcripts involved in the top canonical pathways identified with IPA 7.0. In particular, we focused on genes involved in Nervous System Development and Function, which appeared to be affected by AS changes in either one, or both experimental paradigms. The location of the differentially expressed exon-probe set was manually inspected with the X:map software [START_REF] Okoniewski | An annotation infrastructure for the analysis and interpretation of Affymetrix exon array data[END_REF][START_REF] Yates | X:Map: annotation and visualization of genome structure for Affymetrix exon array analysis[END_REF] to determine its position on the transcripts annotated in Ensembl. Only ASEs occurring in exons for which there is at least one annotated alternative isoform were subjected to experimental validation by RT-PCR. All the amplified fragments were subcloned and sequenced to verify the identity of the splicing isoforms.

The Human Exon 1.0 ST array can identify mRNA variants that differ in the use of exons, introns, promoters and polyadenylation sites. In the case of the RPRD1A gene the microarray data predicted an increased inclusion of exon 8 (identified by the probe set n. 3874170, boxed in Figure 5A). In the Ensembl database there are four transcripts annotated for this gene, one of which contains exon 8. To validate this ASE, RT-PCR analysis was performed with two forward primers (in exons 8 and 9) and a single reverse primer in exon 5 (Figure 5B). As shown in Figure 5C, RT-PCR confirmed the expected inclusion of exon 8 in G93A-SOD1 cells.

Two potential ASE were also predicted to occur in the BIN1 gene. Specifically, microarray data predicted inclusion of exon 7 and skipping of exon 15 in PQ-treated cells. In G93A-SOD1 cells the array data predicted inclusion of exon 15. To validate these ASEs, we amplified by PCR the relevant region of each mRNA using primers in fragments confirmed the inclusion of exon 7. Unfortunately, we could not detect the event affecting exon 15. Instead, we observed a splicing switch affecting exons 14 and 16, which was not predicted by the array. The presence of some discrepancies between microarray data and RT-PCR is due to the limited statistical power of an exon-level analysis. We tried to address this issue by using 5 biological replicates for each microarray experiment and by reducing the number of tested hypotheses, focusing the analysis only on expressed exons associated to annotated mRNA isoforms. Nevertheless, the multiple testing problem remains a critical issue in the exon-level analysis of array data [START_REF] Beffa | Dissecting an alternative splicing analysis workflow for GeneChip Exon 1.0 ST Affymetrix arrays[END_REF]. Additional ASEs in internal exons were confirmed for GNAO1 (Figure 4G) and for NRG1 (data not shown).

Among the probe sets exhibiting changes in expression, several mapped to the 5' end of the transcripts and were considered to be candidate alternative first exons. We considered the case of the ABLIM1 gene for which several different alternatively spliced transcripts have been identified, some of them most likely transcribed from alternative promoters (Figure 6A). The probe set intensities along the entire ABLIM1 gene are shown in Figure 6B. The exon array data predicted the skipping of an internal first exon in both experimental paradigms exon (boxed in Fig. 6B, see also the schematic representations in panels 6A and 6C). To validate this event we performed a semiquantitative PCR with two forward primers and a single reverse primer. This type of competitive PCR reactions have been used previously to detect variations in expression of 5'-and 3'-terminal exons [START_REF] Karni | The gene encoding the splicing factor SF2/ASF is a proto-oncogene[END_REF][START_REF] Yamamoto | Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis[END_REF]. As shown in Figure 6C, both upon PQ treatment and in G93A-SOD1 cells the amount of the PCR fragment corresponding to the mRNA transcribed from Three additional examples of alternative first exon usage that were similarly predicted by the exon array data were verified by RT-PCR (CHN1, LMO3, and NRG1, Figure 6D). In the case of the CHN1 gene, the skipping of an alternative first exon upon PQ treatment and its inclusion in G93A-SOD1 cells was confirmed by qPCR (Figure 6E)

The switches in first exon usage for ABLIM1, CHN1, and NRG1 strongly influence the coding potential of the different mRNA isoforms (Figure 6F). In the case of the ABLIM1 gene, we observed an increase of the longer transcript variant, which encodes a protein that contains four LIM domains. These domains are not present in the isoform encoded by the mRNA variant transcribed from the internal first exon.

Similarly, the short CHN1 isoform that is differentially expressed upon PQ treatment and in SOD1(G93A) cells lacks an SH2 domain. In contrast, the switch of first exons in the LMO3 gene results in different 5' UTR sequences for the different transcripts.

Discussion

In this study we sought to contribute to the understanding of the link between mitochondrial stress and neurodegeneration. To this end we performed a wholegenome analysis using splicing-sensitive microarrays of two different cellular paradigms of mitochondrial dysfunction. First, we profiled human SH-SY5Y neuroblastoma cells untreated or treated with PQ, a redox cycling molecule that induces mitochondrial damage. Second, we compared SH-SY5Y cells stably expressing wild type SOD1 to cells expressing the mutant G93A-SOD1 protein.

Mutations in SOD1 are one of the genetic causes of ALS. (Shi, et al.). The data sets from the two experimental paradigms were then integrated in order to identify common deregulated genes.

Our findings indicate that mitochondrial damage can induce profound changes in the expression of genes involved in relevant pathways for neuronal survival, both at the gene-level and in pre-mRNA splicing. In addition to previously described changes in the expression of cytoskeletal and molecular motor proteins ("Cell Movement"), we identified alterations of a subset of genes involved in Nervous System Development and Function, including axon growth and guidance genes, and genes encoding proteins involved in synaptic vesicles formation. Changes in the expression of a subset of the identified genes were confirmed in the G93A-Sod1 mouse, a model for ALS, supporting the idea that alterations in the expression of this category of genes may contribute to explain the vulnerability of synapses and distal axons observed in many neurodegenerative diseases (for review see [START_REF] Conforti | Neuronal death: where does the end begin?[END_REF].

Cell Movement

Recent studies have shown that neurons in general, and even more so motor neurons, are highly sensitive to defects in axonal transport due to their extreme polarization.

Decreased kinesin-mediated (anterograde) and dynein-mediated (retrograde) axonal transport have been observed both in ALS patients and in transgenic animal models (for review see [START_REF] Vos | Role of axonal transport in neurodegenerative diseases[END_REF][START_REF] Strom | Retrograde axonal transport and motor neuron disease[END_REF]). In G93A-Sod1 transgenic mice, a considerable inhibition of retrograde axonal transport was observed at a very early stage of disease before animals became symptomatic [START_REF] Jiang | Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis[END_REF]. We observed downregulation of several members of the kinesin family in both experimental paradigms. In the G93A-SOD1 experiment is particularly noteworthy KIF1A had already been reported in 75-days old G86R-Sod1 mice [START_REF] Dupuis | Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons[END_REF]. The phenotype of Kif1A knockout mice includes motor and sensory disturbances, a reduction in the density of synaptic vesicles in nerve terminals, and accumulation of clear vesicles in nerve cell bodies [START_REF] Yonekawa | Defect in synaptic vesicle precursor transport and[END_REF]. KIF1A cargos include a subset of precursors for synaptic vesicles: synaptophysin, synaptotagmin, and Rab3A. In this context it should be mentioned that we also observed altered expression of several genes involved in synaptic function including three members of the synaptotagmin family (SYT2, SYT4, and SYT12, which encode integral membrane proteins of synaptic vesicles), synaptojanin-2 (SYNJ2, coding for a phosphoinositide phosphatase implicated in cytoskeletal dynamics during endocytosis) as well as genes encoding components of the presynaptic active zone (RIM1, and RIM3). Consistent with these data, pre-degenerative depletion of synaptic vesicles has been reported in ALS [START_REF] Pun | Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF[END_REF].

In G93A-SOD1 cells, we also observed downregulation myosin X (MYO10) coding for another molecular motor, while PQ treatment induced upregulation of MYLIP (myosin regulatory light chain interacting protein). MYLIP is expressed in the cell bodies and growth cones of rat embryonic hippocampal neurons, and belongs to a family of cytoskeletal effector proteins that link actin to membrane-bound proteins at the cell surface. Interestingly, in rat PC12 neural precursor cells, MYLIP overexpression inhibited NGF-induced neurite outgrowth [START_REF] Olsson | Neuronal expression of the ERM-like protein MIR in rat brain and its localization to human chromosome 6[END_REF][START_REF] Olsson | MIR is a novel ERM-like protein that interacts with myosin regulatory light chain and inhibits neurite outgrowth[END_REF]. Overall, these data reinforce the concept that alteration of axonal transport is involved in neurodegeneration, and suggest that this alteration may descend directly from mitochondrial damage. As mentioned above, pathway analysis of the genes that were differentially expressed in both experimental paradigms highlighted several genes associated to axon guidance (Supp. Table S6). Axon guidance is a partially understood process that has a major role not only in embryonic development [START_REF] O'donnell | Axon growth and guidance: receptor regulation and signal transduction[END_REF] but in the maintenance of the mature peripheral nervous system as well [START_REF] Yoo | Dynamics of axonal mRNA transport and implications for peripheral nerve regeneration[END_REF].

Several lines of evidence support the hypothesis that aberrant expression of axon guidance proteins such as Semaphorins, Ephrins, Netrins and Slits, may induce pathological changes in motor neurons and contribute to the pathogenesis of ALS ( [START_REF] Schmidt | Axon guidance proteins: novel therapeutic targets for ALS?[END_REF]. Both in PQ-treated cells as well as in G93A-SOD1cells we observed downregulation of SEMA6A and of its receptor PLXNA4. Moreover, SEMA3D is among the top upregulated genes in both datasets. This semaphorin is expressed at the dorsal spinal cord midline and appears to guide axons by repulsion and modulation of fasciculation [START_REF] Wolman | Repulsion and attraction of axons by semaphorin3D are mediated by different neuropilins in vivo[END_REF].

Additional genes coding for axon guidance molecules were differentially expressed in either one of the two experimental paradigms. For example, in G93A-SOD1 cells we observed upregulation of neuropilin 2 (NRP2), coding for a selective receptor for class 3 semaphorins that is required for the development of central nervous system and peripheral nervous system projections [START_REF] Giger | Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins[END_REF], and of SEMA3A, a chemorepellant for cortical axons and an inhibitor of axonal regeneration and other regenerative responses after spinal cord injury [START_REF] Kaneko | A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord[END_REF].

Paraquat treatment induced downregulation of SEMA3F, and SEMA6D. Interestingly, an association between SEMA6A polymorphisms and ALS has previously been reported [START_REF] Lesnick | Beyond Parkinson disease: amyotrophic lateral sclerosis and the axon guidance pathway[END_REF]. Also noteworthy is the reduction in NTRK1 expression, a neurotrophin receptor that is thought to play a role in axon guidance and cells. Some axon guidance molecules bind to HSPGs, and this modulates their repulsive properties [START_REF] Jokic | The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model[END_REF]. Thus, the downregulation of SDC2 might suggest a shift to a more repulsive character of the extracellular matrix. This effect is possibly aggravated by the upregulation of SEMA3D and the downregulation of

SEMA6A.

In both experimental paradigms we observed altered expression not only of classical members of the ligand/receptor axon guidance systems but also of genes involved in a broader sense in neuritogenesis, such as genes encoding downstream intracellular signal transduction molecules and upstream transcription factors that control axonal patterning (for review see O 'Donnell, et al., 2009;[START_REF] Polleux | Transcriptional regulation of vertebrate axon guidance and synapse formation[END_REF]. For example, in G93A-SOD1 cells we observed upregulation of RGNEF (Rho-guanine nucleotide exchange factor). RGNEF is a human homologue of mouse p190RhoGEF, a low molecular weight neurofilament (NFL) mRNA stability factor that was shown to be involved in NF aggregate formation in neurons [START_REF] Lin | RNA-binding protein is involved in aggregation of light neurofilament protein and is implicated in the pathogenesis of motor neuron degeneration[END_REF][START_REF] Volkening | Human low molecular weight neurofilament (NFL) mRNA interacts with a predicted p190RhoGEF homologue (RGNEF) in humans[END_REF]. In PQ-treated cells we found the downregulation of three genes (ISL1, LHX8, and LHX9) that belong to the family of LIM homeobox transcription factors. It has been shown that in the developing mouse spinal cord Isl1 and Lhx1 control the dorsoventral specificity of motor axon projections at least in part by controlling the expression of EphA4 [START_REF] Kania | Coordinate roles for LIM homeobox genes in directing the dorsoventral trajectory of motor axons in the vertebrate limb[END_REF]. Interestingly, LHX9 is also downregulated in G93A-SOD1 cells.

A validation of the microarray data in G93A-Sod1 mice confirmed the alterations in the expression of neuritogenesis genes during disease progression. For example, we which has neurotrophic effects in the motor neurons and central nervous system, was found to be upregulated in motor neurons of sporadic ALS patients [START_REF] Jiang | Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis[END_REF]. In addition we found increased expression of Cxcr4 from pre-symptomatic to end-stage. CXCR4 and its ligand SDF1 regulate neural development by modulating cell migration and axon growth and guidance and by promoting cell survival and proliferation [START_REF] Chalasani | A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding[END_REF][START_REF] Stumm | CXC chemokine receptor 4 regulates neuronal migration and axonal pathfinding in the developing nervous system: implications for neuronal regeneration in the adult brain[END_REF][START_REF] Zou | Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development[END_REF] .

Taken together, our data indicate that mitochondrial stress can induce transcriptional alterations of multiple genes involved in neuritogenesis, and are in agreement with the observation that dorsal root ganglia neurons from adult mice expressing G93A-Sod1 in culture grow shorter neurites [START_REF] Perlson | A switch in retrograde signaling from survival to stress in rapid-onset neurodegeneration[END_REF]. The idea that differential expression of genes relating to the growth of neuronal processes, and formation of synapses may play a role in neurodegeneration is supported by a recent micrroarray analysis of spinal motor neurons of Vegf ∂/∂ mice, a different ALS mouse model (Brockington, et al.). Since it has been observed that in neuromuscular diseases such as ALS selective synaptic weakening and denervation start long before the actual motor neuron loss, and prior to the appearance of clinical symptoms [START_REF] Fischer | Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man[END_REF], the expression of repulsive signals could act during the presymptomatic stages and contribute to the retraction of axon terminals from the neuromuscular synapse before the death of motor neurons occurs.

Changes in alternative pre-mRNA splicing

RNA-based mechanisms have been implicated in several neurodegenerative conditions leading to the recognition of new pathogenic pathways (for review see [START_REF] Licatalosi | Splicing regulation in neurologic disease[END_REF][START_REF] Strong | The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS)[END_REF]. In ALS, numerous reports have 28 highlighted differential expression of protein isoforms, which arise from AS events in the affected tissues of patients, as well as in the mouse models [START_REF] Aerbajinai | Increased expression level of the splicing variant of SIP1 in motor neuron diseases[END_REF][START_REF] Munch | Alternative splicing of the 5'sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis[END_REF][START_REF] Pantelidou | Differential expression of molecular motors in the motor cortex of sporadic ALS[END_REF][START_REF] Robertson | A neurotoxic peripherin splice variant in a mouse model of ALS[END_REF][START_REF] Tomiyama | Flip and flop splice variants of AMPA receptor subunits in the spinal cord of amyotrophic lateral sclerosis[END_REF]. RNA processing has been further implicated in ALS pathogenesis by the recent characterization of mutations in two DNA/RNA-binding proteins (TDP43 and FUS/TLS) in familial and sporadic patients (for review see [START_REF] Lagier-Tourenne | Rethinking ALS: the FUS about TDP-43[END_REF][START_REF] Strong | The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS)[END_REF].

Exon-level analysis of our microarray data detected a widespread alteration in pre-mRNA splicing. We selected for experimental validation events affecting genes involved in the pathways mentioned above (i.e. Cell Movement and Nervous System Development and Function). For example, the ABLIM1 gene encodes a cytoskeletal protein that binds to actin filaments. The C. elegans homolog UNC-115 was shown to act as a downstream cytoskeletal effector of Rac signaling in axon pathfinding [START_REF] Gitai | The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM[END_REF][START_REF] Struckhoff | The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C. elegans[END_REF]. Our data indicate that alternative promoter usage modulates the ration between mRNAs encoding the full-length protein or a shorter isoform lacking the LIM zinc-finger domain, which is thought to mediate protein:protein interactions. A second example is provided by CHN1. This gene codes for two different isoforms of a signaling protein implicated in the pathfinding of corticospinal axons in mice brain [START_REF] Brown | Alpha2-chimaerin, cyclin-dependent Kinase 5/p35, and its target collapsin response mediator protein-2 are essential components in semaphorin 3A-induced growth-cone collapse[END_REF]. Upon PQ treatment we observed a decrease of the mRNA encoding the short isoform, and therefore a relative increase in the expression of the long isoform. The two protein isoforms share a RacGAP domain that interacts with and down-regulates Rac activity.

However, the long isoform (alpha2-chimerin) contains an N-terminal SH2 domain that is not present in the short isoform, transcribed from an internal promoter. [START_REF] Shi | Alpha2chimaerin interacts with EphA4 and regulates EphA4-dependent growth cone collapse[END_REF]. Finally, we observed changes in the alternative splicing pattern of BIN1. This was especially intriguing given the known interactions of BIN1 with dynamin in pre-synaptic membranes of the nervous system and its role in endocytic recycling [START_REF] Leprince | Sorting nexin 4 and amphiphysin 2, a new partnership 48[END_REF][START_REF] Nicot | Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy[END_REF][START_REF] Pant | AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling[END_REF][START_REF] Takei | Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis[END_REF]. These findings suggest that mitochondrial dysfunction may have a severe effect not only on transcription but also on pre-mRNA splicing of a subset of genes that participate in biological processes that are relevant for neurodegeneration.

But how do these splicing changes arise? Expression of neuron-specific proteins involved in RNA splicing and metabolism is affected in several neurological disorders [START_REF] Licatalosi | Splicing regulation in neurologic disease[END_REF]. Surprisingly, in our experiments we did not detect any differentially expressed gene encoding a splicing regulatory factor. The only gene coding for an RNA-binding protein, which is differentially expressed in both paradigms, is ELAVL2 (Supp. Table S5),one of the mammalian homologs of ELAV, a protein essential for neurogenesis in Drosophila [START_REF] Robinow | The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs[END_REF]. ELAVL2

encodes the neuronal HuB protein that binds and stabilizes t a r g e t m R N As containing A U -rich e l e m e n t s ( A R E ) i n t h e 3' U T R (for review see M. N. Hinmana and H. Lou, 2008) . Instead, among the common ASEs (Supp. Table S7) we found CUGBP2 , a well-known alternative splicing factor (for review see [START_REF] Barreau | Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions[END_REF]. CUG-BP2 is particularly relevant in the context of neurodegeneration since it has a specific neuromuscular expression and is differentially expressed in several models of SMA [START_REF] Anderson | Expression profiling in spinal muscular atrophy reveals an RNA binding protein deficit[END_REF]. The putative ASE affecting the CUGBP2 transcript occurs in the 3' UTR and may thus influence either intracellular localization or stability of the mRNA. This in turn may affect splicing, stability and/or translation of specific transcripts. The mRNA variant containing the first alternative exon is reduced at least 2fold upon PQ treatment.

F. Graphical view of the PROSITE (Swiss Institute of Bioinformatics, [START_REF] Gasteiger | Mitochondrial dysfunction initiates and propagates neuronal dysfunction, neuronal death and behavioral abnormalities in age-associated neurodegenerative diseases[END_REF]

PQ treatment inhibits cell proliferation and modifies alternative pre-mRNA splicing in SH-SY5Y neuroblastoma cells

PQ is a neurotoxic herbicide that induces Parkinsonian features in animal models (for review see [START_REF] Cicchetti | Environmental toxins and Parkinson's disease: what have we learned from pesticide-induced animal models?[END_REF].). The chemical structure of PQ is similar to the known dopaminergic neurotoxin, N-methyl-4-phenylpridinium ion (MPP+), the active metabolite of MPTP. Thus, it belongs to the class of redox cycling compounds capable of inducing mitochondrial damage, increasing reactive oxygen species (ROS) production and oxidative stress (Birney, et al., 2007;Castello, et al., 2007).

Concentration-response time course studies were conducted to determine an effective range for PQ upon SH-SY5Y cell proliferation. We used the MTT assay, which is mostly based on the activity of a mitochondrial dehydrogenase and therefore is largely used as a measure of mitochondrial activity. Based on the results shown in Figure S1A, we decided to perform subsequent experiments with cells exposed to 0.75 mM PQ for 18 h.

We next evaluated alternative splicing changes in these conditions. We assayed the AS pattern of APAF1, BCL2L1 (BCL-X), CASP2, and CASP9 (Supp.Figure S1B) that we had described in [START_REF] Maracchioni | Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration[END_REF]. Consistent with our previous data, even a treatment with lower PQ concentration and for a shorter incubation time induced a change in the alternative splicing pattern of APAF1, CASP9, and SMN1 but not of BCL2L1 (Supp. Figure S1C).

Experimental validation of the differential expression data for the PQ experiment

Pathway analysis of microarray data identified Cellular Growth and Proliferation, Cell Death, and Cell Cycle as the biological functions that are most affected by PQ. 2 Thus, we first characterized the cell cycle distribution of PQ-treated cells. Flowcytometric analyses of sub-confluent, treated cells showed an increase in the G1-and a decrease in the S-phase populations when compared to untreated control cells (Figure S1A). This observation was consistent with the downregulation of cyclin A2 (CCNA2) and cyclin E2 (CCNE2) required for S/G2 progression, and of cyclin B (CCNB1), required for G2/M transition, as well as with the upregulation of CDKN3, a CDK2-phosphatase, and of p21 (CDKN1B), as detected by microarray analysis (Supp.

Table S1). Western blot analysis confirmed the downregulation of cyclin A and cyclin B (Supp. Figure S1B). In addition, in treated cells we detected the presence of a subG1 population indicating that some cell death occurred upon incubation with PQ (data not shown). The induction of apoptosis was confirmed by Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays (Supp. Figure S1C). While a very low population was found in region 2 in the untreated control samples, treated cells exhibited a TUNEL-positive fraction of ~20 %. Induction of apoptosis in a subset of the cells was further supported by the proteolytic cleavage of PARP a known caspase 1 substrate, observed by Western blotting (Supp. Figure S1D).

Material and Methods

Assessment of cell viability

Cell viability was assessed with the MTT colorimetric assay. The assay is based on the determination of the mitochondrial dehydrogenase activity that reduces 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma-Aldrich). At day 0, SH-SY5Y cells were plated at a density of 5 x 10 4 viable cells per well in 96well plates. The next day, cells were incubated for 24 h at 37°C in the presence of Cytotoxicity was evaluated as percentage of the control untreated with PQ.

Cell cycle analysis

SH-SY5Y neuroblastoma cells were seeded at 1,2x106 cells per well in 60mm dish, and the day after were treated with 0,75mM of PQ for 18 hours. Then untreated and treated cells, both attached and detached populations, were collected and fixed in 70% ethanol in PBS and kept at 4°C before staining. Fixed cells were then washed with cold PBS and resuspended in 1 mL of a solution containing 10 ug/ml of propidium iodide (PI) in PBS and 7 µl RNAse 3 mg/ml in water, and stained overnight at 4°C in the dark. DNA analysis was done on at least 30000 cells for each sample by the FACSCalibur (BD Biosciences -CA USA) and the data were analyzed using ModFIT LTsoftware (Verity Software House -ME USA). Experiment was performed in triplicate.

TUNEL assay

Apoptosis was measured using the TUNEL assay kit (Roche Diagnostics, Basel, Switzerland) following the manufacturers' instruction for dual parameter flow cytometry. Analysis was performed on a FACSCalibur (BD Biosciences -CA USA)

and the data were analyzed using Summit 4.3 software (Coulter -CA USA). and anti-LaminB (Santa Cruz).

F

For the analysis of cyclin expression cell monolayers were washed twice with ice cold PBS and lysed on the tissue culture dish by addition of ice-cold lysis buffer (50mM

Tris-HCl, pH 7.5, 150mM NaCl, and 1% NP-40, and complete protease inhibitor cocktail (Roche Diagnostics). Anti-cyclin A, and anti-cyclin B, were from BD Biosciences, (Pharmingen).

Quality control for RNA and Arrays

RNA quantity was assessed by Ribogreen (Invitrogen). Total RNA integrity was assessed using a Bioanalyzer (Agilent ) and the RNA Integrity Number (RIN) was calculated. RIN values ranged from 8.7 to 10 with a media of 9.6

For target preparation 1.5 µg of total RNA and the GeneChip WT cDNA Amplification kit (Affymetrix) were used. Amounts of ssDNA obtained ranged from 7 to 10 µg with a media of 8.8 µg. ssDNA before and following fragmentation was analysed using a Bioanalyzer (Agilent). Typical ssDNA fragments have a size range from 40-70 nt. The "negative control", "positive control" and "all probe set" metrics are useful in understanding the overall quality of the data from each chip. Metrics based on these categories will reflect the quality of the whole experiment (RNA, target prep, chip, hybridization, scanning, and gridding) and the nature of the data being used in downstream statistical analysis. "All probe set" quality control metric: red line. It represents the intensity signal of all the probe sets analyzed. The quality metrics reported for this category are going to be driven by the main source of content in that particular analysis. Thus the metrics reported for this category will be the most representative of the quality of the data being used downstream.

For a good set of arrays, this intensity value does not have to vary.

"Neg control" quality control metric: blue line. Pos control" quality control metric: green line.

  paraquat (N,N'-dimethyl-4,4'-bipyridinium dichloride, PQ), a neurotoxic herbicide that induces Parkinsonian features in animal models, has

  growth and guidance molecules as a partner in mitochondriamediated neurodegeneration.

  total RNA was isolated from brain and spinal cord with TRIzol®

  of semaphorin 3A expression, 10 6 cells were plated on 60 mm Petri dishes. After 24hrs culture in normal growth medium, cells were shifted in 3ml OPTIMEM (Invitrogen) and cultured for 18hrs. Supernatants were harvested, centrifuged to remove cell debris, and 200ml of cleared supernatants were precipitated with 6 volumes of a 2:1:1 Ethanol/Methanol/Acetone mix and resuspended in Laemmli sample buffer. Equal volumes were subjected to SDS-PAGE and analysed in Western blot with an anti-SEMA3A mouse monoclonal antibody (Santa Cruz Biotechnology Inc.). To normalize the amounts of secreted SEMA3A to the cell number, cells were lysed in RIPA buffer and equivalent amounts of protein extracts were analysed in Western blot with a mouse monoclonal anti-b-actin antibody (Sigma-Aldrich).

  , of these only 50% undergo putive ASEs affecting internal exons.

  downregulation of the neurotrophin receptor NTRK1 in both our models of mitochondrial dysfunction. Taken together these results suggest the formation of an altered, possibly more chemorepulsive cell environment.

  littermates at 150 d. As shown in Figure4A, this study confirmed the differential expression of Hgf, Cxcr4, and Sema3A in the spinal cord of symptomatic G93A-Sod1 mice. Thus, we decided to further characterize the alteration in the expression of eleven genes in the spinal cord of G93A-Sod1 mice during disease progression. We analyzed by RT-qPCR the expression profile of some of the genes identified in the microarray analysis (Cxcr4, Hgf, Ntrk1, Prph1, Sema3A, and Wasf3) and also of additional genes inferred from the literature (Nrp2, and Sept2) at the pre-onset (93, 103, and 113 days), onset (135 days), and symptomatic stage (140 -170 days).Expression of Cxcr4 and Hgf increased significantly over time during disease course (p=0.01 for Cxcr4., and p<0.001 for Hgf, Figure4B), while Wasf3 and Sept2 showed increased expression in spinal cord of G93A-Sod1 mice at early symptomatic stage (Figure4C).

  exons (Figure5, panels D to F). Sequencing of the PCR

  increased relative to the shorter fragment corresponding to the mRNA transcribed from the alternative, internal promoter, thus confirming the microarray prediction.

  KIF1A, an anterograde motor protein. Altered expression of

  with SEMA3A. In addition, we observed reduced expression of SDC2, coding for a member of the heparan sulphate proteoglycan (HSPG) family of extracellular matrix proteins both in PQ-treated and in G93A-SOD1

  increase in the expression of Hgf during disease course. HGF,

Figure 1 .

 1 Figure LegendsFigure 1. Whole genome, splicing-sensitive microarray analysis of the cellular response to mitochondrial dysfunction. A. Diagram of the experimental design. B. Gene-level hierarchical cluster analysis of untreated vs. PQ-samples and WT-SOD1 vs. G93A-SOD1 samples. The two dendrograms (sample level hierarchical clustering with average linkage) represent the relative distance between samples.C. Exon-level hierarchical cluster analysis of untreated vs. PQ-samples and WT-SOD1 vs. G93A-SOD1 samples. D. Venn diagram illustrating the overlap between differentially expressed genes in the PQ-experiment and in the G93A-SOD1 experiment.

Figure 2 .

 2 Figure 2. Experimental validation of the gene-level microarray data in SH-SY5Y cells.qPCR validation of array data from the PQ and G93A-SOD1 experiments. Assays were performed in triplicate from a pool of the RNA samples used for the microarray analysis and from two different RNA preparations.

Figure 3 .

 3 Figure 3. Mitochondrial dysfunction alters expression and splicing of genes involved in axon growth and guidance.A. A gene-interaction network of genes related to axon growth and guidance.Transcriptomics data were overlaid on a genetic network generated with the Ingenuity Pathway Analysis 7.0 software. This diagram is a graphical representation of 24 genes (in green), whose expression is altered in our

Figure 4 .

 4 Figure 4. Analysis of the expression of genes involved in neuritogenesis in the G93A-Sod1 mouse model. A. qPCR analysis of the expression of a subset of axon guidance genes in spinal cord and brain of symptomatic G93A-Sod1 mice. Total RNA was extracted from spinal chord and brain of mice at 150 days of age. Data are presented means±standard error of the mean of three technical replicates of age-matched non-transgenic controls (n=3) and disease (n=4) spinal cord and brain samples. By a 2-tailed t-test Hgf is considered extremely significant (p ≤ 0,0001), Cxcr4 very significant (p = 0.0042), and Sema3A significant (p = 0.0167). B. Time course of Hgf and Cxcr4 mRNA expression in the spinal cord of G93A-Sod1 mice during disease progression. Total RNA was isolated from the spinal cord of G93A-SOD1(closed circles) or age-matched non-transgenic control animals (opened circles) on different days from the pre-onset till the symptomatic stage (93-170 days). Expression was analyzed by RT-qPCR and

Figure 5 .

 5 Figure 5. Experimental validation of the alternative internal exons identified by microarray analysis. A. Identification of differential alternative splicing by exon array probeset-level expression data for the entire RPRD1A gene in the PQ experiment. Plotted are expression levels (y-axis) for each probeset (x-axis). Non-parallel probe set expression levels, highlighted by a box, indicate region-dependent differential splicing of the corresponding exon. Black line untreated; grey line: PQ-treated. B. Exon structure of the human RPRD1A transcripts annotated in the Ensembl database. Indicated are the alternative exon 8 (e8), the position of the PCR primers, and the size of the expected products (359 bp and 968 bp). C. RT-PCR validation of the exon array prediction for the inclusion of exon 8 in

Figure 6 .

 6 Figure 6. Experimental validation of the alternative first exons identified by microarray analysis. A. Exon structure of the human ABLIM1 transcripts annotated in the NCBI database. B. Identification of differential alternative splicing by exon array probe set-level expression data for the entire ABLIM1 gene in the paraquat (left panel) and the G93A-SOD1 (right panel) experiments. Plotted are the average signal intensities (y-axis) for each probe set (x-axis). The box indicates the internal first exon (exon-level probe set id: 3308001, Ensembl id: ENSE00001449337, ENSE00001513698, ENSE00001449334) validated by competitive RT-PCR. Black line untreated or WT-SOD1; red line: PQ-treated or G93A-SOD1. C. RT-PCR validation of the exon array prediction for the ABLIM1 transcript. The indicated splice forms were subcloned and sequenced to verify their identity. Left: analysis of PCR products validating alternative splicing changes. Right: diagrams of the relevant pre-mRNA region. Indicated are: the position of the PCR primers (arrow heads), the splicing pattern, and the size of the expected products. D. RT-PCR validation of the exon array prediction for three additional genes. Common names of the alternatively spliced transcripts are as follows: CHN1, chimerin 1 (GTPase-activating protein for p21-rac and a phorbol ester receptor); LMO3, LIM domain only 3 (neuronal basic helix-loop-helix protein); NRG1, neuregulin 1 (a signaling protein). The indicated fragments were subcloned and sequenced to verify their identity. Left: analysis of PCR

  Experimental validation of the gene-level microarray data in SH-SY5Y cells.180x62mm (600 x 600 DPI)Mitochondrial dysfunction alters expression and splicing of genes involved in axon growth and guidanceExperimental validation of the alternative internal exons identified by microarray analysis. 240x209mm (600 x 600 DPI)Experimental validation of the alternative first exons identified by microarray analysis. 323x269mm (600 x 600 DPI)

  PQ, or for different times in the presence of 0.75mM of PQ. Cells were then exposed to an MTT-containing solution (1 mg/ml in PBS) for 4 h at 37°C and subsequently treated with SDS in dimethylformamide o.n. Absorbance was read at 550 and 655 nm with a Multiscan EX (Thermo Fisher Scientific Inc.).

5

  Hybridization to the Standard array: Fragmented ssDNAs were hybridized to the standard arrays for 17 hours at 45°C; the arrays were then washed and stained using the fluidics station and then scanned using GeneChip Scanner 3000. The images were analyzed using Command Console and comparison analyses were carried out according to the instructions provided by Affymetrix. Standard array quality controls are shown in Supp. FiguresS3-S6

  .

Figure S1 .

 S1 Figure S1. Treatment with paraquat inhibits cell proliferation and modifies alternative pre-mRNA splicing in SH-SY5Y neuroblastoma cells. A. Effect of PQ on cell growth. Dose-(left panel) and time-(right panel) dependent effect of PQ on SH-SY5Y cells. Exponentially dividing cells were treated with increasing concentrations of PQ for 24 h, or with 0,75 mM PQ for

Figure S2 .

 S2 Figure S2. Experimental validation of the gene-level microarray data. A. Analysis of cell cycle phase distribution of SH-SY5Y cells after treatment with 750 µM PQ. Quantifications were performed by flow cytometry after 18 h of treatment on cells stained with propidium iodide. Data are means of three independent experiments; error bars show standard errors. Bars identified by three asterisks have a p value ≤ 0.001 while those labelled with one asterisk have a p value = 0.01 to 0.05. B. Western blot analysis for cyclin A and cyclin B performed on whole cell extracts prepared from untreated SH-SY5Y cells and treated with 0,75 mM PQ for 18 h. C. Representative dot plots of untreated control cells and cells treated with 0,75 mM PQ for 18 h. Cells were stained by the fluorescence based TUNEL reaction and analysed by flow cytometry. The fluorescent cell populations in region 2 (R2) consist of apoptotic cells.

Figure S3 .

 S3 Figure S3. All probe sets, negative and positive controls signal intensities in PQtreated SH-SY5Y cells experiment "All probe set" quality control metric: red line. It represents the intensity signal of all the probe sets analyzed. "Neg control" quality control metric: blue line. It represents the intensity signal of the set of putative intron-based probe sets from putative housekeeping genes. "Pos control" quality control metric: green line. It represents the intensity signal of the set of putative exon based probe sets from putative housekeeping genes.

Figure S4 .

 S4 Figure S4. Positive vs Negative auc line graph in PQ-treated SH-SY5Y cells experiment.Pos vs neg auc is the area under the curve (AUC) for a receiver operating characteristic (ROC) plot comparing signal values for the positive controls to the negative controls. The ROC curve is generated by evaluating how well the probe set signals separate the positive controls from the negative controls with the assumption that the negative controls are a measure of false positives and the positive controls are a measure of true positives. An AUC of 1 reflects perfect separation whereas as an AUC value of 0.5 would reflect no separation. In practice the expected value for this metric is tissue type specific and may be sensitive to the quality of the RNA sample. Values between 0.80 and 0.90 are typical. "Pos vs neg auc" quality control metric: red line.

  Figure S5. "All probe sets", "negative " and "positive" control signal intensities in experiment performed in SOD1 wt vs SOD1(G93A) SH-SY5Y cells.

Figure S6 .

 S6 Figure S6. Positive vs. Negative AUC line graph in the experiment performed in SOD1 wt vs SOD1(G93A) SH-SY5Y cells.. "Pos vs neg auc "is the area under the curve (AUC) for a receiver operating characteristic (ROC) plot comparing signal values for the positive controls to the negative controls. The ROC curve is generated by evaluating how well the probe set signals separate the positive controls from the negative controls with the assumption that the negative controls are a measure of false positives and the positive controls are a measure of true positives. An AUC of 1 reflects perfect separation whereas as an AUC value of 0.5 would reflect no separation. Values between 0.80 and 0.90 are typical. "Pos vs neg auc" quality control metric: red line.

Figure R1 .

 R1 Figure R1. Comparison of the human and mouse BIN1 gene structure (UCSC Genome Browser).

Figure R2 .

 R2 Figure R2. Analysis of the alternative first exon usage in the G93A-Sod1 mouse. A. Comparison of the human and mouse ABLIM1 gene structure (UCSC Genome Browser). B. RT-PCR analysis of the transcripts including the two putative first exons. C. Quantitation of the PCR fragments. The bar graph represents the quantitation of the RT-PCR analysis for the long and the short Ablim1 mRNA variants. The first exon switch was normalized relative to that observed in the respective control (light grey bars). The dark grey bars show the average fold-change of the short mRNA variant transcribed from proximal promoter; error bars indicate the standard error.D. Time course analysis of long Ablim transcript in the spinal cord of G93A-Sod1 mice during disease progression. Plotted is the ration of the long over the short mRNA variant normalized to that of age-matched non-transgenic control animals (opened circles) on different days from the pre-onset till the symptomatic stage (93-170 days).

Table S2

 S2

	analysis"). Analysis of the selected genes with IPA7.0 identified Cellular Movement
	as top affected molecular and cellular function, Nervous System Development as top
	affected function related to "Physiological System Development and Function", and
	Genetic Disorders, Neurological Diseases and Psychological Diseases as the top
	three categories among the affected functions related to "Diseases and Disorders"
	(Table
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	"SOD gene-level
	15

Table S6

 S6

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

). Additional axon guidance genes were differentially expressed in only one of the two dataset. More genes related to different aspects of neuritogenesis (transcription regulation of axon guidance molecules, intracellular

Table 1 . Molecular and Cellular Functions affected by PQ treatment.

 1 patterns predicted for the protein isoforms encoded by

	the alternative transcripts of the ABLIM1, CHN1, NRG1 genes. Domain
	legend: EGF_1, EGF-like domain signature 1; EGF_3, EGF-like domain
	profile; HP, Headpiece domain profile; IG_LIKE, Ig-like domain profile;
	LIM_DOMAIN_2, LIM domain profile; RHOGAP, Rho GTPase-activating
	proteins domain profile; SH2, Src homology 2 (SH2) domain profile;
	ZF_DAG_PE_2, Zinc finger phorbol-ester/DAG-type profile.

Table 3 . Functions affected by the expression of the SOD1(G93A) protein.

 3

	Name

Table 4 . Genes related to the "Nervous System Differentiation and Function" Category

 4

	SYMBOL	DESCRIPTION	Function annotation
	ADORA2A	adenosine A2a receptor	growth of neuritis,
			synaptic transmission
	APBA1	EPH receptor A6	exocytosis of
			synaptic vesicles
	ASCL1	achaete-scute complex homolog 1	neurogenesis
		(Drosophila)
	BMP7	bone morphogenetic protein 7	neurogenesis
	BVES	blood vessel epicardial substance	regeneration of
			satellite cells
	C4ORF6	chromosome 4 open reading frame 6	neurogenesis
	CABLES1	Cdk5 and Abl enzyme substrate 1	neurogenesis
	CDON	Cdon homolog (mouse)	differentiation of
			neurons

Table 5 . Molecular and Cellular Functions affected by putative ASEs in paraquat-treated SH-SY5Y cells.

 5

	Name

Table S6 .Genes associated to Neurological Disorder, Axonal Guidance Signaling, and Neuritogenesis

 S6

	Page 75 of 82	Human Mutation
	DPYSL5	dihydropyrimidinase-like 5	-1,365	-0,529
	EPHA6	EPH receptor A6	-0,702	-0,581
	NTRK1 Neurological Disosrders neurotrophic tyrosine kinase, receptor, type 1 PLXNA4 plexin A4	-1,115 -0,684	-0,749 -0,699
	SYMBOL SDC2 ADORA2A SEMA3D ADRBK2 AKR1B1 SEMA6A ASCL1 CACNG4 CDON CHRM2 CLU DHCR24 GRM7 HSPA2 KIT LYN MSH2 MSRB2 NDRG2 NQO1 NTRK1 PREP SCG2 SERPINE2 SERPINI1 SMARCA2 Glycerolipid Metabolism syndecan 2 DESCRIPTION sema domain, immunoglobulin domain (Ig), short basic adenosine A2a receptor domain, secreted, (semaphorin) 3D adrenergic, beta, receptor kinase 2 sema domain, transmembrane domain (TM), and cytoplasmic aldo-keto reductase family 1, member B1 (aldose reductase) achaete-scute complex homolog 1 (Drosophila) calcium channel, voltage-dependent, gamma subunit 4 Cdon homolog (mouse) cholinergic receptor, muscarinic 2 clusterin 24-dehydrocholesterol reductase glutamate receptor, metabotropic 7 heat shock 70kDa protein 2 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog v-yes-1 Yamaguchi sarcoma viral related oncogene homolog mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) methionine sulfoxide reductase B2 NDRG family member 2 NAD(P)H dehydrogenase, quinone 1 neurotrophic tyrosine kinase, receptor, type 1 prolyl endopeptidase secretogranin II (chromogranin C) serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 serpin peptidase inhibitor, clade I (neuroserpin), member 1 SWI/SNF related, matrix associated, actin dependent domain, (semaphorin) 6A SYMBOL DESCRIPTION AKR1B1 aldo-keto reductase family 1, member B1 (aldose reductase) PPAP2B phosphatidic acid phosphatase type 2B GLB1L2 galactosidase, beta 1-like 2 Glycosphingolipid Biosynthesis SYMBOL DESCRIPTION GLB1L2 galactosidase, beta 1-like 2 ST8SIA2 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 Genes related to neuritogenesis. SYMBOL DESCRIPTION FEZ1 fasciculation and elongation protein zeta 1 (zygin I) ISL1 ISL LIM homeobox 1 FOXD1 forkhead box D1 F o r P e e r R e v i e w FOXM1 forkhead box M1	-0,908 0,999 -1,131 paraquat Log2FC -1.208 -1.020	-1,229 1,136 -0,561 SOD1(G93A) Log2FC -2.047 -1.373
	FOXD4L3	regulator of chromatin, subfamily a, member 2 forkhead box D4-like 3	0,955	0,895
	SPARCL1 LHX9	SPARC-like 1 (hevin) LIM homeobox 9	-1.881	1.685
	ST8SIA2 LMO3	ST8 alpha-N-acetyl-neuraminide alpha-2,8-LIM domain only 3 (rhombotin-like 2)	-3.470	-3.598
	LMO7	sialyltransferase 2 LIM domain 7	-0,553	-0,581
	TIMP1 NEUROD1	TIMP metallopeptidase inhibitor 1 neurogenic differentiation 1	-2.029
	TNFRSF1A NEUROG2	tumor necrosis factor receptor superfamily, member 1A neurogenin 2	-2.631
	TUBB4	tubulin, beta 4
	Axonal Guidance Signaling Supp.	paraquat	SOD1(G93A)
	SYMBOL	DESCRIPTION	Log2FC	Log2FC
	BMP7	bone morphogenetic protein 7	-0.517	-2.050
		John Wiley & Sons, Inc.		2

Table S8 . Main Entrez Gene abbreviations used in the text Page 76 of 82 John Wiley & Sons, Inc.

 S8

	Page 77 of 82		Human Mutation Human Mutation Human Mutation	Page 78 of 82
	Gene Name PLXNA4	GeneID 91584	Official Name plexin A4
	ABLIM1 POLH Supp.	3983 5429	actin binding LIM protein 1 polymerase (DNA directed), eta
	AKR1B1 PPAP2B	231 8613	aldo-keto reductase family 1, member B1 (aldose reductase) phosphatidic acid phosphatase type 2B
	ASCL1 PPM1D	429 8493	achaete-scute complex homolog 1 (Drosophila) protein phosphatase 1D magnesium-dependent, delta isoform
	ATF3 RPRD1A	467 55197	activating transcription factor 3 regulation of nuclear pre-mRNA domain containing 1A
	BIN1 SDC2	274 6383	bridging integrator 1 syndecan 2
	BTG2 CCNA2 CCNB1 CCNE2 CDKN1A CDKN3 CHN1 CXCR4 ERBB3 GADD45A GLB1L2 GNAO1 HGF KIF1A LMO3 SEMA3A SESN1 SESN2 ST8SIA2 TP53INP1 VSNL1 WASF3	7832 890 891 9134 1026 1033 1123 7852 2065 1647 89944 14681 3082 547 55885 10371 27244 83667 8128 94241 7447 10810	F o r P e e r R e v i e w BTG family, member 2 cyclin A2 cyclin B1 cyclin E2 cyclin-dependent kinase inhibitor 1A (p21, Cip1) cyclin-dependent kinase inhibitor 3 chimerin (chimaerin) 1 chemokine (C-X-C motif) receptor 4 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) growth arrest and DNA-damage-inducible, alpha galactosidase, beta 1-like 2 guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O hepatocyte growth factor (hepapoietin A; scatter factor) kinesin family member 1A LIM domain only 3 (rhombotin-like 2) F o r P e e r R e v i e w sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A sestrin 1 sestrin 2 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 tumor protein p53 inducible nuclear protein 1 visinin-like 1 WAS protein family, member 3 F o r P e e r R e v i e w
	LRRK2	120892	leucine-rich repeat kinase 2
	MDM2	4193	Mdm2 p53 binding protein homolog (mouse)
	NEUROG3	50674	neurogenin 3
	NTRK1	4914	neurotrophic tyrosine kinase, receptor, type 1
	NKX2.2	4821	NK2 homeobox 2
	NRG1	3084	neuregulin 1
	PARK7	11315	Parkinson disease (autosomal recessive, early onset) 7
	PAX6	607108	paired box 6
	PINK1	65018	PTEN induced putative kinase 1
			John Wiley & Sons, Inc. John Wiley & Sons, Inc.	3 4 5

Table S9 .

 S9 Oligonucleotide primersNucleotide sequences of the primers employed for RT-PCR real time

	Page 79 of 82 Page 81 of 82		Human Mutation Human Mutation Human Mutation	Page 80 of 82
	VSNL1 Species: Mus musculus GTGATGGCAAGATCACCC	ATCACAGTGCCTACCATT	73
	WASF3	CGAATGGGAATTCAACTGAAA	GTCATTCCCCACTGGCT	75
	ZNF521 Gene ID	GCAGAATCATACAATGACCCA Forward_ Sequence (5'-3') AATCAATTCTCCTTGAGAGACT Reverse_Sequence (5'-3')	65 Product lenght (nt)
	Cdc42	ACAACAAACAAATTCCCATCG	TTGCCCTGCAGTATCAAAAA	114
	Species: Homo sapiens Cxcr4 TGGAACCGATCAGTGTGAGT	GGGCAGGAAGATCCTATTGA	131
	Gene ID Forward_ Sequence (5'-3') Reverse_Sequence (5'-3') Product lenght (nt) ADAMTs1 TTTGATAAATGTGGTGTTTGCG TTTTGCACTAGTAACTGATCCTG 75 AIFM2 CGTCAACTCTGTGAAGCA CATGGACAGGAGGAACG 70 APBA1 CACGAGAAGATCGTCCAC GGCTGGCATTGTCTTCA 66 ASCL1 GACTTTGGAAGCAGGGT CAACGCCACTGACAAGAA 62 CABLES1 GGGTGCTGATGGGAAGACT CAGGTTACGGAACTGGGAGA 115 CHN1 GTATGTAGGCATCTTTCATTCAGA CAAAACACTCAGCATCTCTCTTT 129 CNTN4 CTGTCAGCCATCAGTACAATAAT AGCAAGTCCTTCAGATAACTTT 78 CXCR4 ATTGGGATCAGCATCGACTC CAAACTCACACCCTTGCTTG 61 EPHA6 AAGGAGATTGATCCCTCAAGAA CGCCCACTACAGACTTCTCC 77 GAPDH ACGGATTTGGTCGTATTGGG TGATTTTGGAGGGATCTCGC 231 GPM6B CCTGGAGAACATCTGCAAC GGCCACAATGAACAGGT 61 HGF AGCATGTCCTCCTGCATCTC TTGTATTTCTTCTTTTCCTTTGTCC 78 ITGA9 TGCTCTGGAAGATGGGC GGTTCTTCTCAGCTTCGATAATTT 63 LMO3 CTTTGCATGTCAGCTTTGTAAT CCTTCCTCGTAGTCCGT 96 NRP2 GGACCCCCAACTTGGATT ATGGTTAAAAAGCGCAGGTC 61 NTRK1 GAGGCAATCGACTGCATCA GTAGACCTCTGGTGGGC 69 NUDT11 TTCGGTTAGCATTGGGAGG GCTTGTGGCACTGGAGAA 74 F o r P e e r R e v i e w Fez1 AAGGCATCTCCAACATCCTG AGCATCTGCAGGTCTTCCAC 136 Hgf CACCCCTTGGGAGTATTGTG GGGACATCAGTCTCATTCACAG 66 Hprt1 TCCTCCTCAGACCGCTTTT CCTGGTTCATCATCGCTAATC 90 Nrp2 CGAAGTGAGAAGCCAGCAA GAGGGATAGTCCTGGGGGTA 99 Ntrk1 GGACCTCAACCGTTTCCTC CCATACCAGCAGCCACCT 131 Sema3a GGGAAGAGCCCTTATGATCC CCGCAGCAGTTCCAGAGTA 79 Sept2 CAGTGAAGAAGGGGTTCGAG TCTGGGTAGAGATCAGTCAGGA 103 Sept7 AGGGGATGTCGGTCAGTG CAAGGTTCTTCGGTTGAGCTA 87 Slit1 AACGTGGCAGAGGTGCAA GCAGGAACCGGACGAGAG 93 Slit2 CTGTCATGGGCTGGCACT TCCTCGTGATGTTATTTCCATTC 89 Slit3 ACCGGTTCCAGTGCAAAG TGCAAGTGCCGTTGTTCTT 93 Wasf3 CTGTGGCTGAGGCAAAGC TGCACCTTTTTCAGTTGGATT 106 F o r P e F o r P e e r R e v i e w e r
	SDC2 SEMA3A	GTCCTAGCAGCTGTCATT GCGAAATGAAGAGCGAAAAG	TACACCAACAGCAGGATAAGAAA TTTGTATGAACCCATGTTCCAC R	74 140
	SLIT3 SLITRK6 SMARCA2 TNIK	CTTGTCTCCCTGTCCACCATA TACGCAGTGGTTGGTGTTTT AAACCTGTAGTGAGCGATT AGCTCAAAGGTTAAAGTTTCTATG	CCTCTTCCTCAACCACTTGC TCCACAGCTTCATGTTGTCAT e TCATCATCCGTCCCACTT TTCCTCCAGATCGCACG v i e	90 146 80 71
			John Wiley & Sons, Inc. John Wiley & Sons, Inc. John Wiley & Sons, Inc. w	6 7

Figures for Referee 2

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

This work was supported by grants from Fondazione Cariplo to T.A., S.B., and M.T.C., from MIUR-PRIN to S.B. and M.T.C., from EC 7FPs TOLERAGE 202156 and FIGHT 242210 to F. Zolezzi and D.B., and from Regione Piemonte to R.C.; F.

Cordero is supported by a grant from Regione Piemonte/Università di Torino. We thank R. Ambrosini for help with the statistical analysis of the in vivo data, and D.

Talarico for carefully reading the manuscript and for helpful suggestions.

Supporting Material

File 1. Assessment of RNA quality File 2. Supporting Results and Methods File 3. Assessment of RNA and arrays quality . Table S1. "Paraquat gene-level analysis" Table S2. "SOD1 gene-level analysis" Table S3. "PQ exon-level analysis" Table S4. "SOD1 exon-level analysis" Table S5. "Common gene-level" Table S6. Genes associated to Neurological Disorder, Axonal Guidance Signaling, and Neuritogenesis. Table S7. "Common ASEs" Table S8. Main Entrez Gene abbreviations used in the text Table S9. Oligonucleotide primers