Histopathologically, Alzheimer's disease is characterized by plaques and tangles that develop progressively over time. Experimental data described a statin-induced decrease in beta-amyloid production, a major constituent of the plaques. Others reported data on statin-mediated changes in neuronal survival and cytoskeleton, including the microtubule-associated protein tau, a major constituent of the tangles. However, these latter reports remain contradictory. To clarify and extend our knowledge on the effect of statin on the cytoskeleton, we challenged rat primary neuron cultures by lovastatin and determined the metabolite that is critical for structural integrity and survival of neurons. During the blockade of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the neuritic network was affected and eventually was completely destroyed. This process was not part of the execution phase of apoptosis and was marked by alterations in the microfilament and microtubule system. The distribution and phosphorylation of protein tau changed. Immunoblot analysis and indirect immunofluorescence revealed a transient increase in tau phosphorylation, which ceased during the execution of apoptosis. All of these effects could be linked to the lack of the geranylgeranylpyrophosphate intermediate. Inhibition of the geranylgeranylation of Rho family GTPases (geranylgeranyl-transferase I) evoked similar changes in neurons. These data and our findings that statin treatment reduced the membrane-bound fraction of RhoA-GTPase in neurons suggest that reduced levels of functional small G proteins are responsible for the observed effects. Our data demonstrate that lovastatin concentrations able to suppress not only cholesterol but also geranylgeranylpyrophosphate formation may evoke phosphorylation of tau reminiscent of preclinical early stages of Alzheimer's disease and, when prolonged, apoptosis.

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.

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-3␤ (GSK-3␤), 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-3␤. 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-3␤, and Tau phosphorylation. We found that the coupling of PI3K with mTor signaling, in conjunction with a regulatory interaction between PP2A and GSK-3␤, 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.

Aggregated platelets and occlusive platelet thrombi were found in small myocardial vessels of dogs on electron-microscope examination after prolonged infusion of norepinephrine. The etiology of the myocardial necrosis and fibrosis induced by catecholamines in experimental animals and seen in patients with pheochromocytoma and patients after norepinephrine treatment for shock may be related to this intravascular platelet-aggregating effect of catecholamines. The link between stress and acute myocardial infarction may be via catecholamine-induced intravascular platelet thrombosis. If the thrombogenic theory of atherosclerosis is valid, platelet aggregation induced by catecholamines may be the mechanism whereby arteriosclerotic heart disease is related to stress.

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.

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The present study was undertaken in order to analyse the possibility of culturing post mortem derived human fibroblasts. The combination of post mortem fibroblasts with the autopsy proven and histopathologically staged brain will allow the correlative investigation of dynamic biochemical processes which are systemically underlying or accompanying a neurological and/or psychiatric disorder. These studies are limited in autopsy brain or are uncertain when the neuropathological status is lacking, i.e. when fibroblasts were obtained from living patients. Our examinations of human autopsy fibroblast and those under experimentally controlled post mortem conditions with rats clearly demonstrate that autopsy-derived fibroblasts can be reliably cultured. The cells grown displayed typical morphological and staining characteristics as well as pharmacological responsiveness. Even cells obtained from a 99 years old individual or an individual with a post mortem delay of 48 hours grew in our culture system.