Tri-culture modeling of neuroinflammation, neurodegeneration, and neuroprotection

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Tri-culture modeling of neuroinflammation, neurodegeneration, and neuroprotection

Aswathy Peethambaran Mallika https://orcid.org/0000-0003-0303-5156 apeetha1@jhmi.eduView all authors and affiliations

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Abstract

The study of neurodegenerative diseases, such as Alzheimer's disease (AD), has long been a complex and challenging task. One of the major hurdles in understanding these diseases is the difficulty in recapitulating the complex interactions between neurons, astrocytes, and microglia in a laboratory setting. In recent years, researchers have made significant progress in developing triculture models that combine these three cell types, allowing for a more accurate representation of the cellular context of the human brain. This commentary discusses the recent advancements and importance of using tri-culture model systems in clarifying the pathophysiology of AD and discusses the recent article by Kim et al. (2024) published in the Journal of Alzheimer's Disease.

The human brain contains over 100 billion neurons(Actually, you're out-of-date, it's 80 billion), each with unique morphology, function, and connectivity.^1–3 Understanding the interactions and coordination between these cells during physiological and pathological states is crucial for elucidating the mechanisms underlying neurodegenerative diseases, such as Alzheimer's disease (AD). Neuroinflammation is a hallmark of many neurodegenerative diseases, including AD, and is mediated by microglia, which play a critical role in the initiation and progression of neurodegenerative diseases. The relation between microglia and neuroinflammation is bi-directional.^4–6 These findings have led researchers to study these complex processes in detail and how it contributes to neurodegeneration using model systems combining neurons, astrocytes and microglia.

While microglial monocultures have been a cost effective and valuable tool for studying the responses of these cells to specific inflammatory stimuli or toxins, it has several limitations. One of the main drawbacks is that they do not accurately reflect the in vivo state, where microglia interact with other cell types in a complex and dynamic manner. Therefore, monocultures may not fully capture the microglial behavior and function in the context of disease leading researchers to recognize the need to move beyond monocultures and study microglia in more complex and physiologically relevant settings. Co-cultures, which involve the simultaneous culture of microglia with other cell types, have been developed as means to better mimic the in vivo niche and gain a more comprehensive understanding of microglial function in disease.

To model the cellular context of the human brain, Haenseler et al. co-cultured induced pluripotent stem cell (iPSC)-derived microglia with cortical neurons and reported characteristic microglial features, including expression of microglia-specific markers, dynamic ramifications, and phagocytic capacity, along with enhanced homeostatic gene expression and anti-inflammatory cytokine profiles compared to monocultures.⁷ However, studies have shown that microglia are influenced by the secreted molecules from neurons and astrocytes.⁸ Since then, several groups have developed more advanced triculture models that better mimic the in vivo environment. For example, Park et al. developed a 3D tri-culture model system where neurons and astrocytes were differentiated from human neural progenitor cells first, followed by the addition of adult immortalized human microglia cells mimicking different stages of AD.⁹ This model demonstrated amyloid-β (Aβ) aggregation, phosphorylated tau formation, and increased proinflammatory chemokine/cytokine expression accompanied by microglial recruitment and marked neuron/astrocyte loss, suggesting its validity as a tool to investigate neuroinflammatory mechanisms underlying AD pathophysiology.⁹ In another study, Guttikonda et al. employed a tri-culture system comprising human iPSC-derived neurons, microglia, and astrocytes to investigate the role of complement C3 in AD, revealing a bidirectional signaling between microglia and astrocytes that led to increased C3 levels under pathological conditions.¹⁰ Bassil et al. developed a long-term triculture platform using human iPSC-derived neurons, microglia, and astrocytes, which allowed them to replicate the key features of AD, including Aβ plaques, dystrophic neurites, synaptic loss, dendrite retraction, axon fragmentation, phosphorylated tau induction, and neuronal cell death.¹¹ By adding soluble Aβ species to the system, they found that iPSC-derived microglia were able to provide neuroprotection by internalizing and compacting Aβ, not only through generating but also surrounding plaques.¹¹

Despite their potential, these models can be limited by their complexity and high cost, making them inaccessible to many researchers. Goshi et al. developed a tri-culture model using primary cortical cells from neonatal rats, which could also mimic in vivo neuroinflammatory responses.¹² This model is suggested to be more accurate than traditional mono- and co-cultures as it captured CNS response to lipopolysaccharide exposure and glial scarring in a scratch assay, and neuronal rescue from glutamate-induced excitotoxicity, making it a valuable tool for studying neuroinflammation in vitro.¹² To further advance the knowledge in the context of Aβ induced neurodegeneration and associated neuroinflammatory responses, the same group of researchers report the use of primary rat cortical tri-culture model to study the internalization of Aβ by microglia in the presence of neurons and astrocytes in the latest issue of the Journal of Alzheimer's Disease.¹³ They have used the model to characterize neural and microglial response to Aβ exposure compared to neuronal-astrocyte co-culture and microglial monoculture. They exposed the cultures to fluorescently labeled Aβ (FITC-Aβ) particles and using epifluorescence microscopy, live cell imaging and cytokine profiling, they report that the tri-culture model comprising microglia was able to clear the FITC-Aβ particles more efficiently than that observed in the co-culture model.¹³ This will be important given the growing body of evidence that Aβ clearance by microglia in CNS as well as macrophages (the counterparts of microglia) in the peripheral system, plays crucial role in the clearance of Aβ and the progression of neurodegeneration in AD.^14–17 These findings align with previous research, including studies by Baxter et al.,¹⁸ which showed that the loss of normal microglial function in disease states can be rescued by co-culturing with astrocytes and neurons, the study by Phadke et al.,¹⁹ demonstrating that microglia could suppress neuronal activity and alter synaptic function in a tri-culture model system and the study by Luchena et al.²⁰ reporting that microglia are less inflammatory, astrocytes are less reactive, and neurons display a more mature morphology, in the tri-culture system making it a powerful tool to study neuroinflammation and neurodegeneration in the context of AD and other neurodegenerative diseases. While the study by Kim et al. provides valuable insights into the interactions between microglia and neurons in the context of Aβ exposure, it is crucial to consider the potential influence of genetic variations on these responses. Future studies should also address the response of these cells to tau proteins and should incorporate genetic factors such as APP, PSEN, APOE, TREM2, etc. to provide a more comprehensive understanding of the variability in neuronal and microglial responses.

Moreover, while rodent microglia may not perfectly replicate the human disease context, these studies can still offer valuable insights into the fundamental mechanisms underlying neuroinflammation, neurodegeneration, and neuroprotection during disease progression and could be pivotal in high-throughput screening efforts for target identification, assay development, and therapeutic drug discovery. By understanding the basic mechanisms, researchers can then extrapolate their findings to the human context, potentially leading to the development of new therapeutic strategies for the treatment of neurodegenerative diseases. In conclusion, these tri-culture model systems offer a promising platform for modeling and testing mechanisms associated with microglia in the presence of neurons and astrocytes, providing a more accurate representation of the cellular context of the human brain and AD. However, it is essential to exercise caution when interpreting results, taking into account the origin and condition of cells used, to ensure that findings are accurately translated to the human context.