The Swedish mutation causes early-onset Alzheimer's disease by beta-secretase cleavage within the secretory pathway.

Several missense mutations causing early-onset Alzheimer's disease (AD) have been described in the gene coding for the beta-amyloid precursor protein (beta APP). A double mutation found in a Swedish family is located before the amyloid beta-peptide (A beta) region of beta APP and results in the increased production and secretion of A beta. Here we show that the increased production of A beta results from a cellular mechanism, which differs substantially from that responsible for the production of A beta from wild-type beta APP. In the latter case, A beta generation requires reinternalization and recycling of beta APP. In the case of the Swedish mutation the N-terminal beta-secretase cleavage of A beta occurs in Golgi-derived vesicles, most likely within secretory vesicles. Therefore, this cleavage occurs in the same compartment as the alpha-secretase cleavage, which normally prevents A beta production, explaining the increased A beta generation by a competition between alpha- and beta-secretase.

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.

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.2.29). We measured the specific release of queuine from Q-5'-phosphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC3 colon adenocarcinoma, prompted us to examine a HxGC3-derived cell line, GC3/M. GC3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.

Longitudinal stability of CSF tau levels in Alzheimer patients.

BACKGROUND: Antemortem levels of tau in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients have repeatedly been demonstrated to be elevated when compared to controls. Although CSF tau has been reported to be elevated even in very mild AD, it is unknown how tau levels change during the course of the disease. METHODS: We have followed 29 mild-to-moderately affected AD subjects over 2 years with repeated CSF taps. Clinical measures of dementia severity (Clinical Dementia Rating Scale, Global Deterioration Scale and Mini-Mental Status Examination) were obtained at the start and conclusion of the observation period, and CSF tau was measured with a standard enzyme-linked immunoabsorbent assay (ELISA) using two monoclonal antibodies. RESULTS: Despite significant changes in the clinical measures consistent with progression of the disease, no significant overall change in CSF tau levels (548 +/- 355 vs. 557 +/- 275 pg/mL, NS) was observed. None of the clinical variables was significantly correlated with either baseline measures of CSF tau or delta CSF tau (last-first). Similarly, CSF tau at baseline and changes over time were not significantly related to Apolipoprotein E (APO E) phenotype. CONCLUSIONS: These data suggest that CSF tau levels are stable over extended periods of time in a group of mild-to-moderately demented AD subjects and that CSF tau levels do not predict the severity or rate of progression of AD, at least not during the middle stages of the illness.

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.

Assessment of CSF levels of tau protein in mildly demented patients with Alzheimer's disease.

CSF levels of tau protein are increased in many patients with Alzheimer's disease (AD). Studies disagree on whether the increase is found in moderate or severe AD to a greater extent than in mild AD, and in two reports there was an inverse correlation between tau levels and cognitive scores. To readdress this question, we measured CSF tau in a group of mildly impaired patients with AD (Mini-Mental State Examination [MMSE] scores > or =20/30) and compared their tau levels with those in age-comparable normal and neurologic controls. We found that the mean level of CSF tau was significantly increased in the AD group compared with the controls, and 29 of 36 patients with AD had levels that exceeded a cutoff determined in a previous study. CSF tau levels did not correlate with MMSE scores. These findings and those of previous studies show that elevated CSF tau levels are found in most patients with AD, occur early in the course of dementia, and may be useful in supporting the diagnosis of AD.

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.

Intrahippocampal colchicine injection results in spectrin proteolysis.

Neurons in the hippocampal formation vary markedly in their susceptibility to colchicine toxicity. The present study was directed at evaluating the effects of colchicine on the proteolytic breakdown of the cytoskeletal protein spectrin within the hippocampus in the rat. Quantified by immunoblot analysis of spectrin breakdown products, the extent of proteolysis was found to correlate with the relative vulnerability of different hippocampal subfields to colchicine toxicity. Levels of breakdown products increased dramatically between 1 and 2 days after colchicine injection, peaked between 2 and 4 days, and remained detectably elevated for at least 35 days. Two days after colchicine injection, the spectrin breakdown products were significantly more concentrated in the molecular layer than in the granule cell/hilar region of the dentate gyrus. The colchicine-induced increase in spectrin breakdown products was significantly reduced by pretreatment with the protease inhibitor leupeptin and was significantly elevated by pretreatment with the lysosomal inhibitor chloroquine. Immunohistochemical analyses of the hippocampus at various times after colchicine injection revealed changes in the distribution of spectrin-like immunoreactivity that paralleled the changes observed by Western blot analysis. Thus increased staining was observed in the molecular layer of the dentate gyrus at 2 and 4 days after the injection, while staining in CA3 was only slightly increased. In addition, abnormal staining of reactive astrocytes was prominent at 2 days. The mechanism whereby colchicine results in neuronal death is as yet unknown. However, the results presented here demonstrate that extensive proteolysis of a cytoskeletal protein occurs in response to the drug, suggesting a plausible mechanism for its neurotoxicity. The protease responsible for the effect is likely to be calpain since the process is non-lysosomal, leupeptin-sensitive and produces spectrin breakdown products indistinguishable from those generated by calpain treatment in vitro. These data support the hypothesis that calpain-mediated degradation of cytoskeletal elements is a common and early response to neurodegenerative events and serves as a trigger in the development of various neuropathologies.

Potential treatment opportunities for Alzheimer's disease through inhibition of secretases and Abeta immunization.

Research over the past ten years on Alzheimer's disease has pursued many opportunities. Notable amongst the various approaches are efforts related to the "amyloid hypothesis." This hypothesis posits that the beta amyloid peptide causes the extensive neuropathology and clinical decline associated with the disease. Extensive research in this area has shown that the beta amyloid peptide is produced by proteases termed "secretases" and it has been shown that blockade of secretase functions reduce the amount of beta amyloid peptide produced. An additional approach to reduce beta amyloid, through an increase in clearance mechanisms, is to immunize with the peptide itself and induce an antibody response. The specifically elicited antibodies then bind to and stimulate clearance of the peptide from the brain. These findings have stimulated several approaches to develop novel therapeutic strategies to treat Alzheimer's disease that either are about or have entered the clinic.

Immunotherapy with beta-amyloid for Alzheimer's disease: a new frontier.

Alzheimer's disease (AD) represents the fourth leading cause of death in the U.S. and the leading cause of dementia in the elderly population. Until recently, there was little hope of finding a way to prevent the underlying brain pathology from progressing toward the inevitable conclusion of the disease. However, new immunotherapeutic approaches have been described that are based on vaccination with the beta-amyloid 1-42 peptide (Abeta). The encouraging efficacy and safety of Abeta immunization in reducing neuropathology in animal models of AD has opened up new therapeutic possibilities for patients. Immunization with Abeta is aimed at reducing the Abeta-associated pathology of AD. It is hypothesized that this approach will also reduce the cascade of downstream events leading to neuronal cell loss and, ultimately, dementia. The ensuing articles in this issue describe various aspects of the Abeta immunization strategy and their potential relevance to AD treatment.

Prevention and reduction of AD-type pathology in PDAPP mice immunized with A beta 1-42.

In AD certain brain structures contain a pathological density of A beta protein deposited into plaques. The effect of genetic mutations found in early onset AD patients was an overproduction of A beta 42, strongly suggesting that overproduction of A beta 42 is associated with AD. We hypothesized that an immunological response to A beta 42 might alter its turnover and metabolism. Young PDAPP transgenic mice were immunized with A beta 1-42, which essentially prevented amyloid deposition; astrocytosis was dramatically reduced and there was reduction in A beta-induced inflammatory response as well. A beta 1-42 immunization also appeared to arrest the progression of amyloidosis in older PDAPP mice. A beta immunization appears to increase clearance of amyloid plaques, and may therefore be a novel and effective approach for the treatment of AD.

Ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus.

Transient forebrain ischemia is followed within minutes by accelerated proteolysis of the cytoskeletal protein, spectrin. This effect is most pronounced in the selectively vulnerable CA1 region of hippocampus which also experiences a second proteolytic phase during the terminal stages of neuronal degeneration. Both proteolytic phases are suppressed by MK-801, an NMDA receptor antagonist. Cytoskeletal disruption, via NMDA receptor-linked proteolytic events, is suggested to predispose vulnerable neurons to delayed cell death.

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies.

Lesions of the various afferents to the hippocampus have been widely used to investigate the mechanisms underlying growth and degeneration in adult mammalian CNS. It has been proposed that disturbances in intracellular calcium and activation of calcium-dependent proteases represent key steps in producing come of the consequences of the lesions. In this study, we show that lesions of the entorhinal cortex or of the commissural pathway result in profound changes in the distribution of brain spectrin. At 2 days after lesions of the entorhinal cortex, immunoreactivity to spectrin is markedly increased in the outer molecular layer (OML) of the dentate gyrus; conversely at 2 days after commissural lesions, immunoreactivity to the same antigen is increased in the inner molecular layer. The increase in immunoreactivity to spectrin varies with survival time after lesions of the entorhinal cortex. By 24 h post lesion, the increase is homogeneous across the OML, and becomes more intense by 48 h. Between 1 and 3 weeks the increase is much less than at 48 h and is concentrated at the inner border of the OML. Pretreatment of the animals with the calpain inhibitor leupeptin reduces the increase in spectrin immunoreactivity normally seen 48 h after the lesion of the entorhinal cortex. Changes in the pattern of immunoreactivity to GFAP are very different to that seen with spectrin antibodies and are consistent with the known modifications in astrocytes that follow lesions of hippocampal afferents.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in synaptic transmission and long-term potentiation in hippocampal slices from young and aged PDAPP mice.

Synaptic transmission and plasticity were studied in the CA1 field of hippocampal slices from young and aged transgenic mice over-expressing a mutant form of the human amyloid precursor protein (PDAPP mice). The transgenic mice at 4-5 months of age, prior to the formation of amyloid-beta peptide deposits in these animals, differed from non-transgenic control mice in three respects: (1) paired-pulse facilitation (PPF) was enhanced; (2) responses to high frequency stimulation bursts were distorted; (3) long-term potentiation (LTP) decayed more rapidly. More striking was the profound reduction in the size of synaptic responses and frequent loss of field potentials that were found in the transgenic mice at 27-29 months, an age at which they exhibit numerous amyloid plaques, neuritic dystrophy, and gliosis. Control mice at these ages did not show such dramatic effects. PPF was reduced in aged transgenic mice, compared to aged controls; however, LTP was still in evidence, although direct comparisons of its induction conditions in aged transgenic and control mice were compromised by the profound differences in field potentials between the two groups. These results point to two conclusions: (1) altered synaptic communication appears in PDAPP mice in advance of amyloid plaque formation and probably involves changes in presynaptic calcium kinetics; (2) the disturbances in synaptic transmission that appear when abundant plaques and Alzheimer's-like neuropathology are present in the transgenic mice are not necessarily accompanied by a disproportionate loss of long-term synaptic plasticity.

Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus.

Stimulation of N-methyl-D-aspartate (NMDA) receptors was found to induce proteolysis of brain spectrin in hippocampal slices. The effect was dependent upon extracellular calcium, blocked by the antagonist 2-amino-5-phosphonovalerate (AP5), and was not reproduced by potassium-induced depolarization. These results are consistent with the hypothesis that the involvement of NMDA receptors in plasticity and excitotoxicity is at least partially mediated by calcium-activated proteolysis of cytoskeletal proteins.

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies.

Lesions of the rat entorhinal cortex cause extensive synaptic restructuring and perturbation of calcium regulation in the dentate gyrus of hippocampus. Calpain is a calcium-activated protease which has been implicated in degenerative phenomena in muscles and in peripheral nerves. In addition, calpain degrades several major structural neuronal proteins and has been proposed to play a critical role in the morphological changes observed following deafferentation. In this report we present evidence that lesions of the entorhinal cortex produce a marked increase in the breakdown of brain spectrin, a substrate for calpain, in the dentate gyrus. Two lines of evidence indicate that this effect is due to calpain activation: (i) the spectrin breakdown products observed following the lesion are indistinguishable from calpain-generated spectrin fragments in vitro; and (ii) their appearance can be reduced by prior intraventricular in fusion of leupeptin, a calpain inhibitor. Levels of spectrin breakdown products are increased as early as 4 h post-lesion, reach maximal values at 2 days, and remain above normal to some degree for at least 27 days. In addition, a small but significant increase in spectrin proteolysis is also observed in the hippocampus contralateral to the lesioned side in the first week postlesion. At 2 days postlesion the total spectrin immunoreactivity (native polypeptide plus breakdown products) increases by 40%, suggesting that denervation of the dentate gyrus produces not only an increased rate of spectrin degradation but also an increased rate of spectrin synthesis. These results indicate that calpain activation and spectrin degradation are early biochemical events following deafferentation and might well participate in the remodelling of postsynaptic structures. Finally, the magnitude of the observed effects as well as the stable nature of the breakdown products provide a sensitive assay for neuronal pathology.

Increased spectrin proteolysis in the brindled mouse brain.

Proteolytically generated fragments of the microfilament anchoring protein brain spectrin were found to accumulate in brindled mouse brain. Proteolysis was most extensive in brain regions possessing high concentrations of N-methyl-D-aspartate (NMDA) receptors (e.g. cortex, striatum, hippocampus). The brindle mutation affects copper homeostasis and thus a variety of copper-dependent enzymes needed in intermediary metabolism. The altered mitochondria of these mice are suggested to less efficiently buffer NMDA receptor-gated calcium fluxes, thus promoting activation of calcium-activated proteases and subsequent degradation of the spectrin meshwork.

Increased spectrin proteolysis in fibroblasts from aged and Alzheimer donors.

Since calcium homeostasis is altered in cultured skin fibroblasts from aged and Alzheimer donors, the present study examined the degradation of spectrin, a substrate of the calcium dependent protease calpain. Spectrin proteolysis was estimated as the percentage of spectrin breakdown products (e.g., 150 + 155 kDa bands) per total spectrin immunoreactivity. In the baseline condition (e.g., unstimulated fibroblasts), spectrin breakdown was 53% greater in cells from aged donors when compared to cells from either young or Alzheimer donors. Compared to unstimulated cells, serum increased spectrin breakdown in cells from aged (22.4%) or Alzheimer (92.1%) donors but was ineffective in cells from young donors. Thus, when compared to young donors (100%), serum stimulation increased spectrin proteolysis by 183.9% (aged) or 231.7% (Alzheimer) after serum stimulation. Treatment of unstimulated cells with carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), an uncoupler of mitochondrial function, increased spectrin degradation by 360.6% (young), 242.4% (aged) or 239.7% (Alzheimer) when compared to unstimulated cells of the same group. The combination of FCCP and serum stimulation enhanced spectrin breakdown in cells from aged (123.6%) and Alzheimer (154.0%) donors when compared to young cells (100%). Thus, changes in the regulation of calcium dependent proteases may contribute to decreased cell spreading and may play a role in the altered cytoskeletal dynamics characteristic of Alzheimer's disease.