Copper and aging brain

This review carried the torch of Dr Brewer in trying to shed light on the hazards of using copper (II) sulfate to control pathogens on food crops to the international community. This review cited the literature on links to Alzheimer’s Disease and alternatives to CuSO₄ to control pathogens. This review did not discuss whether the DCuSO₄ used to control pathogens gets incorporated into nicotianamide or some other less toxic compound.

The use of ⁶⁴Cu PET imaging is continuing to teach us things about how copper moves about mammalian bodies, in particular the brain. This particular study was a collaboration between UT Southwestern and Wayne State. Production of the tracer ⁶⁴CuCl₂ required a biomedical cyclotron and ⁶⁴Ni(p,n)⁶⁴Cu at Washington University (St Louis, MO) A 0.1M HCl solution was used to dissolve the radioactive tracer. The goal was to follow ⁶⁴CuCl₂ orally ingested in a common strain of mice, C57BL/6 mice, at various ages.

Figure 1 from Peng 2018. Note that both the copper and glucose analog FDG were given orally.

¹⁸F-FDG, or 2-deoxy-2-[F-18]-fluoro-D-glucose is used to visualize where the glucose is going in the circulation. What makes this study unique is the oral dosing, ⁶⁴CuCl₂ (2 μCi(74 kBq)/g body weight) diluted in normal saline. Delivery was via a blunted oral feeding tube, followed 15min static whole-body imaging at 24 hours. It was the heart and kidney that showed the greatest age-related differences.

Note that the percentages of the initial dose don’t come even close to adding up to 100% Figure 2 gives us a reason as to why.

Note the large amount of the ⁶⁴Cu is in the bowel. The % initial doses do not seem to add up to be 100%. How much of the initial ⁶⁴CuCl₂ was excreted in the urine or feces? Are the blue speckles underneath the red labeled object labeled “Bowel” feces? The liver is green with yellow horizontal stripes. Is this indicative of ⁶⁴Cu being incorporated into a chaperone protein like ceruloplasmin?

The glucose utilization does not seem to follow copper deposition. For some reason muscle and renal utilization seem to increase the most in going from middle to old age.

In going from middle age to old mice, muscle and kidneys showed the greatest proportional increase. The most telling nuance is the whole animal imaging.

The metabolic demands of the liver are fairly high. The liver uptake of glucose might be high when conditions are conducive for production of glycogen. Note that mice were given FDG orally prior to a fast. “Bowel” here likely refers to the small intestine. Also note that there are not little balls going down that would be suggestive of this glucose analog being lost in the feces.

Really no change in Cu uptake in the aging mouse brain?

The apparent no change in the aging mouse brain masks in increase in brain stem and decreases in the hypothalamus/thalamus and basal ganglia.

The FDG measurement of glucose utilization was lower in all regions in the brain in going from 8 to 11 months except the basal ganglia and brain stem. These regions experienced an increase in copper. Is this maintenance of FDG due to maintenance of circulation and dilation of cerebral blood vessels?

Just to round things up, this these are the data for FDG uptake

It would be interesting to see these experiments repeated with a Copper One supplement like Dr George Brewer recommended.

Cu²⁺ handling by presenilin

Ca²⁺ is a natural part of the synaptic cleft function. [3] The presenilin story comes from Ashley Bush’s lab that has long studied how Xu²⁺ modulates ion channels and what not in the synaptic cleft. This is all a good thing with mechanisms of cleaning up the Cu²⁺.

Presnilins, clean up proteins and a protease

The γ-secretase is a multiprotein complex that digests integral memembrane proteins. Presenilins 1 and 2 are aspartate protease members of this complex. The amyloid precursor protein (APP) is the most famous target of the γ-secretase complex. When it comes to PSEN1 and PSEN2 mutations that are associated with Alzheimer’s the mutants exhibit altered processing of APP.

Ctr1 is the main importer of Cu+ to the cell, However, 30% of cellular copper import occurs by uncharacteristic mechanisms according to the Bush group’s review of the literature in 2011..The Bush group mentions transporters for Zn²⁺ and a few of the many enzymes that use Zn²⁺ as a cofactor. We know that divalent cations like Zn²⁺ and Cu²⁺ may catalyze amyloid formation.

Uptake of 0.4MBq of 64CuCl₂ (Australian Radiopharmaceuticals and Industrials) and unlabeled CuCl₂. Copper was determined in Hank’s balanced salt solution. Uptake was stopped by swashing the cell monolayers of mouse embryonic fibroblasts with ice-cold nonlabeled HBSS (with 2 mM L-histidine).

1A The double PSEN1/2 knockout fibroblasts exhibited lower Cu²⁺ uptake compared to the wildtype at 37^oC. The difference was lost at 4oC
1B Time dependent uptake of 2.5 µM ⁶⁴CuCl₂ at 37^oC. Loss of PS1 or PS1/2 results in loss of copper uptake.
1C Actual incorporation of copper into proteins is another matter.
1D Rescuing the double Knock Out with human versions of PS1 does not seem to help.

Figure 3 [4] ..Presenilins influence the activity of Cu/Zn SOD1. The assay used mouse embryonic fibroblasts. The D385A mutant of human PS1 is catalytically inactive when it comes to protease activity, but is capable of doubling the Cu/An SOD1 activity. The double knock PS cells had virtually no SOD1 activity suggestive of inability to metalate the enzyme. Expression of SOD1 was fine (3B). Actin is a housekeeping protein used as a loading control.

Figure 4 CCS is the copper chaperone for SOD1. CCS is responsible for ensuring that Cu⁺ is properly inserted into SOD1. Not having both copies of PS translate into not having as much CCS transcribed as mRNA and/or translated into the CCS protein that can be detected in a Western blot (Figure 3B)

Figure 5 looked at Cu²⁺ and Zn²⁺ content in soluble and insoluble proteins by tissue, eg brain, kidney, heart… in wild type mice and mice having only one of two copies of the PSEN1 gene. While there as a statistically significant drop from about 65 to 55µg Cu per g soluble protein in the brain, it is uncertain if this difference is functionally significant. Likewise, small but significant decreases in were seen in SOD1 activity and CCS protein amounts in PSEN^-/+ mice compared to PSEN^+/+ mice, Fig 6.

The authors took a likely different approach. They started out with a human embryonic kidney cell line, HEK293T and prevented the mRNA transcripts for PS1 and PS2 from being translated into protein by use of silencing RNA (siRNA) Scrambled siRNA is the same complementary sequences scrambled such that it cannot bind to the untranslated mRNA.

The authors demonstrated that silencing of mRNA translation into proteins resulted in a small but significant decrease in Cu loading of proteins (7A) and perhaps a 50% decrease in Zn protein incorporation (7B). Panel 7C shows that the siRNA was successful in knocking down mRNA translation into presenilin proteins. The authors finished up their story in Figure 8 that presented data showing that pharmacological inhibition of protease activity did not affect the Zn and Cu loading.

Concluding statements of the authors brought the reader back to the laboratory’s primary interest: extra-neuronal Cu²⁺ and Zn²⁺ released into the synaptic cleft. They cited evidence supporting hyper metallation of A by divalent cations in the synaptic cleft. In glutamatergic synapses, Zn²⁺ and Cu²⁺ are uniquely high. [3]

Cu distribution tissue and subcellular

Peng and coauthors demonstrated that Cu²⁺ distributes distributes differently in young, middle, and old mice. [2] They did not make a big deal of it, but it seemed that most of the copper was being lost in the feces. A parallel study needs to be performed using a more bio available Cu⁺ source, in our opinion.
Greenough and coauthors made a thoughtful case for presenilin being an alternative way to load proteins with Cu²⁺ in fibrobasts and in synaptic cleft. [4] These results suggest that nature has a clever way of recycling Cu²⁺ from the synaptic cleft before it aggregates with Aβ or otherwise cause problems. Why overload this system by drinking water from copper pipes, eating produce treated with CuSO_4, or taking cupric dietary supplements.

References

Coelho, F. C., Squitti, R., Ventriglia, M., Cerchiaro, G., Daher, J. P., Rocha, J. G., Rongioletti, M., & Moonen, A. C. (2020). Agricultural Use of Copper and Its Link to Alzheimer’s Disease. Biomolecules, 10(6), 897. PMC free article
Peng, F., Xie, F., & Muzik, O. (2018). Alteration of Copper Fluxes in Brain Aging: A Longitudinal Study in Rodent Using ⁶⁴CuCl₂-PET/CT. Aging and disease, 9(1), 109–118. https://doi.org/10.14336/AD.2017.1025 PMC free article
Opazo, C. M., Greenough, M. A., & Bush, A. I. (2014). Copper: from neurotransmission to neuroproteostasis. Frontiers in aging neuroscience, 6, 143. PMC free article
Greenough, M. A., Volitakis, I., Li, Q. X., Laughton, K., Evin, G., Ho, M., Dalziel, A. H., Camakaris, J., & Bush, A. I. (2011). Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity. The Journal of biological chemistry, 286(11), 9776–9786. PMC free article