Full-length ArticleToll-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
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−/−).
Section snippets
Animals
We used 7–8 weeks old C57BL/6N (Charles River) and TLR4−/− male mice (The Jackson Laboratories; strain name C57BL/10ScNJ). A breeder pair of TLR4−/− was kindly provided by Dr. A. Vezzani. TLR4−/− mice have a deletion of the Tlr4 gene that results in the absence of both mRNA and protein, thus causing a lack of response to LPS stimulation. Animals were housed at constant temperature (20 ± 2 °C) and relative humidity (60 ± 5%) with free access to food and water and a fixed 12 h light/dark cycle. The
A single ICV injection of AβO induces glial cell activation and raises cytokines in the hippocampus of C57 naïve mice
On the basis of our previous study showing that a single ICV injection of AβO, but not of monomer or fibrils, impaired memory establishment in mice tested in the NORT (Balducci et al., 2010a, Balducci and Forloni, 2014), and on the assumption that glial cell activation might be involved in the inhibition of memory processing/consolidation (Morris et al., 2013), we investigated whether AβO acutely induced glial cell activation as well. We addressed this question at the hippocampal level which is
Discussion
We report that a single ICV injection of AβO in C57 naïve mice impaired the establishment of new recognition memory, together with rapid glial cell activation and increases in pro-inflammatory cytokines. Anti-inflammatory drugs significantly abolished the effect of AβO on memory. TLR4 appears to play a vital role in AβO’s detrimental action since the Cyp TLR4 antagonist prevented the AβO-mediated memory impairment in naïve mice and TLR4−/− mice no longer had memory establishment deficits and
Conclusions
AβO induced memory impairment in C57 naive mice together with rapid and transient activation of glial cells, within the time frame of memory processing and consolidation. Pre-treatment with anti-inflammatory drugs prevented the memory impairment. We showed that both memory deficit and glial cell activation are TLR4-dependent. Neuroinflammation has been strongly reconsidered in recent years in AD and other neurodegenerative disorders and it is extremely urgent to decipher its role. The present
Competing interests
The authors declare that they have no competing interests.
Funding
This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Acknowledgments
We thank Dr. Annamaria Vezzani for providing a couple of TLR4−/− breeders. We are grateful to F. De Ceglie for technical support in preparing photographs.
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Authors equally contributed to the present study.