Seeding, maturation and propagation of amyloid ß-peptide aggregates in Alzheimer's disease.

Alzheimer's disease is neuropathologically characterized by the deposition of the amyloid ß-peptide (Aß) as amyloid plaques. Aß plaque pathology starts in the neocortex before it propagates into further brain regions. Moreover, Aß aggregates undergo maturation indicated by the occurrence of post-translational modifications. Here, we show that propagation of Aß plaques is led by presumably non-modified Aß followed by Aß aggregate maturation. This sequence was seen neuropathologically in human brains and in amyloid precursor protein transgenic mice receiving intracerebral injections of human brain homogenates from cases varying in Aß phase, Aß load and Aß maturation stage. The speed of propagation after seeding in mice was best related to the Aß phase of the donor, the progression speed of maturation to the stage of Aß aggregate maturation. Thus, different forms of Aß can trigger propagation/maturation of Aß aggregates, which may explain the lack of success when therapeutically targeting only specific forms of Aß.

Aß-induced acceleration of Alzheimer-related τ-pathology spreading and its association with prion protein.

Extracellular deposition of amyloid ß-protein (Aß) in amyloid plaques and intracellular accumulation of abnormally phosphorylated τ-protein (p-τ) in neurofibrillary tangles (NFTs) represent pathological hallmark lesions of Alzheimer's disease (AD). Both lesions develop in parallel in the human brain throughout the preclinical and clinical course of AD. Nevertheless, it is not yet clear whether there is a direct link between Aß and τ pathology or whether other proteins are involved in this process. To address this question, we crossed amyloid precursor protein (APP) transgenic mice overexpressing human APP with the Swedish mutation (670/671 KM → NL) (APP23), human wild-type APP (APP51/16), or a proenkephalin signal peptide linked to human Aß42 (APP48) with τ-transgenic mice overexpressing human mutant 4-repeat τ-protein with the P301S mutation (TAU58). In 6-month-old APP23xTAU58 and APP51/16xTAU58 mice, soluble Aß was associated with the aggravation of p-τ pathology propagation into the CA1/subiculum region, whereas 6-month-old TAU58 and APP48xTAU58 mice neither exhibited significant amounts of p-τ pathology in the CA1/subiculum region nor displayed significant levels of soluble Aß in the forebrain. In APP23xTAU58 and APP51/16xTAU58 mice showing an acceleration of p-τ propagation, Aß and p-τ were co-immunoprecipitated with cellular prion protein (PrPC). A similar interaction between PrPC, p-τ and Aß was observed in human AD brains. This association was particularly noticed in 60% of the symptomatic AD cases in our sample, suggesting that PrPC may play a role in the progression of AD pathology. An in vitro pull-down assay confirmed that PrPC is capable of interacting with Aß and p-τ. Using a proximity ligation assay, we could demonstrate proximity (less than ~ 30-40 nm distance) between PrPC and Aß and between PrPC and p-τ in APP23xTAU58 mouse brain as well as in human AD brain. Proximity between PrPC and p-τ was also seen in APP51/16xTAU58, APP48xTAU58, and TAU58 mice. Based on these findings, it is tempting to speculate that PrPC is a critical player in the interplay between Aß and p-τ propagation at least in a large group of AD cases. Preexisting p-τ pathology interacting with PrPC, thereby, appears to be a prerequisite for Aß to function as a p-τ pathology accelerator via PrPC.

Biochemical stages of amyloid-ß peptide aggregation and accumulation in the human brain and their association with symptomatic and pathologically preclinical Alzheimer's disease.

Alzheimer's disease is characterized by the deposition of amyloid-ß peptide in the brain. N-terminal truncation resulting in the formation of AßN3pE and phosphorylation at serine 8 have been reported to modify aggregation properties of amyloid-ß. Biochemically, soluble, dispersible, membrane-associated, and insoluble, plaque-associated amyloid-ß aggregates have been distinguished. Soluble and dispersible amyloid-ß aggregates are both in mixture with the extracellular or intracellular fluid but dispersible aggregates can be cleared from proteins in solution by ultracentrifugation. To clarify the role of phosphorylated amyloid-ß and AßN3pE in soluble, dispersible, membrane-associated, and plaque-associated amyloid-ß aggregates in the pathogenesis of Alzheimer's disease we studied brains from 21 cases with symptomatic Alzheimer's disease, 33 pathologically preclinical Alzheimer's disease cases, and 20 control cases. Western blot analysis showed that soluble, dispersible, membrane-associated and plaque-associated amyloid-ß aggregates in the earliest preclinical stage of Alzheimer's disease did not exhibit detectable amounts of AßN3pE and phosphorylated amyloid-ß. This stage was referred to as biochemical stage 1 of amyloid-ß aggregation and accumulation. In biochemical amyloid-ß stage 2, AßN3pE was additionally found whereas phosphorylated amyloid-ß was restricted to biochemical amyloid-ß stage 3, the last stage of amyloid-ß aggregation. Phosphorylated amyloid-ß was seen in the dispersible, membrane-associated, and plaque-associated fraction. All cases with symptomatic Alzheimer's disease in our sample fulfilled biochemical amyloid-ß stage 3 criteria, i.e. detection of phosphorylated amyloid-ß. Most, but not all, cases with pathologically preclinical Alzheimer's disease had biochemical amyloid-ß stages 1 or 2. Immunohistochemistry confirmed the hierarchical occurrence of amyloid-ß, AßN3pE, and phosphorylated amyloid-ß in amyloid plaques. Phosphorylated amyloid-ß containing plaques were, thereby, seen in all symptomatic cases with Alzheimer's disease but only in a few non-demented control subjects. The biochemical amyloid-ß stages correlated with the expansion of amyloid-ß plaque deposition and with that of neurofibrillary tangle pathology. Taken together, we demonstrate that AßN3pE and phosphorylated amyloid-ß are not only detectable in plaques, but also in soluble and dispersible amyloid-ß aggregates outside of plaques. They occur in a hierarchical sequence that allows the distinction of three stages. In light of our findings, it is tempting to speculate that this hierarchical, biochemical sequence of amyloid-ß aggregation and accumulation is related to disease progression and may be relevant for an increasing toxicity of amyloid-ß aggregates.

Different aspects of Alzheimer's disease-related amyloid ß-peptide pathology and their relationship to amyloid positron emission tomography imaging and dementia.

Alzheimer's disease (AD)-related amyloid ß-peptide (Aß) pathology in the form of amyloid plaques and cerebral amyloid angiopathy (CAA) spreads in its topographical distribution, increases in quantity, and undergoes qualitative changes in its composition of modified Aß species throughout the pathogenesis of AD. It is not clear which of these aspects of Aß pathology contribute to AD progression and to what extent amyloid positron emission tomography (PET) reflects each of these aspects. To address these questions three cohorts of human autopsy cases (in total n = 271) were neuropathologically and biochemically examined for the topographical distribution of Aß pathology (plaques and CAA), its quantity and its composition. These parameters were compared with neurofibrillary tangle (NFT) and neuritic plaque pathology, the degree of dementia and the results from [18F]flutemetamol amyloid PET imaging in cohort 3. All three aspects of Aß pathology correlated with one another, the estimation of Aß pathology by [18F]flutemetamol PET, AD-related NFT pathology, neuritic plaques, and with the degree of dementia. These results show that one aspect of Aß pathology can be used to predict the other two, and correlates well with the development of dementia, advancing NFT and neuritic plaque pathology. Moreover, amyloid PET estimates all three aspects of Aß pathology in-vivo. Accordingly, amyloid PET-based estimates for staging of amyloid pathology indicate the progression status of amyloid pathology in general and, in doing so, also of AD pathology. Only 7.75% of our cases deviated from this general association.

Modified amyloid variants in pathological subgroups of ß-amyloidosis.

OBJECTIVE: Amyloid ß (Aß) depositions in plaques and cerebral amyloid angiopathy (CAA) represent common features of Alzheimer's disease (AD). Sequential deposition of post-translationally modified Aß in plaques characterizes distinct biochemical stages of Aß maturation. However, the molecular composition of vascular Aß deposits in CAA and its relation to plaques remain enigmatic. METHODS: Vascular and parenchymal deposits were immunohistochemically analyzed for pyroglutaminated and phosphorylated Aß in the medial temporal and occipital lobe of 24 controls, 27 pathologically-defined preclinical AD, and 20 symptomatic AD cases. RESULTS: Sequential deposition of Aß in CAA resembled Aß maturation in plaques and enabled the distinction of three biochemical stages of CAA. B-CAA stage 1 was characterized by deposition of Aß in the absence of pyroglutaminated AßN3pE and phosphorylated AßpS8. B-CAA stage 2 showed additional AßN3pE and B-CAA stage 3 additional AßpS8. Based on the Aß maturation staging in CAA and plaques, three case groups for Aß pathology could be distinguished: group 1 with advanced Aß maturation in CAA; group 2 with equal Aß maturation in CAA and plaques; group 3 with advanced Aß maturation in plaques. All symptomatic AD cases presented with end-stage plaque maturation, whereas CAA could exhibit immature Aß deposits. Notably, Aß pathology group 1 was associated with arterial hypertension, and group 2 with the development of dementia. INTERPRETATION: Balance of Aß maturation in CAA and plaques defines distinct pathological subgroups of ß-amyloidosis. The association of CAA-related Aß maturation with cognitive decline, the individual contribution of CAA and plaque pathology to the development of dementia within the defined Aß pathology subgroups, and the subgroup-related association with arterial hypertension should be considered for differential diagnosis and therapeutic intervention.

Impact of amyloid ß aggregate maturation on antibody treatment in APP23 mice.

INTRODUCTION: The deposition of the amyloid ß protein (Aß) in the brain is a hallmark of Alzheimer's disease (AD). Removal of Aß by Aß-antibody treatment has been developed as a potential treatment strategy against AD. First clinical trials showed neither a stop nor a reduction of disease progression. Recently, we have shown that the formation of soluble and insoluble Aß aggregates in the human brain follows a hierarchical sequence of three biochemical maturation stages (B-Aß stages). To test the impact of the B-Aß stage on Aß immunotherapy, we treated transgenic mice expressing human amyloid precursor protein (APP) carrying the Swedish mutation (KM670/671NL; APP23) with the Aß-antibody ß1 or phosphate-buffered saline (PBS) beginning 1) at 3 months, before the onset of dendrite degeneration and plaque deposition, and 2) at 7 months, after the start of Aß plaque deposition and dendrite degeneration. RESULTS: At 5 months of age, first Aß aggregates in APP23 brain consisted of non-modified Aß (representing B-Aß stage 1) whereas mature Aß-aggregates containing N-terminal truncated, pyroglutamate-modified AßN3pE and phosphorylated Aß (representing B-Aß stage 3) were found at 11 months of age in both ß1- and PBS-treated animals. Protective effects on commissural neurons with highly ramified dendritic trees were observed only in 3-month-old ß1-treated animals sacrificed at 5 months. When treatment started at 7 months of age, no differences in the numbers of healthy commissural neurons were observed between ß1- and PBS-treated APP23 mice sacrificed with 11 months. CONCLUSIONS: Aß antibody treatment was capable of protecting neurons from dendritic degeneration as long as Aß aggregation was absent or represented B-Aß stage 1 but had no protective or curative effect in later stages with mature Aß aggregates (B-Aß stage 3). These data indicate that the maturation stage of Aß aggregates has impact on potential treatment effects in APP23 mice.

The type of Aß-related neuronal degeneration differs between amyloid precursor protein (APP23) and amyloid ß-peptide (APP48) transgenic mice.

BACKGROUND: The deposition of the amyloid ß-peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). It is not yet clear whether Aß always leads to similar changes or whether it induces different features of neurodegeneration in relation to its intra- and/or extracellular localization or to its intracellular trafficking routes. To address this question, we have analyzed two transgenic mouse models: APP48 and APP23 mice. The APP48 mouse expresses Aß1-42 with a signal sequence in neurons. These animals produce intracellular Aß independent of amyloid precursor protein (APP) but do not develop extracellular Aß plaques. The APP23 mouse overexpresses human APP with the Swedish mutation (KM670/671NL) in neurons and produces APP-derived extracellular Aß plaques and intracellular Aß aggregates. RESULTS: Tracing of commissural neurons in layer III of the frontocentral cortex with the DiI tracer revealed no morphological signs of dendritic degeneration in APP48 mice compared to littermate controls. In contrast, the dendritic tree of highly ramified commissural frontocentral neurons was altered in 15-month-old APP23 mice. The density of asymmetric synapses in the frontocentral cortex was reduced in 3- and 15-month-old APP23 but not in 3- and 18-month-old APP48 mice. Frontocentral neurons of 18-month-old APP48 mice showed an increased proportion of altered mitochondria in the soma compared to wild type and APP23 mice. Aß was often seen in the membrane of neuronal mitochondria in APP48 mice at the ultrastructural level. CONCLUSIONS: These results indicate that APP-independent intracellular Aß accumulation in APP48 mice is not associated with dendritic and neuritic degeneration but with mitochondrial alterations whereas APP-derived extra- and intracellular Aß pathology in APP23 mice is linked to dendrite degeneration and synapse loss independent of obvious mitochondrial alterations. Thus, Aß aggregates in APP23 and APP48 mice induce neurodegeneration presumably by different mechanisms and APP-related production of Aß may, thereby, play a role for the degeneration of neurites and synapses.

Dispersible amyloid ß-protein oligomers, protofibrils, and fibrils represent diffusible but not soluble aggregates: their role in neurodegeneration in amyloid precursor protein (APP) transgenic mice.

Soluble amyloid ß-protein (Aß) aggregates have been identified in the Alzheimer's disease (AD) brain. Dispersed Aß aggregates in the brain parenchyma are different from soluble, membrane-associated and plaque-associated solid aggregates. They are in mixture with the extra- or intracellular fluid but can be separated from soluble proteins by ultracentrifugation. To clarify the role of dispersible Aß aggregates for neurodegeneration we analyzed 2 different amyloid precursor protein (APP)-transgenic mouse models. APP23 mice overexpress human mutant APP with the Swedish mutation. APP51/16 mice express high levels of human wild type APP. Both mice develop Aß-plaques. Dendritic degeneration, neuron loss, and loss of asymmetric synapses were seen in APP23 but not in APP51/16 mice. The soluble and dispersible fractions not separated from one another were received as supernatant after centrifugation of native forebrain homogenates at 14,000 × g. Subsequent ultracentrifugation separated the soluble, i.e., the supernatant, from the dispersible fraction, i.e., the resuspended pellet. The major biochemical difference between APP23 and APP51/16 mice was that APP23 mice exhibited higher levels of dispersible Aß oligomers, protofibrils and fibrils precipitated with oligomer (A11) and protofibril/fibril (B10AP) specific antibodies than APP51/16 mice. These differences, rather than soluble Aß and Aß plaque pathology were associated with dendritic degeneration, neuron, and synapse loss in APP23 mice in comparison with APP51/16 mice. Immunoprecipitation of dispersible Aß oligomers, protofibrils, and fibrils revealed that they were associated with APP C-terminal fragments (APP-CTFs). These results indicate that dispersible Aß oligomers, protofibrils, and fibrils represent an important pool of Aß aggregates in the brain that critically interact with membrane-associated APP C-terminal fragments. The concentration of dispersible Aß aggregates, thereby, presumably determines its toxicity.