Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier

Neuronal-specific deficiency of the splicing factor Tra2b causes apoptosis in neurogenic areas of the developing mouse brain.

Alternative splicing (AS) increases the informational content of the genome and is more prevalent in the brain than in any other tissue. The splicing factor Tra2b (Sfrs10) can modulate splicing inclusion of exons by specifically detecting GAA-rich binding motifs and its absence causes early embryonic lethality in mice. TRA2B has been shown to be involved in splicing processes of Nasp (nuclear autoantigenic sperm protein), MAPT (microtubule associated protein tau) and SMN (survival motor neuron), and is therefore implicated in spermatogenesis and neurological diseases like Alzheimer's disease, dementia, Parkinson's disease and spinal muscular atrophy. Here we generated a neuronal-specific Tra2b knock-out mouse that lacks Tra2b expression in neuronal and glial precursor cells by using the Nestin-Cre. Neuronal-specific Tra2b knock-out mice die immediately after birth and show severe abnormalities in cortical development, which are caused by massive apoptotic events in the ventricular layers of the cortex, demonstrating a pivotal role of Tra2b for the developing central nervous system. Using whole brain RNA on exon arrays we identified differentially expressed alternative exons of Tubulinδ1 and Shugoshin-like2 as in vivo targets of Tra2b. Most interestingly, we found increased expression of the cyclin dependent kinase inhibitor 1a (p21) which we could functionally link to neuronal precursor cells in the affected brain regions. We provide further evidence that the absence of Tra2b causes p21 upregulation and ultimately cell death in NSC34 neuronal-like cells. These findings demonstrate that Tra2b regulates splicing events essential for maintaining neuronal viability during development. Apoptotic events triggered via p21 might not be restricted to the developing brain but could possibly be generalized to the whole organism and explain early embryonic lethality in Tra2b-depleted mice

PLS3 Overexpression Delays Ataxia in Chp1 Mutant Mice

Many neurodegenerative disorders share common pathogenic pathways such as endocytic defects, Ca2+ misregulation and defects in actin dynamics. Factors acting on these shared pathways are highly interesting as a therapeutic target. Plastin 3 (PLS3), a proven protective modifier of spinal muscular atrophy across species, is a remarkable example of the former, and thereby offers high potential as a cross-disease modifier. Importantly, PLS3 has been linked to numerous proteins associated with various neurodegenerative diseases. Among them, PLS3 directly interacts with calcineurin like EF-hand protein 1 (CHP1), whose loss-of-function results in ataxia. In this study, we aimed to determine whether PLS3 is a cross-disease modifier for ataxia caused by Chp1 mutation in mice. For this purpose, we generated Chp1 mutant mice, named vacillator mice, overexpressing a PLS3 transgene. Here, we show that PLS3 overexpression (OE) delays the ataxic phenotype of the vacillator mice at an early but not later disease stage. Furthermore, we demonstrated that PLS3 OE ameliorates axon hypertrophy and axonal swellings in Purkinje neurons thereby slowing down neurodegeneration. Mechanistically, we found that PLS3 OE in the cerebellum shows a trend of increased membrane targeting and/or expression of Na+/H+ exchanger (NHE1), an important CHP1 binding partner and a causative gene for ataxia, when mutated in humans and mice. This data supports the hypothesis that PLS3 is a cross-disease genetic modifier for CHP1-causing ataxia and spinal muscular atrophy

Brain malformations are initiated by massive apoptosis in the cortex.

<p>(<b>A</b>) Immunostaining for Caspase-3 on paraffin-embedded coronal sections indicates prominent apoptosis in the proximal cortical layers and in the thalamic area of 14.5 dpc and 15.5 dpc KO embryos (black arrowheads). Remaining cortical tissue does not show apoptosis at 16.5 dpc and later stages (light arrowhead). (<b>B</b>) Immunostaining for Ki-67 shows initial decrease of proliferation at 14.5 dpc which is fully lost at 16.5 dpc in KO animals (black arrowheads). Control and HET animals retain strong Ki-67 signals in the proximal cortical layers at all indicated developmental stages. Scale bar equals 400 µm; ctx, cortex; th, thalamus; cpt, caudoputamen; sn, septal nuclei; 3v, third ventricle; lv, lateral ventricle.</p

<i>p21</i> is upregulated as a response to <i>Tra2b</i> depletion in the mouse brain and in neural stem cells.

<p>(<b>A,B</b>) Semi-quantitative RT-PCR using whole brain RNA of neuronal specific <i>Tra2b</i> KO mice and controls. <i>p21</i> is significantly upregulated by 1.5-fold in KO mice as compared to HET or control mice. <i>p21</i> expression is indifferent between controls and HET mice. <i>p21</i> expression was normalized to <i>Hprt</i>. (<b>C–G</b>) NSC34 neural stem cells were transfected with siRNAs specific to <i>Tra2b</i> or scrambled siRNAs. siRNA treatment but not scr-treatment effectively reduced Tra2b protein and mRNA levels after 24 h, 48 h and 72 h after transfection (<b>C–E</b>). Tra2b function was strongly reduced as the <i>Nasp</i> transcript showed a significantly lower inclusion of the T-exon at 24 h, 48 h and 72 hours after transfection (<b>F</b>). 48 hours after transfection <i>p21</i> expression was found slightly but significantly increased on RNA level (<b>G</b>) but not on protein level (<b>D</b>). 72 hours after transfection p21 was massively and highly significantly upregulated on RNA and protein level by +2.2-fold (<b>D,G</b>). a.u., arbitrary units; nt, non-treated; scr, scrambled siRNA; si, siRNA against <i>Tra2b</i>; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

Conditional ablation of <i>Tra2b</i> causes perinatal lethality and disturbed cortical patterning in mice.

<p>(<b>A</b>) Cross breading of <i>Tra2b^fl/fl</i> with <i>Tra2b^fl/+; Nestin-Cre^tg/0</i> mice allowed generation of neuronal-specific knock-out (KO) animals as well as controls (CRTL) and heterozygous knock-out animals (HET) in one litter. (<b>B</b>) KO animals are born alive and all possible genotypes were detected according to Mendelian law (N = 113). (<b>C</b>) General development of conditional knock-out mice is not impaired as there are no gross morphological differences in embryo appearance. (<b>D</b>) Hematoxylin/Eosin staining of paraffin-embedded coronal sections at indicated developmental stages. KO animals but not controls or HET animals show ventriculomegaly of the third and lateral ventricles starting at around 14.5 dpc. Cortical layers are largely distinguishable at 14.5 dpc but cortical patterning and the ependymal lining of the lateral ventricle appears highly disturbed (black arrowheads) at 16.5 dpc in knock-out brains. (<b>E</b>) Immunostaining of <i>Tra2b</i> on paraffin-embedded coronal sections shows efficient downregulation of <i>Tra2b</i> protein in knock-out brains compared to controls and heterozygote animals. Cells of the ventricular and subventricular zones of the cortex show strongest decrease in staining intensity (black arrowhead). Scale bar equals 400 µm; ctx, cortex; th, thalamus; cpt, caudoputamen; cp, cortical plate; iz, intermediate zone; svz, subventricular zone; vz, ventricular zone; sn, septal nuclei; 3v, third ventricle; lv, lateral ventricle; pc, choroid plexus.</p

Splicing of <i>Sgol2</i> and <i>Tubd1</i> is responsive to changes in Tra2b concentration.

<p>(<b>A</b>) The murine and human versions of the genomic regions comprising the identified exons of <i>Sgol2</i> and <i>Tubd1</i> were cloned into the pSPL3 exon trapping vector. (<b>B</b>) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for <i>Tra2b</i> or a <i>TRA2B-GFP</i> expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. (<b>C</b>) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. (<b>D–G</b>) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human <i>Sgol2</i> is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of <i>Tra2b</i> is insufficient to reduce <i>Sgol2</i> exon4 splicing inclusion. Exon 4 of human but not murine <i>Tubd1</i> is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of <i>Tra2b</i> decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against <i>Tra2b</i>; n.s., non significant; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier

Mouse whole exon array analysis reveals <i>Tubd1</i> exon4 and <i>Sgol2</i> exon4 as <i>in vivo</i> targets of <i>Tra2b</i>.

<p>Whole brain RNA of 4 CTRL animals and 4 KO animals was analyzed on mouse exon array. (<b>A</b>) The inclusion ratio (PSI, percent splicing inclusion) of each identified exon is defined as [PSI_KO]/[PSI_CTRL]. PSI distribution reached from ∼0.2 until ∼4.0. (<b>B</b>) Initial filtering strategies comprised exclusion of PSIs between 0.66 and 1.5 (grey bars) as well as restriction to p-values smaller than 0.05 which yielded a total of 1,006 exons. Exons associated with transcripts identified as being transcriptionally up- or downregulated were excluded from analysis. Ranking of those was further refined using large PSI values and considering presence of putative Tra2b binding sites (AGAA-motifs). Thereby, exons had to contain at least a single AGAA-site and a AGAA-frequency higher than 1.5. (<b>C,D,G,H</b>) Semi-quantitative RT-PCT on whole brain RNA was carried out using isoform specific primers for <i>Sgol2</i> FL (<b>C</b>), <i>Sgol2</i> Δ4 (<b>D</b>), <i>Tubd1</i> FL (<b>G</b>) and <i>Tubd1</i> Δ4 (<b>H</b>) confirming splicing events identified on the microarray. All isoform expression levels were densitometrically measured and normalized against <i>Hprt</i> (<b>E,F,I</b>). The <i>Tubd1</i> Δ4 isoform could not be detected using whole brain RNA, as skipping of exon4 introduces numerous premature termination codons leading to nonsense-mediated decay of the transcript (<b>H</b>). Treatment of wt and <i>Tra2b</i>-depleted murine embryonic fibroblasts with emetine successfully inhibited NMD and the <i>Tubd1</i> Δ4 isoform was detectable in <i>Tra2b</i>-depleted cells only (<b>J</b>). FL, full length; Δ4, transcript lacking exon 4, (−) PCR negative control; a. u., arbitrary units; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZA (TM), has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy