Utility of rapid whole-exome sequencing in the diagnosis of Niemann-Pick disease type C presenting with fetal hydrops and acute liver failure.

Rapid whole-exome sequencing (rWES) is used in critically ill newborn infants to inform about diagnosis, clinical management, and prognosis. Here we report a male newborn infant with hydrops, pancytopenia, and acute liver failure who was listed for liver transplantation. Given the acuity of the presentation, the procedure-related morbidity and mortality, and lack of diagnosis, we used rWES in the proband and both parents with a turnaround time of 10 business days. rWES returned one maternally inherited, likely pathogenic and one paternally inherited, likely pathogenic variant in NPC1, suggestive of a diagnosis of Niemann-Pick disease type C (NPC). Interestingly, a diagnosis of NPC was entertained prior to rWES, but deemed unlikely in light of absent cholesterol storage on liver biopsy and near-normal oxysterol levels in dried blood. The diagnosis of NPC was confirmed on filipin stain in fibroblasts demonstrating defective cholesterol trafficking. NPC is a slowly progressive neurodegenerative disorder that may also affect the liver with overall poor prognosis. It was decided to take the infant off the transplant list and transfer to palliative care, where he died after 4 wk. This case highlights the utility of rWES in an acute clinical setting for several domains of precision medicine including (1) diagnosis, (2) prognosis and outcome, (3) management and therapy, and (4) utilization of resources.

HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration.

Keratinocyte migration plays an important role in cutaneous wound healing by supporting the process of reepithelialisation. During directional migration cells develop a polarised shape with an asymmetric distribution of a variety of signalling molecules in their plasma membrane. Here, we investigated front-to-back differences of the physical properties of the plasma membrane of migrating keratinocyte-like HaCaT cells. Using FRAP and fluorescence lifetime analysis, both under TIR illumination, we demonstrate a reduced viscosity of the plasma membrane in the lamellipodia of migrating HaCaT cells compared with the cell rears. This asymmetry is most likely caused by a reduced cholesterol content of the lamellipodia as demonstrated by filipin staining. siRNA-mediated silencing of the cholesterol transporter ABCA1, which is known to redistribute cholesterol from rafts to non-raft regions, as well as pharmacological inhibition of this transporter with glibenclamide, strongly diminished the viscosity gradient of the plasma membrane. In addition, HaCaT cell migration was inhibited by glibenclamide treatment. These data suggest a preferential role of non-raft cholesterol in the establishment of the asymmetric plasma membrane viscosity.

Fluorescent in situ visualization of sterols in Arabidopsis roots.

Sterols are eukaryotic membrane components with crucial roles in diverse cellular processes. Elucidation of sterol function relies on development of tools for in situ sterol visualization. Here we describe protocols for in situ sterol localization in Arabidopsis thaliana root cells, using filipin as a specific probe for detection of fluorescent filipin-sterol complexes. Currently, filipin is the only established tool for sterol visualization in plants. Filipin labeling can be performed on aldehyde-fixed samples, largely preserving fluorescent proteins and being compatible with immunocytochemistry. Filipin can also be applied for probing live cells, taking into account the fact that it inhibits sterol-dependent endocytosis. The experimental procedures described are designed for fluorescence detection by confocal laser-scanning microscopy with excitation of filipin-sterol complexes at 364 nm. The protocols require 1 d for sterol covisualization with fluorescent proteins in fixed or live roots and 2 d for immunocytochemistry on whole-mount roots.

BAR domain proteins Rvs161 and Rvs167 contribute to Candida albicans endocytosis, morphogenesis, and virulence.

The Candida albicans plasma membrane plays critical roles in growth and virulence and as a target for antifungal drugs. Three C. albicans genes that encode Bin-Amphiphysin-Rvs homology domain proteins were mutated to define their roles in plasma membrane function. The deletion of RVS161 and RVS167, but not RVS162, caused strong defects. The rvs161Delta mutant was more defective in endocytosis and morphogenesis than rvs167Delta, but both were strongly defective in polarizing actin patches. Other plasma membrane constituents were still properly localized, including a filipin-stained domain at the hyphal tips. An analysis of growth under different in vitro conditions showed that the rvs161Delta and rvs167Delta mutants grew less invasively in agar and also suggested that they have defects in cell wall synthesis and Rim101 pathway signaling. These mutants were also more resistant to the antimicrobial peptide histatin 5 but showed essentially normal responses to the drugs caspofungin and amphotericin. Surprisingly, the rvs161Delta mutant was more sensitive to fluconazole, whereas the rvs167Delta mutant was more resistant, indicating that these mutations cause overlapping but distinct effects on cells. The rvs161Delta and rvs167Delta mutants both showed greatly reduced virulence in mice. However, the mutants were capable of growing to high levels in kidneys. Histological analyses of infected kidneys revealed that these rvsDelta mutants grew in a large fungal mass that was walled off by leukocytes, rather than forming disseminated microabscesses as seen for the wild type. The diminished virulence is likely due to a combination of the morphogenesis defects that reduce invasive growth and altered cell wall construction that exposes proinflammatory components to the host immune system.

Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.

OBJECTIVE: Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. METHODS AND RESULTS: AgLDL resides for prolonged periods in surface-connected compartments. Although agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells because of hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. CONCLUSIONS: We describe a novel process that occurs during agLDL-macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathological positive feedback loop for increased catabolism of agLDL and eventual foam cell formation.

Saccades in adult Niemann-Pick disease type C reflect frontal, brainstem, and biochemical deficits.

BACKGROUND: The autosomal recessive disorder Niemannn-Pick type C (NPC) presents in adulthood with psychosis or cognitive deficits associated with supranuclear gaze palsies. While saccadic innervation to the extraocular muscles is generated in the brainstem, the frontal lobes play an integral role in the initiation of volitional saccades and the suppression of unwanted reflexive saccades. No study has examined the frontally driven volitional control of saccadic eye movements in NPC. OBJECTIVE: To examine self-paced and antisaccades as well as reflexive saccades in adult patients with NPC, a disorder known to affect brainstem and frontal cortical function. METHODS: Three biochemically confirmed adult patients with NPC were compared with 10 matched controls on horizontal saccadic and antisaccadic measures using an infrared limbus eye tracker. Patients' cholesterol esterification and filipin staining, Mini-Mental State performance, and NPC symptom level were rated. RESULTS: Reflexive saccade latency ranged from shorter to longer than normal, reflexive saccade gain was reduced, asymptotic peak velocity was reduced, fewer self-paced saccades were generated, and increased errors on antisaccades were made by patients compared to controls. Patients with more severe biochemical, cognitive, and symptom deficits performed most poorly on brainstem and frontal ocular motor measures. Paradoxically, less severe illness was associated with an abnormally reduced saccadic latency. CONCLUSIONS: Ocular motor measures provide an index of disease severity in Niemannn-Pick type C (NPC) and may be a useful adjunct for monitoring the illness progress and medication response. Reduced saccadic latency may result from inadequate fixation input from abnormally functioning frontal eye fields in NPC.

Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts.

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.

[Diagnosis of adult type of Niemann-Pick disease (type C) in two brothers by filipin staining of bone marrow smears].

Niemann-Pick disease, type C (NPC) is a neurometabolic genetic disorder that is distinguished from other types of Niemann-Pick disease by its later onset, more insidious progression, variable visceromegaly, and abnormalities of intracellular cholesterol metabolism. We report cases in 18-year-old and 20-year-old brothers who presented with disinhibition and involuntary movement of their hands. Both brothers presented various signs such as dementia, vertical supranuclear ophthalmoplegia (VSO), dysarthria, axial and limb dystonia, hyperreflexia, pathologic reflex, cerebellar ataxia, as reported. They also presented startle response. Brain MRI showed diffuse cerebral atrophy and abdominal CT reveals hepato-splenomegaly in both patients. These cases were suspected to be NPC based on dementia, VSO, cerebellar ataxia, hepato-splenomegaly and foam cells in the bone marrow. Generally, the diagnosis of NPC is based on deficient cholesterol esterification and excessive lysosomal filipin staining in cultured skin fibroblasts. However, culture of fibroblasts obtained from a biopsied skin samples is slow. We have rapidly made the diagnosis of NPC in our patients by filipin staining of foam cells from bone marrow. This diagnostic process using a bone marrow smear is more convenient and rapid than previous methods using cultured skin fibroblasts.

The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55.

Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication.

Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.

Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization.

Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.

Increased levels of GM2 ganglioside in fibroblasts from a patient with juvenile Niemann-Pick disease type C.

A 15-year-old boy was suffering from splenomegaly and a 10-year history of a neurologic disorder that included mental retardation, vertical supranuclear gaze palsy, dysarthria, ataxia, and dystonia. Bone marrow aspirates revealed foamy cells with storage materials which were positive with filipin staining. Cultured skin fibroblasts derived from the patient showed moderate loss of sphingomyelinase activity and the impairment of cholesterol esterification. The characteristic clinical presentations and typical histochemical findings of this patient met the diagnostic criteria of Niemann-Pick disease type C (NPC). In the fibroblasts from the patient, there was an accumulation of GM2 ganglioside around their cytoplasms. Increased levels of glycolipids. including GM2 ganglioside are reported in the cerebral cortex of NPC, but not in the fibroblasts. The fibroblasts derived from NPC may reflect the abnormal metabolism of glycolipids in the central nervous system of NPC.

Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages.

The free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381-388, 1992) using THP-1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP-1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reduction in cholesterol biosynthesis.

Distribution of filipin-sterol complexes in sperm membranes from hypercholesterolaemic rabbits.

The distribution of membrane filipin-sterol complexes (FSC) was examined ultrastructurally in cauda epididymal sperm from normal and hypercholesterolaemic rabbits. Membrane FSC were quantitatively analysed on replicas of filipin-treated cells. We determined a significant difference in FSC concentration in the plasma membrane of the acrosome region (PMAR) of hypercholesterolaemic animals compared to normal rabbits. Hypercholesterolaemic animals had 0.56 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol diet: Diet 2) in the marginal segment of PMAR; 0.62 +/- 0.05 FSC complex per micron 2 (enriched Cholesterol and fish oil diet: Diet 3) and only 0.28 +/- 0.01 FSC complex per micron 2 for normal animals (Control Diet 1). In the principal (anterior) segment we found 0.54 +/- 0.10 FSC complex per micron 2 (Diet 2), 0.56 +/- 0.03 FSC complex per micron 2 (Diet 3) and 0.30 +/- 0.04 FSC complex per micron 2 (Control Diet 1). We also counted 0.47 +/- 0.1 FSC complex per micron 2 in the equatorial segment of PMAR for Diet 2, 0.27 +/- 0.05 and 0.28 +/- 0.04 FSC complex per micron 2 in Diet 1 and Diet 3 respectively. Diet 4 (fish oil) did not differ from the control. An increase in the Cholesterol (Chol) level in biological membranes or a difference in the Chol membrane domains could cause a variation in the membrane rigidity that could modify the sperm membrane fusion capacity and functionality. The results presented in this paper are in agreement and could explain the decrease in the kinetic of the sperm acrosome reaction that we have observed in experimentally hypercholesterolaemic rabbits (Díaz-Fontdevila & Bustos-Obregón, 1992).

Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus.

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 +/- 44 FSC/microns2 (mean +/- standard deviation, S.D.), 322 +/- 41 FSC/microns2 and 355 +/- 31 FSC/microns2 in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 +/- 39 FSC/microns2) and uterine sperm (243 +/- 50 FSC/microns2). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.

Filipin-sterol complexes in molluscan gill ciliated epithelial cell membranes: intercalation into ciliary necklaces and induction of gap junctional particle arrays.

Freeze-fracture electron microscopy has been used in conjunction with the antibiotic filipin to investigate possible differences in the distribution of sterols in ciliary and somatic cell membranes of scallop and mussel gill epithelial cells. Contrary to previous reports, we find that filipin-sterol lesions can occur among the strands of the ciliary necklace but they are partially excluded from the smooth neck region above the necklace where the membrane is tightly apposed to the axonemal microtubules. No obvious differences in filipin-sterol lesions occur in the membranes of mussel gill cilia of varying mechanical sensitivity. Although abundant in the apical plasma membrane, filipin-sterol complexes are rare within the membranes of microvilli. Filipin-sterol lesions form outside the loosely parallel particle strands of septate junctions, sometimes increasing their relative orderliness. At sufficiently high density, filipin-sterol protrusions within the plasma membrane result in mass aggregation of gap junctions, possibly through recruitment of unorganized connexons.

Type C Niemann-Pick disease: documentation of abnormal LDL processing in lymphocytes.

Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (+/- 5%) and 45% (+/- 16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.

Alignment of cholesterol in the membrane bilayer.

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by "lines." The FCC and "lines" are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in "rhombs," "lines" only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and "lines", as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.

Filipin-labelled complexes are polarized in their distribution in the cytoplasm of meiotically mature mouse eggs.

Unfertilized (germinal vesicle [GV] stage, superovulated and naturally ovulated) and fertilized mouse eggs were treated with the polyene antibiotic filipin, which complexes with unesterified sterols; specimens were observed by fluorescence microscopy and scanning electron microscopy (SEM). In all oocytes examined, filipin fluorescence was localized to the plasma membrane and to subcellular structures of various sizes. In the unfertilized oocyte, polarity was observed both in the plasma membrane stain and in the pattern formed by the subcellular structures. SEM of filipin-treated oocytes had several characteristic features including a specific distribution of heterogeneous microvilli that appears to have a spatial relationship with the fluorescent pattern of the filipin-positive subcellular structures. In GV stage and fertilized eggs the filipin-positive subcellular structures were associated with the germinal vesicle and in fertilized eggs they were associated with the site of polar body abstriction.