Brain copper transport

Menkes Disease is an X-linked infant onset disorder of copper transport caused by mutations in the ATP7A copper transporter. [1] Seizures, hypotonia, gross motor delays were some of the neurological deficits caused by mutations in ATP7A. The authors of Wikipedia list a series of symptoms of Menkes Disease that rads like a list of every cuprous enzyme in the body. Steven Kaler’s group describes a somatic mutation in ATP7A that occurred during embryonic development such that not every organ system was affected. [1]

Lay summary of Stephen Kaler’s work. Mice without functional ATP7A copper transporters cannot transport Cu+ from inside the gastrointestinal enterocyte to the blood. Cu+ goes in the opposite direction in the choroid plexus. ATP7A is needed to get from inside the enterocyte to the CSF where Cu can be delivered to the brain. Dr Kaler has been working on gene therapy targeted to these long-lived cells.

This is the lay summary. Some details of experiments are presented. Some CopperOne thoughts on how cuprous niacin might bypass the need to directly inject copper into the blood because it cannot be transported from inside the enterocytes to the blood.

ATP7A gene therapy plus Cu(II)Cl2 injections

In another Kaler laboratory used a “mottled brown” mo-br mouse mutation at resembles human Menkes disease.  Unlike the human treatment that includes subcutaneous injection of copper, the mo-br mutation on C57BL/6 background is not rescued by peripheral copper administration.  Neonatal  mo-br mice received lateral ventricle injections of either
adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A), copper chloride, or both.  Brain activity of dopamine-β-hydroxylase, a copper-dependent enzyme, and correction of brain pathology.

According to the authors, choroid cells are not replaced in the adult lifespan of the mouse.  It goes without saying that mice were not were not subjected to intraventicular injection of copper for 300 days.  The adenovirus gene therapy was performed on day 2 and the CuCl2 on day 3.  Note that only copper treatments extend the brain copper. ATP7A gene therapy was needed to improve copper utilization.  Injection of copper and adenovirus ATP7A gene therapy augmented brain copper content.   Brain ratios of dihydroxyphenylacetic acid:
dihydroxyphenylglycol (DOPAC: DHPG) were increased in untreated mo-br mutants, reflecting dopamine-β-hydroxylase deficiency.

A few years later the Kaler group conducted a similar study with a Cu(II) histidine chelate that is injected subcutaneously. The idea is to bypass ATP7A in the basolateral membranes of enterocytes. These injections improve the outcome with those who ATP7A mutation(s) result n a partially functional proteins.

Figure 1 (not shown n this post), the authors performed an adenovirus mock infection with green fluorescence protein and then performed immunohistochemistry to show its distribution.  Since this post is about copper, these data will not be presented.  Figure 2 showed data validating their transfection methodology and that the reduced size ATP7A responded to increasing copper concentration by moving to the cell membrane.  Figure 3 showed some more controls.  About 2x as much reduced size ATP7A was expressed in the brains of transfected wild type mice as was full length ATP7A.  The survival curve of panel 3A is perhaps most interesting to a copper supplement company. 

Panels from Haddad (2018) Figures 3, 4, and 7.

It is not enough to perform ATP7A gene therapy on these mo-br mice.  The wild type survive just fine on  copper in the rat chow.  The gene therapy mice required copper histidinate (1 mg/mL)  in a total dose of 15 mg by subcutaneous injection on the back, flank, or neck region, in three 5-mg doses on day 4, day 5, and day 6.  This is because they still lack fully functional ATP7A transporters in the basolateral membranes of their GI eptithelial cells. This study went on for 300 days.  Figure 3 also showed that the gene transfer was mostly in the brain, with a very low cell turnover.  Vectors per mouse genome were less than one in the heart, liver, and muscle.  Data were not presented for the small intestine, with a very high cell turnover.   Seven of the eight of the Cu histidinate plus AT{7A gene therapy did not make it to day 50 whereas the remaining 8 made it to day 300,  Would less have died by day 50 if they had been injected with cuprous histidinate or cuprous glycinate?

What we find interesting about Figure 5 is its relevance to any copper deficiency syndrome that might have causes other than a dysfunctional to marginally functional ATP7A. 

Figure 5 from Hadadd 2018 [3]

Tying this back to cuprous nicotinic acid

We have thought of transdermal copper administration. Would an injected or transdermal copper in the +1 oxidation state be more available to Ctr1, see next image, and therefore more able to restore normal neurotransmitter levels?

Some transporters we think about

Monocarboxylate transporters MCT are primarily known as transporters of lactate, a byproduct of glycolysis. Lactate is transported by the blood back to the liver where it is converted to pyruvate and then back to glucose. MCT1 was observed in the apical cytoplasmic membrane of some epithelial cells in the choroid plexus MCT4 was found in the basolateral cytoplasmic membrane of small number of epithelial cells. [4] A recent PET study with the MCT1 MCT1 inhibitor AZD3965 demonstrated that MCT1 is transporter for 11C labeled niacin more so in the kidney, heart, and liver than the brain [5] A 1979 traced the CSF, choroid plexus, and brain appearance of 14C nicatinamide and niacin injected intravenously in rabbits rabbits. [6] Nicatinamide, but not niacin, rapidly entered these compartments. A 2007 review echoed these results that the nicotinamide vitamer ofr niacin is what is transported across the blood brain barrier. [7] That these unnamed transporters are low specificity, high capacity, and unidirection with the provision that nicotinamide tends to be rapidly converted to NAD. [7]

A mass spec proteomics study

Choroid plexuses were isolated separately from the right-lateral, left-lateral, third, and fourth ventricles of 6 month old porcine brains. [8] Note that this study also used leptomeninges in addition to the choroid plexus: left, right, 3rd, and 4th ventricles. [8] A method was used to separate CSF (aptical) and blood (basolateral (blood) membrane vesicles of the meninges. MDR1 and OAT1 were considered as blood (dura)- and CSF-facing plasma membrane markers at the BAB. [8] This figure has been modified from Uchida 2020 [8}: It is presumed that apical and bsaolatreal orientations of the transporters are the same in the choroid plexus as they are in the meninges. Ctr1 and ATP7A (starred) have been added. Relative expression of these transporters has been totally neglected in this post.

Adapted from Uchida 2020 Figure 2.
  • MCT1, the monocarboxylate tansporter, is involved in transporting, lactate, pyruvate, and branched chain amino acids.
  • MCT8, the thyroid hormone transporter
  • PEPT2 transports peptides of 2-4 amino acids. Is Pept2 bi-directional?
  • MRP3 transports bile salts
  • xCT Sodium-independent, high-affinity exchange of anionic amino acids with high specificity for anionic form of cystine and glutamate.

Future directions?

We’d like to learn more.

References

  1. Donsante, A., Johnson, P., Jansen, L. A., & Kaler, S. G. (2010). Somatic mosaicism in Menkes disease suggests choroid plexus-mediated copper transport to the developing brain. American journal of medical genetics. Part A, 152A(10), 2529–2534. PMC free article
  2. Donsante, A., Yi, L., Zerfas, P. M., Brinster, L. R., Sullivan, P., Goldstein, D. S., Prohaska, J., Centeno, J. A., Rushing, E., & Kaler, S. G. (2011). ATP7A gene addition to the choroid plexus results in long-term rescue of the lethal copper transport defect in a Menkes disease mouse model. Molecular therapy : the journal of the American Society of Gene Therapy, 19(12), 2114–2123. PMC free article
  3. Haddad, M. R., Choi, E. Y., Zerfas, P. M., Yi, L., Martinelli, D., Sullivan, P., Goldstein, D. S., Centeno, J. A., Brinster, L. R., Ralle, M., & Kaler, S. G. (2018). Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model. Molecular therapy. Methods & clinical development, 10, 165–178. PMC free article
  4. Murakami R, Chiba Y, Nishi N, Matsumoto K, Wakamatsu K, Yanase K, Uemura N, Nonaka W, Ueno M. Immunoreactivity of receptor and transporters for lactate located in astrocytes and epithelial cells of choroid plexus of human brain. Neurosci Lett. 2021 Jan 10;741:135479.
  5. Bongarzone, S., Barbon, E., Ferocino, A., Alsulaimani, L., Dunn, J., Kim, J., Sunassee, K., & Gee, A. (2020). Imaging niacin trafficking with positron emission tomography reveals in vivo monocarboxylate transporter distribution. Nuclear medicine and biology, 88-89, 24–33. PMC free article
  6. Spector R. Niacin and niacinamide transport in the central nervous system. In vivo studies. J Neurochem. 1979 Oct;33(4):895-904.
  7. Spector R, Johanson CE. Vitamin transport and homeostasis in mammalian brain: focus on Vitamins B and E. J Neurochem. 2007 Oct;103(2):425-38. Free article
  8. Uchida Y, Goto R, Takeuchi H, Łuczak M, Usui T, Tachikawa M, Terasaki T. (2020) Abundant Expression of OCT2, MATE1, OAT1, OAT3, PEPT2, BCRP, MDR1, and xCT Transporters in Blood-Arachnoid Barrier of Pig and Polarized Localizations at CSF- and Blood-Facing Plasma Membranes. Drug Metab Dispos. 2020 Feb;48(2):135-145. free article

Published by BL

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