CHARACTERIZATION OF CELLULAR AND MOLECULAR MECHANISMS IN CELLULAR MODELS OF NEURONAL CEROID LIPOFUSCINOSES DISEASES

CHARACTERIZATION OF CELLULAR AND MOLECULAR MECHANISMS IN CELLULAR MODELS OF NEURONAL CEROID LIPOFUSCINOSES DISEASES The Neuronal Ceroid Lipofuscinoses (NCLs) represent a progressive group of inherited neurodegenerative diseases affecting children and adults, characterized by retinopathy leading to blindness, ataxia and gait abnormalities, drug-resistant progressive myoclonic epilepsy, mental deterioration, and an early death. NCL diseases were classified according to the age at disease onset (congenital, infantile, late infantile, juvenile, adult). They are considered lysosomal storage disorders, because of their characteristic accumulation of autofluorescent ceroid lipopigments, in lysosomes of most cells, including neurons, skin fibroblasts and blood lymphocytes. The NCL share clinical manifestations, biochemistry, and pathology, hence their classification together. NCL protein deficiencies result in similar cell biologic phenotypes including dysregulation of apoptosis and autophagy, prolonged inflammation, disturbed endoplasmic reticulum–cytosol calcium balance, impaired oxidative metabolism, and abnormal lipid trafficking. There are evidences that NCL proteins are essential components of a common pathway and that they may interact at multiple locations. The possibility that individualized or specific mechanisms are more important in specific subtypes could offer more personalized approaches to cure. Although the genetic underpinnings of most forms of NCL have been identified, the pathway(s) in which neuronal death results in the severe degenerative phenotype remain unknown, and there is no available rational basis for treatments. In this work we attempted to clarify the cellular and molecular mechanisms underlying three different forms of NCLs disease using patients’ material and cell models. Our focus was on the identification of distinctive pathways that could characterize, at least in part, the diseases associated with different genes: CLN1 (causing an Infantile type), CLN5 (determining a Late Infantile form) and CLN13-CTSF (responsible for Adult-NCL). Our data indicate specific roles for the three affected proteins as follow. The CLN1 protein (INCL) seems to be implicated in Oxidative metabolism pathways We analyzed the mitochondrial compartment in CLN1 deficient skin fibroblasts and in an in vitro system of human neuroblastoma cells, SH-SY5Y, reproducing the pathological features seen in “Mediterranean” mutations in CLN1. Mitochondrial morpho/functional analysis showed an involvement of the mitochondrial compartment in both cellular systems. Our data confirmed previous observations reporting abnormalities in number, morphology and intracellular localization pattern of mitochondria in pathological primary cell lines. Moreover, a similar phenotype was seen in a good cellular model with neuronal-like structure. Mitochondrial dysfunctions could be, thus, involved in the pathogenesis of NCLs implying that mechanisms such as oxidative stress or apoptosis-induced via mitochondrial pathway can occur in the brain of CLN1 patients and offers further support to the possibility that small nucleophilic molecules with anti-oxidant properties might have beneficial effects on the neurodegenerative process of the disease. The CLN5 protein (LINCL) is a player in abnormal lipid metabolism The CLN5 disease is a variant form of late-infantile NCL and the exact pathogenetic mechanisms leading gene mutation to cause the disease is still unclear. Clinical and neuropathological similarities in NCL disorders may result from functional redundancy or co-operation of different NCL proteins (Vesa et al., 2002; Sleat et al., 2006) and it is known that the addition of CLN8p (the product of an additional NCL gene) or CERS1 (ceramide synthase) in CLN5-deficient patient fibroblast cells brings correction to the pathological phenotype. We demonstrated the molecular interaction between CLN5, other NCL proteins, several lysosomal hydrolases and ceramides in an in vitro cellular system. In particular we found novel interactions between the CLN5 protein and four ceramide synthases (CERS1, CERS2, CERSS4, and CERS5) that synthesize ceramides, central molecules of sphingolipid metabolism in mammalian cells. Alike CLN8, also CLN5 seems to reside in the ER compartment but the presence of CLN5p in the ER is independent of the CLN8p. Our results confirmed that the ER is an important hub station for the function and/or trafficking of NCL proteins and that these role do not imply a direct connection with mitochondrial inner membrane or mitochondrial bioenergetics. These data foresee a deeper investigation of ceramides (levels, pathway of synthesis or both) in CLN5 disease, the possible connections with the activation of autophagy or mitophagy pathways, and the ensuing consequences on sphingolipid and phospholipid metabolisms. The CLN13 protein (cathepsin F) has a key role in Autophagy mediated processes We characterized at the cellular level a novel c.213+1G>C in CTSF associated with ANCL- Kufs disease (KD) Type B (CLN13). The mutation affects correct splicing, removing exon 1 and predicting a chopped N-terminus of cathepsin F, with a probable loss-of-function mechanism. Overexpression of N-terminus truncated forms of human cathepsin F (Δ-CtsF) in HEK 293T cells has recently been associated with features suggestive of aggresome-like inclusions and multifunctional polyubiquitinated proteins. We demonstrated in CTSF hypofunctioning primary cells from three CLN13 patients ultrastructural features resembling aggresome-like structures in skin biopsies. These features were biochemically replicated in cultured fibroblasts, where we observed an increase of autophagic proteins highly reminiscent of what had been seen in HEK 293T cells (Jerič et al., 2013). All these results were suggestive of dysregulated autophagy, and points to an as yet unexplored connection between autophagy, proteasomal dysfunction and polyUb proteins in triggering neuronal cell death in adult-forms of NCL. To sum up, in this study we dissected at the cell level some of the specificities in three different forms of NCL pathology that are mostly relevant to Italian patients and our results pinpoint different steps in NCL disease and the involvement of specific cellular processes in the pathophysiological function of NCL proteins. In perspective, all these steps and their specificities should be further explored to define steps more relevant as those might represent specific targets for developing more rationale therapeutic options.