Search

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.

The possible link between tau phosphorylation and tau assembly in these neurodegenerative diseases known as tauopathies is described. Additionally, this link is supported by anin vitro experiment showing the higher capacity of phosphotau to assemble in some specific conditions; and, by a recently reported experiment using a tau transgenic mouse model.

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.

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.

Tau protein hyperphosphorylation and aggregation into neurofibrillary tangles are characteristic features of several neurodegenerative disorders referred to as tauopathies. Among them, frontotemporal dementia and Parkinsonism linked to chromosome 17 may be caused by dominant missense mutations in the tau gene. Transgenic mice expressing mutant tau serve as valid model systems to study the ethiopathogenesis of these diseases and assay possible therapeutic interventions. Here we report that chronic lithium treatment of a transgenic mouse strain expressing human tau with three missense mutations results in decreased glycogen synthase kinase-3-dependent-tau phosphorylation and a reduction of filamentous aggregates. These data indicate that lithium, presumably acting through the inhibition of glycogen synthase kinase 3, may be useful to curb neurodegeneration in tauopathies.

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.

The tauopathies, which include Alzheimer‘s disease (AD) and frontotemporal dementias, are a group of neurodegenerative disorders characterized by filamentous Tau aggregates. That Tau dysfunction can cause neurodegeneration is indicated by pathogenic tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To investigate how Tau alterations provoke neurodegeneration we generated transgenic mice expressing human Tau with four tubulin-binding repeats (increased by FTDP-17 splice donor mutations) and three FTDP-17 missense mutations: G272V, P301L, and R406W. Ultrastructural analysis of mutant Tau-positive neurons revealed a pretangle appearance, with filaments of Tau and increased numbers of lysosomes displaying aberrant morphology similar to those found in AD. Lysosomal alterations were confirmed by activity analysis of the marker acid phosphatase, which was increased in both transgenic mice and transfected neuroblastoma cells. Our results show that Tau modifications can provoke lysosomal aberrations and suggest that this may be a cause of neurodegeneration in tauopathies.

It has been extensively described that neuronal differentiation involves the signalling through neurotrophin receptors to a Ras-dependent mitogen-activated protein kinase (MAPK) cascade. However, signalling pathways from other neuritogenic factors have not been well established. It has been reported that cAMP may activate protein kinase (PKA), and it has been shown that PKA-mediated stimulation of MAPK pathway regulates not only neuritogenesis but also survival. However, extracellular regulated kinases (ERKs) mediated pathways are not sufficient to explain all the processes which occur in neuronal differentiation. Our present data show that: in cAMP-mediated neuritogenesis, using the SH-SY5Y human neuroblastoma cell line, there exists a link between the activation of PKA and stimulation of phosphatidylinositol 3-kinase (PI3K). Both kinase activities are essential to the initial elongation steps. Surprisingly, this neuritogenic process appears to be independent of ERKs. While the activity of PI3K is essential for elongation and maintenance of neurites, its inhibition causes retraction. In this neurite retraction process, GSK3 is activated. Using both a pharmacological approach and gene transfer of a dominant negative form of GSK3, we conclude that this induced retraction is a GSK3-dependent process which in turn appears to be a common target for transduction pathways involved in lysophosphatidic acid-mediated and PI3K-mediated neurite retraction.

The recent finding that several point mutations in the gene encoding for the microtubule-binding protein tau correlate with neurological disorders has heightened interest in the mechanisms of destabilization of this protein. In this study the functional consequences of the tau mutation R406W on the interaction of the protein with microtubules have been analyzed. Mutated tau is less phosphorylated than its normal counterpart at serines 396 and 404. Furthermore, the phosphorylated mutant protein is unable to bind to microtubules, and, as a consequence, microtubules assembled after transient nocodazole treatment in the presence of this tau variant contain only unmodified tau and appear to form more and longer bundles than those assembled in the presence of wild-type tau. We propose that phosphorylated tau, unbound to microtubules, could accumulate in the cytoplasm.

The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV-1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV- 1-mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF-responsive by transducing them with the high-affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.