Metallothionein induces a regenerative reactive astrocyte phenotype via JAK/STAT and RhoA signalling pathways.

Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed "reactive" astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

Metallothionein treatment attenuates microglial activation and expression of neurotoxic quinolinic acid following traumatic brain injury.

The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 mug/ml MT almost completely blocking interferon-gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria.

Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-kappaB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.

New insight into the molecular pathways of metallothionein-mediated neuroprotection and regeneration.

There is a large body of evidence demonstrating that metallothioneins (MTs) expressed in astrocytes following CNS injury, exhibit both neuroprotective and neuroregenerative properties and are critical for recovery outcomes. As these proteins lack signal peptides, and have well characterized free radical scavenging and heavy metal binding properties, the neuroprotective functions of MTs have been attributed to these intracellular roles. However, there is an increasing realization that the neuroprotective functions of MTs may also involve an extracellular component. In this issue of Journal of Neurochemistry, Ambjørn et al. reveal considerable insight into this novel function of MTs. In this review, we examine the seminal work of Ambjørn et al. in the context of our current understanding of the role of MT in astrocyte-neuron interactions in the injured brain, and also discuss the significant therapeutic potential of their work.

Excitotoxicity mediated by non-NMDA receptors causes distal axonopathy in long-term cultured spinal motor neurons.

Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long-term culture. Excitotoxicity, mediated principally via non-N-methyl-D-aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.

Metallothionein expression by NG2 glial cells following CNS injury.

Metallothionein (MT) expression is rapidly up-regulated following CNS injury, and there is a strong correlation between the presence or absence of MTand improved or impaired (respectively) recovery from such trauma.We now report that a distinct subset of NG2-positive, GFAP-negative glial cells bordering the injury tract express MT following focal injury to the adult rat neocortex. To confirm the ability of these NG2 glial cells to express MT, we have isolated and cultured them and identified that they can express MT following stimulation with zinc. To investigate the functional importance of MT expression by NG2 glial cells, we plated cortical neurons onto these cells and found that expression of MT enhanced the permissivity of NG2 glial cells to neurite outgrowth. Our data suggest that expression of MT by NG2 glial cells may contribute to the overall permissiveness of these cells to axon regeneration.

Effect of olfactory ensheathing cells on reactive astrocytes in vitro.

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.

Alpha-synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection.

Chronic oxidative stress has been linked to the neurodegenerative changes characteristic of Parkinson's disease, particularly alpha-synuclein accumulation and aggregation. However, it remains contentious whether these alpha-synuclein changes are cytotoxic or neuroprotective. The current study utilised long-term primary neural culture techniques with antioxidant free media to study the cellular response to chronic oxidative stress. Cells maintained in antioxidant free media were exquisitely more vulnerable to acute exposure to hydrogen peroxide, yet exposure of up to 10 days in antioxidant free media did not lead to morphological alterations in neurones or glia. However, a subpopulation of neurones demonstrated a significant increase in the level of alpha-synuclein expressed within the cell body and at synaptic sites. This subset of neurones was also more resistant to apoptotic changes following exposure to antioxidant free media relative to other neurones. These data indicate that increased alpha-synuclein content is associated with neuroprotection from relatively low levels of oxidative stress.

Metallothionein biology in the ageing and neurodegenerative brain.

In recent years metallothionein (MT) biology has moved from investigation of its ability to protect against environmental heavy metals to a wider appreciation of its role in responding to cellular stress, whether as a consequence of normal function, or following injury and disease. This is exemplified by recent investigation of MT in the mammalian brain where plausible roles for MT action have been described, including zinc metabolism, free radical scavenging, and protection and regeneration following neurological injury. Along with other laboratories we have used several models of central nervous system (CNS) injury to investigate possible parallels between injury-dependent changes in MT expression and those observed in the ageing and/or degenerating brain. Therefore, this brief review aims to summarise existing information on MT expression during CNS ageing, and to examine the possible involvement of this protein in the course of human neurodegenerative disease, as exemplified by Alzheimer's disease.

Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors.

Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.

A role for extracellular metallothioneins in CNS injury and repair.

For many years, research focus on metallothioneins, small zinc binding proteins found predominantly within astrocytes in the brain, has centred on their ability to indirectly protect neurons from oxygen free radicals and heavy metal-induced neurotoxicity. However, in recent years it has been demonstrated that these proteins have previously unsuspected roles within the cellular response to brain injury. The aim of this commentary is to provide an overview of the exciting recent experimental evidence from several laboratories including our own suggesting a possible extracellular role for these proteins, and to present a hypothetical model explaining the newly identified function of extracellular metallothioneins in CNS injury and repair.

Olfactory ensheathing cells promote collateral axonal branching in the injured adult rat spinal cord.

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into the spinal cord. The dorsal columns of adult rats were cut at T(8-9) and rats in experimental groups received either EC-filled porous polymer capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2) lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group (P=0.003), while a trend for increased collateral branches was observed in rats that received encapsulated ECs (P=0.07).

Olfactory ensheathing cell phenotype following implantation in the lesioned spinal cord.

Although olfactory ensheathing cells (OECs) are used to promote repair in the injured spinal cord, little is known of their phenotype in this environment. In this study, using quantitative reverse transcriptase-polymerase chain reaction RT-PCR, expression of neuregulin-1 mitogen/survival factors and the axonal growth regulator Nogo was quantified in OECs and compared with other non-neuronal cells. Their expression was also compared with OECs which had previously been encapsulated in a porous polymer tube and implanted into the injured spinal cord. Similar to astrocytes and fibroblasts, OECs expressed various neuregulin subtypes including neu differentiation factor, glial growth factor and sensory and motorneuron-derived factor. Implanted OECs upregulated neu differentiation factor and secreted neuregulin, but downregulated expression of all other variants. OECs and oligodendrocytes expressed Nogo-A, -B and -ABC and were immunopositive for Nogo-A protein. The Nogo-A protein in OECs was found to be cytoplasmic rather than nuclear or cell surface associated. Unlike oligodendrocytes, OECs expressed Nogo-66 receptor (NgR) mRNA. Implanted OECs upregulated Nogo-A and -B, but downregulated Nogo-ABC and NgR.

Localization of alpha-, beta-, and gamma-synuclein during neuronal development and alterations associated with the neuronal response to axonal trauma.

Genetic and protein studies have indicated abnormalities in alpha-synuclein in neurodegenerative diseases. However, the developmental localization and cellular role of synuclein isoforms is contentious. We investigated the cellular localization of alpha-, beta-, and gamma-synuclein in developing cultured rat neurons and following axonal transection of relatively mature neurons, a model that disrupts the axonal cytoskeleton and results in regenerative sprouting. Cortical neurons were grown up to 21 days in vitro (DIV). Axon bundles at 21 DIV were transected and cellular changes examined at 4 and 24 h post-injury. Immunohistochemistry demonstrated that alpha- and beta-synuclein were localized to cellular cytosol and growth cones at 3DIV, with accumulating puncta-like labeling within axons and growth cones by 10-21DIV. In contrast, gamma-synuclein immunoreactivity was limited at all time points. By 21DIV, alpha- and beta-synuclein were present in the same neurons but largely in separate subregions, only 26% of puncta contained both alpha- and beta-synuclein immunoreactivity. Less than 20% of alpha-, beta-, and pan-synuclein immunoreactive puncta directly colocalized to synaptophysin profiles at 10DIV, decreasing to 10% at 21DIV. Both alpha- and beta-synuclein accumulated substantially within damaged axons at 21DIV and were localized to cytoskeletal abnormalities. At latter time points post-injury, alpha- and beta-synuclein immunoreactive puncta were localized to growth cone-like structures in regenerating neurites. This study shows that alpha- and beta-synuclein have a precise localization within cortical neurons and are generally nonoverlapping in their distribution within individual neurons. In addition, synuclein proteins accumulate rapidly in damaged axons and may have a role in regenerative sprouting.

Metallothionein-III inhibits initial neurite formation in developing neurons as well as postinjury, regenerative neurite sprouting.

Human metallothionein-III (MT-III) is an inhibitory factor deficient in the Alzheimer's disease (AD) brain. MT-III has been identified as an inhibitor of neurite sprouting, and its deficiency has been proposed to be involved in the formation of neurofibrillary tangles (NFT) in the neuropathology of AD. However, there has been limited investigation of the proposed neurite growth inhibitory properties of MT-III. We have applied recombinant human MT-III to both single cell embryonic cortical neurons (to investigate initial neurite formation), as well as mature (21 days postplating) clusters of cortical neurons (to investigate the regenerative sprouting response following injury). We report that MT-III inhibited the initial formation of neurites by rat embryonic (E18) cortical neurons. This was based on both the percentage of neurite positive neurons and the number of neurites per neuron (45 and 30% inhibition, respectively). Neurite inhibition was only observed in the presence of adult rat brain extract, and was also reversible following replacement of MT-III-containing medium. MT-III inhibited the formation and growth of both axons and dendrites. Of more physiological significance, MT-III also inhibited the regenerative neurite sprouting response following axonal transection. The morphology of sprouting neurites was also altered, with the distal tip often ending in bulb-like structures. Based on these results, we propose that MT-III, in the presence of brain extract, is a potent inhibitor of neurite sprouting, and may be involved in abnormal sprouting potentially underlying both AD and epilepsy.

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.

Metallothionein is expressed in adipocytes of brown fat and is induced by catecholamines and zinc.

Metallothionein (MT) is thought to have an antioxidant function and is strongly expressed during activation of thermogenesis and increased oxidative stress in brown adipose tissue (BAT). Localization and regulation of MT expression in BAT was therefore investigated in rats and mice. Immunohistochemical analysis of BAT from rats exposed to 4 degrees C for 24 h showed that MT and uncoupling protein 1 (UCP1) were coexpressed in differentiated adipocytes, and both cytoplasmic and nuclear localization of MT was observed. Cold induction of MT-1 expression in BAT was also observed in mice. Administration of norepinephrine to rats and isoproterenol to mice stimulated MT and UCP1 expression in BAT, implying a sympathetically mediated pathway for MT induction. In mice, zinc, and particularly dexamethasone, induced MT-2 expression in BAT and liver. Surprisingly, zinc also induced UCP1 in BAT, suggesting that elevated zinc may induce thermogenesis. We conclude that expression of MT in mature brown adipocytes upon beta-adrenoceptor activation is consistent with a role in protecting against physiological oxidative stress or in facilitating the mobilization or utilization of energy reserves.

Glial growth factor 2 induces proliferation and structural changes in ensheathing cells.

Ensheathing cells were isolated from neonatal rat olfactory bulbs and cultured in the presence of glial growth factor 2 (GGF2). Proliferation assay showed that at concentrations of up to 60 ng/ml GGF2, ensheathing cells underwent a modest increase in proliferation rate. This stimulation was not maintained at high doses of GGF2 at 100 ng/ml or more. Chemotaxis chambers and scanning electron microscopy were used to determine whether GGF2 was a chemoattractant for ensheathing cells. Although the results showed no chemotactic response to GGF2, ensheathing cells demonstrated structural changes when cultured in the presence of 20 ng/ml GGF2. Ultrastructural observations revealed that GGF2 promoted increased deposition of extracellular matrix on the cell membrane, more cytoskeletal elements in the processes and as a possible consequence, contributed to a more rigid support. Ensheathing cells cultured in the absence of GGF2 often extended thinner and curved processes. Reverse transcription-polymerase chain reaction confirmed the presence of GGF2 transcripts in ensheathing cells, suggesting that ensheathing cells themselves are a source of GGF2.

Nonshivering thermogenesis in a marsupial (the tasmanian bettong Bettongia gaimardi) is not attributable to brown adipose tissue.

The Tasmanian bettong (Bettongia gaimardi, a marsupial) is a rat-kangaroo that increases nonshivering thermogenesis (NST) in response to norepinephrine (NE). This study attempted to assess whether brown adipose tissue (BAT), a specialized thermogenic effector, is involved in NST in the bettong. Regulatory NST, indicated by resting oxygen consumption (Vo2) of the whole body, was measured under conscious conditions at 20 degrees C with various stimuli: cold (4 degrees -5 degrees C) or warm (25 degrees C) acclimation, NE injection, and the beta3-adrenoceptor agonist (BRL) 37344. In line with the functional studies in vivo, the presence of BAT was evaluated by examining the expression of the uncoupling protein 1 (UCP1) with both rat cDNA and oligonucleotide probes. Both NE and BRL 37344 significantly stimulated NST in the bettong. After cold acclimation of the animals (at 4 degrees -5 degrees C for 2 wk), the resting Vo2 was increased by 15% and the thermogenic effect of NE was enhanced; warm-acclimated animals showed a slightly depressed response. However, no expression of UCP1 was detected in bettongs either before or after cold exposure (2 wk). These data suggest that the observed NST in the marsupial bettong is not attributable to BAT.

Increased density of metallothionein I/II-immunopositive cortical glial cells in the early stages of Alzheimer's disease.

We have examined the possible role of metallothionein I/II (MT I/II) in Alzheimer's disease (AD), with a focus on the cellular localization of MT I/II relative to the astrocyte marker, glial fibrillary acidic protein (GFAP). In AD and preclinical AD cases, MT I/II immunolabeling was present in glial cells and did not show a spatial relationship with beta-amyloid plaques or neurofibrillary pathology. There was a six- to sevenfold increase in both MT I/II- and GFAP-labeled cells in the gray matter of AD cases, relative to non-AD cases. However, there was a threefold increase in MT I/II-immunoreactive cells, but not GFAP-labeled cells, in the gray matter of preclinical AD cases compared to non-AD cases. Therefore, the specific increase in MT I/II is associated with the initial stages of the disease process, perhaps due to oxidative stress or the mismetabolism of heavy metals.