Francesco Pezzini

Molecular epidemiology of childhood neuronal ceroid-lipofuscinosis in Italy

BackgroundTo review the descriptive epidemiological data on neuronal ceroid lipofuscinoses (NCLs) in Italy, identify the spectrum of mutations in the causative genes, and analyze possible genotype-phenotype relations.MethodsA cohort of NCL patients was recruited through CLNet, a nationwide network of child neurology units. Diagnosis was based on clinical and pathological criteria following ultrastructural investigation of peripheral tissues. Molecular confirmation was obtained during the diagnostic procedure or, when possible, retrospectively.ResultsOne hundred eighty-three NCL patients from 156 families were recruited between 1966 and 2010; 124 of these patients (from 88 families) were tested for known NCL genes, with 9.7% of the patients in this sample having not a genetic diagnosis. Late infantile onset NCL (LINCL) accounted for 75.8% of molecularly confirmed cases, the most frequent form being secondary to mutations in CLN2 (23.5%). Juvenile onset NCL patients accounted for 17.7% of this cohort, a smaller proportion than found in other European countries. Gene mutations predicted severe protein alterations in 65.5% of the CLN2 and 78.6% of the CLN7 cases. An incidence rate of 0.98/100,000 live births was found in 69 NCL patients born between 1992 and 2004, predicting 5 new cases a year. Prevalence was 1.2/1,000,000.ConclusionsDescriptive epidemiology data indicate a lower incidence of NCLs in Italy as compared to other European countries. A relatively high number of private mutations affecting all NCL genes might explain the genetic heterogeneity. Specific gene mutations were associated with severe clinical courses in selected NCL forms only.

Proteomic analysis of the palmitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblastoma cells

UNLABELLED Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway. BIOLOGICAL SIGNIFICANCE The significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.

Variant late infantile ceroid lipofuscinoses associated with novel mutations in CLN6

The neuronal ceroid lipofuscinoses (NCL) are heterogeneous neurodegenerative disorders with typical autofluorescence material stored in tissues. Ten clinical NCL forms and eight causative genes are known. Mutations in CLN6 have been reported in roughly 30 patients, mostly in association with the variant late-infantile NCL (v-LINCL) phenotype. We screened CLN6 in 30 children from a cohort of 53 v-LINCL cases and revised their clinical and ultrastructural features. We detected 11 mutations, eight of which are novel, all predicting a direct impairing of the putative gene function. No clear-cut genotype-phenotype correlations were observed, with inter- and intra-familial variability evident for few recurrent mutations. Ultrastructural findings were suggestive of an impaired regulation of the autophagic vacuoles turnover. While expanding the array of CLN6 mutations, we showed that more than half of our v-LINCL cases lack a DNA confirmation and further molecular etiologies are to be searched.

Pseudo-dominant inheritance of a novel CTSF mutation associated with type B Kufs disease

Neuronal ceroid lipofuscinosis (NCL) has different forms, of which Kufs disease (KD) is the least frequent and the most difficult to diagnose.1 KD can, in turn, be divided into type A, characterized by progressive myoclonus epilepsy and cognitive decline, and type B, characterized by movement and behavioral abnormalities and dementia.2 Mutations in CLN6 and DNAJC5 are responsible for, respectively, the autosomal recessive (AR) (MIM 204300) and autosomal dominant (MIM 162350) forms of type A KD.3 Mutations in cathepsin F (CTSF) have recently been discovered in AR type B KD families of French-Canadian, Australian, and Italian origin.4

Neuronal Ceroid Lipofuscinosis: The Increasing Spectrum of an Old Disease

Neuronal Ceroid Lipofuscinoses (NCL) are genetically heterogeneous heritable neurodegenerative disorders with worldwide distribution. They are considered as childhood diseases; however rare adult onset forms are known. NCL have a progressive course, affecting visual, motor and cognitive functions, and are associated with myoclonic epilepsy; behavioural problems can be observed at the onset. The outcome is invariably fatal, mostly during the second or third decade. The denomination is based on pathological criteria, i.e. the presence of intralysosomal storage of autofluorescent lipopigment of glycoprotein origin with characteristic ultrastructural features. The NCL are autosomal recessive diseases (but a rare autosomal dominant form of adult onset). Thirteen NCL associated genes have been identified so far, which allow a definite diagnosis to be reached and provide genetic counselling to the families. Still unidentified NCL genes are foreseen. Allelic heterogeneity is observed in some mutated genes; likewise phenotypic heterogeneity is seen in several NCL. The gene products are either soluble proteins (such as lysosomal enzymes) or membrane proteins related to lysosomes, endoplasmic reticulum, synaptic vesicles. Little is known about pathogenetic mechanisms, leading to storage formation and cell death. Current research is focusing on intracellular trafficking, neurotransmission and storage removal. No cure is available for any form. Innovative treatments led to some results in mouse models related to lysosome hydrolase defects. Evidences that autophagy, oxidative stress, excitotoxicity play roles in NCL cell pathology raise the possibility that selected steps of these processes might become target of treatments, and therefore modify the disease course.

Involvement of the mitochondrial compartment in human NCL fibroblasts.

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

Quantitative analysis of PPT1 interactome in human neuroblastoma cells

Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015) 42–53).

Phenotype and natural history of variant late infantile ceroid-lipofuscinosis 5

To characterize the phenotypic profile of a cohort of children affected with CLN5, a rare form of neuronal ceroid‐lipofuscinosis (NCL), and to trace the features of the natural history of the disease.

Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

Early infantile neuronal ceroid lipofuscinosis (CLN10 disease) associated with a novel mutation in CTSD

The neuronal ceroid lipofuscinoses (NCLs) are the largest group of neurodegenerative diseases of childhood [1]. In the majority of cases, the clinical presentation occurs at preschool age, and such cases account for around 70 % of the NCL cohort recruited by the Italian network CLNet [2]. Clinical onset of NCL within the first year of life (infantile NCL; INCL) is less frequent; this form is mostly related to mutations in the PPT1/CLN1 gene [3]. Congenital NCLs are extremely rare. To date, five cases, all microcephalic, have been reported; three of these babies carried homozygous mutations in cathepsin D (CTSD), a gene coding for a lysosomal aspartic protease [4–7], and none of them lived longer than 10 days. Biallelic mutations in CTSD have been reported in five patients, who developed either late infantile or juvenile forms of the disease associated with granular osmiophilic deposits (GRODs) in both skin and skeletal muscle [8, 9]. Cases carrying CTSD mutations have been classified, together, as affected by CLN10 disease [10]. We report the case of a female child with INCL, who developed early and dramatic decreased rate of head growth with acquired microcephaly and marked cerebral atrophy (Fig. 1), a relatively late appearance of severely progressive epilepsy (displaying a ‘‘vanishing’’ EEG pattern), GRODs in a skin biopsy, and hypertrophic cardiomyopathy (see Supplementary Material). Defective enzymatic activity of CTSD was detected by a mass spectrometry (MS)-based assay in dried blood spots (DBSs). Sanger sequencing showed that the child harbored a novel homozygous c.205G[A, p.Glu69Lys located in exon 2 of CTSD (Fig. 2). The mutation, absent in a large series of exome polymorphic databases and in a set of ethnically matched healthy controls, was heterozygous in her healthy parents and sister, and it was associated with nearly absent enzymatic activity of CTSD in DBSs and a significant reduction of protein expression in cultured skin fibroblasts. The storage material present in the skin biopsy of this patient has seldom been described in CLN10 disease [5, 8, 9], whereas it is commonly detected in both CNS and peripheral tissues in the CLN1 disease and adult-onset CLN4 [11]. On the basis of the Cln1 mouse model, it is hypothesized that the lack of synergic action of CTSD and PPT1 engulfed lysosomes with undegraded proteins and organelles, which favors the formation of GRODs [12]. The present case presented the atypical association of encephalopathy with hypertrophic cardiomyopathy, already observed in juvenile CLN10 disease (case 2 in [9]), S. Doccini, S. Sartori and S. Maeser contributed equally.