Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45β (GADD45β-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK''s pathological activation. GADD45β-I was engineered by optimizing the domain of the GADD45β, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45β-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen–glucose deprivation in vitro. Moreover, GADD45β-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45β-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45β-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.In many disorders of the nervous system, overactivation of N-methyl-D-aspartate (NMDA) receptors leads to neuronal death and consequent neurological impairment. NMDA-induced neuronal death, that is, excitotoxicity, has been implicated in many neurodegenerative diseases such as stroke, epilepsy, Alzheimer disease, spinal cord injury, traumatic brain injury, hearing loss, Parkinson''s and Huntington diseases.¹ However, the molecular mechanisms underlying excitotoxic neuronal death remain only partially understood.Excitotoxicity triggers complex signal transduction events that induce the neuronal death program. Among them, activation of the c-Jun N-terminal kinase (JNK) pathway has a key role.^{2, 3, 4, 5} There are only two direct upstream activators of JNK: mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7).^{6, 7} In some cell types, MKK4 activates JNK primarily in response to stress stimuli, whereas MKK7 signaling is triggered by release of inflammatory cytokines.^{8, 9, 10} In neurons, however, we showed that MKK7 is mainly responsible for JNK overactivation during excitotoxicity both in vitro³ and in vivo following middle cerebral artery occlusion (MCAo).⁴ Conversely, MKK4 controls JNK physiological role and its activation is not affected by excitotoxic stimuli.³Inhibition of the JNK pathway by the specific JNK inhibitor peptide, D-JNKI1, has been proposed for the treatment of ischemia.² D-JNKI1 induces powerful neuroprotection in in vitro models of excitoxicity^{2, 11} and leads to a 93% reduction in the infarct size in rodent models of ischemia.^{2, 4, 12} Despite the potent and long-lasting neuroprotective effect of D-JNKI1, total inhibition of JNK is not deprived of negative side effects, as it regulates a variety of physiological events¹³ such as cell proliferation, survival and differentiation.¹³ For these reasons, MKK7 may represent a more attractive target for clinical application, as it activates JNK specifically after toxic stimuli. Thus, by targeting MKK7 the physiological role of JNK, regulated by MKK4, will be preserved.Here we designed a set of new cell-permeable inhibitor peptides able to block MKK7 activity and protect against excitotoxic death.We took advantage of the growth arrest and DNA damage-inducible 45β (GADD45β) ability to bind MKK7.^{9, 14, 15} GADD45β is involved in the control of cell stress responses in cell cycle, DNA repair and oncogenesis.^{9, 16} GADD45β binds tightly to MKK7 and inhibits its enzymatic activity¹⁵ by interacting with its catalytic domain.⁹ More importantly, GADD45β inhibition is MKK7-specific and has no effect on MKK4, MKK3/6 and MEK1/2 activity.⁹ The minimal essential domain of interaction between MKK7 and GADD45β has already been defined (GADD45β_60–86 and _69–86 sequences).¹⁵ We here used in silico approaches to design an effector peptide, based on the domain of GADD45β, and optimize its affinity for MKK7. We then linked the effector peptide to a TAT-cargo in order to penetrate neuronal plasma membrane.¹⁷ The selected cell-permeable MKK7 inhibitor peptide (GADD45β-l) confers neuroprotection in vitro against NMDA and oxygen–glucose deprivation (OGD) toxicity, as well as in vivo in two models of MCAo with a clinically relevant post-ischemic temporal window (6 h) at both 24 h and 1 week after lesion. These data shed light on a new approach for the treatment of ischemia.     

Genome-Wide Scan Informed by Age-Related Disease Identifies Loci for Exceptional Human Longevity

We developed a new statistical framework to find genetic variants associated with extreme longevity. The method, informed GWAS (iGWAS), takes advantage of knowledge from large studies of age-related disease in order to narrow the search for SNPs associated with longevity. To gain support for our approach, we first show there is an overlap between loci involved in disease and loci associated with extreme longevity. These results indicate that several disease variants may be depleted in centenarians versus the general population. Next, we used iGWAS to harness information from 14 meta-analyses of disease and trait GWAS to identify longevity loci in two studies of long-lived humans. In a standard GWAS analysis, only one locus in these studies is significant (APOE/TOMM40) when controlling the false discovery rate (FDR) at 10%. With iGWAS, we identify eight genetic loci to associate significantly with exceptional human longevity at FDR < 10%. We followed up the eight lead SNPs in independent cohorts, and found replication evidence of four loci and suggestive evidence for one more with exceptional longevity. The loci that replicated (FDR < 5%) included APOE/TOMM40 (associated with Alzheimer’s disease), CDKN2B/ANRIL (implicated in the regulation of cellular senescence), ABO (tags the O blood group), and SH2B3/ATXN2 (a signaling gene that extends lifespan in Drosophila and a gene involved in neurological disease). Our results implicate new loci in longevity and reveal a genetic overlap between longevity and age-related diseases and traits, including coronary artery disease and Alzheimer’s disease. iGWAS provides a new analytical strategy for uncovering SNPs that influence extreme longevity, and can be applied more broadly to boost power in other studies of complex phenotypes.     

Loss of STK11 expression is an early event in prostate carcinogenesis and predicts therapeutic response to targeted therapy against MAPK/p38

Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To gain insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in an STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients.     

Doxorubicin Impairs the Insulin-Like Growth Factor-1 System and Causes Insulin-Like Growth Factor-1 Resistance in Cardiomyocytes

BackgroundInsulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.ConclusionsDoxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.     

Changes in soil organic carbon under perennial crops

This study evaluates the dynamics of soil organic carbon (SOC) under perennial crops across the globe. It quantifies the effect of change from annual to perennial crops and the subsequent temporal changes in SOC stocks during the perennial crop cycle. It also presents an empirical model to estimate changes in the SOC content under crops as a function of time, land use, and site characteristics. We used a harmonized global dataset containing paired‐comparison empirical values of SOC and different types of perennial crops (perennial grasses, palms, and woody plants) with different end uses: bioenergy, food, other bio‐products, and short rotation coppice. Salient outcomes include: a 20‐year period encompassing a change from annual to perennial crops led to an average 20% increase in SOC at 0–30 cm (6.0 ± 4.6 Mg/ha gain) and a total 10% increase over the 0–100 cm soil profile (5.7 ± 10.9 Mg/ha). A change from natural pasture to perennial crop decreased SOC stocks by 1% over 0–30 cm (?2.5 ± 4.2 Mg/ha) and 10% over 0–100 cm (?13.6 ± 8.9 Mg/ha). The effect of a land use change from forest to perennial crops did not show significant impacts, probably due to the limited number of plots; but the data indicated that while a 2% increase in SOC was observed at 0–30 cm (16.81 ± 55.1 Mg/ha), a decrease in 24% was observed at 30–100 cm (?40.1 ± 16.8 Mg/ha). Perennial crops generally accumulate SOC through time, especially woody crops; and temperature was the main driver explaining differences in SOC dynamics, followed by crop age, soil bulk density, clay content, and depth. We present empirical evidence showing that the FAO perennialization strategy is reasonable, underscoring the role of perennial crops as a useful component of climate change mitigation strategies.     

From ABC genes to regulatory networks,epigenetic landscapes and flower morphogenesis: Making biological sense of theoretical approaches

The ABC model postulates that expression combinations of three classes of genes (A, B and C) specify the four floral organs at early stages of flower development. This classic model provides a solid framework to study flower development and has been the foundation for multiple studies in different plant species, as well as for new evolutionary hypotheses. Nevertheless, it has been shown that in spite of being necessary, these three gene classes are not sufficient for flower organ specification. Rather, flower organ specification depends on complex interactions of several genes, and probably other non-genetic factors. Being useful to study systems of complex interactions, mathematical and computational models have enlightened the origin of the A, B and C stereotyped and robust expression patterns and the process of early flower morphogenesis. Here, we present a brief introduction to basic modeling concepts and techniques and review the results that these models have rendered for the particular case of the Arabidopsis thaliana flower organ specification. One of the main results is the uncovering of a robust functional module that is sufficient to recover the gene configurations characterizing flower organ primordia. Another key result is that the temporal sequence with which such gene configurations are attained may be recovered only by modeling the aforementioned functional module as a noisy or stochastic system. Finally, modeling approaches enable testable predictions regarding the role of non-genetic factors (noise, mechano-elastic forces, etc.) in development. These predictions, along with some perspectives for future work, are also reviewed and discussed.     

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ～60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.