Thalami and corona radiata in juvenile NCL (CLN3): a voxel-based morphometric study.

Juvenile neuronal ceroid lipofuscinosis (CLN3) is characterized by progressive cerebral atrophy. The purpose of this study was to re-evaluate the three-dimensional magnetic resonance (3D-MR) images of patients with CLN3 using voxel-based morphometry (VBM) to achieve a detailed understanding of the affected brain regions. T1-weighted 3D-MR images of 15 patients with CLN3 (age range: 12-25 years, mean age 17.6 years) and 15 age- and sex-matched controls were analyzed using VBM. VBM showed strikingly focal alterations in the brains of CLN3 patients: the gray matter volume was significantly decreased in the dorsomedial part of the thalami of CLN3 patients. In addition, the volume of the white matter was significantly decreased in the corona radiata, containing cortical efferents and afferents in the transition between the internal capsule and the subcortical white matter. These data suggest that the dorsomedial part of the thalamus and the corona radiata may have a central, previously unrecognized role in the pathogenesis of CLN3.

Stress-induced apoptosis is impaired in cells with a lysosomal targeting defect but is not affected in cells synthesizing a catalytically inactive cathepsin D.

The role of cathepsin D in stress-induced cell death has been investigated by using ovine fibroblasts exhibiting a missense mutation in the active site of cathepsin D. The cathepsin D (lysosomal aspartic protease) deficiency did not protect cells against toxicity induced by doxorubicin and other cytotoxic agents, neither did it protect cells from caspase activation. Moreover, the cathepsin D inhibitor, pepstatin A, did not prevent stress-induced cell death in human fibroblasts or lymphoblasts. The possible role of lysosomal ceramide or sphingosine-mediated activation of cathepsin D in apoptosis was also excluded by using human cells either overexpressing or deficient in acid ceramidase. However, a normal lysosomal function seems to be required for efficient cell death, as indicated by the finding that fibroblasts from patients with mucolipidosis II were partially resistant to staurosporine, sphingosine and TNF-induced apoptosis, suggesting a key role of lysosomes in cell death.

Variant late infantile neuronal ceroid-lipofuscinosis: pathology and biochemistry.

The neuronal ceroid-lipofuscinoses (NCL) are among the most common inherited neurodegenerative disorders of childhood. The genomic defect causing a variant late infantile neuronal ceroid-lipofuscinosis (vLINCL, also called CLN-5 or variant Jansky-Bielschowsky disease) has recently been localized to chromosome 13q22, thus delineating this disease as a separate entity. This particular form of NCL is clinically well defined, but lacks pathomorphological and biochemical description. The present analyses indicate that subunit c of the mitochondrial ATP synthase is the major protein in vLINCL brain storage cytosomes. These cytosomes also contain minor amounts of sphingolipid activator proteins (SAPs). The immunohistological distribution of subunit c and SAPs in the central nervous system (CNS) and visceral tissues closely resembles that of classical LINCL. Thus, despite clinical differences and the fact that various forms of NCL are caused by different genetic defects, variant and classical LINCL as well as juvenile NCL are all characterized by pronounced lysosomal accumulation of the same hydrophobic protein, subunit c of the mitochondrial ATP synthase.

Northern epilepsy: a novel form of neuronal ceroid-lipofuscinosis.

Northern epilepsy is an autosomal recessive childhood onset epilepsy syndrome, clinically characterized by generalized tonic-clonic seizures with onset at 5 to 10 years of age and subsequent slowly progressive mental deterioration. The patients may reach 50 or 60 years of age. A mutation responsible for the disease has recently been identified in a novel gene on chromosome 8p23, encoding a putative membrane protein with an unknown function. The present study, based on three autopsied patients, is the first neuropathological analysis of the disease, and showed intraneuronal accumulation of cytoplasmic autofluorescent granules. The granules were strongly stained by the Luxol fast blue, periodic acid-Schiff, and Sudan black B methods in paraffin sections, and were immunoreactive for subunit c of the mitochondrial ATP synthase and sphingolipid activator proteins A and D. The intraneuronal storage was highly selective: the third layer of the isocortex and the hippocampal CA2, CA3, and CA4 sectors were severely affected, while other layers of the isocortex, the CA1 sector, and the cerebellar cortex were only minimally involved. The membrane-bound storage cytosomes showed a curvilinear ultrastructure with admixture of some granular components. Western blotting and N-terminal sequence analysis of purified storage material identified subunit c as the major component. These findings establish Northern epilepsy as a new form of neuronal ceroid-lipofuscinosis with an exceptionally protracted course.

Storage of saposins A and D in infantile neuronal ceroid-lipofuscinosis.

We have isolated storage cytosomes from brain tissue of patients with infantile neuronal ceroid-lipofuscinosis. The purified storage bodies were subjected to compositional analysis which revealed a high content of proteins, accounting for 43% of dry weight. Saposins A and D, also known as sphingolipid activator proteins (SAPs), were shown to constitute a major portion of the accumulated protein using gel electrophoresis and sequence analysis. This is the first time that saposins have been found to be stored in any form of neuronal ceroid-lipofuscinosis.

CSF insulin-like growth factor-1 in infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Infantile neuronal ceroid lipofuscinosis (INCL) is a progressive encephalopathy in which the patients are severely disabled by the age of 3 years. It is characterized by cerebral atrophy, selective loss of cortical neurons, and secondary loss of axons and myelin sheaths of the white matter. INCL has been shown to result from a palmitoyl protein thioesterase deficiency. The authors suggested that insulin-like growth hormones and apoptosis might play a role in the pathogenesis of INCL. METHODS: The authors measured insulin-like growth factor-1 (IGF-1) and IGF binding protein 3 (IGFBP-3) in the CSF of patients with INCL by radioimmunoassay at an early stage when myelin was starting to diminish. RESULTS: The authors found low CSF IGF-1 but normal IGFBP-3 in patients with INCL compared with control subjects. Also, they observed apoptotic cell death in biopsies of INCL patients. CONCLUSIONS: Because the IGF system seems to be important for early brain development, myelination, and neuroprotection, the authors suggest that the pathology in INCL may be associated with low CSF IGF-1.

Sphingolipid activator proteins (SAPs) are stored together with glycosphingolipids in the infantile neuronal ceroid-lipofuscinosis (INCL).

The storage material isolated from the brains of patients with infantile neuronal ceroid-lipofuscinosis (INCL) contains, on average, 43% protein and 35% lipids on a dry weight basis. Recently we identified the major storage proteins as sphingolipid activator proteins (SAPs) A and D by direct sequencing. In the present study we used monospecific anti-sap-B-, anti-sap-C, and anti-sap-D-antisera in immunohistochemical and Western analyses to show that sap-D is, indeed, an integral component of the storage bodies. In contrast, no (or little) immunoreactivity for sap-B or sap-C was detected in the INCL storage granules. This observation is of interest for an understanding of the pathogenesis because the four SAPs are produced from a single precursor protein by proteolytic cleavage. Furthermore, we analysed the stored lipids on high performance thin layer chromatography combined with different staining techniques. In this preliminary analysis we found two glycosphingolipids, yet to be identified, to be common for all INCL patients.

Palmitoyl-protein thioesterase, an enzyme implicated in neurodegeneration, is localized in neurons and is developmentally regulated in rat brain.

Palmitoyl-protein thioesterase (PPT) is an enzyme involved in cleavage of palmitate residues from acylated proteins. Mutations in PPT gene cause a severe neurodegenerative disorder, infantile neuronal ceroid-lipofuscinosis (INCL), characterized by loss of cortical neurons. In order to clarify the role of PPT and palmitoylation/depalmitoylation in the development of CNS and pathogenesis of infantile neuronal ceroid-lipofuscinosis (INCL), we studied the localization and expression of PPT in developing rats. Using immunohistochemical methods, we show for the first time that PPT is truly localized in neurons. Further, using RT-PCR and Western blotting, we show that expression of PPT in rat brain is developmentally regulated, with increasing expression during the maturation of CNS, reaching the maximum in young adulthood. The presented data support the view of PPT being essential for both development and maintenance of cortical neurons.

Hippocampal lesions in the neuronal ceroid lipofuscinoses.

Epilepsy is a common manifestation in all childhood-onset forms of neuronal ceroid lipofuscinosis. In order to document hippocampal lesions and their relationship to epilepsy we studied autopsy specimens from the hippocampi of a series of patients with the infantile (CLN1), classic late infantile (CLN2), Finnish variant late infantile (CLN5), and juvenile (CLN3) neuronal ceroid-lipofuscinosis as well as Northern epilepsy (CLN8), using a battery of histological and immunocytochemical staining methods. Despite striking differences in the overall degree of neocortical neuronal storage and loss, these genetically heterogeneous forms of neuronal ceroid lipofuscinosis showed a common lesional pattern in the hippocampi: a relative sparing of the CA1 sector and severe involvement of the neighbouring CA2 sector, with intermediate degrees of involvement of the CA3 and CA4 sectors. These findings distinguish the hippocampal pathology associated with the neuronal ceroid lipofuscinoses from classical 'mesial temporal sclerosis' and show that the selective lesional pattern in the neuronal ceroid lipofuscinoses is not a secondary anoxic-ischaemic phenomenon. It is rather a consequence of the primary metabolic defects and may be directly involved in the causation of the epileptic discharges.

Congenital ovine neuronal ceroid lipofuscinosis--a cathepsin D deficiency with increased levels of the inactive enzyme.

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.

Incidence of neuronal perikaryal spheroids in neuronal ceroid lipofuscinoses (Batten disease).

The stored material in neuronal ceroid lipofuscinosis (NCL) undergoes, irrespective of the disease type, a uniform modification, altering profoundly its physical and histochemical properties. The process is accompanied by loss of immunodetectable epitopes of subunit c of mitochondrial ATP synthase (SCMAS) in the transformed storage material in NCL2 and NCL6 and of sphingolipid activator proteins (SAPs) A and D in NCL1, NCL2, and NCL6. It is restricted to certain subcortical brain nuclei, typically nucleus niger, dentatus, lentiformis, and thalamus. The process is coupled with progressive enlargement of the deposits caused probably by aggregation and fusion of the storage lysosomes. This ensues in formation of larger pleiomorphic perikaryal corpuscles, the spheroids being only one special form in the spectrum. The process was found to be most intensive in NCL2 brains. As the neuronal unmodified storage deposits tend also to be present in aggregate form, care must be taken to distinguish spheroids composed of modified from those composed of unmodified storage material.

Biochemical aspects of neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCLs) collectively constitute the most common group of progressive brain diseases in children. The childhood forms of NCL are recessively inherited monogenic diseases, resulting in progressive dementia and motor problems, epilepsy, blindness and, finally, early death. Pathologically, the NCLs are characterized by accumulation of autofluorescent storage material in the lysosomes of neurons and other cells. The disease is selectively manifested in the central nervous system, so that there is a progressive loss of neurons. This leads to a dramatic cerebral atrophy typical of the early onset forms of NCL. The present review summarizes the knowledge of the biochemistry of NCLs, and discusses the possible pathogenetic mechanisms involved in the neurodegeneration in NCLs.

The expression of palmitoyl-protein thioesterase is developmentally regulated in neural tissues but not in nonneural tissues.

Mutations in a palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL). INCL is characterized by an extreme and selective neuronal loss in cerebral cortex and retina. Using reverse transcriptase PCR we show that PPT expression is developmentally regulated in rat brain and eyes but not in spleen. The changes in the expression coincide with developmental events in the brain. These data indicate that PPT has an important role in the development of the CNS.

Sphingolipid activator proteins (SAPs) in neuronal ceroid lipofuscinoses (NCL).

Based on the predominant component of the storage material the neuronal ceroid lipofuscinoses (NCL) can be divided into two categories: one storing mitochondrial ATP synthase subunit c and the other storing sphingolipid activator proteins (SAPs). The latter group is represented by the human infantile NCL (INCL), a congenital ovine NCL, and a canine NCL. Small amounts of SAPs also accumulate in most other forms of NCL. The SAPs, their functions and occurrence in different forms of NCL, as well as the relationship between SAPs and palmitoyl protein thioesterase, an enzyme implicated in INCL, are discussed.

Developmental changes in the expression of neuronal ceroid lipofuscinoses-linked proteins.

Neuronal ceroid lipofuscinoses (NCL) form a distinct group of storage diseases where the normal development of the central nervous system is interrupted and neurons of the neocortex begin to degenerate. Mutations in genes encoding three lysosomal enzymes are the causes for three early-onset forms of NCLs: palmitoyl-protein thioesterase 1 (PPT1) is deficient in human infantile NCL, tripeptidyl peptidase 1 (TTP1) in late-infantile NCL, and cathepsin D in congenital ovine NCL. We wanted to compare the developmental expression profiles of these enzymes in rat brain. In conclusion, the PPT1 expression pattern differed from the two other lysosomal enzymes implicated in NCL diseases, thus suggesting a distinctive role for PPT1 in brain development.

Immunological studies on sphingolipid activator proteins in the neuronal ceroid-lipofuscinoses.

The neuronal ceroid-lipofuscinoses constitute an important group of progressive encephalopathies leading to severe psychomotor retardation, blindness, and early death. They are characterized by accumulation of autofluorescent, electron-dense storage bodies within the cytoplasm of neurons and many other cell types. We have recently identified sphingolipid activator proteins A and D as major components of the storage cytosomes in the infantile form of NCL. Using an immunological approach we have shown that sphingolipid activator proteins also constitute an integral component of the storage bodies in the other major forms of the disease.

Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis.

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

Sphingolipid activator proteins in the neuronal ceroid-lipofuscinoses: an immunological study.

The molecular defects underlying neuronal ceroid-lipofuscinoses (NCL) are still unknown. However, more data exist on the composition of the hydrophobic storage material characteristic of NCL. Accumulation of subunit c of the mitochondrial ATP synthase has been shown in most forms of human NCL with the exception of the infantile NCL (INCL) for which we have recently demonstrated storage of sphingolipid activator proteins (SAP). In the present study we raised an antiserum against storage cytosomes purified from INCL brain. Using the anti-INCL antiserum and monospecific SAP antisera, we studied storage material isolated from the brains of patients affected with NCL by Western analysis, and found a 12-kDa protein showing a SAP-like immunoreactivity not only in INCL, but also in all the childhood forms of NCL. Furthermore, using the anti-sap-D antiserum for immunohistochemistry, we observed strong immunoreactivity of the storage cytosomes in all major forms of NCL, and also in tissues of non-neuroectodermal origin. From these data we conclude that the presence of SAP within the storage bodies is a phenomenon common to all major forms of human NCL.

Pig plasma phospholipid transfer protein facilitates HDL interconversion.

Phospholipid transfer protein (PLTP) from pig plasma was purified to homogeneity using ultracentrifugation and a combination of hydrophobic-, heparin-Sepharose-, and anti-pig-PLTP-affinity chromatography techniques. The molecular weight of PLTP is 78,000 as estimated by SDS-PAGE and by gel filtration. The effect of pig plasma PLTP on the particle size distribution of either human or pig high density lipoprotein (HDL) was studied by incubating HDL with PLTP. Incubation of human HDL3 or pig HDL with the highly purified preparations of PLTP induced a conversion of the homogeneous HDL into two main populations of particles. The conversion products were isolated by ultracentrifugation at density 1.21 g/ml. The size changes were evident as analyzed by native gradient gel electrophoresis and by high resolution gel filtration. The diameters of the large and small particles formed were 10.8 nm and 7.6-7.9 nm, respectively. In addition, pig HDL conversion products included a third population of particles with a diameter of 11.5 nm. The degree of conversion was dependent on time and PLTP activity. Neither cholesteryl ester transfer protein nor lecithin:cholesterol acyltransferase activity could be detected in the PLTP preparations. The present study demonstrates that purified pig PLTP can act as a conversion factor: it has the ability to convert HDL into populations of large and small particles. The release of apoA-I is an essential part of the conversion process.

Ca(2+)-modulated phosphorylation of a low-molecular-mass polypeptide in rat liver mitochondria: evidence that it is identical with subunit c of F(0)F(1)-ATPase.

A 3.5-kDa polypeptide associated with the inner membrane of rat liver was found to be phosphorylated by [gamma-(32)P]ATP, presumably via a cAMP-dependent kinase. The phosphorylation was modulated by [Ca(2+)] in the physiological range, with a minimum at 1 microM and rising fourfold toward lower (10 nM) and higher (10 microM) concentrations. Further characterization of the 3.5-kDa component showed that the polypeptide has the same electrophoretic mobility as subunit c of F(0)F(1)-ATPase and that it selectively binds to antibodies against subunit c.