Interpretación de los análisis de procedencia

NCL patients with diseases caused by mutations in different genes have similar clinical, pathological and biochemical phenotypes. Many NCL proteins are trafficked through the ER- Golgi network and located in lysosomes, suggesting that they may share common cross- linked pathways, where different dysfunctions resulted in similar pathologies (Lyly et al., 2009; Getty and Pearce 2011; Palmer et al., 2015). NCL protein interactions have been described in a number of in vitro experiments, which suggest that the mutated growth of

patient cell lines were reversed by transfecting functional NCL proteins (Vesa et al., 2002; Persaud-Sawin et al., 2007; Lyly et al, 2009).

CLN5, a lysosomal protein, may have a central role. It has been suggested to interact with several other NCL proteins in vitro, including PPT1/CLN1, TPP1/CLN2, CLN3, CLN6 and CLN8 (Vesa et al., 2002; Lyly et al., 2009; von Schantz et al., 2009; Getty and Pearce 2011; Palmer et al., 2015). Interactions between CLN5 and CLN2 have been reported to occur after the proteins have left the ER (von Schantz, 2009), possibly in lysosomes or late endosomes (Vesa et al., 2002). CLN5 and CLN1 were suggested to share the same pathway, from the ER to the lysosome, in the CLN5 and CLN1 knockout mice brains (von Schantz et al., 2008; Lyly et al., 2009). Lyly and the co-workers (2009) found that co-expression of CLN1/PPT1 in cell culture could restore CLN5 in the overexpressed COS cells transfected with mutated mouse CLN5. Trafficking of CLN3 was partially affected by the simultaneous expression of mutated CLN5, possibly suggesting that CLN5 and CLN3 proteins interact in the ER (von Schantz, 2009). CLN5 and CLN8 have also been suggested to be related (Haddad et al., 2012).

CLN6, resident in the ER, has been suggested to play a role in protein trafficking pathways (Heine et al., 2004), especially in a network involving other NCL associated proteins (Figure 5.2). An investigation has tested whether mutated ovine CLN6 affected the activity of CLN1, CLN2 and CLN10 proteins (Palmer et al., 2015, 2017). This study indicated that the CLN6 mutation did not disrupt the trafficking of CLN1/PPT1 and CLN2/TPP1 to the lysosome, but CLN10/Cathepsin D enzyme activity in lysosomes was decreased.

Some studies have suggested that CLN5 and CLN6 are likely to interact in the CNS (Lyly et al., 2009; Warrier et al., 2013). Despite having mutations in different genes and having different cellular localisations, the development of clinical and neuropathological disease in CLN5 and

Figure 5.2 Schema of potential NCL protein-protein interactions Arrows indicate reported CLN5 interactions (Lyly et al., 2009; Vesa et al., 2002; von Schantz et al., 2009) and CLN6 interactions (this study; Palmer et al., 2015).

CLN6 affected sheep is very similar. They both result in the accumulation of lysosome- derived storage bodies containing large amounts of the c subunit of ATP synthase, retinal degeneration, seizures and pre-mature death, and follow a similar time-course. Affected sheep brains of both genotypes appear to be normally developed at birth. The masses of CLN5 and CLN6 affected sheep brains peak at 4 months of age, falling behind normal controls at this stage by 11% and 19% respectively, marking the start of progressive brain atrophy (Mitchell, 2016). This similarity is unexpected as the CLN5 and CLN6 genes and mutations are different and unrelated. CLN5 disease is caused by an intronic splice-site mutation resulting in deletion of exon 3 from the mRNA for a soluble lysosomal protein, while CLN6 defects result from a 5’UTR-exon 1 deletion from the gene for an ER membrane bound protein.

Gene therapies provided to small and large animals have shown persistent transgene expression (Jarraya et al., 2009; Lattanzi et al., 2010; Palmer et al., 2015). Affected soluble lysosomal proteins, such as CLN1, CLN2, CLN5 and CLN10, are considered potentially

amenable to viral-mediated gene therapy by “cross-correction” when secreted proteins are taken up by adjacent or distal protein-deficient cells (Fratantoni et al., 1968). Promising results achieved from Western blotting and immunofluorescent studies showed that affected CLN5 foetal neural cells transduced with LV CLN5 expressed CLN5 in vitro (see Chapter 4, Figure 4.8). This is in agreement with the studies of in vivo AAV/lentiviral

injections expressing CLN5 into CLN5 affected Borderdate sheep (Mitchell, 2016). Two years post-injection, the treated CLN5 affected sheep remained disease-free at 27 months of age, with the exception of delayed visual deficits. Compared with untreated animals, they benefited from an improvement in the quality of life, preservation of neurological function, and normalisation of brain structure and volume. Similarly, in vivo viral injections expressing CLN6 into CLN6 affected South Hampshire sheep were studied, but only 1 out of 6 treated animals had inhibited NCL neurobehavioural dysfunction and a normal brain structure at 27 months of age (Mitchell, 2016). The different outcomes to similar treatment by the CLN5 and CLN6 affected animals is likely due to the differences in the proteins. As mentioned before, CLN6 is an intracellular membrane protein that is not secreted like CLN5, and thus is unlikely benefit from extracellular cross-correction.

Co-transduction of LV CLN6 and LV CLN5 restored CLN5 expression in CLN6 _{affected cells}

with both viruses compared with the cells transduced with LV CLN5 alone (Figure 4.9). These results could arise if CLN6 dysfunction caused ER associated protein degradation, therefore impeding the system of folding and glycosylation of CLN5 and preventing CLN5 from being exported out of the ER, or entering the CLN5 M6P trafficking pathway from the ER to lysosomes. Lack of CLN6 could also limit the endocytic pathway returning secreted CLN5 to cells, in agreement with the effect on the endocytic pathway of an exogenous protein, arylsulfatase A (ASA), in CLN6affected cells (Heine et al., 2004).

Other studies are in agreement with these findings. Antibodies specific for the ovine CLN5 protein revealed very little CLN5 expression in CLN6 affected sheep and only intermediate staining in heterozygous CLN6 sheep (Mitchell, 2016). A further quantitative PCR study of brain samples revealed that full length CLN5 mRNA expression was upregulated 3-4 fold in CLN6 affected brains of both South Hampshire and Merino sheep in previous studies, but this does not result in successful CLN5 protein expression (McIntyre, 2014; Palmer et al., 2017). These results indicate that the CLN5 and CLN6 protein and gene expressions may be cross-regulated. If CLN6 is required for the correct processing of the CLN5 protein, then it may be that the majority of the CLN5 protein may not reach the lysosome in its fully

modified and folded mature form in CLN6 affected sheep, thus these CLN5 isoforms cannot function, and their related enzyme activity may be greatly reduced, resulting in an induced lysosomal CLN5 deficiency disease.