Co-expression of truncated and full-length tau induces severe neurotoxicity.

Abundant tau inclusions are a defining hallmark of several human neurodegenerative diseases, including Alzheimer's disease. Protein fragmentation is a widely observed event in neurodegenerative proteinopathies. The relevance of tau fragmentation for the neurodegenerative process in tauopathies has yet remained unclear. Here we found that co-expression of truncated and full-length human tau in mice provoked the formation of soluble high-molecular-weight tau, the failure of axonal transport, clumping of mitochondria, disruption of the Golgi apparatus and missorting of synaptic proteins. This was associated with extensive nerve cell dysfunction and severe paralysis by the age of 3 weeks. When the expression of truncated tau was halted, most mice recovered behaviorally and functionally. In contrast, co-expression of full-length tau isoforms did not result in paralysis. Truncated tau thus induces extensive but reversible neurotoxicity in the presence of full-length tau through the formation of nonfilamentous high-molecular-weight tau aggregates, in the absence of tau filaments. Targeting tau fragmentation may provide a novel approach for the treatment of human tauopathies.

[Mechanisms of Alzheimer's disease : Neuronal hyperactivity and hypoactivity as new therapeutic targets]. / Mechanismen der Alzheimer-Krankheit : Neuronale Hyper- und Hypoaktivität als neue Therapieziele.

Alzheimer's disease (AD) is characterized by the pathological accumulation of amyloid-beta (Abeta) and tau peptides in the brain. Recent evidence suggests that the soluble peptide amyloid-eta (Aeta) may have an additional role in the pathogenesis of AD. The detailed investigation of the cellular and neurophysiological mechanisms underlying AD has revealed surprising results that may become highly relevant for the early diagnosis and treatment of the disease. By analyzing the function of single neurons and large-scale networks in intact brains in vivo it has been shown that A-beta, tau and A-eta abnormally modulate brain activity and obviously unfold contrasting effects: while A-beta promotes neuronal hyperactivity as well as epileptiform activity, tau and A-eta reduce the activity of neurons. Promising new evidence from animal studies and humans with AD indicates that the treatment of hyperactivity may improve cognitive dysfunctions and even slow the underlying disease process.

Exposure to 56Fe-particle radiation accelerates electrophysiological alterations in the hippocampus of APP23 transgenic mice.

Abstract An unavoidable complication of space travel is exposure to high-charge, high-energy (HZE) particles. In animal studies, exposure of the CNS to HZE-particle radiation leads to neurological alterations similar to those seen in aging or Alzheimer's disease. In this study we examined whether HZE-particle radiation accelerated the age-related neuronal dysfunction that was previously described in transgenic mice overexpressing human amyloid precursor protein (APP). These APP23 transgenic mice exhibit age-related behavioral abnormalities and deficits in synaptic transmission. We exposed 7-week-old APP23 transgenic males to brain-only (56)Fe-particle radiation (600 MeV/nucleon; 1, 2, 4 Gy) and recorded synaptic transmission in hippocampal slices at 2, 6, 9, 14 and 18-24 months. We stimulated Schaeffer collaterals and recorded field excitatory postsynaptic potentials (fEPSP) and population spikes (PS) in CA1 neurons. Radiation accelerated the onset of age-related fEPSP decrements recorded at the PS threshold from 14 months of age to 9 months and reduced synaptic efficacy. At 9 months, radiation also reduced PS amplitudes. At 6 months, we observed a temporary deficit in paired-pulse inhibition of the PS at 2 Gy. Radiation did not significantly affect survival of APP23 transgenic mice. We conclude that irradiation of the brain with HZE particles accelerates Alzheimer's disease-related neurological deficits.

Age-dependent time course of cerebral brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 in APP23 transgenic mice.

Neurotrophins, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3), have repeatedly been shown to be involved in the pathophysiology of Alzheimer's disease (AD). Recent studies have claimed that these neurotrophic factors are important tools for therapeutic intervention in neurodegenerative diseases. So far, little is known about the age- and disease-modulated time course of cerebral neurotrophins. Therefore, we have studied protein concentrations of BDNF, NGF, and NT-3 in different brain areas and sciatic nerve, a neurotrophin-transporting peripheral nerve, in a well-characterized AD model of amyloid precursor protein-overexpressing rodents (APP23 mice) at the ages of 5.0, 10.5, and 20.0 months. In APP23 mice, there was a significant increase of BDNF and NGF in the frontal and occipital cortices (for BDNF also in the striatum) of old 20.0-month-old mice (with respect to median values up to 8.2-fold), which was highly correlated with amyloid concentrations of these brain areas. Median values of NGF and NT-3 showed up to a 6.0-fold age-dependent increase in the septum that was not detectable in APP23 mice. Hippocampus, olfactory bulb, and cerebellum (except NT-3) did not show substantial age- or genotype-related regulation of neurotrophins. In the sciatic nerve, BDNF and NGF levels are increased in5-month-old APP23 mice and decrease with age to control levels. In conclusion, APP23 mice show a genotype-dependent increase of cortical BDNF and NGF that is highly correlated with amyloid concentrations and may reflect an amyloid-related glia-derived neurotrophin secretion or an altered axonal transport of these neurotrophic factors.

Aged APP23 mice show a delay in switching to the use of a strategy in the Barnes maze.

Spatial learning and memory deficits in the APP23 transgenic mice have mainly been studied using the Morris water maze (MWM). However learning in the MWM relies on swimming abilities and may be confounded by the stressful nature of this test. We have therefore assessed spatial learning and memory in 12-month-old APP23 using a dry-land maze test developed by Barnes. Mice were given daily learning trials for a total of 41 successive days. After a 12-day interval the mice were re-tested for 4 additional days in order to examine the spatial memory retention. Immediately following this phase, reversal learning was examined for 13 additional days by moving the escape tunnel to the opposite position. During the initial learning phase, APP23 mice showed a significantly longer latency to find the escape tunnel as well as an increased number of errors compared to non-transgenic littermates. These deficits appeared to be due to a delay in switching from a "no strategy" to a spatial strategy. Indeed, this same delay in the use of spatial strategy was observed in the reversal phase of the study. Our results suggest that impairments in APP23 mice in learning and memory maze tests may be due to a specific deficit in the use of spatial strategy.

Neurobehavioral characterization of APP23 transgenic mice with the SHIRPA primary screen.

The SHIRPA primary screen comprises 40 measures covering various reflexes and basic sensorimotor functions. This multi-test battery was used to compare non-transgenic controls with APP23 transgenic mice, expressing the 751 isoform of human beta-amyloid precursor protein and characterized by amyloid deposits in parenchyma and vessel walls. The APP23 mice were distinguishable from controls by pathological limb reflexes, myoclonic jumping, seizure activity, and tail malformation. In addition, this mouse model of Alzheimer's disease was also marked by a crooked swimming trajectory. APP23 mice were also of lighter weight and were less inclined to stay immobile during a transfer arousal test. Despite the neurologic signs, APP23 transgenic mice were not deficient in stationary beam, coat-hanger, and rotorod tests, indicating intact motor coordination abilities.

Spatial learning and exploration of environmental stimuli in 24-month-old female APP23 transgenic mice with the Swedish mutation.

Transgenic mice overexpressing the betaAPP gene with the Swedish mutation under the control of the murine thy1 promoter show Alzheimer-like characteristics such as Abeta plaques in cerebral cortex and vessel walls. By comparison to age-matched non-transgenic controls, 2-year-old female APP23 transgenic mice crossed more segments in the open-field and had a higher number of fast ambulatory and stereotyped movements in a photocell activity chamber. In addition, APP23 mice entered more often and spent more time in the open arms of the elevated plus-maze. The acquisition of place learning in the Morris water maze was impaired in APP23 transgenic mice, but not in probe and visible platform subtasks. These results indicate that hyperactivity and impaired learning abilities characterize this mouse model of Alzheimer's disease and cerebral angiopathy.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease.

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Regional brain cytochrome oxidase activity in beta-amyloid precursor protein transgenic mice with the Swedish mutation.

Cytochrome oxidase activity was examined in a transgenic mouse model of Alzheimer's disease with overexpression of the 751 amino acid isoform of beta-amyloid precursor protein with the Swedish mutation under control of the murine thy-1 promoter. The neuritic plaques, abundantly localized in the hippocampus and anterior neocortical areas, showed a core devoid of enzymatic activity surrounded by higher cytochrome oxidase activity at the sites of the dystrophic neurites and activated glial cells. Quantitative measures, taken only in the healthy-appearing regional areas without neuritic plaques, were higher in numerous limbic and non-limbic regions of transgenic mice in comparison with controls. Enzymatic activity was higher in the dentate gyrus and CA2-CA3 region of the hippocampus, the anterior cingulate and primary visual cortex, two olfactory structures, the ventral part of the neostriatum, the parafascicularis nucleus of the thalamus, and the subthalamic nucleus. Brainstem regions anatomically related with altered forebrain regions were more heavily labeled as well, including the substantia nigra, the periaqueductal gray, the superior colliculus, the medial raphe, the locus coeruleus and the adjacent parabrachial nucleus, as well as the pontine nuclei, red nucleus, and trigeminal motor nucleus. Functional brain organization is discussed in the context of Alzheimer's disease. Although hypometabolism is generally observed in this pathology, the increased cytochrome oxidase activity obtained in these transgenic mice can be the result of a functional compensation on the surviving neurons, or of an early mitochondrial alteration related to increased oxidative damage.

Progressive age-related impairment of cognitive behavior in APP23 transgenic mice.

Transgenic APP23 mice expressing human APP(751) with the K670N/M671L mutation, were compared at ages 3, 18 or 25 months to non-transgenic littermates in passive avoidance and in a small and large Morris maze. The task in the smaller pool habituated their flight response to the platform. Impairments in passive avoidance and small pool performance in APP23 mice were clearly age-related. In the larger Morris maze APP23 mice at all ages were impaired in latency and distance swum before finding the platform. Identical performance of 18-month APP23 and controls in a visible platform condition indicates that the Morris maze performance deficit was not due to sensory, motor or motivational alterations. At age 3 months both groups initially unexpectedly avoided the visible platform, suggesting that in young mice neophobia may contribute significantly to performance in cognitive tests. In conclusion, APP23 mice exhibit both early behavioral impairment in the large Morris maze as well as impairments in passive avoidance and small pool performance that are marked only in old age.

Spatial learning, exploration, anxiety, and motor coordination in female APP23 transgenic mice with the Swedish mutation.

Transgenic mice overexpressing the betaAPP gene with the Swedish mutation under the control of the murine thy-1 promoter show Alzheimer-like characteristics including the accumulation of Abeta protein in the cerebral cortex. Female 16-month-old APP23 transgenic mice were compared to age-matched non-transgenic mice in behavioral tests measuring spatial learning, exploration of environmental stimuli, anxiety, and motor coordination. APP23 transgenic mice had fewer fast ambulatory movements, either fast or slow stereotypy movements, and slow rears in a photocell activity chamber. The acquisition of spatial learning in the Morris water maze was impaired in APP23 transgenic mice, but not during the probe test or while swimming towards a visible platform. Neither were there intergroup differences in tests of anxiety or motor coordination. These results indicate that a learning deficit and hypoactivity, concordant with the early stages of Alzheimer's disease, characterize this mouse model with Abeta accumulation.

Impaired hypoxic tolerance and altered protein binding of NADH in presymptomatic APP23 transgenic mice.

It is being discussed whether impairment of energy metabolism is a final common pathway of neurodegeneration or initiates the neurodegenerative cascade. The goal was to investigate hypoxic tolerance and oxidative energy metabolism in 4-month-old, presymptomatic B6-Tg(ThylAPP)23Sdz (APP23) mice, a transgenic mouse model of Alzheimer's disease. Posthypoxic recovery of the population spike amplitude in hippocampal region CA1 upon stimulation of Schaffer collaterals in region CA3 (15 min hypoxia, 45 min recovery) was 43+/-46% (mean+/-S.D.) vs. 19+/-35% (P<0.05) in slices from wild-type and transgenic animals, respectively. Fluorescence lifetime sensitive spectroscopy of NADH in the CA1 pyramidal cell layer (gate set for detection of protein-bound NADH) showed a wavelength maximum at 455.3+/-1.6 nm (mean+/-S.D.) in controls and 453.5+/-2.4 nm (P<0.05) in mutants. We conclude that hypoxic tolerance is impaired in presymptomatic APP23 mice and occurs prior to extracellular deposition of amyloid plaques. Impaired energy metabolism may thus partake in initiating the neurodegenerative cascade in a transgenic model of Alzheimer's disease. The blue shift of the spectrum of NADH in mutant mice indicates an altered protein microenvironment of energy metabolism under control conditions.

Expression of endothelial and inducible NOS-isoforms is increased in Alzheimer's disease, in APP23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology.

The nitric oxide-synthesizing enzyme nitric oxide synthase (NOS) is present in the mammalian brain in three different isoforms, two constitutive enzymes (i.e., neuronal, nNOS, and endothelial eNOS) and one inducible enzyme (iNOS). All three isoforms are aberrantly expressed in Alzheimer's disease giving rise to elevated levels of nitric oxide apparently involved in the pathogenesis of this disease by various different mechanisms including oxidative stress and activation of intracellular signalling mechanisms. It still is a matter of debate, however, whether the abnormal expression of NOS isoforms has some primary importance in the pathogenetic chain and might thus be a potential therapeutic target or only reflects a secondary effect that occurs at more advanced stages of the disease process. To tackle this question, we analysed the expression of both eNOS and iNOS in patients with sporadic AD, in transgenic mice expressing human amyloid precursor protein (APP) with the Swedish double mutation under control of the Thy1 promotor (APP23 mice), and after electrolytic cortical lesion in rat, an experimental paradigm associated with elevated expression of APP. In all three conditions, an astrocytosis was induced accompanied by a strong increase of both iNOS and eNOS. Both NOS isoforms were frequently though not always colocalized. Thus, based on the expression pattern of NOS isoforms three types of astrocytes, expressing only one of the two isoforms or both together could be distinguished. In both AD and transgenic mice eNOS-expressing astrocytes exceeded iNOS-expressing astrocytes in number. Astrocytes with elevated levels of iNOS or eNOS were constantly seen in direct association with Abeta-deposits in AD and transgenic mice and were found in the vicinity of the lesion site in the rat cortex. The results of the present study show that expression of both iNOS and eNOS is increased in activated astrocytes under experimental conditions associated with elevated expression of APP (electrolytic brain lesion) or Abeta-deposition (APP23 transgenic mice). Therefore, it is suggested that altered expression of these NOS isoforms being part of AD pathology is secondary to the amyloid pathology and might not be primarily involved in the pathogenetic chain though it might contribute to the maintenance, self-perpetuation and progression of the neurodegenerative process.

3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.

Microglia cells are closely associated with compact amyloid plaques in Alzheimer's disease (AD) brains. Although activated microglia seem to play a central role in the pathogenesis of AD, mechanisms of microglial activation by beta-amyloid as well as the nature of interaction between amyloid and microglia remain poorly understood. We previously reported a close morphological association between activated microglia and congophilic amyloid plaques in the brains of APP23 transgenic mice at both the light and electron microscopic levels [25]. In the present study, we have further examined the structural relationship between microglia and amyloid deposits by using postembedding immunogold labeling, serial ultrathin sectioning, and 3-dimensional reconstruction. Although bundles of immunogold-labeled amyloid fibrils were completely engulfed by microglial cytoplasm on single sections, serial ultrathin sectioning and three-dimensional reconstruction revealed that these amyloid fibrils are connected to extracellular amyloid deposits. These data demonstrate that extracellular amyloid fibrils form a myriad of finger-like channels with the widely branched microglial cytoplasm. We conclude that in APP23 mice a role of microglia in amyloid phagocytosis and intracellular production of amyloid is unlikely.

Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy.

A high risk factor for spontaneous and often fatal lobar hemorrhage is cerebral amyloid angiopathy (CAA). We now report that CAA in an amyloid precursor protein transgenic mouse model (APP23 mice) leads to a loss of vascular smooth muscle cells, aneurysmal vasodilatation, and in rare cases, vessel obliteration and severe vasculitis. This weakening of the vessel wall is followed by rupture and bleedings that range from multiple, recurrent microhemorrhages to large hematomas. Our results demonstrate that, in APP transgenic mice, the extracellular deposition of neuron-derived beta-amyloid in the vessel wall is the cause of vessel wall disruption, which eventually leads to parenchymal hemorrhage. This first mouse model of CAA-associated hemorrhagic stroke will now allow development of diagnostic and therapeutic strategies.

Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice.

A microglial response is part of the inflammatory processes in Alzheimer's disease (AD). We have used APP23 transgenic mice overexpressing human amyloid precursor protein with the Swedish mutation to characterize this microglia response to amyloid deposits in aged mice. Analyses with MAC-1 and F4/80 antibodies as well as in vivo labeling with bromodeoxyuridine demonstrate that microglia in the plaque vicinity are in an activated state and that proliferation contributes to their accumulation at the plaque periphery. The amyloid-induced microglia activation may be mediated by scavenger receptor A, which is generally elevated, whereas the increased immunostaining of the receptor for advanced glycation end products is more restricted. Although components of the phagocytic machinery such as macrosialin and Fc receptors are increased in activated microglia, efficient clearance of amyloid is missing seemingly because of the lack of amyloid-bound autoantibodies. Similarly, although up-regulation of major histocompatibility complex class II (IA) points toward an intact antigen-presenting function of microglia, lack of T and B lymphocytes does not indicate a cell-mediated immune response in the brains of APP23 mice. The similar characteristics of microglia in the APP23 mice and in AD render the mouse model suitable to study the role of inflammatory processes during AD pathogenesis.

Comparative analysis of amyloid-beta chemical structure and amyloid plaque morphology of transgenic mouse and Alzheimer's disease brains.

We have undertaken an integrated chemical and morphological comparison of the amyloid-beta (Abeta) molecules and the amyloid plaques present in the brains of APP23 transgenic (tg) mice and human Alzheimer's disease (AD) patients. Despite an apparent overall structural resemblance to AD pathology, our detailed chemical analyses revealed that although the amyloid plaques characteristic of AD contain cores that are highly resistant to chemical and physical disruption, the tg mice produced amyloid cores that were completely soluble in buffers containing SDS. Abeta chemical alterations account for the extreme stability of AD plaque core amyloid. The corresponding lack of post-translational modifications such as N-terminal degradation, isomerization, racemization, pyroglutamyl formation, oxidation, and covalently linked dimers in tg mouse Abeta provides an explanation for the differences in solubility between human AD and the APP23 tg mouse plaques. We hypothesize either that insufficient time is available for Abeta structural modifications or that the complex species-specific environment of the human disease is not precisely replicated in the tg mice. The appraisal of therapeutic agents or protocols in these animal models must be judged in the context of the lack of complete equivalence between the transgenic mouse plaques and the human AD lesions.

The evolution of A beta peptide burden in the APP23 transgenic mice: implications for A beta deposition in Alzheimer disease.

BACKGROUND: High levels of A beta in the cerebral cortex distinguish demented Alzheimer's disease (AD) from nondemented elderly individuals, suggesting that decreased amyloid-beta (A beta) peptide clearance from the brain is a key precipitating factor in AD. MATERIALS AND METHODS: The levels of A beta in brain and plasma as well as apolipoprotein E (ApoE) in brain were investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting at various times during the life span of the APP23 transgenic (Tg) and control mice. Histochemistry and immunocytochemistry were used to assess the morphologic characteristics of the brain parenchymal and cerebrovascular amyloid deposits and the intracellular amyloid precursor protein (APP) deposits in the APP23 Tg mice. RESULTS: No significant differences were found in the plasma levels of A beta between the APP23 Tg and control mice from 2-20 months of age. In contrast, soluble A beta levels in the brain were continually elevated, increasing 4-fold at 2 months and 33-fold in the APP23 Tg mice at 20 months of age when compared to the control mice. Soluble A beta42 was about 60% higher than A beta40. In the APP23 Tg mice, insoluble A beta40 remained at basal levels in the brain until 9 months and then rose to 680 microg/g cortex by 20 months. Insoluble A beta40 was negligible in non-Tg mice at all ages. Insoluble A beta42 in APP23 Tg mice rose to 60 microg/g cortex at 20 months, representing 24 times the control A beta42 levels. Elevated levels of ApoE in the brain were observed in the APP23 Tg mice at 2 months of age, becoming substantially higher by 20 months. ApoE colocalized with A beta in the plaques. Beta-amyloid precursor protein (betaAPP) deposits were detected within the neuronal cytoplasm from 4 months of age onward. Amyloid angiopathy in the APP23 Tg mice increased markedly with age, being by far more severe than in the Tg2576 mice. CONCLUSIONS: We suggest that the APP23 Tg mouse may develop an earlier blockage in A beta clearance than the Tg2576 mice, resulting in a more severe accumulation of A beta in the perivascular drainage pathways and in the brain. Both Tg mice reflect decreased A beta elimination and as models for the amyloid cascade they are useful to study AD pathophysiology and therapy.

Transgenic mouse models of Alzheimer's disease.

Alzheimer's disease (AD) pathology is characterized by A beta peptide-containing plaques, neurofibrillary tangles consisting of hyperphosphorylated tau, extensive neuritic degeneration, and distinct neuron loss. We generated several transgenic mouse lines expressing the human amyloid precursor protein (APP751) containing the AD-linked KM670/671NL double mutation (Swedish mutation) under the control of a neuron-specific Thy-1 promoter fragment. In the best APP-expressing line (APP23), compact A beta deposits can be detected at 6 months of age. These plaques dramatically increase with age, are mostly Congo Red positive, and accumulate typical plaque-associated proteins such as heparansulfate proteoglycan and apolipoprotein E. Activated astrocytes and microglia indicative of inflammatory processes reminiscent of AD accumulate around the deposits. Furthermore, plaques are surrounded by enlarged dystrophic neurites as visualized by neurofilament or Holmes-Luxol staining. Strong staining for acetylcholinesterase activity is found throughout the plaques and is accompanied by local distortion of the cholinergic fiber network. All congophilic plaques contain hyperphosphorylated tau reminiscent of early tau pathology. Modern stereologic methods demonstrate a significant loss of neurons in the hippocampal CA1 region, correlating with an increasing A beta plaque load. Interestingly, APP23 mice develop cerebral amyloid angiopathy in addition to amyloid plaques even though the APP transgene is only expressed in neurons. Crossbreeding of APP23 mice with transgenic mice carrying AD-linked presenilin mutations but not wild-type presenilin resulted in enhanced formation of pathology. In conclusion, our APP transgenic mice present many pathologic features, similar to those observed in AD and therefore offer excellent tools for studying the contribution of A beta to AD pathogenesis.