Brunhilde Wirth - C 16

Unraveling the molecular and cellular mechanism underlying spinal muscular atrophy by use of genetic modifiers

Spinal muscular atrophy (SMA) is caused by homozygous absence of the survival motor neuron gene 1 (SMN1) and inability of SMN2 to compensate for the SMN1 loss. We identified plastin 3 (PLS3), neurocalcin delta (NCALD) and calcineurine-like EF-hand protein 1 (CHP1) to protect against SMA in humans and across species (zebrafish and mice). All three modifiers are able to restore impaired endocytosis in SMA, however the exact underlying mechanisms needs further investigation. Importantly, the suppression of NCALD and CHP1 are currently used to develop novel SMA therapies.


Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for the SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as a SMA protective modifier in humans and confirmed its power to protect against SMA in zebrafish and mice (Oprea et al 2008; Ackermann et al 2013, Hosseinibarkooie et al 2016, Heesen et al 2016). Although the effect of PLS3-mediated protection has been widely studied, the molecular mechanism remains elusive. To unveil the mechanism behind the rescuing effect, we aimed to identify novel PLS3 interacting partners which could be used to modify the disease phenotype and as potential therapeutic targets to treat SMA.

NCALD suppression protects against spinal muscular atrophy in humans and across species by restoring impaired endocytosis

Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish and mouse. Our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

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

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. Here, we identified calcineurin-like EF-hand protein1 (CHP1) as a novel plastin3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and plastin3. 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 presymptomatic 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 dynamin1 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 plastin3, and that CHP1 reduction ameliorates SMA pathology by improving impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

PLS3 and NCALD interacting protein network

To further decipher the molecular pathway of how PLS3 overexpression or NCALD reduction rescues the SMA phenotype, we searched for PLS3 or NCALD interacting partners using tandem tag purification followed by mass spectrometry. HEK293T cell lines stably overexpressing Flag/His-tagged PLS3 and NCALD in comparison to vector alone were generated and Co-IP eluates analysed by mass spectrometry. Moreover, Co-IPs followed by mass spectrometry have been performed from spinal cord of PLS3 overexpressing, WT and Pls3 knockout mice as well as from Ncald knockout compared to WT mice. A large number of highly interesting candidate proteins were identified, which are currently under investigation.


Understanding the interactome of PLS3, NCALD and CHP1 and the proteins and pathways that may contribute to SMA protection is highly relevant to develop novel therapeutic SMN-independent strategies for SMA treatment. In collaboration with IONIS Pharmaceuticals we developed antisense oligonucleotides (ASOs) against Ncald and Chp1 to study the power of combinatorial therapy in SMA.

Selected publications

Janzen, E., … Wirth B. CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis. Brain 2018, 141 (8), 2343-2361

Mendoza-Ferreira N, … Wirth B. Biallelic CHP1 mutation causes human autosomal recessive ataxia by impairing NHE1 function. Neurol Genet 2018, 4: e209

Martinez Carrera LA, …Wirth B. Novel insights into SMALED2: BICD2 mutations increase microtubule stability and cause defects in axonal and NMJ development.
Hum Mol Genet 2018, 27: 1772-1784

Riessland M*, Kaczmarek A*, Schneider S*, ... Wirth B. Neurocalcin delta suppression protects against spinal muscular atrophy in humans and across species by restoring impaired endocytosis. Am J Hum Genet, 2017; 2. Feb. 100 (2): 297-315

Hosseinibarkooie S*, Peters M*, …Wirth B. The power of human protective modifiers: PLS3 and CORO1C unravel impaired endocytosis in spinal muscular atrophy and rescue SMA phenotype. Am J Hum Genet, 2016; 99 (3): 647-665 

Heesen L*, Peitz M*, … Wirth B. Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals. Cell Mol Life Sci, 2016; 73(10):2089-104. 

Patent: B. Wirth, E. Janzen, N. Mendoza-Ferreira, SM Hosseinibarkooie, EP 17172826, Calcineurin B Homologous Protein 1 Inhibitors and Therapeutic and Non-Therapeutic Uses Thereof, 4 filed May. 24, 2017

Prof. Dr. Brunhilde Wirth

Institute for Human Genetics / RG location - CMMC Building

Prof. Dr. Brunhilde Wirth

Principal Investigator C 16
Executive Board Member

Work +49 221 478 86464

Fax (Work) +49 221 478 86465

Institute for Human Genetics
Kerpener Str. 34
50931 Cologne

Publications - Brunhilde Wirth

Link to PubMed

Group Members

Mohsenseyyed Hosseinibarkkoie (PostDoc)
Eva Janzen (PostDoc)
Natalia Mendoza-Ferreira (PostDoc)
Laura Torres-Benito (PostDoc)
Andrea Delle Vedove (MD/PhD student)
Mert Karakaya (MD student)
Anixa Muinos Brühl (PhD student)
Sarah Bachmann (PhD student)
Bryony Ross (MD/PhD student)
Charlotte Veltman (MD student)
Michelle Scharte (Bachelor student
Irmgard Hölker (technician)
Kristina Hupperich (technician)
Roman Rombo (technician)

Figure 1

CMMC Research Wirth
CHP1 downregulation improves endocytosis by increasing calcineurin phosphatase activity (Janzen et al 2018)