María Teresa Moreno-Flores

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.

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.

Up-regulation of Eph tyrosine kinase receptors after excitotoxic injury in adult hippocampus.

The molecular mechanisms underlying the response to injury in the central nervous system are incompletely understood. Many cell activation systems may be involved. Tyrosine kinase receptors and their ligands play key roles in cell activation throughout life. The Eph family of tyrosine kinase receptors/ ligands are developmentally regulated and have been implicated in neural pathfinding. However, nothing is known about their role in the adult brain. We have used a model of central nervous system lesion in the rat, in which intraventricular injection of kainate was performed. This produced neuronal death in the CA3-CA4 fields and glial activation in the hippocampus. Highly degenerate primers, corresponding to the catalytic domain of the tyrosine kinase family, were used for reverse transcription-polymerase chain reaction of pooled RNA extracted from injured hippocampi. The amplified products were cloned and 100 clones (arbitrarily named TK1-TK100) were examined and inserts sequenced. We obtained four clones containing inserts which belong to the Eph receptor family. Two of these inserts (TK17 and TK63) were EphA4 and the other were EphB2 (TK25) and EphA5 (TK23). We performed in situ hybridization, and we found our clones to be present in all fields of the hippocampus, their expression being mainly neuronal. Three days after lesion, prominent expression appeared in CA1 as compared to the same field in the non-treated contralateral hippocampus. We performed northern blot analysis for quantification, and found that, three days after injury, the values decreased to 33 +/- 4%, 33 +/- 1% and 46 +/- 1% of control values for TK63 (EphA4), TK25 (EphB2) and TK23 (EphA5), respectively. Neuronal death in CA3-CA4 might account for this fact. Later, five days post-injury, the expression increased to 63 +/- 3%, 71 +/- 1% and 111 +/- 5% of control values, respectively. This increase was due to an up-regulation of these genes in the hippocampal neurons that survive after the injury, as indicated by in situ hybridization.

Semaphorin 3C preserves survival and induces neuritogenesis of cerebellar granule neurons in culture

Semaphorins (sema) constitute a family of molecules sharing a common extracellular domain (semaphorin domain). This family includes several types of secreted and membrane‐associated molecules that are grouped into eight subclasses (subclasses 1–7 and viral semaphorins). Subclass 3 semaphorins are secreted molecules involved in axonal guidance, mainly through repulsive gradients and induction of growth cone collapse. More recently sema 3 molecules have been identified as positive factors in dependence of the type of neurons. Besides their axonal guidance function, some semaphorins have been implicated in apoptosis and survival. We investigated the effect of sema3C on survival and neurite outgrowth of rat cerebellar granule neurons (CGNs) in culture. 3T3 cells were stably transfected with sema3C. Several clonal lines were established and tested for their neuritogenic activity and one, S3C‐8, was selected for the bulk of experiments. S3C‐8 was co‐cultured with CGNs. Sema3C enhanced CGN viability as assessed in co‐cultures of CGNs with monolayers of S3C‐8 in comparison with co‐cultures of CGNs with control mock‐transfected 3T3 cells. Moreover sema3C induced neuritogenesis of cultured CGNs, which express neuropilin‐1 and ‐2. S3C‐8 cells, overexpressing sema3C, were significantly more neuritogenic for CGN than poly l‐lysine (PLL), a positive substrate for CGNs, as assessed by the measurement of the length of neurites and confirmed by Tau expression along the time of culture. CGNs co‐cultured with S3C‐8, showed up‐regulation of the expression of axonal microtubule‐associated proteins (MAPs) such as Tau, phosphorylated MAP2C and mode I‐phosphorylated MAP1B compared with neurons cultured on control 3T3 cells. We also found increased expression of a specific marker of neuronal cell bodies and dendrites, high molecular weight MAP2 (HMW‐MAP2). Interestingly, there was no accompanying up‐regulation of a marker enriched within the neuronal somatodendritic domain, mode II‐phosphorylated MAP1B. These data support the idea that secreted sema3C favors survival and neuritogenesis of cultured CGNs.

Prevention of Senescence Progression in Reversibly Immortalized Human Ensheathing Glia Permits Their Survival After Deimmortalization

Reversible immortalization holds great potential for primary tissue expansion to develop cell-based therapies as well as for basic research. Human olfactory ensheathing glia (hOEG) are promising candidates for treating spinal cord injury and for studying extrinsic neuroregenerative mechanisms. We used lentivectors with Cre/loxP technology to achieve reversible gene transfer of BMI1, SV40 large T antigen (TAg), a short hairpin RNA against p53 (shp53), and the catalytic subunit of telomerase (TERT) in primary cultures of hOEG from human donor cadaver olfactory bulbs. Several combinations of these genes were able to immortalize hOEG, conserving their antigenic markers and neuroregenerative properties but only those transduced by BMI1/TERT did not accumulate karyotypic alterations or increase senescence marker levels. Strikingly, these were also the only cells which continued to proliferate after transgene removal by Cre recombinase delivery, whereas hOEG immortalized by shp53 or TAg in combination with TERT entered into growth arrest and died. These data support the idea that immortalization and halting senescent changes are separate processes; hOEG immortalized by BMI1/TERT can revert back to their former primary cell replicative state when deimmortalized, whereas those transduced by the other combinations depend on the presence of these transgenes to maintain their aberrant proliferative state.Reversible immortalization holds great potential for primary tissue expansion to develop cell-based therapies as well as for basic research. Human olfactory ensheathing glia (hOEG) are promising candidates for treating spinal cord injury and for studying extrinsic neuroregenerative mechanisms. We used lentivectors with Cre/loxP technology to achieve reversible gene transfer of BMI1, SV40 large T antigen (TAg), a short hairpin RNA against p53 (shp53), and the catalytic subunit of telomerase (TERT) in primary cultures of hOEG from human donor cadaver olfactory bulbs. Several combinations of these genes were able to immortalize hOEG, conserving their antigenic markers and neuroregenerative properties but only those transduced by BMI1/TERT did not accumulate karyotypic alterations or increase senescence marker levels. Strikingly, these were also the only cells which continued to proliferate after transgene removal by Cre recombinase delivery, whereas hOEG immortalized by shp53 or TAg in combination with TERT entered into growth arrest and died. These data support the idea that immortalization and halting senescent changes are separate processes; hOEG immortalized by BMI1/TERT can revert back to their former primary cell replicative state when deimmortalized, whereas those transduced by the other combinations depend on the presence of these transgenes to maintain their aberrant proliferative state.

Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons

Although human olfactory mucosa derived cells (OMC) have been used in animal models and clinical trials with CNS repair purposes, the exact identity of these cells in culture with respect to their tissue of origin is not fully understood and their neuroregenerative capacity in vitro has not yet been demonstrated. In this study we have compared human OMC with human ensheathing glia from olfactory bulb (OB) and human fibroblasts from skin and lung. Our results indicate that these different cultured cell types exhibit considerable overlap of antigenic markers such that it is presently not possible to distinguish them immunocytochemically. However, in rat retinal ganglion neuron coculture assays the axonal regenerative activity of OMC and OB ensheathing glia was dramatically higher than that exhibited by all fibroblast samples, confirming neuroregenerative activity as a unique property shared by cultured cells derived from the human olfactory system.

The Quest to Repair the Damaged Spinal Cord

Spinal cord injuries devastate the lives of those affected. Normally, acute injury leads to chronic injury in the spinal cord, although this has a variable impact on normal sensory and motor functions. Currently the only drug used to treat acute spinal cord injury is methyl-prednisolone, administered in order to prevent secondary inflammatory neural damage. Thus, it is time that alternative and complementary pharmacological, cell and gene therapies be developed. In order to achieve this, several approaches to stimulate spinal cord repair must be considered. Indeed, the main lines of research that have been established in different animal models of spinal cord regeneration are now beginning to produce encouraging results. Several patents have been derived from these studies and hopefully, they will lead to the development of new treatments for human spinal cord injuries. Here is presented a review of the main patents that have been generated by this research, and that can be classified as: - Patents involving the use of different factors that promote axonal regeneration. - Patents aimed at overcoming the activity of glial scar inhibitory molecules that hinder axonal regeneration. These approaches can be further subdivided into those that block Nogo and other myelin components, and those that involve the use of chondroitinase against glial scar chondroitin sulphate proteoglycans. - Patents concerning glial cell therapy, in which glial cells are used to mediate axonal repair in the spinal cord (Schwann cells, olfactory ensheathing cells or astrocytes).

Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single-cell tracking and traction force microscopy.

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but instead a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion, and crossover during cell–cell and cell–matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo receptor complex and that their migration is blocked by myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over myelin. Our data relate the decrease of traction force of OEC with lower migratory capacity over myelin, which correlates with changes in the F-actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo receptor inhibitor NEP1-40.

Increased migration of olfactory ensheathing cells secreting the Nogo receptor ectodomain over inhibitory substrates and lesioned spinal cord

Olfactory ensheathing cell (OEC) transplantation emerged some years ago as a promising therapeutic strategy to repair injured spinal cord. However, inhibitory molecules are present for long periods of time in lesioned spinal cord, inhibiting both OEC migration and axonal regrowth. Two families of these molecules, chondroitin sulphate proteoglycans (CSPG) and myelin-derived inhibitors (MAIs), are able to trigger inhibitory responses in lesioned axons. Mounting evidence suggests that OEC migration is inhibited by myelin. Here we demonstrate that OEC migration is largely inhibited by CSPGs and that inhibition can be overcome by the bacterial enzyme Chondroitinase ABC. In parallel, we have generated a stable OEC cell line overexpressing the Nogo receptor (NgR) ectodomain to reduce MAI-associated inhibition in vitro and in vivo. Results indicate that engineered cells migrate longer distances than unmodified OECs over myelin or oligodendrocyte-myelin glycoprotein (OMgp)-coated substrates. In addition, they also show improved migration in lesioned spinal cord. Our results provide new insights toward the improvement of the mechanisms of action and optimization of OEC-based cell therapy for spinal cord lesion.

Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration.

Olfactory ensheathing glia (OEG) cells are known to facilitate repair following axotomy of adult neurons, although the molecular mechanisms involved are not fully understood. We previously identified plasminogen activator inhibitor‐1 (PAI‐1), proteinase‐activated receptor‐1 (PAR‐1), and thrombomodulin (TM) as candidates to regulate rat OEG‐dependent axonal regeneration. In this study, we have validated the involvement of these proteins in promoting axonal regeneration by immortalized human OEGs. We studied the effect of silencing these proteins in OEGs on their capacity to promote the regeneration of severed adult retinal ganglion cells (RGCs) axons. Our results support the role of glial PAI‐1 as a downstream effector of PAR‐1 in promoting axon regeneration. In contrast, we found that TM inhibits OEG induced‐axonal regeneration. We also assessed the signaling pathways downstream of PAR‐1 that might modulate PAI‐1 expression, observing that specifically inhibiting Gαi, Rho kinase, or PLC and PKC downregulated the expression of PAI‐1 in OEGs, with a concomitant reduction in OEG‐dependent axon regeneration in adult RGCs. Our findings support an important role for the thrombin system in regulating adult axonal regeneration by OEGs.