Upregulation of neuronal nicotinic receptor subunits alpha4, beta2, and alpha7 in transgenic mice overexpressing human acetylcholinesterase

Neuronal nicotinic receptor binding sites as well as mRNA levels encoding for subunits α4, β2, and α7 were analysed in 3-mo-old transgenic mice generated with a neuronal overexpression of human acetylcholinesterase and in age-matched controls. The acetylcholinesterase transgenic mice display progressive cognitive impairment in spatial learning and memory. We here report a significantly increased [³H]epibatidine and [¹²⁵I]αbungarotoxin binding in the cortex and the caudate putamen of these mice. Quantitative in situ hybridization showed significant upregulation of mRNA corresponding to the nicotinic receptor subunits α4, β2, and α7 in various brain regions in the transgenic mice compared to nontransgenic controls. Our results suggest that disruption of balanced cholinergic transmission by constitutive overexpression of acetylcholinesterase is accompanied by variable upregulation of several nicotinic receptor subtypes, in particular these associated with cholinergic terminals participating in compensatory response.     

Unexpected sensitivity of the human alpha7 neuronal nicotinic acetylcholine receptor to aminoglycosides

The wide use of antibiotics and the development of resistance is a major health concern and, despite their relatively severe side effects, aminoglycoside antibiotics are still used in clinics. Effects of seven aminoglycosides were investigated at the human homomeric alpha7 and heteromeric alpha4beta2 neuronal nicotinic acetylcholine receptors. All aminoglycosides tested inhibited the acetylcholine-evoked responses with more pronounced effects at alpha7 than at alpha4beta2. Neomycin displayed higher blockade with a half inhibition in the nanomolar range at low calcium concentration and in the micromolar range in physiological calcium concentration but still exerted blockade below the concentration used in the clinic. These data suggest that some of their side effects may be attributable to their interactions with neuronal nicotinic acetylcholine receptors.     

Increased alpha 7 nicotinic acetylcholine receptor protein levels in Alzheimer's disease patients

We compared the intact alpha7 nicotinic acetylcholine receptor (alpha7nAChR) protein levels in the peripheral blood leukocytes in 15 Alzheimer's disease (AD) patients and 13 normal elderly control subjects. Demographic data and Mini-Mental State Examination (MMSE) scores were obtained. Western blot analysis for alpha7nAChR protein levels in peripheral blood leukocytes was performed. There were no significant differences in sex and age between the AD and control groups. The mean MMSE score of the AD subjects was significantly lower than that of the normal control subjects (15.4 +/- 5.5 vs. 28.5 +/- 1.9 respectively; p < 0.001). The median value of normalized alpha7nAChR protein levels (optical density, arbitrary unit) of the AD group was significantly higher than that of the normal control group (0.6923 vs. 0.4803 respectively; p = 0.045, Mann-Whitney U test). The normalized alpha7nAChR protein levels showed a significant inverse correlation with the MMSE scores (Spearman rho = -0.45; p = 0.016; n = 28). Receiver Operating Characteristic curve analyses showed that the area under curve was 0.72 (95% CI 0.52- 0.87). If the cut-off of the alpha7nAChR protein level was >0.312, the sensitivity, specificity, positive predictive value and negative predictive value would be 80, 39, 60 and 63%, respectively. These findings showed that the alpha7nAChR protein levels would be a potentially useful diagnostic marker for AD.     

BDNF up-regulates alpha7 nicotinic acetylcholine receptor levels on subpopulations of hippocampal interneurons

In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing alpha7 subunits (alpha7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of alpha7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABA(A) receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased alpha7-nAChR clusters were most prominent on interneuron subtypes known to directly innervate excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling alpha7-nAChR levels.     

Nogo receptor interacts with brain APP and Abeta to reduce pathologic changes in Alzheimer's transgenic mice

Pathophysiologic hypotheses for Alzheimer's disease (AD) are centered on the role of the amyloid plaque Abeta peptide and the mechanism of its derivation from the amyloid precursor protein (APP). As part of the disease process, an aberrant axonal sprouting response is known to occur near Abeta deposits. A Nogo to Nogo-66 receptor (NgR) pathway contributes to determining the ability of adult CNS axons to extend after traumatic injuries. Here, we consider the potential role of NgR mechanisms in AD. Both Nogo and NgR are mislocalized in AD brain samples. APP physically associates with the NgR. Overexpression of NgR decreases Abeta production in neuroblastoma culture, and targeted disruption of NgR expression increases transgenic mouse brain Abeta levels, plaque deposition, and dystrophic neurites. Infusion of a soluble NgR fragment reduces Abeta levels, amyloid plaque deposits, and dystrophic neurites in a mouse transgenic AD model. Changes in NgR level produce parallel changes in secreted APP and AB, implicating NgR as a blocker of secretase processing of APP. The NgR provides a novel site for modifying the course of AD and highlights the role of axonal dysfunction in the disease.     

The pathophysiology of traumatic brain injury in alpha7 nicotinic cholinergic receptor knockout mice

The alpha7 nicotinic cholinergic receptor is a ligand-gated ion channel with calcium permeability similar to that of ionotrophic glutamate receptors. Previous studies from our laboratory have implicated changes in expression alpha7 nicotinic cholinergic receptors in the pathophysiology of traumatic brain injury (TBI). In rats, TBI causes a time-dependent and significant decrease in cortical and hippocampal alpha-[(125)I]-bungarotoxin (BTX) binding. We have postulated that deficits in alpha7 expression may contribute to TBI-induced cognitive impairment and that nicotinic receptor agonists can reverse alpha7 binding deficits and result in significant cognitive improvement compared to saline-treated controls. Thus, alpha7 nAChRs could be involved in a form of cholinergically mediated excitotoxicity following brain injury. In the current study, wild-type, heterozygous and null mutant mice were employed to test the hypothesis that genotypic depletion of the alpha7 receptor would render animals less sensitive to tissue loss and brain inflammation following experimental brain injury. Mice were anesthetized and subjected to a 0.5-mm cortical contusion injury of the somatosensory cortex. Brain inflammation, changes in nicotinic receptor expression and cortical tissue sparing were evaluated in wild-type, heterozygous and homozygous mice 1 week following TBI. In wild-type mice, brain injury caused a significant decrease in BTX binding in several hippocampal regions, consistent with what we have measured in rat brain following TBI. However, there were no genotypic differences in cortical tissue sparing or brain inflammation in this experiment. Although the results of this study were largely negative, it is still plausible that changes in the activity/expression of native alpha7 receptors contribute to pathophysiology following TBI. However, when null mutant mice develop in the absence of central alpha7 expression, it is possible that compensatory changes occur that confound the results obtained.     

Effect of oestrogen receptor alpha and beta agonists on brain N-methyl-D-aspartate receptors

Previous studies have shown the oestradiol modulation of brain N-methyl-D-aspartate (NMDA) receptors composed of the NR1/2B subunits. The contribution of oestrogen receptor subtypes in this oestradiol modulation of NMDA receptors and its subunits is not known. The following experiments investigated whether an oestrogenic receptor subtype is involved in the oestradiol effect on NMDA receptor specific binding and subunit mRNA levels. Ovariectomised Sprague-Dawley rats were treated 2 days after ovariectomy for 2 weeks with 17beta-oestradiol, an agonist for oestrogen receptor (ER)alpha 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or an agonist for ER beta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared with control vehicle-treated ovariectomised and intact rats. Uterus weights, used as a peripheral measure of oestrogenic activity, decreased after ovariectomy and increased by oestradiol and PPT but not DPN treatment. In the hippocampal CA1 oriens and CA1 radiatum, [(3)H]Ro 25-6981 specific binding, a NMDA/NR2B ligand, was decreased in ovariectomised compared to intact rats and this was prevented by 17beta-oestradiol or PPT but not DPN treatments; a similar pattern was observed in the CA2/3 and dentate gyrus but did not reach statistical significance. In situ hybridisation of the mRNA of the NMDA/2B subunit in the hippocampus CA1, CA2/3 and dentate gyrus showed a decrease in ovariectomised rats compared to controls and this was also prevented by 17beta-oestradiol and PPT but not DPN treatments. In cingulate and prefrontal cortices, ovariectomy increased [(3)H]Ro 25-6981 specific binding compared to intact controls, which was corrected by 17beta-oestradiol treatment but neither by PPT, nor DPN. In the cortical regions, the lack of effect of the ER alpha or ER beta agonist whereas 17beta-oestradiol was active, suggesting that the oestradiol modulation of cortical NMDA receptors requires both ERs or that this modulation does not involve ERs. In the hippocampus, the results obtained suggest an oestrogenic genomic modulation of NMDA receptors containing the NR2B subunit, implicating an ER alpha.     

Decreased protein level of nicotinic receptor alpha7 subunit in the frontal cortex from schizophrenic brain.

The alpha7 subunit of nicotinic receptor (nAChR) was investigated in post-mortem brain tissue from eight schizophrenics and eight age-matched controls by Western blot. Using anti-alpha7 antibodies a single band with a mol. wt of 42 kDa was detected in human post-mortem brain, which was smaller in size than the single band (52 kDa) detected in SH-SY5Y and PC12 cells. The smaller band specifically bound to [125I]alpha-bungarotoxin, confirming the specificity of the detection in the human brain samples. A significant decrease in the level of alpha7 subunit protein was observed with the same method in the frontal cortex of schizophrenics compared with controls, while no difference was found in the parietal cortex. These findings suggest that a deficit of nAChR alpha7 subunit in the frontal cortex might be involved in the pathophysiology of schizophrenia.     

Functional contribution of alpha3L8' to the neuronal nicotinic alpha3 receptor

The role of position L8', located in transmembrane domain 1 of the neuronal nicotinic alpha3 subunit, was characterized by using two-electrode voltage clamp in Xenopus oocytes. Four amino acids (Ala, Ser, Phe, and Tyr) were inserted at this conserved position, and the mutant subunit was coexpressed with either wild-type beta2 or beta4 subunits. These substitutions led to significant alterations in the pharmacodynamic parameters of cholinergic agents, resulting in loss of function. Ala and Ser substitutions resulted in losses in agonist (ACh, nicotine, and DMPP) potency and intrinsic activity at both alpha3beta2 and alpha3beta4 receptors. Similarly, significant changes in antagonist potency were produced by the Ala and Ser substitutions. Phe and Tyr mutations did not alter the receptor's EC(50) for ACh or nicotine but reduced the EC(50) for DMPP at both receptors. The Phe mutation also reduced the intrinsic activity of all agonists tested at both receptors. The Tyr mutation, though, led to a decrease in intrinsic activity for all agonists at the alpha3beta2 receptor, yet resulted in no changes for DMPP, a decrease for nicotine, and an increase for ACh at the alpha3beta4 receptor. The most dramatic changes in the receptor's functional properties were produced by substitutions that introduced the largest changes in amino acid volume. Additional replacements (Gly, Thr, and Val) suggested an inverse correlation between amino acid volume at position alpha3L8' and EC(50) for alpha3beta4 nAChRs; however, alpha3beta2 nAChRs displayed a nonlinear correlation. These data demonstrate that structural alterations at position alpha3L8' could propagate to the agonist-binding site.     

Development of the alpha7 nicotinic cholinergic receptor in rat hippocampal formation

The alpha7 nicotinic receptor has been implicated in the regulation of a variety of developmental processes. The goal of the present study was to assess whether the alpha7 receptor might participate in the regulation of hippocampal ontogeny by describing the spatiotemporal development of alpha7 mRNA and alpha-bungarotoxin binding in rat hippocampal formation. Message for the alpha7 receptor was initially observed in the hippocampal neuroepithelium at embryonic day 13 and in the anlage of the hippocampal formation on embryonic day 14. Binding of alpha-bungarotoxin was initially seen on embryonic day 15 in the dorsal portion of the anlage of stratum oriens and stratum radiatum-lacunosum moleculare, but was never observed in the neuroepithelium. Dramatic elevations in both alpha7 mRNA and alpha-bungarotoxin binding were observed in most regions of the hippocampal formation neonatally. The levels of both alpha7 message and protein gradually decreased during the first three postnatal weeks to adult levels in most regions. The lack of alpha-bungarotoxin binding in the neuroepithelium suggests that the alpha7 receptor does not influence neurogenesis. The early appearance and complex, prolonged pattern of development of the alpha7 receptor suggest that it may influence processes as diverse as cell migration, dendritic elaboration and apoptosis during hippocampal maturation.     

Abeta levels in serum,CSF and brain,and cognitive deficits in APP + PS1 transgenic mice

We compared beta-amyloid peptide (Abeta) levels in the serum, CSF and brain (hippocampus) and correlated these with spatial learning in APP+PS1 transgenic mice. Compared with non-transgenic littermates, male 14-month-old APP + PS1 mice were impaired in spatial learning in the water maze. Among the APP + PS1 mice, only the hippocampal insoluble Abeta42 level correlated with spatial memory (r = -0.44). The levels of insoluble Abeta40 and Abeta42 were highly correlated (r = 0.92), and also correlated with soluble hippocampal Abeta42 (r = 0.64/0.69), which further correlated with the CSF Abeta42 (r = 0.52). None of these parameters correlated with serum Abeta40 levels. These findings support the role of insoluble Abeta42 in memory dysfunction and suggest a model with several pools (insoluble, extracellular soluble, CSF) of Abeta being in partial equilibrium with each other.     

Effects of Abeta1-42 fibrils and of the tetrapeptide Pr-IIGL on the phosphorylation state of the tau-protein and on the alpha7 nicotinic acetylcholine receptor in vitro

In order to investigate the possible links connecting beta-amyloid (Abeta) accumulation, tau-hyperphosphorylation and nicotinic receptor expression, rat embryonic primary hippocampal cultures were incubated with amyloidogenic peptides. Exposure to 0.5 microm fibrillar Abeta(1-42) for 3 days caused retraction of dendrites, shrinkage of cell bodies and a decrease in the expression of microtubule-associated proteins 2b (MAP2b), without affecting the total number of neurons and their viability. No impact on the tau-phosphorylation sites Ser-202, Thr231/Ser235, Ser262 and Ser396/Ser404 was found. The total number of homomeric alpha7-nicotinic receptors (alpha7-nAChRs) and their affinity for [(125)I]alpha-bungarotoxin remained unaltered. Upon incubation with the putatively protective tetrapeptide propionyl-isoleucine-isoleucine-glycine-leucine (Pr-IIGL), an analogue of the region [31-34] of Abeta, cell bodies were swollen in the region of the apical dendrite. These morphological alterations, different from those elicited by Abeta(1-42), did not involve MAP2 expression changes. In contrast to Abeta(1-42), Pr-IIGL caused a massive hyperphosphorylation of the tau-protein at Ser-202 and at Ser396/Ser404. The total number of homomeric alpha7-nAChRs and their affinity for [(125)I]alpha-bungarotoxin were unaffected. In conclusion, the present results show a toxic effect of Abeta(1-42) on the cytoskeletal structure at concentrations normally present in the brains of Alzheimer's disease patients, but raise some doubts about the role of Abeta(1-42) fibrils as a direct trigger of tau-hyperphosphorylation. The tetrapeptide Pr-IIGL cannot be considered protective with regard to cell morphology. Although it prevents the Abeta(1-42)-induced retraction of dendrites, it exhibits other toxic properties. The homomeric alpha7-nAChRs were not affected either by Abeta(1-42) incubation or by Pr-IIGL-induced tau-hyperphosphorylation.     

Therapeutic implications of a selective alpha7 nicotinic receptor abnormality in schizophrenia

A convergence of preclinical pharmacology, and human autopsy and genetic data support the existence of reduced expression and function of the alpha7 nicotinic receptor in patients with schizophrenia. The alpha7 nicotinic receptor is a member of a family of ligand-gated ion channels. The alpha7 nicotinic receptor may play an essential role in auditory sensory gating and voluntary smooth pursuit eye movements, two psychophysiological functions that are abnormal in patients with schizophrenia and closely related unaffected biological relatives. Diminished expression or function of the alpha7 nicotinic receptor in schizophrenia has stimulated consideration of selective full or partial alpha7 nicotinic receptor agonists as possible therapeutic interventions for this disorder. Further, the availability of positive allosteric modulators of nicotinic receptors that can improve the efficiency of transduction of the acetylcholine signal and prevent the rapid desensitization of the receptor should encourage these novel treatment approaches (e.g., galantamine).     

The effect of nicotine and haloperidol co-treatment on nicotinic receptor levels in the rat brain

Genetic and biological data have suggested a role for the neuronal nicotinic acetylcholine receptors in the neuropathophysiology of schizophrenia. Studies in human postmortem brain demonstrate dose-dependent increases in nicotinic receptor binding in normal smokers. We found this upregulation to be reduced in schizophrenic smokers, many of whom had taken typical neuroleptics during their lifetime. The present study examined the hypothesis that typical antipsychotic drug treatment might modulate nicotinic receptor upregulation in a rat model. Nicotine, administered alone or in combination with haloperidol, increased both high and low affinity neuronal nicotinic receptors in a region specific manner. Haloperidol had no generalized effect on basal levels of nicotinic receptor binding or nicotine induced upregulation of nicotinic receptors. However, haloperidol attenuated high affinity nicotinic receptor upregulation in thalamus and low affinity receptor upregulation in hippocampus. These results suggest that haloperidol is not likely to affect nicotinic receptor regulation by smoking in most brain regions.     

Relationship between the increased cell surface alpha7 nicotinic receptor expression and neuroprotection induced by several nicotinic receptor agonists.

Nicotine and other nicotinic acetylcholine receptor agonists have been shown to exert neuroprotective actions in vivo and in vitro by an as yet unknown mechanism. Even the identification of the subtype of nicotinic receptor(s) mediating this action has not been determined. In neural cell lines, the induction of cytoprotection often requires exposure to nicotine for up to 24 hr to produce a full protective effect. One phenomenon associated with chronic exposure of neural cells to nAChR agonists is the increased expression of nAChRs (upregulation), possibly as a response to desensitization. Because nicotinic receptors desensitize rapidly in the continuous presence of agonist, we investigated whether the neuroprotective actions produced by different nicotinic receptor agonists was related to their ability to induce nicotinic receptor upregulation. Differentiated PC12 cells were preincubated for 24 hr with various nAChR ligands, and the cells were subsequently deprived of both NGF and serum to induce cytotoxicity. Under control conditions cell viability was reduced to 66.5 +/- 5.4% of control by trophic factor withdrawal. For those cells pretreated with nicotine (1 nM-100 microM) cell viability increased from 74.2 +/- 1.5 to 97.3 +/- 4%. The neuroprotective action of nicotine was blocked by co-treatment with either 5 microM mecamylamine or 10 nM methyllycaconitine (MLA). The high potency blockade by MLA suggested that neuroprotection was mediated through the alpha7 nicotinic receptor subtype. For the seven agonists examined for neuroprotective activity, only nicotine was capable of evoking a near maximal (near 100% cell viability) neuroprotective action. The next most effective group included epibatidine, 4OHGTS-21, methycarbamylcholine, and 1,1-dimethyl-4-phenyl-piperazinium iodide. These least effective group included cytisine and tetraethylammonium. Incubation of differentiated PC12 cells with 10 microM nicotine increased the number of [(125)I]alpha bungarotoxin ([(125)I]alphaBGTbinding sites by 41% from 82.6 +/- 3.67 to 117 +/- 10.3 fmol/mg protein). Under similar conditions of incubation, the nicotinic receptor agonist cytisine (that was least effective in terms of neuroprotection) failed to increase the number of [(125)I]alphaBGT binding sites. Cells expressing increased levels of cell surface [(125)I]alphaBGT binding sites received added neuroprotective benefit from nicotine. Thus the induced upregulation of the alpha7 subtype of nicotinic receptors during chronic exposure to nicotine may be responsible for the drug's neuroprotective action.     

Loss of nicotinic acetylcholine receptor subunits alpha4 and alpha7 in the cerebral cortex of Parkinson patients

Cerebral cortical cholinergic deficits, represented by a decrease in choline acetyltransferase activity, severe losses of nicotinic binding sites as well as cell degeneration in the basal forebrain can be observed in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The potential role of nicotinic acetylcholine receptor subunits as pharmacological targets for the treatment of cognitive deficits raises the question as to what extent these subunits are affected in neurodegenerative diseases. We here report on a significant decrease of the alpha4 and the alpha7 nicotinic acetylcholine receptor subunit in cortices of Parkinson patients which turns out to be similar to recent findings in Alzheimer patients.     

Association of the neuronal nicotinic acetylcholine receptor subunit alpha4 polymorphisms with febrile convulsions

PURPOSE: The alpha4-subunit gene of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been identified as the first gene underlying an idiopathic partial epilepsy syndrome in human autosomal-dominant nocturnal frontal lobe epilepsy. Studies provided evidence that the protein coded by CHRNA4 is one of the most abundant subunits of the neuronal nicotinic acetylcholine receptors in mammalian brains, and mutations of CHRNA4 seem to cause neuronal excitation. The CHRNA4 gene may have a role in the development of febrile convulsions (FCs), the majority of childhood seizures. This study assessed the distribution of genotypes of CHRNA4 in patients with FCs. METHODS: A total of 102 children with FCs and 80 normal control subjects were included in the study. Polymerase chain reaction was used to identify the C/T polymorphism of the CHRNA4 gene. Genotypes and allelic frequencies for the CHRNA4 gene polymorphisms in both groups were compared. RESULTS: The number of individuals with heterozygous CHRNA4 (Ser543Ser)-C/T genotype was significantly greater (60.8% vs. 32.5%; p = 0.001), and the CHRNA4 (Ser543Ser)-T allele frequency was significantly higher (p = 0.001), in patients with FCs compared with healthy controls. The odds ratio for developing FCs in individuals with the CHRNA4 (Ser543Ser)-CT genotype was 3.77 compared with individuals with two copies of the CHRNA4 (Ser543Ser)-C allele. CONCLUSIONS: This study demonstrated an association between the CHRNA4 gene and FCs. Individuals with the T allele had a higher incidence of FCs. These data suggest that the CHRNA4 gene or a closely linked gene might be one of the susceptibility factors for FCs.     

Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the α7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine α7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at α7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that α7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.     

Early intraneuronal Abeta deposition in the hippocampus of APP transgenic mice

Increasing evidence suggests that intraneuronal amyloid-beta (Abeta) accumulation may be an early event in Alzheimer's disease (AD) pathogenesis. However direct in vivo evidence regarding initial Abeta seeding is missing. Using an APP transgenic mouse model, our sensitive immunocytochemical procedures revealed a novel intraneuronal Abeta deposition in the somas of hippocampal CA1/subiculum neurons far in advance of the occurrence of extracellular Abetaplaques. These deposits increased exponentially with age and were elevated approximately 4-fold (p < 0.001) by high fat/high cholesterol diet. Abeta40 and Abeta42 were the major constituents of these deposits and were co-localized with lysosomal markers. Our results are consistent with the notion that the earliest Abeta deposition occurs intraneuronally, prior to extracellular amyloid plaque formation.