Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice.

The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.

Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage, Dutch type.

An amyloid protein that precipitates in the cerebral vessel walls of Dutch patients with hereditary cerebral hemorrhage with amyloidosis is similar to the amyloid protein in vessel walls and senile plaques in brains of patients with Alzheimer's disease, Down syndrome, and sporadic cerebral amyloid angiopathy. Cloning and sequencing of the two exons that encode the amyloid protein from two patients with this amyloidosis revealed a cytosine-to-guanine transversion, a mutation that caused a single amino acid substitution (glutamine instead of glutamic acid) at position 22 of the amyloid protein. The mutation may account for the deposition of this amyloid protein in the cerebral vessel walls of these patients, leading to cerebral hemorrhages and premature death.

Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a membrane-spanning glycoprotein that is the source of the beta-amyloid peptide (beta AP) which accumulates as senile plaques in the brains of patients with Alzheimer's disease. beta APP is normally processed such that a cleavage occurs within the beta AP, liberating secreted forms of beta APP (APPss) from the cell. The neuronal functions of these forms are unknown. We now report that APPss have a potent neuroprotective action in cultured rat hippocampal and septal neurons and in human cortical neurons. APPs695 and APPs751 protected neurons against hypoglycemic damage, and the neuroprotection was abolished by antibodies to a specific region common to both APPs695 and APPs751. APPss caused a rapid and prolonged reduction in [Ca2+]i and prevented the rise in [Ca2+]i that normally mediated hypoglycemic damage. APPss also protected neurons against glutamate neurotoxicity, effectively raising the excitotoxic threshold. APPss may normally play excitoprotective and neuromodulatory roles. Alternative processing of APPss in Alzheimer's disease may contribute to neuronal degeneration by compromising the normal function of APPss and by promoting the deposition of beta AP.

Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035].

Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino- or the carboxy-terminal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.

beta-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer's disease.

Recent findings link altered processing of beta-amyloid precursor protein (beta APP) to disruption of neuronal Ca2+ homeostasis and an excitotoxic mechanism of cell death in Alzheimer's disease. A major pathway of beta APP metabolism results in the release of secreted forms of beta APP, APPss. These secreted forms are released in response to electrical activity and can modulate neuronal responses to glutamate, suggesting roles in developmental and synaptic plasticity. beta APP is upregulated in response to neural injury and APPss can protect neurons against excitotoxic or ischemic insults by stabilizing the intracellular Ca2+ concentration [Ca2+]i. An alternative beta APP processing pathway liberates intact beta-amyloid peptide, which can form aggregates that disrupt Ca2+ homeostasis and render neurons vulnerable to metabolic or excitotoxic insults. Genetic abnormalities (e.g. certain beta APP mutations or Down syndrome) and age-related changes in brain metabolism (e.g. reduced energy availability or increased oxidative stress) may favor accumulation of [Ca2+]i-destabilizing beta-amyloid peptide and diminish the release of [Ca2+]i-stabilizing, neuroprotective APPss.

Brain cell nuclear retention of testosterone metabolites, 5alpha-dihydrotestosterone and estradiol-17beta, in adult rats.

Radioactivity was analyzed in tissue homogenates and purified cell nuclear fractions in the pituitary and 9 brain regions 2 h after an iv [7-3H]testosterone infusion into gonadectomized-adrenalectomized adult male and female rats. Unchanged testosterone (T) and its metabolites, 5alpha-dihydrotestosterone (DHT) and estradiol-17beta (E2), were the predominant steroids recovered from cell nuclear fractions in all brain regions examined. Highest levels of E2 as a T metabolite were found in nuclear fractions from the amygdala, followed by the hypothalamus, preoptic area and septum, while levels of DHT as a T metabolite were highest in nuclear fractions from the pituitary, followed by the hypothalamus and the septum. The regional pattern was similar in both sexes. Low levels of DHT and E2 recovered from serum indicated these metabolites were probably formed in situ. Regional distributions of cell nuclear retained DHT and E2 as T metabolites were compared with the respective regional distributions observed after either [1,2-3H]DHT or [6,7-3H]E2 infusion. Cell nuclear levels of E2 as a T metabolite did not correlate well with nuclear retained radioactivity after [3H]E2 injection, but cell nuclear levels of DHT as a T metabolite did correlate with nuclear radioactivity after [3H]-DHT infusion. No apparent sex differences were observed in nuclear-associated radioactivity after [3H]-E2 or [3H]DHT injections. The results emphasize the potential importance of local aromatization in the brains of rats of both sexes, suggest the existence of a DHT receptor site at the brain cell nuclear level, and confirm the existence of estradiol-17beta cell nuclear receptors in the adult male rat brain.

5alpha-Dihydrotestosterone (DHT) receptors in rat brain and pituitary cell nuclei.

Gonadectomized-adrenalectomized (GX-ADX) adult male rats were injected iv with 2-4 mug/kg [1,2-3H]DHT and sacrificed 2 h later. Whole tissue homogenates and purified cell nuclear fractions were prepared from various brain regions and the pituitary and analyzed for radioactivity. Cell nuclei from pituitary and most limbic-hypothalamic regions (but not cerebral cortex) concentrated radioactivity (per unit protein) over whole tissue levels. The highest levels of nuclear-associated radioactivity were present in pituitary, hypothalamic and septal tissues. Analysis of radioactivity by double isotope dilution, chromatography, and recrystallization revealed that unmetabolized DHT represented 96 and 95% of the nuclear-associated radioactivity in pituitary and pooled limbic-hypothalamic structures, respectively. Simultaneously administered 100-fold molar excesses of unlabeled 5betaDHT, progesterone or corticosterone did not reduce nuclear-retained [3H]DHT, while unlabeled 5alphaDHT competed strongly. The anti-androgen, cyproterone acetate, competed at doses capable of of blocking androgen-mediated neuroendocrine effects. Levels of nuclear-retained [3H]DHT were negligible in intact males, but rose markedly 24 h after castration-adrenalectomy, remaining stable for 2 weeks post-operatively. Density gradient centrifugation of pituitary or brain cell nuclear salt extracts (0.4M KCl) revealed the [3H]DHT bound to a macromolecule sedimenting at 3--4S. The characteristics of DHT brain and pituitary cell nuclear binding are compared with the respective characteristics of neural cytosol and ventral prostate nuclear androgen binding components. These preliminary data suggest the existence of the functional, steroid-specific, stereospecific cell nuclear androgen receptor in the adult rat brain and pituitary.

Detection of distinct isoform patterns of the beta-amyloid precursor protein in human platelets and lymphocytes.

Cerebral deposition of the amyloid beta-protein (A beta P), approximately 40 residue fragment of the integral membrane protein, beta-amyloid precursor protein (beta APP), has been implicated as the probable cause of some cases of familial Alzheimer's disease (AD). The parallels between A beta P deposition in AD and the deposition of certain plasma proteins in systemic amyloid diseases has heightened interest in the analysis of beta APP in circulating cells and plasma. Here, we describe distinct isoform patterns of beta APP in peripheral platelets and lymphocytes. PCR-mediated amplification of mRNA from purified platelets demonstrated the expression of all three major beta APP transcripts (beta APP770,751,695). The full-length, approximately 140 kDa form of beta APP751,770 was detected in membranes of resting and activated platelets but very little immature, approximately 122 kDa beta APP751,770 was found, suggesting a different processing of beta APP in platelets than that described in a variety of cultured cells and tissues. Platelets stimulated with thrombin, calcium ionophore, or collagen released the soluble, carboxyl-truncated form of beta APP (protease nexin-II), but no evidence for the shedding of full-length beta APP associated with platelet microparticles was found, in contrast to previous reports. As a positive control marker for microparticles, the fibrinogen receptor subunit, GPIIIa, was readily detected in platelet releasates. Resting and activated platelets contained similar amounts of the approximately 10 kDa carboxyl terminal beta APP fragment that is retained in platelet membranes following the constitutive cleavage of protease nexin-II. Nonstimulated peripheral B and T lymphocytes contained small amounts of membrane-associated mature and immature beta APP751,770. The potentially amyloidogenic full-length beta APP molecules present in circulating platelets and lymphocytes but not in microparticles could serve as a source of the microvascular A beta P deposited during aging and particularly in AD.

Lack of Alzheimer pathology after beta-amyloid protein injections in rat brain.

In order to establish a direct relationship between beta-amyloid protein (beta AP) and in vivo neurotoxicity, we made intraparenchymal injections and Alzet pump infusions of beta AP into the hippocampus and cortex of adult rats. We tested a number of synthetic beta AP peptides (beta AP 1-40, 1-38, and 25-35) and peptide controls (scrambled and reversed 1-40, and scrambled and reversed 25-35) over a wide range of concentrations and in a variety of vehicles. The rats were sacrificed from 2-35 days following the implant, and the brains examined by standard immunohistochemical and histological methods used to evaluate the pathologies associated with Alzheimer's disease. We report the lack of Alzheimer related pathology and no significant morphological differences between the beta AP peptide and the peptide and vehicle control injections. These observations indicate that the simple intraparenchymal injection of beta AP in the rat brain is not an appropriate model of Alzheimer-related neurotoxicity.

Synthetic amyloid beta-protein fails to produce specific neurotoxicity in monkey cerebral cortex.

Because progressive amyloid beta-protein (A beta P) deposition and surrounding neuritic dystrophy occur spontaneously in primates, we evaluated the in vivo effects of synthetic A beta P in monkey cortex. Experimental and control A beta P were stereotactically injected into multiple neocortical sites of adult rhesus monkeys in a vehicle of either artificial cerebrospinal fluid or acetonitrile. After 2 weeks or 3 months, injection sites were identified and characterized histologically and immunocytochemically. A beta P antibodies specifically detected the injected A beta P1-40 peptide. Serial sections stained with silver and antineurofilament protein demonstrated comparable degrees of degenerating neurons, dystrophic neurites, and axonal spheroids associated with both experimental and control peptide injections. Alz 50 staining was sparse or absent in all sites. We conclude that specific cellular changes closely resembling AD pathology were not detected in these experiments, and that control and experimental A beta P peptides produced indistinguishable effects. Methodological concerns regarding the in vivo modeling of A beta P bioactivity are discussed.

High cerebrospinal fluid tau and low amyloid beta42 levels in the clinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype.

OBJECTIVE: To evaluate cerebrospinal fluid (CSF) levels of amyloid beta protein ending at amino acid 42 (Abeta42) and tau as markers for Alzheimer disease (AD) and to determine whether clinical variables influence these levels. DESIGN: Cohort study. SETTING: Six academic research centers with expertise in dementia. SUBJECTS: Eighty-two patients with probable AD, including 24 with very mild dementia (Mini-Mental State Examination score >23/30) (AD group); 60 cognitively normal elderly control subjects (NC group); and 74 subjects with neurological disorders, including dementia (ND group). MAIN OUTCOME MEASURES: Levels of Abeta42 and tau were compared among AD, NC, and ND groups. Relationships of age, sex, Mini-Mental State Examination score, and apolipoprotein E (Apo E) genotype with these levels were examined using multiple linear regression. Classification tree models were developed to optimize distinguishing AD from NC groups. RESULTS: Levels of Abeta42 were significantly lower, and levels of tau were significantly higher, in the AD group than in the NC or ND group. In the AD group, Abeta42 level was inversely associated with Apo E epsilon4 allele dose and weakly related to Mini-Mental State Examination score; tau level was associated with male sex and 1 Apo E epsilon4 allele. Classification tree analysis, comparing the AD and NC subjects, was 90% sensitive and 80% specific. With specificity set at greater than 90%, the tree was 77% sensitive for AD. This tree classified 26 of 74 members of the ND group as having AD. They had diagnoses difficult to distinguish from AD clinically and a high Apo E epsilon4 allele frequency. Markers in CSF were used to correctly classify 12 of 13 patients who later underwent autopsy, including 1 with AD not diagnosed clinically. CONCLUSIONS: Levels of CSF Abeta42 decrease and levels of CSF tau increase in AD. Apolipoprotein E epsilon4 had a dose-dependent relationship with CSF levels of Abeta42, but not tau. Other covariates influenced CSF markers minimally. Combined analysis of CSF Abeta42 and tau levels discriminated patients with AD, including patients with mild dementia, from the NC group, supporting use of these proteins to identify AD and to distinguish early AD from aging. In subjects in the ND group with an AD CSF profile, autopsy follow-up will be required to decide whether CSF results are false positive, or whether AD is a primary or concomitant cause of dementia.

Development of lipophilic anticancer agents for the treatment of brain tumors by the esterification of water-soluble chlorambucil.

The lipophilic derivatives of the anticancer alkylating agent chlorambucil, chlorambucil-methyl, -isopropyl and -tertiary butyl esters, were synthesized and administered i.v. to anesthetized rats. Plasma and brain concentrations of these agents and of their active metabolites, chlorambucil and phenylacetic mustard, then were determined by high-performance liquid chromatography between 5 and 60 min. Whereas large amounts of chlorambucil-tertiary butyl ester entered and were maintained in brain, lower amounts of chlorambucil-isopropyl ester and no chlorambucil-methyl ester were found in brain. The comparative brain/plasma concentration-time integral ratios of the total active agents generated from chlorambucil-tertiary butyl, -isopropyl and -methyl esters were 0.85, 0.12 and 0.06, respectively, compared to a ratio of 0.02 for chlorambucil. In vitro alkylating activity of each ester was compared to that of equimolar chlorambucil, by reaction with 4-(p-nitrobenzyl)pyridine. Each ester possessed high intrinsic alkylating activity, equal to 38.4, 57.0 and 69.9% of chlorambucil activity, for the -tertiary butyl, -isopropyl and -methyl esters, respectively. Therefore each is an active antineoplastic agent irrespective of whether or not chlorambucil is regenerated. The rates of ester hydrolysis of these derivatives to chlorambucil were measured in fresh rat blood and in liver and brain homogenates at 37 degrees C. Chlorambucil-methyl and -isopropyl esters were hydrolysed quickly within 30 s in blood and liver, whereas chlorambucil-tertiary butyl ester was more stable with half-lives of approximately 7 h and 2 h, respectively. All proved to be relatively stable in brain homogenate. Steric hindrance around the ester linkage of chlorambucil-tertiary butyl ester reduces its affinity to and rate of hydrolysis by plasma and liver esterases, and allows it to accumulate within the brain. Chlorambucil-tertiary butyl ester maintains high levels in brain despite rapidly declining plasma concentrations and, due to these favorable pharmacokinetics and to its intrinsic anticancer activity, it possess promising characteristics for the treatment of malignant brain tumors.

Elevation of microtubule-associated protein tau in the cerebrospinal fluid of patients with Alzheimer's disease.

Currently, there is no biochemical marker clinically available to test for the presence of Alzheimer's disease (AD). Recent studies suggest that the core component of AD-associated neurofibrillary tangles (NFTs), the microtubule-associated protein tau, might be present in CSF. This study focuses on establishing both the presence of tau in CSF and its potential utility in the diagnosis of AD. We obtained CSF from 181 individuals; 71 of these were diagnosed as having probable AD by NINCDS-ADRDA criteria. The remaining 110 individuals were divided into three groups: (1) age-matched demented non-AD patients (n = 25), (2) neurologic controls (n = 59), and (3) other controls (n = 26). We developed a sensitive enzyme-linked immunosorbent tau assay using monoclonal antibodies prepared against recombinant human tau. We confirmed specificity of the antibodies by a combination of immunoprecipitation and immunoblot results. By this assay we measured that the AD population has a mean level of tau 50% greater than the non-AD dementia patients. Comparing AD patients with all other groups, the difference in tau levels as analyzed by one-way ANOVA is highly statistically significant (p < 0.001). Postmortem analysis of two AD patients with high levels of CSF tau revealed a high density of NFTs in the hippocampus. There was no significant correlation between tau and age in the non-AD groups. This study suggests that CSF tau is elevated in AD and might be a useful aid in antemortem diagnosis.

Regulated cleavage of Alzheimer beta-amyloid precursor protein in the absence of the cytoplasmic tail.

Alzheimer beta-amyloid precursor protein can be phosphorylated on residues Thr654, Ser655 and Thr668 on its cytoplasmic domain. Proteolytic cleavage of the amyloid precursor protein and release of the amyloid precursor protein ectodomain into the medium of cultured cells can be activated by phorbol esters which stimulate protein kinase C. In the present study, using mutated amyloid precursor protein, we show that phosphorylation of cytoplasmic residues is not required for the phorbol ester-activated cleavage and release of the amyloid precursor protein ectodomain. Remarkably, deletion of the entire amyloid precursor protein cytoplasmic tail had no effect on the phorbol ester-activated cleavage/release. The results indicate that activation of amyloid precursor protein cleavage/release by protein kinase C involves phosphorylation of some component of the processing pathway, instead of or in addition to the cytoplasmic tail of the amyloid precursor protein.

Production and increased detection of amyloid beta protein and amyloidogenic fragments in brain microvessels, meningeal vessels and choroid plexus in Alzheimer's disease.

Recent advances indicate soluble amyloid beta (A beta) protein is produced constitutively during normal metabolism of the amyloid precursor protein (APP). This has not been directly examined in human brain vascular tissues. Using a panel of well-characterized antibodies, here we show that increased amounts of soluble A beta were found in isolated vascular tissues from AD subjects compared to age-matched controls without significant Alzheimer pathology. Immunocytochemical analyses of isolated vessel preparations showed characteristic transverse patterns of A beta deposits in large vessels with smooth muscle, however, fine A beta deposits were apparent even in capillaries. A proportion of such A beta protein and potentially amyloidogenic carboxyl terminal fragments were released by solubilization and disruption of the vascular basement membrane by collagenase treatments. We further demonstrated by in vitro metabolic labelling that soluble A beta or an A beta-like peptide is associated and produced by cerebral microvessels, meningeal vessels and the choroid plexus isolated postmortem from human as well as rat brain. Compared to those from young rats, cerebral microvessels from aging rats showed increased release of carboxyl terminal fragments of APP and A beta-like peptide. Our observations provide the first direct demonstration that human vascular tissues produce soluble A beta, a product of the secretory pathway in APP processing. Our findings also suggest that aging associated alterations in the basement membranes are a factor in A beta accumulation that results in vascular amyloid deposition, the principal feature of cerebral amyloid angiopathy.

Altered calcium signaling and neuronal injury: stroke and Alzheimer's disease as examples.

Several cellular signaling systems have been implicated in the neuronal death that occurs both in development ("natural" cell death) or in pathological conditions such as stroke and Alzheimer's disease (AD). Here we consider the possibility that neuronal degeneration in an array of disorders including stroke and AD arises from one or more alterations in calcium-regulating systems that result in a loss of cellular calcium homeostasis. A long-standing hypothesis of neuronal injury, the excitatory amino acid (EAA) hypothesis, is revisited in light of new supportive data concerning the roles of EAAs in stroke and the neurofibrillary degeneration in AD. Two quite new concepts concerning mechanisms of neuronal injury and death are presented, namely: 1) growth factors normally "stabilize" intracellular free calcium levels ([Ca2+]i) and protect neurons against ischemic/excitotoxic injury, and 2) aberrant processing of beta-amyloid precursor protein (APP) can cause neurodegeneration by impairing a neuroprotective function of secreted forms of APP (APPs) which normally regulate [Ca2+]i. Altered APP processing also results in the accumulation of beta-amyloid peptide which contributes to neuronal damage by destabilizing calcium homeostasis; in AD beta-amyloid peptide may render neurons vulnerable to excitotoxic conditions that accrue with increasing age (e.g., altered glucose metabolism, ischemia). Growth factors may normally protect neurons against the potentially damaging effects of calcium influx resulting from energy deprivation and overexcitation. For example, bFGF, NGF and IGFs can protect neurons from several brain regions against excitotoxic/ischemic insults. Growth factors apparently stabilize [Ca2+]i by several means including: a reduction in calcium influx; enhanced calcium extrusion or buffering; and maintenance or improvement of mitochondrial function. For example, bFGF can suppress the expression of a N-methyl-D-aspartate (NMDA) receptor protein that mediates excitotoxic damage in hippocampal neurons. Growth factors may also prevent the loss of neuronal calcium homeostasis and the increased vulnerability to neuronal injury caused by beta-amyloid peptide. Since elevated [Ca2+]i can elicit cytoskeletal alterations similar to those seen in AD neurofibrillary tangles, we propose that neuronal damage in AD results from a loss of calcium homeostasis. The data indicate that a variety of alterations in [Ca2+]i regulation may contribute to the neuronal damage in stroke and AD, and suggest possible means of preventing neuronal damage in these disorders.

Cells with a familial Alzheimer's disease mutation produce authentic beta-peptide.

Cells overexpressing the beta-amyloid precursor protein possessing a mutation found in familial Alzheimer's disease overproduce beta-amyloid peptide (A beta). Because these findings were based on immunological identification, we have chemically characterized the peptides produced. Purified A beta fragments from the conditioned media of these cells were found to have N-terminal sequence consistent with the A beta found in cerebral plaques. Mass spectrometric data demonstrated a series of A beta fragments consistent with those found in Alzheimer's disease (AD); the major species corresponding to A beta(1-40). Significantly, a longer fragment corresponding to A beta(1-42) was found. These findings suggest that this cellular system may be useful for mechanistic studies of A beta generation and possibly for the development of therapeutic agents to treat AD.

Developmental expression and regional distribution of the scrapie-associated protein mRNA in the rat central nervous system.

The scrapie associated protein (SAP) has been shown to be a normal brain protein of yet undefined function. This study demonstrates that rat brain SAP mRNA levels undergo a transcriptionally dependent increase during normal development. The interregional variation in the adult rat central nervous system (CNS) is roughly 10 fold, with highest levels in the basal ganglion/thalamus and lowest levels in the spinal cord.

Cytoplasmic and nuclear estradiol-17 beta binding in male and female rat brain: regional distribution, temporal aspects and metabolism.

A study was conducted in which we simultaneously measured absolute levels of cell nuclear estradiol-17 beta (E2) retention and cytosol E2 saturation binding capacities in various neural tissues from adult gonadectomized-adrenalectomized (GX-ADX) male and female rats, at various times after a saturating dose of [3H]E2. The results indicate that, with minor exceptions, the basic properties of E2 metabolism and E2 retention kinetics in target brain tissues and corresponding cell nuclear and cytosol fractions are quite similar in GX-ADX male and female rats. The results do not, however, rule out the possibility of subtle sex differences in estrogen binding in at least some brain regions which were studied. The present demonstration of E2 binding in various regions of the male brain is consistent with a physiological role of estrogen in the male; however, the similarities in these E2 binding parameters between the sexes unfortunately shed little light on the marked neuroendocrine sex differences which have been documented. At times of maximal nuclear E2 retention (2 h post-injection), pituitary cytosol was markedly depleted of E2 binding sites, while cytosols obtained from the preoptic area, hypothalamus and amygdala were only moderately depleted. These observations suggest that within the brain the relationship between cytoplasmic and cell nuclear estrogen binding may be different from that observed in non-neural tissues. In all tissues, E2 cytosol binding was replenished to normal levels 24 h after [3H]E2 administration.