Emerging technologies to study glial cells.

Development, physiological functions, and pathologies of the brain depend on tight interactions between neurons and different types of glial cells, such as astrocytes, microglia, oligodendrocytes, and oligodendrocyte precursor cells. Assessing the relative contribution of different glial cell types is required for the full understanding of brain function and dysfunction. Over the recent years, several technological breakthroughs were achieved, allowing "glio-scientists" to address new challenging biological questions. These technical developments make it possible to study the roles of specific cell types with medium or high-content workflows and perform fine analysis of their mutual interactions in a preserved environment. This review illustrates the potency of several cutting-edge experimental approaches (advanced cell cultures, induced pluripotent stem cell (iPSC)-derived human glial cells, viral vectors, in situ glia imaging, opto- and chemogenetic approaches, and high-content molecular analysis) to unravel the role of glial cells in specific brain functions or diseases. It also illustrates the translation of some techniques to the clinics, to monitor glial cells in patients, through specific brain imaging methods. The advantages, pitfalls, and future developments are discussed for each technique, and selected examples are provided to illustrate how specific "gliobiological" questions can now be tackled.

Neuronal Activity and Intracellular Calcium Levels Regulate Intracellular Transport of Newly Synthesized AMPAR.

Regulation of AMPA receptor (AMPAR) trafficking is a key modulator of excitatory synaptic transmission; however, intracellular vesicular transport of newly synthesized AMPARs has been little studied due to technical limitations. By combining molecular tools with imaging strategies in cultured rat hippocampal neurons, we found that vesicles containing newly synthesized, GluA1-subunit-containing AMPARs are transported antero- and retrogradely at a mean speed of 1.5 µm.s-1. Synaptic activity and variations in intracellular calcium levels bidirectionally modulate GluA1 transport. Chemical long-term potentiation (cLTP) initially induces a halt in GluA1 transport, followed by a sustained increase, while acute glutamate uncaging on synaptic spines arrests vesicular movements. GluA1 phosphomimetic mutants preferentially travel to the dendritic tip, probably to replenish extrasynaptic pools, distal to the soma. Our findings indicate that AMPAR intracellular transport is highly regulated during synaptic plasticity and likely controls AMPAR numbers at the plasma membrane.

Apoptosis inducing factor (AIF) mediates lethal redox stress induced by menadione.

Mitochondrial apoptosis inducing factor (AIF) is a redox-active enzyme that participates to the biogenesis/maintenance of complex I of the respiratory chain, yet also contributes to catabolic reactions in the context of regulated cell death when AIF translocates to the cytosol and to the nucleus. Here we explore the contribution of AIF to cell death induced by menadione (2-methyl-1,4-naphtoquinone; also called vitamin K3) in conditions in which this pro-oxidant does not cause the mitochondrial release of AIF, yet causes caspase-independent cell killing. Depletion of AIF from human cancer cells reduced the cytotoxicity of menadione. This cytoprotective effect was accompanied by the maintenance of high levels of reduced glutathione (GSH), which are normally depleted by menadione. In addition, AIF depletion reduced the arylation of cellular proteins induced by menadione. This menadione-triggered arylation, which can be measured by a fluorescence assay, is completely suppressed by addition of exogenous glutathione or N-acetyl cysteine. Complex I inhibition by Rotenone did not mimic the cytoprotective action of AIF depletion. Altogether, these results are compatible with the hypothesis that mitochondrion-sessile AIF facilitates lethal redox cycling of menadione, thereby precipitating protein arylation and glutathione depletion.

Metabolic epistasis among apoptosis-inducing factor and the mitochondrial import factor CHCHD4.

Hypomorphic mutation of apoptosis-inducing factor (AIF) in the whole body or organ-specific knockout of AIF compromises the activity of respiratory chain complexes I and IV, as it confers resistance to obesity and diabetes induced by high-fat diet. The mitochondrial defect induced by AIF deficiency can be explained by reduced AIF-dependent mitochondrial import of CHCHD4, which in turn is required for optimal import and assembly of respiratory chain complexes. Here we show that, as compared to wild type control littermates, mice with a heterozygous knockout of CHCHD4 exhibit reduced weight gain when fed with a Western style high-fat diet. This finding suggests widespread metabolic epistasis among AIF and CHCHD4. Targeting either of these proteins or their functional interaction might constitute a novel strategy to combat obesity.

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation.

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease.

Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

Life with or without AIF.

Apoptosis-inducing factor (AIF) was initially discovered as a caspase-independent death effector. AIF fulfills its lethal function after its release from mitochondria and its translocation to the nucleus of the dying cell. The contribution of AIF to programmed cell death is dependent upon the cell type and apoptotic insult. Recent in vivo data indicate that, in addition to its lethal activity, AIF plays a vital mitochondrial role in healthy cells. A segment of AIF which is dispensable for its apoptotic function carries an NADH-oxidase domain that regulates the respiratory chain complex I and is required for cell survival, proliferation and mitochondrial integrity. Mice that express reduced levels of AIF constitute a reliable model of complex I deficiency. Here we discuss recent reports on the survival-related function(s) of AIF.

AIF: not just an apoptosis-inducing factor.

Since its discovery nearly a decade ago, apoptosis-inducing factor (AIF) has had anything but a staid and uneventful existence. AIF was originally described as a mitochondrial intermembrane protein that, after apoptosis induction, can translocate to the nucleus and trigger chromatin condensation and DNA fragmentation. Over the years, an AIF-mediated caspase-independent cell death pathway has been defined. Rather than functioning as a general component of the cell death machinery, AIF is required for specific cell death pathways, including lethal responses to excitotoxins such as N-methyl-D-aspartate and glutamate, the DNA-alkylating agent N-methyl-N'-nitro-N-nitroso-guanidine, hypoxia-ischemia, or growth factor deprivation. Also, important roles of AIF in mitochondrial metabolism and redox control, and more recently in obesity and diabetes, have been discovered. Much of our knowledge has come from studies of AIF orthologs in model organisms, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mice, which have also highlighted the importance of AIF in animal physiology and human pathology. Here, we discuss the manifold nature of AIF in cell life and death, with particular emphasis of its roles in vivo.

Methods to dissect mitochondrial membrane permeabilization in the course of apoptosis.

In several paradigms of cell death, mitochondrial membrane permeabilization (MMP) delimits the frontier between life and death. Mitochondria control the intrinsic pathway of apoptosis and participate in the extrinsic pathway. Moreover, they have been implicated in nonapoptotic cell death modalities. Irrespective of its initiation at the inner or the outer mitochondrial membrane (IM and OM, respectively), MMP culminates in the functional (dissipation of the mitochondrial transmembrane potential, shutdown of ATP synthesis, redox imbalance) and structural (reorganization of cristae, release of toxic intermembrane space proteins into the cytosol) collapse of mitochondria. This has a profound impact on cellular metabolism, activates caspase-dependent and -independent executioner mechanisms, and finally results in the demise of the cell. However, the partial and/or temporary permeabilization of one or both mitochondrial membranes is not always a prelude to cell death. This chapter proposes a method and several guidelines to discriminate between IM and OM permeabilization and to identify MMP that does indeed precede cell death. This approach relies on the integration of currently available techniques and may be easily introduced in the laboratory routine for a more precise detection of cell death.

Methods for assessing autophagy and autophagic cell death.

Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.