Role of the CaSR during development of cranial sensory neurons

Virtually all studies on the molecular regulation of axonal growth have been carried out on axotomised neurons that regenerate axons in culture and are dependent on neurotrophic factors not only for survival but also for axonal growth. The technical difficulty of obtaining and culturing early, newly differentiated neurons that initiate axonal growth for the first time in culture has meant that developmentally relevant de novo axonal growth has been almost ignored. Numerous studies have shown that axonal regeneration from embryonic and postnatal neurotrophic factor-dependent neurons is regulated by a variety of signalling pathways that influence the assembly and stability of key components of the cytoskeleton in growth cones. Depending on neuron type, these can include MEK, PI3 kinase, GSK3, NF-kB and calcium signalling. The extent to which these pathways are important for de novo axonal growth, and how their importance changes as axonal growth becomes responsive to neurotrophic factors during early development is not known. I have used the experimentally advantageous placode-derived sensory neurons of the chicken embryo to study the molecular basis of de novo, neurotrophic factor independent axonal growth and to compare this neurotrophic factor-dependent axonal growth at later stages of development. These neurons can be dissected from the earliest stages in their development and cultures can be established in which neurons extend axons for the first time. Previous work has shown that at this stage of development, axonal growth is independent of neurotrophic factors and its rate is correlated with target distance. For example, neurons of the nodose ganglion have the most distant targets, the fastest axonal growth rate and survive longest before becoming dependent on the neurotrophic factor BDNF for survival, whereas neurons of the vestibular ganglion have the nearest targets, slowest axonal growth rate and survive for the shortest time before acquiring BDNF dependence. My initial studies focused on the role of the extracellular calcium-sensing receptor (CaSR), a G protein coupled receptor that has recently been shown to regulate axonal growth from sympathetic neurons during the stage when neurons are innervating their targets. I found that during the stage of development when the earliest axons of placode-derived sensory neurons are growing to their targets, nodose ganglion neurons (which have the fastest axon growth rates) express the highest levels of the CaSR, and vestibular neurons (which have the slowest axon growth rates) express the lowest levels of the CaSR. Experimental manipulation of CaSR activation in cultured nodose neurons at the stage in development when their axons are normally growing to their targets markedly affects axon growth rate (enhancing activation increases growth rate whereas reducing activation has the opposite effect). In contrast, similar manipulations of CaSR activation in cultured vestibular neurons have no effects on axonal growth rate. These findings suggest that the CaSR plays an important role in the regulation of de novo axonal growth rate. Manipulating CaSR activation in older, BDNF-dependent nodose neurons at the stage in development when these neurons are innervating their targets also demonstrated a role for the CaSR in promoting axonal growth at this stage. Having demonstrated a role for the CaSR in promoting axonal growth at these two successive stages of development, I then characterised the intracellular signalling pathways that mediate the effects of the CaSR on axonal growth at these stages. Using Western blot analysis and pharmacological inhibitors of PI3-kinase, GSK3 and MEK1/2, I discovered a clear switch in the signalling pathways that are involved in promoting axon elongation between early BDNF-independent stages of de novo axon growth to later BDNF-dependent stages of axon growth. Whereas PI3-kinase signalling plays a pivotal role in transducing CaSR-enhanced, neurotrophin-independent axon growth, GSK3 signalling plays a major role in transducing the growth enhancing effects of CaSR activation on BDNF-promoted axonal growth from older BDNF-dependent nodose neurons. My findings suggest that PI3-Kinase and GSK3 signalling are not linked in developing nodose neurons, but are regulated independently of each other. Furthermore, Western analysis also suggests the operation of a novel activation mechanism of GSK3 in axon growth in BDNF-dependent nodose neurons that involves tyrosine phosphorylation of GSK3 rather than serine phosphoryation following CaSR activation. In all, my studies have revealed several novel and unexpected aspects of regulation of axonal growth by the CaSR during the early stages of neuronal development

Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor

BACKGROUND: The constitutive androstane receptor (CAR) plays a key role in the control of drug metabolism and transport by mediating the phenobarbital-type induction of many phase I and II drug metabolizing enzymes and drug transporters. RESULTS: We identified transcripts generated by four different alternative splicing events in the human CAR gene. Two of the corresponding ligand binding domain isoforms demonstrated novel functional properties: First, CAR(SV3), which is encoded by a transcript containing an lengthened exon 7, differentially transactivated target gene promoters. Second, CAR(SV2), which results from the use of an alternative 3' splice site lengthening exon 8, showed ligand-dependent instead of constitutive interaction with coactivators. Furthermore, alternatively spliced transcripts demonstrated a tissue-specific expression pattern. In most tissues, only transcripts generated by alternative splicing within exon 9 were expressed. The encoded variant demonstrated a loss-of-function phenotype. Correct splicing of exon 8 to exon 9 is restricted to only a few tissues, among them liver and small intestine for which CAR function has been demonstrated, and is associated with the induction of CAR expression during differentiation of intestinal cells. CONCLUSION: Due to their specific activities, CAR variant proteins SV2 and SV3 may modulate the activity of reference CAR(SV1). Furthermore, we propose that transcriptional activation and regulation of splicing of exon 9 may be coupled to ensure appropriate tissue- and differentiation state-specific expression of transcripts encoding functional CAR protein. Altogether, alternative splicing seems to be of utmost importance for the regulation of CAR expression and function

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/ methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LCMS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization

Disrupted neuronal trafficking in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset neurodegenerative disease caused by degeneration of motor neurons in the brain and spinal cord leading to muscle weakness. Median survival after symptom onset in patients is 3–5 years and no effective therapies are available to treat or cure ALS. Therefore, further insight is needed into the molecular and cellular mechanisms that cause motor neuron degeneration and ALS. Different ALS disease mechanisms have been identified and recent evidence supports a prominent role for defects in intracellular transport. Several different ALS-causing gene mutations (e.g., in FUS, TDP-43, or C9ORF72) have been linked to defects in neuronal trafficking and a picture is emerging on how these defects may trigger disease. This review summarizes and discusses these recent findings. An overview of how endosomal and receptor trafficking are affected in ALS is followed by a description on dysregulated autophagy and ER/Golgi trafficking. Finally, changes in axonal transport and nucleocytoplasmic transport are discussed. Further insight into intracellular trafficking defects in ALS will deepen our understanding of ALS pathogenesis and will provide novel avenues for therapeutic intervention

Navigation rules for vessels and neurons: cooperative signaling between VEGF and neural guidance cues.

International audienceMany organs, such as lungs, nerves, blood and lymphatic vessels, consist of complex networks that carry flows of information, gases, and nutrients within the body. The morphogenetic patterning that generates these organs involves the coordinated action of developmental signaling cues that guide migration of specialized cells. Precision guidance of endothelial tip cells by vascular endothelial growth factors (VEGFs) is well established, and several families of neural guidance molecules have been identified to exert guidance function in both the nervous and the vascular systems. This review discusses recent advances in VEGF research, focusing on the emerging role of neural guidance molecules as key regulators of VEGF function during vascular development and on the novel role of VEGFs in neural cell migration and nerve wiring