Justin Dittmann

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.

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

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

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.

Mid-life environmental enrichment increases synaptic density in CA1 in a mouse model of Aβ-associated pathology and positively influences synaptic and cognitive health in healthy ageing

Early‐life cognitive enrichment may reduce the risk of experiencing cognitive deterioration and dementia in later‐life. However, an intervention to prevent or delay dementia is likely to be taken up in mid to later‐life. Hence, we investigated the effects of environmental enrichment in wildtype mice and in a mouse model of Aβ neuropathology (APPSWE/PS1dE9) from 6 months of age. After 6 months of housing in standard laboratory cages, APPSWE/PS1dE9 (n = 27) and healthy wildtype (n = 21) mice were randomly assigned to either enriched or standard housing. At 12 months of age, wildtype mice showed altered synaptic protein levels and relatively superior cognitive performance afforded by environmental enrichment. Environmental enrichment was not associated with alterations to Aβ plaque pathology in the neocortex or hippocampus of APPSWE/PS1dE9 mice. However, a significant increase in synaptophysin immunolabeled puncta in the hippocampal subregion, CA1, in APPSWE/PS1dE9 mice was detected, with no significant synaptic density changes observed in CA3, or the Fr2 region of the prefrontal cortex. Moreover, a significant increase in hippocampal BDNF was detected in APPSWE/PS1dE9 mice exposed to EE, however, no changes were detected in neocortex or between Wt animals. These results demonstrate that mid to later‐life cognitive enrichment has the potential to promote synaptic and cognitive health in ageing, and to enhance compensatory capacity for synaptic connectivity in pathological ageing associated with Aβ deposition.

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.