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Cited by 187Alzheimer's disease research and treatmentsUltrasound and Hyperthermia ApplicationsBarrier Structure and Function Studies

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Combined effects of scanning ultrasound and a tau-specific single chain antibody in a tau transgenic mouse model

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Nisbet, Rebecca M., Van der Jeugd, Ann, Leinenga, Gerhard, Evans, Harrison T., Janowicz, Phillip W., Götz, Jürgen·2017·Brain
DOI 10.1093/brain/awx052PMC PMC5405237
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Abstract

Alzheimer's disease is characterized by the deposition of amyloid-β as extracellular plaques and hyperphosphorylated tau as intracellular neurofibrillary tangles. Tau pathology characterizes not only Alzheimer's disease, but also many other tauopathies, presenting tau as an attractive therapeutic target. Passive tau immunotherapy has been previously explored; however, because only a small fraction of peripherally delivered antibodies crosses the blood-brain barrier, enters the brain and engages with tau that forms intracellular aggregates, more efficient ways of antibody delivery and neuronal uptake are warranted. In the brain, tau exists as multiple isoforms. Here, we investigated the efficacy of a novel 2N tau isoform-specific single chain antibody fragment, RN2N, delivered by passive immunization in the P301L human tau transgenic pR5 mouse model. We demonstrate that, in treated mice, RN2N reduces anxiety-like behaviour and phosphorylation of tau at distinct sites. When administration of RN2N was combined with focused ultrasound in a scanning mode (scanning ultrasound), RN2N delivery into the brain and uptake by neurons were markedly increased, and efficacy was significantly enhanced. Our study provides evidence that scanning ultrasound is a viable tool to enhance the delivery of biologics across the blood-brain barrier and improve therapeutic outcomes and further presents single-chain antibodies as an alternative to full-length antibodies.

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Figure 1 RN2N treatment in combination with SUS reduces anxiety-like behaviour in pR5 tau transgenic mice. (A) Schematic of ultrasound treatment using a transducer in scanning (SUS) mode in order to achieve microbubble-assisted opening of the blood–brain barrier. (B) Female pR5 mice at 4.5 months of age were randomly assigned to one of four groups: pR5, pR5 + SUS, pR5 + RN2N and pR5 + RN2N + SUS and treated as indicated (X) once a week for 4 weeks. A group of wild-type (WT) littermate controls did not undergo any treatment. Upon treatment completion, mice were analysed on the elevated plus maze (EPM), and then sacrificed. (C) The elevated plus maze is an elevated cross-shaped apparatus with a central square and 15 cm long × 5 cm wide closed arms with a 15 cm wall and open arms with unprotected edges. (D) The mean positional heat map within the elevated plus maze for each treatment group. (E) pR5 mice (n = 6) spend significantly less time in the open arms compared to wild-type mice (n = 7) (****P < 0.0001). No difference was observed in the pR5 + SUS group (n = 6) compared to the pR5 mice. However, mice in the pR5 + RN2N group (n = 6) and those in the pR5 + RN2N + SUS group (n = 5) spent significantly more time in the open arms than pR5 mice (*P = 0.03 and ***P = 0.0002, respectively), indicating a reduction in anxiety-like behaviour (mean ± SEM; one-way ANOVA with Dunnett’s multiple comparison test).
Figure 1 RN2N treatment in combination with SUS reduces anxiety-like behaviour in pR5 tau transgenic mice. (A) Schematic of ultrasound treatment using a transducer in scanning (SUS) mode in order to achieve microbubble-assisted opening of the blood–brain barrier. (B) Female pR5 mice at 4.5 months of age were randomly assigned to one of four groups: pR5, pR5 + SUS, pR5 + RN2N and pR5 + RN2N + SUS and treated as indicated (X) once a week for 4 weeks. A group of wild-type (WT) littermate controls did not undergo any treatment. Upon treatment completion, mice were analysed on the elevated plus maze (EPM), and then sacrificed. (C) The elevated plus maze is an elevated cross-shaped apparatus with a central square and 15 cm long × 5 cm wide closed arms with a 15 cm wall and open arms with unprotected edges. (D) The mean positional heat map within the elevated plus maze for each treatment group. (E) pR5 mice (n = 6) spend significantly less time in the open arms compared to wild-type mice (n = 7) (****P < 0.0001). No difference was observed in the pR5 + SUS group (n = 6) compared to the pR5 mice. However, mice in the pR5 + RN2N group (n = 6) and those in the pR5 + RN2N + SUS group (n = 5) spent significantly more time in the open arms than pR5 mice (*P = 0.03 and ***P = 0.0002, respectively), indicating a reduction in anxiety-like behaviour (mean ± SEM; one-way ANOVA with Dunnett’s multiple comparison test).CC BY-NC · PubMed Central
Figure 2 Delivery of RN2N in combination with SUS significantly reduces phosphorylated tau levels in the amygdala. (A) Representative images of the AT8 phosphorylated tau immunoreactivity observed in a full brain section and amygdala of pR5 mice in each treatment group (Scale bar = 100 μm). (B) Representative images of the AT180 phosphorylated tau immunoreactivity observed in the amygdala of pR5 mice in each treatment group (Scale bar = 100 μm). (C) AT8 phosphorylated tau in the amygdala was significantly reduced in all treatment groups compared to the pR5 group (****P ≤ 0.0001). Comparison of the pR5 + RN2N + SUS group to the pR5 + SUS and pR5 + RN2N groups demonstrates a further reduction in AT8 tau-positive area in the amygdala (####P ≤ 0.0001). (D) AT180 phosphorylated tau in the amygdala was significantly reduced in all treatment groups compared to the pR5 group (****P ≤ 0.0001). Comparison of the pR5 + RN2N + SUS group to the pR5 + SUS and pR5 + RN2N groups demonstrates a further reduction in AT180 tau-positive area in the amygdala (###P = 0.0002). (E) 12E8 phosphorylated tau in the amygdala was significantly reduced in the RN2N + SUS group compared to the pR5 group (**P < 0.01), and was reduced in comparison to pR5 + SUS and pR5 + RN2N groups (#P ≤ 0.05). (F) Ser404 phosphorylated tau in the amygdala was not significantly reduced in any treatment group compared to the pR5 group (mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, n = 5).
Figure 2 Delivery of RN2N in combination with SUS significantly reduces phosphorylated tau levels in the amygdala. (A) Representative images of the AT8 phosphorylated tau immunoreactivity observed in a full brain section and amygdala of pR5 mice in each treatment group (Scale bar = 100 μm). (B) Representative images of the AT180 phosphorylated tau immunoreactivity observed in the amygdala of pR5 mice in each treatment group (Scale bar = 100 μm). (C) AT8 phosphorylated tau in the amygdala was significantly reduced in all treatment groups compared to the pR5 group (****P ≤ 0.0001). Comparison of the pR5 + RN2N + SUS group to the pR5 + SUS and pR5 + RN2N groups demonstrates a further reduction in AT8 tau-positive area in the amygdala (####P ≤ 0.0001). (D) AT180 phosphorylated tau in the amygdala was significantly reduced in all treatment groups compared to the pR5 group (****P ≤ 0.0001). Comparison of the pR5 + RN2N + SUS group to the pR5 + SUS and pR5 + RN2N groups demonstrates a further reduction in AT180 tau-positive area in the amygdala (###P = 0.0002). (E) 12E8 phosphorylated tau in the amygdala was significantly reduced in the RN2N + SUS group compared to the pR5 group (**P < 0.01), and was reduced in comparison to pR5 + SUS and pR5 + RN2N groups (#P ≤ 0.05). (F) Ser404 phosphorylated tau in the amygdala was not significantly reduced in any treatment group compared to the pR5 group (mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, n = 5).CC BY-NC · PubMed Central
Figure 3 RN2N inhibits GSK3β phosphorylation of tau in vitro. (A) GSK3β phosphorylates tau at multiple serine and threonine epitopes resulting in a molecular weight shift of tau. Lane 1: molecular weight marker; lanes 2 and 3: tau alone; lanes 4 and 5: tau incubated with GSK3β; lanes 6 and 7: tau incubated with GSK3β in the presence of 10 μM N1 control scFv; lanes 8 and 9: tau incubated with GSK3β in the presence of 1 μM N1 control scFv; lanes 10 and 11: tau incubated with GSK3β in the presence of 10 μM RN2N; lanes 12 and 13: tau incubated with GSK3β in the presence of 1 μM RN2N; and lanes 14 and 15: tau incubated with GSK3β in the presence of 0.1 μM RN2N. Co-treatment of tau and GSK3β with RN2N leads to reduced phosphorylation at the AT8 and AT180 epitopes, quantified in B, with no change to 12E8 or pSer404 epitopes. Blotting for myc indicates the presence of scFv. (B) Quantification of the reduction of phosphorylation of the AT8 and AT180 epitopes with 10 μM RN2N compared to 10 μM N1 control scFv (P < 0.05) (mean ± SEM, t-test).
Figure 3 RN2N inhibits GSK3β phosphorylation of tau in vitro. (A) GSK3β phosphorylates tau at multiple serine and threonine epitopes resulting in a molecular weight shift of tau. Lane 1: molecular weight marker; lanes 2 and 3: tau alone; lanes 4 and 5: tau incubated with GSK3β; lanes 6 and 7: tau incubated with GSK3β in the presence of 10 μM N1 control scFv; lanes 8 and 9: tau incubated with GSK3β in the presence of 1 μM N1 control scFv; lanes 10 and 11: tau incubated with GSK3β in the presence of 10 μM RN2N; lanes 12 and 13: tau incubated with GSK3β in the presence of 1 μM RN2N; and lanes 14 and 15: tau incubated with GSK3β in the presence of 0.1 μM RN2N. Co-treatment of tau and GSK3β with RN2N leads to reduced phosphorylation at the AT8 and AT180 epitopes, quantified in B, with no change to 12E8 or pSer404 epitopes. Blotting for myc indicates the presence of scFv. (B) Quantification of the reduction of phosphorylation of the AT8 and AT180 epitopes with 10 μM RN2N compared to 10 μM N1 control scFv (P < 0.05) (mean ± SEM, t-test).CC BY-NC · PubMed Central
Figure 4 SUS enhances RN2N scFv delivery across the blood–brain barrier and into neurons. pR5 mice under anaesthesia were injected retro-orbitally with either microbubbles only (SUS), RN2N conjugated to Alexa 647 only (RN2N) or a combination of both (RN2N + SUS). SUS was then applied to SUS and RN2N + SUS groups. After 30 min mice were sacrificed and tissue analysed by fluorescence imaging. (A) Fluorescence imaging indicates widespread brain uptake of RN2N in 6-month and 8-month-old RN2N + SUS treated mice. Quantification of the fluorescent intensity demonstrates an approximate 11-fold increase in scFv uptake in the RN2N + SUS treated mice (mean = 662 ± 119) compared to the RN2N only treated mice (mean = 58 ± 32) (grouping 6-month-old mice = red squares; and 8-month-old mice = black circles; mean ± SEM, P < 0.0001, one-way ANOVA with Tukey’s post-test). (B) Post-sectioning, RN2N was only detectable in the RN2N + SUS treated mice as shown for the CA1 region of the hippocampus. Inset: Zoomed-in image of RN2N + SUS treated mouse brain tissue with RN2N observed within neuronal cell bodies and apical dendrites (arrow). (C) Co-immunofluorescence staining for EEA1 (green) and RN2N (magenta) and (D) LAMP1 (green) and RN2N (magenta) indicates that the internalized RN2N is not confined to endosomes or lysosomes (Scale bar = 10 μm). (E) In the RN2N + SUS treated mice, RN2N is also observed in the amygdala where it co-localized with HT7 staining (human tau). Coronal image sourced from Allen Mouse Brain Atlas (2004) (Scale bar = 50 μm).
Figure 4 SUS enhances RN2N scFv delivery across the blood–brain barrier and into neurons. pR5 mice under anaesthesia were injected retro-orbitally with either microbubbles only (SUS), RN2N conjugated to Alexa 647 only (RN2N) or a combination of both (RN2N + SUS). SUS was then applied to SUS and RN2N + SUS groups. After 30 min mice were sacrificed and tissue analysed by fluorescence imaging. (A) Fluorescence imaging indicates widespread brain uptake of RN2N in 6-month and 8-month-old RN2N + SUS treated mice. Quantification of the fluorescent intensity demonstrates an approximate 11-fold increase in scFv uptake in the RN2N + SUS treated mice (mean = 662 ± 119) compared to the RN2N only treated mice (mean = 58 ± 32) (grouping 6-month-old mice = red squares; and 8-month-old mice = black circles; mean ± SEM, P < 0.0001, one-way ANOVA with Tukey’s post-test). (B) Post-sectioning, RN2N was only detectable in the RN2N + SUS treated mice as shown for the CA1 region of the hippocampus. Inset: Zoomed-in image of RN2N + SUS treated mouse brain tissue with RN2N observed within neuronal cell bodies and apical dendrites (arrow). (C) Co-immunofluorescence staining for EEA1 (green) and RN2N (magenta) and (D) LAMP1 (green) and RN2N (magenta) indicates that the internalized RN2N is not confined to endosomes or lysosomes (Scale bar = 10 μm). (E) In the RN2N + SUS treated mice, RN2N is also observed in the amygdala where it co-localized with HT7 staining (human tau). Coronal image sourced from Allen Mouse Brain Atlas (2004) (Scale bar = 50 μm).CC BY-NC · PubMed Central

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Metadata

Authors
Nisbet, R. M., Van der Jeugd, A., Leinenga, G., Evans, H. T., Janowicz, P. W., & Götz, J.
Affiliations
  • The University of Queensland (AU)
  • Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St Lucia Campus, Brisbane, QLD 4072, Australia
Corresponding author
Götz, Jürgen
ORCID
Venue
Brain
Publisher
Oxford University Press (OUP)
Published
2017-04-04
Volume
140
Issue
5
Pages
1220-1230
ISSN
  • 0006-8950 (ISSN-L)
  • 1460-2156
DOI
10.1093/brain/awx052
PMC
PMC5405237
OpenAlex
W2605011026
Cited by
187
References
46
Citations by year
2026: 7 · 2025: 15 · 2024: 22 · 2023: 17 · 2022: 20 · 2021: 31 · 2020: 25 · 2019: 17 · 2018: 25 · 2017: 8
FWCI
15.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Brain (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • estate of Dr Clem Jones AO
  • State Government of Queensland
  • Federal Government of Australia (ACT900116)
  • The Brain Foundation
  • Australian Research Council (DP13300101932)
  • National Health and Medical Research Council of Australia (APP1037746, APP1003150)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Physical Sciences › Engineering › Biomedical Engineering › Ultrasound and Hyperthermia Applications
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
tau pathology, Alzheimer's disease, single-chain antibody, passive immunotherapy, blood-brain barrier, tau phosphorylation, anxiety-like behavior, tau immunotherapy
MeSH terms
Animals, Antibodies, Brain, Combined Modality Therapy, Disease Models, Animal, Humans, Immunization, Passive, Mice, Transgenic, Neurons, Phosphorylation, Ultrasonic Therapy, tau Proteins, Maze Learning, Protein Isoforms, Tauopathies, Mice

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