Ak West

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.

Human metallothionein genes: Structure of the functional locus at 16q13

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.

Metallothionein in the central nervous system: Roles in protection, regeneration and cognition.

Metallothionein (MT) is an enigmatic protein, and its physiological role remains a matter of intense study and debate 50 years after its discovery. This is particularly true of its function in the central nervous system (CNS), where the challenge remains to link its known biochemical properties of metal binding and free radical scavenging to the intricate workings of brain. In this compilation of four reports, first delivered at the 11th International Neurotoxicology Association (INA-11) Meeting, June 2007, the authors present the work of their laboratories, each of which gives an important insight into the actions of MT in the brain. What emerges is that MT has the potential to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions. In this article, the properties and CNS expression of MT are briefly reviewed before Dr Hidalgo describes his pioneering work using transgenic models of MT expression to demonstrate how this protein plays a major role in the defence of the CNS against neurodegenerative disorders and other CNS injuries. His groups work leads to two further questions, what are the mechanisms at the cellular level by which MT acts, and does this protein influence higher order issues of architecture and cognition? These topics are addressed in the second and third sections of this review by Dr West, and Dr Levin and Dr Eddins, respectively. Finally, Dr Aschner examines the ability of MT to protect against a specific toxicant, methylmercury, in the CNS.

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid‐β (Aβ) peptides is a central phenomenon in Alzheimer’s disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ42 fibrillization and initiate formation of non‐fibrillar Aβ42 aggregates, and that the inhibitory effect of Zn(II) (IC50 = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high‐affinity metal chelators including metallothioneins prevented metal‐induced Aβ42 aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ42 fibrillization. Upon prolonged incubation the metal‐induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ42. H13A and H14A mutations in Aβ42 reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross‐β core structure within region 10–23 of the amyloid fibril. Cu(II)‐Aβ42 aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)‐Aβ42 aggregates were non‐toxic. Disturbed metal homeostasis in the vicinity of zinc‐enriched neurons might pre‐dispose formation of metal‐induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal‐chelating therapies for Alzheimer’s disease is discussed in this light.

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.

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.

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.

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 Parkinsons 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.

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).

Characterisation of six additional human metallothionein genes.

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.