BBA - Molecular Basis of Disease (v.1792, #8)

The molecular composition of the postsynaptic density is modified during synaptic plasticity, which forms the molecular basis of learning and memory. Such changes in synaptic composition depends in part on the intricate regulation of phosphorylation of specific proteins via different protein kinases, including a serine/threonine kinase, cyclin-dependent kinase 5 (Cdk5). However, the mechanisms underlying the involvement of Cdk5 in neural plasticity remain elusive. Recently, the identification of a number of synaptic proteins as substrates or interacting proteins with Cdk5 provides important clues on how this kinase modulates the efficacy of synaptic transmission. In this review, we summarize the recent findings to illustrate the multi-faceted roles of Cdk5 in synaptic plasticity through affecting dendritic spine formation, ion channel conductance, protein expression, and transcription in the postsynaptic neurons. Importantly, dysregulation of Cdk5 has been linked to Alzheimer's disease, which involves perturbations in synaptic functions and memory formation. Understanding the mechanisms by which Cdk5 regulates synaptic plasticity may therefore provide important insights in the design of novel therapeutic strategies for neurodegenerative diseases.
Keywords: Cyclin-dependent kinase 5 (Cdk5); Synapse; NMDA receptor; Dendritic spine; Transcription factor; Learning and memory;

TGF-β and fibrosis in different organs — molecular pathway imprints by Dirk Pohlers; Julia Brenmoehl; Ivonne Löffler; Cornelia K. Müller; Carola Leipner; Stefan Schultze-Mosgau; Andreas Stallmach; Raimund W. Kinne; Gunter Wolf (746-756).
The action of transforming-growth-factor (TGF)-β following inflammatory responses is characterized by increased production of extracellular matrix (ECM) components, as well as mesenchymal cell proliferation, migration, and accumulation. Thus, TGF-β is important for the induction of fibrosis often associated with chronic phases of inflammatory diseases. This common feature of TGF-related pathologies is observed in many different organs. Therefore, in addition to the description of the common TGF-β-pathway, this review focuses on TGF-β-related pathogenetic effects in different pathologies/organs, i. e., arthritis, diabetic nephropathy, colitis/Crohn's disease, radiation-induced fibrosis, and myocarditis (including their similarities and dissimilarities). However, TGF-β exhibits both exacerbating and ameliorating features, depending on the phase of disease and the site of action. Due to its central role in severe fibrotic diseases, TGF-β nevertheless remains an attractive therapeutic target, if targeted locally and during the fibrotic phase of disease.
Keywords: TGF-β; Fibrosis; Arthritis; Nephritis; Inflammatory bowel disease; Crohn's disease; Wound-healing; Myocarditis;

Osteoclast heterogeneity: by Vincent Everts; Teun J. de Vries; Miep H. Helfrich (757-765).
The multinucleated osteoclast has a unique function: degradation of mineralized tissues. It is generally taken that all osteoclasts are alike, independent of the skeletal site where they exert their activity. Recent data, however, question this view as they show that osteoclasts at different bony sites appear to differ, for example in the machinery responsible for resorption. Support for the notion that there may be heterogeneity in osteoclasts is obtained from studies in which osteoclast activity is inhibited and from observations in osteopetrosis and inflammatory bone conditions. In this review we discuss the available evidence and propose the existence of bone-site-specific osteoclast heterogeneity.
Keywords: Osteoclast; Osteopetrosis; Heterogeneity; Intramembranous bone; Endochondral bone; Osteoclast subset;

Gene expression of synaptosomal-associated protein 25 (SNAP-25) in the prefrontal cortex of the spontaneously hypertensive rat (SHR) by Qi Li; Jack H. Wong; Gang Lu; Gregory E. Antonio; David K. Yeung; Tzi B. Ng; Lucy E. Forster; David T. Yew (766-776).
Dopamine is believed to play an important role in the etiology of attention-deficit/hyperactivity disorder (ADHD). In our previous study, we showed that gene expression of dopamine D4 receptor decreased in the spontaneously hypertensive rat (SHR) in the prefrontal cortex (PFC). In the present study, we explored the potential causes of dysfunction in the dopamine system in ADHD. It is the first time that neuronal activities in both juvenile SHR and WKY rats have been measured by functional MRI (fMRI). Our results showed that in PFC the Blood Oxygenation Level Dependent (BOLD) signal response in SHR was much higher than WKY under stressful situations. We tested the effects of acute and repeated administration of amphetamine on behavioral changes in SHR combined with the expression of the neuronal activity marker, c-fos, in the PFC. Meanwhile dopamine-related gene expression was measured in the PFC after repeated administration of amphetamine. We found that potential neuronal damage occurred through deficit of D2-like receptor protective functions in the PFC of the SHR. We also measured the expression of synaptosomal-associated protein 25 (SNAP-25) in SHR in PFC. The results showed decreased expression of SNAP-25 mRNA in the PFC of SHR; this defect disappeared after repeated injection of D-AMP.
Keywords: Attention-deficit/hyperactivity disorder (ADHD); Spontaneously hypertensive rat (SHR); Prefrontal cortex (PFC); Functional MRI (fMRI); d-amphetamine; Synaptosomal-associated protein 25 (SNAP-25);

Regulation of the nuclear hormone receptor nur77 in muscle: Influence of exercise-activated pathways in vitro and obesity in vivo by Timo Kanzleiter; Donna Wilks; Elaine Preston; Jiming Ye; Georgia Frangioudakis; Gregory James Cooney (777-782).
Regular physical exercise is well known to improve glucose and lipid metabolism in skeletal muscle. However, the transcription factors regulating these adaptive changes are not well-characterised. Recently the nuclear orphan receptor nur77 was shown to be induced by exercise and linked to regulation of metabolic gene expression in skeletal muscle. In this study we investigated the regulation of nur77 in muscle by different exercise-activated pathways. Nur77 expression was found to be responsive to adrenergic stimulation and calcium influx, but not to activation of the AMP dependent kinase. These results identify the adrenergic-cyclic AMP-PKA pathway to be the most potent activator of nur77 expression in muscle and therefore the likely cause of increased expression after exercise. We also identified nur77 expression to be reduced in the muscle of obese/insulin resistant rats after high fat feeding. Furthermore exposure to fatty acids, insulin or inflammation was not the cause of decreased nur77 expression in insulin resistant muscle. This suggests a reduced responsiveness to adrenergic stimulation as the likely cause of diminished nur77 expression in muscle of high fat fed rats, which has been observed in obese/insulin resistant individuals. Our results suggest adrenergic stimulation as the most important stimulus for nur77 expression and point to a significant role for this transcription factor in adaptive changes in muscle after exercise and in insulin resistant states.
Keywords: Nuclear hormone receptor; Obesity; Insulin resistance; Exercise;

The 4977 bp deletion of mitochondrial DNA (mtDNA), often found in patients with chronic progressive external ophthalmoplegia (CPEO), has been demonstrated to increase the susceptibility to apoptosis of human cells. We investigated the mechanism underlying the apoptotic susceptibility of the Δ4977 cybrid harboring about 80% 4977 bp-deleted mtDNA. The production of hydrogen peroxide (H2O2) and phosphorylation of PKCδ and ERK1/2 were increased in the Δ4977 cybrid, which was more susceptible to UV-induced apoptosis. Moreover, treatment with N-acetyl-l-cysteine (NAC) or blocking of activation of PKCδ by rottlerin or PKCδ-siRNA, and inhibition of ERK1/2 by PD98059 or ERK1/2-siRNA significantly attenuated the susceptibility of the Δ4977 cybrid to apoptosis. Furthermore, the increase of PKCδ expression in the Δ4977 cybrid also amplified the apoptotic signal through caspase 3-mediated proteolytic activation of PKCδ. In addition, PKCδ and ERK1/2 were hyperphosphorylated in skin fibroblasts of CPEO patients harboring 4977 bp-deleted mtDNA. We suggest that the activation of PKCδ and ERK1/2 elicited by 4977 bp-deleted mtDNA-induced oxidative stress plays a role in the susceptibility of the mutant cells to apoptosis. This may explain, at least in part, the degenerative manifestation of brain and muscle in patients with mitochondrial encephalomyopathies such as CPEO syndrome.
Keywords: Apoptosis; ERK1/2; Mitochondrial DNA; Oxidative stress; PKCδ;

The mitochondrial translational machinery allows the genes encoded by mitochondrial DNA (mtDNA) to be translated in situ. Mitochondrial translation requires a number of nucleus-encoded protein factors, some of which have been found to carry mutations in patients affected by mitochondrial encephalomyopathies. We have previously described the first, and so far only, mutation in the mitochondrial elongation factor Tu, mt-EFTu, in a baby girl with polycystic encephalopathy, micropolygyria, and leukodystrophic changes. Despite that the mutant mt-EFTu was present in normal amount in the patient's tissues, mitochondrial translation was severely reduced, determining multiple defects in the amount and activity of mtDNA-dependent respiratory chain complexes. By an in-vitro reconstructed translational system, we here provide evidence that the mutant mt-EFTu variant fails to bind to aminoacylated mitochondrial tRNAs, thus explaining the observed impairment of mitochondrial translation. This is the first analysis on the molecular mechanism of a mtDNA translation defect due to a nuclear gene mutation.
Keywords: Mitochondrial translation; mt-EFTu mutation; mt-EFTu binding activity;

Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney's mitochondria by Egor Y. Plotnikov; Anastasia A. Chupyrkina; Irina B. Pevzner; Nickolaj K. Isaev; Dmitry B. Zorov (796-803).
Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.
Keywords: Rhabdomyolysis; Mitochondria; Reactive oxygen species; Nitric oxide;

Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP+ site and a “structural” NADP+ site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PDWisconsin (R393G) and G6PDNashville (R393H), and G6PDFukaya (G488S) and G6PDCampinas (G488V), in which the mutations are in the vicinity of the “structural” NADP+ site, showed elevated K d values of the “structural” NADP+, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP+ and DTT at 25 °C. The refolding yields of the mutants exhibited strong NADP+-dependence and ranged from 1.5% to 59.4% with 1000 μM NADP+, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP+ can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.
Keywords: Glucose 6-phosphate dehydrogenase deficiency; Structural NADP+; Dissociation constant; Refolding; Stability;

Defects in cell spreading and ERK1/2 activation in fibroblasts with lamin A/C mutations by Lindsay J. Emerson; Mark R. Holt; Matthew A. Wheeler; Manfred Wehnert; Maddy Parsons; Juliet A. Ellis (810-821).
In-frame mutations in nuclear lamin A/C lead to a multitude of tissue-specific degenerative diseases known as the ‘laminopathies’. Previous studies have demonstrated that lamin A/C-null mouse fibroblasts have defects in cell polarisation, suggesting a role for lamin A/C in nucleo-cytoskeletal-cell surface cross-talk. However, this has not been examined in patient fibroblasts expressing modified forms of lamin A/C. Here, we analysed skin fibroblasts from 3 patients with Emery–Dreifuss muscular dystrophy and from 1 with dilated cardiomyopathy. The emerin–lamin A/C interaction was impaired in each mutant cell line. Mutant cells exhibited enhanced cell proliferation, collagen-dependent adhesion, larger numbers of filopodia and smaller cell spread size, compared with control cells. Furthermore, cell migration, speed and polarization were elevated. Mutant cells also showed an enhanced ability to contract collagen gels at early time points, compared with control cells. Phosphotyrosine measurements during cell spreading indicated an initial temporal lag in ERK1/2 activation in our mutant cells, followed by hyper-activation of ERK1/2 at 2 h post cell attachment. Deregulated ERK1/2 activation is linked with cardiomyopathy, cell spreading and proliferation defects. We conclude that a functional emerin–lamin A/C complex is required for cell spreading and proliferation, possibly acting through ERK1/2 signalling.
Keywords: Lamin A/C; Laminopathies; ERK1/2 signalling; Cell adhesion; Cell spreading; Emery–Dreifuss muscular dystrophy;