Sarah K. Fritschi

Soluble Aβ Seeds Are Potent Inducers of Cerebral β-Amyloid Deposition

Cerebral β-amyloidosis and associated pathologies can be exogenously induced by the intracerebral injection of small amounts of pathogenic Aβ-containing brain extract into young β-amyloid precursor protein (APP) transgenic mice. The probable β-amyloid-inducing factor in the brain extract has been identified as a species of aggregated Aβ that is generated in its most effective conformation or composition in vivo. Here we report that Aβ in the brain extract is more proteinase K (PK) resistant than is synthetic fibrillar Aβ, and that this PK-resistant fraction of the brain extract retains the capacity to induce β-amyloid deposition upon intracerebral injection in young, pre-depositing APP23 transgenic mice. After ultracentrifugation of the brain extract, <0.05% of the Aβ remained in the supernatant fraction, and these soluble Aβ species were largely PK sensitive. However, upon intracerebral injection, this soluble fraction accounted for up to 30% of the β-amyloid induction observed with the unfractionated extract. Fragmentation of the Aβ seeds by extended sonication increased the seeding capacity of the brain extract. In summary, these results suggest that multiple Aβ assemblies, with various PK sensitivities, are capable of inducing β-amyloid aggregation in vivo. The finding that small and soluble Aβ seeds are potent inducers of cerebral β-amyloidosis raises the possibility that such seeds may mediate the spread of β-amyloidosis in the brain. If they can be identified in vivo, soluble Aβ seeds in bodily fluids also could serve as early biomarkers for cerebral β-amyloidogenesis and eventually Alzheimers disease.

Multiple factors contribute to the peripheral induction of cerebral beta-amyloidosis

Deposition of aggregated amyloid-β (Aβ) peptide in brain is an early event and hallmark pathology of Alzheimers disease and cerebral Aβ angiopathy. Experimental evidence supports the concept that Aβ multimers can act as seeds and structurally corrupt other Aβ peptides by a self-propagating mechanism. Here we compare the induction of cerebral β-amyloidosis by intraperitoneal applications of Aβ-containing brain extracts in three Aβ-precursor protein (APP) transgenic mouse lines that differ in levels of transgene expression in brain and periphery (APP23 mice, APP23 mice lacking murine APP, and R1.40 mice). Results revealed that beta-amyloidosis induction, which could be blocked with an anti-Aβ antibody, was dependent on the amount of inoculated brain extract and on the level of APP/Aβ expression in the brain but not in the periphery. The induced Aβ deposits in brain occurred in a characteristic pattern consistent with the entry of Aβ seeds at multiple brain locations. Intraperitoneally injected Aβ could be detected in blood monocytes and some peripheral tissues (liver, spleen) up to 30 d after the injection but escaped histological and biochemical detection thereafter. These results suggest that intraperitoneally inoculated Aβ seeds are transported from the periphery to the brain in which corruptive templating of host Aβ occurs at multiple sites, most efficiently in regions with high availability of soluble Aβ.

Aβ seeds resist inactivation by formaldehyde

Cerebral β-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated β-amyloid (Aβ) into young, pre-depositing Aβ precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aβ itself. Here we report that the β-amyloid-inducing activity of Alzheimer’s disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aβ are maintained in fixed tissues. The resistance of Aβ seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aβ aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Highly potent soluble amyloid-β seeds in human Alzheimer brain but not cerebrospinal fluid

The soluble fraction of brain samples from patients with Alzheimers disease contains highly biologically active amyloid-β seeds. In this study, we sought to assess the potency of soluble amyloid-β seeds derived from the brain and cerebrospinal fluid. Soluble Alzheimers disease brain extracts were serially diluted and then injected into the hippocampus of young, APP transgenic mice. Eight months later, seeded amyloid-β deposition was evident even when the hippocampus received subattomole amounts of brain-derived amyloid-β. In contrast, cerebrospinal fluid from patients with Alzheimers disease, which contained more than 10-fold higher levels of amyloid-β peptide than the most concentrated soluble brain extracts, did not induce detectable seeding activity in vivo. Similarly, cerebrospinal fluid from aged APP-transgenic donor mice failed to induce cerebral amyloid-β deposition. In comparison to the soluble brain fraction, cerebrospinal fluid largely lacked N-terminally truncated amyloid-β species and exhibited smaller amyloid-β-positive particles, features that may contribute to the lack of in vivo seeding by cerebrospinal fluid. Interestingly, the same cerebrospinal fluid showed at least some seeding activity in an in vitro assay. The present results indicate that the biological seeding activity of soluble amyloid-β species is orders of magnitude greater in brain extracts than in the cerebrospinal fluid.

Persistence of Aβ seeds in APP null mouse brain.

Cerebral β-amyloidosis is induced by inoculation of Aβ seeds into APP transgenic mice, but not into App−/− (APP null) mice. We found that brain extracts from APP null mice that had been inoculated with Aβ seeds up to 6 months previously still induced β-amyloidosis in APP transgenic hosts following secondary transmission. Thus, Aβ seeds can persist in the brain for months, and they regain propagative and pathogenic activity in the presence of host Aβ.

Progression of Seed-Induced Aβ Deposition within the Limbic Connectome

An important early event in the pathogenesis of Alzheimers disease (AD) is the aberrant polymerization and extracellular accumulation of amyloid‐β peptide (Aβ). In young transgenic mice expressing the human Aβ‐precursor protein (APP), deposits of Aβ can be induced by the inoculation of minute amounts of brain extract containing Aβ aggregates (“Aβ seeds”), indicative of a prion‐like seeding phenomenon. Moreover, focal intracerebral injection of Aβ seeds can induce deposits not only in the immediate vicinity of the injection site, but, with time, also in distal regions of the brain. However, it remains uncertain whether the spatial progression of Aβ deposits occurs via nonsystematic diffusion from the injection site to proximal regions or via directed transit along neuroanatomical pathways. To address this question, we analyzed the spatiotemporal emergence of Aβ deposits in two different APP‐transgenic mouse models that had been previously inoculated with Aβ seeds into the hippocampal formation. The results revealed a specific, neuroanatomically constrained pattern of induced Aβ deposits in structures corresponding to the limbic connectome, supporting the hypothesis that neuronal pathways act as conduits for the movement of proteopathic agents among brain regions, thereby facilitating the progression of disease.

Spectral Discrimination of Cerebral Amyloid Lesions after Peripheral Application of Luminescent Conjugated Oligothiophenes

In vivo imaging of pathological protein aggregates provides essential knowledge of the kinetics and implications of these lesions in the progression of proteopathies, such as Alzheimer disease. Luminescent conjugated oligothiophenes are amyloid-specific ligands that bind and spectrally distinguish different types of amyloid aggregates. Herein, we report that heptamer formyl thiophene acetic acid (hFTAA) passes the blood-brain barrier after systemic administration and specifically binds to extracellular β-amyloid deposits in the brain parenchyma (Aβ plaques) and in the vasculature (cerebral β-amyloid angiopathy) of β-amyloid precursor protein transgenic APP23 mice. Moreover, peripheral application of hFTAA also stained intracellular lesions of hyperphosphorylated Tau protein in P301S Tau transgenic mice. Spectral profiling of all three amyloid types was acquired ex vivo using two-photon excitation. hFTAA revealed a distinct shift in its emission spectra when bound to Aβ plaques versus Tau lesions. Furthermore, a spectral shift was observed for Aβ plaques versus cerebral β-amyloid angiopathy, indicating that different amyloid types and structural variances of a specific amyloid type can be distinguished. In conclusion, by adding spectral signatures to amyloid lesions, our results pave the way for a new area of in vivo amyloid imaging, allowing in vivo differentiation of amyloid (sub)types and monitoring changes of their structure/composition over time.

Aβ seeding potency peaks in the early stages of cerebral β‐amyloidosis

Little is known about the extent to which pathogenic factors drive the development of Alzheimers disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the β‐amyloid peptide (Aβ) is an important factor in AD pathogenesis, we asked whether Aβ seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aβ seeding activity in two mouse models of cerebral Aβ amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aβ deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD50) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aβ, the resulting specific seeding activity sharply peaked at the initial phase of Aβ deposition, which in turn is characterized by a temporary several‐fold increase in the Aβ42/Aβ40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aβ42 than Aβ40 to seed activity. Our findings indicate that the Aβ seeding potency is greatest early in the pathogenic cascade and diminishes as Aβ increasingly accumulates in brain. The present results provide experimental support for directing anti‐Aβ therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

The Prion-Like Aspect of Alzheimer Pathology

Many neurodegenerative disorders are characterized by a predictable spatiotemporal progression of the aggregation of specific proteins in the brain. The most prevalent cerebral proteopathy is Alzheimer’s disease (AD), in which aggregated amyloid-β peptide (Aβ) is deposited in the form of extracellular parenchymal plaques and vascular amyloid. Multiple lines of evidence indicate that β-amyloidosis can be exogenously induced by the application of brain extracts containing aggregated Aβ. The β-amyloid-inducing agent in the extract is likely Aβ itself in a conformation that cannot easily be mimicked with synthetic material. The induced Aβ lesions spread over time within and among brain regions, and they are dependent on the structural and biochemical nature of Aβ in the extract and on the characteristics of the host. We have found that bioactive Aβ seeds exist in both soluble and insoluble forms; some of them are sensitive to proteinase-K digestion and some are not. Observations of similar prion-like induction, spreading, and transmission of tau lesions, the second hallmark of AD pathology, and more recent observations of seeded α-synuclein lesions suggest that the concept of prion-like corruptive templating of proteins may also apply to intracellular lesions in neurodegenerative diseases. The clinical implications of these observations are not yet clear. The finding that the Aβ seeds are partly soluble suggests that such seeds in bodily fluids may have diagnostic value and also that they could represent a novel target for early therapeutic intervention. Furthermore, the possibility that mechanisms exist allowing for the transport of Aβ aggregates (and possibly other seeds) from the periphery to the brain raises the speculation that environmental amyloidogenic seeds might act as risk factors for certain neurodegenerative diseases.

PREVENTION OF CEREBRAL AMYLOID ANGIOPATHY IN A MOUSE MODEL OF HEREDITARY CEREBRAL HEMORRAGE WITH AMYLOIDOSIS-DUTCH TYPE

protofibrils were readily taken up in human AD-derived microglia in an FcgR-independent manner, while BAN2401 facilitated further uptake of protofibrils in FcgR-mediated fashion. BAN2401 facilitated protofibril uptake in EOC-20 microglia cells was blocked with an FcgR blocker or F(ab’)2 fragments (EC501⁄4257661 ng/mL; corrected for background uptake). Conclusions:Ab can be taken up by microglia via a non-specific pattern-recognizing receptor pathway or through an FcgR-mediated process. Previous work has demonstrated that BAN2401 facilitates protofibril clearance, and this clearance was inhibited by an FcgR blocker to levels lower than those seen in the absence of BAN2401, suggesting a shift from non-specific uptake toward microglial activation and FcgR-dependent microglial phagocytosis. The present studies isolate the FcgR-dependent component of BAN2401-mediated protofibril uptake and provide further support for antibody dependent cellular phagocytosis of Ab protofibrils as the primary mechanism of action for BAN2401.