Biomedical subjects

Francisco Wandosell

Publications and source records attributed to Francisco Wandosell.

17 recordsLinked to original sources

PubMed2006

BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons.

Olfactory ensheathing cells (OECs) are the main glial cell type that populates mammalian olfactory nerves. These cells have a great capacity to promote the regeneration of axons when transplanted into the injured adult mammalian CNS. However, little is still known about the molecular mechanisms they employ in mediating such a task. Brain-derived neurotrophic factor (BDNF) was identified as a candidate molecule in a genomic study that compared three functionally different OEC populations: Early passage OECs (OEC Ep), Late passage OECs (OEC Lp) and the OEC cell line TEG3 [Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., Diaz-Nido, J., 2006. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J. Neurosci. 26, 5347-5359]. We have here set out to determine the role played by BDNF in the stimulation of axon outgrowth by OECs. We compared the extracellular BDNF levels in the three OEC populations and show that it is produced in significant amounts by the OECs that can stimulate axon regeneration in adult retinal neurons (OEC Ep and TEG3) but it is absent from the extracellular medium of OEC Lp cells which lack this capacity. Blocking BDNF signalling impaired axonal regeneration of adult retinal neurons co-cultured with TEG3 cells and adding BDNF increased the proportion of adult neurons that regenerate their axons on OEC Lp monolayers. Combining BDNF with other extracellular proteins such as Matrix Metalloproteinase 2 (MMP2) further augmented this effect. This study shows that BDNF production by OECs plays a direct role in the promotion of axon regeneration of adult CNS neurons.

Animals↗

PubMed2006

Cross-talk between estrogen receptors and insulin-like growth factor-I receptor in the brain: cellular and molecular mechanisms.

Accumulating evidence suggests that insulin-like growth factor-I (IGF-I) and estradiol interact to regulate neural function. In this review, we focus on the cellular and molecular mechanisms involved in this interaction. The expression of estrogen receptors (ERs) and IGF-I receptor is cross-regulated in the central nervous system and many neurons and astrocytes coexpress both receptors. Furthermore, estradiol activates IGF-I receptor and its intracellular signaling. This effect may involve classical ERs since recent findings suggest that ERalpha may affect IGF-I actions in the brain by a direct interaction with some of the components of IGF-I signaling. In turn, IGF-I may regulate ER transcriptional activity in neuronal cells. In conclusion, ERs appear to be part of the signaling mechanism of IGF-I, and IGF-I receptor part of the mechanism of estradiol signaling in the nervous system.

Animals↗

PubMed2006

Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2.

The molecular mechanisms used by olfactory ensheathing cells (OECs) to promote repair in the damaged adult mammalian CNS remain unknown. Thus, we used microarrays to analyze three OEC populations with different capacities to promote axonal regeneration in cultured rat retinal neurons. Gene expression in "long-term cultured OECs" that do not stimulate adult axonal outgrowth was compared with that of "primary olfactory ensheathing cells" and the immortalized OEC cell line TEG3. In this way, we identified a number of candidate genes that might play a role in promoting adult axonal regeneration. Among these genes, it was striking that both the matrix metalloproteinase 2 (MMP2) and an inhibitor of this protease were represented. The disruption of MMP2 activity in TEG3 cells impaired their capacity to trigger axon regeneration in cultured adult retinal neurons. Furthermore, the MMP2 protein was detected in grafts of OECs that elicited robust axonal regeneration in the injured spinal cord of adult rats in vivo. These data suggest that MMP2 does indeed participate in adult axonal regeneration induced by OECs.

Animals↗

PubMed2006

A clonal cell line from immortalized olfactory ensheathing glia promotes functional recovery in the injured spinal cord.

Immortalized cell lines of olfactory ensheathing glia (OEG) that maintain the proregenerative properties of primary cultures provide an unlimited source of OEG for both basic and applied studies. Indeed, one specific immortalized rat OEG clonal line (TEG3) proved to be as good as primary OEG in promoting neuritogenesis and axon regeneration in culture models. Thus, we examined the capacity of TEG3 to promote axonal repair in an animal model of spinal cord injury, dorsal column crush. TEG3 cells can acquire astrocyte-like or Schwann cell-like morphology depending on the conditions under which they are cultured. In the injured spinal cord, prelabeled TEG3 survived for at least 10 weeks after grafting and they integrated into the spinal cord, adopting Schwann cell-like, astrocyte-like, or intermediate morphologies. In TEG3-transplanted animals, sensory projection axons grow into the lesion site and there was robust sprouting/axonal growth of the corticospinal tract, both into and beyond the lesion site, after crushing of the spinal cord-dorsal columns. TEG3-transplanted animals also recovered sensory and motor function in tape removal and beam walking behavioral tests. These data indicate that certain immortalized cell lines derived from a single cell can maintain the regenerative properties of primary OEG.

Animals↗

PubMed2005

Binding of microtubule-associated protein 1B to LIS1 affects the interaction between dynein and LIS1.

For neuronal migration to occur, the cell must undergo morphological changes that require modifications of the cytoskeleton. Several different MAPs (microtubule-associated proteins) or actin-binding proteins are proposed to be involved in the migration of neurons. Therefore we have specifically analysed how two members of the MAP family, MAP1B and LIS1 (lissencephaly-related protein 1), interact with one another and participate in neuronal migration. Our results indicate that, in hippocampal neurons, MAP1B and LIS1 co-localize, associate and interact with each another. The interaction between these two MAPs is regulated by the phosphorylation of MAP1B. Furthermore, this interaction interferes with the association between LIS1 and the microtubule-dependent molecular motor, dynein. Clearly, the differential binding of these cytoskeletal proteins could regulate the functions attributed to the LIS1-dynein complex, including those related to extension of the neural processes necessary for neuronal migration.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2004

A role of MAP1B in Reelin-dependent neuronal migration.

The signaling cascades governing neuronal migration are believed to link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the control of the dynamic stability of microtubules and in the cross-talk between microtubules and actin filaments. Here we show that Reelin can induce mode I MAP1B phosphorylation, both in vivo and in vitro, through gsk3 and cdk5 activation. Additionally, mDab1 participates in the signaling cascade responsible for mode I MAP1B phosphorylation. Conversely, MAP1B-deficient mice display an abnormal structuring of the nervous system, especially in brain laminated areas, indicating a failure in neuronal migration. Therefore, we propose that Reelin can induce post-translational modifications on MAP1B that could correlate with its function in neuronal migration.

Animals↗

PubMed2004

MAP1B is required for Netrin 1 signaling in neuronal migration and axonal guidance.

BACKGROUND: The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. RESULTS: Here we show that Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. We also show that map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. CONCLUSIONS: These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway.

Animals↗

PubMed2004

Microtubule-associated protein 1B function during normal development, regeneration, and pathological conditions in the nervous system.

Microtubule-associated protein 1B is the first MAP to be expressed during the development of the nervous system. Several different approaches have revealed that MAP1B function is associated with microtubule and actin microfilament polymerization and dynamics. In recent years, the generation of molecular models to inactivate MAP1B function in invertebrates and mammals has sparked some controversy about the real role of MAP1B. Despite discrepancies between some studies, it is clear that MAP1B plays a principal role in the development of the nervous system. In this article, we summarize the evidence for MAP1B function in a wide variety of cellular processes implicated in the proper construction of the nervous system. We also discuss the role of MAP1B in pathological processes.

Animals↗

PubMed2004

Rat transgenic models with a phenotype of intracellular Abeta accumulation in hippocampus and cortex.

In this communication we report the characterization of several transgenic rat lines expressing human AbetaPP carrying the Swedish and Indiana mutations (coded UKUR28), the human presenilin 1 transgene with the 'Finn' mutation (coded UKUR19) and double transgenic rats expressing both transgenes (coded UKUR25). In these Tg rats, the AbetaPP and PS1 transgene expression was largely restricted to the hippocampus and neocortex. The PS1 transgenic rats did not produce visible changes in Abeta immunoreactivity. The AbetaPP transgenic rats (both the single Tg UKUR28, and double Tg UKUR25) generated a phenotype of intra-neuronalbeta accumulation without plaque formation and with no increased immunoreactivity for AbetaPP amino and carboxyl-terminal epitopes. This phenotype was apparent as early as 6 months of age in the transgenic rat lines carrying the human AbetaPP transgene. No senile plaques of aggregated Abeta were observed in any of the transgenic lines generated, up to 24 months of age. The hAbetaPP single homozygous Tg line (UKUR28) showed an increase in ERK2, without changes in glycogen synthase kinase 3 (GSK3) activity. A preliminary protein analysis of the hippocampus of the double transgenic rat (UKUR25) by mass spectrometry showed differences in the protein profile between this transgenic line and controls.

Alzheimer Disease↗

PubMed2003

Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3.

Prion diseases are characterized by neuronal cell death, glial proliferation and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrP) is considered to be responsible for this neurodegeneration. The PrP 106-126, a synthetic peptide obtained from the amyloidogenic region of the PrP, constitutes a model system to study prion-induced neurodegeneration as it retains the ability to trigger cell death in neuronal cultures. In the present study, we show that the addition of this prion peptide to cultured neurons increases the activity of glycogen synthase kinase 3 (GSK-3), which is accompanied by the enhanced phosphorylation of some microtubule-associated proteins including tau and microtubule-associated protein 2. Prion peptide-treated neurons become progressively atrophic, and die ultimately. Both lithium and insulin, which inhibit GSK-3 activity, significantly decrease prion peptide-induced cell death both in primary neuronal cultures and in neuroblastoma cells. Finally, the overexpression of a dominant-negative mutant of GSK-3 in transfected neuroblastoma cells efficiently prevents prion peptide-induced cell death. These results are consistent with the view that the activation of GSK-3 is a crucial mediator of prion peptide-induced neurodegeneration.

Animals↗

PubMed2003

High level of amyloid precursor protein expression in neurite-promoting olfactory ensheathing glia (OEG) and OEG-derived cell lines.

During all the life of a mammal, olfactory ensheathing glia (OEG) permit the entry and navigation of olfactory neuron axons from peripheral to central nervous system (CNS) territory. This physiological characteristic of OEG has been successfully used for promotion of axonal regeneration after CNS injury in animal models. However, cellular and molecular properties responsible for OEG regenerative ability remain to be unveiled. Two approaches may be followed: to carry out genomic or proteomic analysis to detect secreted and/or membrane bound molecules or to examine the expression of molecules previously described as neuritogenic. This is the case of amyloid precursor protein (APP), a neurite-promoting molecule. We have studied the expression of APP by OEG and OEG-derived clonal lines, immortalised with the large T antigen of SV40 (TEG lines). OEG express high levels of APP in vivo and in culture. TEG lines maintained high expression of APP. Western blot analysis showed the presence of high molecular weight forms of APP in OEG, corresponding probably to glycosylated forms and/or to higher expression of the full length APPs. The main APP isoforms present in OEG cultures were APP770 and 751. L-APP isoforms without the exon 15, which are those corresponding with proteoglycan forms, are predominant in glial cells. Our data showed that OEG had three times as much L-APP as astrocytes, which may correlate with OEG neuritogenic capacity. In conclusion APP, a neurite-promoting molecule, is produced by OEG. Its nexin activity, dependent on the Kunitz family of serine protease inhibitors (KPI) domain and/or in combination with its glycosylation level might contribute with other factors to the ability of these cells to foster axonal elongation.

Amyloid beta-Protein Precursor↗

PubMed2003

Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons.

Olfactory bulb ensheathing glia (OEG) have attracted special attention during the last few years because of their unique properties in promoting regeneration of adult mammalian central nervous system (CNS) components. However the molecular and cellular characteristics responsible for this capacity remain to be revealed. Such studies are presently hindered by the lack of a plentiful source of homogenous OEG. Thus the availability of immortalized OEG lines maintaining the regenerative characteristics of the primary cultures would represent an unlimited source of OEG for use not only in biochemical analyses of neuroregenerative mechanisms but also to characterize their regenerative properties in models in culture and in vivo. We have immortalized primary rat OEG using the SV40 large T antigen expressed from a constitutive cellular promotor, and report here the isolation and characterization of clonal lines. These OEG clonal lines were comparable to primary OEG and Schwann cells in the promotion of axonal regeneration of mature rat retinal ganglion neurons (RGN) but, significantly, this culture assay system more closely reflects the in vivo reparative properties of OEG on transected nerves than other assays of neuritogenesis in that it revealed OEG cells to promote the growth of a larger number of long axons than Schwann cells. Using this assay we were able to grade our OEG lines for their neuroregenerative capacity, opening the possibility of identifying molecules with correlative expression levels in these cells. Our preliminary characterization revealed that the expression level of a classical OEG marker, the p75-NGF receptor, does not correlate with neuroregenerative capacity.

Animals↗

PubMed2002

Glycogen synthase kinase-3 is activated in neuronal cells by Galpha12 and Galpha13 by Rho-independent and Rho-dependent mechanisms.

Glycogen synthase kinase-3 (GSK-3) was generally considered a constitutively active enzyme, only regulated by inhibition. Here we describe that GSK-3 is activated by lysophosphatidic acid (LPA) during neurite retraction in rat cerebellar granule neurons. GSK-3 activation correlates with an increase in GSK-3 tyrosine phosphorylation. In addition, LPA induces a GSK-3-mediated hyperphosphorylation of the microtubule-associated protein tau. Inhibition of GSK-3 by lithium partially blocks neurite retraction, indicating that GSK-3 activation is important but not essential for the neurite retraction progress. GSK-3 activation by LPA in cerebellar granule neurons is neither downstream of Galpha(i) nor downstream of Galpha(q)/phospholipase C, suggesting that it is downstream of Galpha12/13. Overexpression of constitutively active Galpha12 (Galpha12QL) and Galpha13 (Galpha13QL) in Neuro2a cells induces upregulation of GSK-3 activity. Furthermore, overexpression of constitutively active RhoA (RhoAV14) also activates GSK-3 However, the activation of GSK-3 by Galpha13 is blocked by coexpression with C3 transferase, whereas C3 does not block GSK-3 activation by Galpha12. Thus, we demonstrate that GSK-3 is activated by both Galpha12 and Galpha13 in neuronal cells. However, GSK-3 activation by Galpha13 is Rho-mediated, whereas GSK-3 activation by Galpha12 is Rho-independent. The results presented here imply the existence of a previously unknown mechanism of GSK-3 activation by Galpha12/13 subunits.

Animals↗

PubMed2002

Highly efficient and specific gene transfer to Purkinje cells in vivo using a herpes simplex virus I amplicon.

The transduction of cerebellar neurons in vivo with herpes simplex virus 1 (HSV-1) amplicon carrying the lacZ gene has been investigated after injection of the vector in the cerebellar cortex, ventricles, and inferior olive of adult rats. Injection into the cerebellar cortex resulted in transduction of Purkinje cells near the needle tract and injection into the ventricles yielded no transduced neurons. In contrast, high transduction efficiency was achieved by vector injection into the inferior olive, resulting in one of three positive Purkinje cells all over the ipsilateral and contralateral cerebellar hemispheres. Because neurons in the deep cerebellar nuclei are also transduced, we suggest that the vector is delivered from the inferior olive to the cerebellar nuclei and then to Purkinje cells by retrograde axonal transport. Expression of the lacZ gene within Purkinje cells was surprisingly persistent and was maintained at the same level for at least 40 days. Importantly, no signs of either toxicity or inflammation were observed in the cerebellum after vector injection, except for the borders of the needle tract where some reactive astrocytes were detected. Indeed, motor coordination of treated animals was entirely normal, as assessed by the rota-rod test. These results demonstrate that HSV-1 amplicon vectors can effect safe and stable transgene expression in Purkinje cells in vivo, raising the possibility of using these vectors for long-term gene therapy of human cerebellar disorders.

Animals↗

PubMed2002

Ephrin-B1 promotes dendrite outgrowth on cerebellar granule neurons.

Ephrins are developmentally regulated molecules that may contribute to axonal pathfinding through their binding to Eph receptor tyrosine kinases. In many cases, ephrins act as negative molecules that stimulate growth cone collapse, although some forms may promote axonal growth. Here, we have addressed the role played by ephrin-B1 during rat postnatal cerebellar development. Ephrin-B1 is expressed by both granule and Purkinje neurons whereas EphB is present in granule neurons in early postnatal cerebellum at a time coincident with axonal and dendrite outgrowth. Stably transfected 3T3 cells overexpressing ephrin-B1 enhance survival and neurite growth from cultured cerebellar granule neurons, an effect that is inhibited by the presence of an excess of a soluble EphB protein. Ephrin-B1-induced neuritogenesis is correlated with an increased expression of certain neuronal-specific microtubule-associated proteins (MAPs). Cerebellar granule neurons plated on stably transfected 3T3 cells overexpressing ephrin-B1 show an up-regulation of the expression of axonal MAPs such as Tau and phosphorylated MAP2C compared with neurons cultured on control 3T3 cells. The level of expression of these axonal MAPs is similar to that found in neurons plated on poly-L-lysine. Interestingly, there is a noteworthy up-regulation of somatodendritic MAPs such as high-molecular-weight MAP2 and mode II-phosphorylated MAP1B in neurons cultured on stably transfected 3T3 cells overexpressing ephrin-B1 compared with neurons plated on either control 3T3 cells or poly-L-lysine. In view of these data, we suggest that ephrin-B1 favors dendritogenesis of granule neurons during cerebellum development.

3T3 Cells↗

PubMed2002

Glycosaminoglycans and beta-amyloid, prion and tau peptides in neurodegenerative diseases.

Protein aggregation into dense filamentous inclusions is a characteristic feature of many etiologically diverse neurodegenerative disorders including Alzheimer's disease (AD), spongiform encephalopathies, and tauopathies. Thus, beta-amyloid peptide (Abeta) accumulates within senile amyloid plaques in AD, protease-resistant prion protein constitutes the amyloid deposits in spongiform encephalopathies and tau protein gives rise to neurofibrillary tangles (NFT) both in AD and in tauopathies. Curiously, these abnormal protein inclusions contain, in addition to their major peptide components, some associated sulfated glycosaminoglycans (sGAG). Here we discuss the proposal that the binding of sGAG to aggregate-forming peptides may modify the pathogenic process depending on their subcellular localization.

Alzheimer Disease↗

PubMed2002

Olfactory Ensheathing Glia: Drivers of Axonal Regeneration in the Central Nervous System?

Olfactory ensheathing glia (OEG) accompany olfactory growing axons in their entry to the adult mammalian central nervous system (CNS). Due to this special characteristic, considerable attention has been focused on the possibility of using OEG for CNS regeneration. OEG present a large heterogeneity in culture with respect to their cellular morphology and expressed molecules. The specific characteristics of OEG responsible for their regenerative properties have to be defined. These properties probably result from the combination of several factors: molecular composition of the membrane (expressing adhesion molecules as PSA-NCAM, L1 and/or others) combined with their ability to reduce glial scarring and to accompany new growing axons into the host CNS. Their capacity to produce some neurotrophic factors might also account for their ability to produce CNS regeneration.

Journal Article↗