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Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model. Five rhesus macaques were injected with AAV1 or Anc80L65 expressing eGFP using a transmastoid posterior tympanotomy approach to access the round window membrane after making a small fenestra in the oval window. The procedure was well tolerated. All but one animal showed cochlear eGFP expression 7-14 days following injection. Anc80L65 in 2 animals transduced up to 90% of apical inner hair cells; AAV1 was markedly less efficient at equal dose. Transduction for both vectors declined from apex to base. These data motivate future translational studies to evaluate gene therapy for human hearing disorders.

Summary: The adeno-associated virus (AAV) vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy

International audience; Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to the ActRIIB receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo treated mice. Our results suggest that this protection from disuse atrophy is regulated by SGK instead of Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK, and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK, and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non-hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non-hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

Mutations in theleucine-rich repeat kinase 2(LRRK2) gene are the most common known cause of Parkinson's disease (PD). Whether loss of LRRK2 function accounts for neurodegeneration of dopamine neurons in PD is not known, nor is it known whether LRRK2 kinase activity modulates the susceptibility of dopamine (DA) neurons to the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To better understand the role of LRRK2 in DA neuronal survival and its role in the susceptibility of DA neurons to MPTP, we generated LRRK2 knock-out (KO) mice lacking the kinase domain of LRRK2. Here, we show that LRRK2 KO mice are viable and have no major abnormalities and live to adulthood. The dopaminergic system is normal in LRRK2 KO mice as assessed via HPLC for DA and its metabolites and via stereologic assessment of DA neuron number in young and aged mice. Importantly, there is no significant difference in the susceptibility of LRRK2 KO and wild-type mice to MPTP. These results suggest that LRRK2 plays little if any role in the development and survival of DA neurons under physiologic conditions. Thus, PD due to LRRK2 mutations are likely not due to a loss of function. Moreover, LRRK2 is not required for the susceptibility of DA neurons to MPTP.

Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1beta (IL-1beta) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1beta effect on type A gamma-aminobutyric acid receptors (GABA(A)Rs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABA(A)Rs, respectively. Confocal images in both cell types revealed that IL-1beta increases recruitment of GABA(A)Rs to the cell surface. Moreover, brief applications of IL-1beta to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (I(GABA)); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with I(GABA) recording and confocal images of GABA(A) Rs in oocytes showed that IL-1beta stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABA(A)Rs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABA(A)R, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1beta with increased "GABAergic tone." We propose that through this mechanism IL-1beta might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE.

Understanding precisely the functioning of voltage-gated Ca2+ channels and their modulation by signaling molecules will help clarifying the Ca2+-dependent mechanisms controlling exocytosis in chromaffin cells. In recent years, we have learned more about the various pathways through which Ca2+ channels can be up- or down-modulated by hormones and neurotransmitters and how these changes may condition chromaffin cell activity and catecolamine release. Recently, the attention has been focused on the modulation of L-channels (Cav 1), which represent the major Ca2+ current component in rat and human chromaffin cells. L-channels are effectively inhibited by the released content of secretory granules or by applying mixtures of exogenous ATP, opioids, and adrenaline through the activation of receptor-coupled G proteins. This unusual inhibition persists in a wide range of potentials and results from a direct (membrane-delimited) interaction of G protein subunits with the L-channels co-localized in membrane microareas. Inhibition of L-channels can be reversed when the cAMP/PKA pathway is activated by membrane permeable cAMP analog or when cells are exposed to isoprenaline (remote action), suggesting the existence of parallel and opposite effects on L-channel gating by distinctly activated membrane autoreceptors. Here, the authors review the molecular components underlying these two opposing signaling pathways and present new evidence supporting the presence of two L-channel types in rat chromaffin cells (α1C and α1D), which open new interesting issues concerning Ca2+-channel modulation. In light of recent findings on the regulation of exocytosis by Ca2+-channel modulation, the authors explore the possible role of L-channels in the autocontrol of catecholamine release.

Simultaneous recordings of inward whole-cell Ca(2+) channel currents (I(Ca) ) and increments of capacitance as an indication of exocytosis (Delta(Cm)), were performed in voltage-clamped single adrenal chromaffin cells from wild-type and alpha(1A) subunit deficient mice, using the perforated-patch configuration of the patch-clamp technique. Using protocol #1 (one single Ca(2+) channel blocker per cell), to dissect the components of I(Ca), L channels contributed 43%, N channels 35% and P/Q channels 30% to the total I(Ca) of wild-type cells. Using protocol #2 (cumulative sequential addition of 3 microm nifedipine, 1 microm omega-conotoxin GVIA, and 1 microm omega-agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to I(Ca); an R component of around 11% remained. In wild-type mice the changes of Delta(Cm) paralleled those of I(Ca). In alpha(1A) deficient mice the L component of I(Ca) rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, Delta(Cm) associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca(2+) channel subtype, L, N, P/Q, R. Ablation of the alpha(1A) gene led to a loss of P/Q channel current and to a compensatory increase of L channel-associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in alpha(1A) -deficient mice. This may be due to altered Ca(2+) homeostasis in these mice, as compared to wild mouse chromaffin cells.

Skeletal muscle atrophy is a very common clinical challenge in many disuse conditions. Maintenance of muscle mass is crucial to combat debilitating functional consequences evoked from these clinical conditions. In contrast, hibernation represents a physiological state in which there is natural protection against disuse atrophy despite prolonged periods of immobilization and lack of nutrient intake. Even though peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1-α (PGC-1α) is a central mediator in muscle remodeling pathways, its role in the preservation of skeletal muscle mass during hibernation remains unclear. Since PGC-1α regulates muscle fiber type formation and mitochondrial biogenesis, we analyzed muscles of 13-lined ground squirrels. We find that animals in torpor exhibit a shift to slow-twitch Type I muscle fibers. This switch is accompanied by activation of the PGC-1α-mediated endurance exercise pathway. In addition, we observe increased antioxidant capacity without evidence of oxidative stress, a marked decline in apoptotic susceptibility, and enhanced mitochondrial abundance and metabolism. These results show that activation of the endurance exercise pathway can be achieved in vivo despite prolonged periods of immobilization, and therefore might be an important mechanism for skeletal muscle preservation during hibernation. This PGC-1α regulated pathway may be a potential therapeutic target promoting skeletal muscle homeostasis and oxidative balance to prevent muscle loss in a variety of inherited and acquired neuromuscular disease conditions.

Skeletal muscle atrophy can occur as a consequence of immobilization and/or starvation in the majority of vertebrates studied. In contrast, hibernating mammals are protected against the loss of muscle mass despite long periods of inactivity and lack of food intake. Resident muscle-specific stem cells (satellite cells) are known to be activated by muscle injury and their activation contributes to the regeneration of muscle, but whether satellite cells play a role in hibernation is unknown. In the hibernating 13-lined ground squirrel we show that muscles ablated of satellite cells were still protected against atrophy, demonstrating that satellite cells are not involved in the maintenance of skeletal muscle during hibernation. Additionally, hibernating skeletal muscle showed extremely slow regeneration in response to injury, due to repression of satellite cell activation and myoblast differentiation caused by a fine-tuned interplay of p21, myostatin, MAPK, and Wnt signaling pathways. Interestingly, despite long periods of inflammation and lack of efficient regeneration, injured skeletal muscle from hibernating animals did not develop fibrosis and was capable of complete recovery when animals emerged naturally from hibernation. We propose that hibernating squirrels represent a new model system that permits evaluation of impaired skeletal muscle remodeling in the absence of formation of tissue fibrosis.

Sarcopenia, a critical loss of muscle mass and function because of the physiological process of aging, contributes to disability and mortality in older adults. It increases the incidence of pathologic fractures, causing prolonged periods of hospitalization and rehabilitation. The molecular mechanisms underlying sarcopenia are poorly understood, but recent evidence suggests that increased transforming growth factor-β (TGF-β) signaling contributes to impaired satellite cell function and muscle repair in aged skeletal muscle. We therefore evaluated whether antagonism of TGF-β signaling via losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, had a beneficial impact on the muscle remodeling process of sarcopenic mice. We demonstrated that mice treated with losartan developed significantly less fibrosis and exhibited improved in vivo muscle function after cardiotoxin-induced injury. We found that losartan not only blunted the canonical TGF-β signaling cascade but also modulated the noncanonical TGF-β mitogen-activated protein kinase pathway. We next assessed whether losartan was able to combat disuse atrophy in aged mice that were subjected to hindlimb immobilization. We showed that immobilized mice treated with losartan were protected against loss of muscle mass. Unexpectedly, this protective mechanism was not mediated by TGF-β signaling but was due to an increased activation of the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. Thus, blockade of the AT1 (angiotensin II type I) receptor improved muscle remodeling and protected against disuse atrophy by differentially regulating the TGF-β and IGF-1/Akt/mTOR signaling cascades, two pathways critical for skeletal muscle homeostasis. Thus, losartan, a Food and Drug Administration-approved drug, may prove to have clinical benefits to combat injury-related muscle remodeling and provide protection against disuse atrophy in humans with sarcopenia.

Objective: Neuronal nitric oxide synthase (nNOS), normally expressed at the sarcolemmal membrane, is known to be mislocalized to the sarcoplasm in several forms of muscular dystrophy. Our objectives were to characterize further the range of patients manifesting aberrant nNOS sarcolemmal immunolocalization and to study nNOS localization in animal models of nondystrophic myopathy. Methods: We carried out a retrospective cross-sectional study. We performed immunofluorescent staining for nNOS on biopsy specimens from 161 patients with acquired and nondystrophin inherited neuromuscular conditions. The localization of sarcolemmal nNOS correlated with mobility and functional status. Muscle specimens from mouse models of steroid-induced and starvation-related atrophy were studied for qualitative and quantitative nNOS expression. Results: Sarcolemmal nNOS staining was abnormal in 42% of patients with inherited myopathic conditions, 25% with acquired myopathic conditions, 57% with neurogenic conditions, and 93% with hypotonia. Interestingly, we found significant associations between mobility status or muscle function and sarcolemmal nNOS expression. Furthermore, mouse models of catabolic stress also demonstrated mislocalization of sarcolemmal nNOS. Conclusion: Our analyses indicate that nNOS mislocalization is observed in a broad range of nondystrophic neuromuscular conditions associated with impaired mobility status and catabolic stress. Our findings suggest that the assessment of sarcolemmal localization of nNOS represents an important tool for the evaluation of muscle biopsies of patients with a variety of inherited and acquired forms of neuromuscular disorders.

Parkinson's disease (PD) is a common neurodegenerative movement disorder. Whereas the majority of PD cases are sporadic, rare genetic defects have been linked to this prevalent movement disorder. Mutations in DJ-1 are associated with autosomal recessive early-onset PD. The exact biochemical function of DJ-1 has remained elusive. Here we report the generation of DJ-1 knockout (KO) mice by targeted deletion of exon 2 and exon 3. There is no observable degeneration of the central dopaminergic pathways, and the mice are anatomically and behaviorally similar to WT mice. Fluorescent Amplex red measurements of H 2 O 2 indicate that isolated mitochondria from young and old DJ-1 KO mice have a 2-fold increase in H 2 O 2 . DJ-1 KO mice of 2–3 months of age have a 60% reduction in mitochondrial aconitase activity without compromising other mitochondrial processes. At an early age there are no differences in antioxidant enzymes, but in older mice there is an up-regulation of mitochondrial manganese superoxide dismutase and glutathione peroxidase and a 2-fold increase in mitochondrial glutathione peroxidase activity. Mutational analysis and mass spectrometry reveal that DJ-1 is an atypical peroxiredoxin-like peroxidase that scavenges H 2 O 2 through oxidation of Cys-106. In vivo there is an increase of DJ-1 oxidized at Cys-106 after 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine intoxication of WT mice. Taken together these data indicate that the DJ-1 KO mice have a deficit in scavenging mitochondrial H 2 O 2 due to the physiological function of DJ-1 as an atypical peroxiredoxin-like peroxidase.

A small expansion of a CAG repeat domain in exon 47 of the human CACNA1A gene, which codes for the pore-forming α1A subunit of P/Q-type Ca2+ channels, causes spinocerebellar ataxia type-6. Only the human α1A protein has been demonstrated to contain the poly(Q) tract, although this locus has also recently been detected in ape genomes. To our knowledge, no further information has been published on other mammal species. Here, we have cloned the full-length α1A subunit in a non-primate species, the cow. The results have made it possible to explore the exon organization of the bovine CACNA1A gene as well as the splice α1A isoforms expressed by bovine chromaffin cells. We found a splice variant of the protein that, as in humans, also contains a polymorphic poly(Q) tract. Based on this result and using data from different Genome Databases, we performed an interspecies comparison of exon 47 and discovered that the poly(Q) tract is present in all the species studied, with the exception of primitive fish and rodents. Our results provide insight into the evolution of the CAG repeat tract at the C-terminus coding region of the CACNA1A gene. © 2006 Elsevier B.V. All rights reserved., This work was supported by grants to Carmen Montiel from Ministerio de Ciencia y Tecnología (SAF2002-01851, SAF2005-00951), and Fundación Mutua Madrileña, Spain.

Although the specific interaction between synaptic protein SNAP-25 and the α1A subunit of the Cav2.1 channels, which conduct P/Q-type Ca2+ currents, has been confirmed in in vitro-translated proteins and brain membrane studies, the question of how native proteins can establish this association in situ in developing neurons remains to be elucidated. Here we report data regarding this interaction in bovine chromaffin cells natively expressing both proteins. The two carboxyl-terminal splice variants of the α1A subunit identified in these cells share a synaptic protein interaction ('synprint') site within the II/III loop segment and are immunodetected by a specific antibody against bovine α1A protein. Moreover, both α1A isoforms form part of the P/Q-channels-SNARE complexes in situ because they are coimmunoprecipitated from solubilized chromaffin cell membranes by a monoclonal SNAP-25 antibody. The distribution of α1A and SNAP-25 was studied in round or transdifferentiated chromaffin cells using confocal microscopy and specific antibodies: the two proteins are colocalized at the cell body membrane in both natural cell types. However, during the first stages of the cell transdifferentiation process, SNAP-25 migrates alone out to the developing growth cone and what will become the nerve endings and varicosities of the mature neurites; α1A follows and colocalizes to SNAP-25 in the now mature processes. These observations lead us to propose that the association between SNAP-25 and M1A during neuritogenesis might promote not only the efficient coupling of the exocytotic machinery but also the correct insertion of P/Q-type channels at specialized active zones in presynaptic neuronal terminals. © Federation of European Neuroscience Societies., This work was supported by a grant to C.M. from Ministerio de Ciencia y Tecnología, Spain (SAF2002-0151). E.A.M. is a Fellow of the Universidad Autónoma de Madrid; L.M.S.G. and A.M.L.C. are Fellows of Ministerio de Educación y Ciencia, Spain; R.S. is a Fellow of FIS, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain.

Because the presence of a native plasmalemmal Na+/Ca2+ exchange (NCX) activity in Xenopus laevis oocytes remains controversial, its possible functional role in these cells is poorly understood. Here, in experiments on control oocytes and oocytes overexpressing a cloned NCX1 cardiac protein, confocal microscopy combined with electrophysiological techniques reveal that these cells express an endogenous NCX protein forming a functional microdomain with inositol 1,4,5-trisphosphate receptors (InsP3R) that controls intracellular Ca2+ in a restricted subplasmalemmal space. The following data obtained in control denuded oocytes are consistent with this view: (i) reverse transcription-PCR revealed that the oocyte expresses two transcripts for the NCX1 and NCX3 isoforms; (ii) immunofluorescence experiments showed that native NCX1 and InsP3Rs are largely codistributed in discrete areas of the plasma membrane in close apposition to the cortical endoplasmic reticulum shell; (iii) when stimulated by rabbit serum, which elevates intracellular Ca2+ mediated by InsP3, voltage-clamped oocytes display a large and transient inward Ca2+-activated chloride current, ICl(Ca), as a result of the Ca2+ rise at the inner surface membrane; (iv) this current is significantly enhanced by KB-R7943 and by an extracellular sodium-depleted medium, two maneuvers that prevent “Ca2+ extrusion” via NCX; and (v) blocking NCX enhanced the ICl(Ca) elicited by InsP3 but not by Ca2+ photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the “Ca2+ influx” but not for the serum-elicited “Ca2+ efflux” mode of basal exchange activity and does not alter the number of endogenous NCX/InsP3Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP3-mediated Ca2+ signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca2+ microdomains surrounding the native NCX/InsP3Rs complex in the oocyte.

This study aimed at determining the distribution and expression levels of different subtypes of Ca2+ channels in the bovine adrenal medulla, and whether individual subtypes were more abundant in chromaffin cells exhibiting an adrenergic or a noradrenergic phenotype. In situ hybridization using riboprobes specific for the pore-forming Ca2+ channel α1D (L-type channel), α1B (N-type channel), and α1A (P/Q-type channel) subunits of bovine chromaffin cells showed a broad distribution of the three transcripts in adrenal medulla tissue. However, a tissue-specific expression pattern of individual subunits was found; whereas α1B mRNA was homogeneously distributed throughout the medulla, α1D and α1A transcripts were present at higher densities in the internal medullary area, far away from the adrenal cortex. These results were corroborated by comparative analysis of the α1B, α1D, and α1A products amplified by RT-PCR from total RNA extracted from small pieces of tissue dissected out from external or internal medullary areas. Interestingly, immunohistochemical experiments performed in adrenal gland sections, using antidopamine-β-hydroxylase and anti-phenylethanolamine-N-methyltransferase antibodies, indicated a higher density of noradrenergic over adrenergic chromaffin cells in the internal medullary region. These results provide direct evidence in favor of a heterogeneous distribution of Ca2+ channel subtypes in the adrenal medulla, in agreement with previous functional data showing that blockade of the high K+-elicited responses by dihydropyridines was greater in noradrenergic than in adrenergic chromaffin cells. These differences may be relevant for the differential release regulation of each catecholamine under physiological and pathophysiological conditions. Copyright © 2001 S. Karger AG, Basel.

Myostatin, a member of the TGFβ signaling family, acts as a natural inhibitor of skeletal muscle growth via binding to the ActIIb receptor on the cell surface of myofibers. Because myostatin inhibition in a normal mouse induces profound muscle hypertrophy, we investigated the use of a soluble ActIIb receptor as a treatment for immobilization and denervation atrophy in mice. We found that immobilized mice were protected from atrophy after three weeks of administration of ActIIb as compared to the placebo treated group. In contrast, ActIIb treatment did not rescue muscle atrophy in denervated mice. Molecular analysis of the skeletal muscle from immobilized mice demonstrated that ActIIb treatment leads to the protection of several pro-growth pathways. We found that mice treated with ActIIb maintained mTOR signaling while the placebo immobilized group showed a significant decrease in many components of this pathway. The ActIIb treated group was also protected from the loss of anti-apoptotic and pro-growth Jak/Stat signaling markers as compared to placebo immobilized mice. In contrast, and in agreement with the muscle phenotype, molecular analysis of the denervated mice showed a loss of anti-apoptotic and an increase in pro-autophagic markers suggesting that these pathways mediate the loss of muscle mass. In addition, denervated mice demonstrated a pronounced disregulation of pro-growth markers in the Jak/Stat signaling cascade. Surprisingly, denervated mice revealed upregulation of many components of mTOR signaling cascade. As this pathway is associated with hypertrophy, we suggest that this is a compensatory mechanism to prevent further atrophy. These results highlight that myostatin inhibition is a valid therapeutic option for conditions associated with disuse atrophy, but not for denervation induced atrophy. Furthermore, our results indicate that different molecular mechanisms drive loss of muscle mass in immobilization and denervation atrophy.

Disuse atrophy is problematic for individuals of all age; however, it is completely reversible in young muscle while aged muscle lacks the ability to recover. Previously we showed that the exaggerated response to disuse atrophy with age is due to a loss of muscle cells as opposed to smaller muscle cells evident in disuse atrophy of young muscle. Here we show that the loss of muscle mass associated with disuse atrophy in adult muscle is due to a combination of a loss of muscle cells and smaller muscle cells. Additionally, the effects of angiotensin II type I receptor blocker (ARB) losartan on disuse atrophy are age-dependent. Losartan confers no protection against disuse atrophy in young mice and partial protection in adult mice. However, it completely protects against the loss of muscle mass in aged mice. The discrepancies in results are due to the modulation of pathways downstream of renin-angiotensin system (RAS), particularly the TGF-β pathway. Additionally, there appears to be age-dependent regulation of the Akt/mTOR/FoxoO3a pathway known to play an integral role in the loss of muscle mass with disuse. These data show that age-related changes occur in response to disuse atrophy and that the changes in the RAS affect the impact of ARBs on the maintenance of skeletal muscle.

Diabetes mellitus may be associated with intracellular glutathione (GSH) deficiency. Since in vivo studies have shown that plasma intracellular GSH plays a key role in regulating the activation of nuclear factor kappaB (NF-kappaB), we have investigated the relationship between intracellular thiols (GSH, homocysteine, cysteine and cysteinyglycine) and NF-kappaB activity in the peripheral blood mononuclear cells (PBMC) of 63 elderly non-insulin dependent diabetes mellitus (NIDDM) patients (28 microalbuminurics and 35 normoalbuminurics) and 30 healthy age- and sex-matched subjects. In addition, we have measured plasma concentrations of these thiol compounds, serum concentrations of interleukin-6 (IL-6) and vascular cell adhesion molecule-1 (sVCAM-1), that are partly dependent on the NF-kappaB activation, as well as the serum levels of thiobarbituric acid reacting substances (TBARS), as index of lipid peroxidation. Diabetic patients with microalbuminuria (MAB) and normoalbuminuria had NF-kappaB activity 2.1- and 1.5-fold greater, respectively, than the control group. As compared to normoalbuminuric patients, patients with MAB had significantly higher levels of glycemia, plasma homocysteine, and serum concentrations of TBARS, IL-6 and sVCAM-1 (in all cases, p0.01), and significantly lower GSH content in the PBMC (p0.05). The intracellular GSH in PBMC correlated with NF-kappaB activation (r = -0.82; p0.0001), serum TBARS (r = -0.60; p0.001), and with fasting glycemia (r = -0.56; p0.001) in patients with MAB, whereas a weaker association between GSH levels in PBMC and NF-kappaB activation (r = -0.504, p0.001) was seen in patients without MAB. These results suggest that the decrease of intracellular GSH content in elderly NIDDM patients with MAB is strongly associated with enhanced NF-kappaB activation, which could contribute to the development of increased glomerular capillary permeability and its rapid progression.