Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound

Highlights

• Focused ultrasound treatment reduces amyloid-β plaque pathology within 4 days.

• Host immunoglobulins enter the ultrasound-treated cortex where they bind to plaques.

• Microglia and astrocyte markers are increased in the ultrasound-treated cortex.

• Volumes of microglia and astrocytes are increased in the ultrasound-treated cortex.

• Aβ within microglia and astrocytes is increased in the ultrasound-treated cortex.

Abstract

Noninvasive, targeted drug delivery to the brain can be achieved using transcranial focused ultrasound (FUS), which transiently increases the permeability of the blood–brain barrier (BBB) for localized delivery of therapeutics from the blood to the brain. Previously, we have demonstrated that FUS can deliver intravenously-administered antibodies to the brain of a mouse model of Alzheimer's disease (AD) and rapidly reduce plaques composed of amyloid-β peptides (Aβ). Here, we investigated two potential effects of transcranial FUS itself that could contribute to a reduction of plaque pathology, namely the delivery of endogenous antibodies to the brain and the activation of glial cells.

We demonstrate that transcranial FUS application leads to a significant reduction in plaque burden four days after a single treatment in the TgCRND8 mouse model of AD and that endogenous antibodies are found bound to Aβ plaques. Immunohistochemical and western blot analyses showed an increase in endogenous immunoglobulins within the FUS-targeted cortex. Subsequently, microglia and astrocytes in FUS-treated cortical regions show signs of activation through increases in protein expression and changes in glial size, without changes in glial cell numbers. Enhanced activation of glia correlated with increased internalization of Aβ in microglia and astrocytes.

Together these data demonstrate that FUS improved the bioavailability of endogenous antibodies and led to a temporal activation of glial cells, providing evidence towards antibody- and glia-dependent mechanisms of FUS-mediated plaque reduction.

Introduction

The blood–brain barrier (BBB) poses a challenge for the delivery of therapeutics to the brain for treatment of neurological diseases. Chemicals administered intravenously can facilitate the passage of therapeutics from the blood to the brain but produce variability in the extent and duration of BBB opening (Joshi et al., 2010, Patel et al., 2009, Salahuddin et al., 1988). Ideally, only the brain areas affected by disease would be targeted for treatment, minimizing BBB disruption in other brain regions.

In Alzheimer's disease (AD), amyloid-β peptides (Aβ) aggregate and form extracellular plaques. Animal studies delivering anti-Aβ antibodies directly to the cortex have demonstrated a rapid therapeutic response but employed invasive surgical techniques (Kotilinek et al., 2002, Wilcock et al., 2003). The use of transcranial focused ultrasound (FUS) guided by magnetic resonance imaging (MRI) to locally increase the permeability of the BBB has several advantages, including non-surgical application, targeting of specific brain regions, and control of the extent of BBB opening without damaging surrounding tissues when combined with an intravenous injection of microbubbles (Hynynen et al., 2001, Hynynen et al., 2005, McDannold et al., 2005, Sheikov et al., 2004). We previously demonstrated that MRI-guided FUS (MRIgFUS) efficiently delivered systemically administered anti-Aβ antibodies to targeted brain regions of the TgCRND8 mouse model of AD, reducing plaque load within 4 days (Jordão et al., 2010).

Here, we hypothesize that MRIgFUS alone reduces Aβ pathology, considering that it promotes the entry of monomeric endogenous antibodies (Raymond et al., 2008, Sheikov et al., 2004). Previous studies have shown that endogenous antibodies present in the blood can bind to and disaggregate Aβ fibrils (Dodel et al., 2002, Du et al., 2003, Hyman et al., 2001). We first detected the entry of endogenous antibodies at the site of cortical Aβ plaques in MRIgFUS-treated TgCRND8 mice. Then, we investigated whether MRIgFUS allowed pentameric endogenous immunoglobulin M (IgM, ~ 900 kDa), in addition to monomeric immunoglobulin G (IgG, ~ 150 kDa), to pass from the blood to the brain in TgCRND8 and non-transgenic (non-Tg) mice. Finally, we evaluated whether MRI detection of FUS-mediated changes in BBB permeability can predict the amount of endogenous immunoglobulin entering the brain.

In addition, glia have been implicated in the mechanism of antibody-mediated Aβ clearance (Bard et al., 2000, Koenigsknecht-Talboo et al., 2008, Kraft, 2013, Magga et al., 2010, Nicoll et al., 2006, Wilcock et al., 2003, Wilcock et al., 2004). Therefore, we investigated whether MRIgFUS-enhanced BBB permeability activates microglia and astrocytes, and whether these glia contain Aβ, which would suggest their contribution to Aβ internalization and clearance. Glial activation can be characterized by an increase in the expression of certain proteins, such as ionized calcium-binding adaptor molecule 1 (Iba1) in phagocytic microglia (Ito et al., 1998, Ito et al., 2001) and glial fibrillary acidic protein (GFAP) in astrocytes (Pekny and Nilsson, 2005). Activated glial cells undergo morphological changes, including increased volume and surface area. We aimed to establish the glial activation response to MRIgFUS, and the potential role of glia in MRIgFUS-mediated plaque reduction. The temporal response of glial activation following MRIgFUS was characterized using Iba1 and GFAP expression in the cortex of TgCRND8 and non-Tg mice. Changes in glial volume and area, in addition to Aβ internalization by glia were also investigated.

MRIgFUS technology represents a major advance in the field of non-invasive drug delivery to the brain. For validation of this delivery technique for a wide range of applications, establishing the effects of MRIgFUS in animal models under normal and diseased conditions is important. Here, we show that under disease conditions, MRIgFUS alone reduces Aβ plaque load in the targeted cortex of TgCRND8 mice. Additionally, in both TgCRND8 and non-Tg mice, MRIgFUS delivers endogenous antibodies to the brain and activates glial cells.