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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.


Related in: MedlinePlus

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.
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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.

Mentions: Similar to what was observed for the injected monomeric Aβ1-40, analysis of CSF collected from mice injected with LMW oligomeric forms of the peptide displayed, in addition to the full length peptide, a heterogeneous collection of C-terminally truncated species (Figure 4). It should be noted that under the experimental conditions of the mass spectrometry assay, which render non-covalent binding unobservable (Woods et al., 1995), the amyloid molecules are detected at the molecular mass corresponding to the monomeric species, independent of their aggregation state, as we previously reported (Tomidokoro et al., 2010); therefore, the full-length peak is detected with the same experimental mass regardless the use of monomers or LMW oligomers in the experiments. Visual evaluation of the CSF Aβ species resulting from the inoculation of monomeric vs. oligomeric Aβ1-40 at 30 min post-injection (Figure 4, top and bottom panels, respectively) revealed a similar heterogeneity—full-length peptide coexisting with the same C-terminally degraded Aβ fragments—indicative that, irrespective of the aggregation status of the injected Aβ, the same peptide bonds were targeted for enzymatic degradation. Analysis of the relative intensity ratios corresponding to the intact peptide and to each of the major proteolytic fragments generated from the monomeric and the LMW oligomeric species reinforced the similarity of the proteolytic process (Figure 4, normalized intensities). While absolute signal intensity between samples in MALDI-ToF is not reliably quantitative, lower signal intensity for the cleared oligomeric injected forms are consistent with an indication that Aβ1-40 full-length oligomers are not removed as efficiently to the CSF as the monomeric form, in agreement with results illustrated in Figure 2. The lower susceptibility of oligomeric injected forms of Aβ to enzymatic degradation suggested by the lower signal intensity of the C-terminally degraded derivatives—consistent with previously reported studies for most Aβ-degrading enzymes (Morelli et al., 2003; Haass and Selkoe, 2007; De Strooper, 2010)—may also account, at least in part, for the lower clearance rate observed in our experimental paradigm for oligomeric Aβ species shown in Figure 2.


In vivo Differential Brain Clearance and Catabolism of Monomeric and Oligomeric Alzheimer's A β protein
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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037193&req=5

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.
Mentions: Similar to what was observed for the injected monomeric Aβ1-40, analysis of CSF collected from mice injected with LMW oligomeric forms of the peptide displayed, in addition to the full length peptide, a heterogeneous collection of C-terminally truncated species (Figure 4). It should be noted that under the experimental conditions of the mass spectrometry assay, which render non-covalent binding unobservable (Woods et al., 1995), the amyloid molecules are detected at the molecular mass corresponding to the monomeric species, independent of their aggregation state, as we previously reported (Tomidokoro et al., 2010); therefore, the full-length peak is detected with the same experimental mass regardless the use of monomers or LMW oligomers in the experiments. Visual evaluation of the CSF Aβ species resulting from the inoculation of monomeric vs. oligomeric Aβ1-40 at 30 min post-injection (Figure 4, top and bottom panels, respectively) revealed a similar heterogeneity—full-length peptide coexisting with the same C-terminally degraded Aβ fragments—indicative that, irrespective of the aggregation status of the injected Aβ, the same peptide bonds were targeted for enzymatic degradation. Analysis of the relative intensity ratios corresponding to the intact peptide and to each of the major proteolytic fragments generated from the monomeric and the LMW oligomeric species reinforced the similarity of the proteolytic process (Figure 4, normalized intensities). While absolute signal intensity between samples in MALDI-ToF is not reliably quantitative, lower signal intensity for the cleared oligomeric injected forms are consistent with an indication that Aβ1-40 full-length oligomers are not removed as efficiently to the CSF as the monomeric form, in agreement with results illustrated in Figure 2. The lower susceptibility of oligomeric injected forms of Aβ to enzymatic degradation suggested by the lower signal intensity of the C-terminally degraded derivatives—consistent with previously reported studies for most Aβ-degrading enzymes (Morelli et al., 2003; Haass and Selkoe, 2007; De Strooper, 2010)—may also account, at least in part, for the lower clearance rate observed in our experimental paradigm for oligomeric Aβ species shown in Figure 2.

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.


Related in: MedlinePlus