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In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's A β protein

View Article: PubMed Central - PubMed

ABSTRACT

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Aβ oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Aβ influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aβ clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Aβ1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Aβ physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Aβ efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aβ. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

No MeSH data available.


Cellular localization of monomeric and oligomeric Aβ after intracerebral injection. Immunofluorescence microscopy analysis of serial frozen sections from brains of mice injected with monomeric (A) and oligomeric (B) Aβ illustrates the co-localization of Aβ (red signal) with cell specific markers of neurons (neurotubulin), endothelial cells (factor VIII), astrocytes (GFAP), choroid plexus epithelium (E-cadherin), and activated microglia (Iba-1), all in green fluorescence. Co-localization is highlighted by the yellow fluorescence in the merged images. Magnification: bar represents 10 μm in the neurotubulin stainings, 20 μm for the GFAP stainings, and 30 μm in the case of the FVIII, E-cadherin and Iba-1 stainings.
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Figure 5: Cellular localization of monomeric and oligomeric Aβ after intracerebral injection. Immunofluorescence microscopy analysis of serial frozen sections from brains of mice injected with monomeric (A) and oligomeric (B) Aβ illustrates the co-localization of Aβ (red signal) with cell specific markers of neurons (neurotubulin), endothelial cells (factor VIII), astrocytes (GFAP), choroid plexus epithelium (E-cadherin), and activated microglia (Iba-1), all in green fluorescence. Co-localization is highlighted by the yellow fluorescence in the merged images. Magnification: bar represents 10 μm in the neurotubulin stainings, 20 μm for the GFAP stainings, and 30 μm in the case of the FVIII, E-cadherin and Iba-1 stainings.

Mentions: Immunofluorescence confocal microscopy studies in brain tissues harvested 60 min following intracerebral injection of non-labeled Aβ1-40 provided insight into the cellular mechanisms involved in Aβ uptake. Brain tissue sections were co-immunolabeled with antibodies recognizing specific cell types in conjunction with monoclonal anti-Aβ antibody 6E10 and co-localization of the respective signals evaluated at the site of the injection. The images described below were captured from the motor cortex adjacent to the injection site. Although the distance from the injection point varied among the different stainings, the images shown in Figure 5 were typically acquired within a 400 μm range from the injection site. Figure 5A illustrates the localization of injected Aβ monomers to neuronal cells highlighted by neurotubulin staining consistent with recent reports demonstrating the capability of neurons to uptake ISF Aβ through receptor-mediated endocytosis by the low-density lipoprotein receptor-related protein 1 (LRP1; Kanekiyo et al., 2013). Aβ was also present in the cerebral vasculature, as indicated by its co-localization with endothelial cells decorated by their reactivity with Factor VIII, suggestive of Aβ clearance across the BBB, a well study pathway for the removal of Aβ monomers (Shibata et al., 2000; Bell et al., 2007; Deane et al., 2009), or along the peri-/para-vascular tracks, as recently reported (Morris et al., 2016). The immunohistochemical approach also illustrates the co-localization of Aβ with astrocytes surrounding the vasculature—as indicated by the overlapping staining pattern of Aβ and the astrocytic marker GFAP—indicative of the recently described glymphatic pathway involving the aquaporin 4-mediated ISF bulk flow, as one of the mechanisms involved in the clearance of the injected Aβ. Analysis of the choroid plexus epithelium with antibodies recognizing E-cadherin indicates the presence of the intra-cerebrally injected Aβ also at this barrier structure and in accordance to the presence of degraded and intact Aβ in the CSF illustrated in Figures 2–4. Notably, Aβ reactivity was also associated with activated microglia within the choroid plexus, as highlighted by the overlapping signals of Aβ with the Iba-1 microglial marker. The immunoreactivity of 6E10 does not distinguish between monomeric and oligomeric forms of Aβ; thus, we used this antibody in immunohistochemical studies to also assess the localization of the injected oligomeric preparations. As indicated above, the Aβ1–40 oligomers used in the experiments remained stable during the 1 h window of the intra-cerebral injection procedure requiring an additional 24 h incubation to render protofibrillar structures (Fossati et al., 2010). Figure 5B illustrates the comparable overlapping of signals recognizing the injected LMW oligomeric Aβ with neuronal, endothelial, and astrocytic cells as well as within choroid plexus structures and associated activated microglia, suggesting the involvement of comparable routes for the efflux of monomeric and LMW oligomeric Aβ counterparts.


In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's A β protein
Cellular localization of monomeric and oligomeric Aβ after intracerebral injection. Immunofluorescence microscopy analysis of serial frozen sections from brains of mice injected with monomeric (A) and oligomeric (B) Aβ illustrates the co-localization of Aβ (red signal) with cell specific markers of neurons (neurotubulin), endothelial cells (factor VIII), astrocytes (GFAP), choroid plexus epithelium (E-cadherin), and activated microglia (Iba-1), all in green fluorescence. Co-localization is highlighted by the yellow fluorescence in the merged images. Magnification: bar represents 10 μm in the neurotubulin stainings, 20 μm for the GFAP stainings, and 30 μm in the case of the FVIII, E-cadherin and Iba-1 stainings.
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Figure 5: Cellular localization of monomeric and oligomeric Aβ after intracerebral injection. Immunofluorescence microscopy analysis of serial frozen sections from brains of mice injected with monomeric (A) and oligomeric (B) Aβ illustrates the co-localization of Aβ (red signal) with cell specific markers of neurons (neurotubulin), endothelial cells (factor VIII), astrocytes (GFAP), choroid plexus epithelium (E-cadherin), and activated microglia (Iba-1), all in green fluorescence. Co-localization is highlighted by the yellow fluorescence in the merged images. Magnification: bar represents 10 μm in the neurotubulin stainings, 20 μm for the GFAP stainings, and 30 μm in the case of the FVIII, E-cadherin and Iba-1 stainings.
Mentions: Immunofluorescence confocal microscopy studies in brain tissues harvested 60 min following intracerebral injection of non-labeled Aβ1-40 provided insight into the cellular mechanisms involved in Aβ uptake. Brain tissue sections were co-immunolabeled with antibodies recognizing specific cell types in conjunction with monoclonal anti-Aβ antibody 6E10 and co-localization of the respective signals evaluated at the site of the injection. The images described below were captured from the motor cortex adjacent to the injection site. Although the distance from the injection point varied among the different stainings, the images shown in Figure 5 were typically acquired within a 400 μm range from the injection site. Figure 5A illustrates the localization of injected Aβ monomers to neuronal cells highlighted by neurotubulin staining consistent with recent reports demonstrating the capability of neurons to uptake ISF Aβ through receptor-mediated endocytosis by the low-density lipoprotein receptor-related protein 1 (LRP1; Kanekiyo et al., 2013). Aβ was also present in the cerebral vasculature, as indicated by its co-localization with endothelial cells decorated by their reactivity with Factor VIII, suggestive of Aβ clearance across the BBB, a well study pathway for the removal of Aβ monomers (Shibata et al., 2000; Bell et al., 2007; Deane et al., 2009), or along the peri-/para-vascular tracks, as recently reported (Morris et al., 2016). The immunohistochemical approach also illustrates the co-localization of Aβ with astrocytes surrounding the vasculature—as indicated by the overlapping staining pattern of Aβ and the astrocytic marker GFAP—indicative of the recently described glymphatic pathway involving the aquaporin 4-mediated ISF bulk flow, as one of the mechanisms involved in the clearance of the injected Aβ. Analysis of the choroid plexus epithelium with antibodies recognizing E-cadherin indicates the presence of the intra-cerebrally injected Aβ also at this barrier structure and in accordance to the presence of degraded and intact Aβ in the CSF illustrated in Figures 2–4. Notably, Aβ reactivity was also associated with activated microglia within the choroid plexus, as highlighted by the overlapping signals of Aβ with the Iba-1 microglial marker. The immunoreactivity of 6E10 does not distinguish between monomeric and oligomeric forms of Aβ; thus, we used this antibody in immunohistochemical studies to also assess the localization of the injected oligomeric preparations. As indicated above, the Aβ1–40 oligomers used in the experiments remained stable during the 1 h window of the intra-cerebral injection procedure requiring an additional 24 h incubation to render protofibrillar structures (Fossati et al., 2010). Figure 5B illustrates the comparable overlapping of signals recognizing the injected LMW oligomeric Aβ with neuronal, endothelial, and astrocytic cells as well as within choroid plexus structures and associated activated microglia, suggesting the involvement of comparable routes for the efflux of monomeric and LMW oligomeric Aβ counterparts.

View Article: PubMed Central - PubMed

ABSTRACT

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Aβ oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Aβ influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Aβ clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Aβ1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Aβ physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Aβ efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Aβ. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

No MeSH data available.