Reactive astrocytes and alpha1-antichymotrypsin in Alzheimer's disease.

There is ample genetic, biochemical, cellular and molecular evidence to show that the amyloid beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP), plays an important, if not causative role in Alzheimer's disease (AD). An additional hallmark of AD is the neuroinflammatory response that is associated with the amyloid deposition. We discovered that the acute phase protein alpha1-antichymotrypsin (ACT) is overexpressed by reactive astrocytes, and is tightly associated with virtually all amyloid plaques in the AD brain. It has also been shown that Abeta and ACT bind in vitro. Recently, we have reported that astrocytic expression of ACT in APP transgenic mice leads to an increased plaque deposition in ACT/APP doubly transgenic mice compared to the APP mice alone, suggesting that ACT interferes with Abeta clearance. The main objective of this review is to summarize the role of astrocytosis and ACT in the pathogenesis of AD.     

Alterations in beta-amyloid production and deposition in brain regions of two transgenic models

Mutations in the amyloid precursor protein (APP) gene are associated with altered production and deposition of amyloid beta (Abeta) peptide in the Alzheimer's disease (AD) brain. The pathways that regulate APP processing, Abeta production and Abeta deposition in different tissues and brain regions remain unclear. To address this, we examined levels of various APP processing products as well as Abeta deposition in a genomic-based (R1.40) and a cDNA-based (Tg2576) transgenic mouse model of AD. In tissues, only brain generated detectable levels of the penultimate precursor to Abeta, APP C-terminal fragment-beta. In brain regions, holoAPP levels remained constant, but ratios of APP C-terminal fragments and levels of Abeta differed significantly. Surprisingly, cortex had the lowest steady-state levels of Abeta compared to other brain regions. Comparison of Abeta deposition in Tg2576 and R1.40 animals revealed that R1.40 exhibited more abundant deposition in cortex while Tg2576 exhibited extensive deposition in the hippocampus. Our results suggest that AD transgenic models are not equal; their unique characteristics must be considered when studying AD pathogenesis and therapies.     

Is there a future for vaccination as a treatment for Alzheimer's disease?

Vaccination of APP transgenic mice with Abeta has been shown to prevent amyloid deposits. A clinical trial of Abeta vaccination in Alzheimer's disease (AD) was halted due to serious neurological complications developing in some patients. Such complications were not observed in transgenic mice. Since human APP is not a mouse self-protein, vaccination of mice with Abeta should not produce an autoimmune reaction although this would be anticipated in AD. Moreover, mouse C1q poorly recognizes human Abeta so complement activation is much weaker in transgenic mice than in AD. Vaccination will increase complement activation through formation of antigen-antibody complexes. In mice this will enhance phagocytosis. But in AD, where complement is already overactivated, and where the senile plaques are relatively insoluble, this stimulation should increase production of the membrane attack complex, adding to the autodestruction of neurons. The future of vaccination as a therapy for AD will require surmounting the problems of autoimmune reactions generally and autotoxic complement activation specifically.     

Prominent cerebral amyloid angiopathy in transgenic mice overexpressing the london mutant of human APP in neurons

Deposition of amyloid beta-peptide (Abeta) in cerebral vessel walls (cerebral amyloid angiopathy, CAA) is very frequent in Alzheimer's disease and occurs also as a sporadic disorder. Here, we describe significant CAA in addition to amyloid plaques, in aging APP/Ld transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP) exclusively in neurons. The number of amyloid-bearing vessels increased with age, from approximately 10 to >50 per coronal brain section in APP/Ld transgenic mice, aged 13 to 24 months. Vascular amyloid was preferentially deposited in arterioles and ranged from small focal to large circumferential depositions. Ultrastructural analysis allowed us to identify specific features contributing to weakening of the vessel wall and aneurysm formation, ie, disruption of the external elastic lamina, thinning of the internal elastic lamina, interruption of the smooth muscle layer, and loss of smooth muscle cells. Biochemically, the much lower Abeta42:Abeta40 ratio evident in vascular relative to plaque amyloid, demonstrated that in blood vessel walls Abeta40 was the more abundant amyloid peptide. The exclusive neuronal origin of transgenic APP, the high levels of Abeta in cerebrospinal fluid compared to plasma, and the specific neuroanatomical localization of vascular amyloid strongly suggest specific drainage pathways, rather than local production or blood uptake of Abeta as the primary mechanism underlying CAA. The demonstration in APP/Ld mice of rare vascular amyloid deposits that immunostained only for Abeta42, suggests that, similar to senile plaque formation, Abeta42 may be the first amyloid to be deposited in the vessel walls and that it entraps the more soluble Abeta40. Its ability to diffuse for larger distances along perivascular drainage pathways would also explain the abundance of Abeta40 in vascular amyloid. Consistent with this hypothesis, incorporation of mutant presenilin-1 in APP/Ld mice, which resulted in selectively higher levels of Abeta42, caused an increase in CAA and senile plaques. This mouse model will be useful in further elucidating the pathogenesis of CAA and Alzheimer's disease, and will allow testing of diagnostic and therapeutic strategies.     

Antibodies from a DNA peptide vaccination decrease the brain amyloid burden in a mouse model of Alzheimer’s disease

The neuropathology of Alzheimer's disease(AD) is characterized by the accumulation of amyloid peptide Abeta in the brain derived from proteolytic cleavage of the amyloid precursor protein (APP). Vaccination of mice with plasmid DNA coding for the human Abeta42 peptide together with low doses of preaggregated peptide induced antibodies with detectable titers after only 2 weeks. One serum was directed against the four aminoterminal amino acids DAEF and differs from previously described ones. Both immune sera and monoclonal antibodies solubilized preformed aggregates of Abeta42 in vitro and recognized amyloid plaques in brain sections of mice transgenic for human APP. Passive immunization of transgenic AD mice caused a significant and rapid reduction in brain amyloid plaques within 24 h. The combined DNA peptide vaccine may prove useful for active immunization with few inoculations and low peptide dose which may prevent the recently described inflammatory reactions inpatients. The monoclonal antibodies are applicable for passive immunization studies and may lead to a therapy of AD.     

Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis

The E693Q mutation in the amyloid beta precursor protein (APP) leads to cerebral amyloid angiopathy (CAA), with recurrent cerebral hemorrhagic strokes and dementia. In contrast to Alzheimer disease (AD), the brains of those affected by hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) show few parenchymal amyloid plaques. We found that neuronal overexpression of human E693Q APP in mice (APPDutch mice) caused extensive CAA, smooth muscle cell degeneration, hemorrhages and neuroinflammation. In contrast, overexpression of human wild-type APP (APPwt mice) resulted in predominantly parenchymal amyloidosis, similar to that seen in AD. In APPDutch mice and HCHWA-D human brain, the ratio of the amyloid-beta40 peptide (Abeta40) to Abeta42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AbetaDutch40/AbetaDutch42 toward AbetaDutch42 by crossing APPDutch mice with transgenic mice producing mutated presenilin-1 redistributed the amyloid pathology from the vasculature to the parenchyma. The understanding that different Abeta species can drive amyloid pathology in different cerebral compartments has implications for current anti-amyloid therapeutic strategies. This HCHWA-D mouse model is the first to develop robust CAA in the absence of parenchymal amyloid, highlighting the key role of neuronally produced Abeta to vascular amyloid pathology and emphasizing the differing roles of Abeta40 and Abeta42 in vascular and parenchymal amyloid pathology.     

Extracellular amyloid formation and associated pathology in neural grafts

Amyloid precursor protein (APP) processing and the generation of beta-amyloid peptide (Abeta) are important in the pathogenesis of Alzheimer's disease. Although this has been studied extensively at the molecular and cellular levels, much less is known about the mechanisms of amyloid accumulation in vivo. We transplanted transgenic APP23 and wild-type B6 embryonic neural cells into the neocortex and hippocampus of both B6 and APP23 mice. APP23 grafts into wild-type hosts did not develop amyloid deposits up to 20 months after grafting. In contrast, both transgenic and wild-type grafts into young transgenic hosts developed amyloid plaques as early as 3 months after grafting. Although largely diffuse in nature, some of the amyloid deposits in wild-type grafts were congophilic and were surrounded by neuritic changes and gliosis, similar to the amyloid-associated pathology previously described in APP23 mice. Our results indicate that diffusion of soluble Abeta in the extracellular space is involved in the spread of Abeta pathology, and that extracellular amyloid formation can lead to neurodegeneration.     

Cyclooxygenase-2 promotes amyloid plaque deposition in a mouse model of Alzheimer's disease neuropathology

Several epidemiologic studies have reported that cyclooxygenase (COX) inhibitors prevent/delay the onset of Alzheimer's disease (AD). Recent experimental studies suggest that these compounds can also diminish amyloid-beta (Abeta) neuropathology in rodent models of AD. To explore the relationship of COX expression to Abeta neuropathology, we crossed mice expressing both mutant amyloid precursor protein [K670N/M671L (APP(swe)] and mutant PS1 (A246E) with mice expressing human COX-2 selectively in neurons. We show here that human COX-2 expression in APP(swe)/PS1/COX-2 mice induces potentiation of brain parenchymal amyloid plaque formation and a greater than twofold increase in prostaglandin E2 production, at 24 months of age. This increased amyloid plaque formation coincided with a preferential elevation of Abeta1-40 and Abeta1-42 with no change in total amyloid precursor protein (APP) expression/content in the brain. Collectively these data suggest that COX-2 influences APP processing and promotes amyloidosis in the brain.     

Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice

We have shown that interaction of CD40 with CD40L enables microglial activation in response to amyloid-beta peptide (Abeta), which is associated with Alzheimer's disease (AD)-like neuronal tau hyperphosphorylation in vivo. Here we report that transgenic mice overproducing Abeta, but deficient in CD40L, showed decreased astrocytosis and microgliosis associated with diminished Abeta levels and beta-amyloid plaque load. Furthermore, in the PSAPP transgenic mouse model of AD, a depleting antibody against CD40L caused marked attenuation of Abeta/beta-amyloid pathology, which was associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased circulating levels of Abeta. Conversely, in neuroblastoma cells overexpressing wild-type human APP, the CD40-CD40L interaction resulted in amyloidogenic APP processing. These findings suggest several possible mechanisms underlying mitigation of AD pathology in response to CD40L depletion, and validate the CD40-CD40L interaction as a target for therapeutic intervention in AD.     

Hypoxia increases Abeta generation by altering beta- and gamma-cleavage of APP

Environmental factors are significant contributors for the development of Alzheimer's disease (AD). The greatly increased incidence of AD following stroke and cerebral ischemia suggests that hypoxia is a risk factor which may accelerate AD pathogenesis by altering amyloid precursor protein (APP) processing. However, the molecular mechanisms underlying the hypoxia mediated AD pathogenesis have not been fully elucidated. In the present study we demonstrated that repeated hypoxia increased beta-amyloid (Abeta) generation and neuritic plaques formation by elevating beta-cleavage of APP in APP(swe)+PS1(A246E) transgenic mice. We also found that hypoxia enhanced the expression of APH-1a, a component of gamma-secretase complex, which in turn may lead to increase in gamma-cleavage activity. Furthermore, we demonstrated that repeated hypoxia treatment can activate macroautophagy, which may contribute to the increases in Abeta production since pretreatment with macroautophagy inhibitor 3-methyladenine significantly blocked chemical hypoxic condition-induced increase in Abeta production in SH-SY5Y cells. Taken together, our results suggest an important role of hypoxia in modulating the APP processing by facilitating both beta- and gamma-cleavage which may result in a significant increase of Abeta generation.     

Noradrenaline deficiency in brain increases beta-amyloid plaque burden in an animal model of Alzheimer's disease

Loss of Locus coeruleus (LC) noradrenergic (NA) neurons occurs in several neurodegenerative conditions including Alzheimer's disease (AD). In vitro and in vivo studies have shown that NA influences several features of AD disease including inflammation, neurodegeneration, and cognitive function. In the current study we tested if LC loss influenced beta amyloid (Abeta) plaque deposition. LC neuronal degeneration was induced in transgenic mice expressing mutant V717F human amyloid precursor protein (APP) by treatment with the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine DSP4 (5mg/kg every 2 weeks beginning at age 3 months). At 9 months of age, when control mice show low amyloid load, DSP4-treated mice showed an approximately 5-fold increase in the average number of Abeta plaques. This was accompanied by an increase in the levels of APP C-terminal cleavage fragments. DSP4-treatment increased both microglial and astroglial activation. In vivo, DSP4-treatment decreased expression and activity of the Abeta degrading enzyme neprilysin, while in vitro NA increased phagocytosis of Abeta1-42 by microglia. These findings suggest that noradrenergic innervation from LC are needed to maintain adequate Abeta clearance, and therefore that LC degeneration could contribute to AD pathogenesis.     

Spatial and temporal control of age-related APP processing in genomic-based beta-secretase transgenic mice

Genetic mutations associated with Alzheimer's disease (AD) in the Amyloid Precursor Protein (APP) gene specifically alter the production of the APP processing product, amyloid-beta (Abeta) peptide, generated by beta- and gamma-secretases. The accumulation and deposition of Abeta is hypothesized to cause AD pathogenesis, leading to the debilitating neurological deficits observed in AD patients. However, it is unclear how processing of APP to generate Abeta corresponds with the age-dependent pattern of brain-regional neurodegeneration common in AD. We have previously shown that overexpression of BACE1, the primary beta-secretase gene, in mice expressing an AD mutant form of APP leads to significantly elevated regional Abeta levels, which coincide with the regional pattern of Abeta deposition. In the current study, we have used our genomic-based beta-secretase transgenic mice to determine how BACE1 regulates the spatial and temporal pattern of Abeta production throughout post-natal development. Specifically, we observed unique differences in the brain-regional expression pattern between neonatal and adult BACE1 transgenic mice. These alterations in the BACE1 expression profile directly corresponds with age-related differences in regional Abeta production and deposition. These studies indicate that modulation of BACE1 expression leads to dramatic alterations in APP processing and AD-like neuropathology. Furthermore, our studies provide further evidence that BACE1 plays a major role in the regulation of the APP processing pathway, influencing the age-dependent onset of AD pathogenesis.     

Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression

Alzheimer's disease (AD) is a complex, neurodegenerative disease characterized by the impairment of cognitive function in elderly individuals. In a recent global gene expression study of APP transgenic mice, we found elevated expression of mitochondrial genes, which we hypothesize represents a compensatory response because of mitochondrial oxidative damage caused by the over-expression of mutant APP and/or amyloid beta (Abeta). We investigated this hypothesis in a series of experiments examining what forms of APP and Abeta localize to the mitochondria, and whether the presence of these species is associated with mitochondrial dysfunction and oxidative damage. Using immunoblotting, digitonin fractionation, immunofluorescence, and electron microscopy techniques, we found a relationship between mutant APP derivatives and mitochondria in brain slices from Tg2576 mice and in mouse neuroblastoma cells expressing mutant human APP. Further, to determine the functional relationship between mutant APP/Abeta and oxidative damage, we quantified Abeta levels, hydrogen peroxide production, cytochrome oxidase activity and carbonyl proteins in Tg2576 mice and age-matched wild-type (WT) littermates. Hydrogen peroxide levels were found to be significantly increased in Tg2576 mice when compared with age-matched WT littermates and directly correlated with levels of soluble Abeta in Tg2576 mice, suggesting that soluble Abeta may be responsible for the production of hydrogen peroxide in AD progression in Tg2576 mice. Cytochrome c oxidase activity was found to be decreased in Tg2576 mice when compared with age-matched WT littermates, suggesting that mutant APP and soluble Abeta impair mitochondrial metabolism in AD development and progression. An increase in hydrogen peroxide and a decrease in cytochrome oxidase activity were found in young Tg2576 mice, prior to the appearance of Abeta plaques. These findings suggest that early mitochondrially targeted therapeutic interventions may be effective in delaying AD progression in elderly individuals and in treating AD patients.     

Thiamine deficiency induces oxidative stress and exacerbates the plaque pathology in Alzheimer's mouse model

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimer's disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine-dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Abeta(1-42) levels by about three fold, beta-CTF (C99) levels by 33% and beta-secretase (BACE1) protein levels by 43%. TD-induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Abeta and promotes accumulation of plaques independent of neuron loss or neuritic clusters.     

Increased cerebrospinal fluid levels of transforming growth factor-beta1 in Alzheimer's disease

Accumulation of beta-amyloid (Abeta) in senile plaques in specific brain regions is a key event in the development of Alzheimer's disease (AD). Expression of transforming growth factor-beta1 (TGF-beta1), a regulator of brain responses to inflammation and injury, has been correlated with Abeta accumulation, aggregation and clearance in transgenic mice and increased production of amyloid precursor protein (APP) followed by Abeta generation in murine and human astrocyte cultures. Here, we compared TGF-beta1 levels in cerebrospinal fluid (CSF) from 20 AD patients and 20 healthy controls and correlated TGF-beta1 to intrathecal levels of the amyloidogenic 42-amino acid fragment of Abeta (Abeta42). AD patients had higher concentration of TGF-beta1 than controls (P = 0.002). Moreover, TGF-beta1 levels were negatively correlated to Abeta42 levels in the whole material (cases and controls, r = -0.35; P = 0.020), although this correlation failed to reach significance in the AD group alone (r = -0.38; P = 0.099). Taken together, the data indicate that TGF-beta1 plays a role in the processes that affect amyloid metabolism in AD.     

Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice

Transgenic mice with brain amyloid-beta (Abeta) plaques immunized with aggregated Abeta1-42 have reduced cerebral amyloid burden. However, the use of Abeta1-42 in humans may not be appropriate because it crosses the blood brain barrier, forms toxic fibrils, and can seed fibril formation. We report that immunization in transgenic APP mice (Tg2576) for 7 months with a soluble nonamyloidogenic, nontoxic Abeta homologous peptide reduced cortical and hippocampal brain amyloid burden by 89% (P = 0.0002) and 81% (P = 0.0001), respectively. Concurrently, brain levels of soluble Abeta1-42 were reduced by 57% (P = 0.0019). Ramified microglia expressing interleukin-1beta associated with the Abeta plaques were absent in the immunized mice indicating reduced inflammation in these animals. These promising findings suggest that immunization with nonamyloidogenic Abeta derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in Alzheimer's disease, instead of using toxic Abeta fibrils.     

Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase

Amyloid precursor protein (APP) is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides (Abeta40 and 42) that aggregate to form senile plaques in the brains of patients with Alzheimer's disease (AD). The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin (PS 1 or 2). Missense mutations in PS1 (and PS2) occur in patients with early-onset familial AD (FAD), and previous studies in transgenic mice and cultured cell models demonstrated that FAD-PS1 variants shift the ratio of Abeta40 : 42 to favor Abeta42. One hypothesis to explain this outcome is that mutant PS alters the specificity of gamma-secretase to favor production of Abeta42 at the expense of Abeta40. To test this hypothesis in vivo, we studied Abeta40 and 42 levels in a series of transgenic mice that co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain. We demonstrate a direct correlation between the concentration of Abeta42 and the rate of amyloid deposition. We further show that the shift in Abeta42 : 40 ratios associated with the expression of FAD-PS1 variants is due to a specific elevation in the steady-state levels of Abeta42, while maintaining a constant level of Abeta40. These data suggest that PS1 variants do not simply alter the preferred cleavage site for gamma-secretase, but rather that they have more complex effects on the regulation of gamma-secretase and its access to substrates.     

Copper and clioquinol treatment in young APP transgenic and wildtype mice: effects on life expectancy,body weight and metal-ion levels

There is mounting evidence that the amyloid precursor protein (APP), the key protein in Alzheimer's disease (AD) is involved in the copper (Cu) homeostasis in the brain. Conflicting results about the potential use of dietary Cu and clioquinol (CQ), a known Cu chelator, have been reported using APP transgenic mice. Previously, in vitro studies have demonstrated that CQ can act as a Cu transporter. To analyze the potential function of CQ as a Cu transporter in vivo, the nutritional effect of Cu and CQ was analyzed in young APP transgenic mice and nontransgenics with food pellets containing either Cu, CQ, Cu plus CQ (Cu + CQ), or without addition of supplements (control). The offspring were fed with corresponding food pellets until the age of 14 weeks. We observed an increased lethality of APP transgenics upon CQ treatment, which could be rescued by a co-treatment with Cu. The exposure of Cu + CQ led to a modest but significant increase in cerebral Cu levels, most likely due to an enhanced transport of CQ-Cu complexes. In CQ or Cu + CQ treatment groups, the plasma levels of Cu, zinc, and iron were reduced in all animals; moreover, Cu treatment alone reduced only plasma iron levels. We conclude not only that CQ has certain toxicity but also that the chelating effect, perhaps, plays a secondary role with respect to its properties as an intracellular Cu transporter, thus, counteracting the supposed therapeutic effects of CQ as an agent for chelating therapy in AD.     

Reactive astrocytes and α1-antichymotrypsin in Alzheimer’s disease

There is ample genetic, biochemical, cellular and molecular evidence to show that the amyloid β peptide (Aβ), a proteolytic fragment of the amyloid precursor protein (APP), plays an important, if not causative role in Alzheimer’s disease (AD). An additional hallmark of AD is the neuroinflammatory response that is associated with the amyloid deposition. We discovered that the acute phase protein α1-antichymotrypsin (ACT) is overexpressed by reactive astrocytes, and is tightly associated with virtually all amyloid plaques in the AD brain. It has also been shown that Aβ and ACT bind in vitro. Recently, we have reported that astrocytic expression of ACT in APP transgenic mice leads to an increased plaque deposition in ACT/APP doubly transgenic mice compared to the APP mice alone, suggesting that ACT interferes with Aβ clearance. The main objective of this review is to summarize the role of astrocytosis and ACT in the pathogenesis of AD.