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1.
Neuropharmacology ; 151: 159-170, 2019 06.
Article in English | MEDLINE | ID: mdl-31004654

ABSTRACT

Niemann Pick C (NPC) is a fatal hereditary neurovisceral disorder associated with a progressive loss of neurons of unknown mechanism. The disease is caused by mutation in either of two genes, termed npc1 and npc2, accounting for ∼95% and ∼5% of patients, respectively. Recent data suggest a cell-autonomous cause for neuronal cell death. In a former study we could demonstrate that cultured NPC1-deficient (NPC1-/-) neurons are more susceptible to autophagic stress than NPC1-wildtype (wt) neurons. In the present study we tested other stressors for a selective effect on the survival of NPC1-/- neurons. To that end we challenged cultured primary cortical neurons from a NPC mouse model and from wild type littermate mice by a variety of different stressors: glutamate, hydrogen superoxide, osmotic shock and inhibition of HMG-CoA reductase. In all paradigms neurons behave virtually identical with one exception: NPC1 deficient neurons are more vulnerable against a challenge with lovastatin. The analysis of the molecular background provides evidence that statin endangers survival of neurons by interfering in the autophagy of cells.


Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Lovastatin/pharmacology , Neurons/drug effects , Niemann-Pick C1 Protein/metabolism , Animals , Autophagy/drug effects , Cells, Cultured , Glutamic Acid/pharmacology , Mice , Mice, Knockout , Neurons/metabolism , Niemann-Pick C1 Protein/genetics , Superoxides/pharmacology
2.
Neuropharmacology ; 89: 282-9, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25446672

ABSTRACT

A disturbed autophagic pathway leads to chronically increased levels of autophagic vacuoles in Niemann Pick Type-C 1 (NPC1) deficient neurons. Since these accumulations potentially contribute to neuronal cell death associated with the disease, we investigated two pharmacological strategies which potentially reduce the number of autophagic structures under following aspects: efficiency, sustainability and effect on neuronal cell viability. The strategies comprised (i) an interruption of the autophagic flux by the class III PI3K inhibitor 3-methyladenine (3-MA) and (ii) an acceleration of the autophagic execution by 2-hydroxypropyl-ß-cyclodextrin (pCD). Our data show that the inhibition of autophagy with 3-MA only initially reduced the number of autophagic vacuoles in cultured neurons. Prolonged treatments with the PI3K-inhibitor reversed this lowering effect. The re-increase in the number of autophagic vacuoles was combined with a defect in the integrity of lysosomes which endangered further survival of cells. The treatment with pCD evoked a slow but sustained reduction of autophagic structures and had no negative effects on neuronal survival.


Subject(s)
Adenine/analogs & derivatives , Autophagy/drug effects , Enzyme Inhibitors/pharmacology , Neurons/cytology , Proteins/metabolism , Vacuoles/drug effects , beta-Cyclodextrins/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin , Adenine/pharmacology , Animals , Cells, Cultured , Cerebral Cortex/cytology , Cholesterol/metabolism , Embryo, Mammalian , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Intracellular Signaling Peptides and Proteins , Lysosomal-Associated Membrane Protein 2/metabolism , Lysosomes/drug effects , Mice , Mice, Inbred BALB C , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Neurons/drug effects , Neurons/metabolism , Niemann-Pick C1 Protein , Proteins/genetics , Time Factors , Vacuoles/metabolism
3.
J Neurochem ; 133(1): 153-61, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25319340

ABSTRACT

Niemann Pick type C (NPC1) is a rare fatal hereditary cholesterol storage disease associated with a massive Purkinje cells loss. The mechanisms leading to neurodegeneration are still poorly understood. Different laboratories pointed to hypersensitivity to cytotoxic effects of statins (HMG-CoA reductase inhibitors) in NPC1 and suggested an underlying lack of geranylgeranyl pyrophosphate (GGPP). GGPP is a non-sterol isoprenoid essential for cell survival and differentiation. We measured GGPP levels in cerebella of a NPC1 mouse model and of wild-type littermates and found a physiological increase of GGPP levels between post-natal days 21 and 49 in wild-type mice but not in NPC mice. This further supports the hypothesis that Purkinje cell loss may be due to an extremely low level of GGPP. The progressive Purkinje cell loss in NPC starts between p21 and p49. To test the hypothesis, we used long-term organotypic slice cultures of NPC1 mice that display the natural history of NPC1 disease in vitro and tested if chronic administration of GGPP might prevent Purkinje cell loss. We did not see a beneficial effect. This suggests, in contrast to the expectations, that the relative lack of GGPP may not significantly contribute to mechanisms of Purkinje cell loss in NPC1.


Subject(s)
Cell Survival , Neurons/pathology , Niemann-Pick Disease, Type C/metabolism , Niemann-Pick Disease, Type C/pathology , Polyisoprenyl Phosphates/metabolism , Purkinje Cells/pathology , Animals , Cell Count , Cerebellum/metabolism , Cerebellum/pathology , Cholesterol/blood , Mice , Mice, Inbred BALB C , Organ Culture Techniques
4.
J Neuropathol Exp Neurol ; 73(9): 846-54, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25101701

ABSTRACT

Epidemiologic and experimental data suggest the involvement of cholesterol metabolism in the development and progression of Alzheimer disease and Niemann-Pick type C disease, but not of frontotemporal dementias. In these 3 neurodegenerative diseases, however, protein tau hyperphosphorylation and aggregation into neurofibrillary tangles are observed. To elucidate the relationship between cholesterol and tau, we compared sterol levels of neurons burdened with neurofibrillary tangles with those of their unaffected neighbors using semiquantitative filipin fluorescence microscopy in mice expressing P301L mutant human tau (a well-described model of FTDP-17) and in P301L transgenic mice lacking apolipoprotein E (the major cholesterol transporter in the brain). Cellular unesterified cholesterol was higher in neurons affected by tau pathology irrespective of apolipoprotein E deficiency. This argues for an impact of tau pathology on cellular cholesterol homeostasis. We suggest that there is a bidirectional mode of action: Disturbances in cellular cholesterol metabolism may promote tau pathology, but tau pathology may also alter neuronal cholesterol homeostasis; once it is established, a vicious cycle may promote neurofibrillary tangle formation.


Subject(s)
Brain/pathology , Cholesterol/metabolism , Intranuclear Inclusion Bodies/metabolism , Neurons/pathology , Tauopathies/pathology , Animals , Apolipoproteins E/deficiency , Disease Models, Animal , Humans , Intranuclear Inclusion Bodies/pathology , Intranuclear Inclusion Bodies/ultrastructure , Mice , Mice, Transgenic , Microscopy, Confocal , Neurons/ultrastructure , Silver Staining , Tauopathies/genetics , Trinucleotide Repeat Expansion/genetics , tau Proteins/genetics
5.
Neuropathol Appl Neurobiol ; 40(7): 933-45, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24889722

ABSTRACT

AIMS: Niemann-Pick type C (NPC) disease is a fatal hereditary lysosomal lipid storage disease caused by mutations in NPC1 or NPC2. It is still unknown how this disorder evokes clinical signs. Typically, patients develop severe cerebellar ataxia due to progressive Purkinje cell loss. Hitherto, in vitro studies did not allow monitoring the natural process of NPC-associated Purkinje cell degeneration. Aim of this study was to evaluate whether organotypic slice cultures are usable to monitor the natural process of NPC-associated Purkinje-cell degeneration. METHODS: We used organotypic cerebellar slice cultures of a well-established NPC mouse model to display the natural history of cerebellar degeneration in vitro and cultivated them for a prolonged time period of 6 weeks for the first time. Moreover we tested several therapeutic candidates and evaluated their effect on Purkinje-cell survival. RESULTS: Our approach proves that it is possible to monitor and to prevent NPC-related Purkinje cell death reliably in vitro. This is beneficial because in vivo Purkinje cell loss directly translates into clinical signs. Thus, therapeutically interesting compounds can be tested in vitro, not only to correct biochemical abnormalities, but also to show the likelihood of a compound to prevent ataxia. As to be expected from the results of previous animal experiments, 2-hydroxypropyl-ß-cyclodextrin rescued Purkinje cells. We also discovered that 3-methyladenine preserved Purkinje cell numbers by adjusting the autophagic flux in NPC slices. CONCLUSION: We provide evidence that cerebellar slice cultures are a powerful in vitro tool to study NPC-associated Purkinje cell death in an organotypic setting.


Subject(s)
Cerebellar Diseases/pathology , Disease Progression , Niemann-Pick Disease, Type C/complications , Niemann-Pick Disease, Type C/pathology , Purkinje Cells/pathology , Tissue Culture Techniques , Animals , Cell Survival , Cerebellar Diseases/complications , Cerebellar Diseases/drug therapy , Disease Models, Animal , Mice , Mice, Inbred BALB C , Mice, Neurologic Mutants , Niemann-Pick Disease, Type C/drug therapy , Purkinje Cells/drug effects , Purkinje Cells/physiology
6.
Neurobiol Dis ; 64: 88-97, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24412309

ABSTRACT

Niemann-Pick disease type C (NPC) is a fatal, progressive neurovisceral disorder. Several studies report that the autophagic flux is disturbed in NPC1-deficient (NPC1-/-) cells. Since it has been suggested that the autophagic defect may contribute to the neurodegeneration, we used cell cultures of NPC1-deficient and NPC1-wildtype neurons to investigate whether the disturbance influences neuronal survival. We found a genotype-dependent difference in survival, when autophagy is induced during culturing. NPC1-deficient neurons are more sensitive to rapamycin treatment and starvation than wildtype neurons. The subsequent search for defects in regulatory components of the autophagic pathway and the autophagic flux brought up results which differ from previous reports on somatic cells in one essential aspect: we exclude that an enhanced formation of autophagosomes contributes to the imbalanced autophagic flux in NPC1 deficient neurons. We found that solely the clearance of autophagosomes is delayed in these cells, which leads to an accumulation of autophagic vacuoles within the lysosomal compartment. Lowering the abnormal lipid load of the acidic organelles with cyclodextrin is sufficient to correct the autophagic flux and prevents premature death of NPC1-/- neurons under autophagic stress. From our results, we conclude that a pharmacological intervention in the neuropathology of NPC-disease should focus on the restoration of the lysosomal degradation capacity of cells.


Subject(s)
Autophagy/physiology , Neurons/physiology , Niemann-Pick Disease, Type C/physiopathology , Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein-1 Homolog , Beclin-1 , Cell Survival/physiology , Cells, Cultured , Cholesterol/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred BALB C , Mice, Transgenic , Niemann-Pick C1 Protein , Organelles/physiology , Phosphorylation , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , TOR Serine-Threonine Kinases/metabolism , Vacuoles/physiology
7.
J Neuropathol Exp Neurol ; 70(4): 292-301, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21412171

ABSTRACT

Apolipoprotein E (ApoE) is the major cholesterol transporter in the brain. There is epidemiological and experimental evidence for involvement of cholesterol metabolism in the development and progression of Alzheimer disease. A dietary effect on tau phosphorylation or aggregation, or a role of apoE in tau metabolism, has been studied experimentally, but the data are ambiguous. To elucidate the relationship between cholesterol and tau, we studied mice expressing P301L mutant human tau but not apoE (htau-ApoE) and P301L mice with wild-type ApoE (htau- ApoE); both genotypes develop neuron cytoskeletal changes similar to those found in Alzheimer disease. Mice were kept on a cholesterol-enriched diet or control diet for 15 weeks. The numbers of neurons with hyperphosphorylated and conformationally changed tau in the cerebral cortex were assessed by immunohistochemistry, and sterol levels were determined. Highly elevated dietary serum cholesterol levels enhanced ongoing tau pathology in htau-ApoE mice; this effect correlated with elevated brain cholesterol metabolite 27-hydroxycholesterol levels. Apolipoprotein E deficiency promoted significant increases of tau phosphorylation and conformational changes in mice on a control diet. In htau-ApoE mice on the high cholesterol regimen, brain oxysterol levels were less than in htau-ApoE mice, and the numbers of neurons with pathologically altered tau were similar to those in htau-ApoE mice on the high-cholesterol diet.


Subject(s)
Apolipoproteins E/genetics , Cholesterol, Dietary/pharmacology , tau Proteins/metabolism , Animals , Apolipoproteins E/deficiency , Brain Chemistry/drug effects , Cell Count , Cholesterol/blood , Cholesterol/metabolism , Diet , Genotype , Humans , Immunohistochemistry , Lipids/blood , Mice , Mice, Knockout , Mice, Transgenic , Phosphorylation , Protein Conformation , tau Proteins/deficiency , tau Proteins/genetics
8.
J Biol Chem ; 283(1): 100-109, 2008 Jan 04.
Article in English | MEDLINE | ID: mdl-17971449

ABSTRACT

Tau is an important microtubule-stabilizing protein in neurons. In its hyperphosphorylated form, Tau protein loses its ability to bind to microtubules and then accumulates and is part of pathological lesions characterizing tauopathies, e.g. Alzheimer disease. Glycogen synthase kinase-3beta (GSK-3beta), antagonized by protein phosphatase 2A (PP2A), regulates Tau phosphorylation at many sites. Diabetes mellitus is linked to an increased risk of developing Alzheimer disease. This could be partially caused by dysregulated GSK-3beta. In a long term experiment (-16 h) using primary murine neuron cultures, we interfered in the insulin/phosphoinositide 3-kinase (PI3K) (LY294002 treatment and insulin boost) and mammalian target of rapamycin (mTor) (AICAR and rapamycin treatment) signaling pathways and examined consequent changes in the activities of PP2A, GSK-3beta, and Tau phosphorylation. We found that the coupling of PI3K with mTor signaling, in conjunction with a regulatory interaction between PP2A and GSK-3beta, changed activities of both enzymes always in the same direction. These balanced responses seem to ensure the steady Tau phosphorylation at GSK/PP2A-dependent sites observed over a long period of time (>/=6 h). This may help in preventing severe changes in Tau phosphorylation under conditions when neurons undergo transient fluctuations either in insulin or nutrient supply. On the other hand, the investigation of Tau protein at Ser-262 showed that interference in the insulin/PI3K and mTor signaling potentially influenced the Tau phosphorylation status at sites where only one of two enzymes (in this case PP2A) is involved in the regulation.


Subject(s)
Glycogen Synthase Kinase 3/metabolism , Protein Kinases/metabolism , Protein Phosphatase 2/metabolism , Signal Transduction , tau Proteins/metabolism , Animals , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Insulin/pharmacology , Lithium Chloride/pharmacology , Mice , Okadaic Acid/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Sirolimus/pharmacology , TOR Serine-Threonine Kinases
9.
Prog Brain Res ; 163: 723-40, 2007.
Article in English | MEDLINE | ID: mdl-17765747

ABSTRACT

As part of the hippocampus, the dentate gyrus is considered to play a crucial role in associative memory. The reviewed data suggest that the dentate gyrus withstands the formation of plaques, tangles and neuronal death until late stages of Alzheimer's disease (AD). However, changes related to a disconnecting process, and more subtle intrinsic alterations, may contribute to disturbances in memory and learning observed in early stages of AD.


Subject(s)
Alzheimer Disease/pathology , Dentate Gyrus/pathology , Humans , Neurons/pathology , Neurons/physiology
10.
Neurobiol Aging ; 28(5): 677-86, 2007 May.
Article in English | MEDLINE | ID: mdl-17023092

ABSTRACT

The apolipoprotein E genetic polymorphism exerts a well described influence on Alzheimer's disease (AD) risk, although the pathogenetic mechanism is still not clear. Increasing evidence points to a diminished neuroplasticity in apolipoprotein E varepsilon4-allele carriers. But, alternatively or additionally, developmental differences in dendritic geometry may be associated with the polymorphism. We morphometrically examined the dendritic ramification of CA1 Parvalbumin-positive GABAergic hippocampal neurons (n=571) in matched pairs of aged non-demented individuals with different apolipoprotein E genotype. We chose Parvalbumin-positive interneurons since they lack potentially confounding AD-like cytoskeletal changes. To minimize the risk of transneuronal dendritic changes due to significant deafferentation we focused on non-demented individuals. In this chosen paradigm, neither the disease-associated apolipoprotein E varepsilon4-allele nor the apolipoprotein E varepsilon2-allele had a significant impact on dendritic shape when compared to the most common allelic variant apolipoprotein E varepsilon3/3. At least with respect to the studied cell type, the data suggest that the apolipoprotein E polymorphism does not modulate the original formation of dendrites in vivo, contrary to conclusions drawn from in vitro studies on neurite outgrowth.


Subject(s)
Apolipoproteins E/genetics , Dendrites/genetics , Hippocampus/physiology , Interneurons/physiology , Polymorphism, Genetic/physiology , Aged , Aged, 80 and over , Alleles , Cell Shape/genetics , Dendrites/pathology , Female , Hippocampus/cytology , Hippocampus/pathology , Humans , Interneurons/pathology , Male , Middle Aged , Neurites/pathology , Neurites/physiology , Neuronal Plasticity/physiology
11.
Neurobiol Aging ; 27(1): 54-66, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16298241

ABSTRACT

In this study, we investigated the presence and role of immunoproteasome and its LMP2 subunit polymorphism at codon 60 in Alzheimer's disease (AD). Immunoproteasome was present in brain areas such as hippocampus and cerebellum and localized in neurons, astrocytes and endothelial cells. A higher expression of immunoproteasome was found in brain of AD patients than in brain of non-demented elderly, being its expression in young brain negligible or absent. Furthermore, AD affected regions showed a partial decrease in proteasome trypsin-like activity. The study of LMP2 polymorphism (R/H) showed that it does not influence LMP2 expression (neither the mRNA nor mature protein) in brain tissue. However, control brain areas of AD patients carrying the RR genotype showed an increased proteasome activity in comparison with RH carriers. To test whether this effect of the genotype might be related to AD onset we performed a genetic study, which allowed us to exclude an association of LMP2 codon 60 polymorphism with AD onset, despite its influence on the proteasome activity in human brain.


Subject(s)
Aging/genetics , Aging/metabolism , Alzheimer Disease/genetics , Brain/metabolism , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Proteasome Endopeptidase Complex/metabolism , Adult , Aged , Aged, 80 and over , Cadaver , Female , Genetic Predisposition to Disease/genetics , Humans , In Vitro Techniques , Male , Middle Aged , Prevalence , Proteasome Endopeptidase Complex/genetics , Risk Assessment/methods , Risk Factors
13.
Ann N Y Acad Sci ; 1019: 24-8, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15246987

ABSTRACT

Epidemiologic studies indicate that elderly women are at higher risk for Alzheimer disease compared to men. In order to pathologically verify this result, the extent of AD brain lesions (NFT and SP) was compared for men and women at each age, that is, at each decade from 25 years to 95 years, in a large sample of > 5000 routine autopsy cases. Women had more affected brain regions beginning in late middle age. They also had more extensive SP depositions throughout the brain compared to men at each early NFT stage I, II, and III. At later NFT stages IV, V, and VI both men and women had extensive SP deposits. The gender gap in SPs at early NFT stages was large and specific to women who carried the APOE4 allele (P <.001) and in addition to the acceleration in NFT stage also found for APOE4+ women.


Subject(s)
Apolipoproteins E/genetics , Brain/pathology , Neurofibrillary Tangles/pathology , Plaque, Amyloid/pathology , Polymorphism, Genetic , Adult , Age Factors , Aged , Aged, 80 and over , Alleles , Female , Humans , Male , Middle Aged , Neurons/metabolism , Sex Factors
14.
Neurobiol Aging ; 25(6): 697-711, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15165691

ABSTRACT

One histological hallmark in Alzheimer's disease is the tangle. The other is the plaque. A widely discussed hypothesis is the "amyloid cascade" assuming that tangle formation is a direct consequence of amyloid plaque formation. The aim of this study was to examine plaques and tangles in a highly defined neuronal circuitry in order to determine their detailed spatial and temporal relationships. We investigated serial sections of the whole hippocampal formation of brains with early Braak-stages (0-III) for tangles only, i.e. one case at stage 0, six at stage I, six at stage II, and nine at stage III. Most cases displayed both plaques and tangles. Four cases of stages 0 and I, three cases with stage II, and even one with stage III, however, did not display plaques. In turn, no plaque was found in the absence of tangles. The spatial relationship indicates that plaques lay in the terminal fields of tangle-bearing neurons. Our analysis suggests that tangles either antecede plaques or--less likely--are independently formed.


Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Hippocampus/pathology , Neurofibrillary Tangles/metabolism , Plaque, Amyloid/metabolism , Temporal Lobe/pathology , Aged , Aged, 80 and over , Brain Mapping , Female , Humans , Immunohistochemistry/methods , Male , Middle Aged , Temporal Lobe/metabolism
15.
Neurochem Res ; 28(11): 1715-23, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14584825

ABSTRACT

Tangles are a major histopathological feature of Alzheimer's disease and their regional location and number correlate significantly with the individual's cognitive decline. Intriguingly, these tangles are formed only in a small subset of nerve cell types and are practically absent in most animal species examined so far. In humans, tangle formation seemingly starts decades before clinical signs of dementia are seen and spread over cortical areas in a regular manner described by the Braak classification. In the present article the role of plasticity-related molecules and mechanisms are discussed considering their putative role in neuronal vulnerability and spread of tangles. Special emphasis is given to some aspects of lipid metabolism, that is, apolipoprotein E polymorphism, statin effects, and lysosomal dysfunction in Alzheimer's and Niemann-Pick C's diseases.


Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Neural Pathways/metabolism , Neural Pathways/pathology , Neuronal Plasticity , Alzheimer Disease/classification , Alzheimer Disease/physiopathology , Animals , Apolipoproteins E/metabolism , Cholesterol/metabolism , Humans , Lipid Metabolism , Nerve Degeneration/physiopathology , Neural Pathways/physiopathology , Species Specificity
16.
Neuroreport ; 14(11): 1523-7, 2003 Aug 06.
Article in English | MEDLINE | ID: mdl-12960778

ABSTRACT

G-protein alpha-subunits (Galphao, Galphai, Galphas, Galphaq) and adenylyl cyclase (AC) I and II isoforms were quantified in hippocampus and entorhinal cortex from 22 cases staged for Alzheimer's disease (AD) pathologies according to Braak and Braak. Hippocampal Galphai levels declined significantly with neurofibrillary staging, whereas AC I levels in this region increased. Significant amyloid stage-related reductions of Galphai were seen in both the hippocampus and entorhinal cortex. The hippocampus also showed a significant reduction of Galphao with amyloid staging. It is concluded that levels of inhibitory G-protein subunits Galphao, and in particular Galphai, decrease in parallel to the extent of AD pathology.


Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Brain Chemistry/physiology , Entorhinal Cortex/metabolism , Entorhinal Cortex/pathology , GTP-Binding Protein alpha Subunits/metabolism , Hippocampus/metabolism , Hippocampus/pathology , Neurofibrillary Tangles/pathology , Plaque, Amyloid/pathology , Adolescent , Adult , Child , Electrophoresis, Polyacrylamide Gel , Female , Humans , Immunoblotting , Male
17.
J Pathol ; 200(1): 95-103, 2003 May.
Article in English | MEDLINE | ID: mdl-12692847

ABSTRACT

Niemann-Pick type C (NPC) disease is a fatal hereditary neurovisceral disorder with diagnostically relevant intracellular accumulation of cholesterol in non-brain tissue, for example the spleen and fibroblasts. In the brain, many ballooned neurons are seen. Using filipin microfluorodensitometry, significant accumulations of free cholesterol in specified neurons have been described in NPC patients. The present study demonstrates spatial and temporal accumulation of free cholesterol in the brains of homozygous NPC (-(npc)/-(npc)) mice, a widely acknowledged mouse model, and in primarily cultured neurons therefrom. Intraneuronal storage of free cholesterol was already prominent at a pre-clinical stage in various grey matter areas of the murine cerebral cortex. Hippocampal areas showed differential development of the pathological distribution of free cholesterol. The pyramidal cells in the CA3 sector of Ammon's horn were affected much earlier than in CA1. Some of the deeper cerebral nuclei were affected only slightly, even at the final stage. Neurons (E15-E17) cultured in a cholesterol-free medium also showed massive accumulation of intracellular free cholesterol. In addition, brains from the murine NPC model for Alzheimer's disease (AD)-like changes in the microtubule-associated protein tau were tested using the Gallyas silver technique and AT8-immunolabelling, since both human diseases are accompanied by intraneuronal tangles made up of tau protein aggregations. Although the analysis failed to show classical silver-stainable tangles of the AD type in the NPC mice, tau protein phosphorylated at epitopes considered to represent early stages of AD was found. This further strengthens the concept that an alteration in cholesterol metabolism may play an important role in AD. The NPC mouse model may thus serve as a tool to analyse the role of cholesterol in initial changes of tau that eventually lead to the formation of tangles in both NPC and AD.


Subject(s)
Brain Chemistry , Cholesterol/analysis , Disease Models, Animal , Niemann-Pick Diseases/metabolism , tau Proteins/metabolism , Alzheimer Disease/metabolism , Animals , Blotting, Western/methods , Cells, Cultured , Cerebellum/metabolism , Cerebral Cortex/metabolism , Hippocampus/metabolism , Humans , Intracellular Fluid/metabolism , Mice , Mice, Inbred BALB C , Neurofibrillary Tangles/metabolism , Neurons/metabolism , Phosphorylation , Pons/metabolism
18.
J Pathol ; 200(1): 104-11, 2003 May.
Article in English | MEDLINE | ID: mdl-12692848

ABSTRACT

Niemann-Pick type C disease is an inherited neurovisceral storage disorder with intracellular accumulation of cholesterol. In affected brains, many ballooned neurons are seen. Considerable nerve cell loss of unknown pathogenesis leads to neurological deterioration and dementia. Chemical examination of brains has failed to demonstrate increased levels of cholesterol. Using filipin fluorometry of neuronal cells in tissue slices, we found massive accumulation of cholesterol in neurons in four out of five human Niemann-Pick type C cases including adult patients. Neurofibrillary tangles composed of aggregates of the otherwise highly soluble protein tau were present in three Niemann-Pick type C cases and were also immunologically identical to those associated with Alzheimer's disease. However, only a thin slab of spinal cord or a tiny piece of isocortex was available for examination in the two cases without tangles. In a further semi-quantitative analysis of 576 neurons, we determined higher cholesterol content in tangle-bearing neurons than in adjacent tangle-free neurons. The association of cholesterol accumulation with neurofibrillary degeneration in Niemann-Pick type C disease and Alzheimer's disease awakens interest in the role of impaired cholesterol metabolism in the development of neurofibrillary tangles in both diseases.


Subject(s)
Brain Chemistry , Cholesterol/analysis , Niemann-Pick Diseases/metabolism , Tauopathies/metabolism , Adolescent , Adult , Alzheimer Disease/metabolism , Cerebral Cortex/metabolism , Female , Filipin , Humans , Immunohistochemistry/methods , Male , Neurofibrillary Tangles/metabolism , Neurons/metabolism , Neuropil Threads/metabolism , tau Proteins/metabolism
19.
J Neurochem ; 84(1): 35-42, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12485399

ABSTRACT

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.


Subject(s)
Cholesterol/metabolism , Membrane Proteins/metabolism , Synaptophysin/metabolism , Animals , Anticholesteremic Agents/pharmacology , Brain/metabolism , CHO Cells , Cholesterol/pharmacology , Cricetinae , Cyclodextrins/pharmacology , Detergents , Filipin/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Lovastatin/pharmacology , Membranes/metabolism , Mice , Mice, Inbred BALB C , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/metabolism , Octoxynol , Protein Transport , R-SNARE Proteins , Rats , Solubility , Synaptic Vesicles/metabolism , Synaptophysin/chemistry , Up-Regulation
20.
Acta Neuropathol ; 103(4): 363-9, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11904756

ABSTRACT

Recent stereological studies documented a severe loss of hippocampal neurons in end-stage Alzheimer's disease. The development of the disease, however, is progressive and slow, over clinically inconspicuous decades. The Braak-staging system distinguishes six histopathological stages some of which are not accompanied by clinical symptoms. We analyzed hippocampal cell loss in correlation to Braak stages. Neuron numbers were determined with unbiased stereological principles in a defined subportion of the hippocampus of 28 subjects. There were no age-dependent neuronal losses in any of the hippocampal subdivisions examined. Compared to stage I, pyramidal cell loss in CA1 was reduced by 33% in stage IV ( P<0.02) and by 51% in stage V ( P<0.0002). In the subiculum, considerable neuron loss was seen only in stage V (22%; P<0.09). Other subdivisions of the Ammon's horn showed no neuron loss. Neuron loss was greater than volume loss, e.g., neuron loss of 51% between stages I and V in CA1 was accompanied by volume loss of only 25%. Our findings indicate (i) that neuronal loss is sector and stage dependent, (ii) that neuronal loss in CA1 and the subiculum is related to the formation of neurofibrillary tangles, and (iii) that neuron loss makes a weak contribution to the observed volume loss.


Subject(s)
Alzheimer Disease/pathology , Hippocampus/pathology , Neurons/pathology , Aged , Aged, 80 and over , Cell Count , Female , Humans , Male , Middle Aged
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