Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life.

Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

Early Aß reduction prevents progression of cerebral amyloid angiopathy.

OBJECTIVE: Clinical trials targeting ß-amyloid peptides (Aß) for Alzheimer disease (AD) failed for arguable reasons that include selecting the wrong stages of AD pathophysiology or Aß being the wrong target. Targeting Aß to prevent cerebral amyloid angiopathy (CAA) has not been rigorously followed, although the causal role of Aß for CAA and related hemorrhages is undisputed. CAA occurs with normal aging and to various degrees in AD, where its impact and treatment is confounded by the presence of parenchymal Aß deposition. METHODS: APPDutch mice develop CAA in the absence of parenchymal amyloid, mimicking hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). Mice were treated with a ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. We used 3-dimensional ultramicroscopy and immunoassays for visualizing CAA and assessing Aß in cerebrospinal fluid (CSF) and brain. RESULTS: CAA onset in mice was at 22 to 24 months, first in frontal leptomeningeal and superficial cortical vessels followed by vessels penetrating the cortical layers. CSF Aß increased with aging followed by a decrease of both Aß40 and Aß42 upon CAA onset, supporting the idea that combined reduction of CSF Aß40 and Aß42 is a specific biomarker for vascular amyloid. BACE1 inhibitor treatment starting at CAA onset and continuing for 4 months revealed a 90% Aß reduction in CSF and largely prevented CAA progression and associated pathologies. INTERPRETATION: This is the first study showing that Aß reduction at early disease time points largely prevents CAA in the absence of parenchymal amyloid. Our observation provides a preclinical basis for Aß-reducing treatments in patients at risk of CAA and in presymptomatic HCHWA-D. ANN NEUROL 2019;86:561-571.

The sleep-wake cycle regulates brain interstitial fluid tau in mice and CSF tau in humans.

The sleep-wake cycle regulates interstitial fluid (ISF) and cerebrospinal fluid (CSF) levels of ß-amyloid (Aß) that accumulates in Alzheimer's disease (AD). Furthermore, chronic sleep deprivation (SD) increases Aß plaques. However, tau, not Aß, accumulation appears to drive AD neurodegeneration. We tested whether ISF/CSF tau and tau seeding and spreading were influenced by the sleep-wake cycle and SD. Mouse ISF tau was increased ~90% during normal wakefulness versus sleep and ~100% during SD. Human CSF tau also increased more than 50% during SD. In a tau seeding-and-spreading model, chronic SD increased tau pathology spreading. Chemogenetically driven wakefulness in mice also significantly increased both ISF Aß and tau. Thus, the sleep-wake cycle regulates ISF tau, and SD increases ISF and CSF tau as well as tau pathology spreading.

Aß seeding potency peaks in the early stages of cerebral ß-amyloidosis.

Little is known about the extent to which pathogenic factors drive the development of Alzheimer's disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the ß-amyloid peptide (Aß) is an important factor in AD pathogenesis, we asked whether Aß 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 Aß seeding activity in two mouse models of cerebral Aß amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aß 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 Aß, the resulting specific seeding activity sharply peaked at the initial phase of Aß deposition, which in turn is characterized by a temporary several-fold increase in the Aß42/Aß40 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 Aß42 than Aß40 to seed activity. Our findings indicate that the Aß seeding potency is greatest early in the pathogenic cascade and diminishes as Aß increasingly accumulates in brain. The present results provide experimental support for directing anti-Aß therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

Persistence of Aß seeds in APP null mouse brain.

Cerebral ß-amyloidosis is induced by inoculation of Aß 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 Aß seeds up to 6 months previously still induced ß-amyloidosis in APP transgenic hosts following secondary transmission. Thus, Aß seeds can persist in the brain for months, and they regain propagative and pathogenic activity in the presence of host Aß.

Progression of Seed-Induced Aß Deposition within the Limbic Connectome.

An important early event in the pathogenesis of Alzheimer's disease (AD) is the aberrant polymerization and extracellular accumulation of amyloid-ß peptide (Aß). In young transgenic mice expressing the human Aß-precursor protein (APP), deposits of Aß can be induced by the inoculation of minute amounts of brain extract containing Aß aggregates ("Aß seeds"), indicative of a prion-like seeding phenomenon. Moreover, focal intracerebral injection of Aß 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 Aß 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 Aß deposits in two different APP-transgenic mouse models that had been previously inoculated with Aß seeds into the hippocampal formation. The results revealed a specific, neuroanatomically constrained pattern of induced Aß 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.

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

The soluble fraction of brain samples from patients with Alzheimer's disease contains highly biologically active amyloid-ß seeds. In this study, we sought to assess the potency of soluble amyloid-ß seeds derived from the brain and cerebrospinal fluid. Soluble Alzheimer's disease brain extracts were serially diluted and then injected into the hippocampus of young, APP transgenic mice. Eight months later, seeded amyloid-ß deposition was evident even when the hippocampus received subattomole amounts of brain-derived amyloid-ß. In contrast, cerebrospinal fluid from patients with Alzheimer's disease, which contained more than 10-fold higher levels of amyloid-ß 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-ß deposition. In comparison to the soluble brain fraction, cerebrospinal fluid largely lacked N-terminally truncated amyloid-ß species and exhibited smaller amyloid-ß-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-ß species is orders of magnitude greater in brain extracts than in the cerebrospinal fluid.

Aß seeds resist inactivation by formaldehyde.

Cerebral ß-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated ß-amyloid (Aß) into young, pre-depositing Aß 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 Aß itself. Here we report that the ß-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 Aß are maintained in fixed tissues. The resistance of Aß 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 Aß aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Multiple factors contribute to the peripheral induction of cerebral ß-amyloidosis.

Deposition of aggregated amyloid-ß (Aß) peptide in brain is an early event and hallmark pathology of Alzheimer's disease and cerebral Aß angiopathy. Experimental evidence supports the concept that Aß multimers can act as seeds and structurally corrupt other Aß peptides by a self-propagating mechanism. Here we compare the induction of cerebral ß-amyloidosis by intraperitoneal applications of Aß-containing brain extracts in three Aß-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-Aß antibody, was dependent on the amount of inoculated brain extract and on the level of APP/Aß expression in the brain but not in the periphery. The induced Aß deposits in brain occurred in a characteristic pattern consistent with the entry of Aß seeds at multiple brain locations. Intraperitoneally injected Aß 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 Aß seeds are transported from the periphery to the brain in which corruptive templating of host Aß occurs at multiple sites, most efficiently in regions with high availability of soluble Aß.

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 ß-amyloid deposits in the brain parenchyma (Aß plaques) and in the vasculature (cerebral ß-amyloid angiopathy) of ß-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 Aß plaques versus Tau lesions. Furthermore, a spectral shift was observed for Aß plaques versus cerebral ß-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.

Soluble Aß seeds are potent inducers of cerebral ß-amyloid deposition.

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