The cln-3 genes of Caenorhabditis elegans : making C. elegans models for Juvenile Neuronal Ceroid Lipofuscinosis.
Voer, G. de
Citation
Voer, G. de. (2008, May 7). The cln-3 genes of Caenorhabditis elegans : making C. elegans models for Juvenile Neuronal Ceroid Lipofuscinosis. Retrieved from
https://hdl.handle.net/1887/12840
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CHAPTER 7
Summary
170 Summary | C H A P T E R 7 The cln-3 genes of Caenorhabditis elegans
Summary
This thesis describes our exploration of the potential to use the nematode C. elegans as a model organism for juvenile neuronal ceroid lipofuscinosis (JNCL), the most common neurodegenerative genetically inherited disease of childhood. No treatment is available for this devastating lysosomal storage disorder, which is caused by mutations in the CLN3 gene. In lysosomes of patients material with the characteristics of ceroid and lipofuscin accumulates and Subunit c of the mitochondrial ATP synthase (Subunit c) is the major component of this material. How mutations in CLN3 lead to the neuronal symptoms, such as loss of vision, epileptic seizures, and dementia is unknown.
Homologues of the CLN3 protein are being investigated in model organisms, such as baker’s yeast Saccharomyces cerevisiae and mice, to elucidate the mechanisms by which these genetic lesions cause the symptoms. The research into the CLN3 homologues in these model organisms has proven useful as they have provided insight into the protein function at the molecular and organismal levels, respectively, but this has not resulted in a complete understanding of JNCL pathology. Therefore, the study of CLN3 homologues in relatively simple multicellular model organisms that have a nervous system would most likely complement research performed in other models.
A model organism that has proven to be useful for research into proteins involved in neurodegenerative diseases is the nematode Caenorhabditis elegans and the data obtained with this model organism can be extrapolated to more complex organisms, such as humans (Chapter 2).
C. elegans nematodes have three proteins, designated CLN-3.1, CLN-3.2, and CLN- 3.3, that are homologous to the human CLN3 protein (Chapter 3). For each of the genes that encode these proteins nematode knock-outs were generated (Chapter 3, 4).
The cln-3.1 deletion mutant has a slightly decreased life span compared to wildtype animals, and the cln-3.2 deletion mutant has a little decrease in brood size (Chapter 4).
Since no neurological or other robust phenotypes were present in the cln-3 mutants, which could be due to redundancy, the mutations were combined into a triple mutant strain. This cln-3 triple mutant is viable, indicating that the cln-3 genes are not essential for nematode life under standard laboratory conditions. Cln-3 expression analysis was performed using GFP reporter constructs to identify specific nematode life stages and cells or tissues on which the phenotypic analysis of the cln-3 mutants could be focused (Chapter 4). The cln-3.1 promoter regulated GFP expression in the intestine, cln-3.2 was expressed in the hypoderm, and the cln-3.3 reporter indicated expression in the intestinal muscle, male specific posterior muscle cells and hypoderm.
The cln-3 triple mutant was thoroughly analyzed, but no neurological phenotype could be found and no stored material could be identified on electron micrographs of triple mutant nematodes. An explanation for the absence of accumulated materials could be the short life span of C. elegans. Therefore, transgenic nematodes were generated in
C H A P T E R 7 | Summary 171
Making C. elegans models for Juvenile Neuronal Ceroid Lipofuscinosis
which Subunit c of the mitochondrial ATP synthase, the main component of the stored material in patients, was inducibly overexpressed and these transgenes were crossed into the cln-3 triple mutant background (Chapter 5). Overexpression of Subunit c is deleterious to wildtype nematodes and appears to affect mitochondrial ultra-structure.
The effect of Subunit c overexpression was not different in cln-3 mutant nematodes.
Perhaps, the overexpressed protein could be targeted to lysosomes or overexpression induction could be optimized to be able to observe differences between wildtype and cln-3 triple mutant background.
Although a neuronal phenotype was not found, the nematode model presented here can be used for additional experiments, which may increase our understanding of the function of the cln-3 genes.
