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In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Aβ Protein

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In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's Aβ Protein

Farron L McIntee et al. Front Aging Neurosci.

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.

Keywords: Aβ brain efflux; Aβ brain homeostasis; cerebrospinal fluid; local proteolytic degradation; stereotaxic intra-cerebral injection; targeted mass spectrometric analysis.

Figures

Figure 1
Figure 1
Aβ1-40 radioiodination, assessment of peptide oligomerization and intra-cerebral injection. (A) Amino acid sequence of Aβ1-40 highlighting the location of tyrosine 10, the target of the radioiodination procedure (red arrow). (B) Representative separation of [125I]-labeled Aβ1-40 from free iodine using a desalting 1.8 kDa cut-off polyacrylamide column. (C) Autoradiogram following electrophoretic separation of monomeric and oligomeric preparations of radiolabeled Aβ1-40 on 16.5% SDS-polyacrylamide gels (top) and EM images illustrating the differential conformational assemblies negatively stained with uranyl acetate (bottom). Magnification: bar represents 100 nm. (D) Schematic representation of the needle location for the intra-cerebral injection of Aβ1-40 preparations (top panel) and immunostaining with monoclonal anti-Aβ 6E10 followed by Alexafluor 488 conjugated secondary antibody and DAPI counterstain at the injection site demonstrating minimal—although unavoidable—tissue disruption (bottom right panel). The absence of Aβ signal in the contralateral site in animals sacrificed immediately after Aβ injection corroborates the specificity of the immunostaining (bottom left panel). Magnification: bar represents 100 μm.
Figure 2
Figure 2
Brain clearance of radiolabeled Aβ species. (A) Clearance of monomeric (black bars) and oligomeric (gray bars) [125I]Aβ1-40 species removed from the brain at three different time-points. (B) Monomeric (black bars) and oligomeric (gray bars) [125I]Aβ1-40 species cleared to the CSF evaluated at three different time-points. In all cases bars illustrate percentage cleared relative to total injected radioactivity; values represent mean ± SD obtained from inoculation of 5–7 mice (ANOVA and Tukey post-hoc test; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
Figure 3
Figure 3
Aβ species cleared to the CSF assessed by a combination of immunoprecipitation and mass spectrometry. MALDI-ToF spectra and normalized ion counts of the monomeric Aβ1-40 used for the experiments prior injection (top). MS profile of the material immunoprecipitated from CSF at different time points (5, 30, and 60 min) post injection identify multiple C-terminally degraded proteolytic fragments. Immunoprecititation was performed in pooled CSF samples from 4 mice injected under the same experimental conditions in 2 independent experiments (n = 8) and spotted in duplicate in the mass spectrometer aluminum plates.
Figure 4
Figure 4
Comparative analysis of Aβ species cleared to the CSF after intracerebral injection of monomeric and oligomeric Aβ1-40. MALDI-ToF spectra and normalized ion counts on CSF collected 30 min after intracerebral injection of comparable amounts of monomeric (top panel) and oligomeric (bottom panel) Aβ1-40 immunoprecipitated with anti-Aβ monoclonal antibody 6E10. For the immunoprecipitation, CSF was pooled from 4 mice injected under the same experimental conditions. Data are representative of 2 independent experiments (n = 8) spotted in duplicate in the mass spectrometer aluminum plates.
Figure 5
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.
Figure 6
Figure 6
The Aβ-peptidome in normal human CSF. The spectrum highlights the C-terminal heterogeneity of the Aβ species present in the CSF, extending far beyond the classic Aβ1-42/Aβ1-40 dichotomy and reflecting the proteolytic action of local resident enzymes.

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