Cerebral β-Amyloidosis in Mice Investigated by Ultramicroscopy

Alzheimer´s disease (AD) is the most common neurodegenerative disorder. AD neuropathology is characterized by intracellular neurofibrillary tangles and extracellular β-amyloid deposits in the brain. To elucidate the complexity of AD pathogenesis a variety of transgenic mouse models have been generated. An ideal imaging system for monitoring β-amyloid plaque deposition in the brain of these animals should allow 3D-reconstructions of β-amyloid plaques via a single scan of an uncropped brain. Ultramicroscopy makes this possible by replacing mechanical slicing in standard histology by optical sectioning. It allows a time efficient analysis of the amyloid plaque distribution in the entire mouse brain with 3D cellular resolution. We herein labeled β-amyloid deposits in a transgenic mouse model of cerebral β-amyloidosis (APPPS1 transgenic mice) with two intraperitoneal injections of the amyloid-binding fluorescent dye methoxy-X04. Upon postmortem analysis the total number of β-amyloid plaques, the β-amyloid load (volume percent) and the amyloid plaque size distributions were measured in the frontal cortex of two age groups (2.5 versus 7-8.5 month old mice). Applying ultramicroscopy we found in a proof-of-principle study that the number of β-amyloid plaques increases with age. In our experiments we further observed an increase of large plaques in the older age group of mice. We demonstrate that ultramicroscopy is a fast, and accurate analysis technique for studying β-amyloid lesions in transgenic mice allowing the 3D staging of β-amyloid plaque development. This in turn is the basis to study neural network degeneration upon cerebral β-amyloidosis and to assess Aβ -targeting therapeutics.     

Apolipoprotein E Attenuates β-Amyloid-Induced Astrocyte Activation

Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β-amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ-induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain.     

β-Amyloid Protein Precursor and τ mRNA Levels Versus β-Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain

Abstract: To learn whether or not the levels of β-amyloid protein precursor (APP) and τ mRNAs are related to the formation of β-amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto-chemically for β-amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β-amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β-protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β-amyloid and/or tangle formation in the aged human brain.     

Generation of C5a in the absence of C3: a new complement activation pathway

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.     

Aggregation State-Dependent Activation of the Classical Complement Pathway by the Amyloid β Peptide

Abstract: Activation of the classical complement pathway has been widely investigated in recent years as a potential mechanism for the neuronal loss and neuritic dystrophy characteristic of Alzheimer's disease (AD) pathogenesis. We have previously shown that amyloid β peptide (Aβ) is a potent activator of complement, and recent evidence suggesting that the assembly state of Aβ is crucial to the progress of the disease prompted efforts to determine whether the ability of Aβ to activate the classical complement pathway is a function of the aggregation state of the peptide. In this report, we show that the fibrillar aggregation state of Aβ, as determined by thioflavin T fluorometry, electron microscopy, and staining with Congo red and thioflavine S, is precisely correlated with the ability of the peptide to induce the formation of activated fragments of the complement proteins C4 and C3. These results suggest that the classical complement pathway provides a mechanism whereby complement-dependent processes may contribute to neuronal injury in the proximity of fibrillar but not diffuse Aβ deposits in the AD brain.     

Signal transduction in Alzheimer disease: p21-activated kinase signaling requires C-terminal cleavage of APP at Asp664

The deficits in Alzheimer disease (AD) stem at least partly from neurotoxic β-amyloid peptides generated from the amyloid precursor protein (APP). APP may also be cleaved intracellularly at Asp664 to yield a second neurotoxic peptide, C31. Previously, we showed that cleavage of APP at the C-terminus is required for the impairments seen in APP transgenic mice, by comparing elements of the disease in animals modeling AD, with (platelet-derived growth factor B-chain promoter-driven APP transgenic mice; PDAPP) versus without (PDAPP D664A) a functional Asp664 caspase cleavage site. However, the signaling mechanism(s) by which Asp664 contributes to these deficits remains to be elucidated. In this study, we identify a kinase protein, recently shown to bind APP at the C-terminus and to contribute to AD, whose activity is modified in PDAPP mice, but normalized in PDAPP D664A mice. Specifically, we observed a significant increase in nuclear p21-activated kinase (isoforms 1, 2, and or 3; PAK-1/2/3) activation in hippocampus of 3 month old PDAPP mice compared with non-transgenic littermates, an effect completely prevented in PDAPP D664A mice. In contrast, 13 month old PDAPP mice displayed a significant decrease in PAK-1/2/3 activity, which was once again absent in PDAPP D664A mice. Similarly, in hippocampus of early and severe AD subjects, there was a progressive and subcellular-specific reduction in active PAK-1/2/3 compared with normal controls. Interestingly, total PAK-1/2/3 protein was increased in early AD subjects, but declined in moderate AD and declined further, to significantly below that of control levels, in severe AD. These findings are compatible with previous suggestions that PAK may be involved in the pathophysiology of AD, and demonstrate that both early activation and late inactivation in the murine AD model require the cleavage of APP at Asp664.     

Thrombin Attenuates Neuronal Cell Death and Modulates Astrocyte Reactivity Induced by β-Amyloid In Vitro

Abstract: β-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 n M , thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 p M and 1 µ M . In contrast, thrombin potentiates rather than attenuates the β-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of β-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.     

Morphological and Biochemical Analyses of Amyloid Plaque Core Proteins Purified from Alzheimer Disease Brain Tissue

Abstract— Amyloid plaque cores were purified from Alzheimer disease brain tissue. Plaque core proteins were solubilized in formic acid which upon dialysis against guan-idinium hydrochloride (GuHCI) partitioned into soluble (∼15%) and insoluble (∼85%) components. The GuHCI-soluble fraction contained β-amyloid_1-40, whereas the GuHCI-insoluble fraction was fractionated into six components by size exclusion HPLC: S1 (>200 kDa), S2 (200 kDa), S3 (45 kDa), S4 (15 kDa), S5 (10 kDa), and S6 (5 kDa). Removal of the GuHCI reconstituted 10-nm filaments composed of two intertwined 5-nm strands. Fractions S5 and S6 also yielded filamentous structures when treated similarly, whereas fractions S1–S4 yielded amorphous aggregates. Chemical analysis identified S4–S6 as multimeric and monomeric β-amyloid. Immunochemical analyses revealed α₁-antichymotrypsin and non-β-amyloid segments of the β-amyloid precursor protein within fractions S1 and S2. Several saccharide components were identified within plaque core protein preparations by fluorescence and electron microscopy, as seen with fluores-cein isothiocyanate-and colloidal gold-conjugated lectins. We have shown previously that this plaque core protein complex is more toxic to neuronal cultures than β-amyloid. The non-β-amyloid components likely mediate this additional toxicity, imposing a significant influence on the pathophysiology of Alzheimer disease.     

Modeling Alzheimer's disease and other proteopathies in vivo: is seeding the key?

Summary.  Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of β-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5–6 months prior to the usual onset of β-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1) Premature deposition of β-amyloid in eight month-old, β-amyloid precursor protein (βAPP)-transgenic mice (Kane et al., 2000); 2) augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of β-amyloid in vivo by AD brain extracts suggests pathogenic similarities between β-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. Received June 28, 2001 Accepted August 6, 2001 Published online June 26, 2002     

Complement component C1q inhibits beta-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and calpain-independent mechanisms

Alzheimer's disease is a neurodegenerative disorder characterized by neuronal loss, β-amyloid (Aβ) plaques, and neurofibrillary tangles. Complement protein C1q has been found associated with fibrillar Aβ deposits, however the exact contributions of C1q to Alzheimer's disease is still unknown. There is evidence that C1q, as an initiator of the inflammatory complement cascade, may accelerate disease progression. However, neuronal C1q synthesis is induced after injury/infection suggesting that it may be a beneficial response to injury. In this study, we report that C1q enhances the viability of neurons in culture and protects neurons against Aβ- and serum amyloid P (SAP)-induced neurotoxicity. Investigation of potential signaling pathways indicates that caspase and calpain are activated by Aβ, but C1q had no effect on either of these pathways. Interestingly, SAP did not induce caspase and calpain activation, suggesting that C1q neuroprotection is in distinct from caspase and calpain pathways. In contrast to Aβ- and SAP-induced neurotoxicity, neurotoxicity induced by etoposide or FCCP was unaffected by the addition of C1q, indicating pathway selectivity for C1q neuroprotection. These data support a neuroprotective role for C1q which should be further investigated to uncover mechanisms which may be therapeutically targeted to slow neurodegeneration via direct inhibition of neuronal loss.     

Complement activation in amyloid plaques in Alzheimer's dementia

Amyloid plaques in Alzheimer's dementia contain complement factors C1q, C4 and C3. In the present study we demonstrate complement activation in amyloid plaques using immunoenzymatical techniques and specific antibodies against subunits of individual complement components and activated complement products. Amyloid plaques contain C1q and activated C3 fragments (C3c and C3d, g) but no C1s and C3a. These findings demonstrate that the complement components are not passively bound to the amyloid plaque structures but are the result of an activation process. The role of complement activation in the genesis of senile plaques is discussed.     

Guinea Pigs as a Nontransgenic Model for APP Processing in Vitro and in Vivo

Alzheimer's disease (AD) is characterized, amongst others, by the appearance of vascular and parenchymal -amyloid deposits in brain. Such aggregates are mainly composed of -amyloid peptides, which are derived by proteolytic processing of a larger amyloid precursor protein (APP). APP is highly conserved among mammalian species, but experimental studies in rodents are often hampered by the humble APP-processing in the amyloidogenic pathway and by the inability of rodent -amyloid peptides to form higher molecular aggregates such as soluble oligomers and insoluble -amyloid plaques. Thus, there is need for in vitro and in vivo model systems that allow identification of factors that increase amyloidogenic APP processing and accelerate -amyloid plaque formation and testing the potency of pharmacological manipulations to ameliorate -amyloid load in brain. Transgenic mice that overexpress human APP containing AD-associated mutations that favor the amyloidogenic pathway of APP processing represent such a model. However, mutations of the APP gene are not frequent in AD and, therefore, the mechanisms of -amyloid plaque formation, the composition of -amyloid plaques, and the accompanying tissue response in brain of these animals may be different from that in AD. In contrast, guinea pigs express -amyloid peptides of the human sequence and appear to represent a more physiological model to examine the long-term effects of experimental manipulations on APP processing and -amyloid plaque formation in vivo. Additionally, APP processing in guinea pig primary neuronal cultures has been shown to be similar to cultures of human origin. In this article we highlight the advantages and limitations of using guinea pigs as experimental models to study APP processing.     

Inhibition of β-Amyloid Production by Activation of Protein Kinase C

The cellular factors regulating the generation of β-amyloid from the amyloid precursor protein (APR) are unknown. Activation of protein kinase C (PKC) by phorbol ester treatment inhibited the generation of the 4-kDa β-amyloid peptide in transfected COS cells, a human glioma cell line, and human cortical astrocytes. An analogue of diacylglycerol, the endogenous cellular activator of PKC, also inhibited the generation of β-amyloid. Activation of PKC increased the level of secreted APP in transfected COS cells but did not significantly affect the level of secreted APP in primary human astrocytes or in the glioma cell line. Cell-associated APP and the secreted APP derivative, but not β-amyloid, were phosphorylated on serine residues. Activation of PKC did not increase the level of APP phosphorylation, suggesting that PKC modulates the proteolytic cleavage of APP indirectly by phosphorylation of other substrates. These results indicate that PKC activation inhibits β-amyloid production by altering APP processing and suggest that β-amyloid production can be regulated by the phospholipase C-diacylglycerol signal transduction pathway.     

Reduced Membrane Lipids in the Cortex of Alzheimer’s Disease Transgenic Mice

Accumulating evidence suggests that the conversion of Aβ peptides to soluble, neurotoxic polymers is the key event in the development of Alzheimer’s disease (AD). Moreover, interactions between Aβ peptides and neuronal membrane lipids likely play a vital role in developing the neurotoxicity associated with AD. The aim of this study is to assess whether lipid matrix of neuronal membranes is affected by the accumulation of Aβ peptides in double transgenic mouse model of AD expressing both mutant human β-amyloid precursor protein (APP) and presenilin 1 (PS1). We apply high pressure liquid chromatography with an evaporative light scattering detector to compare levels of cholesterol, galactocerebrosides, and phospholipid subclasses simultaneously in cortex samples between AD double transgenic mice at 4 months of age when Aβ production and amyloid plaque deposition is just beginning and at 9 months, when there is advanced Aβ levels and plaque deposition compared to age-matched wild-type (B6/SJL) mice. Both cholesterol (CL) and phospholipids (PL) are significantly lower in 9-month-old AD mice than the same age of B6/SJL mice. Among PL subclasses, phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) are selectively reduced in 9-month-old AD mice. The molar ratios of CL to PL in 9-month-old AD mice (1.19 ± 0.27) were significantly higher than those of 9-month-old B6/SJL mice (0.81 ± 0.08). In keeping with decreased levels of PL, there are also significant reductions of very long-chain n-3 fatty acids (docosahexaenoic acid) and n-6 fatty acid (arachidonic acid) in 9-month-old AD mice. On the other hand, ratios of total n-6 to total n-3 fatty acids were significantly higher in 9-month-old AD mice than in the same age of B6/SJL mice. Taken together, our present data support a role for the interactions of amyloid-β peptide and neuronal membranes in the subsequent development of AD. Special issue article in honor of Dr. George DeVries.     

Dysregulation of Calcium Homeostasis in Alzheimer’s Disease

The accumulation of oligomeric species of β-amyloid protein in the brain is considered to be a key factor that causes Alzheimer’s disease (AD). However, despite many years of research, the mechanism of neurotoxicity in AD remains obscure. Recent evidence strongly supports the theory that Ca²⁺ dysregulation is involved in AD. Amyloid proteins have been found to induce Ca²⁺ influx into neurons, and studies on transgenic mice suggest that this Ca²⁺ influx may alter neuronal excitability. The identification of a risk factor gene for AD that may be involved in the regulation of Ca²⁺ homeostasis and recent findings which suggest that presenilins may be involved in the regulation of intracellular Ca²⁺ stores provide converging lines of evidence that support the idea that Ca²⁺ dysregulation is a key step in the pathogenesis of AD. Special issue article in Honor of Dr. Graham Johnston.     

Immunotherapy for Alzheimer disease

Immunotherapy approaches for Alzheimer disease currently are among the leading therapeutic directions for the disease. Active and passive immunotherapy against the β-amyloid peptides that aggregate and accumulate in the brain of those afflicted by the disease have been shown by numerous groups to reduce plaque pathology and improve behavior in transgenic mouse models of the disease. Several ongoing immunotherapy clinical trials for Alzheimer disease are in progress. The background and ongoing challenges for these immunological approaches for the treatment of Alzheimer disease are discussed.Key words: Alzheimer disease, amyloid, tau, immunotherapy, vaccineThe publication in Nature on a vaccine approach for Alzheimer disease (AD) by Schenk and colleagues in 1999 initiated a push for treatment for this major disease of aging. AD neuropathology is characterized by the progressive loss of synapses and neurons, and the aberrant accumulation in the brain of β-amyloid peptides in plaques and the microtubule associated protein tau in neurofibrillary tangles. Mutations in familial forms of AD have been associated with elevated β-amyloid levels, whereas mutations in tau have been linked to familial forms of frontotemporal dementia. Remarkably, injection of β-amyloid peptides with Freund''s adjuvant into transgenic mice harboring a human AD mutation that develop AD-like neuropathology and progressive cognitive decline led to reduced β-amyloid plaque pathology.¹ This study was subsequently confirmed and extended by multiple groups to show also behavioral improvement in AD transgenic mice with active β-amyloid immunization.^2,3 Passive immunotherapy with antibodies directed at β-amyloid were similarly effective in reducing plaques and improving behavior in AD transgenic mice.⁴ A temporary setback occurred when the first clinical trial with β-amyloid vaccination was halted after 6% of patients developed an inflammatory reaction in the brain (chemical meningoencephalitis). A subsequent study supported clinical benefits among patients in this active vaccination trial.⁵ A more recent postmortem study on a subset of patients who had participated in the aborted trial supported active removal of β-amyloid plaques by inflammatory cells, but also indicated that 7 of the 8 patients who were studied at autopsy continued to have progressive cognitive decline despite the removal of amyloid plaques.⁶The critical mechanisms whereby active or passive vaccination against β-amyloid can affect the disease process remain uncertain. Recruitment and activation of microglia, the macrophage of the central nervous system, by β-amyloid antibodies is thought to lead to β-amyloid plaque removal. At the same time, fibrillar β-amyloid containing plaques, formerly viewed as the major toxic entities in AD, are increasingly viewed as potentially only pathological remnants of the disease. Smaller assemblies, particularly of two to twelve β-amyloid peptides (oligomers), are considered pathogenic, although the site of pathogenesis remains controversial. Secreted, extracellular β-amyloid oligomers have been shown to damage synapses.⁷ Some groups stress the aberrant accumulation of β-amyloid within neurons and synapses leading to subsequent extracellular localization following destruction of neurites and synapses.⁸ Evidence has been presented that antibodies targeting β-amyloid peptides up to 42–43 amino acids can block the toxic effects of extracellular β-amyloid oligomers on synapses.⁷ Interestingly, β-amyloid immunotherapy was also shown to clear intraneuronal β-amyloid in an AD transgenic mouse; the intraneuronal variety is a pool of β-amyloid that correlates with the onset of cognitive decline prior to plaques and tangles in these mice.⁹ Intriguingly, antibodies directed at the β-amyloid domain exposed to the extracellular space within the amyloid precursor protein (APP) were shown to be internalized by neurons, where they reduced the intraneuronal pool of β-amyloid and protected against synaptic damage in neurons cultured from AD transgenic mice.^10,11 It is possible that inefficient clearance of the intracellular pool of β-amyloid played a role in the continued cognitive decline in the seven of eight patients in the aborted active vaccination clinical trial studied at autopsy who showed clearance of β-amyloid plaques.Work on β-amyloid immunotherapy in AD contributed to a reevaluation of the role of the immune system in the brain. Previously, it was considered that the brain was immune privileged, and that antibodies entered the brain only with the breakdown of the blood brain barrier. Rare neuroimmunological disorders had suggested more complex interactions. Pathological antibodies directed at neuronal proteins could be found localizing to specific types of neurons in paraneoplastic diseases linked to diverse systemic cancers^12,13 or collagen-vascular diseases such as lupus.¹⁴ Such pathological antibodies can be directed at synaptic or even intracellular proteins in selective neurons in the brain, leading to localized neurological symptoms. For paraneoplastic diseases it is hypothesized that antibodies directed at the cancer cells cross-react with neuronal antigens. Since titers of antibodies can be higher in brain than in the blood, intrathecal synthesis of antibodies from sequestration of B cells has been proposed to occur in the brain.¹⁵ The interaction between the immune system and the brain is therefore viewed as increasingly complex, with antibodies not only gaining access to the brain but also nerve cells, where they can even alter intracellular biology.¹⁰ These findings open up new possibilities for antibody-directed therapies for diseases of the nervous system.Currently, leading concerns for β-amyloid immunotherapy are the potential development of chemical meningoencephalitis and micro-hemorrhages in the brain. Involvement of T cells in damage to the brain vasculature is considered to contribute to these potential side effects. In addition, the β-amyloid released upon antibody-induced removal of plaques may damage blood vessels as β-amyloid is cleared from the brain via the vasculature.¹⁶ Recently, a phase 2 Elan/Wyeth study using passive β-amyloid immunotherapy with a humanized monoclonal antibody described (at the 2008 International Conference on Alzheimer''s disease) significant benefits in patients not harboring the apolipoprotein E4 (apoE4) allele genetic risk factor for late onset AD. In contrast, no clear therapeutic benefit and more cases with brain inflammation occurred in those with the apoE4 allele linked with an increased risk for AD. Why apoE4 carriers did not benefit in this β-amyloid immunotherapy trial is unknown, but has prompted separation of patients into E4 negative and positive groups in subsequent clinical trials. The less robust than hoped for effects even in the apoE4 negative patients has further dampened expectations. The reason for why the human studies are not showing the protection seen in the transgenic mouse studies could relate to β-amyloid playing less of a role in the more typical late onset AD than it does in the rare autosomal dominant familial forms used to generate the AD transgenic mice. It is also not clear which β-amyloid epitope(s) should be targeted by antibodies to maximize potential benefits while minimizing side effects in AD patients. Optimizing antibody specificity for immunotherapy is further complicating by the varied conformations of different β-amyloid aggregation states. In addition, β-amyloid immunotherapy may be more challenging in patients with AD because it is not effective in reducing tau tangle pathology.⁶ Most immunotherapy studies were done on transgenic AD mouse models that deposit β-amyloid plaques, but not tau tangles. In the more recently generated triple transgenic AD mouse that develops both plaques and tangles, β-amyloid antibodies reversed β-amyloid pathology and early pre-tangle tau pathology, but not hyperphosphorylated tau aggregates.⁸ Recent evidence supports that β-amyloid neurotoxicity acts synergistic with tau,¹⁷ and that both pathologies begin at synapses.¹⁸ Interestingly, tau immunotherapy was reported to protect against tau pathology in transgenic mice harboring mutant tau.¹⁹ Thus, dual immunotherapy targeting of both β-amyloid and tau can be considered. Finally, immunotherapy at earlier stages of the disease process may be more effective.In summary, the β-amyloid vaccine heralded a new era of therapeutic research for AD and despite some setbacks is actively being pursued in several ongoing clinical trials. It continues to be among the leading hopes in the AD research community. Another major effort to specifically block the generation of β-amyloid is also progressing, although not without setbacks along the way. For example, the protease involved in the final cleavage to liberate β-amyloid was found to be involved in multiple other important activities, such as cleavage of Notch. Antibody approaches are also being applied in efforts to block secretase cleavage to generate β-amyloid.²⁰ Finally, there remains some worry that β-amyloid peptides have an as yet unknown normal biological function, although cumulative immunotherapy and other therapeutic studies in animal models have provided sufficient support for the continued pursuit of β-amyloid lowering as a treatment for AD.     

Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation

We have investigated the production of C3, C4, and factor B complement components in primary cultures of murine astrocytes and in clonal cell lines belonging to the astrocytic lineage by immunoprecipitation of secreted labeled polypeptides. Although C4 has not been detected, C3 appeared to be constitutively synthesized both by two transformed astroblastic cell lines and by astrocytes in primary cultures. In contrast, factor B was only secreted upon lipopolysaccharide stimulation both in astroglial primary cultures and in an immortalized astrocytic cell line. The eventual physiologic relevance of an endogenous brain production of components of the alternative pathway of complement activation is discussed.     

Behavioral stress accelerates plaque pathogenesis in the brain of Tg2576 mice via generation of metabolic oxidative stress

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by genetic and non-genetic factors. Most AD cases may be triggered and promoted by non-genetic environmental factors. Clinical studies have reported that patients with AD show enhanced baseline levels of stress hormones in the blood, but their physiological significance with respect to the pathophysiology of AD is not clearly understood. Here we report that AD mouse models exposed to restraints for 2 h daily on 16 consecutive days show increased levels of β-amyloid (Aβ) plaque deposition and commensurable enhancements in Aβ(1–42), tau hyperphosphorylation, and neuritic atrophy of cortical neurons. Repeated restraints in Tg2576 mice markedly increased metabolic oxidative stress and down-regulated the expression of MMP-2, a potent Aβ-degrading enzyme, in the brain. These stress effects were reversed by blocking the activation of the hypothalamus-pituitary-adrenal gland axis with the corticotropin-releasing factor receptor antagonist NBI 27914, further suggesting that over-activation of the hypothalamic-pituitary-adrenal axis is required for stress-enhanced AD-like pathogenesis. Consistent with these findings, corticosteroid treatments to cultured primary cortical neurons increased metabolic oxidative stress and down-regulated MMP-2 expression, and MMP-2 down-regulation was reversed by inhibition of oxidative stress. These results suggest that behavioral stress aggravates AD pathology via generation of metabolic oxidative stress and MMP-2 down-regulation.     

Fibrillar Amyloid Plaque Formation Precedes Microglial Activation

In Alzheimer’s disease (AD), hallmark β-amyloid deposits are characterized by the presence of activated microglia around them. Despite an extensive characterization of the relation of amyloid plaques with microglia, little is known about the initiation of this interaction. In this study, the detailed investigation of very small plaques in brain slices in AD transgenic mice of the line APP-PS1(dE9) revealed different levels of microglia recruitment. Analysing plaques with a diameter of up to 10 μm we find that only the half are associated with clear morphologically activated microglia. Utilizing in vivo imaging of new appearing amyloid plaques in double-transgenic APP-PS1(dE9)xCX3CR1^+/- mice further characterized the dynamic of morphological microglia activation. We observed no correlation of morphological microglia activation and plaque volume or plaque lifetime. Taken together, our results demonstrate a very prominent variation in size as well as in lifetime of new plaques relative to the state of microglia reaction. These observations might question the existing view that amyloid deposits by themselves are sufficient to attract and activate microglia in vivo.