The method presented here combines biochemical subcellular fractionation with the precision of GF-AAS to detect zinc present in synaptic vesicles. The zinc concentration was found to be increased in the extracellular enriched and synaptic vesicle fractions isolated from the hippocampus of AD patients as compared to age-matched controls. Furthermore, this approach is particularly amenable to autopsy tissue where traditional techniques such as histological and fluorescence detection of zinc are often unsatisfactory.
The method described here allows for more informative zinc measurements to be made as compared to previous approaches measuring metals in whole tissue pieces or total homogenates. Many metals, such as zinc, act as cofactors bound to various proteins. Zinc is essential for correct function of many proteins such as transcription factors, ion channels, enzymes and receptors (Bitanihirwe and Cunningham, 2009
). In addition, the expression of metallothioneins, which are important functional zinc-binding proteins (Chung and West, 2004
), is tightly regulated by zinc itself (isoforms 1 and 2) (Bitanihirwe and Cunningham, 2009
) in that the expression of this class of proteins increases when zinc concentration is increased. Consequently, overall increases of the bound zinc pool levels may not be dishomeostatic as bound zinc can trigger mechanisms (e.g., increased expression of metallothioneins) that modulate its own impact/availability. On the other hand, increases in the levels of chelatable (releasable) zinc pool can quickly perturb homeostasis, especially free zinc that is released from synaptic vesicles into the microenvironment of the synaptic cleft during synaptic stimulation. Under these conditions, abnormal zinc interactions with the postsynaptic element can profoundly affect neurotransmitter receptor function and thus alter synaptic transmission (Quinta-Ferreira and Matias, 2005
; Weiss et al., 1993
). Furthermore, compelling evidence suggests that released free zinc can coordinate with amyloid beta, stabilize the toxic oligomeric form, and promote their pathological targeting of the postsynaptic element (Deshpande et al., 2009
; Noy et al., 2008
). Therefore, by isolating the synaptic vesicles where the releasable zinc is stored as described here, accurate measurements of the zinc pool most significant to AD neuropathology can be achieved.
Only around 5 % of the zinc in the brain is releasable into the synaptic cleft, and therefore detecting changes in these levels is difficult (Frederickson et al., 2000
). Histochemical techniques, such as the various Timm’s methods for silver staining, or fluorescent dyes can be utilized to measure this zinc population. However, these methods work optimally only in fresh tissue. Silver staining techniques can be used in autopsy tissue for qualitative detection of which cell type/areas contain chelatable zinc, but cannot measure the concentration (Stoltenberg et al., 2005
) and staining quality becomes unsatisfactory as soon as 6 hr postmortem (Danscher et al., 1997b
). On the other hand, zinc fluorescent dyes, such as TSQ, are highly sensitive but staining varies substantially depending on the PMI and freezing conditions of the tissue (Suh et al., 1999
). This severely limits the number of samples that can be compared as the PMI is one condition that varies substantially within a cohort of autopsy tissue specimens. As shown in , such limitations is less pressing when using the method described here in that there is no correlation between PMI and variability of vesicular zinc concentration in the different sample tissue. Furthermore, our tests show that there is no apparent contamination of the synaptic vesicles zinc from other zinc pools released in the homogenate during the fractionation procedure. This is of particular importance when working with AD brain samples that contain zinc-enriched amyloid plaques from which substantial amounts of zinc may be released during tissue homogenization/processing.
The increase in zinc concentrations that we observed in the extracellular enriched fractions prepared from the AD hippocampi, which include zinc released from the zinc-rich amyloid plaques (Suh et al., 2000
), is consistent with findings previously reported (Danscher et al., 1997a
; Deibel et al., 1996
; Religa et al., 2006
). More significantly, we found consistently higher concentrations of releasable zinc in synaptic vesicle isolated from AD brain tissue. Notably, when comparing AD to age-matched controls, such an increase in zinc in the synaptic vesicles (+178%) was much more pronounced than the increase in zinc levels determined in the extracellular fraction (+45%). This could suggest that during synaptic activity, more zinc may be released in the synaptic cleft in the AD hippocampus and thus negatively impact the post-synaptic element and/or bind soluble amyloid beta within the synaptic space. This collective evidence strongly indicates that zinc regulation is altered in AD and suggests that such disregulation may play a key role in AD pathology. Indeed, recent studies have highlighted the importance of the maintenance of proper zinc homeostasis and that upsetting the balance of zinc can have profound effects on cognitive integrity. For example, zinc supplementation has been shown to delay memory deficits in the transgenic mouse model of AD, 3xTg-AD, and increases levels of the neurotrophic factor brain-derived growth factor, BDNF (Corona et al., 2010
). On the other hand, the zinc transporter 3 (ZnT3) KO mouse, which cannot transport releasable zinc into synaptic vesicles, has significant age-dependent learning and memory deficits (Adlard et al., 2010
In conclusion, we have here described a method to measure releasable, synaptic vesicle-associated zinc that is quantitative, reproducible, and has minimal sensitivity to factors such as variability of PMI among different samples. This method is particularly useful to studies concerned with determining the pathological significance of brain zinc levels in neurodegenerative conditions such as AD where releasable synaptic zinc has been proposed to play a key role in toxic amyloid beta oligomer formation and their dysfunctional targeting to post-synaptic elements.
►Zinc homeostasis is altered in Alzheimer’s disease. ►Synaptic vesicles were isolated from Alzheimer’s disease (AD) and age-matched frozen hippocampal autopsy tissue. ►Graphite furnace atomic absorption spectrophotometry (GF-AAS) was used to measure zinc in the synaptic vesicles. ►Synaptic vesicle zinc or chelatable zinc was significantly increased in AD hippocampal samples. ►The combination of biochemical fractionation and GF-AAS allows quantitative analysis of chelatable zinc concentration from autopsy tissue.