hyl-2(gnv1) and the sensitivity to anoxia

To better understand by which mechanisms the hyl-2(gnv1) mutation could lead to disturbed production of sphingolipids in C. elegans, we used both genetic and biochemical approaches. The genetic approach consisted of the search for suppressors using EMS random mutagenesis. This screen allowed us to identify a suppressor mutation that plays a role in the ceramide pathway. The biochemical

approach consisted of a lipidomic profiling of the suppressor to analyse any possible changes in lipid content compared to hyl-2(gnv1). The lipidomic profiling will be discussed later in the discussion.

The suppressor screen allowed us to find the ttc-1(gnv3) mutation that perfectly restored a normal resistance to the hyl-2(gnv1) mutant by influencing the ceramide homeostasis of the worm. Our results showed that ttc-1(gnv3) consisted of two substitutions: one substitution of the nucleotide 512 in exon 4 (GGA to GAA) and one substitution of the nucleotide 3781 exon 16 (CAG to TAG) resulting in Gly¹⁷¹ to Glu¹⁷¹ and Gln¹²⁶¹ to STOP¹⁶²¹ respectively. We know that downregulation of ttc-1 expression using RNAi can recapitulate the effects provided by the mutations in ttc-1 as described above. To date, we do not know whether mutation of the glycine into glutamic acid is responsible for loss of function and protection against anoxia. This would indicate that the glycine lies into a critical domain of the protein. Alternatively, the loss of function may be due to the premature stop codon due to mutation at position 1621. To know whether both mutations could be independently responsible for the loss of function it would be necessary to produce two transgenic hyl-2(gnv1) mutant strains containing the first or the second mutations respectively, and to test them in anoxia.

The suppressor screen is a powerful approach since it allows for mutation of a very large population of worms at the same time. In addition, since hyl-2(gnv1) has a strong phenotype to anoxia compared to N2, it was simple to screen and to discriminate potential suppressors from sensitive worms. However, we realized in

mutation. The percentage of revertants was around 20%. This observation led us to hypothesise that the site of the allele gnv1 could be a mutation hotspot region.

Consequently, we also performed a suppressor screen in the hyl-2(ok1766) deletion mutant in case only revertants were found with the first suppressor screen. However, in the end, this was not necessary.

To find potential interactors of hyl-2 through another method, we could have performed a reverse genetic screen. For this, it would have been first necessary to generate a list of potential interactors implicated in ceramide homeostasis using an in silico approach. Then, we would have downregulated each candidate gene using RNA interference to test for their ability to restore resistance to anoxia within hyl-2(gnv1).

2.1 Implication of sphingolipid homeostasis in the development of C. elegans and in the protection to anoxia

The hyl-2 gene appears to be important for resistance to anoxia but also seems to play a major role in aging or during development ((Menuz et al., 2009), full publication in appendix 1). We have recently observed that the hyl-2 mutant had an egg-laying defect compared to the wild type animals. Interestingly this defect was almost completely restored by ttc-1(gnv3) (Figure 10). This observation indicates that the ceramides produced by HYL-2 are not only important for resisting anoxia-induced stress but probably play also important roles in various tissues of the worm, including during normoxia.

Figure 10: Brood size of hyl-2(gnv1) in comparison with N2 and ttc-1(gnv3);hyl-2(gnv1), n = 3.

The correlation between defective egg-laying and resistance to stress is a matter of debate. For instance the daf-2-related mutant, rta-1, which is described in the appendix 2, is a long-lived mutant and has a defective brood size. In addition, daf-2(e1370) mutants that have an increased resistance to anoxia also have a defective brood size (Mendenhall et al., 2006; Mendenhall et al., 2009; Pickett and Kornfeld, 2013). It has been hypothesized that decreased embryo-production could allow the worm to save energy and to better resist stresses such as anoxia. However, our observations do not validate this hypothesis since the hyl-2(gnv1) mutant has both an egg-laying defect and a sensitivity to anoxia. Our results suggest that the worm is rather intrinsically weakened by a disturbed sphingolipids homeostasis, which can be compensated by ttc-1(gnv3). The lipidomic analysis of N2, hyl-2(gnv1), ttc-1(gnv3);hyl-2(gnv1) and ttc-1(gnv3) strains after a period of anoxia could be highly informative. Indeed, it might help learning whether anoxia triggers changes in the lipidome that hyl-2(gnv1) could not achieve or whether the sensitivity to anoxia could be due to a negative preconditioning induced by the disturbed sphingolipid homeostasis of hyl-2(gnv1). This is an analysis that we would like to perform but it

strain together at the same time. This is required to obtain a reliable lipidomic analysis. However, the anoxic chamber that we use has a volume that is too small to perform such an experiment.

2.1.1 Complex sphingolipid accumulation might be the cause of the sensitivity to anoxia

Importantly, the results that we obtained during my thesis work first confirmed the observation previously made by Menuz et al, which showed the absence of certain ceramides with C22 fatty acyl chains in hyl-2(gnv1). This suggested a protective role of these ceramides for C. elegans in anoxic conditions. Other important additional results that we obtained during my thesis work revealed that the levels of certain sphingolipid species, C24 and C25 GlcCers and C24, C25 and C26 SMs, which were noticeably increased in hyl-2(gnv1), were normal in the suppressor ttc-1(gnv3);hyl-2(gnv1). These results suggest an important role of ttc-1(gnv3) in influencing sphingolipid homeostasis and suggest that accumulation of sphingolipid species is responsible for the impaired resistance to anoxia in the hyl-2(gnv1) mutant.

This hypothesis can be supported by studies that show the importance of ceramides and sphingolipids in several degenerative disorders. In fact, as previously mentioned, abnormal metabolism of ceramides has been reported to be involved in different neuronal related disorders such as Farber, Niemman Pick’s, Alzheimer and Parkinson diseases (Brady et al., 1966b; Fabelo et al., 2011; Han et al., 2002; He et al., 2010; Levade et al., 1995; Satoi et al., 2005; Sugita et al., 1972). Interestingly a recent study revealed that the accumulation of GlcCer could trigger neuroinflammation and neurodegeneration in specific brain areas in a mouse model

of neuropathic Gaucher disease (Farfel-Becker et al., 2014). Still using the mouse, another group was able to counteract the negative effect of SM accumulation in a Niemann Pick type A model by injecting myristoylated alanine-rich C-kinase substrate (MARCKS) (Trovo et al., 2014). Thus, these studies support a possible toxic effect of GlcCer and SM accumulation in C. elegans lacking the HYL-2 ceramide synthase. It would be interesting to know whether the absence of HYL-2 sensitizes the worm to other stresses, in particular those involving ER stress, since ER membranes are particularly enriched in ceramides.