Liver X Receptor Genes Variants Modulate ALS Phenotype

Amyotrophic lateral sclerosis (ALS) is one of the most severe motor neuron (MN) disorders in adults. Phenotype of ALS patients is highly variable and may be influenced by modulators of energy metabolism. Recent works have implicated the liver X receptors α and β (LXRs), either in the propagation process of ALS or in the maintenance of MN survival. LXRs are nuclear receptors activated by oxysterols, modulating cholesterol levels, a suspected modulator of ALS severity. In a cohort of 438 ALS patients and 330 healthy controls, the influence of LXR genes on ALS risk and phenotype was studied using single nucleotide polymorphisms (SNPs). The two LXRα SNPs rs2279238 and rs7120118 were shown to be associated with age at onset in ALS patients. Consistently, homozygotes were twice more correlated than were heterozygotes to delayed onset. The onset was thus delayed by 3.9 years for rs2279238 C/T carriers and 7.8 years for T/T carriers. Similar results were obtained for rs7120118 (+2.1 years and +6.7 years for T/C and C/C genotypes, respectively). The LXRβ SNP rs2695121 was also shown to be associated with a 30% increase of ALS duration (p = 0.0055, FDR = 0.044). The tested genotypes were not associated with ALS risk. These findings add further evidence to the suspected implication of LXR genes in the disease process of ALS and might open new perspectives in ALS therapeutics.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Characterization of the ion channel activity in planar bilayers containing IgE-Fc epsilon receptor and the cromolyn-binding protein.

Electric conductance was studied across micropipette-supported planar lipid bilayers, reconstituted with IgE-Fc epsilon receptor and the cromolyn-binding protein (CBP) isolated from membranes of rat basophilic leukemia cells (RBL-2H3). Currents were observed following the addition of aggregating agents, specific for either of the two proteins. The results show that the two proteins are necessary and sufficient for the opening of cation channels. Both aggregation of Fc epsilon receptor via IgE with a specific antigen and of CBP by anti-CBP induce channels with similar conductances and open-time distributions. In the presence of 1.8 mM calcium, the most frequently observed channels have a conductance of 1-2 pS. At 100 mM calcium conductance increased to 4-5 pS. Channels induced by antigen were susceptible to blocking by the anti-allergic drug cromolyn. These results suggest that CBP acts as the core of the cation channel and that the channel conductance and open-time characteristics are independent of the mode of aggregation.     

Chromatographic micro-procedure for trace determination of phenobarbital in blood serum

Volumes of 100 μl of serum were sufficient for the determination of therapeutic levels of phenobarbital. The isolation procedure was performed using a column method with a hydrophobic adsorbent, graphitized carbon black (Carbopack B). With this method the quantitative (98.1%) recovery of phenobarbital was measured. By suitable choice of experimental conditions, a highly selective purification of the drug can be obtained, thus eliminating various sources of error during quantitation due to the presence in the final samples of endogenous compounds. For the quantitation procedure, another type of graphitized carbon black (Carbopack C) suitably modified was used for gas chromatography. Calibration curves showed no chemisorption effect along the column even on injecting 5 ng of phenobarbital. Some practical aspects of the procedure for improving the reliability of the results are discussed.     

1H-NMR-based metabolomic profiling of CSF in early amyotrophic lateral sclerosis

Background

Pathophysiological mechanisms involved in amyotrophic lateral sclerosis (ALS) are complex and none has identified reliable markers useful in routine patient evaluation. The aim of this study was to analyze the CSF of patients with ALS by ¹H NMR (Nuclear Magnetic Resonance) spectroscopy in order to identify biomarkers in the early stages of the disease, and to evaluate the biochemical factors involved in ALS.

Methodology

CSF samples were collected from patients with ALS at the time of diagnosis and from patients without neurodegenerative diseases. One and two-dimensional ¹H NMR analyses were performed and metabolites were quantified by the ERETIC method. We compared the concentrations of CSF metabolites between both groups. Finally, we performed principal component (PCA) and discriminant analyses.

Principal Findings

Fifty CSF samples from ALS patients and 44 from controls were analyzed. We quantified 17 metabolites including amino-acids, organic acids, and ketone bodies. Quantitative analysis revealed significantly lower acetate concentrations (p = 0.0002) in ALS patients compared to controls. Concentration of acetone trended higher (p = 0.015), and those of pyruvate (p = 0.002) and ascorbate (p = 0.003) were higher in the ALS group. PCA demonstrated that the pattern of analyzed metabolites discriminated between groups. Discriminant analysis using an algorithm of 17 metabolites revealed that patients were accurately classified 81.6% of the time.

Conclusion/Significance

CSF screening by NMR spectroscopy could be a useful, simple and low cost tool to improve the early diagnosis of ALS. The results indicate a perturbation of glucose metabolism, and the need to further explore cerebral energetic metabolism.     

Elevated Serum Ferritin Is Associated with Reduced Survival in Amyotrophic Lateral Sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the loss of motor neurons. Its etiology remains unknown, but several hypothesis have been raised to explain motor neuron death, including oxidative stress. Dysregulation of cellular iron metabolism can lead to increased oxidative stress, and existing data argue for a role of iron metabolism in ALS pathophysiology.

Methods

We performed a retrospective analysis of iron metabolism (IM) variables (serum levels of iron, transferrin, ferritin, and TSC for Transferrin Saturation Coefficient) in a cohort of 694 ALS patients and 297 healthy controls.

Results

Serum ferritin levels and TSC were higher, whereas serum transferrin levels were lower in ALS patients than controls. In addition, patients with a high level serum ferritin had a shorter survival time compared to those with low level serum ferritin (618 days versus 921 days for men subgroup; p = .007). Site of onset and ALS-FRS score were not associated with IM variables.

Conclusion

This study suggests that ALS patients may have increased iron storage, as measured by increased serum ferritin and TSC. Elevated serum ferritin may also have a deleterious impact on survival in ALS.     

Distribution physiologique cérébrale et corps entier du 18F-DPA-714 en TEP/TDM

ObjectiveTo investigate the physiological biodistribution of N,N-diethyl-2-(2 – (4 – (2-fluoroethoxy) phenyl) -5,7-dimethylpyrazolo [1,5] pyrimidin-3-yl) acetamide labeled with fluorine 18 (¹⁸F-DPA-714) in humans, by PET/CT in the brain and the whole body. The DPA-714 is a ligand of the translocator protein (Translocator Protein kDa or TSPO), protein overexpressed by microglia in case of neuroinflammation.Materials and methodsDynamic PET/CT brain acquisitions were performed in six healthy volunteers for 90 minutes after intravenous injection of ¹⁸F-DPA-714. Brain biodistribution of ¹⁸F-DPA-714 was assessed visually and using regions of interest (ROI), according to MNI AAL guidelines in order to obtain the activity/time curves for each brain region predefined. One of the subjects was also included whole body PET/CT acquisitions 1 hour after injection of ¹⁸F-DPA-714, allowing visual analysis and semi-quantitative distribution of the tracer, by definition of ROI and SUVs max computation.ResultsThe maximum brain uptake of ¹⁸F-DPA-714 was visualized at 3.5 minutes after injection, gray matter, mostly thalamic. This peak was followed by two elimination phases: an initial rapid phase (3.5 to 35 minutes) and a slower phase until the end of recording. Uptake of ¹⁸F-DPA-714 was generally consistent across brain structures analyzed. The whole body images show significant activity in the gallbladder, spine and salivary glands under the jaw, in accordance with previous published studies using other radioligands for TSPO.ConclusionThis very preliminary study confirms that the brain biodistribution of ¹⁸F-DPA-714 makes it an interesting marker of neuroinflammation. This work allows to recommend a PET protocol acquisition. However, it now seems necessary to implement these findings in patients referred for brain conditions.     

Hyponatraemia and death or permanent brain damage in healthy children.

OBJECTIVE--To determine if hyponatraemia causes permanent brain damage in healthy children and, if so, if the disorder is primarily limited to females, as occurs in adults. DESIGN--Prospective clinical case study of 16 affected children and a review of 24,412 consecutive surgical admissions at one medical centre. PATIENTS--16 children (nine male, seven female; age 7 (SD 5) years) with generally minor illness were electively hospitalised for primary care. Consultation was obtained for the combination of respiratory arrest with symptomatic hyponatraemia (serum sodium concentration less than or equal to 128 mmol/l). MAIN OUTCOME MEASURES--Presence, gender distribution, and classification of permanent brain damage in children with symptomatic hyponatraemia in both prospective and retrospective studies. RESULTS--By retrospective evaluation the incidence of postoperative hyponatraemia among 24,412 patients was 0.34% (83 cases) and mortality of those afflicted was 8.4% (seven deaths). In the prospective population the serum sodium concentration on admission was 138 (SD 2) mmol/l. From three to 120 inpatient hours after hypotonic fluid administration patients developed progressive lethargy, headache, nausea, and emesis with an explosive onset of respiratory arrest. At the time serum sodium concentration was 115 (7) mmol/l and arterial oxygen tension 6 (1.5) kPa. The hyponatraemia was primarily caused by extrarenal loss of electrolytes with replacement by hypotonic fluids. All 16 patients had cerebral oedema detected at either radiological or postmortem examination. All 15 patients not treated for their hyponatraemia in a timely manner either died or were permanently incapacitated by brain damage. The only patient treated in a timely manner was alive but mentally retarded. CONCLUSIONS--Symptomatic hyponatraemia can result in high morbidity in children of both genders, which is due in large part to inadequate brain adaptation and lack of timely treatment.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)