Plastin 3 influences bone homeostasis through regulation of osteoclast activity.

Over 200 million people suffer from osteoporosis worldwide, one third of which will develop osteoporotic bone fractures. Unfortunately, no effective cure exists. Mutations in plastin 3 (PLS3), an F-actin binding and bundling protein, cause X-linked primary osteoporosis in men and predisposition to osteoporosis in postmenopausal women. Moreover, the strongest association so far for osteoporosis in elderly women after menopause was connected to a rare SNP in PLS3, indicating a possible role of PLS3 in complex osteoporosis as well. Interestingly, 5% of the general population are overexpressing PLS3, with yet unknown consequences. Here, we studied ubiquitous Pls3 knockout and PLS3 overexpression in mice and demonstrate that both conditions influence bone remodeling and structure: while Pls3 knockout mice exhibit osteoporosis, PLS3 overexpressing mice show thickening of cortical bone and increased bone strength. We show that unbalanced PLS3 levels affect osteoclast development and function, by misregulating the NFκB pathway. We found upregulation of RELA (NFκB subunit p65) in PLS3 overexpressing mice-known to stimulate osteoclastogenesis-but strikingly reduced osteoclast resorption. We identify NFκB repressing factor (NKRF) as a novel PLS3 interactor, which increasingly translocates to the nucleus when PLS3 is overexpressed. We show that NKRF binds to the NFκB downstream target and master regulator of osteoclastogenesis nuclear factor of activated T cells 1 (Nfatc1), thereby reducing its transcription and suppressing osteoclast function. We found the opposite in Pls3 knockout osteoclasts, where decreased nuclear NKRF augmented Nfatc1 transcription, causing osteoporosis. Regulation of osteoclastogenesis and bone remodeling via the PLS3-NKRF-NFκB-NFATC1 axis unveils a novel possibility to counteract osteoporosis.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

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 SPINRAZATM, 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.

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.

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.

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.