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In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

Nabuurs RJ, Rutgers KS, Welling MM, Metaxas A, de Backer ME, Rotman M, Bacskai BJ, van Buchem MA, van der Maarel SM, van der Weerd L - PLoS ONE (2012)

Bottom Line: In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection.Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ.Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. R.J.A.Nabuurs@LUMC.nl

ABSTRACT
This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

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In vivo Aβ imaging after direct brain application.Topical application of ni3A- or pa2H-Alexa594 (red) as visualized over time by intravital multiphoton microscopy in APP/PS1 mice clearly shows the specific in vivo labeling of different Aβ deposits. In the left, vascular and parenchymal Aβ deposits, detected by prior labeling with Methoxy-X04 (blue), colocalize with ni3A-Alexa594 (red) directly following topical application. One day later, labeling of the plaques has diminished to almost none with some residual left bound to CAA. With interpretation hampered by Methoxy-X04, middle images show a similar experiment. Colocalization with Aβ deposits based upon autofluorescence (green) gave comparable results and almost complete wash out after two days. Pa2H-Alexa594 (red), as shown in the right images, remains bound to vascular Aβ even two days after application, when the plaques remained undetected. All images are maximum intensity projections of a 3D cortical volume with a field of view 615×615 µm.
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pone-0038284-g003: In vivo Aβ imaging after direct brain application.Topical application of ni3A- or pa2H-Alexa594 (red) as visualized over time by intravital multiphoton microscopy in APP/PS1 mice clearly shows the specific in vivo labeling of different Aβ deposits. In the left, vascular and parenchymal Aβ deposits, detected by prior labeling with Methoxy-X04 (blue), colocalize with ni3A-Alexa594 (red) directly following topical application. One day later, labeling of the plaques has diminished to almost none with some residual left bound to CAA. With interpretation hampered by Methoxy-X04, middle images show a similar experiment. Colocalization with Aβ deposits based upon autofluorescence (green) gave comparable results and almost complete wash out after two days. Pa2H-Alexa594 (red), as shown in the right images, remains bound to vascular Aβ even two days after application, when the plaques remained undetected. All images are maximum intensity projections of a 3D cortical volume with a field of view 615×615 µm.

Mentions: After direct application onto the exposed mouse brain, fluorescent VHH were followed up for at least 48 hours by in vivo multiphoton microscopy. (Figure 3) Specific in vivo labeling of Aβ plaques by ni3A-Alexa594 was initially confirmed by colocalization with Methoxy-X04, a known in vivo amyloid targeting fluorophore. Beside possible binding competition with the VHH, Methoxy-X04 hampered good validation due to signal cross-over into the red channel. However, colocalization based on the typical autofluorescence patterns of the different Aβ deposits resulted in similar findings. Selectivity was confirmed by lack of nonspecific background signal. Although both VHH were capable of targeting Aβ in vivo, only pa2H-Alexa594 was detectable after two days, mainly bound to vascular amyloid.


In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

Nabuurs RJ, Rutgers KS, Welling MM, Metaxas A, de Backer ME, Rotman M, Bacskai BJ, van Buchem MA, van der Maarel SM, van der Weerd L - PLoS ONE (2012)

In vivo Aβ imaging after direct brain application.Topical application of ni3A- or pa2H-Alexa594 (red) as visualized over time by intravital multiphoton microscopy in APP/PS1 mice clearly shows the specific in vivo labeling of different Aβ deposits. In the left, vascular and parenchymal Aβ deposits, detected by prior labeling with Methoxy-X04 (blue), colocalize with ni3A-Alexa594 (red) directly following topical application. One day later, labeling of the plaques has diminished to almost none with some residual left bound to CAA. With interpretation hampered by Methoxy-X04, middle images show a similar experiment. Colocalization with Aβ deposits based upon autofluorescence (green) gave comparable results and almost complete wash out after two days. Pa2H-Alexa594 (red), as shown in the right images, remains bound to vascular Aβ even two days after application, when the plaques remained undetected. All images are maximum intensity projections of a 3D cortical volume with a field of view 615×615 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366949&req=5

pone-0038284-g003: In vivo Aβ imaging after direct brain application.Topical application of ni3A- or pa2H-Alexa594 (red) as visualized over time by intravital multiphoton microscopy in APP/PS1 mice clearly shows the specific in vivo labeling of different Aβ deposits. In the left, vascular and parenchymal Aβ deposits, detected by prior labeling with Methoxy-X04 (blue), colocalize with ni3A-Alexa594 (red) directly following topical application. One day later, labeling of the plaques has diminished to almost none with some residual left bound to CAA. With interpretation hampered by Methoxy-X04, middle images show a similar experiment. Colocalization with Aβ deposits based upon autofluorescence (green) gave comparable results and almost complete wash out after two days. Pa2H-Alexa594 (red), as shown in the right images, remains bound to vascular Aβ even two days after application, when the plaques remained undetected. All images are maximum intensity projections of a 3D cortical volume with a field of view 615×615 µm.
Mentions: After direct application onto the exposed mouse brain, fluorescent VHH were followed up for at least 48 hours by in vivo multiphoton microscopy. (Figure 3) Specific in vivo labeling of Aβ plaques by ni3A-Alexa594 was initially confirmed by colocalization with Methoxy-X04, a known in vivo amyloid targeting fluorophore. Beside possible binding competition with the VHH, Methoxy-X04 hampered good validation due to signal cross-over into the red channel. However, colocalization based on the typical autofluorescence patterns of the different Aβ deposits resulted in similar findings. Selectivity was confirmed by lack of nonspecific background signal. Although both VHH were capable of targeting Aβ in vivo, only pa2H-Alexa594 was detectable after two days, mainly bound to vascular amyloid.

Bottom Line: In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection.Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ.Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. R.J.A.Nabuurs@LUMC.nl

ABSTRACT
This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits.

Show MeSH
Related in: MedlinePlus