Lead induced differences in bone properties in osteocalcin +/+ and -/- female mice.

Lead (Pb) toxicity is a major health problem and bone is the major reservoir. Lead is detrimental to bone, affects bone remodeling and is associated with elderly fractures. Osteocalcin (OC) affects bone remodeling, improves fracture resistance and decreases with age and in some diseases. The effect of lead in osteocalcin depleted bone is unknown and of interest. We compared bone mineral properties of control and Pb exposed (from 2 to 6 months) femora from female adult C57BL6 OC+/+ and OC-/- mice using Fourier Transform Infrared Imaging (FTIRI), Micro-computed tomography (uCT), bone biomechanical measurements and serum turnover markers (P1NP, CTX). Lead significantly increased turnover in OC+/+ and in OC-/- bones producing increased total volume, area and marrow area/total area with decreased BV/TV compared to controls. The increased turnover decreased mineral/matrix vs. Oc+/+ and increased mineral/matrix and crystallinity vs. OC-/-. PbOC-/- had increased bone formation, cross-sectional area (Imin) and decreased collagen maturity compared OC-/- and PbOC+/+. Imbalanced turnover in PbOC-/- confirmed the role of osteocalcin as a coupler of formation and resorption. Bone strength and stiffness were reduced in OC-/- and PbOC-/- due to reduced material properties vs. OC+/+ and PbOC+/+ respectively. The PbOC-/- bones had increased area to compensate for weaker material properties but were not proportionally stronger for increased size. However, at low lead levels osteocalcin plays the major role in bone strength suggesting increased fracture risk in low Pb2+ exposed elderly could be due to reduced osteocalcin as well. Years of low lead exposure or higher blood lead levels may have an additional effect on bone strength.

Osteocalcin affects bone mineral and mechanical properties in female mice.

Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC-/-) may vary on different backgrounds and with sex. Previous studies using adult female (OC-/-) mice on a mixed genetic background (129/B6) showed osteocalcin inhibited bone formation leading to weaker bone in wild-type (OC+/+). Yet on a pure (B6) genetic background male mice revealed osteocalcin improved fracture resistance and OC-/- bones were more prone to fracture. Osteocalcin is decreased with age and in some diseases (diabetes) where bone weakness is observed. The effect of osteocalcin in adult female bone from mice on a pure B6 background is unknown. We investigated differences in bone mineral properties and bone strength in female adult (6 months) (OC+/+) and (OC-/-) mice on a pure C57BL/6J background using Fourier Transform Infrared Imaging (FTIRI), micro-computed tomography (uCT), biomechanical measurements, histomorphometry and serum turnover markers (P1NP, CTX). Similar to female age matched mice on the (129/C57) background we found B6 OC-/- mice had a higher bone formation rate, no change in bone resorption, more immature mineral, decreased crystallinity and increased trabecular bone as compared to OC+/+. In contrast, the OC-/- mice on a pure B6 background had a lower bone mineral density, lower mineral to matrix ratio resulting in reduced stiffness and weaker bone strength. Our results demonstrate some properties of the OC-/- phenotype are dependent on genetic background. This may suggest that reduced osteocalcin may contribute to fracture and weaker bone in some groups of elderly and adults with diseases where osteocalcin is low.

Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus.

Amyloid-beta, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-beta, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.

Biochemical and immunocytochemical characterization of calsenilin in mouse brain.

Mutations in the presenilin 1 and 2 genes cause the majority of early onset familial forms of Alzheimer's disease. Here we describe the biochemical and immunohistochemical characterization of calsenilin, a novel calcium binding protein that we have previously shown to interact with presenilins 1 and 2, in mouse brain. The co-immunoprecipitation of endogenous calsenilin and presenilin 1 demonstrates that these proteins are physiologic binding partners. Although calsenilin has been predicted to be a soluble protein, we have found that the majority of it is tightly associated with the cytoplasmic face of intracellular membranes and that it can only be dissociated using harsh treatments such as urea. In addition, we have demonstrated that calsenilin is a developmentally regulated protein that is mainly present in the brain, where it localizes to both the hippocampus and cerebellum. Calsenilin staining co-localized with the somatodendritic marker microtubule-associated protein-2 primarily in the granular cell layer of the cerebellum, indicating that calsenilin expression is primarily neuronal. In primary cultured neurons, calsenilin immunoreactivity was observed in cell bodies as well as in some neuronal processes. Co-localization experiments using specific axonal and dendritic markers indicate that these processes were mainly axonal in nature, although a smaller subset of dendrites also appears to contain calsenilin. In summary, we have established that calsenilin and presenilin 1 can interact at physiologic levels, and that calsenilin is a developmentally regulated protein that is expressed primarily in the cerebellum and hippocampus. Although calsenilin is a soluble protein, it is tightly associated with the membrane. Finally, the expression pattern of calsenilin, which is similar to that of the presenilin(s), suggests that the common locations of these two proteins provide an opportunity for physical interaction in vivo.

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide.

The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.

Notch1 and amyloid precursor protein are competitive substrates for presenilin1-dependent gamma-secretase cleavage.

Proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of a highly amyloidogenic Abeta peptide, which deposits in the brains of Alzheimer's disease patients. Similar gamma-secretase processing occurs in another transmembrane protein, Notch1, releasing a potent signaling molecule, the Notch C-terminal domain. It has been shown that both events are dependent on a presenilin-dependent protease. We now test the hypothesis that activated Notch1 and APP are competitive substrates for the same proteolytic activity in neurons. Treatment of neurons with the native Notch ligand, Delta, induces endogenous Notch1 intramembraneous cleavage and diminishes Abeta production in a dose-dependent manner. Complementary experiments showed that the converse was also true. Overexpressing human APP (APP(695Sw)) in neurons leads to a decrease in endogenous Notch1 signal transduction, as assessed by a CBF1 luciferase transcription assay, by Notch C-terminal domain nuclear translocation in vitro and by analysis of Notch C-terminal domain generation and Notch1 staining in vivo. In summary, two complementary approaches suggest that APP and Notch1 are physiologically relevant competitive substrates for gamma-secretase activity.

Block copolymers modify the internalization of micelle-incorporated probes into neural cells.

An important therapeutic concern is rate and extent of internalization of drugs into cells. Hydrophilic agents often internalize poorly and slowly, and highly lipophilic ones too rapidly. The incorporation of drugs into micelles allows regulation of their internalization parameters, and newly-described block copolymers can be selectively tailored to suit specific drugs. This report compares internalization of Cell Tracker CM-DiI (DiI), a highly lipophilic non-cytotoxic fluorescent probe in common use in biology, from the freely-presented (non-micelle-incorporated) and micelle-incorporated states. DiI was effectively incorporated (>60%) into 25-50 nm diameter spherical micelles made from polycaprolactone-b-polyethylene oxide block copolymer. Confocal microscopy was used to evaluate the internalization of DiI into mixed neuron-glia cultures (2-14 days in vitro, 2DIV-14DIV). Incorporation of DiI into micelles strikingly reduced the rate and extent of its internalization in both 2DIV and 14DIV cultures. Both the age of the cultures and the block copolymer employed to construct the micelles significantly influence the internalization of micelle-incorporated probe.

Effect of PS1 deficiency and an APP gamma-secretase inhibitor on Notch1 signaling in primary mammalian neurons.

Presenilin1 (PS1) has been implicated in normal Notch1 processing and signaling in several experimental systems. In the present study, the relationship between PS1 and Notch1 in mammalian neurons is studied by analyzing Notch1 cleavage and C-terminal nuclear translocation as well as Notch1 signaling via the transactivation of a CBF1-luciferase reporter construct. We show that full-length Notch1 [N1(FL)] transfected into wild type (WT) primary neurons is cleaved in the presence of its biological ligand Delta (Dl) and translocated to the nucleus within 1--3 min of ligand addition. PS1 deficient neurons show normal Notch1 insertion into the cellular membrane, yet lack Notch1 activation resulting in markedly inhibited nuclear translocation of the C-terminal Notch fragment (NICD). PS1 deficient neurons also have impaired Notch1 signaling which can be restored fully or partially to levels seen in WT littermates by transfection with WT or familial Alzheimer's disease-associated M146L mutant PS1, respectively. We also show that pharmacological inhibition of PS1-associated gamma-secretase activity parallels the effects of genetic PS1 deficiency in these assays. These results support the hypothesis that PS1 deficiency blocks neuronal Notch1 processing and signaling.

Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notch1 proteolysis and nuclear translocation with relative preservation of notch1 signaling.

It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma-secretase processing of APP to reduce Abeta production.

Rapid Notch1 nuclear translocation after ligand binding depends on presenilin-associated gamma-secretase activity.

Recent data suggest an intimate relationship between the familial Alzheimer disease gene presenilin 1 (PS1) and proteolytic processing of both the amyloid precursor protein (APP) and the important cell signaling molecule, Notch1. We now show, using mammalian cells transfected with full-length Notch1, that the C terminal domain of Notch1 rapidly translocates to the nucleus upon stimulation with the physiologic ligand Delta and initiates a CBF1-dependent signal transduction cascade. Using this assay, we demonstrate that the same aspartate mutations in PS1 that block APP processing also prevent Notch1 cleavage and translocation to the nucleus. Moreover, we show that two APP gamma-secretase inhibitors also diminish Notch1 nuclear translocation in a dose-dependent fashion. However, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream gene, was reduced but not completely abolished in the presence of either aspartate mutations or gamma-secretase inhibitors. Our results support the hypothesis that similar PS1-related enzymatic activity is necessary for both APP and Notch1 processing, yet suggest that Notch signaling may remain relatively preserved with moderate levels of gamma-secretase inhibition.

Expression of colony stimulating factor-1 receptor (CSF-1R) by CNS neurons in mice.

We report that neurons in the central nervous system express colony stimulating factor-1 receptor (CSF-1R) mRNA and protein and that the expression has regional specificity. The presence of CSF-1R in neurons was demonstrated by the use of four different types of antibodies to CSF-1R and the presence of CSF-1R mRNA by in situ hybridization using oligonucleotide probe. In the steady state in most areas of the brain, CSF-1R is weakly expressed in only a few neurons. In the cerebellum, brainstem, and spinal cord, however, CSF-1R is expressed constitutively in greater numbers of neurons. After cerebral cortex ischemic injury, neurons in the area next to the ischemic lesion markedly upregulate CSF-1R. It is also upregulated in the contralateral cortex and in many other areas of the brain and spinal cord. We demonstrated that in cultures the ligand CSF-1 binds to its receptor (CSF-1R) in neurons and that reduction of the number of apoptotic neurons and potentiation of neuron survival is CSF-1 dose dependent. We propose that CSF-1/CSF-1R signaling is an important regulatory pathway between neurons, microglia, and astrocytes.

Notch1 inhibits neurite outgrowth in postmitotic primary neurons.

Notch plays an important role in cell fate decisions in uncommitted proliferative cells, including neurogenesis, but is believed to not have a role in postmitotic cells. We have shown previously that Notch1 is highly expressed in embryonal mouse and human brain, but surprisingly it continues to be expressed at low levels in the adult brain. The function of Notch1 in postmitotic neurons in mammals is unknown. To better understand the potential role of Notch1 in mature central nervous system neurons we studied the effect of Notch1 transfection on neurite outgrowth in primary neocortex hippocampal neurons. Transfection at two days in vitro with full length Notch1 inhibited neurite outgrowth. Transfection at five to six days in vitro, after neurite outgrowth was established, led to apparent regression of neurites. These effects were enhanced when truncated constitutively active forms of Notch1 were introduced. Co-transfection with Numb, a physiological inhibitor of Notch, blocked Notch's effect on neurite outgrowth. We also examined whether Notch1 could activate C-promoter binding factor (CBF1) transcription factor using C-promoter binding factor-luciferase constructs, and demonstrated that this signal transduction pathway is present and can be activated in postmitotic neurons. Our results show that in postmitotic neurons Notch1 influences neurite morphology, and can activate its native signal transduction pathway. These data strongly suggest that Notch1 may play a physiologically important role in the central nervous system beyond neurogenesis.

The Alzheimer-related gene presenilin 1 facilitates notch 1 in primary mammalian neurons.

The normal functional neurobiology of the Alzheimer's disease (AD) related gene presenilin 1 (PS1) is unknown. One clue comes from a genetic screen of Caenorhabditis elegans, which reveals that the presenilin homologue sel-12 facilitates lin-12 function [D. Levitan, I. Greenwald, Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene, Nature 377 (1995) 351-355]. The mammalian homologue of lin-12, Notch1, is a transmembrane receptor that plays an important role in cell fate decisions during development, including neurogenesis, but does not have a known function in fully differentiated cells. To better understand the potential role of Notch1 in mammalian postmitotic neurons and to test the hypothesis that Notch and PS 1 interact, we studied the effect of Notch1 transfection on neurite outgrowth in primary cultures of hippocampal/cortical neurons. We demonstrate that Notch1 inhibits neurite extension, and thus has a function in postmitotic mature neurons in the mammalian CNS. Furthermore, we present evidence demonstrating that there is a functional interaction between PS1 and Notch1 in mammalian neurons, analogous to the sel-12/lin-12 interaction in vulval development in C. elegans [D. Levitan, T. Doyle, D. Brousseau, M. Lee, G. Thinakaran, H. Slunt, S. Sisodia, I. Greenwald, Assessment of normal and mutant human presenilin function in Caenorhabditis elegans, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 14940-14944; D. Levitan, I. Greenwald, Effect of Sel-12 presenilin on Lin-12 localization and function in Caenorhabditis elegans, Development, 125 (1998) 3599-3606]. The inhibitory effect of Notch1 on neurite outgrowth is markedly attenuated in neurons from PS1 knockout mice, and enhanced in neurons from transgenic mice overexpressing wild type PS1, but not mutant PS1. These data suggest that PS1 facilitates Notch1 function in mammalian neurons, and support the hypothesis that a functional interaction exists between PS1 and Notch1 in postmitotic mammalian neurons.

Notch is expressed in adult brain, is coexpressed with presenilin-1, and is altered in Alzheimer disease.

In C. elegans, the Notch family member lin-12 has been shown to have a genetic interaction with sel-12, the homologue of the Alzheimer disease-associated presenilin (PS) genes in humans. Mutations in PS genes cause autosomal dominant Alzheimer disease, with age of onset frequently in the 40s. Notch is known as a developmental protein that plays an important role in lateral inhibition and specifying cell fate decisions in proliferating immature cells, and is not known to be present in adult neurons. We reasoned that, if Notch1/PS-1 interaction is relevant in Alzheimer disease, Notch1 would also need to be expressed in neurons in adult brain and colocalized with PS-1. We found that Notch1, Notch2, and a Notch ligand, Jagged1, are expressed in adult brain in mouse and in human, with strongest expression in the hippocampal formation and Purkinje cells of the cerebellum. Double immunofluorescent staining demonstrates neuronal colocalization of Notch1 with PS-1. Moreover, Notch1 expression in sporadic Alzheimer disease hippocampus is elevated more than 2-fold in comparison to that in control human hippocampus by both immunohistochemistry and Western blot analysis (p < 0.007). These results support the hypothesis that Notch1 continues to play a role in terminally differentiated neurons, and that Notch1/PS-1 interactions may occur in adult mammalian brain. The alteration in Notch1 expression in sporadic Alzheimer disease raises the possibility that disruption of Notch1/PS-1 functional interactions may occur in Alzheimer disease.

Lack of specific association of presenilin 1 (PS-1) protein with plaques and tangles in Alzheimer's disease.

Missense mutations in the presenilin-1 (PS-1) gene are causally related to the majority of familial early-onset Alzheimer's disease (FAD). PS-1 immunohistochemical expression in normal human brain and in brains with Alzheimer's disease (AD) has so far been controversial. Here, we report a study of PS-1 expression in brains, cell lines and peripheral blood mononuclear cells using a panel of well characterized PS-1-specific antibodies. These antibodies were characterized by immunofluorescent staining of PS-1 transfectants followed by flow cytometric analysis. In human brain, widespread neuronal staining was observed. PS-1 immunoreactivity was primarily confined to neuronal cell bodies and proximal dendrites. Weaker staining of microglia was also detected, in accord with the finding of PS-1 immunoreactivity in monocytes. PS-1 expression is not particularly associated with neurons either containing or spared from neurofibrillary tangles, nor with senile plaques. The level of PS-1 expression does not differ between normal and AD brains. Immunoprecipitation from AD, FAD and control brains revealed only a 32 kDa N-terminal fragment and an 18-20 kDa C-terminal fragment. Little or no full length PS-1 was detected. The enriched presence of PS-1 in neurons implies an important role in neuronal function, however, the lack of apparent association of its expression with AD pathology signifies the need for a better understanding of its pathophysiological role.

Structure and functions of human cerebrospinal fluid lipoproteins from individuals of different APOE genotypes.

Recent data have implicated apolipoprotein E (apoE) in neuritic outgrowth, synaptic stability, and Alzheimer's disease; these data led us to examine the normal role of apoE-containing lipoproteins in the central nervous system (CNS). We isolated lipoproteins from human cerebrospinal fluid (CSF) in order to examine their composition and potential functions. CSF particles were composed of approximately one-third protein, one-third phospholipid, and one-third cholesterol. ApoE3 formed homodimers and heterodimers with apoA-II, while apoE4, as expected, was monomeric. We addressed the function of CSF lipoproteins with assays of cholesterol efflux and cholesterol influx. CSF lipoproteins decreased intracellular levels of cholesterol in cholesterol-loaded fibroblasts, suggesting these particles can act to remove excess lipids from cells. CSF lipoproteins competed for 125I-labeled LDL degradation by fibroblasts, suggesting they can also interact with the LDL receptor. Furthermore, CSF lipoproteins labeled with the fluorescent dye Dil were internalized by neuroglioma cells and primary neurons and astrocytes in culture. Together, these data support a model of CSF lipoproteins acting to remove lipids from degenerating cells and delivering lipids to cells for new membrane synthesis or storage.

Changes in the numbers of osteoclasts in newts under conditions of microgravity.

Intensity of osteoclastic resorption and calcium content were investigated in intact limb bones of the newts flown on board of a biosatellite Cosmos-2229 after amputation of their forelimbs and tail. Using X-ray microanalysis it was shown an increase in calcium content in the bones on 20th day after operation. Histological study revealed an activation of osteoclastic resorption on endosteal surface of long bones. The newts exposed after surgery on a biosatellite had the same level of bone mineralisation as operated ground control ones, but the increase in number of polynuclear osteoclasts was lower.

Developmental regulation of presenilin mRNA expression parallels notch expression.

Mutations in the presenilin (PS) 1 and 2 genes are associated with autosomal dominant Alzheimer disease. PS1 shares striking homology with sel-12, a C. elegans gene implicated in the function of lin-12/Notch, a protein important in neurogenesis. We studied mRNA expression using RT-PCR and in situ hybridization techniques during neural development in mouse and rat. Strong expression of PSs and Notch1 was observed in embryos, especially in the ventricular zone, which decreased gradually as embryos developed. Very low levels of PSs and Notch were present in adulthood, their signals present primarily in the hippocampus and cerebellum. These observations show that, like Notch, PS1 and PS2 are strongly developmentally regulated, and support the plausibility of an interaction between PSs and Notch.