Meng Inn Chuah

Cultured olfactory ensheathing cells express nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor and their receptors ☆

In the primary olfactory pathway axons of olfactory neurons (ONs) are accompanied by ensheathing cells (ECs) as the fibres course towards the olfactory bulb. Ensheathing cells are thought to play an important role in promoting and guiding olfactory axons to their appropriate target. In recent years, studies have shown that transplants of ECs into lesions in the central nervous system (CNS) are able to stimulate the growth of axons and in some cases restore functional connections. In an attempt to identify a possible mechanism underlying EC support for olfactory nerve growth and CNS axonal regeneration, this study investigated the production of growth factors and expression of corresponding receptors by these cells. Three techniques immunohistochemistry, enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess growth factor expression in cultured ECs. Immunohistochemistry showed that ECs expressed nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF). ELISA confirmed the intracellular presence of NGF and BDNF and showed that, compared to BDNF, about seven times as much NGF was secreted by ECs. RT-PCR analysis demonstrated expression of mRNA for NGF, BDNF, GDNF and neurturin (NTN). In addition, ECs also expressed the receptors trkB, GFRalpha-1 and GFRalpha-2. The results of the experiments show that ECs express a number of growth factors and that BDNF in particular could act both in a paracrine and autocrine manner.

Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Genetic Expression Profile of Olfactory Ensheathing Cells Is Distinct From That of Schwann Cells and Astrocytes

Olfactory ensheathing cells (OECs) accompany the axons of olfactory receptor neurons, which regenerate throughout life, from the olfactory mucosa into the olfactory bulb. OECs have shown widely varying efficacy in repairing the injured nervous system. Analysis of the transcriptome of OECs will help in understanding their biology and will provide tools for investigating the mechanisms of their efficacy and interactions with host tissues in lesion models. In this study, we compared the transcriptional profile of cultured OECs with that of Schwann cells (SCs) and astrocytes (ACs), two glial cell types to which OECs have similarities. Two biological replicates of RNA from cultured OECs, SCs, and ACs were hybridized to long oligo rat 5K arrays against a common reference pool of RNA (50% cultured fibroblast RNA and 50% neonatal rat brain RNA). Transcriptional profiles were analyzed by hierarchical clustering, Principal Components Analysis, and the Venn diagram. The three glial cell types had similarly increased or decreased expression of numerous transcripts compared with the reference. However, OECs were distinguishable from both SCs and ACs by a modest number of transcripts, which were significantly enriched or depleted. Furthermore, OECs and SCs were more closely related to each other than to ACs. Expression of selected transcripts not previously characterized in OECs, such as Lyz, Timp2, Gro1 (Cxcl1), Ccl2 (MCP1), Ctgf, and Cebpb, was validated by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR); immunohistochemistry in cultured OECs, SCs, and ACs, and adult tissues was performed to demonstrate their expression at the protein level.

Morphological plasticity of olfactory ensheathing cells is regulated by cAMP and endothelin-1

Olfactory ensheathing cells (ECs) are a promising tool for the repair of injury in the adult central nervous system. However, important aspects of the cell biology of ECs remain unclear, such as whether ECs exist as a single population or as two subpopulations with Schwann cell‐like and astrocyte‐like characteristics. The morphologies of these subpopulations are used as defining characteristics, yet ECs are known to be morphologically plastic. To elucidate this apparent inconsistency, we investigated the morphological plasticity of ECs in culture. We defined purified ECs as immunopositive for both p75 neurotrophin receptor and glial fibrillary acidic protein. In MEM D‐valine modification + 10% dialyzed fetal calf serum, 87%–90% of ECs displayed a flat morphology. In three different serum‐free media (N2 medium, neurobasal medium + B27 supplement, and DMEM/F‐12 medium + G5 supplement), 78%–84% of ECs displayed process‐bearing morphology. Ensheathing cells switched reversibly between these morphologies within a day of the serum conditions being changed. Exposure to 1 nM endothelin‐1 in serum‐free medium prevented the switch from flat to process‐bearing morphology, while 1 mM dibutyryl cAMP accelerated this change. The effects of both agents were completely reversible and similar to that reported for astrocytes. Both flat and process‐bearing ECs were immunopositive for brain‐derived neurotrophic factor, nerve growth factor, neurotrophin‐4, and TrkB but not TrkA. Together, these results suggest that ECs exist as a single morphologically plastic population. GLIA 41:393–403, 2003.

Neuron-glia communication: metallothionein expression is specifically up-regulated by astrocytes in response to neuronal injury

Recent data suggests that metallothioneins (MTs) are major neuroprotective proteins within the CNS. In this regard, we have recently demonstrated that MT‐IIA (the major human MT‐I/‐II isoform) promotes neural recovery following focal cortical brain injury. To further investigate the role of MTs in cortical brain injury, MT‐I/‐II expression was examined in several different experimental models of cortical neuron injury. While MT‐I/‐II immunoreactivity was not detectable in the uninjured rat neocortex, by 4 days, following a focal cortical brain injury, MT‐I/‐II was found in astrocytes aligned along the injury site. At latter time points, astrocytes, at a distance up to several hundred microns from the original injury tract, were MT‐I/‐II immunoreactive. Induced MT‐I/‐II was found both within the cell body and processes. Using a cortical neuron/astrocyte co‐culture model, we observed a similar MT‐I/‐II response following in vitro injury. Intriguingly, scratch wound injury in pure astrocyte cultures resulted in no change in MT‐I/‐II expression. This suggests that MT induction was specifically elicited by neuronal injury. Based upon recent reports indicating that MT‐I/‐II are major neuroprotective proteins within the brain, our results provide further evidence that MT‐I/‐II plays an important role in the cellular response to neuronal injury.

Bacteria and PAMPs activate nuclear factor κB and Gro production in a subset of olfactory ensheathing cells and astrocytes but not in Schwann cells

The primary olfactory nerves provide uninterrupted conduits for neurotropic pathogens to access the brain from the nasal cavity, yet infection via this route is uncommon. It is conceivable that olfactory ensheathing cells (OECs), which envelope the olfactory nerves along their entire length, provide a degree of immunological protection against such infections. We hypothesized that cultured OECs would be able to mount a biologically significant response to bacteria and pathogen‐associated molecular patterns (PAMPs). The response of OECs to Escherichia coli (E. coli) and various PAMPs was compared to that of Schwann cells (SCs), astrocytes (ACs), and microglia (MG). A subset of OECs displayed nuclear localization of nuclear factor κB), an inflammatory transcription factor, after treatment with E. coli (20% ± 5%), lipopolysacchride (33% ± 9%), and Poly I:C (25% ± 5%), but not with peptidoglycan or CpG oligonucleotides. ACs displayed a similar level of activation to these treatments, and in addition responded to peptidoglycan. The activation of OECs and ACs was enhanced by coculture with MG (56% ± 16% and 85% ± 13%, respectively). In contrast, SCs did not respond to any treatment or to costimulation by MG. Immunostaining for the chemokine Gro demonstrated a functional response that was consistent with NFκB activation. OECs expressed mRNA for Toll‐like receptors (TLRs) 2 and 4, but only TLR4 protein was detected by Western blotting and immunohistochemistry. The results demonstrate that OECs possess the cellular machinery that permits them to respond to certain bacterial ligands, and may have an innate immune function in protecting the CNS against infection.

α-Internexin immunoreactivity reflects variable neuronal vulnerability in Alzheimer's disease and supports the role of the β-amyloid plaques in inducing neuronal injury

This study investigated the role of α-internexin in the neuronal alterations associated with β-amyloid plaque formation in Alzheimers disease (AD). Cortical neurons could be defined by their variable content of neurofilament (NF) triplet and α-internexin proteins, with a distinct population of supragranular pyramidal cells containing α-internexin alone. Both NF triplet and α-internexin were localized to reactive axonal structures in physically damaged neurons in experimental trauma models. Similarly, NF triplet and α-internexin immunoreactive neurites were localized to plaques densely packed with β-amyloid fibrils in preclinical AD cases, indicating that certain plaques may cause structural injury or impediment of local axonal transport. However, α-internexin, and not NF triplet, ring-like reactive neurites were present in end-stage AD cases, indicating the relatively late involvement of neurons that selectively contain α-internexin. These results implicate the expression of specific intermediate filament proteins in a distinct hierarchy of differential neuronal vulnerability to AD.

Protective role of metallothioneins in the injured mammalian brain.

Metallothioneins (MTs) are small cysteine-rich proteins which are found widely throughout the mammalian body, including the CNS. There are extensive data on the structure and expression of MTs, and many basic properties pertinent to MT biology in the CNS appear to be well established. As discussed in this review, one isoform class (MT-I/II) is rapidly induced following many types of CNS insult, and is strongly neuroprotective, whilst another isoform class (MT-III) shows major differences in its expression profile and physiological properties. As in other tissues, there is no clear consensus on the mechanism of MT action in the CNS and how it exerts its protective role, despite a number of excellent animal and cell culture models of MT expression in the brain, and a large literature on the physico-chemical properties of MTs extending over several decades. This review is therefore an attempt to summarise the recent literature on the expression of MTs in the adult mammalian brain and how MTs possibly act to protect the brain following physical or chemical insult. One exciting finding from recent work is that perturbing the levels of MT in the brain has an effect that extends beyond cells which normally express MT to other cell types including neurons, microglia and cells of the immune system. These observations were made mainly using animal models in which MT action can be observed in its normal cellular context, and this review focuses particularly on work conducted in animal models of physical and chemical injury in the brain.

Cytokines and olfactory bulb microglia in response to bacterial challenge in the compromised primary olfactory pathway

BackgroundThe primary olfactory pathway is a potential route through which microorganisms from the periphery could potentially access the central nervous system. Our previous studies demonstrated that if the olfactory epithelium was damaged, bacteria administered into the nasal cavity induced nitric oxide production in olfactory ensheathing cells. This study investigates the cytokine profile of olfactory tissues as a consequence of bacterial challenge and establishes whether or not the bacteria are able to reach the olfactory bulb in the central nervous system.MethodsThe olfactory epithelium of C57BL/6 mice was damaged by unilateral Triton X-100 nasal washing, and Staphylococcus aureus was administered ipsilaterally 4 days later. Olfactory mucosa and bulb were harvested 6 h, 24 h and 5 days after inoculation and their cytokine profile compared to control tissues. The fate of S. aureus and the response of bulbar microglia were examined using fluorescence microscopy and transmission electron microscopy.ResultsIn the olfactory mucosa, administered S. aureus was present in supporting cells of the olfactory epithelium, and macrophages and olfactory nerve bundles in the lamina propria. Fluorescein isothiocyanate-conjugated S. aureus was observed within the olfactory mucosa and bulb 6 h after inoculation, but remained restricted to the peripheral layers up to 5 days later. At the 24-h time point, the level of interleukin-6 (IL-6) and tumour necrosis factor-α in the compromised olfactory tissues challenged with bacteria (12,466 ± 956 pg/ml and 552 ± 193 pg/ml, respectively) was significantly higher than that in compromised olfactory tissues alone (6,092 ± 1,403 pg/ml and 80 ± 2 pg/ml, respectively). Immunohistochemistry confirmed that IL-6 was present in several cell types including olfactory ensheathing cells and mitral cells of the olfactory bulb. Concurrently, there was a 4.4-, 4.5- and 2.8-fold increase in the density of iNOS-expressing cells in the olfactory mucosa, olfactory nerve and glomerular layers combined, and granule layer of the olfactory bulb, respectively.ConclusionsBacteria are able to penetrate the immunological defence of the compromised olfactory mucosa and infiltrate the olfactory bulb within 6 h even though a proinflammatory profile is mounted. Activated microglia may have a role in restricting bacteria to the outer layers of the olfactory bulb.

Effect of olfactory ensheathing cells on reactive astrocytes in vitro

Abstract.Olfactory ensheathing cells have been used in several studies to promote repair in the injured spinal cord. However, cellular interaction between olfactory ensheathing cells and glial cells induced to be reactive in the aftermath of injury site has not been investigated. Using an in vitro model of astrogliosis, we show that reactive astrocytes expressed significantly less glial fibrillary acidic protein (GFAP) when cultured both in direct contact with olfactory ensheathing cells and when the two cell types were separated by a porous membrane. Immunofluorescence staining also suggested that reactive astrocytes showed decreased chondroitin sulfate proteoglycans in the presence of olfactory ensheathing cells, although the reduction was not statistically significant. No down-regulation of GFAP was observed when reactive astrocytes were similarly cultured with Schwann cells. Cell viability assay and bromodeoxyuridine uptake showed that proliferation of reactive astrocytes was significantly increased in the presence of olfactory ensheathing cells and Schwann cells.