Universität zu Köln

Veltman, Charlotte (2024). Characterization of novel potential binding partners of Plastin 3, a protective modifier of Spinal Muscular Atrophy. PhD thesis, Universität zu Köln.

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Abstract

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease caused by mutations in the survival motor neuron 1 (SMN1) gene, resulting in progressive loss of motor neurons (MNs) and severe muscle atrophy. SMN2, a copy gene of SMN1, produces only 10% functional SMN and its copy number is inversely correlated with the severity of the SMA phenotype in humans. Besides SMN2, as the main modifier of SMA, several SMN-independent modifiers have been identified in humans and different SMA models. Plastin 3 (PLS3), an F-actin binding- and bundling protein, has been identified as one of the most effective modifiers of SMA in humans. Further characterization of PLS3 in various SMA mouse models shows that PLS3 not only improves the morphology and function of neuromuscular junctions (NMJs) but also rescues the survival of a severe SMA mouse model, whose phenotype has been slightly improved with a low dose of SMN-antisense- oligonucleotides (ASO) (nusinersen). PLS3 overexpression, by restoring the F-actin dynamics, rescues reduced endocytosis in SMN knockdown cells and SMA mouse model. PLS3 and its role in endocytosis, led to unveiling of endocytosis as one of the major pathways disrupted in SMA and restored by PLS3 overexpression. However, the mechanisms and signaling pathways underlying the rescuing function of PLS3 are not yet fully understood and require further investigation. This could lead to new combinatorial therapeutic approaches. To gain further insights into the role of PLS3 in vivo, an interactome study using PLS3 co- immunoprecipitation (co-IP) from murine spinal cord samples of Pls3 knockout, PLS3 overexpressing and wildtype animals was performed, followed by mass spectrometry analysis. Based on different criteria such as pathway analysis and abundance, several potential PLS3 binding partners were selected and cloned into pcDNATM3.1⁄CT-GFP-TOPO vectors. The interaction of PLS3 with several selected candidates including paralemmin-1 (PALM1), lunapark (LNP), sodium/potassium-transporting ATPase subunit beta-2 (ATP1B2), sorting nexin-3 (SNX3), clathrin light chain B (CLTB), stromal interaction molecule 1 (STIM1), syntaphilin (SNPH), syntaxin 16 (STX16) and guanine nucleotide-binding protein G(s) complex locus (GNAS) was confirmed using independent co-IP assays. For further characterization of the identified binding partners, we next examined a possible colocalization of selected candidates with PLS3 in primary mouse embryonic fibroblasts (MEFs). Furthermore, we analyzed the level of the identified candidates upon PLS3 overexpression, Pls3 knockout and SMA condition in several tissues and primary motor neurons by Western Blotting and immunofluorescence staining. Most interestingly, we found two binding partners colocalizing with PLS3 and altered protein levels upon disease relevant conditions. First, we showed a colocalization of PLS3 with SNPH in lamellipodia of MEFs and reduced levels of SNPH in brain and spinal cord of SMA mice. SNPH has an inhibitory role in vesicle endo- and exocytosis and it further functions in axonal mitochondria dynamics. More specifically, SNPH anchors mitochondria to microtubules. Mitochondrial transport dysfunction has been implicated in various neurodegenerative and neurological disorders, such as Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS) and nerve injury, showing a multifaceted involvement of SNPH. Interestingly, mitochondrial dysfunction in motor neurons and muscle has been demonstrated in SMA. Based on our results, we suggest an involvement of SNPH in mitochondrial dysfunction in SMA, displaying a highly potent candidate for further studies. Secondly, we found that STIM1 colocalizes with PLS3 in the lamellipodia and is upregulated upon PLS3 overexpression in spinal cord and motor neurons derived from PLS3 overexpressing mice. Remarkably, this is in line with the finding that STIM1 was detected as an interaction partner of PLS3 only in PLS3 overexpressing spinal cord samples in the initial co-IP and mass spectrometry analysis. Analysis of STIM1 levels in Transversus abdominis muscle (TVA) of SMA mice showed a significant downregulation of STIM1. STIM1 is a calcium sensor in the ER and regulates store operated calcium entry (SOCE). Disturbances or changes in this signaling pathway are known in several neurodegenerative disorders including ALS and Familial Alzheimer’s Disease (FAD). In SMA, disturbances in calcium homeostasis have been demonstrated in both neurons and astrocytes. Based on our results and the importance of calcium regulation, we hypothesize that the interaction between PLS3 and STIM1 could be an important regulatory mechanism potentially leading to improved calcium homeostasis in SMA upon PLS3 overexpression. Future studies are needed to further elucidate the functional role of STIM1 and further proteins of SOCE in relation to the rescue function of PLS3 in SMA.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Veltman, Charlotte UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-722005
Date:	2024
Language:	English
Faculty:	Faculty of Medicine
Divisions:	Faculty of Medicine > Humangenetik > Institut für Humangenetik
Subjects:	Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language Spinal Muscular Atrophy English
Date of oral exam:	8 November 2023
Referee:	Name Academic Title Wirth, Brunhilde Universitätsprofessorin Dr. rer. nat. Trifunovic, Aleksandra Universitätsprofessorin Dr. rer. nat.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/72200