Sj Fung

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.

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.

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.

Metallothionein expression by NG2 glial cells following CNS injury

Abstract.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.

Interactions between metallothionein and cortical neurons in vitro

Bi-directional signaling between Eph receptor tyrosine kinases and their membrane-bound ephrin ligands is required for the regulation of numerous physiological processes including neuronal differentiation and axon guidance. Though it is well established that ephrins possess Eph-receptor independent signaling activities, the signaling pathways that they employ remain largely unknown. Our central hypothesis is that the highly conserved regions of the cytoplasmic domain of transmembrane Bclass ephrins are required to induce a signalling event. Notably, the most well conserved region between all three B-class ephrins is found within the carboxy-terminal 33 amino acid cytoplasmic tail. This region contains five conserved tyrosine residues that when phosphorylated may recruit phospho-tyrosine binding proteins. In addition, there is a short polyproline stretch, as well as a PDZ-binding motif at the extreme carboxy-terminus. To directly investigate the function of the highly conserved cytosolic tail in B-class ephrin reverse signalling, we have constructed a number of ephrin B1 deletion mutants in this region and are currently testing their function both in vitro and in vivo – via virus – and electroporation-mediated gene transfer in NIH3T3 cells and the developing chick neural tube respectively. Intriguingly, in vitro functional analysis of wildtype protein has led to the serendipitous observation that ephrin B1 undergoes proteolytic processing via a gamma-secretase-like cleavage to produce a signalling competent fragment(s). We have therefore also constructed and are similarly testing a construct that lacks the majority of the extracellular domain of ephrin B1 but retains the entire transmembrane and intracellular domains. These experiments will reveal novel insight into the function of the cytosolic regions of ephrin B1 in the development of the chick neural tube.