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João Relvas
Institute for Research and Innovation in Health (i3S) – University of Porto, Portugal
The actin cytoskeleton dynamically controls many different aspects of cell biology. Its reorganization likely underlies critical microglial function(s), including phagocytosis, process extension required for surveillance and synaptic interactions, and whole-cell migration notably towards pathological foci. The ubiquitously expressed GTPases of the Rho family, including the most well-characterized members RhoA, Rac1, and Cdc42, are critical orchestrators of cytoskeleton reorganization, making them essential players to govern microglial function. Recent studies from our lab highlighted essential and distinct roles for RhoGTPases RhoA and Rac1 in controlling the microglial cytoskeleton and function. While RhoA balances microglial reactivity during neuroinflammation, and its ablation in adult microglia results in microglia dysfunction leading to neurodegeneration, Rac1 is required for crucial microglia-synapse crosstalk driving experience-dependent plasticity. During my talk, I will present and discuss these and other recent unpublished data related to regulation of microglia function by the actin cytoskeleton.
Izabela Lepiarz-Raba1, Taufik Hidayat1, Weronika Tomaszewska1, Sandra Binias2, Bartłmiej Gielniewski2, Jacek Miłek3, Magdalena Dziembowska3, Ali Jawaid1
1Translational Research in Exposures and Neuropsychiatric Disorders (TREND), Nencki Institute of Experimental Biology PAS, Warsaw, Poland
2Laboratory of Sequencing, Nencki Institute of Experimental Biology PAS, Warsaw, Poland
3Department of Biology, University of Warsaw, Warszawa, Poland
Alzheimer's disease (AD), the leading cause of dementia, is characterized by abnormal accumulation of amyloid-β (Aβ) in the brain. Aβ clearance is primarily the function of microglia, the brain-resident immune cells that are highly sensitive to environmental stimuli and respond to homeostatic changes via altering the release of inflammatory cytokines and phagocytosis. These functional adaptations in microglia are intricately linked to their metabolism, which provides a unique opportunity to harnessing microglial phagocytosis for selective Aβ clearance in AD without substantially harming healthy neurons. To investigate this further, we tested Aβ phagocytosis in HMC3 human microglia after various metabolic manipulations. Starving HMC3 microglia of lipids via delipidation of the medium, as well as, overall nutrients through serum starvation increased the uptake of Aβ. However, efficient degradation of the internalized Aβ was only observed upon lipid starvation, whereas only a minor fraction of the Aβ internalized upon serum starvation degraded over 24 hours. Furthermore, both lipid and serum starvation induced transcriptomic changes in HMC3 microglia in molecular cascades relevant to cholesterol biosynthesis, sterol response element binding factors (SREBF) signalling and steroid metabolism. Notably, knock down of SREBF2 abolished the effect of lipid starvation on Aβ phagocytosis by HMC3 microglia without impacting their phagocytosis of healthy neurosynaptosomes. These results represent microglial SREBF signalling as a novel therapeutic target in AD that can be targeted for preferential microglial clearance of Aβ. In vivo validation of these findings, as well as, clinical correlations based on serum samples from AD patients are subjects of our ongoing investigations.
Natalia Malek
Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wroclaw, Poland
TMicroglia, as integral immune cells within the central nervous system (CNS), play a pivotal role in maintaining immune balance in response to infections or injuries. However, prolonged activation of microglia can lead to neuroinflammation, with microglial pyroptosis emerging as a potential contributor to neuronal inflammation. Despite this, research on microglial pyroptosis remains limited, prompting our investigation into the activation of NLRP3 pathways in these cells. Therapies for microglia-derived neuroinflammation are lacking, yet modulation of cannabinoid receptor type 2 (CB2) shows promise. While the exact mechanism of CB2 agonists in exerting anti-inflammatory effects is unclear, recent research highlights the involvement of inflammasomes, particularly the NLRP3 inflammasome. Our study aimed to elucidate the downstream targets of CB2 stimulation within the canonical and non-canonical components of the NLRP3 inflammasome pathway. Using a murine cell line lacking CB2 receptor expression, we observed that CB2 agonist treatment reduced microglial pyroptosis and neuroinflammation induced by inflammatory stimuli. Specifically, we observed decreased expression of Caspase 4, indicative of involvement of the non-canonical pyroptosis pathway in the absence of CB2 signaling. Furthermore, we detected increased and prolonged calcium influx in the absence of CB2 receptor, leading to activation of the calpain-dependent pyroptotic pathway. Additionally, CB2 activation resulted in reduced release of IL1β and IL18, characteristic of pyroptosis, suggesting a neuroprotective role for CB2 receptor stimulation in mitigating NLRP3 inflammasome-mediated pyroptosis in microglia.
Funding: Supported by National Science Center, Poland grant OPUS 2020/37/B/NZ7/03411
Natalia Stelmach1, Wioletta Rut1, Marcin Drąg1, Natalia Małek-Chudzik1
1Department of Chemistry, Department of Chemical Biology and Bioimaging, Wroclaw Institute of Science and Technology, Wroclaw, Poland
Proteostasis is crucial for maintaining the proper synthesis, folding, and degradation of proteins within cells. Disruption of this balance can contribute to the development of neurological conditions characterized by the formation of inclusion bodies, such as Alzheimer’s and Parkinson’s disease. The dysregulation of proteostasis can be influenced by a decrease in the catalytic activity of the 20S proteasome, leading to the accumulation of various proteins and the promotion of chronic inflammation, particularly involving microglial cells. In this study, we examined how inflammation induced by lipopolysaccharide (LPS) activation affects the expression of the β1, β2, and β5 subunits of the 20S proteasome in a human microglial cell line (HMC3). Our investigation involved analyzing the transcriptome using RT-qPCR, the proteome via Western Blotting, and the activome using Activity Based Probes (ABP) staining. Transcriptomic analysis revealed upregulation of β1 and β5 subunits in the presence of LPS, while protein-level analysis showed elevated levels of β1 and β2 subunits along with a decrease in β5 subunit levels. Activome studies indicated higher activity of β1 and β2 subunits and lower activity of β5 in the presence of LPS, consistent with the protein-level findings. Our comprehensive investigation sheds light on the dynamic changes occurring during inflammation in microglial cell proteostasis, suggesting that targeting the activity of β subunits of the 20S proteasome could be a viable approach for therapeutic intervention in neurodegenerative diseases.
Funding: Supported by National Science Center, Poland grant OPUS 2020/37/B/NZ7/03411.
