Abstract
The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a heterogeneous group of inherited neurodegenerative disorders, clinically characterised by blindness, myoclonic epilepsy, cerebral atrophy, and progressive cognitive and motor decline. To date, there are 13 genetically-identified variants, which collectively represent the most common neurodegenerative disorders of childhood. Despite diverse genetic aetiologies, the NCLs are grouped together based on common principle features, namely progressive neurodegeneration and the lysosomal accumulation of autofluorescent storage material. Currently there is no curative therapy, although there are multiple strategies for controlling disease symptoms.
The majority of the NCL-associated genes encode proteins residing in the endosomal/lysosomal pathways, although in many cases the primary functions of these proteins remain largely undefined. Two NCL-related genes of unknown function are CLN5, a soluble lysosomal protein, and CLN6, a transmembrane endoplasmic reticulum (ER) protein. The aims of this work were to identify molecular changes underpinning CLN5 and CLN6 disease and, as a result, help to inform potential therapeutic strategies.
In order to investigate the cellular and molecular changes occurring in CLN5 and CLN6 disease, several naturally occurring animal disease models were utilised. Firstly, isolated primary neural cultures from CLN5-/- and CLN6-/- ovine disease models identified perturbations of endosomal, autophagic and lysosomal networks. Using these diseaseassociated alterations as biomarkers, several candidates were trialled for therapeutic efficacy. Firstly, lentiviral mediated gene therapy was used to introduce a wild-type (WT) copy of the mutant gene. Secondly, due to the previously observed autophagic and lysosomal defects, compounds were tested to increase autophagic activity. Both gene therapy and autophagy induction appeared to restore biomarker activity in both the CLN5-/- and CLN6-/- culture models, suggesting disease correction and the possibility of a common therapeutic.
Based upon the success of ameliorating autophagy-related disease biomarkers in ovine cell culture models, two therapeutic approaches were tested further in the Cln6nclf mouse model. CLN6 therapy was focused on, as membrane-protein deficiencies are invariably harder to treat with traditional routes, such as gene therapy. In correlation with the sheep model, the Cln6nclf mouse showed autophagy changes, with altered expression of both phagophore and lysosomal markers. Firstly, an alternate approach to CLN6 gene therapy was explored, which looked at modulating the expression of secretable factors lost in disease, alternate to CLN6. This work indicated aberrant regulation of Cln5 and Cln2 in the Cln6nclf mouse, as well as an altered secretome that may be potentiating negative effects to healthy cells. As a second approach, a Food and Drug Administration (FDA) approved compound, gemfibrozil, was used to upregulate autophagy in the Cln6nclf mouse. Treatment reduced the presence of autofluorescent storage material, and decreased inflammatory phenotypes, suggesting gemfibrozil may be of therapeutic benefit in CLN6 Batten disease.
Collectively, this data identifies novel common endosomal, autophagy and lysosomal phenotypes, in several models of CLN5 and CLN6, and suggests that modulating the secretome and/or increasing autophagy gene expression may be beneficial for treating CLN6. Further determining the mechanisms underlying disease, as well as the positive gemfibrozil related-changes, will be key to improving and tailoring future therapeutic strategies.