Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein.

Recent data suggest that intraneuronal accumulation of metabolites of the amyloid-ß-precursor protein (APP) is neurotoxic. We observed that transgenic mice overexpressing in neurons a human APP gene harboring the APPE693Q (Dutch) mutation have intraneuronal lysosomal accumulation of APP carboxylterminal fragments (APP-CTFs) and oligomeric amyloid ß (oAß) but no histological evidence of amyloid deposition. Morphometric quantification using the lysosomal marker protein 2 (LAMP-2) immunolabeling showed higher neuronal lysosomal counts in brain of 12-months-old APPE693Q as compared with age-matched non-transgenic littermates, and western blots showed increased lysosomal proteins including LAMP-2, cathepsin D and LC3. At 24 months of age, these mice also exhibited an accumulation of α-synuclein in the brain, along with increased conversion of LC3-I to LC3-II, an autophagosomal/autolysosomal marker. In addition to lysosomal changes at 12 months of age, these mice developed cholinergic neuronal loss in the basal forebrain, GABAergic neuronal loss in the cortex, hippocampus and basal forebrain and gliosis and microgliosis in the hippocampus. These findings suggest a role for the intraneuronal accumulation of oAß and APP-CTFs and resultant lysosomal pathology at early stages of Alzheimer's disease-related pathology.

Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Protein topology of presenilin 1.

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.

Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo.

The majority of early-onset cases of familial Alzheimer's disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.

Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. We now document that the Abeta1-42(43)/Abeta1-40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Abeta1-42(43)/Abeta1-40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Abeta peptides terminating at 42(43), species that foster Abeta deposition.

Protective role of superoxide dismutase against diabetogenic drugs.

Copper-zinc superoxide dismutase (SOD) is present in relatively high concentrations in the beta-cells of human islets. The activity of the extracted enzyme is partially inhibited upon incubation with the diabetogenic drugs alloxan, streptozotocin, or Vacor. The role of this enzyme in protecting beta-cells against chemically induced diabetes was further investigated. Incubation of intact canine islets with alloxan (0.2 mg/ml) and 4 mM glucose decreased the insulin secretory response by 87% during subsequent exposure to 28 mM glucose. Concomitantly the SOD-specific activity (units of enzyme activity per milligram immunoreactive SOD) decreased 50% in alloxan-exposed islets. When islets were protected from alloxan toxicity by including 28 mM glucose with alloxan, the insulin secretory response and SOD specific activity remained identical to controls. Thus, SOD specific activity correlates with maintenance of beta-cell function. To test the effectiveness of SOD against streptozotocin in vitro, canine islets were incubated 10 min with or without streptozotocin (0.1 mg/ml) with 4 mM glucose; their functional integrity was tested subsequently as the insulin secretory response to 28 mM glucose. Exposure to streptozotocin alone decreased the response by 70%; inclusion of SOD (1.5 mg/ml) before and during exposure to streptozotocin completely prevented this effect. Cyanide-inactivated SOD was not effective. The potential of SOD to prevent streptozotocin-induced diabetes was tested in rats in vivo. SOD injected 10 s or 50 min before streptozotocin prevented or significantly attenuated diabetes. Injection of SOD and streptozotocin simultaneously was much less effective, and cyanide-inactivated SOD was ineffective. No protection was afforded by injection of SOD 12 or 24 h before streptozotocin. Our results support hypotheses that (a) oxygen radicals mediate the beta-cell toxicity of both alloxan and streptozotocin, and (b) beta-cells may be particularly vulnerable to oxygen radical damage.

Alzheimer's disease amyloid-beta peptide modulates apolipoprotein E isoform specific receptor binding.

The major protein component of the extracellular deposits in Alzheimer's disease (AD) is a 4 kDa peptide termed amyloid-beta (Abeta). This peptide is known to bind apolipoprotein E (apoE), a key mediator of lipoprotein transport, in an isoform specific manner. Whilst these isoform specific effects on apoE are well recognized, the functional significance of this interaction is poorly understood. Here, we investigated the influence of Abeta on apoE-mediated lipoprotein binding to cells using fluorescently tagged lipoprotein-like emulsions. Using this approach, we demonstrate that Abeta enhanced the normally poor binding of apoE2 lipoprotein-like particles to fibroblasts in culture, whilst markedly reducing the binding of apoE3 and apoE4. This suggests that the action of apoE isoforms on cellular lipoprotein or cholesterol metabolism is differentially modulated by Abeta. This also suggests that Abeta may also compromise apoE function in the Alzheimer disease affected brain.

Neurological soft signs are associated with APOE genotype, age and cognitive performance.

Neurodegeneration is associated with increased frequency of neurological soft signs (NSS). We designed the present study to investigate the association between NSS and subjective memory complaints, cognitive function and apolipoprotein E genotype in a community-dwelling sample of volunteers participating in an ongoing longitudinal program investigating predictors of cognitive decline. NSS were found to be associated with apolipoprotein E (APOE) epsilon4 genotype (p = 0.015), age (p = 0.012) and poor cognitive performance, as assessed by the Mini Mental State Examination (p = 0.053). There was no significant difference between subjects with and without memory complaints in relation to the frequency of NSS (p = 0.130). The association with age and the APOE epsilon4 genotype suggests that the systematic investigation of NSS may contribute to identify subjects at risk of clinically significant cognitive decline in later life.

The nature and metabolism of potentially amyloidogenic carboxyl-terminal fragments of the Alzheimer beta/A4-amyloid precursor protein: some technical notes.

The proteolytic processing and secretion of APP are regulated by protein phosphorylation, especially via protein kinase C and protein phosphatases 1 and/or 2A. Our studies of these regulatory mechanisms have led us to perform extensive experimentation on the metabolism of APP carboxyl-terminal fragments, using as our system either untransfected, undifferentiated rat pheochromocytoma (PC12) cells or APP-baculovirus infected Sf9 cells. We have not assayed APP fragments for biological activity in either system. However, we have made potentially relevant observations regarding APP carboxyl-terminal fragment trafficking. In this note, we review our published and unpublished data in relation to published reports from other laboratories using related systems.

Presenilin 1 is actively degraded by the 26S proteasome.

The metabolic pathways governing the turnover of presenilin 1 (PS1) have been incompletely worked out. The PS1 holoprotein has low abundance in many cells and appears to undergo endoproteolytic cleavage near residue 298. We provide evidence that one mechanism by which the PS1 holoprotein is degraded is through the action of the 26S proteasome. We also show that the proteasome does not participate in the endoproteolytic cleavage.

Immunocytochemical localization of amyloid precursor protein in rat brain.

The localization of amyloid precursor protein (APP) in rat brain was studied with a cytoplasmic domain-specific antibody. Light microscopic immunocytochemistry demonstrated that APP is present in most neurons, in some oligodendrocytes, and in a population of cells with diameters less than 10 microns that may be glial. Marked differences in immunoreactivity among neurons were observed, and the strongest immunoreactivity was contained in larger neurons. Neurons with scant cytoplasm, such as granule cells in the olfactory bulb, dentate gyrus, and cerebellum, were weakly immunoreactive. Differences in neuropil immunoreactivity were also observed; this type of staining was strongest in the caudatoputamen, lateral septum, medial habenula, nucleus reticularis of the dorsal thalamus, and the lateral portion of the ventroposterior nucleus. Neuropil immunostaining was weakest in layer IV of cortex and in areas containing granule cells. The fact that APP seems to be present in the vast majority of neurons suggests that this protein plays a role common to all neurons. The fact that there is a great difference in the steady-state amount of APP among different types of neurons suggests that APP may play a specific role in the function of certain classes of neurons.

"Early" initiation of levodopa treatment does not promote the development of motor response fluctuations, dyskinesias, or dementia in Parkinson's disease.

We reviewed the histories of patients seen in a large Parkinson's disease clinic from 1983 to 1989 to determine if there is a relationship between the timing of initiation of levodopa therapy and the development of motor response fluctuations, dyskinesias, and dementia. There were no factors predisposing to the development of response fluctuations or dementia. Younger age at disease onset predisposed to the development of dyskinesia. Dyskinesias occurred in a greater proportion of patients in whom the initiation of levodopa therapy was delayed by more than 2 years from disease diagnosis than among those in whom treatment was started earlier. We thus failed to identify any adverse consequences of early levodopa treatment in our patient population.

MRI demonstrates descending transtentorial herniation.

Descending cerebral transtentorial herniation (DTH) is a serious and often fatal complication of intracranial mass lesions. The condition can be inferred from clinical neurologic signs, but has not been visualized during life. Using midsagittal magnetic resonance images (MRIs), we compared vertical brainstem position on 50 images from normals and 21 images from 15 clinically stable patients with large supratentorial tumors. The length of Twining's line (T), the perpendicular distance from T to the pontomesencephalic junction (T-PMJ), and from T to the apex of the midbrain aqueduct (T-A) were measured. We also measured lateral shifts of the diencephalon and midbrain on axial images. T-PMJ decreased from 2.04 +/- 0.06 mm in normals to 0.94 +/- 0.2 mm in patients with large cerebral tumors (p less than 0.0001). Similarly, T-A decreased from 6.35 +/- 0.13 mm in normals to 4.83 +/- 0.35 mm in patients (p = 0.001). Lateral diencephalic-midbrain shifts often accompanied DTH but to an unpredictable degree. Either lateral or downward brainstem shift could occur alone and did not necessarily produce specific neurologic signs or an altered state of consciousness. Anatomic DTH occurs in life, it can be quantified with MRI, and in slowly developing cerebral mass lesions the process can precede the appearance of neurologic signs and symptoms that indicate lower-diencephalic or midbrain dysfunction.

Phosphorylation of Alzheimer amyloid precursor protein by protein kinase C.

The beta/A4 amyloid precursor protein is a membrane protein with one transmembrane domain. The accumulation and deposition of beta/A4 amyloid protein in Alzheimer's disease is thought to be brought about by altered processing of beta/A4 amyloid precursor protein. Activation of protein kinase C and/or inhibition of protein phosphatases 1 and 2A results in an increase in the proteolytic processing and secretion of beta/A4 amyloid precursor protein. These effects might result either from phosphorylation of beta/A4 amyloid precursor protein by protein kinase C or from phosphorylation of components of the beta/A4 amyloid precursor protein processing apparatus. We have previously reported phosphorylation by protein kinase C of a synthetic peptide corresponding to part of the cytoplasmic domain of beta/A4 amyloid precursor protein. However, it was not known whether beta/A4 amyloid precursor protein holoprotein was phosphorylated in its native conformation in the cell membrane. Using a PC12 (rat pheochromocytoma) semi-intact cell system, we now report that mature isoforms of beta/A4 amyloid precursor protein are phosphorylated by protein kinase C at Ser655. Five COOH-terminal fragments which are generated by processing of mature beta/A4 amyloid precursor protein were also phosphorylated by protein kinase C at Ser655. The results support the idea that the beta/A4 amyloid precursor protein haloprotein is a physiological substrate for protein kinase C. These observations should facilitate our understanding of the relationship between altered protein phosphorylation and beta/A4 amyloid production.

Apolipoprotein E promotes the binding and uptake of beta-amyloid into Chinese hamster ovary cells in an isoform-specific manner.

The epsilon4 allele of apolipoprotein E gene is a major risk factor for Alzheimer's disease. However, the mechanism by which the E4 isoform of apolipoprotein E increases the risk of Alzheimer's disease is poorly understood. To determine whether the isoform-specific effects of apolipoprotein E may be mediated via clearance of bound beta-amyloid, we examined the uptake of beta-amyloid 1-40 into Chinese hamster ovary cells in the presence or absence of the apolipoprotein E isoforms E2, E3 and E4. Apolipoprotein E2 and E3 treatments were associated with higher association of beta-amyloid with cells as compared to treatment with E4. Heparin blocked the association of beta-amyloid with cells, as did an antibody to one of the apolipoprotein E receptors (the low-density lipoprotein receptor-related protein). Thus, the apolipoproteins E2 and E3, but not E4, may play important roles in the clearance of beta-amyloid from the extracellular space via the low-density lipoprotein receptor-related protein.

The effect of insulin and glucose on the plasma concentration of Alzheimer's amyloid precursor protein.

The deposition of beta amyloid is a critical event in the pathogenesis of Alzheimer's disease. This peptide is a metabolite of the amyloid precursor protein. Recent research suggests that there is a correlation between plasma insulin and glucose concentrations and memory performance in Alzheimer's disease sufferers. Additionally, in vitro evidence suggests that both insulin and glucose may affect the metabolism of amyloid precursor protein and therefore the production of beta amyloid--however, to our knowledge no in vivo data have yet been published. We investigated the effect of elevated plasma levels of glucose and insulin on the plasma concentration of amyloid precursor protein in non-Alzheimer's disease subjects. As would be expected following ingestion of a glucose drink, blood insulin and glucose levels significantly increased. Interestingly, however, plasma amyloid precursor protein concentration decreased. Whilst no correlation was observed between insulin or glucose levels and plasma amyloid precursor protein concentration, the decrease in plasma amyloid precursor protein concentration was affected by the apolipoprotein E genotype of the subject. Possession of an epsilon4 allele resulted in a reduced decrease in plasma amyloid precursor protein in response to glucose ingestion when compared to non-epsilon4 subjects. We conclude that glucose ingestion, and the subsequent elevation of plasma levels of glucose and insulin leads to a decrease in plasma amyloid precursor protein concentration. Further studies are required to determine the clinical significance of these physiological changes in plasma amyloid precursor protein and the implications for Alzheimer's disease pathogenesis.

Beta-amyloid protein protein precursor expression in lacrimal glands and tear fluid.

PURPOSE: Proteases in tear fluid play important roles in the regulation of corneal wound healing. Inhibitors of proteolytic activity are major modulators of the associated events. Although it is known that various enzyme inhibitors exist in tear fluid, it is not known whether certain isoforms of the beta-amyloid protein precursor (beta-APP), a potent inhibitor of serine proteases, are present in tear fluid. The purpose of this study was to investigate whether beta-APP can be detected in human tear fluid and, if so, to determine the isoform composition and cellular origin. METHODS: Tear fluid was collected from healthy volunteers. The beta-APP was identified and characterized by immunoblotting using antibodies specific for domains of the beta-APP. The protein was characterized further by ion exchange chromatography. Expression of the beta-APP gene was studied using in situ hybridization and RNA-RNA solution hybridization assay. RESULTS: beta-APP with protease inhibitory properties was identified in all samples of human tear fluid. Immunologic analysis revealed that it had been processed proteolytically before secretion. Gene expression studies showed that the beta-APP gene was expressed in lacrimal glands, particularly in acinar cells. The gene transcript almost exclusively corresponded to beta-APP containing the protease inhibitor insert. CONCLUSIONS: beta-APP is expressed in lacrimal glands and subsequently is secreted into tear fluid. Because the bulk of the beta-APP contained the protease inhibitor insert, the authors propose that beta-APP is an important regulator of proteolysis in tear fluid and that possibly it plays a role in the events associated with corneal wound healing. This suggests a novel physiological function of beta-APP in addition to those previously described-regulation of blood coagulation and cell growth.

Oestrogen replacement therapy may improve memory functioning in the absence of APOE epsilon4.

There is currently intense controversy regarding the use of hormone replacement therapy (HRT) in postmenopausal women, in relation to its therapeutic efficacy in Alzheimer's disease (AD). It has been suggested that the benefits of HRT may be modified by apolipoprotein E (APOE) genotype (the major genetic risk factor for AD). Here we report the findings of the first study designed to systematically explore the interaction of (a) oestrogen replacement therapy (ERT) and (b) possession of an epsilon4 allele of APOE on specific elements of episodic learning and memory that are commonly used indices of age-related cognitive decline. This data represents a cross-sectional analysis of the interaction of ERT and APOE genotype on learning and memory in a cohort of 181 healthy postmenopausal women [ERT users (n = 101, mean age 65.40 +/- 6.34); ERT non-users (n = 80, mean age 67.03 +/- 6.80)] residing in Perth, Western Australia. The highest level of learning (trials 2-5; P < 0.05) and memory (e.g. total number of items recalled; P < 0.05) performance was observed in women taking ERT who were not carriers of the APOE epsilon4 allele. APOEepsilon4 carriers receiving ERT performed no better on episodic memory testing than APOE epsilon4 carriers who were not receiving ERT. These cognitive differences related to genetic profile, were noted on both recall and recognition (P = 0.005) tests of memory. The findings have significance for evaluating whether and when ERT may be clinically indicated. Specifically, ERT may benefit the cognitive functioning of women not carrying the APOE epsilon4 allele.

Protein phosphorylation regulates relative utilization of processing pathways for Alzheimer beta/A4 amyloid precursor protein.

The Alzheimer amyloid precursor protein (APP) is a phosphoprotein, and the phosphorylation state of APP at Ser655 can be regulated by protein kinase C, calcium/calmodulin-dependent protein kinase II, and okadaic acid-sensitive protein phosphatases. Other enzymes may also play a role at Ser655 of APP and, perhaps, at other residues. Signal transduction via protein phosphorylation regulates APP metabolism. In particular, APP processing via the nonamyloidogenic secretory cleavage pathway is increased following the activation of protein kinase C or the inactivation of okadaic acid-sensitive protein phosphatases. The mechanism(s) by which protein phosphorylation regulates APP secretory cleavage include (among others): substrate activation, substrate redistribution, protease activation and/or protease redistribution. Current experimental evidence will be discussed, addressing the relative importance of each of these possibilities and the implications for these events in the modulation of beta/A4-amyloidogenesis.

Thiamine deficiency alters APP but not presenilin-1 immunoreactivity in vulnerable brain regions.

Presenilin-1 (PS-1) and amyloid precursor protein (APP) have been linked to the pathogenesis of Alzheimer's disease. While APP accumulation is well documented in several models of brain injury, the role of PS-1 levels in neurodegeneration, if any, remains to be elucidated. The current studies examined PS-1 and APP expression in brain following thiamine deficiency (TD), a nutritional model associated with impaired oxidation and selective neurodegeneration. TD did not alter PS-1 immunoreactivity in any region of rodent brain before or after cell loss. In contrast, APP immunoreactivity accumulated in swollen neurites within, or around lesions in rats, or in abnormal clusters in mice. Thus, alterations in APP but not PS-1 levels are involved in TD-induced neurodegeneration.