Amyloid precursor protein (APP) processing genes and cerebrospinal fluid APP cleavage product levels in Alzheimer's disease.

The aim of this exploratory investigation was to determine if genetic variation within amyloid precursor protein (APP) or its processing enzymes correlates with APP cleavage product levels: APPα, APPß or Aß42, in cerebrospinal fluid (CSF) of cognitively normal subjects or Alzheimer's disease (AD) patients. Cognitively normal control subjects (n = 170) and AD patients (n = 92) were genotyped for 19 putative regulatory tagging SNPs within 9 genes (APP, ADAM10, BACE1, BACE2, PSEN1, PSEN2, PEN2, NCSTN and APH1B) involved in the APP processing pathway. SNP genotypes were tested for their association with CSF APPα, APPß, and Aß42, AD risk and age-at-onset while taking into account age, gender, race and APOE ε4. After adjusting for multiple comparisons, a significant association was found between ADAM10 SNP rs514049 and APPα levels. In controls, the rs514049 CC genotype had higher APPα levels than the CA, AA collapsed genotype, whereas the opposite effect was seen in AD patients. These results suggest that genetic variation within ADAM10, an APP processing gene, influences CSF APPα levels in an AD specific manner.

Abeta vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease.

The authors report a patient with Alzheimer disease (AD) without encephalitis who was immunized with AN-1792 (an adjuvanted formulation of Abeta-42). There were no amyloid plaques in the frontal cortex and abundant Abeta-immunoreactive macrophages, but tangles and amyloid angiopathy were present. The white matter appeared normal and minimal lymphocytic infiltration in the leptomeninges was observed. This case illustrates the effects of an Abeta-based immunization on AD pathogenesis in the absence of overt meningoencephalitis and leukoencephalopathy.

Encapsulation in biodegradable microparticles enhances serum antibody response to parenterally-delivered beta-amyloid in mice.

Poly(lactide-co-glycolide) (PLG) microspheres were tested as a parenteral delivery system for human beta-amyloid (1-42) (Abeta), a potential immunotherapeutic undergoing assessment in Phase 1 studies for Alzheimer's disease (AD). Abeta was successfully encapsulated in PLG microspheres of average sizes of 3 or 15 microm diameter. Swiss Webster (SW) mice were injected by the sub-cutaneous (s.c.) or intra-peritoneal (i.p.) routes with 3-33 microg Abeta. Abeta-PLG microparticles (3 microm) induced dose-dependent antibody responses, which were maximal at 33 microg Abeta, while Abeta in phosphate-buffered saline (PBS) produced weak antibody responses at the same doses by both routes. Significantly increased antibody responses were seen for both small and large particle formulations given by the i.p. route in comparison to the s.c route. It was previously reported that passive immunisation with Abeta-specific antibodies cleared amyloid plaques in a mouse model of AD (Bard F, Cannon C, Barbour R, et al. Peripherally administered antibodies against amyloid beta-peptide enter the nervous system and reduce pathology in a mouse model of Alzheimer disease. Nature Med 2000;6:916-19), an indication that induction of serum antibody is a prerequisite for efficacy.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

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.

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.

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.

Effects of time and cholinesterase inhibitor treatment on multiple cerebrospinal fluid parameters in Alzheimer's disease.

Development of neuropathology in Alzheimer's disease (AD) cannot be studied directly in living patients. Therefore, concentrations in cerebrospinal fluid (CSF) of the proteins tau, A beta 42, alpha 1-ACT, apoE and other molecules have been analyzed to elucidate their possible role in degeneration and as biomarkers of the disease. To date, however, studies have not analyzed multiple markers in the same patients over time and as a function of pharmacological interventions. In the present investigation we measured CSF tau, A beta 42, alpha 1-ACT, apoE, total protein and electrophoretic fractions, and leukocytes, as well as MMSE, in 12 AD patients of known APOE phenotype. Two or three CSF examinations were performed during periods of up to 2 1/2 years, while subjects were on and off treatment with the cholinesterase inhibitor (ChEI) metrifonate (MTF). CSF A beta 42 and tau levels were in agreement with clinical diagnosis of AD in all patients. Abnormally high proportions of monocytes were found in CSF at baseline, and these proportions correlated positively with plasma alpha 1-ACT and MMSE scores. A small but significant increase in CSF alpha 1-ACT, which correlated with peripheral alpha 1-ACT, was associated with 6 months' MTF treatment, though alpha 1-ACT levels did not change further when treatment continued for 2 years. Monocyte proportions in CSF declined over time in both treated and untreated patients. Among 5 of 6 patients treated for 2 years or more with MTF, CSF measures remained relatively stable. One patient had changes in CSF parameters apparently associated with a transient ischemic attack. Our findings did not indicate that slowed cognitive decline with MTF treatment is associated with systematic change in any CSF marker analyzed. The results suggest that further investigations of the relationship of tau, A beta 42 and cellular abnormalities in CSF early in the course of AD are warranted.

Purification and cloning of amyloid precursor protein beta-secretase from human brain.

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

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.

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.

Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.

Amyloid-beta peptide (Abeta) seems to have a central role in the neuropathology of Alzheimer's disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Abeta42), which is the predominant form found in the amyloid plaques of Alzheimer's disease. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner. In the present study, transgenic animals were immunized with Abeta42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-beta deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of beta-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-beta may be effective in preventing and treating Alzheimer's disease.

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi.

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.

Apolipoprotein E epsilon 4 allele in association with global cognitive performance and CSF markers in Alzheimer's disease.

To better define the influence of apolipoprotein E (ApoE) epsilon 4 genotype on the cognitive and biochemical features of Alzheimer's disease (AD), cross-sectional analysis of global cognitive measures and cerebrospinal fluid studies gathered on AD subjects at a tertiary care facility between 1986 and 1997 was carried out. The 112 AD patients examined included 62 women and 50 men with a mean (SD) age of 64.2 (9.2) years. Patient demographics; illness onset age and duration, education level and global cognitive measures were recorded systematically. Genetic analysis for ApoE allele type and biochemical characterization of CSF, including total tau concentration, was performed. Descriptive statistics of demographics, cognitive and CSF measures were performed by chi-square, ANOVA and Tukey's tests. Overrepresentation of the epsilon 4 allele was found, with 45.5% of AD patients heterozygous and 20.5% homozygous for ApoE epsilon 4. Overall, ApoE epsilon 4 status had no effect on mean onset age of AD (F = 1.56; p = 0.214), but an earlier mean onset age of AD (F = 4.10; p = 0.02) was seen in the late-onset subjects. No differences were found with regard to ApoE epsilon 4 status and measures of disease, duration of illness or global cognitive performance. Although CSF tau was elevated in our sample (575.4 +/- 290.3 pg/ml), ApoE epsilon 4 status did not influence total CSF tau or neurotransmitter metabolite levels. ApoE epsilon 4 genotype had no impact on a variety of illness severity, cognitive and CSF examinations in the largest cross-sectional analysis of AD subjects yet reported.

Cerebrospinal fluid tau protein is not elevated in HIV-associated neurologic disease in humans. HIV Neurobehavioral Research Center Group (HNRC).

We measured the concentrations of the neuron-specific protein, tau, in the cerebrospinal fluid (CSF) of 32 neurologically characterized HIV-infected (HIVpos) subjects and nine matched seronegative (HIVneg) controls using a sensitive ELISA assay. Of 32 HIVpos subjects, nine had HIV-associated neurocognitive disorders, and nine had clinically diagnosed peripheral neuropathies. CSF tau levels in subjects with HIV-associated neurocognitive disorders were similar to those in HIVneg subjects (185 +/- 83 vs. 223 +/- 106 pg/ml; P = 57). CSF tau levels in HIVpos subjects with peripheral neuropathies did not differ from those without neuropathies (320 +/- 190 vs. 251 +/- 185; P = 23). In summary, CSF tau levels were not elevated in patients with HIV-associated neurologic disease.

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.

A novel presenilin-1 mutation: increased beta-amyloid and neurofibrillary changes.

The prevalence of known mutations in presenilin genes (PS1 and PS2) causing early-onset familial Alzheimer's disease (FAD) was assessed in a population of 98 singleton early-onset AD cases, 29 early-onset FAD cases, and 15 late-onset FAD cases. None of the cases tested positive for the eight mutations initially reported, and none of these mutations were observed in 60 age-matched controls. A novel mutation (R269H) in PS1 was found in a single case of early-onset AD but not in any other AD or control case. Thus, the PS mutations tested are quite rare in early-onset AD. Amyloid beta protein (A beta) deposition was investigated in the temporal cortex of the R269H mutation case using end-specific monoclonal antibodies to detect the presence of A beta x-40 and A beta x-42 subspecies. Stereologically unbiased tangle and neuropil thread counts were obtained from the same region. R269H PS1 mutation was associated with early age of dementia onset, higher amounts of total A beta and A beta x-42, and increased neuronal cytoskeletal changes. Thus, if the changes observed on this case prove to be typical of PS1 mutations, PS1 mutations may impact both amyloid deposition and neurofibrillary pathology.