Liraglutide is neurotrophic and neuroprotective in neuronal cultures and mitigates mild traumatic brain injury in mice

Traumatic brain injury (TBI), a brain dysfunction for which there is no present effective treatment, is often caused by a concussive impact to the head and affects an estimated 1.7 million Americans annually. Our laboratory previously demonstrated that exendin‐4, a long‐lasting glucagon‐like peptide 1 receptor (GLP‐1R) agonist, has neuroprotective effects in cellular and animal models of TBI. Here, we demonstrate neurotrophic and neuroprotective effects of a different GLP‐1R agonist, liraglutide, in neuronal cultures and a mouse model of mild TBI (mTBI). Liraglutide promoted dose‐dependent proliferation in SH‐SY5Y cells and in a GLP‐1R over‐expressing cell line at reduced concentrations. Pre‐treatment with liraglutide rescued neuronal cells from oxidative stress‐ and glutamate excitotoxicity‐induced cell death. Liraglutide produced neurotrophic and neuroprotective effects similar to those of exendin‐4 in vitro. The cAMP/PKA/pCREB pathway appears to play an important role in this neuroprotective activity of liraglutide. Furthermore, our findings in cell culture were well‐translated in a weight drop mTBI mouse model. Post‐treatment with a clinically relevant dose of liraglutide for 7 days in mice ameliorated memory impairments caused by mTBI when evaluated 7 and 30 days post trauma. These data cross‐validate former studies of exendin‐4 and suggest that liraglutide holds therapeutic potential for the treatment of mTBI.

     

Genome modifications in plant cells by custom-made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.     

The stimulatory effect of cannabinoids on calcium uptake is mediated by Gs GTP-binding proteins and cAMP formation

Cannabinoids are neurodepressive drugs that convey their cellular action through G(i/o) GTP-binding proteins which reduce cAMP formation and Ca(2+) influx. However, a growing body of evidence indicates that the stimulatory effects of cannabinoids include the elevation in cAMP and cytosolic Ca(2+) concentration. The present study expands our previous findings and demonstrates that, in N18TG2 neuroblastoma cells, the cannabinoid agonist desacetyllevonantradol (DALN) stimulates both cAMP formation and Ca(2+) uptake. The stimulatory effect of DALN on cAMP formation was not eliminated by blocking Ca(2+) entry to the cells, while its stimulatory effect on Ca(2+) uptake was abolished by blocking cAMP-dependent protein kinase. Furthermore, elevating cAMP by forskolin stimulated calcium uptake, while elevating the intracellular Ca(2+) concentration by ionomycin or KCl failed to stimulate cAMP formation. These findings suggest that cAMP production precedes the influx of Ca(2+) in the cannabinoid stimulatory cascade. The stimulatory effect of DALN on calcium uptake resisted pertussis toxin treatment, and was completely blocked by introducing anti-G(s) antibodies into the cells, indicating that the stimulatory activity of cannabinoids is mediated by G(s) GTP-binding proteins. The relevance of the cellular stimulatory activity of DALN to the pharmacological profile of cannabinoid drugs is discussed.     

Intracellular Ca2+ regulates the phosphorylation and the dephosphorylation of ciliary proteins via the NO pathway

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO synthase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.     

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.     

Parents’ posthumous use of daughter’s ovarian tissue: Ethical dimensions

In recent years, progress in cancer treatment has greatly increased the chances of recovery. Yet, treatment may have irreversible effects on patients’ fertility. In order to protect future fertility, preservation of ovarian tissue may be offered today even to very young girls, involving a surgical procedure that may be performed by minimally invasive laparoscopy, under general anesthesia. However, in the tragic event of a girl’s death, questions may arise regarding the possible use of the preserved ovarian tissue by her parents. Should posthumous reproductive use of ovarian tissue without the girl’s prior consent (due to her young age) be considered a violation of her rights? On the other hand, can it be argued that it is in the interest of a child who died young to leave a genetic trace through posthumous reproduction, because genetic continuity is in the interest of every human being? After presenting the relevant clinical facts, we explore the ethical dimensions of this possible practice through an analysis of the interests of the deceased, her parents, and the child that may be born posthumously.