Age-dependent increase in desmosterol restores DRM formation and membrane-related functions in cholesterol-free DHCR24-/- mice.

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24(-/-) mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased beta-secretase activity and diminished Abeta generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24(-/-) mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24(-/-) mouse.

Modulation of Alzheimer's pathology by cerebro-ventricular grafting of hybridoma cells expressing antibodies against Abeta in vivo.

Accumulation in brain of the beta-amyloid peptide (Abeta) is considered as crucial pathogenic event causing Alzheimer's disease (AD). Anti-Abeta immune therapy is a powerful means for Abeta clearance from the brain. We recently showed that intravenous injections of anti-Abeta antibodies led to reduction, elevation or no change in brain Abeta42 concentrations of an AD mouse model. We report here, in a second passive immunization protocol, a different bioactivity of same antibodies to alter brain Abeta42 concentrations. Comparing the bioactivity of anti-Abeta antibodies in these two passive immunization paradigms underscores the potential of immune therapy for AD treatment and suggests that both the epitope recognized by the antibody and the mode of antibody administration are crucial for its biological activity.

Prosurvival effect of DHCR24/Seladin-1 in acute and chronic responses to oxidative stress.

DHCR24/seladin-1, a crucial enzyme in sterol synthesis, is of lower abundance in brain areas affected by Alzheimer's disease. While high levels of DHCR24/seladin-1 exert antiapoptotic function by conferring resistance against oxidative stress, the molecular mechanism for this protective effect is not fully understood. Here we show that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a chronic response to oxidative stress. High levels of DHCR24/seladin-1 were associated with elevated cholesterol concentrations and a general increase in cholesterol biosynthesis upon oxidative stress exposure in neuroblastoma SH-SY5Y cells. DHCR24/seladin-1 overexpression conferred resistance to oxidative stress in a cholesterol-dependent manner. Mutating the reductase activity within DHCR24/seladin-1 abolished this protective effect. Conversely, DHCR24/seladin-1 levels diminished upon chronic exposure to oxidative stress. Low levels of DHCR24/seladin-1 were associated with reduced p53 levels, independent of DHCR24 activity and cholesterol concentrations. Additionally, ablation of DHCR24/seladin-1 prevented apoptosis of primary neurons in a p53-dependent manner and reduced the response of critical p53 targets due to deficient stabilization of p53 and therefore elevated p53 ubiquitination and degradation. Our findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically independent prosurvival effects, exerting an acute response and a chronic response to oxidative stress.

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo.

The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of beta-secretase (BACE) from DRMs to APP-containing membrane fractions, increased beta-cleavage of APP and high levels of Abeta peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Abeta formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Abeta levels. Pharmacological enhancement of seladin-1 activity may be a novel Abeta-lowering approach for the treatment of AD.

Intact spatial memory in mice with seizure-induced partial loss of hippocampal pyramidal neurons.

We generated defined neuronal loss in hippocampus of genetically identical mice by pilocarpine injections and studied the impact of these seizures on the performance of mice in spatial learning and memory. The numbers of TUNEL-positive degenerating cells paralleled the severity of the seizures. When compared to the numbers found for not-seizured control mice, mild, moderate, and severe seizures produced significant increases in TUNEL-positive neurons in CA1 and CA3 regions by 19, 25, and 63%, respectively. Water maze learning was abolished after the severe seizures. However, spatial learning was normal after mild or moderate seizures. Therefore, there was no linear correlation between the impairment of learning and memory performance with the number of degenerating neurons in hippocampus. Our data suggest that normal spatial learning and memory can be achieved without the full number of hippocampal pyramidal neurons in partially lesioned hippocampus.

Assessment of the bioactivity of antibodies against beta-amyloid peptide in vitro and in vivo.

The accumulation of the beta-amyloid peptide (Abeta) is a central event in the pathogenesis of Alzheimer's disease (AD). Abeta removal from the brain by immune therapy shows promising potential for the treatment of patients with AD, although the mechanisms of the antibody action are incompletely understood. In this study we compared the biological activities of antibodies raised against various Abeta fragments for Abeta reduction in vitro and in vivo. Antibodies against Abeta enhanced the uptake of Abeta42 aggregates up to 6-fold by primary microglial cells in vitro. The kinetics of Abeta42 uptake varied considerably among antibodies. Based on the activity to mediate Abeta42 uptake by microglial cells, we identified a bioactive antibody that significantly reduced Abeta42 levels in the brains of transgenic mice with neuronal expression of an AD-related mutated amyloid precursor protein. This effect depended on the epitopes recognized by the antibody. Our data suggest that the ability to facilitate Abeta42 uptake by primary microglia cells in vitro can be used to predict the biological activity of the antibody by passive immunization in vivo. This protocol may prove useful for the rapid validation of the activity of antibodies designed to be used in immune therapy of AD.