Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy.

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.

Subsarcolemmal and interfibrillar mitochondria display distinct superoxide production profiles.

Cardiac subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) subpopulations display distinct biochemical, morphological, and functional characteristics. Moreover, they appear to be differently influenced during cardiac pathologies or toxic injuries. Although mitochondrial reactive oxygen species seem to play a critical role in cardiac function and diseases, limited information exists about the superoxide production characteristics of these mitochondrial subpopulations. In this work, using direct measurement of superoxide by electron paramagnetic resonance, we showed that differences in superoxide production profiles were present between cardiac IFM and SSM, in terms of intensity and major sites of superoxide generation. In SSM incubated with glutamate plus malate as substrates, the total observed superoxide levels were significantly higher than those observed with IFM, with an important contribution of the NADH-oxidizing site of complex I (site If) and the quinol-oxidizing site of complex III (site IIIQ0). In both IFM and SSM, succinate leads to similar rates of total superoxide levels with a substantial role for contribution of reverse electron transfer. Finally, using two spin probes with different membrane permeabilities, our data on complex III showed direct intra- and extra-mitochondrial superoxide release whereas complex I- and II-dependent superoxide were exclusively released inside the mitochondria, confirming previous studies. Feasibility of this approach to measure intra- and extra-mitochondrial superoxide levels and to characterize distinct superoxide production profiles of cardiac IFM and SSM has been demonstrated.

Direct targeting of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocorticoid receptor activation.

An important question arising from previous observations in vivo is whether glucocorticoids can directly influence neuronal survival in the hippocampus. To this end, a primary postnatal hippocampal culture system containing mature neurons and expressing both glucocorticoid (GR) and mineralocorticoid (MR) receptors was developed. Results show that the GR agonist dexamethasone (DEX) targets neurons (microtubule-associated protein 2-positive cells) for death through apoptosis. GR-mediated cell death was counteracted by the MR agonist aldosterone (ALDO). Antagonism of MR with spironolactone ([7alpha-(acetylthio)-3-oxo-17alpha-pregn-4-ene-21 carbolactone] (SPIRO)) causes a dose-dependent increase in neuronal apoptosis in the absence of DEX, indicating that nanomolar levels of corticosterone present in the culture medium, which are sufficient to activate MR, can mask the apoptotic response to DEX. Indeed, both SPIRO and another MR antagonist, oxprenoate potassium ((7alpha,17alpha)-17-hydroxy-3-oxo-7-propylpregn-4-ene-21-carboxylic acid, potassium salt (RU28318)), accentuated DEX-induced apoptosis. These results demonstrate that GRs can act directly to induce hippocampal neuronal death and that demonstration of their full apoptotic potency depends on abolition of survival-promoting actions mediated by MR.

Fractalkine protein localization and gene expression in mouse brain.

Few chemokines are expressed constitutively in the brain at detectable levels; amongst them is fractalkine. We analyzed the distribution of fractalkine in the mouse brain with the aim of giving a neuroanatomical support to the study of its physiological function. To this end, we carried out an analysis of fractalkine protein localization and gene expression. An anti-fractalkine antibody was produced and used to perform an immunohistochemical study. The results indicated a high level of fractalkine protein in cortex, hippocampus, basal ganglia, and olfactory bulb. In particular, the presence of abundant immunoreactive neurons was observed in layers II, III, V, and VI of the cortex. In the hippocampus, the CA1 region was the most intensely labeled, but immunoreactive neurons were present also in CA2 and CA3, whereas in the basal ganglia, immunoreactive cells were observed in the caudate putamen. Other brain structures such as the brainstem showed a few scattered immunoreactive cells. The presence of fractalkine immunoreactive fibers was revealed only in the olfactory bulb and in the anterior olfactory nuclei. Gene expression study results, obtained by both semiquantitative PCR and in situ hybridization, matched protein localization with the highest levels of fractalkine transcript detected in the hippocampus, cortex, and striatum. The present study showed that fractalkine protein and mRNA are constitutively expressed at a high level in forebrain structure, but are almost absent in the hindbrain. Furthermore, localization at the cellular body level would suggest a paracrine or cell-to-cell interaction role for fractalkine more than a neurotransmission modulatory function.

Differential induction of NF-kappaB activity and neural cell death by antidepressants in vitro.

Tricyclic antidepressants and selective serotonin reuptake inhibitors are here shown to induce cell death in a neural cell line. The exposure to these drugs led to increased generation of reactive oxygen species and a concomitant reduction of intracellular glutathione levels. Furthermore, these antidepressants induced DNA fragmentation and increased the transcriptional and DNA-binding activity of NF-kappaB. In contrast, treatment with type A and B monoamine oxidase inhibitors did not induce changes in NF-kappaB activity and did not exert a detrimental influence on cell viability. These results indicate that some antidepressant drugs may cause both oxidative stress and changes in cellular antioxidative capacity, resulting in altered NF-kappaB activity and, ultimately, cell death.

Subtle shifts in the ratio between pro- and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate.

Glucocorticoid receptor (GR) activation induces apoptosis of granule cells in the hippocampus. In contrast, neuroprotection is seen after mineralocorticoid receptor (MR) activation. To date there is no in vivo evidence for direct interactions between corticosteroids and any of the key regulatory molecules of programmed cell death. In this report, we show that the opposing actions of MR and GR on neuronal survival result from their ability to differentially influence the expression of members of the bcl-2 gene family; specifically, in the rat hippocampus, activation of GR induces cell death by increasing the ratio of the proapoptotic molecule Bax relative to the antiapoptotic molecules Bcl-2 or Bcl-x(L); the opposite effect is observed after stimulation of MR. The same results were obtained in both young and aged animals; however, older subjects (which were more susceptible to GR-mediated apoptosis) tended to express the antiapoptotic genes more robustly. Using a loss-of-function mouse model, we corroborated the observations made in the rat, demonstrating Bax to be essential in the GR-mediated cell death-signaling cascade. In addition, we show that GR activation increases and MR activation decreases levels of the tumor suppressor protein p53 (a direct transcriptional regulator of bax and bcl-2 genes), thus providing new information on the early genetic events linking corticosteroid receptors with apoptosis in the nervous system.

PACAP type I receptor activation promotes cerebellar neuron survival through the cAMP/PKA signaling pathway.

The molecular nature, transduction pathways, and neurotrophic functions of pituitary adenylate cyclase activating peptide (PACAP) receptors were studied in primary culture of rat cerebellar granule cells. We show that cerebellar neurons express several PACAP type I receptor (PVR I) isoforms, including the short (PVR Is) and the Hop (PVR I-Hop) splice variants, the latter being restricted to neurons and not found in cerebellar glial cell cultures. In vitro, cerebellar granule cells die rapidly in the absence of a high concentration of K+ (25 mM), as demonstrated by TUNEL histochemistry, which shows that K+ deprivation induces massive neuronal apoptosis within 12 hr. This effect was reversed by PACAP 27 and 38. Both forms of PACAP prevent DNA fragmentation and allow long-term neuronal survival in the absence of high K+ (as shown by MAP2 immunostaining) and stimulate a reporter gene driven by the full-length c-fos promoter. These effects of PACAP are fully abolished upon transient transfection of cells with a dominant inhibitory mutant of the cAMP-dependent protein kinase (PKA). Taken together, these results show that in cerebellar granule neurons, PACAP type I receptors regulate gene expression and promote neuronal survival through the cAMP/PKA pathway.