Capetillo-Zarate, E. | Search bioRxiv

bioRxiv2021

A neuron, microglia, and astrocyte triple coculture model to study Alzheimer disease

Glial cells are essential to understand Alzheimers disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as cocultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated a triple co-culture with neurons, microglia and astrocytes. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric A{beta} (oA{beta}) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory markers Arginase I and TGF-{beta}1, and reduced pro-inflammatory iNOS and IL-1{beta}, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oA{beta} in the triple coculture reduced synaptic markers and increased microglial activation, which are hallmarks of AD. Consequently, we developed a reliable model, where cells better resemble physiological conditions: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology than in individual primary cultures. Moreover, we were able to recapitulate A{beta}-induced synaptic loss and inflammation. This model emerges as a powerful tool to study neurodegeneration and inflammation in the context of AD and other neurodegenerative diseases. Table of content image O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Main pointsO_LIIn our model, microglia and astrocytes are less reactive, and neurons have a more mature morphology than in primary cultures. C_LIO_LIoA{beta} reduced synaptic markers and increased microglial activation. C_LIO_LIThis triple co-culture is a reliable tool to study neurodegeneration and gliosis in vitro. C_LI

neuroscience↗

bioRxiv2021

Microglial phagocytosis dysfunction during stroke is prevented by rapamycin

Microglial phagocytosis is rapidly emerging as a therapeutic target in neurodegenerative and neurological disorders. An efficient removal of cellular debris is necessary to prevent buildup damage of neighbor neurons and the development of an inflammatory response. As the brain professional phagocytes, microglia are equipped with an array of mechanisms that enable them to recognize and degrade several types of cargo, including neurons undergoing apoptotic cell death. While microglia are very competent phagocytes of apoptotic cells under physiological conditions, here we report their dysfunction in mouse and monkey (Macaca fascicularis and Callithrix jacchus) models of stroke by transient occlusion of the medial cerebral artery (tMCAo). The impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrients deprivation (OND), which led to reduced process motility, lysosomal depletion, and the induction of a protective autophagy response in microglia. Basal autophagy, which is in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using knock-out models of autophagy genes and the autophagy inhibitor MRT68921. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro. These results suggest a more complex role of microglia in stroke than previously acknowledged, classically related to the inflammatory response. In contrast, here we demonstrate the impairment of apoptotic cell phagocytosis, a microglial function critical for brain recovery. We propose that phagocytosis is a therapeutic target yet to be explored and provide evidence that it can be modulated in vivo using rapamycin, setting the stage for future therapies for stroke patients.

neuroscience↗