Rebeca Mejias

Losartan restores skeletal muscle remodeling and protects against disuse atrophy in sarcopenia.

Losartan improves muscle remodeling and protects against immobilization atrophy by mediating pathways critical for muscle homeostasis. Losartan Comes of Age The Little Old Lady from Pasadena describes a diminutive woman of advanced years who aggressively drives her Dodge around a southern California city. In popular culture, people link long life spans with being “little”; yet, shortened stature is only one physical change associated with aging. Another, less jocular, transformation is loss of muscle mass and strength—called sarcopenia—which can cause disability and predicts impending death in older adults. Burks et al. now identify losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, as a new drug candidate for treating sarcopenia. Although the causes of sarcopenia are poorly understood, transforming growth factor–β (TGF-β) may contribute to faulty repair in aged muscle. Burks et al. used losartan to antagonize TGF-β signaling in an aged mouse model of sarcopenia. Losartan treatment improved muscle remodeling after injury and protected sarcopenic muscle from further loss of muscle mass caused by immobilization; these effects were mediated via two signaling circuits critical for skeletal muscle homeostasis: the TGF-β and insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathways. These observations suggest that treatment with losartan, a Food and Drug Administration (FDA)–approved drug, may benefit sarcopenia patients and allow little old ladies everywhere to continue their street racing for many years to come. Go granny go. 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.

Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent 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.

Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells

We studied the regulation of glucose‐6‐phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose‐ and time‐dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia‐induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia‐inducible factor (HIF)‐dependent glycolytic enzyme. Hypoxic‐G6PD induction was almost negligible in non‐excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO2. Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N‐acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose‐metabolizing enzymes.

Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism

Glutamate receptor interacting protein 1 (GRIP1) is a neuronal scaffolding protein that interacts directly with the C termini of glutamate receptors 2/3 (GluA2/3) via its PDZ domains 4 to 6 (PDZ4–6). We found an association (P < 0.05) of a SNP within the PDZ4-6 genomic region with autism by genotyping autistic patients (n = 480) and matched controls (n = 480). Parallel sequencing identified five rare missense variants within or near PDZ4–6 only in the autism cohort, resulting in a higher cumulative mutation load (P = 0.032). Two variants correlated with a more severe deficit in reciprocal social interaction in affected sibling pairs from proband families. These variants were associated with altered interactions with GluA2/3 and faster recycling and increased surface distribution of GluA2 in neurons, suggesting gain-of-function because GRIP1/2 deficiency showed opposite phenotypes. Grip1/2 knockout mice exhibited increased sociability and impaired prepulse inhibition. These results support a role for GRIP in social behavior and implicate GRIP1 variants in modulating autistic phenotype.

Neuroprotection by Transgenic Expression of Glucose-6-Phosphate Dehydrogenase in Dopaminergic Nigrostriatal Neurons of Mice

Oxidative damage to dopaminergic nigrostriatal (DNS) neurons plays a central role in the pathogenesis of Parkinsons disease (PD). Glucose-6-phosphate dehydrogenase (G6PD) is a key cytoprotective enzyme that provides NADPH, the major source of the reducing equivalents of a cell. Mutations of this enzyme are the most common enzymopathies worldwide. We have studied in vivo the role of G6PD overexpressed specifically in the DNS pathway and show that the increase of G6PD activity in the soma and axon terminals of DNS neurons, separately from other neurons or glial cells, protects them from parkinsonism. Analysis of DNS neurons by histological, neurochemical, and functional methods showed that even a moderate increase of G6PD activity rendered transgenic mice more resistant than control littermates to the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The neuroprotective action of G6PD was also observed in aged animals despite that they had a greater susceptibility to MPTP. Therefore, overexpression of G6PD in dopaminergic neurons or pharmacological activation of the native enzyme should be considered as potential therapeutic strategies to PD.

Developmental changes of chromaffin cell secretory response to hypoxia studied in thin adrenal slices

Adrenomedullary chromaffin (AMC) cells are sensitive to hypoxia in the newborn, but whether this property is lost during postnatal maturation is a matter of controversy. We have developed a rat adrenal slice preparation that allows the study of neonatal and adult AMC cell sensitivity to hypoxia in almost optimal physiological conditions. Responses to secretagogues can be quantitatively and noninvasively monitored in intact cells by amperometry. We have found hypoxia “responsive” (R) and “non-responsive” AMC cells in both neonatal (P0–P8) and juvenile/adult (P12–P60) adrenal glands. However, in the neonate, the proportion of R cells and the magnitude of the response to hypoxia were larger than in the adult. This developmental change of hypoxia responsiveness did not seem to depend on a decrease of the AMC cell’s excitability. Spontaneous secretory activity in slices from adult rats was even increased with respect to neonatal animals. The analysis of the secretory events suggests that changes in spike frequency, rather than in vesicle size, account for the increased basal secretion rate in adult AMC cells. Thus, we report a major, but not complete, loss of direct hypoxia sensitivity in adult AMC cells. The adrenal slice appears to be a valuable technique to study acute O2 sensing and its modifications in pathophysiological states.

Hibernating squirrel muscle activates the endurance exercise pathway despite prolonged immobilization

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.

GluA3-deficiency in Mice is Associated with Increased Social and Aggressive Behavior and Elevated Dopamine in Striatum

Glutamate signaling has been implicated in the regulation of social behavior. AMPA-glutamate receptors are assembled from four subunits (GluA1-4) of mainly GluA1/2 and GluA2/3 tetramers that form ion channels of distinct functional properties. Mice lacking GluA1 showed a reduced anxiety and male aggression. To understand the role of GluA3 in modulating social behavior, we investigated GluA3-deficient mice (Gria3-/Y) on C57BL/6J background. Compared to wild type (WT) littermates (n=14), Gria3-/Y mice (n=13) showed an increase in isolation-induced male aggression (p=0.011) in home cage resident-intruder test; an increase in sociability (p=0.01), and increase in male-male social interactions in neutral arena (p=0.005); an increase in peripheral activities in open field test (p=0.037) with normal anxiety levels in elevated plus maze and light-dark box; and minor deficits in motor and balance function in accelerating rotarod test (p=0.016) with normal grip strength. Gria3-/Y mice showed no significant deficit in spatial memory function in Morris-water maze and Y-maze tests, and normal levels of testosterone. Increased dopamine concentrations in stratum (p=0.034) and reduced serotonin turnover in olfactory bulb (p=0.002) were documented in Gria3-/Y mice. These results support a role of GluA3 in the modulation of social behavior through brain dopamine and/or serotonin signaling and different AMPA receptor subunits affect social behavior through distinct mechanisms.

Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

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 ActRIIB, a 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 serum and glucocorticoid-induced kinase (SGK) rather than by 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.