Peters, Miriam (2016). Combined therapie of SMN-ASO and Plastin 3 overexpression rescues severe SMA in mice. PhD thesis, Universität zu Köln.


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Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by the loss of α-motor neurons in the anterior horns of the spinal cord. This leads to atrophy of proximal symmetrical muscles and paralysis. Patients suffering from SMA in almost all cases show homozygous absence of the survival of motor neuron 1 gene (SMN1) and the severity of the disease is inversely correlated with the copy number of the main disease modifying gene survival motor neuron 2 (SMN2). Rarely, individuals with homozygous SMN1-deletion remain asymptomatic, suggesting the action of modifying gene(s). Plastin 3 (PLS3) – a F-actin binding and bundling protein - has been identified as a protective modifier showing high expression in SMN1-deleted asymptomatic siblings of SMA patients. PLS3 is highly expressed in spinal cord and axonal growth cones. PLS3 overexpression rescues the axonal outgrowth phenotype in SMA motor neuron cultures and SMA zebrafish (Oprea et al. 2008). Severely affected SMA mice carrying heterozygously a transgenic PLS3 overexpressing allele show improved motoric abilities as a consequence of improved innervation at neuromuscular junctions (NMJ), increased NMJs and muscle fibre size as well as an increase of proprioceptive input on motor neuron (MN) soma. Most importantly, PLS3 overexpression delays axon pruning and improves connectivity of the presynapse to motor endplates. Nonetheless, survival is only marginally increased by a few days, as a consequence of massive impairment of inner organs in SMA mice, which is not rescued by PLS3 overexpression (Ackermann et al. 2013). As PLS3 overexpression ameliorates some SMA hallmarks in the severe SMA mouse model, but its modifying function is likely diminished by the multi-organ impairment, we decided to study the effect of elevated PLS3 in an intermediate SMA mouse model, which would more faithfully model the condition of initially identified asymptomatic individuals. To do so, we made use of a low dose SMN-antisense oligonucleotide (ASO) that allows SMN2 splice correction (Hua et al. 2011). In this study, (1) an intermediately-affected SMA mouse model was produced by presymptomatic subcutaneous injection of low dose SMN-ASOs into the severely-affected SMA mouse model; (2) SMN-ASO treated SMA mice on congenic FVB/N background showed a much larger variability in survival than on C57BL/6N background. (3) The optimal conditions turned out to be 30 μg SMN-ASO, injected twice at P2 and P3 in C57BL/6N SMA mice. These mice showed an elongated lifespan from 14 to 28 days. (4) The suboptimal doses of SMN-ASO considerably decreased inner organ defects including intestine, lung and heart. (5) Additional overexpression of the PLS3 transgene either heterozygously or homozygously rescued survival from 28 to >250 days, weight and motoric abilities (grip strength force and tube test). These data proved that PLS3 is a protective modifier of SMA if elevated in an animal model that resembles the situation in the asymptomatic SMN1-deleted individuals. (6) In this low dose SMN-ASO injected intermediate SMA mouse model, PLS3 counteracts the defects within the neuronal circuitry by increasing NMJ size and thereby stabilizing AChR clustering at the postsynaptic site. Presynaptically, PLS3 increases the number of proprioceptive inputs at the MN soma and leads to enlarged MN soma size. (7) Even in MNs isolated from the severely affected SMA mouse model, PLS3 overexpression led to an increased length of the main axon. (8) As PLS3 knockout in yeast impairs endocytosis (Kubler and Riezman 1993), 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 in murine embryonic fibroblasts, which was restored to control levels by overexpression of PLS3. Taken together, our findings in transgenic SMA mice demonstrate the efficacy of a combined treatment with SMN-ASO in addition to PLS3 overexpression, rather than solely increasing SMN levels. Hence, we suggest that compounds, which can restore endocytosis in combination with SMN-ASO therapy might not only improve the SMA phenotype but maybe even result in a complete rescue, and this possibility will be tested in future studies.

Item Type: Thesis (PhD thesis)
CreatorsEmailORCIDORCID Put Code
Peters, Miriampeters-miriam@web.deUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-73785
Date: 14 November 2016
Language: English
Faculty: Faculty of Mathematics and Natural Sciences
Divisions: Zentrum für Molekulare Medizin
Subjects: Natural sciences and mathematics
Life sciences
Uncontrolled Keywords:
Date of oral exam: 16 January 2017
NameAcademic Title
Wirth, BrunhildeProf. Dr. rer. nat.
Refereed: Yes


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