It has long been known that the homeostasis of metal ions is a critical requirement for normal cellular health. In the last few decades the role of metals in pathological ageing has also gained increasing attention, with strong preclinical evidence for their role in the onset and progression of numerous neurodegenerative diseases such as Huntington’s disease, schizophrenia, Parkinson’s disease and Alzheimer’s disease [18
]. More recently, utilising the ZnT3 KO mouse line and other methods, we and others have also demonstrated a key role for zinc in “normal” synaptic plasticity and learning/memory via interactions on key neuronal signalling cascades and proteins [7
]. Ultimately, the role of zinc in higher order cognitive function may well intersect with its effect on these and other age- and disease-related pathways. In order to further characterize the ZnT3 KO mouse line, which lacks zinc at the glutamatergic synapse, we have utilised SEC-ICP-MS to undertake an examination of the age-dependent changes in the metalloproteome in these mice. We demonstrate for the first time here that there are distinct changes not only in zinc, but also in the iron and to a lesser extent the copper, metalloproteome across age in the ZnT3 KO animals.
Changes to the normal metal content of a cell or tissue can occur through a variety of mechanisms that may be related to dietary, genetic or other pathways, which can also include metal:metal interactions that affect levels of a given element. Such direct and indirect changes in metals can all precipitate or potentiate biological outcomes. Whilst the cellular levels and distribution of metal is an important factor, recent evidence has also highlighted the notion that the amount of metal associated with a protein, i.e., the metalation state, may be an important determinant of protein function/toxicity. Two examples include metallothionein (MT) and superoxide dismutase. Metallothioneins are a family of proteins that have a capacity to bind various metals, including copper, zinc and cadmium. This sequestration of metals gives MTs a role in metal ion homeostasis and protection from the toxic effects of metals such as arsenic and lead. They also help regulate oxidative stress, and have reported roles in various diseases. One such disease is AD, where it has been reported that there is a “metal-swap” event with zinc-bound MT-3 (Zn7
MT-3) such that MT-3 removes copper from aggregated and soluble β-amyloid-copper complexes (in turn exchanging metal species within MT-3, going from Zn7
MT-3 to Cu4
MT-3; β-amyloid is considered a primary toxic moiety in AD, and is the primary constituent of the β-amyloid plaque that characterizes the AD brain) to then prevent ROS production and subsequent cellular toxicity [23
]. These effects are not seen with fully copper-laden MT-3. Thus, the metal content of MT-3 is a critical determinant of its ability to interact in this AD-related pathway that may be involved in disease pathogenesis. It is a similar scenario for the antioxidant enzyme copper-zinc superoxide dismutase (Cu, Zn-SOD), which exists as two isoforms (SOD1 and SOD3, having activity primarily in the cytoplasm and extracellular space respectively). SOD1 is implicated in the pathogenesis of amyotrophic lateral sclerosis [24
], a rare disease that primarily results in the degeneration of upper and lower motor neurons leading to muscle paralysis and atrophy, but which is also associated with other non-motor symptoms [25
]. SOD1 mutations account for ~15%–30% of familial ALS cases, depending upon the specific population. Whilst there is good evidence that the level of mutant SOD1 correlates well with disease in preclinical models, recent evidence suggests a more complex scenario. Specifically, treatment of the SOD1G37R ALS mouse model with CuII
(atsm), a copper-targeting compound, resulted in improvements in survival and motor function in parallel with an elevation in mutant SOD1 protein levels [14
]. Whilst seemingly paradoxical, the SOD1 was actually converted from metal-deficient to a more stable copper-replete form (holo-SOD1). These data supported the notion that the toxicity of SOD1 may be driven more by its metal content rather than by the level of the mutant protein itself. Together with other literature demonstrating that the pro-oxidant effect of zinc-deficient SOD1 can be modulated by the removal of copper [27
], these data suggest that the metalation state of SOD1 is a key determinant of its function/toxicity.
In this body of work then, we have explored the metalloproteome across age in the ZnT3 KO mouse model, which may represent a phenocopy of AD or advanced ageing. There are a number of limitations of this work. Firstly, these data are snapshots in time and do not necessarily reflect the dynamic change in metals or the metalloproteome that may be occurring across age. One related consideration in this regard is the potential confound introduced by dietary metal content. The long-term modulation of metal intake via the food/water can impact peripheral and central levels of that metal, as well as have flow-on effects for the levels of other metal species (discussed later). Such alterations can then impact both “normal” and “disease-related” cellular chemistry (including the regulation of metal binding proteins and specific pathological proteins), biological pathways and downstream behavioural function (e.g., [28
]). In this study, however, where all animals have received the same standardized metal-replete diet, then this is unlikely to impact our current findings. Secondly, due to the concentration of zinc and its transporters, together with the relative functional significance of zinc in this area, then we have focused only on the hippocampus. Thirdly, we have not performed formal protein identification, and so our interpretation of the data is limited to known elution profiles/standards. Future work would be focused on understanding the specific protein/s represented by the specific peaks we have identified, and would also examine other brain regions. Finally, once proteins have been identified, expression levels will need to be assessed to understand whether the differences observed in this study are driven by a change in metalation of a given protein/s and/or a change in levels of a specific metal binding protein.
Despite these limitations, the data presented provide insight into the metallobiology of the ZnT3 KO animal. As anticipated, there are significant deficits in zinc associated proteins, with all three peaks (P1–P3) identified at three months of age showing significant deficits in zinc load in the ZnT3 KO animals, as compared to the age-matched wildtypes. There were only small and non-significant trends in these same peaks at six months of age. At 18 months however, the P1 fraction, which demonstrated the highest zinc load and greatest difference to wildtype at three months, again showed a robust and statistically significant difference with wildtype animals. Such potential changes in the metalation state of proteins may, together with changes in protein expression, have significant impact on zinc-dependent pathways, such as those related to synaptic plasticity that we have previously published [7
]. Our historical data suggested that there were significant deficits in hippocampal zinc levels at both 3 and 6 months of age in the ZnT3 KO mice [8
], as well as significant and dynamic alterations in a number of different proteins (with some showing increases/decreases at 3 months that were then either exacerbated or reversed at 6 months; these proteins aren’t necessarily “zinc binding” proteins, and so may not contribute to the SEC-ICP-MS traces reported here). In the absence of peaks with elution times that may correspond to “classic” high abundance zinc binding proteins such as SOD and metallothionein [15
], then identification of the possible protein/s in the P1-P3 fractions is not possible without further characterisation.
Whilst changes in the zinc metalloproteome were anticipated, what is perhaps more surprising is that there were changes in the iron, and to a lesser extent the copper, metalloproteome as well. In the iron proteome it is likely that ferritin (P1) is altered at both six and eighteen months of age; whereas for copper the change in fraction P1 (unidentified protein) was only observed at three months of age. What is interesting is that, in contrast to zinc, both the iron and copper levels associated with specific metalloproteins were elevated in the ZnT3 KO mice. As noted earlier, this may reflect an increase in iron and copper binding to specific proteins (such as ferritin and transferrin for iron; and ceruloplasmin, SOD and metallothionein for copper) and/or an increase in expression of those specific proteins.
An interaction between metals, such as zinc/ copper and zinc/ iron, has been widely reported in the past. In the case of copper for example, there are several reports in the clinical literature where zinc supplementation in coeliac disease, sickle cell anemia, acrodermatitis enteropathica and Wilson’s disease results (potentially via an effect on proteins such as metallothionein) in severe copper deficiency [34
]. Similarly, a study in Caco-2 cells demonstrated that there is an inverse relationship between zinc and iron such that increased levels of extracellular zinc applied to the cells results in decreasing intracellular iron levels [38
]. The converse of this finding, where high iron levels impact on zinc absorption, have also been reported in humans (reviewed in [39
]). In our case, the facile explanation for the data presented is that the loss of ZnT3, which results in decreased brain zinc concentrations, has resulted in a compensatory upregulation in specific ZIP proteins that are responsible for the import of zinc into the cytosol and which are known to be rapidly regulated in response to cellular zinc levels (increased under zinc-deficient conditions and vice versa; it should be noted however, that not all ZIPs are responsive to zinc in this way—ZIP5, for example, is regulated in the opposite way [40
]). In the context of the ZnT3 KO mice then, as the ZIP transporters may not be exclusive transporters of zinc (with some of them also handling iron, manganese, copper and cadmium for example) [40
], then the ZnT3 KO mice may have a parallel increase in other metals (such as iron and copper). As the levels and localization of such metals are tightly controlled in a cell and tissue-specific way, then an upregulation of their associated regulatory proteins is also not unexpected. If there is a long-term change in iron in the ZnT3 KO mouse, this may add further complexity to the interpretation of the apparent age-dependent phenotype present in these animals (particularly given that iron is associated with aging and cellular senescence [42
]) and this may all have long-term implications for zinc deficiency conditions. Further study is required to characterize the metalloproteome changes that occur in response to brain zinc deficiency, both from the perspective of zinc and its regulatory partners, but also more broadly for effects on other metals such as iron.