Molecular interactions underlying neuronal ceroid lipofuscinoses CLN1 and CLN5

NCL disorders, neuronal ceroid lipofuscinoses, comprise the most common group of neurodegenerative diseases in children. The incidence of these hereditary disorders is approximately 1 : 12 500 worldwide. Features common to all NCLs include accumulation of the autofluorescent intracellular storage material, ceroid lipofuscine, in the tissues of patients, and progressive neuronal death, leading to blindness, psychomotor retardation, and premature death. Disease-causing mutations have been characterized in eight different genes so far, but the cellular mechanisms leading to neurodegeneration are still largely unknown. Infantile NCL (INCL), together with congenital NCL, is the most severe form of the NCL disorders. The clinical manifestations include progressive visual failure, epileptic seizures, and ataxia. INCL patients die approximately at the age of ten. The CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme is mutated in INCL. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. CLN5 is a Finnish variant of late infantile NCL (vLINCLFin). The defective gene, CLN5, encoding for the CLN5 protein, was found in 1998. The first symptoms of vLINCLFin, visual failure and motor deterioration, are observed before school age. Progressive mental retardation leads to death at early adulthood. The function of CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases enriched in Finland. The focus has been on the molecular interactions of the defective proteins. It was previously known that the intracellular localization of PPT1 is different in neurons than in other cell types. In neurons, it localizes mainly to axonal presynaptic compartments instead of lysosomes, where it is found in other cell types. In this study we observed that the processing of PPT1 was different between neuronal cells and connective tissue cells, fibroblasts. Furthermore, the intracellular trafficking of overexpressed PPT1 was found to differ from the classic trafficking pathway utilized by another lysosomal enzyme, AGA. These findings implicate novel properties for the PPT1 enzyme in neurons, and are significant when designing therapeutic strategies for INCL. PPT1 was also found in high molecular weight complexes and its activity was separated into two fractions. The protein that formed a complex with PPT1 and purified with it was identified as the -subunit of the mitochondrial ATP synthase. Later we found that PPT1 interacted with the entire F1-complex that includes the -subunit. The F1-complex has been localized to the cell surface in addition to mitochondria, and in hepatocytes it has been shown to participate in HDL metabolism. The connection between PPT1 and the localization of F1-subunits was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. Our results show that in Ppt1-deficient neurons, the amount of F1-subunits was specifically increased on the cell surface, while this was not detected in fibroblasts. Studies conducted with neuronal cells and serum samples further showed several changes in lipid metabolism both at the cellular and systemic levels, since the cellular uptake of apolipoproteins and the lipoprotein particle size were altered in Ppt1-deficient mice compared to controls. Thus, the identification of the first interaction partner for PPT1 led on to the studies connecting the altered neuronal function and disturbancies in lipid metabolism in INCL. In the last part of the thesis, the focus was to elucidate the interactions between different NCL proteins. The reasoning behind this was the possible common metabolic route affected in NCL disorders, leading to similar clinical and neuropathological manifestations. CLN5 was chosen as the center molecule, as it was already known to interact with two other NCL proteins, CLN2 and CLN3, and it represents an NCL protein with an entirely elusive function. We detected three novel interactions between CLN5 and other NCL proteins, including an interaction with PPT1. The interaction between CLN5 and PPT1 turned out to be especially strong, as both proteins affected the intracellular transport of each other. In addition, we detected an interaction between CLN5 and the F1-complex, further supporting a functional connection between CLN5 and PPT1. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level, where they possibly modify the phenotypes of each other. This is supported by the bidirectional interconnection of CLN5 and PPT1 in intracellular protein sorting. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism is strengthened by the characterization of the first interaction partner for PPT1, introducing a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and the development of therapies for these diseases.