Anterior thalamic nuclei neurons sustain memory.

A hippocampal-diencephalic-cortical network supports memory function. The anterior thalamic nuclei (ATN) form a key anatomical hub within this system. Consistent with this, injury to the mammillary body-ATN axis is associated with examples of clinical amnesia. However, there is only limited and indirect support that the output of ATN neurons actively enhances memory. Here, in rats, we first showed that mammillothalamic tract (MTT) lesions caused a persistent impairment in spatial working memory. MTT lesions also reduced rhythmic electrical activity across the memory system. Next, we introduced 8.5 Hz optogenetic theta-burst stimulation of the ATN glutamatergic neurons. The exogenously-triggered, regular pattern of stimulation produced an acute and substantial improvement of spatial working memory in rats with MTT lesions and enhanced rhythmic electrical activity. Neither behaviour nor rhythmic activity was affected by endogenous stimulation derived from the dorsal hippocampus. Analysis of immediate early gene activity, after the rats foraged for food in an open field, showed that exogenously-triggered ATN stimulation also increased Zif268 expression across memory-related structures. These findings provide clear evidence that increased ATN neuronal activity supports memory. They suggest that ATN-focused gene therapy may be feasible to counter clinical amnesia associated with dysfunction in the mammillary body-ATN axis.

Optogenetic stimulation: Understanding memory and treating deficits.

Technology allowing genetically targeted cells to be modulated by light has revolutionized neuroscience in the past decade, and given rise to the field of optogenetic stimulation. For this, non-native, light activated proteins (e.g., channelrhodopsin) are expressed in a specific cell phenotype (e.g., glutamatergic neurons) in a subset of central nervous system nuclei, and short pulses of light of a narrow wavelength (e.g., blue, 473 nm) are used to modulate cell activity. Cell activity can be increased or decreased depending on which light activated protein is used. We review how the greater precision provided by optogenetics has transformed the study of neural circuits, in terms of cognition and behavior, with a focus on learning and memory. We also explain how optogenetic modulation is facilitating a better understanding of the mechanistic underpinnings of some neurological and psychiatric conditions. Based on this research, we suggest that optogenetics may provide tools to improve memory in neurological conditions, particularly diencephalic amnesia and Alzheimer's disease.

Epac and the high affinity rolipram binding conformer of PDE4 modulate neurite outgrowth and myelination using an in vitro spinal cord injury model.

BACKGROUND AND PURPOSE: cAMP and pharmacological inhibition of PDE4, which degrades it, are promising therapeutic targets for the treatment of spinal cord injury (SCI). Using our previously described in vitro SCI model, we studied the mechanisms by which cAMP modulators promote neurite outgrowth and myelination using enantiomers of the PDE4-specific inhibitor rolipram and other modulators of downstream signalling effectors. EXPERIMENTAL APPROACH: Rat mixed neural cell myelinating cultures were cut with a scalpel and treated with enantiomers of the PDE4-specific inhibitor rolipram, Epac agonists and PKA antagonists. Neurite outgrowth, density and myelination were assessed by immunocytochemistry and cytokine levels analysed by qPCR. KEY RESULTS: Inhibition of the high-affinity rolipram-binding state (HARBS), rather than the low-affinity rolipram binding state (LARBS) PDE4 conformer promoted neurite outgrowth and myelination. These effects were mediated through the activation of Epac and not through PKA. Expression of the chemokine CXCL10, known to inhibit myelination, was markedly elevated in astrocytes after Rho inhibition and this was blocked by inhibition of Rho kinase or PDE4. CONCLUSIONS AND IMPLICATIONS: PDE4 inhibitors targeted at the HARBS conformer or Epac agonists may provide promising novel targets for the treatment of SCI. Our study demonstrates the differential mechanisms of action of these compounds, as well as the benefit of a combined pharmacological approach and highlighting potential promising targets for the treatment of SCI. These findings need to be confirmed in vivo.

KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but functional consequences of these biochemical changes have not been previously addressed. We show that in MCF10A cells, loss of KIBRA expression displays epithelial-to-mesenchymal transition (EMT) features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the serine 127 phosphorylation site and we show that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Finally, reduced KIBRA expression in primary breast cancer specimens correlates with the recently described claudin-low subtype, an aggressive sub-group with EMT features and a poor prognosis.

Repair of central nervous system lesions by transplantation of olfactory ensheathing cells.

Clinical conditions affecting the central nervous system (CNS) fall into two main categories - degenerative conditions in which nerve cells are lost (Alzheimer's, Parkinson's, Huntington's disease, etc.), and traumatic insults which sever nerve fibers but leave their cell bodies and initial parts of the severed axons intact (spinal cord injury, cerebrovascular accidents, or tumors affecting fiber tracts). After injuries of this second type, the survival of the nerve cell bodies and the local sprouting at the severed ends of the proximal stumps of the axons raise the tantalizing possibility of one day learning how to induce these severed fibers to regenerate to their original targets and restore lost functions. This chapter gives an overview of current research into the strategy of transplantation of olfactory ensheathing cells into axotomizing injuries.

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture.

In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled-down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell-seeding procedures for static cultures might not be applicable, therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro-grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture.

Myelinated, synapsing cultures of murine spinal cord--validation as an in vitro model of the central nervous system.

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

Implantation of pure cultured olfactory ensheathing cells in an animal model of parkinsonism.

BACKGROUND: Implantation of neural cells has been proposed as a therapeutic strategy for repairing the injured or diseased brain. In the present study we have examined the potential of olfactory ensheathing cells (OEC) to promote brain repair after surgical implantation in a rodent model of parkinsonism. METHODS: Neonatal OECs were implanted in the striatum after a 6-hydroxydopamine lesion of the ipsilateral substantia nigra. Amphetamine-induced rotational asymmetry scores were determined 48 hours before and 4, 6 and 8 weeks after OEC implantation. The density of immunostaining for tyrosine hydroxylase and synaptophysin in the striatum and the number of tyrosine hydroxylase-positive cells remaining in the substantia nigra were also determined. RESULTS: Rotational asymmetry scores were similar in OEC-implanted and vehicle-treated groups at all time points examined, and at each time were similar to those observed prior to implantation. Levels of striatal tyrosine-hydroxylase and synaptophysin immunoreactivity were similar in OEC- and vehicle-treated groups. The number of tyrosine-hydroxylase-positive cells in the substantia nigra was similar in both groups indicating that severity of the lesion was similar. Visualisation of GFP-labelled OECs one week after implantation in a separate group of animals revealed the cells to be located in the area immediately surrounding the needle tract. CONCLUSION: This study demonstrates that implantation of OECs alone is not sufficient to promote tissue repair and functional recovery in a rodent model of parkinsonism. The results add to a growing number of studies that propose a caveat for the use of pure OECs as a neurosurgical strategy for the treatment of brain disease or injury.

Cryopreserved cells isolated from the adult canine olfactory bulb are capable of extensive remyelination following transplantation into the adult rat CNS.

Naturally occurring spinal cord injury in dogs provides a potentially powerful intermediate model for testing the efficacy of therapeutic strategies developed in experimental rodent models before phase 1 trials in human patients. A particularly promising strategy involves transplantation of olfactory ensheathing cells (OECs) that both promote axon regeneration and generate new myelin sheaths. As a first step in developing OEC transplantation in the canine intermediate model we describe the isolation, purification, and characterization of OECs from adult dog olfactory bulb. We also show that the canine OEC behaves in a manner similar to its rodent counterpart following transplantation into demyelinating lesions in rat spinal cord and that these properties are retained following cryopreservation.

SCIP/Oct-6, Krox-20, and desert hedgehog mRNA expression during CNS remyelination by transplanted olfactory ensheathing cells.

Olfactory ensheathing cells (OECs), although having a separate developmental origin to Schwann cells, are able to generate myelin sheaths following transplantation into areas of CNS demyelination that are remarkably similar to those made by Schwann cells. The transcriptional control of Schwann cell myelination has been well documented, in particular the role of SCIP/Oct-6 and Krox-20. It is not known, however, whether these transcription factors are also expressed when OECs assume a myelinating phenotype. In this study, we addressed this question by using a transplantation approach to generate myelinating OECs and then examined the expression of SCIP/Oct-6 and Krox-20 mRNA by in situ hybridization using oligonucleotide probes. We also examined the expression of desert hedgehog (Dhh), a Schwann cell-derived signaling molecule that is responsible for regulating the development of the connective tissue elements in peripheral nerve, which bear similarities to the morphologies adopted by nonmyelinating transplanted cells. Our results indicate that both Krox-20 and Dhh mRNA are strongly expressed by transplanted OECs, with SCIP mRNA present at much lower levels. The expression of Krox-20 relative to the expression of P0 mRNA by the transplanted OECs is consistent with its playing a similar role in OEC myelination to that in Schwann cell myelination, while the expression of Dhh suggests a possible mechanism for the diverse morphologies that cells adopt following OEC transplantation into the damaged CNS. Taken together, our results provide further evidence for the close similarity of OECs and Schwann cells and suggest that, despite their separate origins, the manner in which they generate a peripheral-type myelin sheath involves similar regulatory mechanisms.

Gap junctional communication and connexin expression in cultured olfactory ensheathing cells.

The olfactory ensheathing cell (OEC) is a unique glial cell able to support neurite outgrowth in the CNS throughout life. The OEC has been described as having both Schwann cell-like and astrocyte-like characteristics. The purpose of this study was to compare gap junctional communication and connexin (Cx) expression in cultured olfactory ensheathing cells with both astrocytes and Schwann cells to establish which of these two cells types they most closely resemble. We examined the Cx mRNA profile of OECs, astrocytes, and Schwann cells using primers to Cx26, Cx32, Cx37, Cx43, Cx46, and Cx50. All connexins tested except Cx50 were expressed by all three cell types when initially cultured. However, we observed differences in the levels of expression of Cx32 and Cx26 between astrocytes, Schwann cells, and OECs that became pronounced with time. All three cell types show limited and variable gap junctional communication in culture as assessed by the transfer of microinjected Lucifer yellow. OECs had limited coupling compared with Schwann cells and astrocytes, although the extent of the dye spread through OECs was more comparable to that seen with Schwann cells than astrocytes. Thus, OECs display a profile of Cx expression that more closely resembles the Cx expression of Schwann cells rather than astrocytes.

Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.

Transplanted olfactory ensheathing cells (OECs) are able to remyelinate demyelinated axons and support regrowth of transected axons after transplantation into the adult CNS. Transplanted Schwann cells (SCs) share these repair properties but have limitations imposed on their behavior by the presence of astrocytes (ACs). Because OECs exist alongside astrocytes in the olfactory bulb, we have hypothesized that they have advantages over SCs in transplant-mediated CNS repair due to an increased ability to integrate and migrate within an astrocytic environment. In this study, we have tested this hypothesis by comparing the interactions between astrocytes and either SCs or OECs, using a range of in vitro assays. We have shown that (1) astrocytes and SCs segregate into defined non-overlapping domains in co-culture, whereas astrocytes and OECs freely intermingle; (2) both SCs and OECs will migrate across astrocyte monolayers, but only OECs will migrate into an area containing astrocytes; (3) SCs spend less time in contact with astrocytes than do OECs; and (4) astrocytes undergo hypertrophy when in contact with SCs, but not with OECs. Expression of N-cadherin has been implicated as a key mediator of the failure of SCs to integrate with astrocytes. However, we found no differences in the intensity of N-cadherin immunoreactivity between SCs and OECs, suggesting that it is not the adhesion molecule that accounts for the observed differences. In addition, the number of astrocytes expressing chondroitin sulfate proteoglycans (CSPG) is increased when astrocytes are co-cultured with Schwann cells compared with the number when astrocytes are grown alone or with OECs. Taken together, these data support the hypothesis that OECs will integrate more extensively than Schwann cells in astrocytic environments and are therefore better candidates for transplant-mediated repair of the damaged CNS.

AP-1 activity during the growth, differentiation, and death of O-2A lineage cells.

Oligodendrocyte differentiation has been correlated with AP-1 activity, being low in progenitors and high in differentiated cells. In this study we have carried out a detailed temporal analysis of AP-1 activity in oligodendrocyte-type-2 astrocyte (O-2A) lineage cells. We show that low AP-1 activity in progenitor cells depended on the application of growth factors. Treatment of cells with B104-conditioned medium induced high AP-1 activity, increased process length, and improved growth. The role of AP-1 in proliferation and process extension was emphasized when progenitor cells overexpressing a c-Jun dominant-negative mutant had impaired growth and shortened processes. AP-1 DNA-binding activity during O-2A differentiation in vitro showed an initial down-regulation followed by up-regulation after 2 days. Increased AP-1 levels in oligodendrocytes were inhibited by overexpression of bcl-2, indicating that AP-1 in mature oligodendrocytes is involved in the regulation of apoptosis. Prevention of cell death by bcl-2 in oligodendrocytes was accompanied by progressive differentiation and expression of MOG and PLP.

Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons.

The olfactory ensheathing cell (OEC) has attracted much interest recently because of its potential for transplantation-based therapy of CNS disease. Rat OECs are able to remyelinate demyelinated axons and support regeneration of damaged axons. Although OECs can be grown readily from the rat, a macrosmatic species, it has been uncertain whether it would be similarly straightforward to obtain these cells from the human, a microsmatic species with a relatively poorly developed olfactory system. In this study, we have identified a human OEC which shares many properties with its rat counterpart, including expression of the low-affinity nerve growth factor receptor (L-NGFr) and similar growth factor requirements. Purified populations of human OECs obtained by selection with L-NGFr antibodies have extremely high viability in tissue culture, and are capable of remyelinating persistently demyelinated CNS axons following transplantation into experimentally induced demyelinating lesions in the rat spinal cord. Thus, the human OEC represents an important new cell for the development of transplant therapy of CNS diseases.

Comparison of neuregulin-1 expression in olfactory ensheathing cells, Schwann cells and astrocytes.

Recently we demonstrated that a member of the neuregulin-1 (NRG-1) family of growth factors is a mitogen and survival factor for olfactory ensheathing cells (OECs). OECs are specialized glial cells within the olfactory system that are believed to play a role in the continual nerve re-growth of this tissue. OECs share properties with both astrocytes and Schwann cells but are likely to be a distinct glial cell type. NRG-1s have been found to be important regulators of Schwann cells in vivo, but the role of NRG-1 for OECs is less clear. The nrg-1 gene produces at least 12 different isoforms, that are likely to have different functions, due to alternative splicing of its mRNA. In this study, the expression of NRG-1 mRNAs in OECs was compared with other glial cells and their corresponding tissue sources. Cultured glial cells, unlike their tissue sources, expressed NRG-1 mRNAs containing the alpha EGF-like domain and expressed only the type 1beta isoform that lacks the glycosylated spacer domain. This correlated with expression of these isoforms during olfactory nerve degeneration in vivo. Although OECs expressed mRNA for all NRG-1 isoforms, the protein could not be detected in concentrated supernatant, or on the cell surface by immunofluorescence, but was detected in the nucleus or cytoplasm (depending on the isoform). These data support the hypothesis that NRG-1s play a functional role in OEC biology.

Neuregulin is a mitogen and survival factor for olfactory bulb ensheathing cells and an isoform is produced by astrocytes.

The rat olfactory bulb is an exceptional CNS tissue. Unlike other areas of the brain, growing axons are able to enter the olfactory bulb and extend within this CNS environment throughout adult life. It appears that the glial cells of the olfactory system, known as olfactory bulb ensheathing cells (OBECs), may have an important role in this remarkable process of CNS neural regeneration. OBECs are unusual glial cells, possessing properties of both astrocytes and Schwann cells. In this study we show that astrocytes (in the form of astrocyte-conditioned medium; ACM) produce two critical regulatory functions for OBECs: mitogenic activity and a survival factor. Interestingly, the ACM-derived activity for OBECs appears to reside in a signalling protein(s) belonging to the neuregulin (NRG) family of growth factors, and specifically appears to coincide with one or more products of the nrg-1 gene. Our observations provide evidence for the following: recombinant human neu differentiation factors (NDFbeta1, -2 and -3) are mitogenic to OBECs; the activity in ACM can be neutralized by NDF antibodies; these same antibodies detect a 50-kDa, non-heparin binding protein in concentrated ACM; astrocytes express detectable nrg-1 transcripts; and OBECs express functional NRG receptors erbB2 and erbB4.

Adhesion molecule expression and phenotype of glial cells in the olfactory tract.

These data illustrate that OBECs have a highly plastic nature in keeping with their need to respond rapidly to changing environmental cues. This relates to their required function in supporting axonal extension throughout life. Future studies using antibodies to PSA-NCAM and L-NGFr together with FACS sorting to purify the two types of OBEC should give us a clearer understanding of the lineage relationship of the two phenotypes. With purified populations of the astrocyte-like and Schwann cell-like OBEC we should be able to determine if these cells have different functions in vivo, using several approaches namely: i) identifying the growth factors that regulate their growth and differentiation, ii) measuring the ability of the purified cells to remyelinate the experimentally-created CNS lesions and iii) carry out more detailed cellular and molecular comparisons of the two phenotypes.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells transformed with c-myc and H-ras form high-grade glioma after stereotactic injection into the rat brain.

The oligodendrocyte-type-2 astrocyte lineage (O-2A) comprises a progenitor cell that is able to differentiate into an oligodendrocyte or astrocyte in vitro. The lineage was originally identified in the neonatal rat central nervous system but evidence suggests that the equivalent O-2A lineage also exists in humans. Apart from its putative and widely studied role in glial repair, this cell type could potentially be involved in malignant glioma formation. In this study we demonstrate that a rat O-2A progenitor cell line carrying the bacterial beta-galactosidase reporter gene and transformed with the c-myc and H-ras oncogenes which has lost its differentiation capacity in vitro generates glioma-like growth after stereotactic injection into the adult rat brain. Tumour pathology was similar to human glioblastoma, suggesting that one of the pathways in the generation of human glioblastomas may be the transformation of adult O-2A progenitor cells. Parallel studies demonstrated the presence of a DNA-binding protein complex, termed APprog, in a panel of human glioma cell lines. This protein was initially identified in O-2A progenitor cells and not their differentiated progeny. These data lead us to propose that APprog could be used as an indicator of the lineage origin of gliomas.

Expression of the dm-20 isoform of the plp gene in olfactory nerve ensheathing cells: evidence from developmental studies.

The olfactory bulb is a specialized area of the CNS with well-defined areas containing both myelinated, and non-myelinated axons. The presence of plp gene transcripts has been demonstrated previously in the olfactory bulb, but no detailed description of plp gene activity in this complex area of the CNS is available. In this study we describe the developmental expression of the two plp gene isoforms, plp and dm-20, and their products in the mouse olfactory bulb. plp gene activity was present in the non-myelinated olfactory nerve layer of the bulb from E14 through to adult ages. Expression in the deeper layers of the bulb, associated with myelination of the second order axons, was apparent from P10 onwards. dm-20 was the predominant isoform expressed at the transcript level in the olfactory nerve layer and was the only isoprotein demonstrable by immunostaining. This expression was associated with the resident glial cell of the non-myelinated olfactory nerve layer, the olfactory nerve ensheathing cell. Selective expression of the DM-20 isoprotein in the non-myelin forming olfactory nerve ensheathing cells implies a role for DM-20 other than as a structural myelin protein. Further study of this specialized glial cell may prove useful in elucidating the specific functions(s) of the DM-20 isoprotein.