Toll-like receptor 4-dependent glial cell activation mediates the impairment in memory establishment induced by β-amyloid oligomers in an acute mouse model of Alzheimer’s disease

Highlights

• An ICV injection of AβO impairing memory leads also to a rapid glial activation.

• Anti-inflammatory drugs prevent the AβO-mediated memory impairment.

• AβO-mediated effects are abolished by TLR4 antagonist or in TLR4^−/− mice.

• Concluding, TLR4-mediated glial activation is involved in AβO-induced memory impairment.

Abstract

Background: Amyloid-β oligomers (AβO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer’s disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AβO-mediated mouse model of AD, we explored whether the memory alteration AβOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved. Methods: C57 naïve mice were given a single intracerebroventricular injection of synthetic AβO-containing solution (1 μM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1β receptor antagonist) to prevent impairment in the novel object recognition task, and compared AβO’s effects in TLR4 knockout mice. Results: A single AβO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AβO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AβO’s action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation. Conclusions: These data provide new information on AβO’s mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well.

Introduction

Amyloid-β oligomers (AβO) are soluble aggregates involved in the induction of synaptic and cognitive dysfunction in Alzheimer’s disease (AD) (Balducci et al., 2010a, Mucke and Selkoe, 2012), and their levels correlate with disease severity better than the number of senile plaques (Kuo et al., 1996, McLean et al., 1999). Although there are numerous theories about AβO’s actions, the precise mechanism of cognitive and synaptic dysfunction is still not clear. Most likely they act in a multifactorial manner involving several neuronal systems and central nervous system (CNS) cells (Benilova et al., 2012). Although their action is mostly at neuronal level, inflammatory cells might also be involved. New data do in fact support the concept that immune system-related modifications drive AD pathogenesis (Heneka et al., 2015).

AβO induce in vitro glial cell activation and pro-inflammatory cytokine release to a greater extent than fibrillar forms of Aβ (He et al., 2012), and the classic hallmarks of inflammation are all seen in the brain tissues of AD patients and animal models (Akiyama et al., 2000). Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) protects individuals from AD (Vlad et al., 2008). Microglia and astrocytes, when in a resting/ramified state, control synaptic and structural plasticity as well as learning and memory (Morris et al., 2013, Ota et al., 2013). Therefore alterations in their functional state might affect cognitive functions. The therapeutic efficacy of inhibiting inflammation for improving learning and memory in AD animal models has been described (Bernardi et al., 2012, Craft et al., 2004, Imbimbo et al., 2009, Richardson et al., 2002).

We hypothesized that by activating immune cells, AβO would deprive neurons and synapses of glial control, precluding the appropriate processing of new memories.

Different Aβ species injected into the animal brain, eliciting an inflammatory response, neuronal degeneration and synaptic loss, have been reported (Craft et al., 2004, O’Hare et al., 1999), with concomitant learning and memory impairment (Carrero et al., 2012, He et al., 2012, Perez et al., 2010). However, to our knowledge no study has thoroughly investigated whether AβO’s interference with memory involves glial cell activation.

We employed our recently developed acute mouse model of AD (Balducci et al., 2010a, Balducci and Forloni, 2014), with which we can specifically decipher and monitor AβO’s early actions and follow their progression over time in different brain cell populations. This is a useful complement to transgenic AD mice, where it is difficult to distinguish the specific action of AβO.

We investigated whether a single intracerebroventricular (ICV) injection of well-characterized AβO solutions, leading to impairment of memory establishment (Balducci et al., 2010a, Balducci and Forloni, 2014), concomitantly fostered pro-inflammatory processes, and whether anti-inflammatory drugs prevented their detrimental action on memory. In addition, with a view to clarifying the immune molecular pathway implicated, we investigated the involvement of Toll-like receptor 4 (TLR4), a member of a well-known family of pathogen- or damage-associated molecular pattern receptors (PAMPs, DAMPs) of innate immunity, responsible for the initiation of an inflammatory response. Despite the fact that their role in AD is still extremely controversial, there is a large amount of data in favor of TLR4’s harmful effects over its benefits (Trotta et al., 2014). We antagonized TLR4 or tested the effects of AβO on memory and glial cells in TLR4 knockout mice (TLR4^−/−).