DF3016A induces increased BDNF transcription in ischemic neuroinflammation injury

Highlights

• C5aR is involved in the neuroinflammatory process induced by hypoxia/ischemia.

• The DF3016A, a C5aR antagonist, promotes protection in ischemic/hypoxic neurons.

• The DF3016A induces neuroprotection by activating BDNF pathway.

Abstract

C5a is a crucial terminal effector of the C cascade, mostly involved in pain and neuroinflammatory conditions. DF3016A is a novel potent and selective C5a receptor (C5aR) inhibitor that crosses the blood-brain barrier (BBB) and may have pharmacological properties. We have previously demonstrated a protective effect of DF3016A on injured primary cortical neurons by oxygen-glucose deprivation-reoxygenation (OGD/R) model to mimic the neuroinflammatory process. Here, we investigated the molecular pathway and factors involved in the neuroprotection previously reported. Our findings show that DF3016A protects against the neuroinflammatory insult by activating brain-derived neurotrophic factor (BDNF) transcription pathway, which involves methyl CpG-binding protein 2 (MeCP2) and microRNA-132 (miR-132) regulatory factors, both required in nociceptive signaling and neuroinflammation. Further in vivo investigations will confirm the functionality of the DF3016A molecule as a therapeutic resource in neuroinflammation and pain injuries.

Introduction

The complement (C) cascade is a part of the innate immune system that enhances the ability of antibodies and phagocytic cells to remove microbes, promote inflammation and kill the pathogen cells. The complement system consists of a number of small proteins that circulate in the blood as inactive precursors. The Central Nervous System (CNS) constitutively expresses complement cascade proteins and receptors. C5a is a crucial protein of the C cascade, mostly involved in pain and neuroinflammatory conditions. When activated, C5a initiates an amplifying cascade of further cleavages that ends in the stimulation of phagocytes and inflammation. In the CNS, sensitization and increased neuronal responsiveness is a consequence of neuroinflammation in the peripheral and central nervous system. Persistent nociceptive input induces central sensitization characterized by increased neuronal activity, synaptic and molecular adjustments that involve neurotransmitters, growth factors and protein activation through signal transduction (Ji et al., 2018). Increased neuronal activity may trigger the concerted actions of neighboring neurons and immunocompetent molecules release, increase in the permeability of the blood-spinal cord barrier (BSCB) and the blood-brain barrier (BBB) causing neuroinflammation within the CNS (Beggs et al., 2010, Skaper et al., 2012).

The persistent sensitization in CNS induces neuroinflammation state that is a major cause of neuronal degeneration, and chronic neuroinflammation is critical for pain development and maintenance (Fusco et al., 2017). However, the primary goal of the inflammatory process is to repair the injured tissues and preserve the integrity of the neuronal cells. Growth factors are primarily implicated in neuronal mechanisms involving recovery processes.

The brain-derived neurotrophic factor (BDNF) and its regulatory factors fit perfectly into this context. The role of BDNF to promote survival, differentiation and synaptic plasticity in the central nervous system is well-known (Berninger and Poo, 1996). In adulthood, BDNF has a critical role in central pain modulation (Merighi et al., 2008). Accumulating evidence suggests that BDNF transcription is regulated by different regulatory factors that act synergically according to different physiological or in response to detrimental processes. MicroRNAs (miRNAs), particularly microRNA-132 (miR-132) and methyl CpG-binding protein 2 (MeCP2), are the main regulatory factors of BDNF transcription (Carlezon et al., 2005, Xie et al., 2019).

MiRNAs, a highly conserved non-coding single-strand RNAs with about 22 nucleotides, regulate the gene expression at the post-transcriptional level. Specifically, miRNAs can exert their effects directly on the messenger RNA (mRNA) via interacting with the 3′-untranslated region (UTR) of mRNA of the target gene, leading to protein translation inhibition (Bartel, 2004, Mendell and Olson, 2012). Neuronal stress can induce de novo protein synthesis, and miRNAs regulate the protein translation by mediating the neuronal plasticity. Moreover, miRNAs are involved in many cellular processes like cell apoptosis, growth, differentiation, and many pathological events (Mendell and Olson, 2012). In mammals, differentiated neurons from CNS highly express miR-132 that induce bdnf transcription in a cAMP response element-binding protein (CREB) -dependent manner (Vo et al., 2005). Chronic mild stress can induce changes in bdnf expression and miRNAs expressions together with MeCP2 modifications (Su et al., 2015).

MiR-132 tightly regulates the expression of MeCP2 (Klein et al., 2007) by selectively binding to bdnf promoter III (Chen et al., 2003). MeCP2 transcriptional factors can modulate DNA methylation and chromatin remodeling. Thus, miR-132 appears to connect the synaptic activity to the neuronal structural/functional plasticity.

Using oxygen-glucose deprivation-reoxygenation (OGD/R), an experimental model of the neuroinflammatory process (Goldberg and Choi, 1993), we have previously demonstrated a protective effect of DF3016A in a model of injured cortical neurons obtained by oxygen-glucose deprivation-reoxygenation (OGD/R), and which mimics the neuroinflammatory process. We showed that DF3016A protects neuronal viability by restoring the intracellular calcium homeostasis, and the pro-inflammatory cytokine levels (Brandolini et al., 2019). However, the molecular mechanism involved in the neuroprotection is still unknown.

In this study we investigated how the DF3016A exerts its protection under the neuroinflammatory condition, and we discovered that BDNF, a major neurotrophic factor, is tightly involved in the protective pathway. We provided the evidence that miR-132 and MeCP2 are also involved in the process. These findings highlighted the possible therapeutic resource of the DF3016A molecule as a potential candidate in neuroinflammation and pain treatment.