Niemann-Pick C Disease Gene Mutations and Age-Related Neurodegenerative Disorders

Niemann-Pick type C (NPC) disease is a rare autosomal-recessively inherited lysosomal storage disorder caused by mutations in NPC1 (95%) or NPC2. Given the highly variable phenotype, diagnosis is challenging and particularly late-onset forms with predominantly neuropsychiatric presentations are likely underdiagnosed. Pathophysiologically, genetic alterations compromising the endosomal/lysosomal system are linked with age-related neurodegenerative disorders. We sought to examine a possible association of rare sequence variants in NPC1 and NPC2 with Parkinson''s disease (PD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), and to genetically determine the proportion of potentially misdiagnosed NPC patients in these neurodegenerative conditions. By means of high-resolution melting, we screened the coding regions of NPC1 and NPC2 for rare genetic variation in a homogenous German sample of patients clinically diagnosed with PD (n = 563), FTLD (n = 133) and PSP (n = 94), and 846 population-based controls. The frequencies of rare sequence variants in NPC1/2 did not differ significantly between patients and controls. Disease-associated NPC1/2 mutations were found in six PD patients (1.1%) and seven control subjects (0.8%), but not in FTLD or PSP. All rare variation was detected in the heterozygous state and no compound heterozygotes were observed. Our data do not support the hypothesis that rare NPC1/2 variants confer susceptibility for PD, FTLD, or PSP in the German population. Misdiagnosed NPC patients were not present in our samples. However, further assessment of NPC disease genes in age-related neurodegeneration is warranted.     

Pro-inflammatory cytokine and chemokine release by human brain microvascular endothelial cells stimulated by Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a world-wide agent of diseases among pigs including meningitis, septicemia and arthritis. This microorganism is also recognized as an important zoonotic agent. The pathogenesis of the meningitis caused by S. suis is poorly understood. We have previously shown that S. suis is able to adhere to human brain microvascular endothelial cells (BMEC), but not to human umbilical vein endothelial cells (HUVEC). The objective of this work was to study the ability of S. suis serotype 2 to induce the release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1); IL-6 and the chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) by human BMEC and HUVEC, using a sandwich enzyme-linked immunosorbent assay. S. suis was able to stimulate the production of IL-6, IL-8 and MCP-1 by BMEC but not HUVEC, in a time- and concentration-dependent manner. Bacterial cell wall components were largely responsible for such stimulation. The human and pig origin of strains does not seem to affect the intensity of the response; indeed, a very heterogeneous pattern of cytokine and chemokine production was observed for the different strains tested in this study. In situ production of cytokines and chemokines by BMEC may be the result of specific adhesion of S. suis to this cell type, with several consequences such as increased recruitment of leukocytes and an increase in the blood-brain barrier permeability.     

Oscillating Ca2+-Induced Channel Activity Obtained in BLM with a Mitochondrial Membrane Component

Oscillations in ion fluxes and membrane potential may be observed in cells and in mitochondria as well. We obtained Ca²⁺-induced oscillations in channel activity in black-lipid membranes reconstituted with hydrophobic components extracted from mitochondria. Mitoplasts prepared from purified rat liver mitochondria were extracted with ethanol followed by Folch extraction and further partial purification by silicic acid chromatography. Channel activity was measured in lipid bilayers formed from bovine brain lipids and 10% cardiolipin with addition of the purified tractions. The conductance with 10 mM Ca²⁺ was 100 pS or its multiples. Ca²⁺ gradients of 4 : 1 induced oscillating channel activity for several hours, with initial open states of 40 s and closed states of 56 s; the open times gradually decreasing to 8.6 s. No channel activity was seen without added fractions. The channel activity was associated with a Ca²⁺-binding lipid, nonpolar, low-molecular-weight fraction that in gel electrophoresis was not stained with Coomassie Blue and did not contain carbohydrate-staining material. ¹H-Nuclear magnetic resonance spectra of the substance showed the presence of aliphatic chains and carbonyls, but the detailed structure remains to be elucidated.     

Deficiencies in sex-regulated expression and levels of two hepatic sterol carrier proteins in a murine model of Niemann-Pick type C disease.

Hepatic sterol carrier protein-2 (SCP2) and sterol carrier protein-X (SCPx) levels in normal and in mutant Niemann-Pick Type C mice were determined by immunoblotting with antiserum against rat SCP2. A 14-kDa protein (SCP2) was detected in the cytosol fraction and a 58-kDa protein (SCPx) was found in both cytosolic and organellar fractions. Expression of hepatic SCPx protein was developmentally regulated in a sex-specific pattern. The amounts of organelle-associated SCPx increased 4-fold during sexual development of normal males but decreased dramatically during development of normal females. Levels of hepatic SCP2 increased much less dramatically during sexual maturation of normal males and females. Adult Niemann-Pick Type C mice were deficient in both hepatic SCPx and SCP2. The deficit in SCPx in affected males reflected a failure to increase hepatic SCPx levels during sexual maturation. In affected males SCPx remained at levels found in immature mice. Affected male and female mice were also unable to maintain levels of hepatic SCP2. The level of SCP2 was near normal in affected immature males and subnormal in affected immature females. During sexual maturation hepatic SCP2 declined in affected animals.     

Lipid Changes in Niemann-Pick Disease Type C Brain: Personal Experience and Review of the Literature

Niemann-Pick disease type C (NPC) is a neurovisceral disorder characterized by lysosomal sequestration of endocytosed LDL-cholesterol, premature and abnormal enrichment of cholesterol in trans Golgi cisternae and accompanying anomalies in intracellular sterol trafficking. In addition to cholesterol, the NPC lesion has also been shown to impact the metabolism of sphingolipids. Lipids, more particularly glycolipids, were studied in brain tissue from eight cases with proven NPC, ranging from 21 fetal weeks to 19 years of age (one case with rapidly fatal neonatal cholestatic icterus, three cases with infantile neurological onset, one late infantile and two juvenile neurological cases). In gray matter, the concentrations of total cholesterol, sphingomyelin and total gangliosides were within the normal range in all cases. In white matter, a severe loss of galactosylceramide and other myelin lipids (including cholesterol) was prominent in patients with the neurological severe infantile form (levels similar to those in 6–8 month-old infants) or the late infantile form of the disease, but only a slight decrease was observed in patients with a juvenile neurological onset. Analysis of the ganglioside profiles and study of minor neutral glycolipids revealed striking abnormalities, although not present at the fetal stage. In cerebral cortex, gangliosides GM3 and GM2 showed a significant increase, 10–15 fold and 3–5-fold the normal level, respectively, with already some abnormalities in a 3-month-old patient. Except in the latter patient, a prominent storage of glucosylceramide, lactosylceramide and gangliotriaosylceramide (asialo-GM2) was observed, with 10–50-fold increases from the normal concentration. The fatty acid composition of these glycolipids suggests that they have a neuronal origin. A slight increase of globotriaosyl- and globotetraosyl-ceramide and of more complex neutral glycolipids also occurred. While ganglioside changes were essentially similar in gray and white matter, changes of the neutral glycolipids were only minimal in the latter. Our data are in good accordance with previous studies and provide additional information. They emphasize that, apart a varying demyelinating process (most pronounced in children with a severe infantile neurological form) brain lipids abnormalities are essentially located to the gray matter. They confirm and give more precise information on the glycolipid nature of the neuronal storage, and establish that a similar type of changes occurs in the different neurological forms of the disease. Yet, our study indicates that glycolipid changes in brain do not occur before a few months after birth, possibly at a period concomitant with the onset of neurological symptoms, in contrast to the very early glycolipid abnormalities observed in non-neural organs. Glycolipid changes rather similar to those seen in NPC brain, in particular for gangliosides, have been described for other lysosomal disorders such as Niemann-Pick type A and mucopolysaccharidoses. The glucosyl-and lactosylceramide accumulation, however, is more striking in NPC, especially taking into account that there is no other known storage in NPC brain. Some neuropathological changes, such as ectopic neurites, could be related to the glycolipid changes. Metabolic studies in cultured fibroblasts combined to the observation that no lipids other than glycolipids accumulate in brain suggest that the NPC gene products possibly participate in intracellular transport or regulate metabolism of glycolipids.     

Long‐term response of a Mojave Desert winter annual plant community to a whole‐ecosystem atmospheric CO2 manipulation (FACE)

Desert annuals are a critically important component of desert communities and may be particularly responsive to increasing atmospheric (CO₂) because of their high potential growth rates and flexible phenology. During the 10‐year life of the Nevada Desert FACE (free‐air CO₂ enrichment) Facility, we evaluated the productivity, reproductive allocation, and community structure of annuals in response to long‐term elevated (CO₂) exposure. The dominant forb and grass species exhibited accelerated phenology, increased size, and higher reproduction at elevated (CO₂) in a wet El Niño year near the beginning of the experiment. However, a multiyear dry cycle resulted in no increases in productivity or reproductive allocation for the remainder of the experiment. At the community level, early indications of increased dominance of the invasive Bromus rubens at elevated (CO₂) gave way to an absence of Bromus in the community during a drought cycle, with a resurgence late in the experiment in response to higher rainfall and a corresponding high density of Bromus in a final soil seed bank analysis, particularly at elevated (CO₂). This long‐term experiment resulted in two primary conclusions: (i) elevated (CO₂) does not increase productivity of annuals in most years; and (ii) relative stimulation of invasive grasses will likely depend on future precipitation, with a wetter climate favoring invasive grasses but currently predicted greater aridity favoring native dicots.     

Structure and function of the NPC2 protein

Somatic cell hydridization and linkage studies indicated the implication of a second gene as a cause of Niemann-Pick C disease in a minority (5%) of patients. A study of the lysosomal proteome led to the identification of a previously known gene, HE1, as the NPC2 gene. The mature NPC2/HE1 protein is a ubiquitous soluble small 132-amino-acid glycoprotein, first characterized as a major secretory protein in the human epididymis, but also detected in most tissues. Seventeen families with mutations in the NPC2 gene are known. Good genotype-phenotype correlations were observed. No distinction can be made between the biochemical phenotypes of NPC1 or NPC2 mutants. The NPC2 protein binds cholesterol with submicromolar affinity at neutral and acidic pH. The bovine protein has been crystallized, and the cholesterol-binding site assigned to a hydrophobic loosely packed region. There is strong evidence that the NPC1 and NPC2 proteins must function in a closely related fashion. Current data have led to the hypothesis that NPC2 would bind cholesterol from internal lysosomal membranes, enabling a physical interaction with NPC1 (or another protein) and allowing postlysosomal export of cholesterol. In this model, the activity of NPC1 would depend on that of NPC2. The precise function of the NPC2 protein has, however, not been fully elucidated.     

The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.