Geranylgeranyl pyrophosphate is crucial for neuronal survival but has no special role in Purkinje cell degeneration in Niemann Pick type C1 disease.

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.

The autophagic defect in Niemann-Pick disease type C neurons differs from somatic cells and reduces neuronal viability.

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.

The natural history of cerebellar degeneration of Niemann-Pick C mice monitored in vitro.

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.

NPC1-deficient neurons are selectively vulnerable for statin treatment.

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.

How to reduce the accumulation of autophagic vacuoles in NPC1-deficient neurons: a comparison of two pharmacological strategies.

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.

Dietary cholesterol and its effect on tau protein: a study in apolipoprotein E-deficient and P301L human tau mice.

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.

Coupling of mammalian target of rapamycin with phosphoinositide 3-kinase signaling pathway regulates protein phosphatase 2A- and glycogen synthase kinase-3 -dependent phosphorylation of Tau.

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.

Spatial and temporal distribution of intracellular free cholesterol in brains of a Niemann-Pick type C mouse model showing hyperphosphorylated tau protein. Implications for Alzheimer's disease.

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.

Cholesterol storage and tau pathology in Niemann-Pick type C disease in the brain.

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.

Plasticity and the spread of Alzheimer's disease-like changes.

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.