Michal Arbel-Ornath

Amyloid β Induces the Morphological Neurodegenerative Triad of Spine Loss, Dendritic Simplification, and Neuritic Dystrophies through Calcineurin Activation

Amyloid β (Aβ)-containing plaques are surrounded by dystrophic neurites in the Alzheimers disease (AD) brain, but whether and how plaques induce these neuritic abnormalities remain unknown. We tested the hypothesis that soluble oligomeric assemblies of Aβ, which surround plaques, induce calcium-mediated secondary cascades that lead to dystrophic changes in local neurites. We show that soluble Aβ oligomers lead to activation of the calcium-dependent phosphatase calcineurin (CaN) (PP2B), which in turn activates the transcriptional factor nuclear factor of activated T cells (NFAT). Activation of these signaling pathways, even in the absence of Aβ, is sufficient to produce a virtual phenocopy of Aβ-induced dystrophic neurites, dendritic simplification, and dendritic spine loss in both neurons in culture and in the adult mouse brain. Importantly, the morphological deficits in the vicinity of Aβ deposits in a mouse model of AD are ameliorated by CaN inhibition, supporting the hypothesis that CaN–NFAT are aberrantly activated by Aβ and that CaN–NFAT activation is responsible for disruption of neuronal structure near plaques. In accord with this, we also detect increased levels of an active form of CaN and NFATc4 in the nuclear fraction from the cortex of patients with AD. Thus, Aβ appears to mediate the neurodegeneration of AD, at least in part, by activation of CaN and subsequent NFAT-mediated downstream cascades.

Interstitial fluid drainage is impaired in ischemic stroke and Alzheimer’s disease mouse models

The interstitial fluid (ISF) drainage pathway has been hypothesized to underlie the clearance of solutes and metabolites from the brain. Previous work has implicated the perivascular spaces along arteries as the likely route for ISF clearance; however, it has never been demonstrated directly. The accumulation of amyloid β (Aβ) peptides in brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD), and it is likely related to an imbalance between production and clearance of the peptide. Aβ drainage along perivascular spaces has been postulated to be one of the mechanisms that mediate the peptide clearance from the brain. We therefore devised a novel method to visualize solute clearance in real time in the living mouse brain using laser guided bolus dye injections and multiphoton imaging. This methodology allows high spatial and temporal resolution and revealed the kinetics of ISF clearance. We found that the ISF drains along perivascular spaces of arteries and capillaries but not veins, and its clearance exhibits a bi-exponential profile. ISF drainage requires a functional vasculature, as solute clearance decreased when perfusion was impaired. In addition, reduced solute clearance was observed in transgenic mice with significant vascular amyloid deposition; we suggest the existence of a feed-forward mechanism, by which amyloid deposition promotes further amyloid deposition. This important finding provides a mechanistic link between cerebrovascular disease and Alzheimer disease and suggests that facilitation of Aβ clearance along the perivascular pathway should be considered as a new target for therapeutic approaches to Alzheimer disease and cerebral amyloid angiopathy.

Lymphatic Clearance of the Brain: Perivascular, Paravascular and Significance for Neurodegenerative Diseases.

The lymphatic clearance pathways of the brain are different compared to the other organs of the body and have been the subject of heated debates. Drainage of brain extracellular fluids, particularly interstitial fluid (ISF) and cerebrospinal fluid (CSF), is not only important for volume regulation, but also for removal of waste products such as amyloid beta (Aβ). CSF plays a special role in clinical medicine, as it is available for analysis of biomarkers for Alzheimer’s disease. Despite the lack of a complete anatomical and physiological picture of the communications between the subarachnoid space (SAS) and the brain parenchyma, it is often assumed that Aβ is cleared from the cerebral ISF into the CSF. Recent work suggests that clearance of the brain mainly occurs during sleep, with a specific role for peri- and para-vascular spaces as drainage pathways from the brain parenchyma. However, the direction of flow, the anatomical structures involved and the driving forces remain elusive, with partially conflicting data in literature. The presence of Aβ in the glia limitans in Alzheimer’s disease suggests a direct communication of ISF with CSF. Nonetheless, there is also the well-described pathology of cerebral amyloid angiopathy associated with the failure of perivascular drainage of Aβ. Herein, we review the role of the vasculature and the impact of vascular pathology on the peri- and para-vascular clearance pathways of the brain. The different views on the possible routes for ISF drainage of the brain are discussed in the context of pathological significance.

Gene transfer of human apoe isoforms results in differential modulation of amyloid deposition and neurotoxicity in mouse brain

Introduction of different APOE isoforms modulates Aβ peptide aggregation and neurotoxicity after amyloid deposition in mouse brain. Variants and Risk in Alzheimer’s Disease Certain genes help to determine the chance that someone will develop Alzheimer’s disease (AD). In the case of the APOE gene, one form of the gene increases the risk of developing AD, whereas another form decreases the risk. Hudry et al. use a transgenic mouse model of AD and advanced microscopy techniques to examine one mechanism that might help to explain how these different APOE isoforms affect the risk of developing AD. The authors used gene therapy to deliver the APOE4 high-risk human gene variant or the APOE2, protective human gene variant to transgenic mice with amyloid plaques, a pathological characteristic of AD. Introduction of the APOE4 variant increased the rate at which amyloid plaques developed and increased plaque-associated damage to brain neurons. In contrast, the APOE2 variant did the opposite with shrinking of some amyloid plaques and plaque-associated damage in the brains of mice receiving human APOE2. These results confirm a major role for APOE4 in amyloid deposition and may help to guide development of therapies aimed at mitigating APOE4 risk or enhancing APOE2-mediated protection. Inheritance of the ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor associated with the sporadic form of Alzheimer’s disease (AD), whereas the rare APOE ε2 allele has the opposite effect. However, the mechanisms whereby APOE confers risk and protection remain uncertain. We used a gene transfer approach to bathe the cortex of amyloid plaque–bearing transgenic mice with virally expressed human APOE. We monitored amyloid-β (Aβ) with multiphoton imaging, in vivo microdialysis, and postmortem array tomography to study the kinetics of human APOE-mediated changes in Aβ-related neurotoxicity in a mouse model of AD. We observed that human APOE4 increased the concentrations of oligomeric Aβ within the interstitial fluid and exacerbated plaque deposition; the converse occurred after exposure to human APOE2. Peri-plaque synapse loss and dystrophic neurites were also worsened by APOE4 or attenuated by APOE2. Egress of Aβ from the central nervous system (CNS) into the plasma was diminished by APOE3 and APOE4 compared to APOE2, in accord with isoform-specific retention of Aβ in the CNS. Overall, our data show a differential effect of human APOE isoforms on amyloid deposition and clearance in transgenic mice and, more importantly, on Aβ-mediated synaptotoxicity. These results suggest that the APOE genetic risk is mediated by Aβ, and that therapeutic approaches aimed at decreasing APOE4, or increasing APOE2, may be beneficial in AD.

INHIBITION OF THE NFAT PATHWAY ALLEVIATES AMYLOID BETA NEUROTOXICITY IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

Amyloid β (Aβ) peptides, the main pathological species associated with Alzheimers disease (AD), disturb intracellular calcium homeostasis, which in turn activates the calcium-dependent phosphatase calcineurin (CaN). CaN activation induced by Aβ leads to pathological morphological changes in neurons, and overexpression of constitutively active calcineurin is sufficient to generate a similar phenotype, even without Aβ. Here, we tested the hypothesis that calcineurin mediates neurodegenerative effects via activation of the nuclear transcription factor of activated T-cells (NFAT). We found that both spine loss and dendritic branching simplification induced by Aβ exposure were mimicked by constitutively active NFAT, and abolished when NFAT activation was blocked using the genetically encoded inhibitor VIVIT. When VIVIT was specifically addressed to the nucleus, identical beneficial effects were observed, thus enforcing the role of NFAT transcriptional activity in Aβ-related neurotoxicity. In vivo, when VIVIT or its nuclear counterpart were overexpressed in a transgenic model of Alzheimers disease via a gene therapy approach, the spine loss and neuritic abnormalities observed in the vicinity of amyloid plaques were blocked. Overall, these results suggest that NFAT/calcineurin transcriptional cascades contribute to Aβ synaptotoxicity, and may provide a new specific set of pathways for neuroprotective strategies.

Abnormal synaptic Ca2+ homeostasis and morphology in cortical neurons of familial hemiplegic migraine type 1 mutant mice

Migraine is among the most common and debilitating neurological conditions. Familial hemiplegic migraine type 1 (FHM1), a monogenic migraine subtype, is caused by gain‐of‐function of voltage‐gated CaV2.1 calcium channels. FHM1 mice carry human pathogenic mutations in the α1A subunit of CaV2.1 channels and are highly susceptible to cortical spreading depression (CSD), the electrophysiologic event underlying migraine aura. To date, however, the mechanism underlying increased CSD/migraine susceptibility remains unclear.

Topological analyses in APP/PS1 mice reveal that astrocytes do not migrate to amyloid-β plaques

Significance We sought to identify forces shaping the interaction between astrocytes and amyloid-β plaques by performing a spatial analysis, using tools from statistical physics, of 3D images from living transgenic mice. We discovered that astrocytes are repelled by other astrocytes and by plaques. The implications are (i) that a tight balance of repulsive factors maintains the highly territorial astrocyte organization and (ii) that, contrary to a belief widely held by Alzheimer’s researchers, astrocytes do not break this order, migrate to plaques, and phagocytose them. We conclude that astrocytes do sense something within the plaque microenvironment, as shown by the dramatic up-regulation of their key molecule, GFAP, but there is little change in position. These small positional changes are directed away from plaques. Although the clustering of GFAP immunopositive astrocytes around amyloid-β plaques in Alzheimer’s disease has led to the widespread assumption that plaques attract astrocytes, recent studies suggest that astrocytes stay put in injury. Here we reexamine astrocyte migration to plaques, using quantitative spatial analysis and computer modeling to investigate the topology of astrocytes in 3D images obtained by two-photon microscopy of living APP/PS1 mice and WT littermates. In WT mice, cortical astrocyte topology fits a model in which a liquid of hard spheres exclude each other in a confined space. Plaques do not disturb this arrangement except at very large plaque loads, but, locally, cause subtle outward shifts of the astrocytes located in three tiers around plaques. These data suggest that astrocytes respond to plaque-induced neuropil injury primarily by changing phenotype, and hence function, rather than location.

Matrix metalloproteinase 9–mediated intracerebral hemorrhage induced by cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA), the deposition of amyloid-β in cerebrovascular walls, is the most common cause of lobar hemorrhagic stroke. Previous studies show that cerebrovascular amyloid-β induces expression and activation of matrix metalloproteinase 9 (MMP-9) in cerebral vessels of amyloid precursor protein transgenic mice. Here, we extended these findings and evaluated MMP-9 expression in postmortem brain tissues of human CAA cases. MMP-9 colocalized with CAA, correlated with the severity of the vascular pathology, and was detected in proximity to microbleeds. We characterized a novel assay using longitudinal multiphoton microscopy and a novel tracer to visualize and quantify the magnitude and kinetics of hemorrhages in three dimensions in living mouse brains. We demonstrated that topical application of recombinant MMP-9 resulted in a time- and dose-dependent cerebral hemorrhage. Amyloid precursor protein mice with significant CAA developed more extensive hemorrhages which also appeared sooner after exposure to MMP-9. Our data suggest an important role for MMP-9 in development of hemorrhages in the setting of CAA. Inhibition of MMP-9 may present a preventive strategy for CAA-associated hemorrhage.

Reducing available soluble β-amyloid prevents progression of cerebral amyloid angiopathy in transgenic mice.

Abstract Cerebral amyloid angiopathy (CAA), the accumulation of &bgr;-amyloid (A&bgr;) in the walls of leptomeningeal and cortical blood vessels of the brain, is a major cause of intracerebral hemorrhage and cognitive impairment and is commonly associated with Alzheimer disease. The progression of CAA, as measured in transgenic mice by longitudinal imaging with multiphoton microscopy, occurs in a predictable linear manner. The dynamics of A&bgr; deposition in and clearance from vascular walls and their relationship to the concentration of A&bgr; in thebrain are poorly understood. We manipulated A&bgr; levels in the brain using 2 approaches: peripheral clearance via administration of the amyloid binding “peripheral sink” protein gelsolin and direct inhibition of its formation via administration of LY-411575, a small-molecule &ggr;-secretase inhibitor. We found that gelsolin and LY-411575 both reduced the rate of CAA progression in Tg2576 mice from untreated rates of 0.58% ± 0.15% and 0.52% ± 0.09% to 0.11% ± 0.18% (p = 0.04) and −0.17% ± 0.09% (p < 0.001) of affected vessel per day, respectively, in the absence of an immune response. The progression of CAA was also halted when gelsolin was combined with LY-411575 (−0.004% ± 0.10%, p < 0.003). These data suggest that CAA progression can be prevented with non-immune approaches that may reduce the availability of soluble A&bgr; but without evidence of substantial amyloid clearance from vessels.

An acute functional screen identifies an effective antibody targeting amyloid-β oligomers based on calcium imaging

Soluble amyloid β oligomers (AβOs) are widely recognized neurotoxins that trigger aberrant signaling in specific subsets of neurons, leading to accumulated neuronal damage and memory disorders in Alzheimer’s disease (AD). One of the profound downstream consequences of AβO-triggered events is dysregulation of cytosolic calcium concentration ([Ca2+]i), which has been implicated in synaptic failure, cytoskeletal abnormalities, and eventually neuronal death. We have developed an in vitro/in vivo drug screening assay to evaluate putative AβO-blocking candidates by measuring AβO-induced real-time changes in [Ca2+]i. Our screening assay demonstrated that the anti-AβO monoclonal antibody ACU3B3 exhibits potent blocking capability against a broad size range of AβOs. We showed that picomolar concentrations of AβOs were capable of increasing [Ca2+]i in primary neuronal cultures, an effect prevented by ACU3B3. Topical application of 5 nM AβOs onto exposed cortical surfaces also elicited significant calcium elevations in vivo, which was completely abolished by pre-treatment of the brain with 1 ng/mL (6.67 pM) ACU3B3. Our results provide strong support for the utility of this functional screening assay in identifying and confirming the efficacy of AβO-blocking drug candidates such as the human homolog of ACU3B3, which may emerge as the first experimental AD therapeutic to validate the amyloid oligomer hypothesis.