Fasudil inhibits the activation of microglia and astrocytes of transgenic Alzheimer's disease mice via the downregulation of TLR4/Myd88/NF-κB pathway

Highlights

• Fasudil could inhibit the activation of microglia in the brain tissue of APP/PS1 Tg mice.

• Fasudil could shift astrocytes from an A1 to an A2 phenotype in the brain tissue of APP/PS1 Tg mice.

• Fasudil could reverse microglial-induced neurotoxic astrocyte(A1) to A2 in vitro.

• The study provides preclinical evidence that Fasudil treatment ameliorated memory deficits in APP/PS1 Tg mice.

Abstract

Emerging evidence suggests an association of Alzheimer's Disease (AD) with microglial and astrocytic dysregulation. Recent studies have proposed that activated microglia can transform astrocytes to a neurotoxic A1 phenotype, which has been shown to be involved in the promotion of neuronal damage in several neurodegenerative diseases, including AD. In the present study, we observed an obvious microglial activation and A1-specific astrocyte response in the brain tissue of APP/PS1 Tg mice. Fasudil treatment improved the cognitive deficits of APP/PS1 Tg mice, inhibited microglial activation and promoted their transformation to an anti-inflammatory phenotype, and further shifted astrocytes from an A1 to an A2 phenotype. Our experiments suggest Fasudil exerted these functions by inhibing the expression of TLR4, MyD88, and NF-κB, which are key mediators of inflammation.

Using in vitro experiments, we further validated in vivo findings. Our cell experiments indicated that Fasudil induces a shift of inflammatory microglia towards an anti-inflammatory phenotype. LPS-induced microglia-conditioned medium promotes A1 astrocytic polarization, but Fasudil treatment resulted in a direct transformation of A1 astrocytes to A2. To summarize, our results show that Fasudil inhibits the neurotoxic activation of microglia and shifts astrocytes towards a neuroprotective A2 phenotype, representing a promising candidate for AD treatment.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid-β (Aβ) plaque deposition, neurofibrillary tangles, neuronal death, abnormal synaptic function, and cognitive deficits (Fulop et al., 2018; Gulisano et al., 2018). Although the amyloid and tau hypotheses are the most recognized hypotheses for the pathogenesis of AD, several other mechanisms of pathogenesis have been proposed, including mitochondrial dysfunction, oxidative stress, metabolic disruption, and neuroinflammation, amongst others (Kim and Mook-Jung, 2019; Tobore TO, 2019; Ryu et al., 2019; Calsolaro and Edison, 2016). Unfortunately, so far, there are no available treatments capable of curing or preventing AD, and only few treatments have been found to slow the progression and alleviate symptoms of AD (Gao et al., 2016). An estimated 35.6 million people worldwide have AD-related dementia in 2010, and this number is expected to reach 115.4 million by 2050 and AD has become a serious health and economic problem burden (Prince et al., 2013). Therefore, it is as urgent as ever to elucidate the molecular and cellular mechanisms underlying the AD pathology and develop effective reagents and innovative therapeutic strategies to cure this disease.

Neuroinflammation is an important pathological process closely related to the occurrence and development of AD (Saito and Saido, 2018). Microglia and astrocytes contribute to neuroinflammatory process and the investigation of microglia-astrocyte crosstalk recently became a focus of glial research (Ahmad et al., 2019; Jha et al., 2019). Studies have shown that Aβ-mediated excessive microglial activation can significantly promote the occurrence and development of AD by releasing pro-inflammatory cytokines, complement components, chemokines, and free radicals (Cai et al., 2014; Yao and Zu, 2020). Astrocytes are the most numerous glial cells in the CNS and perform several physiological functions involved in a range of homeostatic maintenance functions. These include the regulation of neurotransmitter transmission, the maintenance of the blood-brain barrier (BBB), the provision of nutrition for neurons, and the promotion of synapse formation and synaptic plasticity (Acosta et al., 2017). Furthermore, similar to microglia, astrocytes have been shown to play a crucial role in the regulation of neuroinflammation (Colombo and Farina, 2016). Recent evidence suggests that neuroinflammation promotes a neurotoxic astrocytic polarization, termed the A1 phenotype, while ischemia leads to a neuroprotective astrocyte polarization (A2) (Liddelow et al., 2017). A1 astrocytes contribute to nerve damage by highly upregulating many classical complement cascade genes, in particular complement component 3 (C3). A shift to A1 astrocytic activation has been shown to require microglial activation, and both A1 astrocytes and activated inflammatory microglia coexist in the prefrontal cortex of AD patients. In contrast, A2 astrocytes play a protective role by upregulation of a variety of neurotrophic factors, including S100 calcium-binding protein A10 (S100A10) and brain-derived neurotrophic factor (BDNF) (Zamanian et al., 2012; Liddelow et al., 2017). Therefore, inhibition of an astrocytic shift to the A1 phenotype, or reversing A1 astrocytes to an A2 phenotype, may represent potential therapeutic options for neurodegenerative diseases.

Toll-like receptor 4 (TLR4) plays an important role in the process of neuroinflammation. In the context of AD, TLR4 and CD14 act together to bind fibrillary Aβ, which causes activation of TLR4, recruitment of MyD88, and activation of NF-κB, ultimately resulting in microglial activation (Gambuzza et al., 2014). Activation of the TLR4/MyD88/NF-κB pathway in microglia increases the levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and IL-6 (Azam et al., 2019; Rahimifard et al., 2017). In turn, this induces the activation of astrocytes, thus affecting amyloid-dependent neuronal death.

There is increasing evidence suggesting that the Rho/Rho kinase (ROCK) signaling pathway is involved in inflammation and oxidative stress. ROCK inhibitors have been shown to prevent neuroinflammation, neuronal damage, and demyelination, and may therefore represent an appropriate treatment option for AD (LoGrasso and Feng, 2009; Yan et al., 2019). Fasudil, a potent ROCK inhibitor, is widely used for treatment of cerebral vasospasm induced by subarachnoid hemorrhage (Sayama et al., 2006). In several of our previous studies we have demonstrated that Fasudil can regulate the polarization of activated microglia and inhibit neuroinflammation in mouse models of Parkinson's disease (PD), experimental autoimmune encephalomyelitis (EAE), and AD (He et al., 2016; Hou et al., 2012; Yu et al., 2017). However, it was unknown whether Fasudil is also able to regulate astrocytic polarization.

In order to further elucidate the cellular and molecular mechanisms underlying the therapeutic actions of Fasudil, the aim of this study was therefore to investigate the effects of Fasudil on both microglial activation and astrocyte polarization in the APP/PS1 double transgenic mouse.