BBA - Molecular Basis of Disease (v.1586, #2)

Genetic defects in hepatobiliary transport by Ronald Oude Elferink; Albert K Groen (129-145).
Bile formation, the exocrine function of the liver, represents a process that is unique to the hepatocyte as a polarized epithelial cell. The generation of bile flow is an osmotic process and largely depends on solute secretion by primary active transporters in the apical membrane of the hepatocyte. In recent years an impressive progress has been made in the discovery of these proteins, most of which belong to the family of ABC transporters. The number of identified ABC transporter genes has been exponentially increasing and the mammalian subfamily now counts at least 52. This development has been of crucial importance for the elucidation of the mechanism of bile formation, and it is therefore not surprising that the development in this field has run in parallel with the discovery of the ABC genes. With the identification of these transporter genes, the background of a number of inherited diseases, which are caused by mutations in these solute pumps, has now been elucidated. We now know that at least six primary active transporters are involved in canalicular secretion of biliary components (MDR1, MDR3, BSEP, MRP2, BCRP and FIC1). Four of these transporter genes are associated with inherited diseases. In this minireview we will shortly describe our present understanding of bile formation and the associated inherited defects.
Keywords: Bile; ABC transporter; P-type ATPase; Cholestasis; Bile salt; Bile acid; Hepatocyte;

Events upstream of mitochondrial protein import limit the oxidative capacity of fibroblasts in multiple mitochondrial disease by Arne A. Rungi; Andy Primeau; Lorraine Nunes Christie; Joseph W. Gordon; Brian H. Robinson; David A. Hood (146-154).
To investigate whether protein import is defective in mitochondrial disease, we compared the rate of import and the expression of protein import machinery components in skin fibroblasts from control subjects and a patient with multiple mitochondrial disease (MMD). The patient exhibited a 35% decrease in cytochrome c oxidase activity and a 59% decrease in cellular oxygen consumption compared to control. Western blot analyses revealed that patient levels of MDH, mtHSP70, HSP60, and Tom20 protein were 57%, 20%, 75% and 100% of control cells, respectively. MDH and Tom20 mRNA levels were not different from control levels, whereas mtHSP70 mRNA were 50% greater than control. Radiolabeled MDH was imported into mitochondria with equal efficiency between patient (44% of total synthesized) and control (43%) cells, although the total MDH synthesized in patient cells was reduced by about 40%. The unaffected levels of mRNA and post-translational import into mitochondria, combined with reduced protein levels of MDH, mtHSP70, and HSP60 suggest a translational defect in this patient with MMD. This was verified by the 50% reduction in overall cellular protein synthesis in the patient compared to control. Further, the similar import rates between patient and control cells suggest an important role for Tom20, but a lesser role for mtHSP70 in regulating protein import into mitochondria.
Keywords: Mitochondrial disease; Protein import; Malate dehydrogenase; Tom20; Heat shock protein;

Effects of aluminum and zinc on the oxidative stress caused by 6-hydroxydopamine autoxidation: relevance for the pathogenesis of Parkinson’s disease by Estefanı́a Méndez-Álvarez; Ramón Soto-Otero; Álvaro Hermida-Ameijeiras; Ana Marı́a López-Real; José Luis Labandeira-Garcı́a (155-168).
Aluminum and zinc have been related to the pathogenesis of Parkinson’s disease (PD), the former for its neurotoxicity and the latter for its apparent antioxidant properties. 6-Hydroxydopamine (6-OHDA) is an important neurotoxin putatively involved in the pathogenesis of PD, its neurotoxicity often being related to oxidative stress. The potential effect of these metals on the oxidative stress induced by 6-OHDA autoxidation and the potential of ascorbic acid (AA), cysteine, and glutathione to modify this effect were investigated. Both metals, particularly Al3+, induced a significant reduction in OH production by 6-OHDA autoxidation. The combined action of AA and a metal caused a significant and sustained increase in OH generation, particularly with Al3+, while the effect of sulfhydryl reductants was limited to only the first few minutes of the reaction. However, both Al3+ and Zn2+ provoked a decrease in the lipid peroxidation induced by 6-OHDA autoxidation using mitochondrial preparations from rat brain, assessed by TBARS formation. In the presence of AA, only Al3+ induced a significant reduction in lipid peroxidation. After intrastriatal injections of 6-OHDA in rats, tyrosine hydroxylase immunohistochemistry revealed that Al3+ reduces 6-OHDA-induced dopaminergic lesion in the striatum, which corroborates the involvement of lipid peroxidation in 6-OHDA neurotoxicity and appears to discard the participation of this mechanism on PD by Al3+ accumulation. The previously reported antioxidant properties of Zn2+ appear to be related to the induction of Zn2+-containing proteins and not to the metal per se.
Keywords: 6-Hydroxydopamine; Aluminum; Zinc; Antioxidant; Oxidative stress; Parkinson’s disease;

α-Mannosidosis is a lysosomal storage disorder caused by deficient activity of the lysosomal α-mannosidase. We report here the sequencing and expression of the lysosomal α-mannosidase cDNA from normal and α-mannosidosis guinea pigs. The amino acid sequence of the guinea pig enzyme displayed 82–85% identity to the lysosomal α-mannosidase in other mammals. The cDNA of the α-mannosidosis guinea pig contained a missense mutation, 679C>T, leading to substitution of arginine by tryptophan at amino acid position 227 (R227W). The R227W allele segregated with the α-mannosidosis genotype in the guinea pig colony and introduction of R227W into the wild-type sequence eliminated the production of recombinant α-mannosidase activity in heterologous expression studies. Furthermore, the guinea pig mutation has been found in human patients. Our results strongly indicate that the 679C>T mutation causes α-mannosidosis and suggest that the guinea pig will be an excellent model for investigation of pathogenesis and evaluation of therapeutic strategies for human α-mannosidosis.
Keywords: Animal model; Lysosomal storage; Missense; α-Mannosidosis; Guinea pig;

Oxidative stress increases internal calcium stores and reduces a key mitochondrial enzyme by Gary E. Gibson; Hui Zhang; Hui Xu; Larry C.H. Park; Thomas M. Jeitner (177-189).
Fibroblasts from patients with genetic and non-genetic forms of Alzheimer’s disease (AD) show many abnormalities including increased bombesin-releasable calcium stores (BRCS), diminished activities of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC), and an altered ability to handle oxidative stress. The link between genetic mutations (and the unknown primary event in non-genetic forms) and these other cellular abnormalities is unknown. To determine whether oxidative stress could be a convergence point that produces the other AD-related changes, these experiments tested in fibroblasts the effects of H2O2, in the presence or absence of select antioxidants, on BRCS and KGDHC. H2O2 concentrations that elevated carboxy-dichlorofluorescein (c-H2DCF)-detectable ROS increased BRCS and decreased KGDHC activity. These changes are in the same direction as those in fibroblasts from AD patients. Acute treatments with the antioxidants Trolox, or DMSO decreased c-H2DCF-detectable ROS by about 90%, but exaggerated the H2O2-induced increases in BRCS by about 4-fold and did not alter the reduction in KGDHC. Chronic pretreatments with Trolox more than doubled the BRCS, tripled KGDHC activities, and reduced the effects of H2O2. Pretreatment with DMSO or N-acetyl cysteine diminished the BRCS and either had no effect, or exaggerated the H2O2-induced changes in these variables. The results demonstrate that BRCS and KGDHC are more sensitive to H2O2 derived species than c-H2DCF, and that oxidized derivatives of the antioxidants exaggerate the actions of H2O2. The findings support the hypothesis that select abnormalities in oxidative processes are a critical part of a cascade that leads to the cellular abnormalities in cells from AD patients.
Keywords: Alzheimer’s disease; Calcium; Reactive oxygen species; Fibroblast; α-Ketoglutarate dehydrogenase complex; Antioxidant;

Role of glycine-33 and methionine-35 in Alzheimer’s amyloid β-peptide 1–42-associated oxidative stress and neurotoxicity by Jaroslaw Kanski; Sridhar Varadarajan; Marina Aksenova; D.Allan Butterfield (190-198).
Recent theoretical calculations predicted that Gly33 of one molecule of amyloid β-peptide (1–42) (Aβ(1–42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Aβ(1–42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Aβ(1–42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Aβ(1–42) was substituted by Val (Aβ(1–42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Aβ(1–42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Aβ(1–42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence – a measure of reactive oxygen species (ROS) – in comparison to native Aβ(1–42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-α-phenylnitrone (PBN). While Aβ(1–42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Aβ(1–42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Aβ(1–42G33V) does not form fibrils nearly as well as Aβ(1–42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Aβ(1–42) and that contribute to the peptide’s toxicity in Alzheimer’s disease brain.
Keywords: Alzheimer’s disease; Reactive oxygen species; Amyloid β-peptide; Methionine free radical; Glycine free radical; Fibrilogenesis;

γ-Secretase is an enzymatic activity responsible for the final cleavage of the amyloid precursor protein leading to the production of the amyloid β-peptide (Aβ). γ-Secretase is likely an aspartyl protease, since its activity can be inhibited by both pepstatin and active-site directed aspartyl protease inhibitors. Recent work has indicated that presenilins 1 and 2 may actually be the γ-secretase enzymes. Presenilin (PS) mutations, which lead to an increase in the production of a longer form of Aβ, are also the most common cause of familial Alzheimer’s disease (FAD). Therefore, in an attempt to better characterize the substrate preferences of γ-secretase, we performed experiments to determine how FAD-linked mutations in PS1 would affect the generation of Aβ peptides from full length precursor substrates that we have previously demonstrated to be proteolytically cleaved at alternative sites and/or by enzymatic activities that are pharmacologically distinct. Presenilin mutations increased the production of Aβ peptides from sites distal to the primary cleavage site (‘longer’ peptides) and in several cases also decreased production of ‘shorter’ peptides. These results support a model in which the FAD-linked mutants subtly alter the conformation of the γ-secretase complex to favor the production of long Aβ.
Keywords: Alzheimer’s disease; Amyloid β-peptide; Amyloid precursor protein; Mass spectrometry; Presenilin 1; γ-Secretase;

Magnetic investigations of human mesencephalic neuromelanin by F. Bolzoni; S. Giraudo; L. Lopiano; B. Bergamasco; M. Fasano; P.R. Crippa (210-218).
Pigmentation of neurons in substantia nigra is due to neuromelanin, a pigment that stores large amounts of iron. Human mesencephalic neuromelanin has been investigated by means of magnetic susceptibility measurements as a function of temperature. Magnetic measurements provide a physico-chemical characterization of the iron cluster buried in the organic melanin matrix and support the view that iron is not simply chelated, but rather is organized in a three-dimensional network. The paramagnetism of isolated iron ions is observed, in agreement with electron paramagnetic resonance spectroscopy. Furthermore, antiferromagnetic grains with a large size distribution function are present. These grains contain N spins coupled antiferromagnetically; however, N 1/2 spins are decoupled from the grain bulk and parallelly aligned. The latter subgrains are superparamagnetic with a blocking temperature ranging between 5 K and room temperature. This behavior has not been observed in synthetic melanin, where the paramagnetic contribution is strongly enhanced. Preliminary results on pigment isolated from patients affected by Parkinson’s disease, a neurodegenerative pathology involving primarily pigmented neurons in substantia nigra pars compacta, show a lower total magnetization compared to control neuromelanin. The temperature behavior of zero field cooling and field cooling magnetizations is similar for both. The significant depletion of iron content in Parkinson’s disease neuromelanin could indicate a progressive Fe migration from its storage environment to the cytosol.
Keywords: Antiferromagnetism; Oxidative stress; Neuromelanin; Parkinson’s disease; Substantia nigra; Superparamagnetism;

Lactate accumulation rather than ATP depletion predicts ischemic myocardial necrosis by Achim M Vogt; Cordula Ackermann; Murat Yildiz; Wolfgang Schoels; Wolfgang Kübler (219-226).
In ischemia, the myocardial metabolic status determines the expansion of necrosis. Decreased ATP levels and increased lactate contents in ischemic myocardium undergoing lethal injury are known to be related to the expansion of irreversible damage. However, their individual contributions have not yet been firmly established. Using two differently effective protocols of ischemic preconditioning (IP short and IP long), ischemic cardioplegic arrest (CP) and their combination (IP+CP) to directly influence the metabolic status of porcine myocardium, graded preservations in ATP content and decreases in lactate accumulation during 45 min ischemia could be achieved (control: ATP, 0.15±0.03; lactate, 60.53±4.89 μmol/g wet weight; IP short, 0.33±0.10/27.42±3.90; IP long, 0.60±0.10/17.49±2.14; CP, 0.98±0.12/11.82±0.96; IP+CP, 2.24±0.28/10.88±0.89; all P<0.001 vs. control). At the same time, a graded reduction of myocardial necrosis was observed (90.0±3.1 vs. 31.7±4.55 vs. 5.05±2.1 vs. 0.0 [isolated patchy necroses] vs. none). Regression analysis revealed only a weak correlation of infarct size and ATP preservation (r=0.567). In fact, there was a biphasic relation: with ATP levels above 1 μmol/g wet weight, no infarction occurred. ATP levels below this threshold value were associated with steep increase in infarct size. However, even for this latter range, the regression coefficient remained low (r=0.654). Instead, over the entire range, there was a close, rectilinear correlation of infarct size and lactate accumulation (r=0.939). These data indicate that lactate accumulation rather than ATP depletion determines the development of lethal myocardial injury. However, the biphasic relation between ATP depletion and infarct size suggests the latter to play a permissive role, since above a threshold value of 1 μmol/g wet weight neither substantial lactate accumulation nor infarction was observed. Below this threshold, however, infarct size increased as lactate accumulated.
Keywords: ATP; Lactate; Energy metabolism; Myocardial infarction; Myocardial ischemia; Infarct size;