Daniela Merlo

Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat–containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type–specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

4-Aminopyridine-induced epileptogenesis depends on activation of mitogen-activated protein kinase ERK

Extracellular signal‐regulated kinases such as ERK1 [p44 mitogen‐activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the CNS under physiological and pathological conditions such as ischemia and epilepsy. Here, we studied the activation state of ERK1/2 in rat hippocampal slices during application of the K+ channel blocker 4‐aminopyridine (4AP, 50 µm), a procedure that enhances synaptic transmission and leads to the appearance of epileptiform activity. Hippocampal slices superfused with 4AP‐containing medium exhibited a marked activation of ERK1/2 phosphorylation that peaked within about 20 min. These effects were not accompanied by changes in the activation state of c‐Jun N‐terminal kinase (JNK), another member of the MAP kinase superfamily. 4AP‐induced ERK1/2 activation was inhibited by the voltage‐gated Na+ channel blocker tetrodotoxin (1 µm). We also found that application of the ERK pathway inhibitors U0126 (50 µm) or PD98059 (100 µm) markedly reduced 4AP‐induced epileptiform synchronization, thus abolishing ictal discharges in the CA3 area. The effects induced by U0126 or PD98059 were not associated with changes in the amplitude and latency of the field potentials recorded in the CA3 area following electrical stimuli delivered in the dentate hylus. These data demonstrate that activation of ERK1/2 accompanies the appearance of epileptiform activity induced by 4AP and suggest a cause‐effect relationship between the ERK pathway and epileptiform synchronization.

Neuroprotective Effects of Thymosin β4 in Experimental Models of Excitotoxicity

Abstract:  The aim of this study was to evaluate the possible neuroprotective effects of thymosin β4 in different models of excitotoxicity. The application of thymosin β4 significantly attenuated glutamate‐induced toxicity both in primary cultures of cortical neurons and in rat hippocampal slices. In in vivo experiments, the intracerebroventricular administration of thymosin β4 significantly reduced hippocampal neuronal loss induced by kainic acid. These results show that thymosin β4 induced a protective effect in models of excitotoxicity. The mechanisms underlying such an effect, as well as the real neuroprotective potential of thymosin β4, are worthy of further investigations.

In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression

PURPOSE Mucins released into the tear film are crucial to maintaining a healthy ocular surface. Alterations in goblet cell numbers and mucin secretion are observed in chronic ocular surface inflammatory diseases. Nerve growth factor (NGF) plays a crucial role in healing and inflammation of the ocular surface. The aim of this study was to evaluate in vitro the effect of NGF on conjunctival goblet cell differentiation and mucin production and secretion. METHODS Human conjunctival epithelial cells were exposed to increasing NGF concentrations (1 to 250 ng/mL) and analyzed to quantify cell growth (MTT/Ki67/BrdU), goblet cell differentiation (PAS/MUC5AC confocal staining), and mucin mRNA expression (real-time PCR). Secreted and cellular MUC5AC were also analyzed by sandwich-ELISA and FACS, respectively. To confirm the biological effects of NGF, the same evaluations were performed on primary cultures, and changes in markers of stemness (p63) and commitment (14-3-3 sigma) were also investigated. RESULTS In cell cultures, NGF induced a dose-dependent increase of goblet cell numbers, MUC5AC production, storage, and release. Additionally, in primary cultures, NGF induced an increase of abortive colonies and 14-3-3 sigma protein, and a decrease of p63 mRNA and protein, suggesting a differentiating effect of NGF on human conjunctival epithelium. CONCLUSIONS These findings show that NGF might play a role in the complex mechanism leading to conjunctival epithelium differentiation and mucin secretion. In addition to the known roles of NGF in promoting ocular surface healing and sensitivity, its effects on conjunctival goblet cells support a rationale to investigate the therapeutic effectiveness of NGF in dry eye disease.

Thymosin β4 targeting impairs tumorigenic activity of colon cancer stem cells

Thymosin β4(Tβ4) is an actin‐binding peptide overexpressed in several tumors, including colon carcinomas. The aim of this study was to investigate the role of Tβ4 in promoting the tumorigenic properties of colorectal cancer stem cells (CR‐CSCs), which are responsible for tumor initiation and growth. We first found that CR‐CSCs from different patients have higher Tβ4 levels than normal epithelial cells. Then, we used a lentiviral strategy to down‐regulate Tβ4 expression in CR‐CSCs and analyzed the effects of such modulation on proliferation, survival, and tumorigenic activity of CR‐CSCs. Empty vector‐transduced CR‐CSCs were used as a control. Targeting of the Tβ4 produced CR‐CSCs with a lower capacity to grow and migrate in culture and, interestingly, reduced tumor size and aggressiveness of CR‐CSC‐based xenografts in mice. Moreover, such loss in tumorigenic activity was accompanied by a significant increase of phosphatase and tensin homologue (PTEN) and a concomitant reduction of the integrin‐linked kinase (ILK) expression, which resulted in a decreased activation of protein kinase B (Akt). Accordingly, exogenous expression of an active form of Akt rescued all the protumoral features lost after Tβ4 targeting in CR‐CSCs. In conclusion, Tβ4 may have important implications for therapeutic intervention for treatment of human colon carcinoma.—Ricci‐Vitiani, L., Mollinari, C., di Martino, S., Biffoni, M., Pilozzi, E., Pagliuca, A., Chiara de Stefano, M., Circo, R., Merlo, D., De Maria, R., Garaci, E. Thymosin β4 targeting impairs tumorigenic activity of colon cancer stem cells. FASEB J. 24, 4291–4301 (2010). www.fasebj.org

GABAA receptors present higher affinity and modified subunit composition in spinal motor neurons from a genetic model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the selective degeneration of motor neurons in the spinal cord, brainstem and cerebral cortex. In this study we have analysed the electrophysiological properties of GABAA receptors and GABAA alpha1 and alpha2 subunits expression in spinal motor neurons in culture obtained from a genetic model of ALS (G93A) and compared with transgenic wild type SOD1 (SOD1) and their corresponding non transgenic litter mates (Control). Although excitotoxic motor neuron death has been extensively studied in relation to Ca2+‐dependent processes, strong evidence indicates that excitotoxic cell death is also remarkably dependent on Cl− ions and on GABAA receptor activation. In this study we have analysed the electrophysiological properties of GABAA receptors and the expression of GABAAα1 and α2 subunits in cultured motor neurons obtained from a genetic model of amyotrophic lateral sclerosis (G93A) and compared them with transgenic wild‐type Cu,Zn superoxide dismutase and their corresponding non‐transgenic littermates (Control). In all tested motor neurons, the application of γ‐aminobutyric acid (GABA) (0.5–100 μm) evoked an inward current that was reversibly blocked by bicuculline (100 μm), thus indicating that it was mediated by the activation of GABAA receptors. Our results indicate that the current density at high GABA concentrations is similar in control, Cu,Zn superoxide dismutase and G93A motor neurons. However, the dose‐response curve significantly shifted toward lower concentration values in G93A motor neurons and the extent of desensitization also increased in these neurons. Finally, multiplex single‐cell real‐time polymerase chain reaction and immunofluorescence revealed that the amount of GABAAα1 subunit was significantly increased in G93A motor neurons, whereas the levels of α2 subunit were unchanged. These data show that the functionality and expression of GABAA receptors are altered in G93A motor neurons inducing a higher Cl− influx into the cell with a possible consequent neuronal excitotoxicity acceleration.

Systematic review of randomised clinical trials on topical ciclosporin A for the treatment of dry eye disease.

Aims Topical ciclosporin A (CsA) is a therapeutic option for dry eye disease (DED) to control ocular surface inflammation and improve tear function. The aim of this study is to systematically review data from randomised clinical trials (RCTs) evaluating efficacy and safety of topical CsA treatment for DED. Methods Articles published up to December 2012 were identified from Medline, Embase and the Cochrane Controlled Trials Register. A total of 18 RCTs that evaluated the efficacy and safety of different topical CsA formulations for the treatment of DED were selected according to the set criteria. The Jadad score was calculated to assess RCT quality. Results The mean Jadad score of the included RCTs was 2.8±0.6. All CsA formulations proved safe for the treatment of DED. Symptoms improved in 100% (9/9) RCTs, tear function improved in 72% (13/18) RCTs and ocular surface damage was ameliorated in 53% (9/17) RCTs in patients with DED. No improvements with CsA treatment versus control were observed in DED resulting from surgical procedures, contact lens use and thyroid orbitopathy. Statistical comparison of CsA efficacy through a meta-analysis of data was not possible due to a lack of standardised criteria and comparable outcomes among studies. Conclusions Although topical CsA appears to be a safe treatment for DED, evidence emerging from RCTs is limited, and this affects the strength of recommendations to healthcare providers and policymakers for optimal management. Standardised diagnostic criteria to assess the efficacy of topical CsA are recommended to improve the design of future RCTs in DED.

Sublethal Doses of β-Amyloid Peptide Abrogate DNA-dependent Protein Kinase Activity

Background: Accumulation of DNA damage and deficiency in DNA repair may contribute to neuronal loss in Alzheimer disease. Results: Sublethal concentrations of aggregated β-amyloid peptides inhibit DNA-PK kinase activity in PC12 cells. Conclusion: DNA-PK inhibition may contribute to neurodegeneration by impairing DNA repair capability, inducing DNA damage accumulation. Significance: This represents a novel mechanism by which Aβ exerts its neurotoxic effects in Alzheimer disease. Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25–35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1–42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD.

LiCl promotes survival of GABAergic neurons from cerebellum and cerebral cortex: LiCl induces survival of GABAergic neurons.

Previous studies showed that LiCl promotes short-term survival of PC12 cells after NGF or serum deprivation. In the present work, we investigate the survival effect of lithium on cerebellar granule primary cultures. While the total population of cerebellar neurons, mainly granule cells, showed only a short-term survival (about 20 h) in the presence of LiCl, the survival of 65-100% of the GABAergic interneurons originating from cerebellum and cerebral cortex at two different developmental stages was prolonged by 1-2 weeks. Optimal activity was elicited between 5 and 7 mM LiCl. The action of lithium required the presence of serum and persisted also after medium renewal. By direct visual inspection, LiCl promoted neuronal survival without apparently altering the morphological differentiation of the cells. Our studies thereby suggest a means to obtaining enriched populations of GABAergic neurons.

Identification of an ectokinase activity in cerebellar granule primary neuronal cultures.

Abstract: Primary neuronal cultures from 8‐day‐old rat cerebellum were incubated in the presence of exogenously added 16 nM [γ‐32P]ATP. Phosphorylation of a 45‐kDa endogenous protein was detected within 1 min and increased linearly for ∼20 min. Unlike what was seen with [γ‐32P]ATP, in the presence of [32P] orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45‐kDa protein was reduced by 80–90% in the presence of 1 µM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO43−. Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 µM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45‐kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45‐kDa protein by 80–90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45‐kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto‐protein enzymes and intracellular kinases.