Increased Amyloid Burden Research Articles

Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor

Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD.

L-Type Calcium Channel CaV 1.2 in Transgenic Mice Overexpressing Human AβPP751 with the London (V717I) and Swedish (K670M/N671L) Mutations

Cumulative evidence indicates that amyloid-beta peptides exert some of their neurodegenerative effects through modulation of L-type voltage gated calcium channels, which play key roles in a diverse range of CNS functions. In this study we examined the expression of CaV1.2 L-type voltage gated calcium channels in transgenic mice overexpressing human AbetaPP751 with the London (V717I) and Swedish (K670M/N671L) mutations by immunohistochemistry in light and electron microscopy. In hippocampal layers of wild type and transgenic mice, CaV1.2 channels were predominantly localized to somato-dendritic domains of neurons, and to astrocytic profiles with an age-dependent increase in labeling density. In transgenic animals, CaV1.2-like immunoreactive clusters were found in neuronal profiles in association with amyloid-beta plaques. Both the number and density of these clusters depended upon age of animals and number of plaques. The most striking difference between wild type and transgenic mice was the age-dependent expression of CaV1.2 channels in reactive astrocytes. At the age of 6 month, CaV1.2 channels were rarely detected in reactive astrocytes of transgenic mice, but an incremental number of CaV1.2 expressing reactive astrocytes was found with increasing age of animals and number of amyloid-beta plaques. This study demonstrates that CaV1.2 channels are highly expressed in reactive astrocytes of 12-months of age transgenic mice, which might be a consequence of the increasing amyloid burden. Further studies should clarify which functional implications are associated with the higher availability of CaV1.2 channels in late stage Alzheimer's disease.

APOE ε4 and bapineuzumab

Therapeutic development for Alzheimer disease (AD) is in a transitional period. Neurotransmitter-based approaches to date have been moderately beneficial, but only palliative. Strategies aimed at reducing the burden of amyloid-β peptide (Aβ) offer the prospect of modifying the underlying disease processes and ultimately slowing the course of neurodegenerative decline. However, results with amyloid-targeted agents have been disappointing so far. Among the many unknowns confronting the development of successful therapeutic interventions, the role of genetic background looms large. To date, APOE is the most common among a half dozen genetic loci that implicate aberrant metabolism of Aβ in AD as a primary etiologic agent and therapeutic target. Roughly half to two-thirds of patients with AD carry the e4 allele of APOE that conveys an increased risk for the disease. Early work with the cholinesterase inhibitor tacrine raised the possibility that treatment response may be reduced in APOE e4 carriers,1 heralding the routine use of APOE genotyping in subsequent AD trials. Since the APOE e4 allele is associated with increased amyloid burden,2 it theoretically may have a greater influence on Aβ-directed therapies. With this as background, Schenk and colleagues3 formulated the first Aβ vaccine, AN-1792, using a synthetic Aβ peptide. However, problems achieving a robust active immune response in the elderly, coupled with the allergic complications of AN-1792, …

Optimal parameters for near infrared fluorescence imaging of amyloid plaques in Alzheimer's disease mouse models

Amyloid-β plaques are an Alzheimer's disease biomarker which present unique challenges for near-infrared fluorescence tomography because of size (<50 µm diameter) and distribution. We used high-resolution simulations of fluorescence in a digital Alzheimer's disease mouse model to investigate the optimal fluorophore and imaging parameters for near-infrared fluorescence tomography of amyloid plaques. Fluorescence was simulated for amyloid-targeted probes with emission at 630 and 800 nm, plaque-to-background ratios from 1–1000, amyloid burden from 0–10%, and for transmission and reflection measurement geometries. Fluorophores with high plaque-to-background contrast ratios and 800 nm emission performed significantly better than current amyloid imaging probes. We tested idealized fluorophores in transmission and full-angle tomographic measurement schemes (900 source–detector pairs), with and without anatomical priors. Transmission reconstructions demonstrated strong linear correlation with increasing amyloid burden, but underestimated fluorescence yield and suffered from localization artifacts. Full-angle measurements did not improve upon the transmission reconstruction qualitatively or in semi-quantitative measures of accuracy; anatomical and initial-value priors did improve reconstruction localization and accuracy for both transmission and full-angle schemes. Region-based reconstructions, in which the unknowns were reduced to a few distinct anatomical regions, produced highly accurate yield estimates for cortex, hippocampus and brain regions, even with a reduced number of measurements (144 source–detector pairs).

Decreased behavioral impairments in an Alzheimer mice model by interfering with TNF-alpha metabolism

The performance of mice expressing PDAPP (+/+ or +/−) was studied in the Morris place navigation task. Different lines of questions were investigated using PDAPP+/− mice in which the activity of the cytokine Tumor Necrosing Factor α (TNFα) was attenuated by chronic treatment with anti-TNF or deleting TNFα (TNF−/−). Two different categories of behavior were analyzed in adult (6 months) and middle aged (15 months) subjects. Classically, the cognitive performance was assessed from the escape efficacy and quantitative bias toward the training position in a Morris water maze. Second, stereotyped circling was quantified, along with more qualitative behavioral impairments such as self-mutilation or increased reactivity. Our results can be summarized as follows. (1) All of the PDAPP mice expressed reduced cognitive performance in the Morris task, but only those with a clear-cut amyloid burden in the hippocampus showed behavioral abnormalities such as stereotyped circling. (2) Chronic treatment with anti-TNF prevented the development of pathological circling in the 6-month-old mice but not in the 15-month-old mice and had no significant effect on amyloid burden. (3) The absence of TNFα prevented the development of stereotyped circling in 6- and 15-month-old mice but increased amyloid burden after 15 months. These data indicate that PDAPP mice express cognitive impairments disregarding absence of TNF. The pathological behavioral anomalies related to the PDAPP mutation seem reduced by treatments interfering with TNFα.

Why lipids are important for Alzheimer disease?

Several lines of evidence suggest that dysregulated lipid metabolism may participate in the pathogenesis of Alzheimer's disease (AD). Epidemiologic studies suggest that elevated mid-life plasma cholesterol levels may be associated with an increased risk of AD and that statin use may reduce the prevalence of AD. Cellular studies have shown that the levels and distribution of intracellular cholesterol markedly affect the processing of amyloid precursor protein into A beta peptides, which are the toxic species that accumulate as amyloid plaques in the AD brain. Most importantly, genetic evidence identifies apolipoprotein E, the major cholesterol carrier in the central nervous system, as the primary genetic risk factor for sporadic AD. In humans, apoE exists as three major alleles (apoE2, apoE3, and apoE4), and inheritance of the apoE4 allele increases the risk of developing AD at an earlier age. However, exactly how apoE functions in the pathogenesis of AD remains to be fully determined. Our studies have identified that the cholesterol transporter ABCA1 is a crucial regulator of apoE levels and lipidation in the brain. Deficiency of ABCA1 leads to the loss of approximately 80% of apoE in the brain, and the residual 20% that remains is poorly lipidated. Several independent studies have shown this poorly lipidated apoE increases amyloid burden in mouse models of AD, demonstrating that apoE lipidation by ABCA1 affects key steps in amyloid deposition or clearance. Conversely, robust overexpression of ABCA1 in the brain promotes apoE lipidation and nearly eliminates the formation of mature amyloid plaques. These studies show that the lipid binding capacity of apoE is a major mechanism of its function in the pathogenesis of AD, and suggest that increasing apoE lipidation may be of therapeutic importance for this devastating disease.

PET Amyloid Ligand [11C]PIB Uptake and Cerebrospinal Fluid β-Amyloid in Mild Cognitive Impairment

Background: In mild cognitive impairment (MCI), Alzheimer’s disease (AD)-type cerebrospinal fluid (CSF) biomarker profiles predict rapid progression and conversion to AD. An increased brain amyloid burden in AD and MCI has been demonstrated with PET using [¹¹C]PIB (Pittsburgh compound B). Little is known about the relationship between these biomarkers in MCI. Methods: We studied 15 patients with amnestic MCI and 22 controls with PET using [¹¹C]PIB. In MCI patients, CSF levels of Aβ42, pTAU, totalTAU and the Aβ42/pTAU ratio were measured. Results: In MCI patients, CSF Aβ42 was abnormal in 53% of patients, totalTAU in 67%, pTAU in 64% and the Aβ42/pTAU ratio in 64%. A composite neocortical [¹¹C]PIB uptake score was increased in 87% of the MCI patients. Only 54% of [¹¹C]PIB-positive subjects showed AD-type Aβ42 values. During a 2-year follow-up, 6 MCI patients converted to AD, all of them had increased neocortical PIB scores at the MCI stage. Abnormal CSF Aβ42 was found in 3 patients, pTAU in 3 patients and Aβ42/pTAU ratio in 4 patients. Conclusion: Follow-up studies are needed to confirm whether [¹¹C]PIB uptake might be more sensitive than CSF Aβ42 concentration in detecting increased amyloid burden in MCI, as suggested by the results of this study.

Cycloxygenase-2 activity promotes cognitive deficits but not increased amyloid burden in a model of Alzheimer’s disease in a sex-dimorphic pattern

Administration of non-steroidal anti-inflammatory agents reduces the risk of developing Alzheimer’s disease in normal aging populations, an effect that may occur from inhibition of the cyclooxygenases, the rate-limiting enzymes in the formation of prostaglandins. In this study, we investigated whether increased activity of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, potentiates disease progression in a transgenic mouse model of Alzheimer’s disease. To study the functional effects of COX-2 activity, male and female bigenic mice (amyloid precursor protein with Swedish mutation [APPswe]–presenilin-1 protein with deletion of exon 9 [PS1dE9] and trigenic COX-2/APPswe-PS1dE9) were behaviorally tested ±administration of the selective COX-2 inhibitor celecoxib. Behavioral testing included a three-trial Y maze that measures spatial working and recognition memories and an open field task that tested levels of hyperactivity. Overexpression of COX-2 in APPswe-PS1dE9 mice resulted in specific deficits in spatial working memory in female but not male mice. These sex-specific deficits were abolished by pharmacological inhibition of COX-2 activity. Importantly, COX-2-associated deficits were dependent on co-expression of all three transgenes since COX-2 single transgenic and APPswe-PS1dE9 bigenic mice showed normal memory. Quantification of amyloid plaque load and total Aβ 40 and 42 peptides did not reveal significant differences in trigenic versus bigenic mice treated with either vehicle or celecoxib. Taken together, these data indicate an interaction between the effects of COX-2 and Aβ peptides on cognition that occurs in a sex-specific manner in the absence of significant changes in amyloid burden. These findings suggest that pathological activation of COX-2 may potentiate the toxicity of Aβ peptides, particularly in females, without significantly affecting Aβ accumulation.

Propentofylline attenuates tau hyperphosphorylation in Alzheimer's Swedish mutant model Tg2576

Key pathological hallmarks of Alzheimer's disease (AD) are the deposition of amyloid plaques containing Aβ-peptides and the formation of neurofibrillary tangles containing hyperphosphorylated tau. Propentofylline (PPF) is a synthetic xanthine derivative that inhibits phosphodiesterase and adenosine uptake. These effects of PPF influence many cellular functions including stimulating synthesis/release of nerve growth factor. We tested the effects of PPF on disease progression in transgenic mice overexpressing the Swedish mutant human APP (Tg2576). The untreated Tg mice show, together with increased amyloidogenesis, increased levels of tau hyperphosphorylation and increased ratios of the activated to inactivated GSK-3β, one of the key kinases that can phosphorylate tau. One month of PPF feeding (40 mg/kg per day) reduced the burden of amyloid plaques and the levels of hyperphosphorylated tau and immunoreactive IL-1β. In parallel with these changes, PPF reduced the activated form of GSK-3β and increased the inactivated form of GSK-3β, restoring their ratio almost to normal values. These results demonstrate that PPF can exert multiple protective effects on both amyloidogenesis and tau hyperphosphorylation in an animal model of AD. Our earlier report [Neurochem. Int. 43(3) (2003) 225] demonstrated that Tg2576 animals show decreased levels of mRNA for NGF with increased amyloid burden while feeding of PPF results in a major shift from β-amyloidogenic to α-secretory processing of APP together with increased expression of NGF mRNA. The current new data enlarge our understanding of PPF effects in brain and of tau hyperphosphorylation in Tg animals and are consistent with the hypothesis that GSK-3β is a nodal point linking amyloid and tau pathology. Therapeutic interventions directed toward multiple pathological processes may be more protective than treatments directed toward a single process. The new results reported here indicate that further testing of PPF as a potential therapy in AD is warranted.

Immunization for Alzheimer's disease

AbstractThe recent termination of a Phase II clinical trial in which volunteers with Alzheimer's disease (AD) were vaccinated with Amyloid‐β (Aβ)1‐42, has cast doubt on the feasibility of this therapeutic approach. While the exact reasons for the cerebral inflammation in these patients is being determined, it is difficult to evaluate the cause of these adverse effects. The most likely reasons are Aβ1‐42 toxicity and/or autoimmunity. Aβ vaccination approaches are based on the hypothesis that Aβ deposition and toxicity are central to the pathogenesis of AD. Therefore, it is counterintuitive to use the whole Aβ peptide for human vaccination. Aβ1‐40/42 is a major plaque component that forms inflammatory/toxic fibrils as observed in many in vitro and in vivo studies. Furthermore, numerous studies have shown that Aβ1‐40/42 bidirectionally crosses the blood–brain barrier (BBB) in experimental animals. Additionally, in vitro and in vivo studies indicate that minute amounts of Aβ1‐42 may seed fibril/amyloid formation. The elderly, a target population for AD therapy, often have a poor immune response to vaccines, which enhances the gravity of these safety concerns. In these patients with an attenuated immune reaction, injected Aβ1‐42 may initiate and/or enhance congophilic angiopathy, which eventually may result in reduced cerebral blood flow and/or intracerebral bleeding. Aβ1‐42 may also cross the BBB and once within the brain parenchyma it may contribute to plaque formation and/or co‐deposit on plaques. Together, these effects within blood vessels and/or brain parenchyma may actually enhance the progression of AD. Given the potential serious side effects of Aβ1‐42 vaccination, it is safer to use immunogenic Aβ derivatives, which are less likely to be toxic. The main immunogenic epitopes of Aβ1‐42 are contained within the first 30 amino acids of the peptide. Taking this into account, we have developed soluble antigenic Aβ derivatives, which are nonfibrillogenic and nontoxic in human cell culture. Our prototype peptide, K6Aβ1‐30‐NH2, diminishes amyloid burden to a similar extent as reported for Aβ1‐42. Additionally, ramified IL‐1β‐positive microglia as well as phagocytes, associated with the Aβ plaques, were absent in the immunized mice, indicating reduced inflammation in these animals at the time point examined. Autoimmunity may be the culprit if follow‐up studies reveal that the brain inflammation is related to antibody interactions with Aβ and/or amyloid precursor protein (APP). In such a scenario, any vaccination approach targeting Aβ can have similar consequences, although preventive treatment initiated prior to amyloid deposition may not result in these adverse reactions. T–cell‐related autoimmunity may also be involved and can be expected to be less with Aβ derivatives not containing certain T‐cell epitopes. An alternative to the active vaccination approach is passive immunization, which is associated with a lower risk of irreversible autoimmunity. This approach may also be used in patients with a muted immune response to the vaccine. However, in a chronic disease such as AD repeated antibody injections may lead to an anti‐idiotype response and the resulting serum immune complexes can cause vasculitis and/or glomerulonephritis. Reduction of soluble Aβ within the peripheral system may be a critical part of the pathway that reduces cerebral plaque burden in Tg mice and ultimately in AD patients. Overall, the use of nontoxic Aβ derivatives and/or antibodies with very limited access into the CNS, such as IgM, may prove to have reduced side effects compared to Aβ1‐40/42 and/or IgG‐based immunization. Any therapeutic approach will be more effective when used prophylactically because of neuronal loss and increased amyloid burden in the later stages of AD. Reversal of clinical symptoms cannot be expected and early diagnosis of AD may be needed for effective therapy. Drug Dev. Res. 56:135–142, 2002. © 2002 Wiley‐Liss, Inc.

Apolipoprotein E affects amyloid formation but not amyloid growth in vitro: Mechanistic implications for ApoE4 enhanced amyloid burden and risk for Alzheimer's disease

The transition from the partially folded soluble Aβ monomer to insoluble Aβ amyloid fibrils is seminal to the formation and growth of amyloid plaques in Alzheimer's disease (AD). A detailed understanding of the role of AD risk factors in these processes is essential to understanding the physiochemical nature of this conformational rearrangement- The apolipoprotein E ϵ4 allele, a risk factor for AD, affects AD pathology by increasing amyloid burden relative to the much more common ϵ3 allele. In the present study, in vitro models were employed to probe the effect of these proteins on kinetically distinct steps in Aβ fibrillogenesis. Formation of Aβ amyloid was faster in the presence of apo E4 than apoE3, while growth of existing plaques was unaffected by either isoform. Further, experiments with Aβ stereoiso-mers establish that this effect ofapoE3 is mediated through interaction with oligomeric fibrillogenic intermediates rather than through specific contacts with monomeric Aβ. Consistent with the altered pathology and enhanced risk for AD associated with inheritance of the ϵ4 allele, we conclude that APOE ϵ4 is a risk factor for AD not due to a pathological gain of function of apo E4 but to a loss of protective function of apo E3.

Permeability of Proteins at the Blood–Brain Barrier in the Normal Adult Mouse and Double Transgenic Mouse Model of Alzheimer's Disease

The permeability of albumin, insulin, and human Aβ 1–40 at the blood–brain barrier (BBB) was determined in the normal adult mouse (B6/SJL) and in the double transgenic Alzheimer mouse (APP, PS1) by using an I.V. bolus injection technique to quantify the permeability coefficient-surface area (PS) product for each protein after correction for the residual plasma volume (Vp) occupied by the protein in the blood vessels of different brain regions using a second aliquot of the same protein radiolabeled with a different isotope of iodine (125I vs 131I) as a vascular space marker. This technology for quantifying BBB permeability of proteins was adapted from the rat to the mouse and involved catheterizing the femoral artery and vein of the mouse instead of the brachial artery and vein as for the rat. Because of the smaller blood volume in the mouse, serial sampling (20 μl) of blood from the femoral artery of the mouse was performed and directly TCA precipitated to generate a whole blood washout curve for the intact protein. When similar blood sampling techniques were used in the rat, the PS values for albumin and insulin at the BBB were similar in these two species. In the double transgenic mouse, the Vp values for albumin were significantly increased 1.4- to 1.6-fold in five of six brain regions compared to the normal adult mouse, which indicated increased adherence of albumin to vessel walls. As a result, the PS values were significantly decreased, from 1.4- to 3.2-fold, which likely reflected decreased transport of albumin by passive diffusion. In contrast, insulin, which is taken up into the brain by a receptor-mediated transport mechanism at the BBB, showed no significant difference in the Vp values but a significant increase in the PS values in four of six brain regions. This suggests a compensatory mechanism in the Alzheimer's transgenic brain whereby there is an increased permeability to insulin at the BBB. Surprisingly, there was no significant difference in the Vp or PS values for human Aβ 1–40 at the BBB in the double transgenic Alzheimer mouse at 24, 32, or 52 weeks of age, when there is both significant Aβ levels in the plasma and amyloid burden in the brains of these animals. These data suggest that there is not an alteration in permeability to human Aβ 1–40 at the BBB with increasing amyloid burden in the double transgenic Alzheimer mouse. Although these observations suggest structural alterations at the BBB, they do not support the concept of extensive BBB damage with substantial increases in BBB permeability in Alzheimer's disease.

Accelerated decline in apolipoprotein E-epsilon4 homozygotes with Alzheimer's disease.

The apolipoprotein E-epsilon4 (APOE-epsilon4) allele is a powerful genetic risk factor for the development of Alzheimer's disease (AD). AD patients who are APOE-epsilon4 homozygotes have an earlier age at onset, increased amyloid burden, and decreased acetylcholine levels--findings that suggest differences in disease severity or rate of progression. Studies of genotype differences in rate of decline, however, have produced negative results that may be due to methodologic biases. The current study examined rate of decline in the largest sample of APOE-genotyped AD patients for whom longitudinal cognitive data have been reported. Newly diagnosed patients with probable AD (n = 201) comprised four genotype groups: epsilon2/3 (n = 14), epsilon3/3 (n = 75), epsilon3/4 (n = 82), and epsilon4/4 (n = 30). The Dementia Rating Scale (DRS) was administered at baseline and then annually for 1 to 6 years (mean, 2.5 years). For each subject, a DRS slope was calculated reflecting annual rate of decline. Rate of decline as measured by DRS slope differed according to genotype, with the effect modified by DRS score (p < 0.014). At the mean DRS score observed in our sample (DRS = 105), the epsilon4/4 group had an increased rate of decline (11.9 points per year) relative to the epsilon2/3 (5.8 points per year; p < 0.003), epsilon3/3 (9.3 points per year; p < 0.076), and epsilon3/4 (9.6 points per year; p < 0.055) groups. At a lower DRS score (DRS = 80), even larger differences were observed among genotypes; the epsilon4/4 group had a increased rate of decline (22.2 points per year) relative to the epsilon2/3 (9.7 points per year; p < 0.0006), epsilon3/4 (15.8 points per year; p < 0.020), and epsilon3/3 (18.2 points per year; p < 0.173) groups. The epsilon2/3 group had a significantly slower rate of decline than all other groups at DRS scores of 80 or 105. APOE-epsilon4 homozygosity is associated with a faster rate of cognitive decline, whereas the epsilon2 allele slows disease progression. These findings suggest that APOE plays a mechanistic role in the progression of AD, and is not simply related to disease onset.

The limbic system: an anatomic, phylogenetic, and clinical perspective.

The limbic system is the border zone where psychiatry meets neurology. The authors provide a model of limbic function that combines phylogenetic, anatomic, functional, and clinical data to interpret diseases relevant to neuropsychiatry. They provide evidence supporting two major divisions in the limbic system: a paleocortical division with the amygdala and orbitofrontal cortex at its center, and an archicortical division with the hippocampus and cingulate cortex at its center. The implicit integration of affect, drives, and object associations is the function of the paleocortical limbic division; explicit sensory processing, encoding, and attentional control is the function of the archicortical limbic division. The two work in concert to integrate thought, feeling, and action. Understanding their development and organization informs us about how best to care for our patients.