Protocols for assessing neurodegenerative phenotypes in Alzheimer’s mouse models

AUTHORS

Jongkyun Kang, Hirotaka Watanabe, Jie Shen

ABSTRACT

Quantitative assessment of neuropathological changes is essential for the characterization of animal models of neurodegenerative disease. Here, we describe a detailed protocol for the detection and quantification of key neuropathological changes in Alzheimer's mouse models. The protocol covers detailed methods including perfusion, dissection, and paraffinization of the brain, preparation of serial brain sections, immunohistochemical analysis, stereological quantification, and sample coding methods for genotype blind analysis. This protocol may be applied to the analysis of neuropathological changes of other neurological disorders.

Focused ultrasound with anti-pGlu3 Aβ enhances efficacy in Alzheimer's disease-like mice via recruitment of peripheral immune cells

AUTHORS

TaoSun, Qiaoqiao Shi, Yongzhi Zhang, Chanikarn Power, Camilla Hoesch, Shawna Antonelli, Maren K. Schroeder, Barbara J. Caldarone, Nadine Taudte, Mathias Schenk, Thore Hettmann, Stephan Schilling, Nathan J. McDannold, Cynthia A.Lemere

ABSTRACT

Pyroglutamate-3 amyloid-β (pGlu3 Aβ) is an N-terminally modified, pathogenic form of amyloid-β that is present in cerebral amyloid plaques and vascular deposits. Here, we used focused ultrasound (FUS) with microbubbles to enhance the intravenous delivery of an Fc-competent anti-pGlu3 Aβ monoclonal antibody, 07/2a mAb, across the blood brain barrier (BBB) in an attempt to improve Aβ removal and memory in aged APP/PS1dE9 mice, an Alzheimer's disease (AD)-like model of amyloidogenesis.

First, we demonstrated that bilateral hippocampal FUS-BBB disruption (FUS-BBBD) led to a 5.5-fold increase of 07/2a mAb delivery to the brains compared to non-sonicated mice 72 h following a single treatment. Then, we determined that three weekly treatments with 07/2a mAb alone improved spatial learning and memory in aged, plaque-rich APP/PS1dE9 mice, and that this improvement occurred faster and in a higher percentage of animals when combined with FUS-BBBD. Mice given the combination treatment had reduced hippocampal plaque burden compared to PBS-treated controls. Furthermore, synaptic protein levels were higher in hippocampal synaptosomes from mice given the combination treatment compared to sham controls, and there were more CA3 synaptic puncta labeled in the APP/PS1dE9 mice given the combination treatment compared to those given mAb alone. Plaque-associated microglia were present in the hippocampi of APP/PS1dE9 mice treated with 07/2a mAb with and without FUS-BBBD. However, we discovered that plaque-associated Ly6G+ monocytes were only present in the hippocampi of APP/PS1dE9 mice that were given FUS-BBBD alone or even more so, the combination treatment. Lastly, FUS-BBBD did not increase the incidence of microhemorrhage in mice with or without 07/2a mAb treatment.

Our findings suggest that FUS is a useful tool to enhance delivery and efficacy of an anti-pGlu3 Aβ mAb for immunotherapy either via an additive effect or an independent mechanism. We revealed a potential novel mechanism wherein the combination of 07/2a mAb with FUS-BBBD led to greater monocyte infiltration and recruitment to plaques in this AD-like model. Overall, these effects resulted in greater plaque removal, sparing of synapses and improved cognitive function without causing overt damage, suggesting the possibility of FUS-BBBD as a noninvasive method to increase the therapeutic efficacy of drugs or biologics in AD patients.

Ultrasound-mediated blood-brain barrier disruption improves anti-pyroglutamate3 Aβ antibody efficacy and enhances phagocyte infiltration into brain in aged Alzheimer’s disease-like mice

AUTHORS

Qiaoqiao Shi, Tao Sun, Yongzhi Zhang, Chanikarn Power, Camilla Hoesch, Shawna Antonelli, Maren K. Schroeder, Barbara J. Caldarone, Nadine Taudte, Mathias Schenk, Thore Hettmann, Stephan Schilling, Nathan J. McDannold, Cynthia A. Lemere

ABSTRACT

Pyroglutamate-3 amyloid-β (pGlu3 Aβ) is an N-terminally modified, toxic form of amyloid-β that is present in cerebral amyloid plaques and vascular deposits. Using the Fc-competent murine anti-pGlu3 Aβ monoclonal antibody (mAb), 07/2a, we present here a nonpharmacological approach using focused ultrasound (FUS) with intravenous (i.v.) injection of microbubbles (MB) to facilitate i.v. delivery of the 07/2a mAb across the blood brain barrier (BBB) in order to improve Aβ removal and restore memory in aged APP/PS1 mice, an Alzheimer’s disease (AD)-like model of amyloidogenesis.

Compared to sham-treated controls, aged APP/PS1 mice treated with 07/2a immediately prior to FUS-mediated BBB disruption (mAb + FUS-BBBD combination treatment) showed significantly better spatial learning and memory in the Water T Maze. FUS-BBBD treatment alone improved contextual fear learning and memory in aged WT and APP/PS1 mice, respectively. APP/PS1 mice given the combination treatment had reduced Aβ42 and pGlu3 Aβ hippocampal plaque burden compared to PBS-treated APP/PS1 mice.

Hippocampal synaptic puncta density and synaptosomal synaptic protein levels were also higher in APP/PS1 mice treated with 07/2a just prior to BBB disruption. Increased Iba-1+ microglia were observed in the hippocampi of AD mice treated with 07/2a with and without FUS-BBBD, and APP/PS1 mice that received hippocampal BBB disruption and 07/2a showed increased Ly6G+ monocytes in hippocampal CA3. FUS-induced BBB disruption did not increase the incidence of microhemorrhage in mice with or without 07/2a mAb treatment.

Our findings suggest that FUS is useful tool that may enhance delivery of an anti-pGlu3 Aβ mAb for immunotherapy. FUS-mediated BBB disruption in combination with the 07/2a mAb also appears to facilitate monocyte infiltration in this AD model. Overall, these effects resulted in greater sparing of synapses and improved cognitive function without causing overt damage, suggesting the possibility of FUS as a noninvasive method to increase the therapeutic efficacy in AD patients.