Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Abstract

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique -described here -is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis 13,14 . Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer's or Parkinson's disease. Video LinkThe video component of this article can be found at

Living Neurons with Tau Filaments Aberrantly Expose Phosphatidylserine and Are Phagocytosed by Microglia

“…We noted several examples of IB4 + phagocytes in close proximity to cultured pFTAA + neurons and some internalization of pFTAA + aggregates ( Figure 4C ). To examine whether endogenous phagocytes were responsible for the loss of tau aggregate-containing neurons that occurs over 2–3 weeks, we eliminated the phagocytes by treatment (4 hr) with leucine methyl ester (LME) ( Jebelli et al, 2015 ), followed by an overnight incubation to ensure death of phagocytes. We found that ~90% of the pFTAA + neurons were maintained after phagocytes were eliminated from the cultures, whereas only ~70% of the neurons remained in untreated cultures after 14 days ( Figure 4D ; p < 0.0001).…”

Astrocytes in juvenile neuronal ceroid lipofuscinosis (CLN3) display metabolic and calcium signaling abnormalities

“…; Jebelli et al . ), in the absence of growth factor supplementation. Therefore, the presence of growth factors during the culture period and initial contact with other glia may contribute to the differences in astrocyte glutamate phenotypes between the studies.…”

Soluble Fibrinogen Triggers Non-cell Autonomous ER Stress-Mediated Microglial-Induced Neurotoxicity

“…Microglia present in the ENGCs were depleted with 25 mM leucine-methyl-ester (LME), as previously described ( Jebelli et al, 2015 ).…”

“…Conversely, ER stress can also mediate upregulation of pro-inflammatory cytokines ( Chen et al, 2013 ), suggesting the possibility of a self-perpetuating mechanism of detrimental signaling in neurodegenerative diseases centered on ER stress and aberrant, or chronic, inflammatory signaling. With this in mind, we aimed to identify if exposure to FG modulated inflammatory signaling and subsequent neuronal toxicity in a well-characterized in vitro neuronal cell culture model, which can be pharmacologically manipulated to eliminate the presence of microglia ( Piers et al, 2011 ; Jebelli et al, 2015 ). Here, we identify for the first time, a FG-induced increase in neurotoxicity via a non-cell autonomous mechanism involving a microglial TNFα-mediated induction of neuronal ER stress signaling.…”