Integrated analysis of transcriptomic and proteomic alterations in mouse models of ALS/FTD identify early metabolic adaptions with similarities to mitochondrial dysfunction disorders.

OBJECTIVE: Sporadic and familial amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease that results in loss of motor neurons and, in some patients, associates with frontotemporal dementia (FTD). Apart from the accumulation of proteinaceous deposits, emerging literature indicates that aberrant mitochondrial bioenergetics may contribute to the onset and progression of ALS/FTD. Here we sought to investigate the pathophysiological signatures of mitochondrial dysfunction associated with ALS/FTD. METHODS: By means of label-free mass spectrometry (MS) and mRNA sequencing (mRNA-seq), we report pre-symptomatic changes in the cortices of TDP-43 and FUS mutant mouse models. Using tissues from transgenic mouse models of mitochondrial diseases as a reference, we performed comparative analyses and extracted unique and common mitochondrial signatures that revealed neuroprotective compensatory mechanisms in response to early damage. RESULTS: In this regard, upregulation of both Acyl-CoA Synthetase Long-Chain Family Member 3 (ACSL3) and mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) were the most representative change in pre-symptomatic ALS/FTD tissues, suggesting that fatty acid beta-oxidation and mitochondrial protein translation are mechanisms of adaptation in response to ALS/FTD pathology. CONCLUSIONS: Together, our unbiased integrative analyses unveil novel molecular components that may influence mitochondrial homeostasis in the earliest phase of ALS.

Metabolic adaption of cancer cells toward autophagy: Is there a role for ER-phagy?

Autophagy is an evolutionary conserved catabolic pathway that uses a unique double-membrane vesicle, called autophagosome, to sequester cytosolic components, deliver them to lysosomes and recycle amino-acids. Essentially, autophagy acts as a cellular cleaning system that maintains metabolic balance under basal conditions and helps to ensure nutrient viability under stress conditions. It is also an important quality control mechanism that removes misfolded or aggregated proteins and mediates the turnover of damaged and obsolete organelles. In this regard, the idea that autophagy is a non-selective bulk process is outdated. It is now widely accepted that forms of selective autophagy are responsible for metabolic rewiring in response to cellular demand. Given its importance, autophagy plays an essential role during tumorigenesis as it sustains malignant cellular growth by acting as a coping-mechanisms for intracellular and environmental stress that occurs during malignant transformation. Cancer development is accompanied by the formation of a peculiar tumor microenvironment that is mainly characterized by hypoxia (oxygen < 2%) and low nutrient availability. Such conditions challenge cancer cells that must adapt their metabolism to survive. Here we review the regulation of autophagy and selective autophagy by hypoxia and the crosstalk with other stress response mechanisms, such as UPR. Finally, we discuss the emerging role of ER-phagy in sustaining cellular remodeling and quality control during stress conditions that drive tumorigenesis.

SGPL1 stimulates VPS39 recruitment to the mitochondria in MICU1 deficient cells.

OBJECTIVE: Mitochondrial "retrograde" signaling may stimulate organelle biogenesis as a compensatory adaptation to aberrant activity of the oxidative phosphorylation (OXPHOS) system. To maintain energy-consuming processes in OXPHOS deficient cells, alternative metabolic pathways are functionally coupled to the degradation, recycling and redistribution of biomolecules across distinct intracellular compartments. While transcriptional regulation of mitochondrial network expansion has been the focus of many studies, the molecular mechanisms promoting mitochondrial maintenance in energy-deprived cells remain poorly investigated. METHODS: We performed transcriptomics, quantitative proteomics and lifespan assays to identify pathways that are mechanistically linked to mitochondrial network expansion and homeostasis in Caenorhabditis elegans lacking the mitochondrial calcium uptake protein 1 (MICU-1/MICU1). To support our findings, we carried out biochemical and image analyses in mammalian cells and mouse-derived tissues. RESULTS: We report that micu-1(null) mutations impair the OXPHOS system and promote C. elegans longevity through a transcriptional program that is independent of the mitochondrial calcium uniporter MCU-1/MCU and the essential MCU regulator EMRE-1/EMRE. We identify sphingosine phosphate lyase SPL-1/SGPL1 and the ATFS-1-target HOPS complex subunit VPS-39/VPS39 as critical lifespan modulators of micu-1(null) mutant animals. Cross-species investigation indicates that SGPL1 upregulation stimulates VPS39 recruitment to the mitochondria, thereby enhancing mitochondria-lysosome contacts. Consistently, VPS39 downregulation compromises mitochondrial network maintenance and basal autophagic flux in MICU1 deficient cells. In mouse-derived muscles, we show that VPS39 recruitment to the mitochondria may represent a common signature associated with altered OXPHOS system. CONCLUSIONS: Our findings reveal a previously unrecognized SGPL1/VPS39 axis that stimulates intracellular organelle interactions and sustains autophagy and mitochondrial homeostasis in OXPHOS deficient cells.

Effects of the autophagy modulators d-limonene and chloroquine on vimentin levels in SH-SY5Y cells.

The molecular target and mechanism by which d-limonene induces LC3 lipidation and autophagosome formation remain elusive. Here, we report that this monoterpene rapidly enhances Ca2+ levels in SH-SY5Y cells; yet this effect does not lead to calpain- or caspase-mediated proteolysis of α-spectrin, nor calpain activity is required for the established enhancement of LC3-II levels by d-limonene. However, d-limonene rapidly reduced vimentin levels, an unexpected effect also induced by the autophagy inhibitor chloroquine (CQ). The magnitude of vimentin reduction parallels accumulation of LC3-II caused by a brief incubation with d-limonene or CQ. For longer exposure (48 h), d-limonene does not reduce vimentin, nor it increases LC3-II levels; conversely, a clear reduction of vimentin along with a massive accumulation of LC3-II is evident in cells treated with CQ. Vimentin participates in organelle positioning and in other cellular processes that have linked this intermediate filament protein to various diseases, including cancer, inflammatory and autoimmune disorders, and to virus replication and internalization. Our findings suggest an inverse relationship between vimentin reduction and LC3-II accumulation, whose causal link needs to be examined. Further experiments are needed to dissect the role of vimentin reduction in the mechanisms through which CQ impairs fusion of autophagosome with lysosomes as well as in other effects of this drug.

Early LC3 lipidation induced by d-limonene does not rely on mTOR inhibition, ERK activation and ROS production and it is associated with reduced clonogenic capacity of SH-SY5Y neuroblastoma cells.

BACKGROUND: d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. PURPOSE: To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. METHODS: Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. RESULTS: LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and stimulates ROS generation, yet none of these events is implicated in lipidation of LC3, which was only partly reduced by chelation of intracellular calcium. The early LC3 lipidation induced by d-limonene is associated with inhibition of clonogenic capacity which is reverted by the autophagy inhibitor chloroquine. CONCLUSIONS: d-Limonene rapidly stimulates the autophagic flux in cultured cancer cells, which could be usefully exploited for therapeutic purposes.