Francesc X. Soriano

Evidence for a Mitochondrial Regulatory Pathway Defined by Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α, Estrogen-Related Receptor-α, and Mitofusin 2

Mitofusin 2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion and regulates mitochondrial metabolism in mammalian cells. Here, we show that Mfn2 gene expression is induced in skeletal muscle and brown adipose tissue by conditions associated with enhanced energy expenditure, such as cold exposure or β3-adrenergic agonist treatment. In keeping with the role of peroxisome proliferator–activated receptor-γ coactivator (PGC)-1α on energy expenditure, we demonstrate a stimulatory effect of PGC-1α on Mfn2 mRNA and protein expression in muscle cells. PGC-1α also stimulated the activity of the Mfn2 promoter, which required the integrity of estrogen-related receptor-α (ERRα)-binding elements located at −413/−398. ERRα also activated the transcriptional activity of the Mfn2 promoter, and the effects were synergic with those of PGC-1α. Mfn2 loss of function reduced the stimulatory effect of PGC-1α on mitochondrial membrane potential. Exposure to cold substantially increased Mfn2 gene expression in skeletal muscle from heterozygous Mfn2 knock-out mice, which occurred in the presence of higher levels of PGC-1α mRNA compared with control mice. Our results indicate the existence of a regulatory pathway involving PGC-1α, ERRα, and Mfn2. Alterations in this regulatory pathway may participate in the pathophysiology of insulin-resistant conditions and type 2 diabetes.

The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells

Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity‐dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Mitochondrial fragmentation in excitotoxicity requires ROCK activation

Mitochondria morphology constantly changes through fission and fusion processes that regulate mitochondrial function, and it therefore plays a prominent role in cellular homeostasis. Cell death progression is associated with mitochondrial fission. Fission is mediated by the mainly cytoplasmic Drp1, which is activated by different post-translational modifications and recruited to mitochondria to perform its function. Our research and other studies have shown that in the early moments of excitotoxic insult Drp1 must be nitrosylated to mediate mitochondrial fragmentation in neurons. Nonetheless, mitochondrial fission is a multistep process in which filamentous actin assembly/disassembly and myosin-mediated mitochondrial constriction play prominent roles. Here we establish that in addition to nitric oxide production, excitotoxicity-induced mitochondrial fragmentation also requires activation of the actomyosin regulator ROCK. Although ROCK1 has been shown to phosphorylate and activate Drp1, experiments using phosphor-mutant forms of Drp1 in primary cortical neurons indicate that in excitotoxic conditions, ROCK does not act directly on Drp1 to mediate fission, but may act on the actomyosin complex. Thus, these data indicate that a wider range of signaling pathways than those that target Drp1 are amenable to be inhibited to prevent mitochondrial fragmentation as therapeutic option.

Excitotoxicity in the pathogenesis of neurological and psychiatric disorders: Therapeutic implications:

Neurological and psychiatric disorders are leading contributors to the global disease burden, having a serious impact on the quality of life of both patients and their relatives. Although the molecular events underlying these heterogeneous diseases remain poorly understood, some studies have raised the idea of common mechanisms involved. In excitotoxicity, there is an excessive activation of glutamate receptors by excitatory amino acids, leading to neuronal damage. Thus, the excessive release of glutamate can lead to a dysregulation of Ca2+ homeostasis, triggering the production of free radicals and oxidative stress, mitochondrial dysfunction and eventually cell death. Although there is a consensus in considering excitotoxicity as a hallmark in most neurodegenerative diseases, increasing evidence points to the relevant role of this pathological mechanism in other illnesses affecting the central nervous system. Consequently, antagonists of glutamate receptors are used in current treatments or in clinical trials in both neurological and psychiatric disorders. However, drugs modulating other aspects of the excitotoxic mechanism could be more beneficial. This review discusses how excitotoxicity is involved in the pathogenesis of different neurological and psychiatric disorders and the promising strategies targeting the excitotoxic insult.

Synaptic activity‐induced glycolysis facilitates membrane lipid provision and neurite outgrowth

The formation of neurites is an important process affecting the cognitive abilities of an organism. Neurite growth requires the addition of new membranes, but the metabolic remodeling necessary to supply lipids for membrane expansion is poorly understood. Here, we show that synaptic activity, one of the most important inducers of neurite growth, transcriptionally regulates the expression of neuronal glucose transporter Glut3 and rate‐limiting enzymes of glycolysis, resulting in enhanced glucose uptake and metabolism that is partly used for lipid synthesis. Mechanistically, CREB regulates the expression of Glut3 and Siah2, the latter and LDH activity promoting the normoxic stabilization of HIF‐1α that regulates the expression of rate‐limiting genes of glycolysis. The expression of dominant‐negative HIF‐1α or Glut3 knockdown blocks activity‐dependent neurite growth in vitro while pharmacological inhibition of the glycolysis and specific ablation of HIF‐1α in early postnatal mice impairs the neurite architecture. These results suggest that the manipulation of neuronal glucose metabolism could be used to treat some brain developmental disorders.

Absence of ERK5/MAPK7 delays tumorigenesis in Atm -/- mice

Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm−/− mice. Compared with Atm−/− thymocytes, Mapk7−/−Atm−/− thymocytes exhibited an improved response to DNA damage (increased phosphorylation of H2AX) and a restored apoptotic response after treatment of mice with ionizing radiation. These findings define an antagonistic function of ATM and MAPK7 in the thymocyte response to DNA damage, and suggest that the lack of MAPK7 inhibits thymic lymphoma growth in Atm−/− mice by partially restoring the DNA damage response in thymocytes.