William R. Randall

Activity-dependent nuclear translocation and intranuclear distribution of NFATc in adult skeletal muscle fibers

TTranscription factor nuclear factor of activated T cells NFATc (NFATc1, NFAT2) may contribute to slow-twitch skeletal muscle fiber type–specific gene expression. Green fluorescence protein (GFP) or FLAG fusion proteins of either wild-type or constitutively active mutant NFATc [NFATc(S→A)] were expressed in cultured adult mouse skeletal muscle fibers from flexor digitorum brevis (predominantly fast-twitch). Unstimulated fibers expressing NFATc(S→A) exhibited a distinct intranuclear pattern of NFATc foci. In unstimulated fibers expressing NFATc–GFP, fluorescence was localized at the sarcomeric z-lines and absent from nuclei. Electrical stimulation using activity patterns typical of slow-twitch muscle, either continuously at 10 Hz or in 5-s trains at 10 Hz every 50 s, caused cyclosporin A–sensitive appearance of fluorescent foci of NFATc–GFP in all nuclei. Fluorescence of nuclear foci increased during the first hour of stimulation and then remained constant during a second hour of stimulation. Kinase inhibitors and ionomycin caused appearance of nuclear foci of NFATc–GFP without electrical stimulation. Nuclear translocation of NFATc–GFP did not occur with either continuous 1 Hz stimulation or with the fast-twitch fiber activity pattern of 0.1-s trains at 50 Hz every 50 s. The stimulation pattern–dependent nuclear translocation of NFATc demonstrated here could thus contribute to fast-twitch to slow-twitch fiber type transformation.

Nicotine at concentrations found in cigarette smokers activates and desensitizes nicotinic acetylcholine receptors in CA1 interneurons of rat hippocampus.

Behavioral effects of cigarette smoking are attributed to the interactions of nicotine with brain nicotinic acetylcholine receptors (nAChRs). However, the mechanisms by which nAChR function in developing and mature brain is affected by a smokers level of nicotine (50-500 nM) remain unclear. Thus, the objective of this study was to determine the concentration- and time-dependent effects of nicotine on alpha7 and alpha4beta2 nAChRs, the two major brain subtypes, natively expressed in CA1 interneurons of rat hippocampal slices. Only at concentrations > or =5 microM did nicotine (applied for 6-60 s) elicit action potentials or measurable whole-cell currents (EC(50)=158 microM) in stratum radiatum interneurons that express alpha7 nAChRs. Continuous exposure for 10-15 min of the neurons to nicotine (0.5-2.5 microM) inhibited alpha7 nAChR-mediated currents (IC(50)=640 nM) evoked by choline (10 mM). Nicotine (> or =0.125 microM) applied to the neurons for 1-5 min induced slowly desensitizing whole-cell currents (EC(50)=3.2 microM) in stratum lacunosum moleculare interneurons; this effect was mediated by alpha4beta2 nAChRs. Also via activation of alpha4beta2 nAChRs, nicotine (0.125-0.5 microM) increased the frequency and amplitude of GABAergic postsynaptic currents (PSCs) in stratum radiatum interneurons. However, exposure of the neurons for 10-15 min to nicotine (0.25-0.5 microM) resulted in desensitization of alpha4beta2 nAChRs. It is suggested that nanomolar concentrations of nicotine after acute intake suppress inhibitory inputs to pyramidal cells through a disinhibitory mechanism involving activation of alpha4beta2 nAChRs and desensitization of alpha7 nAChRs, and after chronic intake leads to up-regulation of both receptor subtypes via desensitization. These findings have direct implications to the actions of nicotine in cigarette smokers.

Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle.

Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2). Here, we show that repetitive slow fiber type electrical stimulation, but not fast fiber type stimulation, caused HDAC4-GFP, but not HDAC5-GFP, to translocate from the nucleus to the cytoplasm in cultured adult skeletal muscle fibers. HDAC4-GFP translocation was blocked by calmodulin-dependent protein kinase (CaMK) inhibitor KN-62. Slow fiber type stimulation increased MEF2 transcriptional activity, nuclear Ca2+ concentration, and nuclear levels of activated CaMKII, but not total nuclear CaMKII or CaM-YFP. Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca2+-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4. Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

Targeted Deletion of the Kynurenine Aminotransferase II Gene Reveals a Critical Role of Endogenous Kynurenic Acid in the Regulation of Synaptic Transmission via α7 Nicotinic Receptors in the Hippocampus

It has been postulated that endogenous kynurenic acid (KYNA) modulates α7* nicotinic acetylcholine receptor (nAChR) and NMDA receptor activities in the brain.a To test this hypothesis, α7* nAChR and NMDA receptor functions were studied in mice with a targeted null mutation in the gene encoding kynurenine aminotransferase II (mKat-2-/- mice), an enzyme responsible for brain KYNA synthesis. At 21 postnatal days, mKat-2-/- mice had lower hippocampal KYNA levels and higher spontaneous locomotor activity than wild-type (WT) mice. At this age, α7* nAChR activity induced by exogenous application of agonists to CA1 stratum radiatum interneurons was ∼65% higher in mKat-2-/- than WT mice. Binding studies indicated that the enhanced receptor activity may not have resulted from an increase in α7* nAChR number. In 21-d-old mKat-2-/- mice, endogenous α7* nAChR activity in the hippocampus was also increased, leading to an enhancement of GABAergic activity impinging onto CA1 pyramidal neurons that could be reduced significantly by acute exposure to KYNA (100 nM). The activities of GABAA and NMDA receptors in the interneurons and of α3β4* nAChRs regulating glutamate release onto these neurons were comparable between mKat-2-/- and WT mice. By 60 d of age, KYNA levels and GABAergic transmission in the hippocampus and locomotor activity were similar between mKat-2-/- and WT mice. Our findings that α7* nAChRs are major targets for KYNA in the brain may provide insights into the pathophysiology of schizophrenia and Alzheimers disease, disorders in which brain KYNA levels are increased and α7* nAChR functions are impaired.

Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer’s disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning.

Signaling pathways in activity-dependent fiber type plasticity in adult skeletal muscle

Adult fast- and slow-twitch skeletal muscle fibers exhibit characteristic differences in functional properties due to differences in the isoforms and quantities of expression of most muscle proteins. However, these differences may be reversed by chronic electrical stimulation of denervated muscle with the pattern typical of the other fiber type. Here, we review three possible signaling pathways that may contribute to fast to slow fiber type transformation. The first pathway involves cytosolic activation of the Ca2+ sensitive posphatase calcineurin (CaN) due to elevated cytosolic [Ca2+], resulting in dephosphorylation of cytoplasmic NFATc, translocation of dephosphorylated NFATc from cytoplasm into the nucleus and activation of slow fiber gene expression by NFATc in the nucleus. The second pathway involves elevated intranuclear [Ca2+] causing the activation of nuclear calmodulin dependent protein kinase, which phosphorylates HDAC within the nucleus and thereby permits nuclear efflux of HDAC, thus decreasing the HDAC suppression of MEF2 activation of slow fiber gene expression. The third possible pathway involves nuclear entry of CaN, dephosphorylation of intranuclear MEF2 and consequent increased activation of slow fiber type gene expression by dephosphorylated MEF2. Evidence for the first two pathways from our studies on adult fast twitch skeletal muscle fibers is briefly reviewed.

The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Regulates Assembly of Titin at the Z-Disk through Interactions with Ran Binding Protein 9

Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 microM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.

The opioid antagonist naltrexone inhibits activity and alters expression of α7 and α4β2 nicotinic receptors in hippocampal neurons : implications for smoking cessation programs

This study was designed to investigate whether naltrexone, an opioid antagonist that has been evaluated clinically as a co-adjuvant in smoking cessation programs, affects function and expression of neuronal nicotinic receptors (nAChRs). Whole-cell current recordings from rat hippocampal neurons in culture and in slices demonstrated that α7 nAChRs can be inhibited non-competitively by naltrexone (IC50∼25 μM). The voltage dependence of the effect suggested that naltrexone acts as an open-channel blocker of α7 nAChRs. Naltrexone also inhibited activation of α4β2 nAChRs in hippocampal neurons; however its IC50 was higher (∼141 μM). At a concentration as high as 300 μM (which is sufficient to block by 100% and 70% the activity of α7 and α4β2 nAChRs, respectively), naltrexone had no effect on kainate and AMPA receptors, blocked by no more than 20% the activity of NMDA and glycine receptors, and reduced by 35% the activity of GABAA receptors. A 3-day exposure of cultured hippocampal neurons to naltrexone (30 μM) or nicotine (10 μM, a concentration that fully desensitized α7 nAChRs) resulted in a 2-fold increase in the average amplitude of α7 nAChR-subserved currents. Naltrexone did not augment the maximal up-regulation of α7 nAChRs induced by nicotine, indicating that both drugs act via a common mechanism. In addition to increasing α7 nAChRs-mediated responses per neuron, nicotine increased the number of neurons expressing functional non-α7 nAChRs (probably α4β2 nAChRs); this effect was blocked by naltrexone (0.3 and 30 μM). Therefore, naltrexone may affect dependence on cigarette smoking by differentially altering function and expression of α7 and α4β2 nAChRs in the central nervous system.

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1‐day‐old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110‐kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72‐kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58‐kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110‐kDa subunit were reactive with it in im‐munoblots. Sucrose density gradient analysis of the antibody‐enzyme complexes showed that the anti‐110‐kDa subunit mAbs cross‐link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2–3S increase in the sedimentation constant with the mAbs specific for the 72‐kDa or for the 58‐kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross‐linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross‐reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.

Regulation of the nuclear export of the transcription factor NFATc1 by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibres

The transcription factor nuclear factor of activated T cells (NFAT)c1 has been shown to be involved in turning on slow skeletal muscle fibre gene expression. Previous studies from our laboratory have characterized the stimulation pattern‐dependent nuclear import and resting shuttling of NFATc1–green fluorescent protein (GFP) in flexor digitorum brevis (FDB) muscle fibres from adult mouse. In this study, we use viral expression of the transcription factor NFATc1–GFP fusion protein to investigate the mechanisms underlying the nuclear export of the NFATc1–GFP that accumulated in the nuclei of cultured dissociated adult mouse FDB muscle fibres during slow‐twitch fibre type electrical stimulation. In these studies, we found that inhibition of either glycogen synthase kinase 3β (GSK3β) or casein kinase 1 or 2 (CK1/2) markedly slowed the decay of nuclear NFATc1–GFP after cessation of muscle fibre electrical stimulation, whereas inhibition of casein kinase 1δ, p38 mitogen‐activated protein kinase, c‐Jun N‐terminal kinase and protein kinase A had little effect. Simultaneous inhibition of GSK3β and CK1/2 completely blocked the nuclear export of NFATc1–GFP after muscle activity. We also developed a simplified model of NFATc1 phosphorylation/dephosphorylation and nuclear fluxes, and used this model to simulate the observed time courses of nuclear NFATc1–GFP with and without NFATc1 kinase inhibition. Our results suggest that GSK3β and CK1/2 are the major protein kinases that contribute to the removal of NFATc1 that accumulates in muscle fibre nuclei during muscle activity, and that GSK3β and CK1/2 are responsible for phosphorylating NFATc1 in muscle nuclei in a complementary or synergistic fashion.