ABSTRACT
Studying the intrinsic properties of microglia, astrocytes, and neurons is essential to our understanding of brain function. Here, we present a protocol to isolate and culture these neural cells from the same mouse brain. Using immunocapture magnetic beads, we describe steps for dissociating, cleaning, and sequentially separating brains from 9-day-old mice into microglia, astrocytes, and neurons. Following these detailed procedures for seeding and culturing of isolated cells, we can address critical questions related to brain function.
Subject(s)
Astrocytes , Microglia , Mice , Animals , Neurons/physiology , Brain , Immunomagnetic SeparationABSTRACT
The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer.
ABSTRACT
Mitochondria have a pivotal role in the maintenance of cell homeostasis and survival. Mitochondria are involved in processes such as ATP production, reactive oxygen species production, apoptosis induction, calcium homeostasis and protein degradation. Thus, mechanisms that regulate the intrinsic quality of mitochondria have a crucial role in dictating overall cell fate. The importance of these well-regulated mechanisms is highlighted in disease scenarios such as neurodegeneration, cancer and neuromuscular atrophy. How mitochondria senses and regulates their intrinsic quality control, and consequently cell survival, is still not fully understood. In this review, we discuss the pathways that are at present considered as state-of-the-art for mitochondria quality control regulation, and highlight a mitochondrial protein-PINK1-that has revealed to act as a mitochondrial gatekeeper able to sense the presence of healthy or damaged mitochondria.
Subject(s)
Mitochondria/genetics , Mitophagy/genetics , Muscular Atrophy/genetics , Neoplasms/genetics , Neurodegenerative Diseases/genetics , Protein Kinases/genetics , Apoptosis/genetics , Dynamins , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Gene Expression Regulation , Homeostasis/genetics , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Neoplasms/metabolism , Neoplasms/pathology , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Protein Kinases/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
PINK1 is mutated in Parkinson's disease (PD), and mutations cause mitochondrial defects that include inefficient electron transport between complex I and ubiquinone. Neurodegeneration is also connected to changes in lipid homeostasis, but how these are related to PINK1-induced mitochondrial dysfunction is unknown. Based on an unbiased genetic screen, we found that partial genetic and pharmacological inhibition of fatty acid synthase (FASN) suppresses toxicity induced by PINK1 deficiency in flies, mouse cells, patient-derived fibroblasts, and induced pluripotent stem cell-derived dopaminergic neurons. Lower FASN activity in PINK1 mutants decreases palmitate levels and increases the levels of cardiolipin (CL), a mitochondrial inner membrane-specific lipid. Direct supplementation of CL to isolated mitochondria not only rescues the PINK1-induced complex I defects but also rescues the inefficient electron transfer between complex I and ubiquinone in specific mutants. Our data indicate that genetic or pharmacologic inhibition of FASN to increase CL levels bypasses the enzymatic defects at complex I in a PD model.
