Universität zu Köln

Kaczmarek, Anna (2016). Analysis of reduced Neurocalcin delta (NCALD) as a protective modifier in mouse models of Spinal Muscular Atrophy (SMA). PhD thesis, Universität zu Köln.

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Abstract

Spinal muscular atrophy (SMA) is a common and devastating genetic disease characterized by degeneration of spinal alpha motor neurons and muscle atrophy. SMA is caused by homozygous deletions or rarely other mutations of the SMN1 gene, resulting in a functional loss of the Survival of Motor Neuron (SMN) protein. SMA severity is determined by a nearly identical copy gene, SMN2, which encodes an identical SMN protein but produces only ~10% correctly spliced transcripts. Rarely, individuals with SMN1 deletion are fully asymptomatic, suggesting a protection by other modifying genes. Identification of these modifiers would allow us to better understand the cellular pathways affected by SMN deficiency and to develop new therapies. Here, we report a novel modifier of SMA, Neurocalcin delta (NCALD), that showed a reduced expression in SMN1-deleted asymptomatic individuals as compared to other affected family members or unrelated SMA patients. NCALD is a neuronal calcium sensor mainly expressed in CNS. Further preliminary studies in vitro and in non-mammalian SMA animal models (C. elegans, zebrafish) have shown that NCALD knock-down ameliorates SMA-related symptoms. Therefore, we proceeded to study the protective effect of NCALD suppression in SMA mouse models. First, we followed a strategy of generating an inducible Ncald knock-down mouse. We identified efficient shRNA sequences that suppressed NCALD expression and cloned them into the targeting vector, which was integrated into mouse embryonic stem cells by electroporation. This approach was discontinued when an Ncaldko/ko mouse line became available at the Jackson Laboratory. Next, we crossed the Ncaldko allele heterozygously with a severely affected SMA mouse model and performed extensive phenotypic analysis. Reduced NCALD expression improved many characteristics related to SMN deficiency, such as axonal length of cultured motor neurons, size of acetylcholine receptor (AChR) clusters at the neuromuscular junction and spinal motor neuron circuitry as assessed by the number of glutamatergic inputs on motor neuron soma. However, due to very low SMN levels, other internal organs were impaired, which resulted in early death of the SMA mice irrespective of their NCALD levels. Therefore, we hypothesized that increasing peripheral SMN levels is needed for the modifier to fully exert its function. Indeed, in SMA-Ncaldko/wt mice injected with a suboptimal dose of SMN-ASO (antisense oligonucleotides), all characteristics listed above were reproducibly improved; additionally, also the muscle fiber size was increased. Unexpectedly, also in this intermediate model the survival of the SMA-Ncaldko/wt+ASO mice was not rescued, presumably because NCALD is mainly expressed in neurons and unable to counteract SMN deficiency in other inner organs. Currently, we are performing further studies of NCALD suppression in an even milder SMA model, which shows only a motoric impairment but without a shortened lifespan; this model resembles better the phenotype of asymptomatic individuals, who all carry four SMN2 copies and would hypothetically develop a milder SMA type without compromised survival. Indeed, first results show that mild SMA mice have an improved motoric behavior upon reduced NCALD levels. Finally, we anticipated that NCALD suppression would be eligible for a combinatorial therapy using a mix of ASOs to increase SMN and simultaneously downregulate NCALD. In collaboration with Ionis Pharmaceuticals (USA), a walk along the Ncald gene was performed to identify the most efficient Ncald-ASOs. In our laboratory two ASOs were subsequently tested by intracerebroventricular injections in neonatal mice. While we achieved a satisfying NCALD knock-down, we encountered some toxicity issues; therefore, the therapeutic potential of NCALD suppression requires further examination considering its biosafety and clinical efficiency.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Kaczmarek, Anna kaczmara@smail.uni-koeln.de UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-71196
Date:	31 December 2016
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Zentrum für Molekulare Medizin
Subjects:	Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language Spinal muscular atrophy, modifier genes, Neurocalcin delta UNSPECIFIED
Date of oral exam:	28 November 2016
Referee:	Name Academic Title Wirth, Brunhilde Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/7119