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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

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.
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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.

Mentions: In order to biochemically analyze the Aβ species cleared to the CSF, a targeted mass spectrometric analysis of Aβ was conducted on CSF collected from a parallel set of mice 5, 30, and 60 min after intra-cerebral injection of non-radiolabeled monomeric and oligomeric forms of Aβ1-40. Immunoprecipitation of CSF samples with paramagnetic beads coated with monoclonal anti-Aβ 6E10 antibodies followed by MALDI-ToF mass spectrometry analysis revealed the presence of heterogeneous C-terminally truncated Aβ species in addition to full-length Aβ1-40, suggestive of a fast enzymatic degradation by brain resident enzymes. Figure 3 depicts the differential pattern exhibited by the intact injected peptide—a single peak with an experimental mass of 4329.1Da (top panel)—in comparison with the multiple degradation fragments retrieved in CSF at the different time-points (lower panels). The relative intensity ratios between the intact peptide and each of the major proteolytically- generated fragments assessed at the different time points indicate that the CSF truncated Aβ species became comparatively less abundant with time while the peak pertaining to the intact peptide, a relatively minor component at 5 min, increased as the time after injection progressed and was the predominant form at 60 min (Figure 3). These differences in the CSF Aβ heterogeneity—highly dependent on the time of sample collection following the intra-cerebral Aβ injection—strongly suggest that C-terminally degraded Aβ fragments are cleared into and out of the CSF more efficiently than the full length peptide—likely reflecting their higher solubility and lower tendency to aggregate (Cabrera et al., unpublished)—and supporting the beneficial role for local catabolism in brain Aβ efflux. Whether a decreased ratio of Aβ-truncated fragments at longer time points after injection also reflects a preferential involvement of other Aβ-removal mechanisms including perivascular drainage, glymphatic pathway or meningeal lymphatic vessels, and/or simply results from the focal exhaustion of available local enzymes remain to be elucidated.


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

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

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.
Mentions: In order to biochemically analyze the Aβ species cleared to the CSF, a targeted mass spectrometric analysis of Aβ was conducted on CSF collected from a parallel set of mice 5, 30, and 60 min after intra-cerebral injection of non-radiolabeled monomeric and oligomeric forms of Aβ1-40. Immunoprecipitation of CSF samples with paramagnetic beads coated with monoclonal anti-Aβ 6E10 antibodies followed by MALDI-ToF mass spectrometry analysis revealed the presence of heterogeneous C-terminally truncated Aβ species in addition to full-length Aβ1-40, suggestive of a fast enzymatic degradation by brain resident enzymes. Figure 3 depicts the differential pattern exhibited by the intact injected peptide—a single peak with an experimental mass of 4329.1Da (top panel)—in comparison with the multiple degradation fragments retrieved in CSF at the different time-points (lower panels). The relative intensity ratios between the intact peptide and each of the major proteolytically- generated fragments assessed at the different time points indicate that the CSF truncated Aβ species became comparatively less abundant with time while the peak pertaining to the intact peptide, a relatively minor component at 5 min, increased as the time after injection progressed and was the predominant form at 60 min (Figure 3). These differences in the CSF Aβ heterogeneity—highly dependent on the time of sample collection following the intra-cerebral Aβ injection—strongly suggest that C-terminally degraded Aβ fragments are cleared into and out of the CSF more efficiently than the full length peptide—likely reflecting their higher solubility and lower tendency to aggregate (Cabrera et al., unpublished)—and supporting the beneficial role for local catabolism in brain Aβ efflux. Whether a decreased ratio of Aβ-truncated fragments at longer time points after injection also reflects a preferential involvement of other Aβ-removal mechanisms including perivascular drainage, glymphatic pathway or meningeal lymphatic vessels, and/or simply results from the focal exhaustion of available local enzymes remain to be elucidated.

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