Roger S. Chung

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.

Characterisation of the Expression of NMDA Receptors in Human Astrocytes

Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS). However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs) in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN). Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH) activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B) are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.

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 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.

Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer's disease brain.

To assess the role of the kynurenine pathway in the pathology of Alzheimers disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

Recent rodent models for Alzheimer’s disease: clinical implications and basic research

Alzheimer’s disease (AD) is the most common origin of dementia in the elderly. Although the cause of AD remains unknown, several factors have been identified that appear to play a critical role in the development of this debilitating disorder. In particular, amyloid precursor protein (APP), tau hyperphosphorylation, and the secretase enzymes, have become the focal point of recent research. Over the last two decades, several transgenic and non-transgenic animal models have been developed to elucidate the mechanistic aspects of AD and to validate potential therapeutic targets. Transgenic rodent models over-expressing human β-amyloid precursor protein (β-APP) and mutant forms of tau have become precious tools to study and understand the pathogenesis of AD at the molecular, cellular and behavioural levels, and to test new therapeutic agents. Nevertheless, none of the transgenic models of AD recapitulate fully all of the pathological features of the disease. Octodon degu, a South American rodent has been recently found to spontaneously develop neuropathological signs of AD in old age. This review aims to address the limitations and clinical relevance of transgenic rodent models in AD, and to highlight the potential for O. degu as a natural model for the study of AD neuropathology.

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.

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.