Robert Rousson

Type C Niemann-Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing

To investigate biochemical heterogeneity within Niemann-Pick type C disease (NPC), the two most characteristic abnormalities, namely (1) kinetics of LDL-stimulated cholesteryl ester formation and (2) intravesicular accumulation of LDL-derived unesterified cholesterol, evaluated by histochemical filipin staining, were studied in cultured skin fibroblasts from a population of 125 NPC patients. Profound alterations (esterification rates less than 10% of normal, very numerous and intensely fluorescent cholesterol-filipin granules) were demonstrated in 86% of the cases, depicting the classical NPC phenotype. The remaining cell lines showed a graded less severe impairment and more transient delay in the induction of LDL-mediated cholesteryl esterification, along with an attenuated accumulation of unesterified cholesterol. In particular, cells from a small group (7%) of patients, which have been individualized as representative of a variant phenotype, showed only slight alterations of esterification, restricted to the early phase of LDL uptake and undistinguishable from those in heterozygotes. In these cells, an abnormal cytochemical distribution of LDL-derived cholesterol, although moderate, was still evident provided rigorous experimental conditions were followed. A third, less clearly individualized group (7%), differing from the classical phenotype mostly by higher rates of cholesteryl ester formation, has been designated as an intermediary phenotype to reflect a more difficult diagnosis of such patients. These findings have an important bearing with regard to diagnosis and genetic counselling, although the significance of such a phenotypic variation in terms of genetic heterogeneity has still to be demonstrated. A given biochemical phenotype was however a constant observation within a family (14 pairs of siblings tested so far). The unique feature of LDL-cholesterol processing alterations in NPC has been further established from comparative studies in Wolman disease and I-cell disease, showing normal or different intracellular distribution of unesterified LDL-derived cholesterol in the latter disorders. Correlation between biochemical and clinical NPC phenotypes was only partial, but a correlation between the severity of alterations in cholesterol processing and sphingomyelin catabolism could be established.

Niemann-Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterification: A collaborative study on 70 patients

Seventy patients were selected to cover the range of variability in clinical expression of Niemann‐Pick disease group C (NP‐C). Their individual main clinical features and course of the disease (age at discovery and type of visceromegaly, age at onset and first neurological manifestation, later neurological symptoms) are schematically described. In cultured skin fibroblasts from these patients, sphingomyelinase activities measured in vitro showed decreased values only in approximately half of the cases, and when the metabolic fate of [14C]‐sphingomyelin was studied in living cell cultures, still 20% of the cases had a normal hydrolysis rate. Esterification of exogenous cholesterol was investigated in cell lines from these and 5 additional patients and in 21 of their parents. Using a non‐lipoprotein [3H]cholesterol source, very low esterification rates were obtained in more than 90% of the cases. All the numerous other pathological conditions studied, including Niemann‐Pick disease types A and B, gave normal results. A more sensitive method was elaborated, in which the cells were challenged with pure human low density lipoproteins (LDL) and the early rate of esterification studied. With the latter procedure, a pronounced deficiency could also be demonstrated in the few cases which had shown a milder impairment using a [3H]cholesterol source, and intermediate rates of esterification were obtained in heterozygotes. Discrimination of these difficult cases and of heterozygotes could also be achieved replacing LDL with total unfrozen human serum. Correlations were established between given clinical phenotypes and the severity of the biochemical lesion. Defective intracellular cholesterol esterification is further established as an intrinsic feature of NP‐C, and demonstration of this metabolic alteration appears as a major advance in diagnosing the condition.

Type C Niemann-Pick Disease: Biochemical Aspects and Phenotypic Heterogeneity

Within Niemann-Pick diseases, type C has now been demonstrated to be a nosological entity totally distinct from types A and B, and is best characterized at present by unique abnormalities of intracellular translocation of exogenous cholesterol, which are briefly reviewed. Although the primary defect is still unknown in type C Niemann-Pick disease, this discovery has had immediate medical applications, by providing the first strategy for reliable prenatal detection of the disorder and easy diagnosis of patients. From our personal experience of 134 cases, diagnosis is best reached by the combined demonstration of a deficient induction of esterification and of an intravesicular cholesterol storage by cytochemistry after filipin staining. The prevalence of the various clinical forms observed is given, together with a brief report of 6 adult-onset cases. The spectrum of phenotypic heterogeneity in relation to abnormal LDL processing has been defined, resulting in the delineation of three biochemical groups, classical (86%), variant (7%) and intermediate (7%). Correlations between clinical and biochemical phenotypes have been studied. To get further insight into genetic heterogeneity, complementation studies were performed. Preliminary results have yet given no evidence of several complementation groups within type C Niemann-Pick disease. The recognition of the three biochemical phenotypes is however critical for diagnosis and genetic counselling.

Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease

Addition of low-density lipoprotein (LDL) to cholesterol-deprived human skin fibroblast cultures treated by imipramine at a 20 microM concentration induced a significant intracellular accumulation of unesterified cholesterol. Intracytoplasmic inclusions were already visible by histochemical filipin staining after 2 h of LDL uptake and were progressively mobilized towards the perinuclear region within 24 h. At this concentration of the drug, the rate of proteolytic 125I-LDL hydrolysis was similar in treated and untreated cells. Treated cells maintained in lipoprotein-deficient medium showed no abnormality, indicating the exogenous origin of the accumulated sterol. Further, the drug induced a drastic dose-dependent impairment of LDL-stimulated cholesterol esterification, not related to an inhibition of acyl CoA:cholesterol acyltransferase, and a significant delay in down-regulation of de novo cholesterol synthesis. However, imipramine did not affect 25-hydroxycholesterol-mediated regulation of the two latter processes. These results resemble those observed in Niemann-Pick type C disease and suggest an impaired mobilization of LDL-derived cholesterol in imipramine-treated cells.

Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders

The 20-fold increase of free sphingoid bases found in liver from a murine model of Niemann-Pick type C (NPC) combined to the NPC-like phenotype induced by addition of sphinganine to normal fibroblast cultures prompted us to investigate the potential involvement of these compounds in the human disease. The contents of sphingosine and sphinganine were measured in liver, spleen, brain and skin fibroblast cultures by a sensitive HPLC method. In liver and spleen from NPC patients, a 6- to 24-fold elevation of sphingosine and sphinganine already prominent at the fetal stage of the disease was observed, while no clear increase could be evidenced in brain tissue. A significant increase, not modulated by the intralysosomal content of free cholesterol, also occurred in skin fibroblast cultures. To investigate the specificity of these findings, other lysosomal storage disorders were studied. A striking accumulation was found in liver and spleen (24- to 36-fold) from patients with Niemann-Pick disease type A and B (sphingomyelinase-deficient forms), and in cerebral cortex of type A Niemann-Pick disease. A significant storage also occurred in Sandhoff disease, while several other sphingolipidoses showed a moderate elevation. In all cases but Sandhoff disease brain, the sphingosine/sphinganine ratio remained unchanged, suggesting that the accumulated free sphingoid bases derived from sphingolipid catabolism. Formation of complexes between sphingosine and the lipid material accumulated in lysosomes might be a general mechanism in lysosomal lipidoses. In NPC, however, an increase of free sphingoid bases disproportionate to the degree of lysosomal storage and a specific involvement of cultured fibroblasts suggested a more complex or combined mechanism.

Niemann-Pick Disease Type C: An Update

SummaryThe concept of Niemann-Pick disease type C as a secondary sphingomyelin storage disorder (in contrast to the sphingomyelinase-deficient types A and B) has become more and more prevalent, in view of the complex lipid storage pattern and variable sphingomyelinase activities. Although the primary lesion is still unknown, studies conducted over the past six years have led to a breakthrough by showing that this disorder is characterized by unique abnormalities of intracellular translocation of exogenous cholesterol. In cultured fibroblasts of patients, this block leads to a delayed induction of the homeostatic responses to exogenous cholesterol, in particular cholesteryl ester formation, and to the accumulation of unesterified cholesterol in a vesicular, essentially lysosomal, compartment. The transport of endogenous cholesterol is apparently unaffected. The spectrum of phenotypic heterogeneity in relation to abnormal LDL-processing has been defined in a large patient population. Clinical presentation of the disease is also reviewed and biochemical correlations are discussed. This discovery has had immediate medical applications, by providing the first strategy for reliable prenatal diagnosis of the disorder and easy diagnosis of patients. To date, the exact implication of the cholesterol transport defect in the pathogenesis of Niemann-Pick type C is not known; recent observations have opened up new possible approaches for the understanding of this lesion. Although final classification of Niemann-Pick disease type C must await elucidation of the primary defect(s), present knowledge already establishes that the disease is a nosological entity distinct from Niemann-Pick disease type A and B, and suggests that it might be the model for a new molecular concept of neurolipidosis — and even of inherited metabolic disease.

Deletion of arginine (608) in acid sphingomyelinase is the prevalent mutation among Niemann-Pick disease type B patients from northern Africa.

There is a high incidence of Niemann-Pick type B disease in the Maghreb region of North Africa, which includes Morocco, Algeria and Tunisia. A hypothesis that there may well be a common, predominant mutant acid sphingomyelinase allele responsible for the type B phenotype in this population has been tested. A deletion of an arginine codon at amino acid residue 608 was found in one patient. The same mutation was also observed in another of our cases. An original screening procedure using 3′end digoxigenin-labeled allele-specific oligonucleotides and chemiluminescent detection was developed and used parallel to the conventional assay with 5′-end radiolabeled oligonucleotides. Of the 15 non-related, non-Jewish North African type B patients studied, 12 were homozygous and two compound heterozygous for this deletion (26 ΔR608 alleles/30 mutant alleles). Among type B patients from other geographic regions (France, UK, Italy, Czechoslovakia), this mutation was observed in only one of the 16 alleles studied. Our results indicate that deletion of arginine 608 in the acid sphingomyelinase gene is the highly prevalent mutation underlying Niemann-Pick type B disease in the population of Maghreb. A varying severity of the clinical and enzymatic expression within the non-neuronopathic phenotype has however been observed in patients homozygous for the mutation.

Increasing endogenous ceramide using inhibitors of sphingolipid metabolism maximizes ionizing radiation-induced mitochondrial injury and apoptotic cell killing.

To enhance the killing effects of ionizing radiation, we amplified the endogenous ceramide signal in Jurkat cell cultures using 3 different inhibitors of sphingolipid metabolism: DL‐PDMP, D‐MAPP and imipramine. Of the various possible drug combinations, only DL‐PDMP (20 μM) + imipramine (20 μM) and DL‐PDMP (20 μM) + imipramine (20 μM) + D‐MAPP (5 μM) induced a major increase in ceramide levels, reaching 240% and 340% of control values, respectively, after incubation for 48 hr. With these models, we demonstrate that endogenously formed ceramide triggers time‐ and concentration‐dependent apoptosis through induction of mitochondrial injury and activation of the caspase pathway. Cellular dysfunction includes alterations to the cellular redox potential, as assessed by the generation of ROS and total glutathione depletion, and a drop in ΔΨm. A parallel elevation of mitochondrial ceramide levels was also observed. The combination of DL‐PDMP + imipramine ± D‐MAPP with 10 Gy irradiation produced cumulative effects leading to apoptosis via mitochondrial collapse and activation of the caspase cascade. The association efficiency was confirmed in normal and acid sphingomyelinase‐deficient lymphoid cell lines. Taken together, these results suggest that increasing endogenous ceramide levels may potentially be very valuable when combined with ionizing radiation in tumor therapy.

Biochemical studies in Niemann-Pick disease. III. In vitro and in vivo assays of sphingomyelin degradation in cultured skin fibroblasts and amniotic fluid cells for the diagnosis of the various forms of the disease.

Sphingomyelinase activities were assayed in vitro in cultured skin fibroblasts of 61 patients with Niemann‐Pick disease (NPD). Residual activities found in type A and B were 1% and 4%, respectively, of the mean control values, i.e. significantly higher in type B. In 27 cases with NPD type C, the mean activity was 42% of that in controls, with residual activities ranging from 15% up to normal. Fifteen pregnancies at risk for NPD type A and B were monitored; 4 affected foetuses were found.

Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to γ-radiation but activation of this sequence is defective in radioresistant SQ20B cells

Purpose : To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to γ-rays. Materials and methods : Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (ΔΨm) , reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry. Results : In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in ΔΨm and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, γ-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway. Conclusions : Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.