Mechanisms underlying the initiation of cerebral betaamyloidosis and neurofibrillary tau pathology : new insights form transgenic mice

Numerous neurodegenerative disorders result from the aggregation of proteins that misfold and accumulate as fibrillar amyloid deposits in selectively vulnerable regions of the central nervous system. Alzheimer’s disease (AD) is one of these protein conformational diseases and the leading cause of dementia in the Western world. Postmortem, it is characterized by two major neuropathological features: extracellular deposition of Abeta (Aβ) peptide and intracellular aggregates of neurofibrillary lesions made of hyperphosphorylated tau protein. The observation that early-onset familial forms of AD are caused by mutations in three genes (the amyloid precursor protein (APP), presenilin-1 (PS1), and presenilin-2 (PS2)), all of which increase the production of Aβ, led to the so-called amyloid cascade hypothesis. This hypothesis proposed that the aggregation of polymerized forms of Aβ in soluble multimeric and/or insoluble senile plaque deposits in the brain is an early and critical event that triggers a cascade of pathological events leading to hyperphosphorylation and somatodendritic segregation of tau, formation of neurofibrillary lesions, neuroinflammation, neurodegeneration and, finally, dementia.
The generation of transgenic (tg) mice that exhibit cerebral Aβ-amyloidosis through expression of mutated human APP gene has provided new opportunities to explore pathogenic mechanisms and treatments of AD. The studies presented below were done in part using the well-established APP23 tg mouse model that carries human KM670/671NL mutated APP under the control of a neuron specific Thy-1 promoter and develops amyloid deposits progressively with age. We also generated and made use of a novel tg model, the APPPS1 mouse. This model coexpresses KM670/671NL mutated APP and L166P mutated PS1 that lead to accelerated cerebral amyloidosis concomitantly with additional AD-like pathologies such as local neuron loss, hyperphosphorylated tau-positive neuritic structures, dystrophic synaptic boutons and robust cortical gliosis. Because of the early onset of amyloid pathology, APPPS1 mice are well suited for studying the mechanism and impact of cerebral amyloidosis and to test therapeutic amyloid-targeting strategies.
The aggregation of Aβ protein is an established pathogenic mechanism in Alzheimer’s disease, however little is known about the initiation of this process in vivo. The first set of experiments were therefore performed to characterize the induction of cerebral Aβ-
amyloidosis in vivo, and to clarify which factors are involved in the seeding process. We show that aggregation of Aβ can be exogenously induced in a time- and concentration-dependent manner by injecting Aβ-containing brain extracts from humans with Alzheimer's disease or APP23 tg mice into the brains of young APP23 tg host mice. By injecting extracts from APPPS1 tg mice into APP23 hosts and vice versa, our results suggest that characteristics of both the brain extract and the host are important in governing the phenotype of the induced amyloid. Intriguingly, intracerebral injections of synthetic fibrillar Aβ preparations as well as cell culture-derived Aβ in concentrations similar to brain extract levels were not able to seed Aβ aggregation in vivo. And yet the seeding requires an aggregated form of Aβ, because formic acid denaturation and Aβ-immunodepletion abolished the Aβ-inducing activity of the extract. Last but not least, we show that β-amyloid-induction is halted by Aβ-immunization of the host, demonstrating that the earliest stages of cerebral Aβ-amyloidosis deposition are amenable to therapeutic intervention.
AD presents morphologically with abundant neurofibrillary lesions, but the events that initiate tau pathogenesis in vivo remain unclear. Thus, the second series of experiments were performed to characterize the induction of tau pathology in vivo, as well as to identify potential tau-inducing candidates. We show that intracerebral injection of Aβ-containing brain extract from human with AD or β-amyloid-laden APP23 mouse induces neurofibrillary pathology in transgenic mice expressing human tau with the P301L mutation. Importance of soluble Aβ species for exogenous induction of tau pathogenesis was demonstrated by intracerebral injection of human brain extract with low Aβ level. Notably, intracerebral injection of brain extract from aged tangle-bearing P301L transgenic mouse induced only limited deposition of tau in brains. Our results show that neurofibrillary pathology is exogenously induced by extracts from Aβ-containing brains, and provide further support for the hypothesis that Aβ is a causative pathogenic factor of AD.
It has long been recognized that microglial cells react to β-amyloid deposition as to other brain injuries, but various aspects of their reactive kinetics and ability to phagocytose amyloid in vivo remain hypothetical, despite their potential as therapeutic target. To resolve these controversies, we have designed in a third series of experiments a set of robust
analysis tools centered around intracranial multiphoton imaging to characterize the time-course and extend of microglia’s response to amyloid deposition in tg mice. By following individual plaques and microglia over time in the living brain, we have shown that plaque formation was accompanied by microglial process extension and subsequent migration to the site of injury where individual microglia then exhibited signs of uptake of the Aß peptide. Furthermore, by infusing fluorescent Aß-antibody into the brain parenchyma we demonstrated that the interaction of microglia with plaques can be stimulated, leading to further internalization of Aß and at least partial clearance of existing dense deposits. Together, these results demonstrate an ongoing dynamic homeostasis between plaques and microglia that is amenable to therapeutic intervention.
In summary, the experiments described herein have increased our understanding of the mechanisms underlying the initiation of cerebral Aβ-amyloidosis and neurofibrillary pathology in vivo. We have shown that cerebral Aβ-amyloidosis can be induced by exogenous, Aβ-rich brain extract, and that intrinsic properties of the Aβ in the extract as well as of the host are crucial for the induction of Aβ accumulation in brain. We also demonstrated that neurofibrillary pathology can be induced in vivo by intracerebral infusion of Aβ-rich brain extract into a susceptible transgenic host, and that soluble Aβ species are potent effectors of tau pathology in the brain. Moreover, our observations that interaction of microglial cells with amyloid plaques can be stimulated, such that these cells are induced to clear existing Aβ deposits, provide the mechanistic clues necessary for development of additional therapeutic targets for amyloid clearance.