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Background: The beta-amyloid (Aβ) peptide comprises the amyloid plaques that characterise Alzheimer's disease (AD), and is thought to significantly contribute towards disease pathogenesis. Oxidative stress is elevated in the AD brain, and there is substantial evidence that the interaction between Aβ and redox-active copper is a major contributing factor towards oxidative stress in AD. Results: The major findings of this study are that redox-active Cu(II)–Aβ causes pronounced axonal pathology in long-term neuronal cultures, including axonal fragmentation and the formation of hyperphosphorylated tau-immunoreactive axonal swellings. Notably, MAP-2 expressing dendritic processes remain largely un-affected by Cu(II)–Aβ treatment. These dystrophic axonal manifestations resemble some of the characteristic neuritic pathology of the AD brain. We show that Cu(II)–Aβ directly causes formation of intra-axonal swellings via the generation of free radicals and subsequent efflux of K + out of neurons. Conclusion: In summary, we report that redox-active Cu(II)–Aβ can induce substantial neurodegenerative changes in mature neurons, and may have an important role to play in the slowly progressing pathogenesis of AD.

There are currently few clinical strategies in place, which provide effective neuroprotection and repair, despite an intense international effort over the past decades. One possible explanation for this is that a deeper understanding is required of how endogenous mechanisms act to confer neuroprotection. This mini-review reports the proceedings of a recent workshop >Neuroprotection and Neurorepair: New Strategies> (Iguazu Falls, Misiones, Argentina, April 11-13, 2011, Satellite Symposium of the V Neurotoxicity Society Meeting, 2011) in which four areas of active research were identified to have the potential to generate new insights into this field. Topics discussed were (i) metallothionein and other multipotent neuroprotective molecules; (ii) oxidative stress and their signal mediated pathways in neuroregeneration; (iii) neurotoxins in glial cells, and (iv) drugs of abuse with neuroprotective effects. © 2011 Springer Science+Business Media, LLC.

Human metallothionein (MT) genes are clustered in a locus on chromosome 16, and this report presents the characterisation of the remaining six univestigated members of the family. Nucleotide sequencing in whole or part suggested that four of these genes, MT1I, MT1J, MT1K and MT1L do not encode expressed MT proteins, based on the presence of structural faults or atypical amino acid assignments. On the other hand, the structures of MT1H and MT1X are consistent with these genes being functional and encoding unique type 1 isoforms. The promoters of both genes conferred activity to CAT expression constructs when transfected into HeLa cells, and showed differential responses to inducers MT synthesis. Endogenous MT1H and MT1X genes were expressed at the mRNA level in HeLa cells following cadmium treatment. This work brings the number of functional class 1 and 2 MT genes in the human to eight, and confirms that each encodes structurally unique proteins.

We have investigated the expression of the protooncogene c- myc in rat hearts following exposure to norepinephrine, both in vivo and in isolated perfused preparations. Both chronic and acute norepinephrine treatment produced a rapid, transient elevation of c- myc mRNA in adult rat hearts, but chronic infusion produced a second, larger increase. This expression profile was characteristic for c- myc since it was not found for four other protooncogenes. In the isolated, perfused heart, addition of norepinephrine to the perfusion buffer and elevation of perfusion pressure separately increase c- myc mRNA suggesting both direct hormonal and hemodynamic factors might be important in vivo. Immunocytochemistry showed that Myc protein accumulated predominately in the nuclei of non-myocyte cells following norepinephrine treatment indicating that expression at the mRNA level culminated in protein synthesis. These findings suggest that the c- myc expression observed in the hypertrophying adult heart following exposure to norepinephrine may be associated with proliferating cells like fibroblasts rather than cardiomyocytes.

Background Metallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system. Methods Wild type and MT-I/II-/- mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay. Results Comparison of MT-I/II-/- mice with wild type controls following cryolesion brain injury revealed that the MT-I/II-/- mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II-/- mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II-/- mice that was independent of brain injury. Conclusions These results contribute to the evidence that MT-I/II-/- mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II-/- mice after brain injury relative to wild type mice.

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism.

Burn injury can lead to abnormal sensory function at both the injury and at distant uninjured sites. Here, we used a mouse model to investigate return of nociceptive function and reinnervation of the skin at the wound and uninjured distant sites following a 3% total burn surface area full-thickness burn injury. We have previously shown that topical application of zinc-metallothionein-IIA (Zn(7) -MT-IIA) accelerates healing following burn injury, and here, we investigated the potential of Zn(7) -MT-IIA to enhance reinnervation and sensory recovery. In all burn-injured animals, there was a significant reduction in nociceptive responses (Semmes-Weinstein filaments) at locations near and distant to the wound up to 8 weeks following injury. Cutaneous nerve reinnervation (assessed using protein gene product 9.5 immunohistochemistry) of the wound center was slow in the epidermis but rapid in the dermis. In the dermis, nerves subsequently degenerated both at the wound center and in distant uninjured areas. In contrast, epidermal nerve densities in the distant uninjured areas returned to normal, uninjured levels. Zn(7) -MT-IIA did not influence return of nociceptive function nor reinnervation. We conclude that burn injury compromises nociceptive function and nerve regeneration both at the injury site and systemically; thus, therapies in addition to Zn(7) -MT-IIA should be explored to return normal sensory function.

BackgroundOne of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time.ResultsIn this study, we report that daily exposure to a sublethal concentration of Aβ(1-40) (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ(1-40) treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K(+) homeostasis following Aβ treatment. Furthermore, blocking K(+) efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aβ(1-40) caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions.ConclusionsOur data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K(+)-dependent neurodegeneration that has pathological characteristics similar to AD.

Metallothionein has a well-documented protective and proregenerative effect in the mammalian brain, particularly following physical trauma and ischemia or during the onset of neurodegenerative disease. A range of mechanisms have been established for this, including metallothionein’s metal binding properties and its ability to scavenge free radicals. In recent years it has become apparent that metallothionein is present in the extracellular compartment of the central nervous system and that it can interact with cell surface receptors of the lipoprotein-receptor-related protein family, including lipoprotein-receptor-related protein 1 (LRP1) and megalin. These interactions activate intracellular pathways which are consistent with many of the observed effects of metallothionein in the central nervous system, including its effects on neurons, glial cells, and cells of the immune system. The evidence describing the release, receptor interactions, and subsequent physiological consequences of metallothionein is discussed in this review.

We have investigated the cellular and regional localization of Fos-like immunoreactivity (FLI) in the rat heart in response to the hypertrophic hormone norepinephrine (NE). Previous studies have demonstrated elevated c-fos mRNA levels in the rat heart following this treatment but have not shown which cell type(s) or specific chamber(s) of the heart contribute to the response in vivo, or whether Fos protein is actually produced. Administration of a single injection of NE (2.5 mg/kg) or chronic infusion of NE (100 micrograms/kg/h) led to an increase in Fos-like immunoreactivity in the nuclei of cardiac myocytes and vascular smooth muscle cells compared to control tissue. The response was transient with maximal immunoreactivity observed 2-3h following treatment, falling to near basal levels in most regions of the heart after 6 h. Although all chambers of the heart contributed to the response, greatest Fos-like immunoreactivity was observed in the left atrium and left ventricle, with intermediate levels found in the septum and right ventricle, and lowest levels in the right atrium. Fos-like immunoreactivity observed in the left atrium was accompanied by elevated mRNA levels of fra-1 and fra-2 but not c-fos itself indicating that a related gene product other than Fos contributed to the observed response. Experiments with the Langendorff perfused rat heart showed that NE and elevated perfusion pressure independently increased both c-fos mRNA and FLI. This work is the first evidence for a direct action of NE on Fos expression in adult, as opposed to neonatal, cardiomyocytes. These results lend further support to the notion that Fos and related gene products mediate some of the hypertrophic actions of norepinephrine.

Neuronal growth-inhibitory factor, later renamed metallothionein-3, is one of four members of the mammalian metallothionein family. Metallothioneins are a family of ubiquitous, low-molecular-weight, cysteine-rich proteins. Although neuronal growth-inhibitory factor shares metal-binding and reactive oxygen species scavenging properties with the other metallothioneins, it displays several distinct biological properties. In this review, we examine the recent developments regarding the function of neuronal growth-inhibitory factor within the brain, particularly in response to brain injury or during neurodegenerative disease progression.

Nuclear factor kappaB (NFκB) is a key transcriptional regulator of inflammatory genes. We investigated the modulatory effects of olfactory ensheathing cells (OECs), microglia and meningeal fibroblasts on translocation of NFκB to astrocyte nuclei. The percentage of activated astrocytes in co-cultures with OECs was significantly less than for co-cultures with microglia (p

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.

Severe injury to the epidermal barrier often results in scarring and life-long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn(7)-MT-IIA), a naturally occurring small cysteine-rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT-I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT-I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn(7)-MT-IIA significantly increased cell viability by up to 30% (p0.05), decreased apoptosis by 13% (p0.05) and promoted keratinocyte migration by up to 14% (p0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7-MT-IIA did not affect fibroblast motility or contraction (p0.05). Topical administration of exogenous Zn(7)-MT-IIA (2 microg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate-buffered saline (PBS), 8.9+/-0.3 mm diameter vs. MT-I/II, 7.1+/-0.7 mm; 7 days: PBS 5.8+/-0.98 mm vs. MT-I/II, 3.6+/-1.0 mm, p0.05) and reduced epidermal thickness (MT-I/II: 45+/-4 microm vs. PBS: 101+/-19 microm, p0.05) compared with controls. Our data suggest that exogenous Zn(7)-MT-IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 micrograms/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2 h. The response of both cellular oncogenes to norepinephrine was reduced significantly by alpha blockade but beta blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2 x 10(-7) M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.

The olfactory pathway represents a vulnerable route for pathogens such as pneumococcus, meningococcus, mumps, rabies, poliovirus, Herpes Simplex 1, West Nile Virus and Borna Disease Virus to access the central nervous system from the nasal cavity. Olfactory ensheathing cells (OECs), glial cells which ensheath the olfactory nerves from the nasal cavity to the olfactory bulb are in a prime position to assist with host immunity. This project investigates possible mechanisms relating to OECs' hypothesised role in host immunity, including the production of nitric oxide (NO), a potent antibacterial and antiviral agent. OECs were incubated with Escherichia coli and Staphylococcus aureus. Nitrite and NO production were analyzed using high performance liquid chromatography and live cell imaging, mRNA levels were assessed using Real-time RT-PCR, and inducible nitric oxide synthase (iNOS) expression by immunocytochemistry. An in vivo rat model was established to investigate iNOS expression in the compromised olfactory pathway. We show that elevated levels of nitrite were detected in bacteria-treated OECs, compared to untreated OECs and this was attenuated by the NO synthase inhibitor L-NMMA. Bacteriatreated OECs produced elevated levels of NO using the diaminofluorescein dye DAF2-DA (also attenuated by LNMMA). mRNA was detected for iNOS in OECs but not for neuronal nitric oxide synthase or endothelial nitric oxide synthase. Expression of iNOS was elevated in bacteria-incubated OECs compared to untreated OECs. It was found that in the zinc sulphate treated rat olfactory pathway, bacteria were able to infiltrate the compromised olfactory tissue and that iNOS expression was up regulated in OECs. The potential immune capacity of OECs, as indicated by NO production, is significant in the context of OECs being trialed as an agent of neural repair because of the damaging inflammatory reactions present in injury. from Infectious diseases of the nervous system: pathogenesis and worldwide impact Paris, France. 10–13 September 2008

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.

The human metallothionein gene complex on chromosome 16 has been remapped to 16q13 using high-resolution in situ hybridization. The complex is not disrupted by the rearrangement breakpoint on the long arm of chromosome 16 in patients with myelomonocytic leukemia with abnormal eosinophils, as had been previously reported. The locus order on 16q is cen-MT-FRA16B-D16S4-inversion breakpoint-HP-tel.

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.

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.

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 kappaB), 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 NF kappaB 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.

Metallothionein (MT)-I/II has been shown to be neuroprotective and neuroregenerative in a model of rat cortical brain injury. Here we examine expression patterns of MT-I/II and its putative receptor megalin in rat retina. At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not glial or amacrine cells; megalin was present throughout the retina. Whilst MT-I/II was absent from adult RGC in normal animals and after optic nerve transection, the constitutive megalin expression in RGCs was lost following optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth: more RGCs grew neurites longer than 25 μm (P < 0.05) in dissociated retinal cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA treatment of mixed retinal cultures increased megalin expression in RGCs, and pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth in RGCs and may do so via the megalin receptor; we propose that neurite extension is triggered via signal transduction pathways activated by the NPxY motifs of megalin’s cytoplasmic tail.

Metallothioneins (MTs) are ubiquitous low molecular weight proteins characterized by their abundance of the thiol (SH)-containing amino acid, cysteine. To date four MT isoforms have been identified and cloned in mammals. MT-I and MT-II, the most widely expressed isoforms are generally coordinately regulated in all mammalian tissues; MT-III, is predominantly expressed in zinc (Zn)-containing neurons of the hippocampus; MT-IV is not expressed in brain tissue. The MT proteins have been implicated in gene expression regulation, homeostatic control of cellular metabolism of metals, and cellular adaptation to stress, including oxidative stress. MTs therefore impact on transcription, replication, protein synthesis, metabolism, and numerous other Zn-dependent biological processes. Disordered MT homeostasis leads to changes in brain concentrations of Zn. Since intracellular concentration of Zn are mediated by complexing with apothionein to form MT, there has been great interest in ascertaining whether disordered MT regulation plays a role in the etiology of neurodegenerative disorders. Though abnormalities in MT and/or Zn homeostasis have been reported in multiple neurological disorders a definitive link between MTs and the above disorders remains to be established. The chapter will commence with a brief discussion on the various MT isoforms, their structure and abundance (in brain), followed by a survey on the ability of MTs to potentiate or attenuate neurodegenerative process, with major emphasis on the role of MTs in the etiology of Alzheimer disease (AD).

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. © 2005 Wiley-Liss, Inc.

The precise role of beta-amyloid plaque formation in the cascade of brain cell changes that lead to neurodegeneration and dementia in Alzheimer's disease has been unclear. Studies have indicated that neuronal processes surrounding and within plaques undergo a series of biochemical and morphological alterations. Morphological alterations include reactive, degenerative and sprouting-related 'dystrophic' neuritic structures, derived principally from axons, which involve specific changes in cytoskeletal proteins such as tau and NF triplet proteins. More compact and fibrous plaques are associated with more extensive neuritic pathology than non-fibrillar, diffuse beta-amyloid deposits. Cortical apical dendritic processes are either 'clipped' by plaque formation or are bent around more compact plaques. Examination of cases of 'pathological' brain ageing, which may represent a preclinical form of Alzheimer's disease, demonstrated that the earliest neuritic pathology associated with plaques was similar to the reactive changes that follow structural injury to axons. In vivo and in vitro experimental models of structural injury to axons produce identical reactive changes that subsequently lead to an attempt at regenerative sprouting by damaged axons. Thus, beta-amyloid plaque formation may cause structural injury to axons that is subsequently followed by an aberrant sprouting response that presages neurodegeneration and dementia. Identification of the key neuronal alterations underlying the pathology of Alzheimer's disease may provide new avenues for therapeutic intervention.

In the primary olfactory pathway, olfactory ensheathing cells (OECs) extend processes to envelop bundles of olfactory axons as they course towards their termination in the olfactory bulb. The expression of growth-promoting adhesion and extracellular matrix molecules by OECs, and their spatially close association with olfactory axons are consistent with OECs being involved in promoting and guiding olfactory axon growth. Because of this, OECs have been employed as a possible tool for inducing axonal regeneration in the injured adult CNS, resulting in significant functional recovery in some animal models and promising outcomes from early clinical applications. However, fundamental aspects of OEC biology remain unclear. This brief review discusses some of the experimental data that have resulted in conflicting views with regard to the identity of OECs. We present here recent findings which support the notion of OECs as a single but malleable phenotype which demonstrate extensive morphological and functional plasticity depending on the environmental stimuli. The review includes a discussion of the normal functional role of OECs in the developing primary olfactory pathway as well as their interaction with regenerating axons and reactive astrocytes in the novel environment of the injured CNS. The use of OECs to induce repair in the injured nervous system reflects the functional plasticity of these cells. Finally, we will explore the possibility that recent microarray data could point to OECs assuming an innate immune function or playing a role in modulating neuroinflammation.

One of the primary hallmarks of glutamate excitotoxicity is degradation of the neuronal cytoskeleton. Using a tissue culture approach, we have investigated the relationship between excitotoxicity and cytoskeletal degradation within axons, with particular reference to the axon specific neurofilament proteins. Neurofilaments were rapidly lost from axons over a 24-h period in response to excitotoxic insult (as observed by immunocytochemistry and western blotting), while other axonal cytoskeletal markers (such as betaIII-tubulin) remained intact. Treatment with kainic acid and NMDA, or complementary experiments using the pharmacological glutamate receptors blockers CNQX (kainate/AMPA receptor antagonist) and MK-801 (NMDA receptor antagonist), demonstrated that neurofilament degeneration was mediated primarily by NMDA receptor activity. This work suggests that excitotoxicity triggers a progressive pathway of cytoskeletal degeneration within axons, initially characterised by the loss of neurofilament proteins.

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.

This study investigated the expression of uncoupling proteins 2 and 3 (UCP2 and UCP3) in the carnivorous marsupial Sarcophilus harrisii. The current study used molecular techniques to ascertain whether this species expresses UCP2 and/or UCP3. This species increases nonshivering thermogenesis in response to cold exposure and norepinephrine, although our previous study was unable to demonstrate the presence of brown adipose tissue or uncoupling protein 1. Samples of skeletal muscle and white adipose tissues were taken from five S. harrisii pre- and post-cold acclimation (2 degrees -3 degrees C for 2 wk). The tissues were examined for UCP2 and UCP3 expression through Western blots and reverse transcriptase polymerase chain reaction, with subsequent sequencing to ensure identification of the desired gene. These data suggest that S. harrisii expresses UCP2 but not UCP3. The sequencing of the amplified S. harrisii UCP2 cDNA has revealed a 76% homology with human UCP2 cDNA and a 72% homology with rat UCP2 cDNA. The expression of UCP2 but not UCP3 suggests that UCP2 is conserved from a common ancestor to both the Marsupialia and the Eutheria taxa.

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.

Metallothioneins (MTs) are small, cysteine-rich, metal binding proteins. Their function has often been considered as stress-related proteins capable of protecting cells from heavy metal toxicity and oxidative free radicals. However, recent interest has focused on the brain-specific MT-III isoform, which has neurite-inhibitory properties. To investigate the effect of another MT isoform, human MT-IIA, on neurite growth, we used rat cortical neuron cultures. MT-IIA promoted a significant increase in the rate of initial neurite elongation of individually plated neurons. We also investigated the effect of MT-IIA on the neuronal response to axonal transection in vitro. MT-IIA promoted reactive axonal growth after injury, and, by 18 hr after transection, MT-IIA had promoted axonal growth across the injury tract. Exogenous application of MT-IIA after cortical brain injury promoted wound healing, as observed by a significant decrease in cellular degradation at 4 d after injury. Furthermore, MT-IIA-treated rats exhibited numerous SMI-312-immunoreactive axonal processes within the injury tract. This was in contrast to vehicle-treated animals, in which few axonal sprouts were observed. By 7 d after injury, MT-IIA treatment resulted in a total closing over of the injury tract by microglia, astrocytes, and reactive axonal processes. However, although some reactive axonal processes were observed within the injury tract of vehicle-treated rats, the tract itself was almost never entirely enclosed. These results are discussed in relation to a possible physiological role of metallothioneins in the brain, as well as in a therapeutic context.

This study has identified the expression of uncoupling proteins in a marsupial using molecular techniques. The Tasmanian bettong, Bettongia gaimardi, increases non-shivering thermogenesis (NST) in response to cold exposure and norepinephrine, although previous studies have been unable to demonstrate the presence of brown adipose tissue or uncoupling protein 1 (UCP1). This study used molecular techniques to confirm the absence of UCP1 as well as ascertain if this species expresses UCP2 and/or UCP3. Tissue samples from four B. gaimardi were taken prior to and post-cold exposure at 4-5 degrees C for 2 weeks. The tissues were then examined for UCP1, UCP2 and UCP3 expression using Western blotting. UCP2 and UCP3 were amplified through RT-PCR and subsequently sequenced to confirm molecular identity. Our work confirms that B. gaimardi does not express UCP1 and that this species expresses both uncoupling proteins 2 and 3. The sequencing of the amplified B. gaimardi UCP2 and UCP3 cDNAs have revealed a 74% homology with rat UCP2 cDNA, and 65% homology with rat UCP3 cDNA. Although this work has not yet characterised the functional properties of these proteins in the marsupial, it does suggest a possible mechanism to explain the existence of NST in B. gaimardi.

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.

Inheritance of a particular apolipoprotein E gene polymorphism, the epsilon4 allele, has been associated with elevated risk for Alzheimer's disease and a poor outcome following head injury. The neuronal injury associated with Alzheimer's disease and brain injury may have a number of similarities with the nerve cell changes associated with glaucoma. Thus, we have investigated the association of inheritance of apolipoprotein E allelic isoforms (epsilon2, [epsilon]3, and epsilon4) with relative risk for different forms of glaucoma.Apolipoprotein E genotype was examined in a Tasmanian population sample comprised of glaucoma sufferers with elevated or normal intraocular pressure and compared to a control sample of elderly Tasmanians without glaucoma.Approximately twice as many normal tension (38.0%) and high tension (34.2%) glaucoma cases possessed an epsilon4 allele compared to control cases (18.9%). The odds of epsilon4 carriers having normal tension glaucoma were significantly greater than for epsilon3 homozygotes (odds ratio 2.45, 95% confidence interval [1.02-5.91]) even after adjusting for age and gender (odd ratio 2.87 [1.02-8.05]). The increased odds of high tension glaucoma among [epsilon]4 allele carriers were not significant (adjusted odds ratio 1.53 [0.64-3.68]).The data indicate that, in the Tasmanian population, inheritance of the [epsilon]4 allele is associated with elevated risk for glaucomatous changes that are not related to increased intraocular pressure.

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.

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.

The expression of metallothionein (MT) in the mammalian brain is of great current interest, and several reports localising MT immunoreactivity to specific regions of the adult brain have appeared [1-5]. These have found that MT-I and MT-II expression is widespread in the CNS and is mainly in astrocytes. It appears that both astrocytes [5] and neurons [6] are able to express MT-III, and these findings have been confirmed independently [7].

UNLABELLED: Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II(-/-)) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II(-/-) mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver. CONCLUSION: MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.

BACKGROUND: A major pathological hallmark of AD is the deposition of insoluble extracellular beta-amyloid (Abeta) plaques. There are compelling data suggesting that Abeta aggregation is catalysed by reaction with the metals zinc and copper. METHODOLOGY/PRINCIPAL FINDINGS: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Abeta1-40 and Abeta1-42. This action of MT-2A appears to involve a metal-swap between Zn7MT-2A and Cu(II)-Abeta, since neither Cu10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Abeta aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Abeta induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. CONCLUSIONS/SIGNIFICANCE: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Abeta aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Abeta leaving a metal-free Abeta that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Abeta leaves the Abeta in a Zn-bound, relatively inert form.

The functional human metallothionein (MT) genes are located on chromosome 16q13. We have physically mapped the functional human MT locus by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This analysis defines the genetic limits of metallothionein functional diversity in the human genome.

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.

Ensheathing cells are the glial cells that envelop olfactory axons as they course from the olfactory epithelium to the bulb. They are derived from the olfactory placode and differ from the typical glia in terms of sharing the phenotypes of both astrocytes and Schwann cells. The aims of this study are to review (1) cellular characterisation of ensheathing cells in vivo and in vitro, (2) molecular insight into their growth promoting properties, and (3) their role in olfactory development and potential function as a therapeutic agent for nerve repair. Much of the characterisation of ensheathing cell property has developed from immunohistochemical studies that have been supplemented with new molecular methodologies in recent years. Many pieces of evidence clearly indicate that ensheathing cells actively produce growth-promoting molecules, which act in a paracrine and, in some cases, autocrine manner. However, a review of the available literature also suggests that there is a great deal that remains to be elucidated regarding the cell biology of ensheathing cells, for example, their rate of formation and turnover. In addition, the apparent antigenic heterogeneity as revealed by numerous in vitro studies warrants further analysis, particularly in view of the fact that in recent years these cells have been touted as a possible agent for central nerve repair. New molecular methodologies such as the microarray techniques will prove to be crucial for defining the unique characteristics of ensheathing cells.

Chloroplast DNA based phylogenetic studies using PCR-amplification and digestion (40 species) combined with Southern blotting (23 species) suggest that Angophora and some representatives of the Eucalyptus subgenera Corymbia and Blakella form a monophyletic group. The subgenera Eudesmia, Monocalyptus, Gaubaea, Idiogenes, Symphyoinyrtus and Telocalyptus appear to form a well defined monophyletic group encompassing most of Eucalyptus, but the exact sister taxon to this group remains unresolved. The results suggest that subgenera Corymbia and Blakella are paraphyletic and that Telocalyptus should be submerged within the subgenus Symnphyoinyrtus.

Random amplified polymorphic DNA (RAPD) studies of a natural hybrid swarm between Eucalyptus amygdalina Labill. and E. risdonii Hook.f. and nearby allopatric stands revealed that, despite clear morphological differences, all bands were shared between species. However, frequency differences revealed genetic divergence between species, populations within species, and individuals within populations. Variation was greatest between individuals within populations and lowest between species. For both species, the direction of variation which distinguished the two populations was in a different direction to that which separated the two species, suggesting population differences were not due to introgression but were the result of genetic isolation and/or strong localised selection. Several morphologically typical individuals with intermediate RAPD profiles were detected in the hybrid swarm and nearby allopatric samples of both species, suggesting that some cryptic introgression may be occurring. Controlled F1 crosses generally had closer genetic affinity to E. risdonii, raising the possibility that some parents used may have been advanced generation hybrids. While natural hybrids selected for their intermediate leaf phenotype were usually also intermediate between the two species using RAPD markers, some deviated markedly toward E. risdonii. The study suggests that morphological appearance does not necessarily reflect genetic (RAPD) status and in some cases detectable RAPD differences between spatially close populations of the same species may be as great or greater than the differences between species.

The potential use of restriction fragment length polymorphisms (RFLPs) of chloroplast DNA to determine relationships at higher taxonomic levels in the genus Eucalptus was examined. Chloroplast DNA from 24 species, encompassing representatives of all the subgenera of Eucalyptus as well as one representative of the genus Angophora, was analysed using four 6-base restriction endonucleases. Eighty-four polymorphisms were obtained (twenty-eight autopomorphic) and the data matrix analysed using both cladistic and phenetic approaches. Results provided relatively good congruence with taxonomic perceptions based on morphological traits. Eucalyptus subgenera Blakella and Corymbia appear to be genetically similar to each other and to Angophora, although their phylogenetic relationships are not resolved in this study. Using Angophora alone, or together with the bloodwoods Blakella and Corynthia, as the outgroup for cladistic analysis, the two representatives of Eudesmia examined form a distinctly separate monophyletic group, which appears to be the sister taxon to Idiogenes, Gaubaen, Monocalyptus and Symphyomyrtus. The results provide some support for the close association of Idiogenes, Gauhaea and Monocalyptus and the hypothesis that they are the sister group of Symphyomyrtus. Taxonomically problematic species Eucalyptus guilfoylei, E. microcorys and E. deglupta were included in the study and it was found that E. guilfoylei appeared to diverge prior to the rest of the Symphyomyrtus, E. microcorys near the root of this clade, while the Telocalyptus representative E. deglupta fell within Symplgvontyrtus. The results obtained from the chloroplast DNA data provided independent support for previous morphological studies while generating new hypotheses and highlighting areas requiring closer examination.

Chloroplast DNA was extracted from six species of Eucalyptus (E. perriniana, E. nitens, E. ovata, E. regnans, E. amygdalina and E. risdonii). Digests with four restriction enzymes (Hind III, Xho I, Nco I and Eco RV) revealed restriction fragment length polymorphisms (RFLPs) between subgenera, between species and within species. However, no variation in fragment pattern was detected with Sac II or Pst I. The subgenera Monocalyptus and Symphyomyrtus were clearly differentiated by their RFLP patterns where, with the exception of one outlying specimen of E. amygdalina, 45% of all polymorphic fragments were specific to one or other subgenus. While species from different subgenera and series were well differentiated, it was more difficult to differentiate species within series with the low sample sizes used. However, the average net divergence between species increased with increasing taxonomic distance between species, from 0.02% within series and 0.20% between species from different series within subgenera, to 0.99% of nucleotides per nucleotide site for species from different subgenera. Based on Eco RV digests, the eucalypt chloroplast genome was estimated at 143 kb.