Palmitoyl Protein Thioesterase Research Articles

Adeno-associated virus 2-mediated gene therapy decreases autofluorescent storage material and increases brain mass in a murine model of infantile neuronal ceroid lipofuscinosis

Infantile neuronal ceroid lipofuscinosis (INCL) is the earliest onset form of a class of inherited neurodegenerative disease called Batten disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Autofluorescent storage material accumulates in virtually all tissues in INCL patients, including the brain, and leads to widespread neuronal loss and cortical atrophy. To determine the efficacy of viral-mediated gene therapy, we injected a recombinant adeno-associated virus 2 vector encoding human PPT1 (rAAV–PPT1) intracranially (I.C.) into a murine model of INCL. INCL mice given four I.C. injections of rAAV–PPT1 as newborns exhibited PPT1 activity near the injection sites and decreased secondary elevations of another lysosomal enzyme. In addition, storage material was decreased in cortical, hippocampal, and cerebellar neurons, and brain weights and cortical thicknesses were increased. These data demonstrate that an adeno-associated virus 2 (AAV2)-mediated gene therapy approach may provide some therapeutic benefit for INCL.

Regional and cellular neuropathology in the palmitoyl protein thioesterase-1 null mutant mouse model of infantile neuronal ceroid lipofuscinosis

Infantile neuronal ceroid lipofuscinosis (INCL) is one of a group of fatal hereditary lysosomal storage disorders. Palmitoyl protein thioesterase 1 null mutant mice (PPT1−/−) now exist that accurately recapitulate many important disease features. The severely affected PPT1−/− mouse CNS exhibited reduced volume of both cortical and subcortical regions, but with sparing of the cerebellum. Pronounced differences existed in the extent of cortical thinning between different regions, due to lamina-specific effects upon neuronal survival. A dramatic reduction in cortical and hippocampal interneuron number was also evident, with different extents of specific interneuron loss depending upon the region and phenotypic marker. These neuronal changes were accompanied by widespread astrocytosis and localized microglial activation in restricted cortical and subcortical regions. This characterization of PPT1−/− mice not only provides defined pathological landmarks for understanding disease pathogenesis, but also provides an invaluable resource for subsequently judging the efficacy of therapeutic strategies.

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.

Current state of clinical and morphological features in human NCL.

The neuronal ceroid lipofuscinoses (NCL) are a large group of autosomal recessive lysosomal storage disorders with both enzymatic deficiency and structural protein dysfunction. Previously, diagnosis of NCL was based on age at onset and clinicopathological (C-P) findings described 4 forms, classified as infantile (INCL) (2), late-infantile (LINCL) (5), juvenile (JNCL) (6), and adult (ANCL) (12). Most patients with NCL have progressive ocular and cerebral dysfunction, including cognitive/motor dysfunction and uncontrolled seizures. After reviewing 520 patients with NCL, we found that about 104 (20%) did not fit this classification of NCL. With further research, 4 additional forms have been recognized: Finnish (13), Gypsy/Indian (14), Turkish (15)--variants of LINCL, and Northern epilepsy (16), also known as progressive epilepsy with mental retardation. These eight NCL forms resulted from 151 different mutations in genes CLN1 to CLN8 causing different phenotypes (http://www.ucl.ac.uk/ncl). The genes CLN1 and CLN2 encode lysosomal palmitoyl protein thioesterase and tripeptidyl peptidase 1. The diagnosis of NCL is based on clinicopathological (C-P) findings, enzymatic assay, and molecular genetic testing. Ultrastructural studies must be performed to confirm the presence and nature of lysosomal storage material (fingerprint or curvilinear profiles, or granular osmiophilic deposits) before doing biochemical testing. Pheno/genotypic correlation studies are discussed.

Autosomal Dominant Adult Neuronal Ceroid Lipofuscinosis: a Novel Form of NCL with Granular Osmiophilic Deposits without Palmitoyl Protein Thioesterase 1 Deficiency

We describe the neuropathological and biochemical autopsy findings in 3 patients with autosomal dominant adult neuronal ceroid lipofuscinosis (ANCL, Parry type; MIM 162350), from a family with 6 affected individuals in 3 generations. Throughout the brain of these patients, there was abundant intraneuronal lysosomal storage of autofluorescent lipopigment granules. Striking loss of neurons in the substantia nigra was found. In contrast, little neuronal cell loss occurred in other cerebral areas, despite massive neuronal inclusions. Visceral storage was present in gut, liver, cardiomyocytes, skeletal muscle, and in the skin eccrine glands. The storage material showed highly variable immunoreactivity with antiserum against subunit c of mitochondrial ATP synthase, but uniform strong immunoreactivity for saposin D (sphingolipid activating protein D). Protein electrophoresis of isolated storage material revealed a major protein band of about 14 kDa, recognized in Western blotting by saposin D antiserum (but not subunit c of mitochondrial ATPase (SCMAS) antiserum). Electron microscopy showed ample intraneuronal granular osmiophilic deposits (GRODs), as occurs in CLN1 and congenital ovine NCL. These forms of NCL are caused by the deficiencies of palmitoyl protein thioesterase 1 and cathepsin D, respectively. However, activities of these enzymes were within normal range in our patients. Thus we propose that a gene distinct from the cathepsin D and CLN1-CLN8 genes is responsible for this autosomal dominant form of ANCL.

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform non-redundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 A. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix alpha4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (beta3-alphaA and beta8-alphaF) located immediately above the lipid-binding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

Characterization of Drosophila palmitoyl-protein thioesterase 1

Batten disease or neuronal ceroid lipofuscinoses (NCL) are a group of genetic neurodegenerative diseases that primarily afflict infants and children and are characterized by progressive loss of brain functions caused by the death of central nervous system (CNS) neurons. The most severe form of the disease is infantile NCL (INCL). INCL is caused by mutations in the palmitoyl-protein thioesterase 1 ( PPT1) gene, which encodes a palmitoyl-protein thioesterase 1 enzyme that cleaves long-chain fatty acids from S-acylated proteins within the lysosome. How the loss of this activity causes the death of CNS neurons is not known. A PPT1 homolog and palmitoyl-protein thioesterase 1 enzyme activity were characterized in Drosophila melanogaster as an initial step in developing Drosophila as a model system for studying the etiology of INCL. Predicted gene CG12108 in region 8A2 of the X chromosome is 55% identical and 72% similar to human PPT1 and contains conserved catalytic residues and sites of glycosylation. Northern-blot hybridizations revealed a major 1.5 kb CG12108 transcript in unfertilized eggs, embryos, larvae, pupae, adult head and thorax, ovary, testis, and S2 tissue culture cells, as well as several minor mRNA species in some tissues. Levels of the 1.5 kb transcript were fairly uniform among tissues except in testis, where the transcript was enriched 5-fold. The same tissues also contained palmitoyl-protein thioesterase 1 enzyme activity measured using the fluorometric substrate 4-methylumbelliferyl-6-thiopalmitoyl-β- d-glucoside. Enzyme activity was highest in testis and varied among the other tissues to a greater extent than did CG12108 message, suggesting that CG12108 is subjected to post-transcriptional regulation. Finally, flies homozygous for a deletion that removes CG12108 and three unrelated neighboring genes had less than 3% of wildtype levels of enzyme activity, consistent with CG12108 encoding functional palmitoyl-protein thioesterase 1 activity and being the fly ortholog of human PPT1. CG12108 has been appropriately renamed Ppt1.

Rapid and simple assay for the determination of tripeptidyl peptidase and palmitoyl protein thioesterase activities in dried blood spots.

Neuronal ceroid lipofuscinoses constitute a group of at least eight inherited, progressive encephalopathies that are characterized by lipofuscin-like inclusions in various tissues and that have recently been classified as CLN1 to CLN8 according to their genetic defects (1). A form with mostly infantile manifestation (CLN1) and the classical late infantile form (CLN2) are caused by deficiencies of the lysosomal enzymes palmitoyl protein thioesterase 1 (PPT1) and tripeptidyl peptidase 1 (TPP1), respectively. The basic defects underlying the other forms are still ill-defined or unknown. To date, more than 30 mutations have been reported in the PPT1 and TPP1 genes, rendering molecular genetic analysis impractical as a primary means of diagnosis. Electron microscopy of characteristic cellular inclusions remains an important diagnostic method, but it is also tedious and not readily available. Enzymatic assays based on fluorescent substrates have recently been reported for PPT1 (2) and TPP1(3). These assays can also be used with isolated leukocytes. However, because few laboratories are experienced in diagnosing these rare disorders, blood samples must be sent to specialized centers by expensive express mail services to avoid loss of enzymatic activity. In our experience, samples for this kind of study frequently arrive in poor condition. As an alternative to the use of leukocytes, methods for determining several lysosomal enzymes from dried blood spots (DBS) have recently been described (4). DBS are also used routinely for enzyme measurements within newborn-screening programs for the detection of biotinidase and galactose-uridyltransferase deficiencies in neonates. DBS require only minute amounts of blood, are easy to …

An over-expression system for characterizing Ppt1 function in Drosophila.

The infantile onset form of Neuronal Ceroid Lipofuscinoses (INCL) is the earliest and most severe form of NCL, with neurological symptoms that reflect massive neurodegeneration in the CNS and retina. INCL is due to recessively inherited mutations at the CLN1 locus. This locus encodes the evolutionarily conserved enzyme palmitoyl-protein thioesterase 1 (PPT1), indicating an essential role for protein palmitoylation in normal neuronal function. To begin to elucidate the specific role that Ppt1 plays in neuronal cells, we have developed a Ppt1 over-expression system in Drosophila. We report that over-expression of DmPpt1 in the developing Drosophila visual system leads to the loss of cells through apoptotic cell death. This DmPpt1 over-expression phenotype is suppressed by DmPpt1 genomic deficiencies. Moreover, over-expression of DmPpt1S123A, which bears a catalytic site serine 123 to alanine mutation, does not lead to the severe eye phenotype observed with over-expression of wild-type DmPpt1. Thus, cell loss in DmPpt1 flies is directly related to the dosage of wildtype DmPpt1. Although INCL is due to the loss of PPT1; increased levels of DmPpt1 also lead to neurodegeneration possibly via a detrimental effect on some aspect of PPT1's normal function. This suggests that the precise levels of PPT1 activity are important for neuronal cell survival. The Drosophila DmPpt1 over-expression system provides a resource for genetic experiments that aim to identify the processes by which PPT1 regulates the palmitoylation-state of its essential protein substrates.

Palmitoyl protein thioesterase 1 is targeted to the axons in neurons.

Palmitoyl protein thioesterase 1 (PPT1) is a depalmitoylating enzyme whose deficiency leads to infantile neuronal ceroid lipofuscinosis. The disease is characterized by early loss of vision and massive neuronal death. Although PPT1 is expressed in many tissues, a deficiency of PPT1 damages neurons only in the cerebral and cerebellar cortexes and retina; other cell types remain relatively unaffected. We previously demonstrated that PPT1 is present in the synaptosomes and synaptic vesicles of neurons. To understand the crucial role of PPT1 for neuronal cells, we further investigated the expression and targeting of PPT1 in retinal, hippocampal, and cortical neurons during their maturation in culture. We found that PPT1 activity increases by neuronal maturation and is highest in retinal neuron cultures. In retinal neurons the expression of PPT1 precedes that of the synaptic vesicle protein 2 and synaptophysin, indicating a significant role for PPT1 in the early development of neuronal cells. We also found by quantitative confocal immunofluorescence microscopy that PPT1 is targeted preferably to axons in mature neurons, as indicated by its colocalization with the axonal marker microtubule-associated protein 1. In axons PPT1 is targeted specifically to axonal varicosities and presynaptic terminals, as indicated by its significant colocalization with growth-associated protein 43 and synaptophysin. Axonal localization of PPT1 was confirmed by double labeling with synaptophysin and postembedding immunoelectron microscopy. The polarized axonal targeting of PPT1 may well indicate a role for PPT1 in the exocytotic pathway of neurons.

Prostate epithelial expression of a novel androgen target gene.

To better understand the role of androgens in prostate development and disease it is important to characterize androgen-regulated genes in the prostate. Using suppression subtractive hybridization between congenitally androgen-deficient (hpg) and androgen-replaced hpg mouse prostates, we have cloned a novel androgen up-regulated gene from mouse prostate (AUMP). The messenger RNA sequence of AUMP consists of 805 nucleotides with an open reading frame of 408 base pairs. In non-hpg mice with normal androgen levels, AUMP is selectively expressed in the prostate, as shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis of 9 organs. Depletion of androgens via castration of mature mice resulted in loss of AUMP expression, whereas testosterone replacement restored it. Tissue in situ hybridization localized AUMP expression to the luminal epithelial cells of the androgen-sufficient prostate. Database searches indicate that AUMP codes for a novel protein that shares approximately 65% similarity and 35% identity to palmitoyl protein thioesterase of human, rat, mouse, and bovine. A motif for protein-transport protein, which promotes translocation as well as integration of secretory proteins into membrane, is also present. Further efforts will be made to obtain the human homologue of AUMP that will enable evaluation of its role in normal and diseased human prostate.

Status Epilepticus Induces Changes in the Expression and Localization of Endogenous Palmitoyl-Protein Thioesterase 1

Kainic acid (KA)-induced experimental epilepsy, a model of excitotoxicity, leads to selective neuronal death and synaptic restructuring. We used this model to investigate the effects of neuronal hyperactivation on palmitoyl-protein thioesterase 1 (PPT1), the deficiency of which causes drastic neurodegeneration. Immunological stainings showed that epileptic seizures in adult rats led to a progressive and remarkable increase of PPT1 in limbic areas of the brain. Within 1 week, the maximal expression was observed in CA3 and CA1 pyramidal neurons of the hippocampus. In the surviving pyramidal neurons, PPT1 localized in vesicular structures in cell soma and neuritic extensions. After seizures, colocalization of PPT1 with synaptic membrane marker (NMDAR2B) was enhanced. Further, synaptic fractionation revealed that after seizures PPT1 was readily observed on the presynaptic side of synaptic junction. These data suggest that PPT1 may protect neurons from excitotoxicity and have a role in synaptic plasticity.

Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2).

Infantile and classical late infantile neuronal ceroid lipofuscinoses (NCL) are two recent additions to the expanding spectrum of lysosomal storage disorders caused by deficiencies in lysosomal hydrolases. They are latecomers to the lysosomal storage disorders, probably because of the heterogeneous nature of the storage material, which precluded meaningful biochemical analysis. Infantile NCL is caused by deficiency in palmitoyl-protein thioesterase, an enzyme that hydrolyzes fatty acids from cysteine residues in lipid-modified proteins. Classical late-infantile NCL is caused by a deficiency in tripeptidyl amino peptidase-I, a lysosomal peptidase that removes three amino acids from the free amino terminus of peptides or small proteins. Late-onset forms of these disorders have been described. The clinical, biochemical, and molecular genetic aspects of these two latest lysosomal storage disorders are discussed in this review. In addition, approaches to treatment and future directions for research are examined.

Profound loss of GABAergic interneurons in the PPT1 knockout mouse model of infantile neuronal ceroid lipofuscinosis

Introduction: The neuronal ceroid lipofuscinoses (NCL) are progressive neurodegenerative disorders with onset from infancy to adulthood that are manifested by blindness, seizures and dementia. In infantile NCL (INCL), a mutation in the palmitoyl protein thioesterase (PPT1) gene results in loss of PPT1 activity and lysosomal accumulation of autofluorescent proteolipid in the brain and other tissues. We have generated a PPT1 knockout mouse model of INCL (PPT1–/–) and characterized pathological changes in the CNS of these mice, which die by 8 months of age.Results: 7‐month‐old PPT1–/– exhibited NCL‐like pathology with prominent accumulation of autofluorescent lipopigment throughout the CNS, together with pronounced cerebral atrophy. Staining for phenotypic markers normally present in subpopulations of interneurons in the cortex and hippocampus revealed progressive loss of staining in the cortex and hippocampus, with persisting interneurons exhibiting pronounced hypertrophy and abnormal dendritic morphology.Conclusions: Taken together with our findings in the other mouse models of NCL and preliminary data from NCL patient derived tissue, these results provide further substantive evidence for the involvement of interneurons in the NCLs.Acknowledgements: Supported by The Batten's Disease Support and Research Association, The Natalie Fund, The Remy Fund; Batten's Disease Family Association.

The effects of lysosomotropic agents on normal and INCL cells provide further evidence for the lysosomal nature of palmitoyl-protein thioesterase function

Fatty acylation of proteins on cysteine residues is a common post-translational modification that plays roles in protein–membrane and protein–protein interactions. Recently, we described a lysosomal palmitoyl-protein thioesterase that removes long-chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in palmitoyl-protein thioesterase results in a human lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis (INCL), which primarily affects the central nervous system. The pathological hallmark of the disorder is the accumulation of granular osmiophilic deposits (GROD) that resemble lipofuscin, or aging pigment. In previous work, we have shown that [ 35S]cysteine-labeled lipid thioesters derived from fatty acylated proteins accumulate in cultured cells derived from palmitoyl-protein thioesterase-deficient patients. In the present work, we show that the lipid cysteine thioesters accumulate in the lysosomal fraction, and we further show that the appearance of these compounds in the organic phase is blocked by inhibitors of lysosomal proteolysis, demonstrating through biochemical means the lysosomal nature of the site of palmitoyl-protein thioesterase action. Furthermore, substrates for palmitoyl-protein thioesterase accumulate even in normal cells after leupeptin or chloroquine treatment. This was demonstrated by subjecting extracts of treated cells to exhaustive proteolysis to release protein-bound cysteine lipid for analysis. Cysteamine, a lysosomotropic drug recently proposed for the treatment of INCL, was found to have effects similar to leupeptin and chloroquine, suggesting that its mechanism of action may be more complex than previously understood.

The Peroxisome Proliferator-induced Cytosolic Type I Acyl-CoA Thioesterase (CTE-I) Is a Serine-Histidine-Aspartic Acid α/β Hydrolase

Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317-34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.

Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice.

PPT1 and PPT2 encode two lysosomal thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create "knockout" mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a "clasping" phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.

Pheno/genotypic correlations of neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCL) are a large group of autosomal recessive lysosomal storage disorders with both enzymatic deficiency and structural protein dysfunction. Previously, diagnosis of NCL was based on age at onset and clinicopathologic (C-P) findings, classified as 1) infantile (INCL), 2) late infantile (LINCL), 3) juvenile (JNCL), and 4) adult (ANCL). Most patients with NCL have progressive ocular and cerebral dysfunction, including cognitive/motor dysfunction and uncontrolled seizures. After reviewing 319 patients with NCL, the authors found that 64 (20%) did not fit into this classification of NCL. With research progress, four additional forms have been recognized: 5) Finnish, 6) Gypsy/Indian, and 7) Turkish variants of LINCL and 8) northern epilepsy, also known as progressive epilepsy with mental retardation. These eight NCL forms resulted from 100 different mutations on genes CLN1to CLN8 causing different phenotypes (http://www.ucl.ac.uk/ncl). The genes CLN1 and CLN2 encode lysosomal palmitoyl protein thioesterase and tripeptidyl peptidase 1. The function of CLN3, CLN5, and CLN8 gene-encoded products is unknown, although their predicted amino acid sequences suggest they have a transmembrane topology. The diagnosis of NCL is based on C-P findings, enzymatic assay, and molecular genetic testing. Before biochemical and genetic tests are conducted, ultrastructural studies (i.e., blood [buffy coat] or punch biopsies [skin, conjunctiva]) must be performed to confirm the presence and nature of lysosomal storage material (fingerprint or curvilinear profiles or granular osmiophilic deposits). The recognition of variable onset from infancy to middle age supersedes the traditional emphasis on age-related NCL forms.

Neuronal Trafficking of Palmitoyl Protein Thioesterase Provides an Excellent Model to Study the Effects of Different Mutations Which Cause Infantile Neuronal Ceroid Lipofuscinocis

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative storage disorder in children caused by mutations in the palmitoyl protein thioesterase gene (PPT1). We have investigated here four naturally occurring previously described PPT1 mutations and show that all cause severe effects on PPT1 enzyme activity in transiently transfected COS-1 cells. Two of the mutations (delPhe84 and insCys45) cause a classical INCL phenotype and two (Thr75Pro and Leu219Gln) result in a late onset disease phenotype. All these mutated PPT1 molecules have severely altered intracellular localization in transiently transfected BHK-cells, whereas in mouse primary neuron cultures different effects were observed. In neurons the delPhe84 and insCys45 mutant polypeptides were targeted to the ER. Interestingly the Thr75Pro and Leu219Gln mutations had only minor effects on the neuronal trafficking of PPT1 and the mutated polypeptides were observed in neuronal shafts and showed colocalization with the presynaptic marker SV2. Our data indicates that neuronal cells provide an excellent model to study the genotype-phenotype correlation in INCL.

Biochemical analysis of mutations in palmitoyl-protein thioesterase causing infantile and late-onset forms of neuronal ceroid lipofuscinosis.

Deficiency in a recently characterized lysosomal enzyme, palmitoyl-protein thioesterase (PPT), leads to a severe neurodegenerative disorder of children, infantile neuronal ceroid lipofuscinosis (NCL). Over 36 different mutations in the PPT gene have been described, and missense mutations have been interpreted in the light of the recently solved X-ray crystallographic structure of PPT. In the current study, we assessed the biochemical impact of mutations through the study of cells derived from patients and from the expression of recombinant PPT enzymes in COS and Sf9 cells. All missense mutations associated with infantile NCL showed no residual enzyme activity, whereas mutations associated with late-onset phenotypes showed up to 2.15% residual activity. Two mutations increased the K(m) of the enzyme for palmitoylated substrates and were located in positions that would distort the palmitate-binding pocket. An initiator methionine mutation (ATG-->ATA) in two late-onset patients was expressed at a significant level in COS cells, suggesting that the ATA codon may be utilized to a clinically important extent in vivo. The most common PPT nonsense mutation, R151X, was associated with an absence of PPT mRNA. Mannose 6-phosphate modification of wild-type and mutant PPT enzymes was grossly normal at the level of the phosphotransferase reaction. However, mutant PPT enzymes did not bind to mannose 6-phosphate receptors in a blotting assay. This observation was related to the failure of the mutant expressed enzymes to gain access to 'uncovering enzyme' (N-acetylglucosamine-1-phosphodiester alpha-N-acetyl glucosaminidase), presumably due to a block in transit out of the endoplasmic reticulum, where mutant enzymes are degraded.