Palmitoyl protein thioesterase 1 (PPT1) deficiency causes endocytic defects connected to abnormal saposin processing

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of the childhood caused by mutations in the gene encoding palmitoyl protein thioesterase 1 (PPT1). PPT1 localizes to late endosomes/lysosomes of non-neuronal cells and in neurons also to presynaptic areas. PPT1-deficiency causes massive death of cortical neurons and most tissues show an accumulation of saposins A and D. We have here studied endocytic pathways, saposin localization and processing in PPT1-deficient fibroblasts to elucidate the cellular defects resulting in accumulation of specific saposins. We show that PPT1-deficiency causes a defect in fluid-phase and receptor-mediated endocytosis, whereas marker uptake and recycling endocytosis remain intact. Furthermore, we show that saposins A and D are more abundant and relocalized in PPT-deficient fibroblasts and mouse primary neurons. Metabolic labeling and immunoprecipitation analyses revealed hypersecretion and abnormal processing of prosaposin, implying that the accumulation of saposins may result from endocytic defects. We show for the first time a connection between saposin storage and a defect in the endocytic pathway of INCL cells. These data provide new insights into the metabolism of PPT1-deficient cells and offer a basis for further studies on cellular processes causing neuronal death in INCL and other neurodegenerative diseases.     

Palmitoyl-protein thioesterase 1 deficiency in Drosophila melanogaster causes accumulation of abnormal storage material and reduced life span

Human neuronal ceroid lipofuscinoses (NCLs) are a group of genetic neurodegenerative diseases characterized by progressive death of neurons in the central nervous system (CNS) and accumulation of abnormal lysosomal storage material. Infantile NCL (INCL), the most severe form of NCL, is caused by mutations in the Ppt1 gene, which encodes the lysosomal enzyme palmitoyl-protein thioesterase 1 (Ppt1). We generated mutations in the Ppt1 ortholog of Drosophila melanogaster to characterize phenotypes caused by Ppt1 deficiency in flies. Ppt1-deficient flies accumulate abnormal autofluorescent storage material predominantly in the adult CNS and have a life span 30% shorter than wild type, phenotypes that generally recapitulate disease-associated phenotypes common to all forms of NCL. In contrast, some phenotypes of Ppt1-deficient flies differed from those observed in human INCL. Storage material in flies appeared as highly laminar spherical deposits in cells of the brain and as curvilinear profiles in cells of the thoracic ganglion. This contrasts with the granular deposits characteristic of human INCL. In addition, the reduced life span of Ppt1-deficient flies is not caused by progressive death of CNS neurons. No changes in brain morphology or increases in apoptotic cell death of CNS neurons were detected in Ppt1-deficient flies, even at advanced ages. Thus, Ppt1-deficient flies accumulate abnormal storage material and have a shortened life span without evidence of concomitant neurodegeneration.     

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.     

RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis

Palmitoyl-protein thioesterase-1 (PPT1) deficiency causes infantile neuronal ceroid lipofuscinosis (INCL), a devastating childhood neurodegenerative storage disorder. We previously reported that neuronal apoptosis in INCL is mediated by endoplasmic reticulum-stress. ER-stress disrupts Ca²⁺-homeostasis and stimulates the expression of Ca²⁺-binding proteins. We report here that in the PPT1-deficient human and mouse brain the levels of S100B, a Ca²⁺-binding protein, and its receptor, RAGE (receptor for advanced glycation end-products) are elevated. We further demonstrate that activation of RAGE signaling in astroglial cells mediates pro-inflammatory cytokine production, which is inhibited by SiRNA-mediated suppression of RAGE expression. We propose that RAGE signaling contributes to neuroinflammation in INCL.     

Pathogenesis and therapies for infantile neuronal ceroid lipofuscinosis (infantile CLN1 disease)

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative diseases. Infantile neuronal ceroid lipofuscinosis (INCL, infantile Batten disease, or infantile CLN1 disease) is caused by a deficiency in the soluble lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1) and has the earliest onset and fastest progression of all the NCLs. Several therapeutic strategies including enzyme replacement, gene therapy, stem cell-mediated therapy, and small molecule drugs have resulted in minimal to modest improvements in the murine model of PPT1-deficiency. However, more recent studies using various combinations of these approaches have shown more promising results; in some instances more than doubling the lifespan of PPT1-deficient mice. These combination therapies that target different pathogenic mechanisms may offer the hope of treating this profoundly neurodegenerative disorder. Similar approaches may be useful when treating other forms of NCL caused by deficiencies in soluble lysosomal proteins. Different therapeutic targets will need to be identified and novel strategies developed in order to effectively treat forms of NCL caused by deficiencies in integral membrane proteins such as juvenile neuronal ceroid lipofuscinosis. Finally, the challenge with all of the NCLs will lie in early diagnosis, improving the efficacy of the treatments, and effectively translating them into the clinic. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

The Batten disease Palmitoyl Protein Thioesterase 1 gene regulates neural specification and axon connectivity during Drosophila embryonic development

Palmitoyl Protein Thioesterase 1 (PPT1) is an essential lysosomal protein in the mammalian nervous system whereby defects result in a fatal pediatric disease called Infantile Neuronal Ceroids Lipofuscinosis (INCL). Flies bearing mutations in the Drosophila ortholog Ppt1 exhibit phenotypes similar to the human disease: accumulation of autofluorescence deposits and shortened adult lifespan. Since INCL patients die as young children, early developmental neural defects due to the loss of PPT1 are postulated but have yet to be elucidated. Here we show that Drosophila Ppt1 is required during embryonic neural development. Ppt1 embryos display numerous neural defects ranging from abnormal cell fate specification in a number of identified precursor lineages in the CNS, missing and disorganized neurons, faulty motoneuronal axon trajectory, and discontinuous, misaligned, and incorrect midline crossings of the longitudinal axon bundles of the ventral nerve cord. Defects in the PNS include a decreased number of sensory neurons, disorganized chordotonal neural clusters, and abnormally shaped neurons with aberrant dendritic projections. These results indicate that Ppt1 is essential for proper neuronal cell fates and organization; and to establish the local environment for proper axon guidance and fasciculation. Ppt1 function is well conserved from humans to flies; thus the INCL pathologies may be due, in part, to the accumulation of various embryonic neural defects similar to that of Drosophila. These findings may be relevant for understanding the developmental origin of neural deficiencies in INCL.     

pdf1, a palmitoyl protein thioesterase 1 Ortholog in Schizosaccharomyces pombe: a yeast model of infantile Batten disease

Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H(+)-ATPase (vma) mutants in S. cerevisiae. Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for the study of human infantile Batten disease.     

Characterization of candidate genes for neuronal ceroid lipofuscinosis in dog

The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of monogenic autosomal recessive inherited progressive neurodegenerative diseases characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Until today, eight forms of NCL have been classified in humans by clinical criteria, which result from mutations in at least six different genes (TPP1, CLN2, PPT1, CLN5, CLN6, and CLN8). NCL has also been reported in various domestic animal species including cattle, goat, sheep, cat, and certain dog breeds. In this report, the experimental analysis of canine PPT1, CLN5, CLN6, and CLN8 full-length cDNA sequences is described, and the current whole genome sequence assembly was used for gene structure analyses. Characterization of the four canine genes revealed a conserved organization with respect to the human orthologs. In general the gene size in dog is smaller compared to the human sequence due to shorter intron length. Using four individuals of Tibetan terrier with NCL, and a single affected Polish Owczarek Nizinny (PON) dog, we excluded the complete coding region of canine PPT1 and CLN8 and three of four exons of CLN5 and six of seven exons of CLN6 harboring disease-causing mutations.     

Thematic review series: lipid posttranslational modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.     

CLN6, which is associated with a lysosomal storage disease, is an endoplasmic reticulum protein

The neuronal ceroid lipofuscinoses (NCLs) are severe inherited neurodegenerative disorders affecting children. In this disease, lysosomes accumulate autofluorescent storage material and there is death of neurons. Five types of NCL are caused by mutations in lysosomal proteins (CTSD, CLN1/PPT1, CLN2/TTPI, CLN3 and CLN5), and one type is caused by mutations in a protein that recycles between the ER and ERGIC (CLN8). The CLN6 gene underlying a variant of late infantile NCL (vLINCL) was recently identified. It encodes a novel 311 amino acid transmembrane protein. Antisera raised against CLN6 peptides detected a protein of 30 kDa by Western blotting of human cells, which was missing in cells from some CLN6 deficient patients. Using immunofluorescence microscopy, CLN6 was shown to reside in the endoplasmic reticulum (ER). CLN6 protein tagged with GFP at the C-terminus and expressed in HEK293 cells was also found within the ER. Investigation of the effect of five CLN6 disease mutations that affect single amino acids showed that the mutant proteins were retained in the ER. These data suggest that CLN6 is an ER resident protein, the activity of which, despite this location, must contribute to lysosomal function.     

Defective Self-Renewal and Differentiation of GBA-Deficient Neural Stem Cells Can Be Restored By Macrophage Colony-Stimulating Factor

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene (GBA), which encodes the lysosomal enzyme glucosylceramidase (GCase). Deficiency in GCase leads to characteristic visceral pathology and lethal neurological manifestations in some patients. Investigations into neurogenesis have suggested that neurodegenerative disorders, such as GD, could be overcome or at least ameliorated by the generation of new neurons. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are potential candidates for use in the treatment of neurodegenerative disorders because of their ability to promote neurogenesis. Our objective was to examine the mechanism of neurogenesis by BM-MSCs in GD. We found that neural stem cells (NSCs) derived from a neuronopathic GD model exhibited decreased ability for self-renewal and neuronal differentiation. Co-culture of GBA-deficient NSCs with BM-MSCs resulted in an enhanced capacity for self-renewal, and an increased ability for differentiation into neurons or oligodendrocytes. Enhanced proliferation and neuronal differentiation of GBA-deficient NSCs was associated with elevated release of macrophage colony-stimulating factor (M-CSF) from BM-MSCs. Our findings suggest that soluble M-CSF derived from BM-MSCs can modulate GBA-deficient NSCs, resulting in their improved proliferation and neuronal differentiation.     

Palmitoyl-protein thioesterase gene expression in the developing mouse brain and retina: implications for early loss of vision in infantile neuronal ceroid lipofuscinosis

Mutations in the palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL), the clinical manifestations of which include the early loss of vision followed by deterioration of brain functions. To gain insight into the temporal onset of these clinical manifestations, we isolated and characterized a murine PPT (mPPT)-cDNA, mapped the gene on distal chromosome 4, and studied its expression in the eye and in the brain during development. Our results show that both cDNA and protein sequences of the murine and human PPTs are virtually identical and that the mPPT expression in the retina and in the brain is temporally regulated during development. Furthermore, the retinal expression of mPPT occurs much earlier and at a higher level than in the brain at all developmental stages investigated. Since many retinal and brain proteins are highly palmitoylated and depalmitoylation by PPT is essential for their effective recycling in the lysosomes, our results raise the possibility that inactivating mutations of the PPT gene, as occur in INCL, are likely to cause cellular accumulation of lipid-modified proteins in the retina earlier than in the brain. Consequently, the loss of vision occurs before the deterioration of brain functions in this disease.     

In a Model of Batten Disease,Palmitoyl Protein Thioesterase-1 Deficiency Is Associated with Brown Adipose Tissue and Thermoregulation Abnormalities

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disorder caused by a deficiency of palmitoyl-protein thioesterase-1 (PPT1). We have previously shown that children with INCL have increased risk of hypothermia during anesthesia and that PPT1-deficiency in mice is associated with disruption of adaptive energy metabolism, downregulation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and mitochondrial dysfunction. Here we hypothesized that Ppt1-knockout mice, a well-studied model of INCL that shows many of the neurologic manifestations of the disease, would recapitulate the thermoregulation impairment observed in children with INCL. We also hypothesized that when exposed to cold, Ppt1-knockout mice would be unable to maintain body temperature as in mice thermogenesis requires upregulation of Pgc-1α and uncoupling protein 1 (Ucp-1) in brown adipose tissue. We found that the Ppt1-KO mice had lower basal body temperature as they aged and developed hypothermia during cold exposure. Surprisingly, this inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with an adequate upregulation of Pgc-1α and Ucp-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. In addition, during baseline conditions, brown adipose tissue of Ppt1-KO mice had less vacuolization (lipid droplets) compared to wild-type animals. After cold stress, wild-type animals had significant decreases whereas Ppt1-KO had insignificant changes in lipid droplets compared with baseline measurements, thus suggesting that Ppt1-KO had less lipolysis in response to cold stress. These results uncover a previously unknown phenotype associated with PPT1 deficiency, that of altered thermoregulation, which is associated with impaired lipolysis and neurotransmitter release to brown adipose tissue during cold exposure. These findings suggest that INCL should be added to the list of neurodegenerative diseases that are linked to alterations in peripheral metabolic processes. In addition, extrapolating these findings clinically, impaired thermoregulation and hypothermia are potential risks in patients with INCL.     

PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.     

Genetic modifiers of Drosophila palmitoyl-protein thioesterase 1-induced degeneration

Infantile neuronal ceroid lipofuscinosis (INCL) is a pediatric neurodegenerative disease caused by mutations in the human CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase 1 (PPT1), suggesting an important role for the regulation of palmitoylation in normal neuronal function. To further elucidate Ppt1 function, we performed a gain-of-function modifier screen in Drosophila using a collection of enhancer-promoter transgenic lines to suppress or enhance the degeneration produced by overexpression of Ppt1 in the adult visual system. Modifier genes identified in our screen connect Ppt1 function to synaptic vesicle cycling, endo-lysosomal trafficking, synaptic development, and activity-dependent remodeling of the synapse. Furthermore, several homologs of the modifying genes are known to be regulated by palmitoylation in other systems and may be in vivo substrates for Ppt1. Our results complement recent work on mouse Ppt1(-/-) cells that shows a reduction in synaptic vesicle pools in primary neuronal cultures and defects in endosomal trafficking in human fibroblasts. The pathways and processes implicated by our modifier loci shed light on the normal cellular function of Ppt1. A greater understanding of Ppt1 function in these cellular processes will provide valuable insight into the molecular etiology of the neuronal dysfunction underlying the disease.     

Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.