The research conducted by the Andis Klegeris Laboratory of Cellular and Molecular Pharmacology focuses on the fundal mechanisms of intercellular communication and neuroimmune responses of the central nervous system (CNS) as well as the identification of novel therapeutic targets and key signaling processes in neurodegenerative diseases, notably Alzheimer’s disease (AD). We examine the effects of exogenous and endogenous pro- and anti-inflammatory compounds on immune-stimulated model cells of the CNS, and monitor the responses elicited by these molecules using a variety of biochemical assays. By unravelling the intricate signaling processes between neuronal and non-neuronal cells in the healthy brain and under disease conditions, we gain valuable insights into the elusive molecular mechanisms of normal CNS functions as well as neurodegenerative processes.
Neuroimmune Signaling
There are two primary cell types in the CNS; neurons, responsible for transmitting electrical and chemical signals, and glial cells, which maintain homeostasis in the brain environment, and include microglia and astrocytes. Microglia –the resident macrophages of the CNS– support neurodevelopment by secreting neurotrophic factors and clearing cellular debris, protein aggregates, and dying cells. Additionally, microglia can adopt a reactive phenotype in response to endogenous or exogenous stimuli and elicit neuroimmune responses by secreting cytotoxic compounds such as nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. While acute activation of microglia is required for immune defense, these cells are found to sustain a chronically activated state in many neurodegenerative conditions. Microglia in diseased brains release cytotoxic levels of pro-inflammatory mediators into the extracellular space, resulting in neuronal death, cognitive impairment, and memory deficits – hallmark characteristics of AD. Astrocytes play a multifaceted role in maintaining the health of neurons by upkeeping the blood-brain barrier, assisting with synapse formation and elimination, engaging in phagocytic activity, and contributing to inflammatory responses. Maintaining homeostasis in the brain involves complex communication mechanisms between neuronal and non-neuronal cells, and the precise roles of microglia and astrocytes in modulating neuroinflammation remains poorly understood. Therefore, research towards better understanding the pathophysiology of disease-associated microglia and astrocytes is fundamental for identifying effective therapeutic strategies and slowing the progression of neurodegenerative disease.
Microglia and astrocytes are established targets and sources of signaling molecules, including chemokines, cytokines, and growth factors, that are necessary for the crosstalk between the different CNS cell types. To this end, we have extensively studied the role of a variety of signaling molecules in neuroimmunomodulation by these cell types. Our findings reveal distinct responses by murine microglia to a variety of immune stimulants, including exogenous lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), and zymosan A, and endogenous interferon (IFN)-ß and IFN-γ (Bernath et al., 2023). Additionally, we have characterized LPS, IFN-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and cytochrome c (CytC) as key differential regulators of human and murine astrocyte phagocytic activity (Yang et al., 2023). These results demonstrate the dependence of neuroimmune responses on the microenvironment of cells and highlight existing differences between glial cells derived from different species.
Damage-associated molecular patterns
In addition to the known exogenous triggers of CNS neuroinflammation, our lab has identified additional key players in the molecular mechanisms of neurodegeneration in the form of damage-associated molecular patterns, or DAMPs. DAMPs are intracellular compounds, that when released into the extracellular space by damaged or dying cells, act as signaling molecules to elicit an acute inflammatory response in immune cells. In disease conditions, the accumulation of dying neurons result in toxic levels of DAMPs present in the extracellular milieu, exacerbating the inflammatory cycle (Klegeris, 2020). Therefore, identifying novel DAMPs in the CNS is essential for understanding the pathogenesis of AD.
Histones are a family of highly conserved DNA binding proteins that play an essential role in epigenetic regulation. Histones are recognised to be released into the extracellular space by dying cells, where they act as DAMPs on peripheral immune cells. Our lab has identified linker histone H1 and core histone H3 as modulators of neuroimmune responses in the CNS, which upregulate the production of NO, TNF-α, and IFN-γ-inducible protein (IP)-10, in both human and murine microglia-like cells. Moreover, histone H1 downregulates phagocytic activity –an important homeostatic process– in murine microglia and is directly cytotoxic to both human and murine neuronal cells (McRae et al., 2024).
Cytochrome c (CytC) is a metalloprotein normally located in the mitochondrial intermembrane space, where it facilitates electron transport necessary for adenosine triphosphate (ATP) production. When released into the cytosol by damaged mitochondria, CytC acts as a signaling molecule for the initiation of apoptosis. Recent evidence indicates that CytC can be released into the extracellular space by dying cells, suggesting potential DAMP-like properties. Recently we established that CytC exhibits DAMP activity within the CNS, where upon recognition by microglia, it elicits a pro-inflammatory response characterized by increased ROS and NO production, and enhanced cytotoxicity of immune stimulated microglia-like cells (Gouveia et al., 2017). Furthermore, extracellularly applied CytC increases immune activation of primary human astrocytes, by upregulating secretion of IL-1β, IL-12 p70, and granulocyte-macrophage colony stimulating factor (GM-CSF) (Wenzel et al., 2019).
Anti-inflammatory mediators
Our group has contributed to the evidence supporting a hypothesis that neuronal death stems from the adverse activation of microglia, in part by studies demonstrating the neuroprotective mechanism of non-steroidal anti-inflammatory drugs (NSAIDs) and other anti-inflammatory compounds. These investigations reinforce the hypothesis that normalizing immune responses of disease-associated microglia provides a viable target for slowing the progression of neurodegenerative disease. To this end, our laboratory has identified low molecular weight compounds capable of crossing the blood-brain barrier that hold promise as therapeutic agents for attenuating chronic neuroinflammation (Alford et al., 2019; McKenzie et al., 2019).
Kainic acid (KA) is a natural analog of the excitatory neurotransmitter glutamate. Recent studies have demonstrated KA to modulate microglial function and prevent chronic activation. However, KA and glutamate are known neurotoxins at high concentrations. Our group has characterized the neuroprotective potential of eight novel kainic acid analogs, four of which possess anti-inflammatory activity towards murine and human microglia-like cells by attenuating the production of cytotoxic ROS, NO, and monocyte chemoattractant protein (MCP)-1, in part by targeting aldose reductase (Alford et al., 2019). We are continuing to study new anti-inflammatory compounds that hold promise as inhibitors of chronic inflammation in the CNS.
Significance
At present, only symptomatic treatments exist for AD, with no disease modifying therapies available. Therefore, continued study of the molecular mechanisms are critical for gaining a broader understanding of disease progression and establishing viable treatment strategies. If you are interested in joining the research effort, please contact Dr. Andis Klegeris (andis.klegeris@ubc.ca) for more information.