Spinal muscular atrophy (SMA) is caused by homozygous absence of the survival motor neuron gene 1(SMN1) and inability of SMN2to compensate for the SMN1loss. We found that increased levels of plastin 3 (PLS3) and reduced levels of neurocalcin delta (NCALD) protect against SMA in humans and across species (zebrafish, worm and mice). However, the molecular genetic cause for the modified expression of these modifiers in asymptomatic SMN1-deleted individuals is not understood, but relevant for future therapies. Moreover, these protective modifiers helped us to uncover – 20 years after gene discovery- impaired endocytosis as a main cellular mechanism disturbed in SMA; the exact underlying mechanisms is unknown.
The motor neuron (MN) disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, whose function is critical for assembly of the spliceosome and other protein complexes. Despite the housekeeping function, SMN deficit in SMA patients primarily affects the function of spinal MN and especially of the neuromuscular junction (NMJ), resulting in dysfunction, degeneration and consequent muscular atrophy.
We identified two human-protective SMA modifiers - PLS3 upreg-ulation and NCALD downregulation - and proved their ability to counteract SMA in vitroand in vivousing SMA zebrafish and mouse models. Both proteins and their interactome helped us to uncover endocytosis as a major cellular pathway disturbed in SMA and rescued by these modifiers. Here, we address the following two questions:
We found that Plastin 3 (PLS3) is upregulated in genetically predisposed but phenotypically asymptomatic women, and acts protective against SMA (Oprea et al 2008). The protective effect of PLS3 upregulation has been confirmed in various genetically-modified SMA models (worm, fly, zebrafish and mice) as well as by gene therapy using AAV9-PLS3 (Oprea et al 2008; Ackermann et al 2013; Hosseinibarkooie et al 2016; Kaifer et al 2017).
PLS3is upregulated in approximately 5% of the healthy population, while the reason is still elusive. As PLS3is localized on the X-chromosome, we hypothesised that PLS3might escape the X-chromosomal inactivation (XCI), since no other variants within PLS3 were identified that may explain this unusual finding (Oprea et al 2008).
Using a multi-OMICS approach, including genome sequencing, transcriptome (RNASeq) from various cell lines (lymphoblastoid cell lines, fibroblasts and iPSC-derived motor neurons), epigenetic analysis as well as proteome analysis, we aim to unveil the underlying cis and trans-regulatory network leading to the cell-type specific PLS3 upregulation. We found that PLS3 in asymptomatic individuals is upregulated in lymphoblastoid cell lines and MN-derived from iPCSs, but not in the fibroblast cell line from which these originated. This suggest a cell type-specific regulation. Transcriptome and SNP analyses of single cell clones of iPSC-derived MNs showed biallelic expression of PLS3but not of most other X-linked genes in asymptomatic but not SMA individuals, indicating that PLS3 escapes XCI in asymptomatic individuals. PLS3is located adjacent to the unique macrosatellite DXZ4. This macrosatellite has a highly variable number of a 3 kb repeat monomer in humans, and is essential for XCI. Indeed, DXZ4is associated with heterochromatic epigenetic features on the active X-chromosomes, while it has euchromatic features on the inactive X-chromosomes and the locus of DXZ4 is therefore open for transcription. We hypothesised that the repeat number of DXZ4modulates the expression of PLS3and other adjacent genes on the inactivated X-chromosome facilitating their escape from XCI. To determine the repeat number of DXZ4, we established a novel technique called molecular combing and compared women that differ in their PLS3expression. This method uses multi-colour DNA probes to mark repeat regions on linear stretched DNA molecules.
By this, we quantified the repeat number of DXZ4in SMA affected and asymptomatic women and compared the results to PLS3 expression levels measured by qRT-PCR. Asymptomatic women with a high PLS3 expression showed at least one allele with a high copy number repeat as compared to the symptomatic one with none or low PLS3 levels. Currently, we perform 4C-sequencing to identify the regulatory landscape of PLS3and DXZ4. Moreover, we are currently establishing a methylation sensitive assay to verify the skewed XCI. Overall, this is a highly challenging, but promising and exciting project, for which a DFG-funding will be submitted soon.
NCALD shows a Ca2+-dependent interaction with clathrin, an important protein, coating synaptic vesicles during endocytosis. We found that NCALD binds clathrin only when the Ca2+level is low. Using patch clamp and Ca2+imaging, we demonstrate that the Ca2+influx is reduced in Smn-depleted PC12 cells. However, this was not restored by simultaneous NCALD reduction. Thus, we assumed that another mechanism must be responsible for the positive effect of NCALD reduction on endocytosis. Indeed, we show that NCALD binds clathrin only in the presence of low calcium levels, as is the case in SMA cells. Hence, a reduced amount of NCALD releases clathrin, resulting in an increased endocytosis rate.
Moreover, under stimulation of different cell types as well as ex vivoin NMJs, endocytosis was strongly impaired in SMA, whereas NCALD reduction restored it (Riessland et al 2017). Moreover, we identified calcineurin-like EF-hand protein1 (CHP1) as a third SMA protective modifier (Janzen et al 2018). In NSC34 cells, Chp1knockdown tripled macro-pinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1knockdown 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 hyperphos-phorylation of dynamin1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1mutant allele (Janzen et al 2018).
In collaboration with IONIS Pharmaceuticals we test antisense oligonucleotides (ASOs) against Ncaldand Chp1to study the power of combinatorial therapy in SMA.
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Martinez Carrera LA, Gabriel E, Donohoe CD, Holker I, Mariappan A, Storbeck M, Uhlirova M, Gopalakrishnan J, and Wirth B (2018). Novel insights into SMALED2: BICD2 mutations increase microtubule stability and cause defects in axonal and NMJ development. Hum Mol Genet 27, 1772-1784.
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Storbeck M, Horsberg Eriksen B, Unger A, Holker I, Aukrust I, Martinez-Carrera LA, Linke WA, Ferbert A, Heller R, Vorgerd M, Houge G, and Wirth B (2017). Phenotypic extremes of BICD2-opathies: from lethal, congenital muscular atrophy with arthrogryposis to asymptomatic with subclinical features. Eur J Hum Genet 25, 1040-8.
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Targeting CHP1, an SMN-independent pathway to counteract spinal muscular atrophy in mice and human iPSCs
Institute of Human Genetics / Location: CMMC Building
CMMC - PI - C 16
CMMC - Executive Board Membershow more…
Mohsenseyyed Hosseinibarkkoie (PostDoc)
Eva Janzen (PostDoc
Natalia Mendoza-Ferreira (PostDoc)
Svenja Schneider (Postdoc)
Laura Torres-Benito (PostDoc)
Andrea delle Vedove (MD/PhD student)
Mert Karakaya (MD student)
Anixa Muinos Brühl (PhD student)
Ilka Müller (PhD student)
Bryony Ross (MD/PhD student)
Tamas Schmidt (PhD student)
Eike Strathmann (PhD student)
Aaradhita Upaydhyay (PhD student)
Charlotte Veltman (MD student
Lisa Wolff (Master student)
Charlotte Dresen (Bachelor student)
Michelle Scharte (Bachelor student)
Irmgard Hölker (TA)
Kristina Hupperich (TA)