Turn up the power –pharmacological activation of mitochondrial biogenesis in mouse models

The oxidative phosphorylation (OXPHOS) system in mitochondria is responsible for the generation of the majority of cellular energy in the form of ATP. Patients with genetic OXPHOS disorders form the largest group of inborn errors of metabolism. Unfortunately, there is still a lack of efficient therapies for these disorders other than management of symptoms. Developing therapies has been complicated because, although the total group of OXPHOS patients is relatively large, there is enormous clinical and genetic heterogeneity within this patient population. Thus there has been a lot of interest in generating relevant mouse models for the different kinds of OXPHOS disorders. The most common treatment strategies tested in these mouse models have aimed to up-regulate mitochondrial biogenesis, in order to increase the residual OXPHOS activity present in affected animals and thereby to ameliorate the energy deficiency. Drugs such as bezafibrate, resveratrol and AICAR target the master regulator of mitochondrial biogenesis PGC-1α either directly or indirectly to manipulate mitochondrial metabolism. This review will summarize the outcome of preclinical treatment trials with these drugs in mouse models of OXPHOS disorders and discuss similar treatments in a number of mouse models of common diseases in which pathology is closely linked to mitochondrial dysfunction. In the majority of these studies the pharmacological activation of the PGC-1α axis shows true potential as therapy; however, other effects besides mitochondrial biogenesis may be contributing to this as well.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Mitochondrial function and energy metabolism in neuronal HT22 cells resistant to oxidative stress

Background and Purpose

The hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc−. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction.

Experimental Approach

We compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line.

Key Results

Glutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery.

Conclusions and Implications

These results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Synthesis and Biological Evaluation of Pyrazolo[1,5-a]pyrimidine Compounds as Potent and Selective Pim-1 Inhibitors

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FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

The large family of fibroblast growth factors (FGFs) has been known for its important roles in regulating critical cellular processes during embryonic development and homeostasis of normal tissues (1–3). While most FGFs act as cytokines or hormonelike proteins that mediate their pleiotropic cellular processes by binding to cell surface receptors endowed with intrinsic tyrosine kinase activity (FGFRs), a subfamily of FGFs (FGF 11–14) was shown to be uniquely expressed intracellularly. The mechanism of action and physiological roles of intracellular FGFs are poorly understood (4–6).In contrast to most receptor tyrosine kinases (RTKs) that are activated by a single ligand molecule that binds with high affinity to the extracellular domain of its cognate RTK with a dissociation constant in the subnanomolar range, the binding affinities of FGFs to FGFRs are, at least, 1,000–10,000 fold weaker with dissociation constants in the submicromolar range (7–9). The weak binding affinities toward FGFRs of the largest subfamily of FGF molecules designated canonical FGFs are offset by interactions with cell surface heparan sulfate proteoglycans (HSPGs). Both biochemical and structural studies revealed how multiple interactions between heparin or HSPG with both FGF and FGFR mediate tight association enabling robust receptor dimerization and tyrosine kinase activation (10, 11).The three endocrine FGFs, FGF19, 21, and 23 are part of an additional subfamily of FGF molecules. Endocrine FGFs function as circulating hormones that play essential roles in the control of various metabolic processes (12). In addition to the conserved FGF-domain found in all FGF ligands, endocrine FGFs contain unique C-terminal tails (CTs) composed of 46 (FGF19), 34 (FGF21), or 89 (FGF23) amino acids that serve as specific and high-affinity ligands for the two members of the Klotho family of surface receptors. It was shown that KLA serves as a high-affinity receptor for FGF23 while β-Klotho (KLB) functions as a high-affinity surface receptor for both FGF19 and FGF21 (13–16). Structural analyses of free and ligand-occupied Klotho proteins revealed the molecular basis underlying the specificity and high affinity of KLA and KLB toward endocrine FGFs. It also showed that Klotho proteins function as the primary receptors for endocrine FGFs whereas FGFR functions as a catalytic subunit that mediates cell signaling via its tyrosine kinase domain (8, 17, 18). Accordingly, endocrine FGFs stimulate their cellular responses by forming a ternary complex with Klotho proteins and FGFRs to induce receptor dimerization, tyrosine kinase activation, and cell signaling. Unlike FGFRs that are ubiquitously expressed, the expressions of KLA and KLB are restricted to specific tissues and organs to enable precise targeting of endocrine FGFs to stimulate their physiological responses in specific cells and tissues (19–22). The ability of endocrine FGFs to circulate is attributed to the loss of conserved heparin binding sites that are essential for the function of canonical FGFs (23).FGF23 is a 32-kDa glycoprotein, mainly produced in the bone by osteoblasts and osteocytes, that serve as a key hormone in regulating phosphate homeostasis, vitamin D, and calcium metabolism (24, 25). Circulating levels of physiologically active FGF23 are regulated by proteolytic cleavage to produce a FGF23 molecule lacking its unique CT (26, 27). The cleavage resulting in FGF23 inactivation prevents assembly and stimulation of the FGF23/FGFR/KLA complex. Additionally, the processing of FGF23 includes several posttranslational modifications which affect its stability and susceptibility toward proteolysis. Secreted FGF23 was shown to be O-glycosylated in its C-terminal cleavage site (28, 29) to protect the protein from C-terminal cleavage. In order for the cleavage site to be exposed, FGF23 has to be first phosphorylated in this region (30). Phosphorylation prevents glycosylation and exposes the cleavage site to proteolysis.In this paper, we demonstrate that the CT of FGF23 contains two tandem repeats and that each repeat binds with high affinity to KLA. This contrasts with FGF19 and FGF21, whose CTs contain a single binding site to KLB. Engineered FGF23 variants containing each of the two repeats individually or both repeats bind specifically to KLA and stimulate cell signaling to a similar extent. We also demonstrate that two cysteine residues flanking the second repeat form a disulfide bridge in FGF23 secreted by mammalian cells. However, both oxidized or unbridged forms of FGF23 exhibit similar KLA binding characteristics and similar cellular stimulatory activities. We also show that FGF23-WT induces mitogen-activated protein kinase (MAPK) activation in cells expressing chimeric KLA-FGFR proteins and use TIRFM imaging of individual KLA molecules on the cell surface to demonstrate that FGF23 has the capacity for simultaneous binding to two KLA molecules. These insights reveal the complexity of FGF23 regulation and its role in assembling the FGF23/FGFR/KLA signaling complex.     

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2alpha or CK2alpha') and regulatory (CK2beta) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2alpha or CK2alpha') and the CK2beta regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5- to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2alpha that eliminate phosphorylating activity [CK2alpha(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2alpha(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.     

Effect of a toxic in vivo dose of ouabain on guinea-pig heart mitochondria

The effects of toxic doses of ouabain on two parameters of mitochondrial activity, oxidative phosphorylation and calcium uptake were examined. Ouabain was injected intraperitoneally into guinea-pigs until signs of severe intoxication appeared. State 3 oxygen consumption (Q_O2, State 3, in natom oxygen/mg/min) of isolated heart mitochondria was 314 ± 16 and 281 ± 16 (glutamate-malate) for treated and control group, respectively; 225 ± 21 and 207 ± 23 (pyruvate-malate), and 251 ± 12 and 230 ± 13 (succinate), respectively. The rate of calcium uptake was 411 nmol Ca²⁺/min/mg for treated and 329.6 nmol Ca²⁺/min/mg for control. The rate of calcium release was the same in control and treated groups.The data suggest that increases of respiration and calcium uptake in vitro, if they reflect similar increases in vivo, may contribute to digitalis intoxication by intracellular redistribution of calcium.     

Phosphorylation of connexin in functional regulation of the cardiac gap junction

In cardiac muscle, the gap junction contributes to electrical cell-to-cell coupling. This physiological function of the gap junction depends on the phosphorylation state of the connexin molecule, which comprises the gap junction channel. The effects of intracellular Ca²⁺ overload, acidosis, activation of protein kinase (PK) A, PKC and PKG on the phosphorylation and expression of connexin 43 (Cx43) were examined in animal hearts with reference to physiological function. Activation of PKA promotes cell-to-cell coupling due to augmentation of the PKA-mediated phosphorylation of Cx43, with a rise in the quantity of and an increase in the expression of Cx43. A rise in the ionic strength of Ca²⁺ and H⁺ impaired cell communication, with the inhibition of PKA-mediated Cx43 phosphorylation. Activation of PKC reduces the quantity and expression of Cx43 despite augmentation of PKC-mediated phosphorylation of the protein. The effects of PKG activation are similar to those of PKC activation. It is suggested that PKA activation upregulates and PKC activation downregulates Cx43. The role of connexin phosphorylation in the regulation of gap junction function is discussed.     

Factors affecting directional migration of bone marrow mesenchymal stem cells to the injured spinal cord

Microtubule-associated protein 1B plays an important role in axon guidance and neuronal migration. In the present study, we sought to discover the mechanisms underlying microtu- bule-associated protein 1B mediation of axon guidance and neuronal migration. We exposed bone marrow mesenchymal stem cells to okadaic acid or N-acetyl-D-erythro-sphingosine （an inhibitor and stimulator, respectively, of protein phosphatase 2A） for 24 hours. The expression of the phosphorylated form of type I microtubule-associated protein 1B in the cells was greater after exposure to okadaic acid and lower after N-acetyl-D-erythro-sphingosine. We then injected the bone marrow mesenchymal stem cells through the ear vein into rabbit models of spinal cord contusion. The migration of bone marrow mesenchymal stem cells towards the injured spinal cord was poorer in cells exposed to okadaic acid- and N-acetyl-D-erythro-sphingosine than in non-treated bone marrow mesenchymal stem cells. Finally, we blocked phosphatidylinosi- tol 3-kinase （PI3K） and extracellular signal-regulated kinase 1/2 （ERK1/2） pathways in rabbit bone marrow mesenchymal stem cells using the inhibitors LY294002 and U0126, respectively. LY294002 resulted in an elevated expression of phosphorylated type I microtubule-associated protein 1B, whereas U0126 caused a reduction in expression. The present data indicate that PI3K and ERKI/2 in bone marrow mesenchymal stem cells modulate the phosphorylation of micro- tubule-associated protein 1B via a cross-signaling network, and affect the migratory efficiency of bone marrow mesenchymal stem cells towards injured spinal cord.     

Tau-induced neurodegeneration: mechanisms and targets

The accumulation of hyperphosphorylated tau is a common feature of several dementias. Tau is one of the brain microtubule-associated proteins. Here we discuss tau’s functions in microtubule assembly and stabilization and with regard to its interactions with other proteins. We describe and analyze important post-translational modifications: hyperphosphorylation, ubiquitination, glycation, glycosylation, nitration, polyamination, proteolysis, acetylation, and methylation. We discuss how these post-translational modifications can alter tau’s biological function. We analyze the role of mitochondrial health in neurodegeneration. We propose that microtubules could be a therapeutic target and review different approaches. Finally, we consider whether tau accumulation or its conformational change is related to tau-induced neurodegeneration, and propose a mechanism of neurodegeneration.     

Disruption of dopamine D1 receptor phosphorylation at serine 421 attenuates cocaine-induced behaviors in mice

Dopamine D1 receptors（D1Rs） play a key role in cocaine addiction, and multiple protein kinases such as GRKs, PKA, and PKC are involved in their phosphorylation. Recently, we reported that protein kinase D1 phosphorylates the D1 R at S421 and promotes its membrane localization. Moreover, this phosphorylation of S421 is required for cocaineinduced behaviors in rats. In the present study, we generated transgenic mice over-expressing S421A-D1 R in the forebrain. These transgenic mice showed reduced phospho-D1R（S421） and its membrane localization, and reduced downstream ERK1/2 activation in the striatum. Importantly, acute and chronic cocaine-induced locomotor hyperactivity and conditioned place preference were significantly attenuated in these mice. These findings provide in vivo evidence for the critical role of S421 phosphorylation of the D1 R in its membrane localization and in cocaine-induced behaviors. Thus, S421 on the D1 R represents a potential pharmacotherapeutic target for cocaine addiction and other drug-abuse disorders.