The Journal of Steroid Biochemistry and Molecular Biology, 2021
Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer&... more Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer's disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analy...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2019
Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity... more Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.
Background: Acute infection is a well-established risk factor of cardiovascular inflammation incr... more Background: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide (LPS) derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here we utilize a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation. Methods: Acute infection was mimicked by injection of a single dose of LPS into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. Results: LPS treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional m...
Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucl... more Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucleinopathies. The existence of various intracellular and extracellular αSyn species, including cleaved αSyn, complicates the quest for an appropriate therapeutic target. Hence, to develop efficient disease-modifying strategies, it is fundamental to achieve a deeper understanding of the relevant spreading and toxic αSyn species. Here, we describe comparative and proof-of-principle approaches to determine the involvement of αSyn fragments in intercellular spreading. We demonstrate that two different αSyn fragments (1–95 and 61–140) fulfill the criteria of spreading species. They efficiently instigate formation of proteinase-K-resistant aggregates from cell-endogenous full-length αSyn, and drive it into different aggregation pathways. The resulting aggregates induce cellular toxicity. Strikingly, these aggregates are only detectable by specific antibodies. Our results suggest that αSyn fragm...
Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion chan... more Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages ...
Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-secretase is implica... more Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimers disease pathogenesis. Since conformational flexibility of a di-glycine hinge in the C99 transmembrane domain (TMD) might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. CD, NMR and hydrogen/deuterium exchange measurements as well as MD simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the TMD. However, although helix destabilization/unfolding was not observed at the initial ε-cleavage sites of C99, both mutants impaired γ-secretase cleavage and altered its cleavage specificity. Moreover, helix flexibility enabled by the di-glycine hinge translated to motions of other helix parts. Our data suggest that both local helix stabilization and destabilization in the di-glycine hinge may decrease the occurrence of enzyme...
Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patient... more Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the CNS. We analyzed their pathogenic activity by affinity-purifying these Abs from patients and transferring them to experimental animals. Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and determined the recognized MOG-epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in two models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically. We identified 17 patients with MOG Abs from our outpatient clinic and selected two with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs f...
Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug fo... more Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether bexarotene induces removal of Aβ plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aβ in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited ...
Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with front... more Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates wit...
Dissociation of fatty acids (FA) from and transbilayer movement (flip-flop) in small unilamellar ... more Dissociation of fatty acids (FA) from and transbilayer movement (flip-flop) in small unilamellar phosphatidylcholine vesicles (SUV) were monitored by measuring the pH inside the vesicle with an entrapped water-soluble fluorophore, pyranin. With a pH gradient imposed upon SUV preloaded with FA, the rate of flip-flop of saturated very long chain FA (C20:0, C:22:0, and C24:0) was shown to be fast (t1/2 < 1 s); previously, we showed by stopped flow measurements that flip-flop of long chain (14-18 carbons) FA is very fast [t1/2 < 10 ms; Kamp, F., et al. (1995) Biochemistry 34, 11928-11937]. The rates of dissociation of FA from SUV were evaluated by incorporating FA into donor vesicles and measuring transfer to acceptor vesicles. The transfer was followed by changes in internal pH of either donor or acceptor vesicles with stopped flow (C14:0, C16:0, C17:0, C18:0, C18:1, and C18:2) or on-line (C20:0, C22:0, and C24:0) fluorescence. All FA showed a single-exponential transfer process that was slower than the lower limits established for the rate of flip-flop, with t1/2 of dissociation ranging from 20 ms for C14:0 to 1900 s for C24:0. The pseudo-unimolecular rate constant (koff) for dissociation of C14:0 to C26:0 showed a 10-fold decrease for each addition of two CH2 groups to the acyl chain and a delta (delta G) of -740 cal/CH2. The dissociation rate constants for oleic acid (18:1) and linoleic acid (18:2) were 5 and 10 times faster, respectively, than that of C18:0. The rates of dissociation for typical dietary FA are sufficiently rapid that complex mechanisms (e.g. protein-mediated) may not be required for their desorption from biological membranes. The very slow dissociation rates for C24:0 and C26:0 may accentuate their pathological effects in diseases in which they accumulate in tissues.
The Journal of Steroid Biochemistry and Molecular Biology, 2021
Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer&... more Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer's disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analy...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2019
Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity... more Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.
Background: Acute infection is a well-established risk factor of cardiovascular inflammation incr... more Background: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide (LPS) derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here we utilize a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation. Methods: Acute infection was mimicked by injection of a single dose of LPS into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. Results: LPS treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional m...
Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucl... more Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucleinopathies. The existence of various intracellular and extracellular αSyn species, including cleaved αSyn, complicates the quest for an appropriate therapeutic target. Hence, to develop efficient disease-modifying strategies, it is fundamental to achieve a deeper understanding of the relevant spreading and toxic αSyn species. Here, we describe comparative and proof-of-principle approaches to determine the involvement of αSyn fragments in intercellular spreading. We demonstrate that two different αSyn fragments (1–95 and 61–140) fulfill the criteria of spreading species. They efficiently instigate formation of proteinase-K-resistant aggregates from cell-endogenous full-length αSyn, and drive it into different aggregation pathways. The resulting aggregates induce cellular toxicity. Strikingly, these aggregates are only detectable by specific antibodies. Our results suggest that αSyn fragm...
Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion chan... more Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages ...
Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-secretase is implica... more Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimers disease pathogenesis. Since conformational flexibility of a di-glycine hinge in the C99 transmembrane domain (TMD) might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. CD, NMR and hydrogen/deuterium exchange measurements as well as MD simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the TMD. However, although helix destabilization/unfolding was not observed at the initial ε-cleavage sites of C99, both mutants impaired γ-secretase cleavage and altered its cleavage specificity. Moreover, helix flexibility enabled by the di-glycine hinge translated to motions of other helix parts. Our data suggest that both local helix stabilization and destabilization in the di-glycine hinge may decrease the occurrence of enzyme...
Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patient... more Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the CNS. We analyzed their pathogenic activity by affinity-purifying these Abs from patients and transferring them to experimental animals. Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and determined the recognized MOG-epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in two models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically. We identified 17 patients with MOG Abs from our outpatient clinic and selected two with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs f...
Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug fo... more Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether bexarotene induces removal of Aβ plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aβ in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited ...
Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with front... more Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates wit...
Dissociation of fatty acids (FA) from and transbilayer movement (flip-flop) in small unilamellar ... more Dissociation of fatty acids (FA) from and transbilayer movement (flip-flop) in small unilamellar phosphatidylcholine vesicles (SUV) were monitored by measuring the pH inside the vesicle with an entrapped water-soluble fluorophore, pyranin. With a pH gradient imposed upon SUV preloaded with FA, the rate of flip-flop of saturated very long chain FA (C20:0, C:22:0, and C24:0) was shown to be fast (t1/2 < 1 s); previously, we showed by stopped flow measurements that flip-flop of long chain (14-18 carbons) FA is very fast [t1/2 < 10 ms; Kamp, F., et al. (1995) Biochemistry 34, 11928-11937]. The rates of dissociation of FA from SUV were evaluated by incorporating FA into donor vesicles and measuring transfer to acceptor vesicles. The transfer was followed by changes in internal pH of either donor or acceptor vesicles with stopped flow (C14:0, C16:0, C17:0, C18:0, C18:1, and C18:2) or on-line (C20:0, C22:0, and C24:0) fluorescence. All FA showed a single-exponential transfer process that was slower than the lower limits established for the rate of flip-flop, with t1/2 of dissociation ranging from 20 ms for C14:0 to 1900 s for C24:0. The pseudo-unimolecular rate constant (koff) for dissociation of C14:0 to C26:0 showed a 10-fold decrease for each addition of two CH2 groups to the acyl chain and a delta (delta G) of -740 cal/CH2. The dissociation rate constants for oleic acid (18:1) and linoleic acid (18:2) were 5 and 10 times faster, respectively, than that of C18:0. The rates of dissociation for typical dietary FA are sufficiently rapid that complex mechanisms (e.g. protein-mediated) may not be required for their desorption from biological membranes. The very slow dissociation rates for C24:0 and C26:0 may accentuate their pathological effects in diseases in which they accumulate in tissues.
Uploads
Papers