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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

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.
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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.

Mentions: Monomeric and oligomeric of Aβ1-40 preparations were labeled with Na[125I] at the tyrosine residue located at position 10 of Aβ (Figure 1A), as described above in Material and Methods. The use of a 1.8 kDa cut off desalting column allowed the removal of free iodine from radio-iodinated Aβ, as illustrated in Figure 1B by the clear separation between peaks in a representative experiment. Autoradiography after SDS-gel electrophoresis confirmed the respective monomeric or oligomeric composition of each respective preparation. As indicated in the pertinent autoradiograms shown in Figure 1C, monomeric preparations displayed a single 4 kDa band whereas oligomeric preparations consisted of low molecular mass Aβ species, exhibiting monomeric, dimeric, trimeric, and tetrameric components with predominance of dimeric forms. The monomeric or oligomeric nature of the preparations was corroborated via EM in parallel experiments performed using non-labeled counterparts. As illustrated in Figure 1C, monomeric preparations rendered scattered globular structures 4–6 nm in diameter whereas oligomeric preparations contained a higher number of the same globular structures in many cases associated in small groups containing 2–4 globular components. No evidence of protofibrillar formation was noticed at this time-point, in agreement with our previous reports (Solito et al., 2009; Viana et al., 2009; Fossati et al., 2010). Classic protofibrils (short rods of < 150 nm in length and 4–6 nm in diameter) did not appear before an additional 24 h incubation in our experimental conditions, providing the necessary safety net of structural stability for the 1 h window of our intra-cerebral injection experiments. Both monomeric and oligomeric preparations were intra-cerebrally inoculated into C57BL/6 mice to evaluate their respective efflux from brain. The schematic representation in Figure 1D depicts the injection coordinates and the needle position whereas immunostaining at the actual injection site using anti-Aβ monoclonal 6E10 and Alexafluor 488 conjugates demonstrates minimal—although unavoidable—tissue disruption (right panel). Specificity of the immunostaining is indicated by the absence of Aβ signal in the contralateral site in animals sacrificed immediately after inoculation (left panel).


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

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

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.
Mentions: Monomeric and oligomeric of Aβ1-40 preparations were labeled with Na[125I] at the tyrosine residue located at position 10 of Aβ (Figure 1A), as described above in Material and Methods. The use of a 1.8 kDa cut off desalting column allowed the removal of free iodine from radio-iodinated Aβ, as illustrated in Figure 1B by the clear separation between peaks in a representative experiment. Autoradiography after SDS-gel electrophoresis confirmed the respective monomeric or oligomeric composition of each respective preparation. As indicated in the pertinent autoradiograms shown in Figure 1C, monomeric preparations displayed a single 4 kDa band whereas oligomeric preparations consisted of low molecular mass Aβ species, exhibiting monomeric, dimeric, trimeric, and tetrameric components with predominance of dimeric forms. The monomeric or oligomeric nature of the preparations was corroborated via EM in parallel experiments performed using non-labeled counterparts. As illustrated in Figure 1C, monomeric preparations rendered scattered globular structures 4–6 nm in diameter whereas oligomeric preparations contained a higher number of the same globular structures in many cases associated in small groups containing 2–4 globular components. No evidence of protofibrillar formation was noticed at this time-point, in agreement with our previous reports (Solito et al., 2009; Viana et al., 2009; Fossati et al., 2010). Classic protofibrils (short rods of < 150 nm in length and 4–6 nm in diameter) did not appear before an additional 24 h incubation in our experimental conditions, providing the necessary safety net of structural stability for the 1 h window of our intra-cerebral injection experiments. Both monomeric and oligomeric preparations were intra-cerebrally inoculated into C57BL/6 mice to evaluate their respective efflux from brain. The schematic representation in Figure 1D depicts the injection coordinates and the needle position whereas immunostaining at the actual injection site using anti-Aβ monoclonal 6E10 and Alexafluor 488 conjugates demonstrates minimal—although unavoidable—tissue disruption (right panel). Specificity of the immunostaining is indicated by the absence of Aβ signal in the contralateral site in animals sacrificed immediately after inoculation (left panel).

View Article: PubMed Central - PubMed

ABSTRACT

Amyloid &beta; (A&beta;) 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 A&beta; 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 A&beta; influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced A&beta; 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 A&beta;1-40 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 A&beta; 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 A&beta; efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of A&beta;. 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