Elisa Tamariz

Semaphorins 3A, 3C, and 3F in mesencephalic dopaminergic axon pathfinding

By analyzing the mechanisms that govern dopaminergic axon pathfinding from the midbrain to the striatum in embryonic rat brains, we identified neuroepithelial regions that exert chemotropic effects on mesencephalic dopaminergic axons. Explants from the pretectum and the striatum showed an attractive effect, whereas those from the midhindbrain boundary, the dorsal thalamus, and the ventral thalamus had no effect. Expression of semaphorin (Sema) 3C and Sema3F in the pretectum and of Sema3A in the striatum suggested a role for these axon guidance molecules in dopaminergic axon pathfinding. When expressed in HEK293 cell aggregates, Sema3C had an attractive effect and enhanced axon growth, Sema3A enhanced axon growth, and Sema3F had a repulsive effect on dopaminergic axons. Antineuropilin‐1 and antineuropilin‐2 antibodies reduced attraction by the pretectum, whereas attraction by the striatum was not affected by the presence of antineuropilin‐1 antibodies. Moreover, neuropilin‐1‐ and neuropilin‐2‐soluble Fc chimeras reduced the attraction by the pretectum. These results suggest that semaphorins may help to establish the dopaminergic projection from the midbrain to the striatum during embryonic development. J. Comp. Neurol. 506:387–397, 2008.

LIM-Homeobox Gene Lhx5 Is Required for Normal Development of Cajal–Retzius Cells

Cajal–Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.

Axon responses of embryonic stem cell-derived dopaminergic neurons to Semaphorins 3A and 3C

Class 3 Semaphorins are a subfamily of chemotropic molecules implicated in the projection of dopaminergic neurons from the ventral mesencephalon and in the formation of the nigrostriatal pathway (NSP) during embryonic development. In humans, loss of mesencephalic dopaminergic neurons leads to Parkinsons disease (PD). Cell replacement therapy with dopaminergic neurons generated from embryonic stem cells (ES‐TH+) is being actively explored in models of PD. Among several requisites for this approach to work are adequate reconstruction of the NSP and correct innervation of normal striatal targets by dopaminergic axons. In this work, we characterized the response of ES‐TH+ neurons to semaphorins 3A, 3C, and 3F and compared it with that of tyrosine hidroxylase‐positive neurons (TH+) obtained from embryonic ventral mesencephalon (VM‐TH+). We observed that similar proportions of ES‐TH+ and VM‐TH+ neurons express semaphorin receptors neuropilins 1 and 2. Furthermore, the axons of both populations responded very similarly to semaphorin exposure: semaphorin 3A increased axon length, and semaphorin 3C attracted axons and increased their length. These effects were mediated by neuropilins, insofar as addition of blocking antibodies against these proteins reduced the effects on axonal growth and attraction, and only TH+ axons expressing neuropilins responded to the semaphorins analyzed. The observations reported here show phenotypic similarities between VM‐TH+ and ES‐TH+ neurons and suggest that semaphorins 3A and 3C could be employed to guide axons of grafted ES‐TH+ in therapeutic protocols for PD.

Delivery of chemotropic proteins and improvement of dopaminergic neuron outgrowth through a thixotropic hybrid nano-gel

Chemotropic proteins guide neuronal projections to their final target during embryo development and are useful to guide axons of neurons used in transplantation therapies. Site-specific delivery of the proteins however is needed for their application in the brain to avoid degradation and pleiotropic affects. In the present study we report the use of Poly (ethylene glycol)-Silica (PEG-Si) nanocomposite gel with thixotropic properties that make it injectable and suitable for delivery of the chemotropic protein semaphorin 3A. PEG-Si gel forms a functional gradient of semaphorin that enhances axon outgrowth of dopaminergic neurons from rat embryos or differentiated from stem cells in culture. It is not cytotoxic and its properties allowed its injection into the striatum without inflammatory response in the short term. Long term implantation however led to an increase in macrophages and glial cells. The inflammatory response could have resulted from non-degraded silica particles, as observed in biodegradation assays.

Recovery From Experimental Parkinsonism by Semaphorin-guided Axonal Growth of Grafted Dopamine Neurons

Cell therapy in animal models of Parkinsons disease (PD) is effective after intrastriatal grafting of dopamine (DA) neurons, whereas intranigral transplantation of dopaminergic cells does not cause consistent behavioral recovery. One strategy to promote axonal growth of dopaminergic neurons from the substantia nigra (SN) to the striatum is degradation of inhibitory components such as chondroitin sulphate proteoglycans (CSPG). An alternative is the guidance of DA axons by chemotropic agents. Semaphorins 3A and 3C enhance axonal growth of embryonic stem (ES) cell-derived dopaminergic neurons in vitro, while Semaphorin 3C also attracts them. We asked whether intranigral transplantation of DA neurons, combined with either degradation of CSPG or with grafts of Semaphorin 3-expressing cells, towards the striatum, is effective in establishing a new nigrostriatal dopaminergic pathway in rats with unilateral depletion of DA neurons. We found depolarization-induced DA release in dorsal striatum, DA axonal projections from SN to striatum, and concomitant behavioral improvement in Semaphorin 3-treated animals. These effects were absent in animals that received intranigral transplants combined with Chondroitinase ABC treatment, although partial degradation of CSPG was observed. These results are evidence that Semaphorin 3-directed long-distance axonal growth of dopaminergic neurons, resulting in behavioral improvement, is possible in adult diseased brains.

The discovery of the growth cone and its influence on the study of axon guidance

For over a century, there has been a great deal of interest in understanding how neural connectivity is established during development and regeneration. Interest in the latter arises from the possibility that knowledge of this process can be used to re-establish lost connections after lesion or neurodegeneration. At the end of the XIX century, Santiago Ramón y Cajal discovered that the distal tip of growing axons contained a structure that he called the growth cone. He proposed that this structure enabled the axon’s oriented growth in response to attractants, now known as chemotropic molecules. He further proposed that the physical properties of the surrounding tissues could influence the growth cone and the direction of growth. This seminal discovery afforded a plausible explanation for directed axonal growth and has led to the discovery of axon guidance mechanisms that include diffusible attractants and repellants and guidance cues anchored to cell membranes or extracellular matrix. In this review the major events in the development of this field are discussed.

Shock Waves and DNA-Cationic Lipid Assemblies: A Synergistic Approach to Express Exogenous Genes in Human Cells

Cationic lipid/DNA complexes (lipoplexes) represent a powerful tool for cell transfection; however, their use is still limited by important concerns, including toxicity and poor internalization into deep tissues. In this work, we investigated the use of shock wave-induced acoustic cavitation in vitro for the transfection of lipoplexes in human embryo kidney 293 cells. We selected shock waves with the ability to internalize 10-kDa fluorescein isothiocyanate-dextran into cells while maintaining survival rates above 50%. Cell transfection was tested using the green fluorescent protein-encoding plasmid pCX::GFPGPI2. Confocal microscopy and fluorescence-assisted cell sorting analyses revealed successful transfection after treatments ranging from 1 to 3 min using 60 to 180 shock waves at peak amplitudes of 12.3 ± 1.5 MPa. Interestingly, the combination of shock waves and lipoplexes induced a 3.1- and 3.8-fold increase in the expression of the reporter gene compared with the use of lipoplexes or shock waves alone, respectively. These results indicate that cationic DNA assembly and shock waves act in a synergistic manner to promote transfection of human cells, revealing a potential approach for non-invasive site-specific gene therapy.

Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cell line and in primary cultures of bovine myoblasts

Ghrelin is an acylated hormone that influences food intake, energy metabolism and reproduction, among others. Ghrelin may also stimulate proliferating myoblast cell differentiation and multinucleated myotube fusion. The aim of this work was to assess the effect of human ghrelin (hGHRL) and human ghrelin fragment 1-18 (hGHRL1-18) on myoblast differentiation by means of mRNA expression and protein level. Two types of cells were tested, the cell line i28 obtained from mouse skeletal muscle and primary cultures of bovine myoblasts. Both ghrelin and its N-terminal fragment hGHRL1-18 were used at concentrations of 0, 0.01, 0.1, 1, 10 and 100 nm. Treatments were applied to pre-confluent cultures and were maintained for 4 days. We determined that between 0.1 and 100 nm, hGHRL and hGRHL1-18 had similar effects on myogenic differentiation of i28 cells (p < 0.01). On the other hand, only the higher concentrations (10 and 100 nm) of hGHRL stimulated bovine myoblast differentiation. These results could be attributed to the presence, in both i28 cells and in bovine myoblasts, of the mRNA for GHS-R1a and CD36 receptors. The use of ghrelin in livestock production is still questionable because of the limited effects shown in this study, and additional research is needed in this field.

Biodegradation of Medical Purpose Polymeric Materials and Their Impact on Biocompatibility

The use of polymeric materials in medical devices and pharmaceutical applications has been extended in the last decades. Biodegradable implantable polymers for tissue engineering and drug release have the advantage to avoid a permanent and chronic immune response, and to avoid removal surgery; moreover the versatility of polymeric materials aloud the de‐ sign of specific biodegradable characteristics to control drug release, to develop resorbable devices, and to improve cell integration.

Ascending midbrain dopaminergic axons require descending GAD65 axon fascicles for normal pathfinding

The Nigrostriatal pathway (NSP) is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.