BackgroundOncogene activation plays a role in metabolic reprogramming of cancer cells. We have previously shown that K-ras transformed fibroblasts have a stronger dependence on glycolysis and a reduced oxidative phosphorylation ability as compared to their normal counterparts. Another metabolic adaptation of cancer cells, that has long been established, is their propensity to exhibit increased glutamine consumption, although the effects induced by glutamine deprivation on cancer cells are still controversial.Methodology and Principal FindingsHere, by using nutritional perturbations and molecular physiology, we show that reduction or complete depletion of glutamine availability in K-ras transformed fibroblasts causes a strong decrease of proliferation ability and a slower re-entry of synchronized cells into the cell cycle. The reduced proliferation is accompanied by sustained expression of cyclin D and E, abortive S phase entrance and is dependent on Ras signalling deregulation, since it is rescued by expression of a dominant negative guanine nucleotide exchange factor. The growth potential of transformed cells as well as the ability to execute the G1 to S transition is restored by adding the four deoxyribonucleotides, indicating that the arrest of proliferation of K-ras transformed cells induced by glutamine depletion is largely due to a reduced supply of DNA in the presence of signalling pathways promoting G1 to S transition.Conclusions and SignificanceOur results suggest that the differential effects of glutamine and glucose on cell viability are not a property of the transformed phenotype per se, but rather depend on the specific pathway being activated in transformation. For instance, myc-overexpressing cells have been reported to die under glutamine depletion and not under glucose shortage, while the opposite holds for ras-transformed fibroblasts as shown in this paper. These different responses of transformed cells to nutritional stress should be taken into account when designing anti-cancer therapies that aim to exploit metabolic differences between normal and transformed cells.

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons. STEM CELLS 2016;34:124-134 SIGNIFICANCE STATEMENTMotor neurons carry information from the brain to the peripheral musculature and loss of motor neurons in neurodegenerative conditions leads to paralysis and ultimately death. Hence, there is great interest in understanding the development of these neurons and how they degenerate in disease. In this study, we have identified a small RNA called miR-375 that is required for motor neuron development and further protects motor neurons from stress. Importantly, we find that diseased motor neurons have reduced amounts of miR-375 suggesting that loss of this RNA may lead to neurodegeneration. Our study opens up a new avenue for therapeutic intervention to rescue degenerating neurons.

The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKβ and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground‐breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington's disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brainderived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity. We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down. We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse. These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.