ALS TDP-43 disulfides

We have been asked, “Can CopperOne help Amyotrophic lateral sclerosis (ALS) patients given the rumored success of CuATSM clinical trials? This Australian study was accepting patients with familial or sporadic ALS without specifying which proteins (SOD1, TDP-43, or others) are mutated in the familial version when this post was initiated. As of July 25 2023 the results on clincialtials.gov were returned to quality control issues.

Methods: The ALD pilot study was just that. All 12 patient remained on the ALS drug riluzole. Many of the analyses were performed after the patients had died. [1]

• CuATSM and riluzole ALS-TDP (n=5) and ALS-SOD1 (n=1)
• riluzole only ALS-TDP (n=4) and ALS-SOD1 (n=2)

Results: Our results revealed no significant difference in neuron density or TDP-43 burden in the motor cortex and spinal cord of patients that had received CuATSM compared to patients that had not. In patients that had received CuATSM, p62-immunoreactive astrocytes were observed in the motor cortex and reduced Iba1 density was found in the spinal cord. However, no significant difference in measures of astrocytic activity and SOD1 immuno reactivity was found with CuATSM treatment. [1]

Discussion: CuATSM does not significantly alleviate neuronal pathology or astrogliosis in patients with ALS.

Let’s go back to the original CopperOne customer question, “Can CopperOne help patients with ALS?” We have not clue! We need to first understand what this protein TPD-43 is, why it mis folds, and what may be done to prevent it’s mis folding. We’ll meet a few new copper proteins along the way.

1. Exploring TPD-43 misfolding
   1. The structure of TDP-43
   2. TDP-43 Disulfides!
2. DJ-1 proteolysis revisited
   1. A thought experiment summary…
3. References

Exploring TPD-43 misfolding

Mutant, misfolded super oxide dismutase 1(SOD1) protein is commonly associated with ALS. An earlier study suggested that sporadic cases were associated with TDP-43 aggregates that tended not to appear in aggregates familial ALS caused by mutations in Cu/Zn SOD1. [2] Misfolded Tar DNA binding protein (TDP-43) is found in many, as well as some familial, cases of ALS. A search of the protein database of crystal structures reveals few structures with single stranded DNA and many aggregates.

The structure of TDP-43

TDP-43 is normally found in the nucleus. A recent review describes its antics in the mitochondria. [2] “TDP-43 proteinopathies” is a term used to describe detergent resistant uniquitinated (figure 2) protein aggregates. As an added bonus, these protein aggregates also tend to be hyper phosphorylated, ubiquitin tags for destruction, and truncated (Figure 1). The accumulation of cytoplasmic inclusion bodies is generally thought of as a toxic gain of function. Formation of these inclusion bodies is accompanied by loss of normal functions that include RNA metabolism,translation of mRNA into protein, stress-induced responses, and mitochondrial function. [3] Studies have demonstrated the association of TDP-43 with mitochondria in motor neuron-like cells. At least a portion of TDP-43 could localize in the inner membrane of mitochondria; full-length and truncated forms of TDP‐43 could differential reside in the matrix and inter membrane space of mitochondria. [3]

1. TDP-43 controls mitochondrial trafficking along axons [3], Figure 2
2. Motor neuron like cells over expressing TDP-43 have reduced mitochondrial complex I activity, mitochondrial transmembrane potential difference, and increased expression of mitochondrial uncoupling protein 2 (UCP2) that functions to decrease the mitochondrial membrane potential.[3]
3. TDP-43 interacts with mitochondrial RNA
4. TDP-43 may also facilitate autophagy (self eating) destruction of damaged mitochondria. [2]. The authors mention the Dj-1 protease. As we shall see later, Cu²⁺ is toxic to proteolytic function.
5. DJ-1, aka Park7,is a copper cofactor protease localized both in cytoplasm and mitochondria that protects against oxidative stress-induced cell death through the suppression of cytoplasmic TDP-43 aggregation. It was proposed that that the copper enzyme DJ1 may alleviate TDP-43-caused toxicity by degrading both cytoplasmic and mitochondrial TDP-43. [2]
6. Mitochondrial fission and fusion were discussed in this review [3].

In a cell culture model Dj-1 co-expression with TDP-43 reduces protein aggregation in response to the mitochondrial toxin and free radical generator paraquat. [4]

TDP-43 Disulfides!

A disulfide is formed when sulfur atoms between two cysteine amino acids form an S-S bond. These disulfide bonds tend to be structural and can be thought of as structural as the buttons and button holes in a dress shirt. Sometimes disulfides form correctly as the new protein comes off the ribosome. In other instances protein disulfide isomerase “unbuttons and rebuttons” disulfides of misfolded proteins in the endoplasmic reticulum. Aberrant disulfides can be formed when sulfur of cysteine is oxidized to cysteine sulfenic acid. This intermediate plays a role in activation of the IL-1β producing inflammasomes.

Let’s take a look at some of the aberrant disulfide bonds of TDP-43 that have been reported in the literature.

• Lue71m=,Val72 have been shown to be responsible for non-covalent dimer formation and ability to splice the mRNA for the cystic fibrosis chloride channel CFTR. [5] Two cysteines , Cys39 and Cys50, in this N-terminal domain were shown to be for tetramer formation. [6]
• Two forms of oxidative stress, H₂O₂ and sodium arsenite, have been shown to induce TDP-43 disulfide cross-linking in cultured hippocampus neurons and a cell line expressing TDP-43. Mutants in which Cys198 and Cys244 have been mutated to other amino acids cannot form disulfide bonds. [5]
• Cys173 and Cys175 were found to produce intra chain disulfides whereas the others formed inter chain disulfide bonds. [7] Sodium arsentite was used as an indirect means of generating reactive species from the mitochondria.
• A mechanism has been proposed by which H₂O₂ oxidizes one of the two cysteines, Cys198 and Cys244, in the second RNA recognition motif (RRM2). [8]
• An extraordinarily through review was published in 2019 by Prasad and coauthors. [9] These authors even covered some publications that suggested TNP-43 aggregation could be protective.
• The LOPAC®1280 library, a collection of pharma-developed compounds and approved drugs covering most signaling pathways and major drug target classes was applied at 10 µM concentration to the transfected mouse neuroblastoma Neuro2a (N2a) cells for the TDP-43 self-interaction assay. [10] Auranofin was one compound found to prevent aggregate formation. According to PubChem Auranofin is an inhibitor of thioredoxin reductase, an enzyme thought to give tumor cells resistance to the oxidative stress of their environment.[9] On many levels these results are counter intuitive. Have we not been discussing how inter and intra chain disulfide bond formation can seed aggregation?
• The ability of protein disulfide isomerase to prevent aggregation of SOD1 and TDP-43 mutants associated with human ALS adds clarity. [11] Co-expression with functional PDI prevented non-nuclear localization and aggregation in a manner that was dependent on a supply of reduced glutathione.

DJ-1 proteolysis revisited

Protease activity of DJ‐1 lacking C‐terminal α‐helix (DJ‐1ΔH9) that is cleaved under oxidative stress conditions was stronger than that of full‐sized DJ‐1 [12] The most susceptible sequence digested by DJ‐1ΔH9 was valine–lysine–valine-alanine (VKVA).

The optimal conditions of pH 5.5 and 0 mM NaCl. [12]. Nanomolar Cu²⁺ was inhibitory to DJ‐1’s protease activity. The authors did not comment on monovalent Cu⁺. Mutation of the first Cu binding site [13] abolished proteolytic activity[12]. DJ-1 loaded with Cu(II) via CuSO₄ was shown to photo oxidize to Cu(I). [13] Transfer between Cu(I) from site 1 to site 2 as well as Cu deficient Cu/Zn SOD1 has also been demonstrated. [13] An unanswered question is if Cu(I), but not Cu(II), can be transferred from site 1 to site 2 so as to enable proteolysis.

A thought experiment summary…

Let’s imagine dress shirts in a wash with all sorts of oxidative stress agents that produce cysteine sulfenic acids that act as button hooks facilitating aberrant disulfide bond (button) formation. The pin stripe dress shirt becomes cross-linked to the paisley, which becomes cross-linked to the plain white shirt, which becomes cross-linked to the plaid. .. To make matters worse, the sleeves of the dress shirts become intertwined and form amyloids like the C-termini of TDP-43 in figure 1. The first response would be to add some thioredoxin to unbutton the mess. Partial unbuttoning would only allow the plaid dress shirt to become cross-linked, button to the plain white dress shirt…Thioredoxin inhibitor Auranofin could be added to the mix to keep the dress shirt aggregate from growing larger. PDI, with its thioredoxin like domains, could be added to our dress shirt wash. PDI unbuttons the aberrant buttoning and allows for the right button-button hole matches. Protease DJ-1 reduces our dress shirt aggregation by ripping some of the dress shirts down the back into two pieces. The role of Cu(I) in this process is kind of murky. Cu(I) in DJ-1 [13] can load Cu/Zn SOD with its Cu cofactor so that the aberrant buttoning does not occur in our wash in the first place.

References

1. Yang Y, Rowe D, McCann H, Shepherd CE, Kril JJ, Kiernan MC, Halliday GM, Tan RH. Treatment with the copper compound CuATSM has no significant effect on motor neuronal pathology in patients with ALS. Neuropathol Appl Neurobiol. 2023 Jun 15:e12919. PubMed
2. Mackenzie IR, Bigio EH, Ince PG, Geser F, Neumann M, Cairns NJ, Kwong LK, Forman MS, Ravits J, Stewart H, Eisen A, McClusky L, Kretzschmar HA, Monoranu CM, Highley JR, Kirby J, Siddique T, Shaw PJ, Lee VM, Trojanowski JQ. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol. 2007 May;61(5):427-34. Cross Ref
3. Gao J, Wang L, Yan T, Perry G, Wang X. (2019) TDP-43 proteinopathy and mitochondrial abnormalities in neurodegeneration. Mol Cell Neurosci. 2019 Oct;100:103396. Free PMC article
4. Loganathan S, Lehmkuhl EM, Eck RJ, Zarnescu DC.(2020) To Be or Not To Be…Toxic-Is RNA Association With TDP-43 Complexes Deleterious or Protective in Neurodegeneration? Front Mol Biosci. 2020 Jan 10;6:154. Free PMC article.
5. Lei Y, Zhang ZF, Lei RX, Wang S, Zhuang Y, Liu AC, Wu Y, Chen J, Tang JC, Pan MX, Liu R, Liao WJ, Feng YG, Wan Q, Zheng M. DJ-1 Suppresses Cytoplasmic TDP-43 Aggregation in Oxidative Stress-Induced Cell Injury. J Alzheimers Dis. 2018;66(3):1001-1014
6. Jiang LL, Xue W, Hong JY, Zhang JT, Li MJ, Yu SN, He JH, Hu HY.(2017)The N-terminal dimerization is required for TDP-43 splicing activity. Sci Rep. 2017 Jul 21;7(1):6196. Free PMC article.
7. Cohen TJ, Hwang AW, Unger T, Trojanowski JQ, Lee VM. EMBO J. (2012) Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. Embo J.31(5):1241-52. doi: 10.1038/emboj.2011.471. Epub 2011 Dec 23. PMID: 22193716 Free PMC article.
8. Rabdano SO, Izmailov SA, Luzik DA, Groves A, Podkorytov IS, Skrynnikov NR.(2017) Onset of disorder and protein aggregation due to oxidation-induced intermolecular disulfide bonds: case study of RRM2 domain from TDP-43. Sci Rep. 2017 Sep 11;7(1):11161. Free PMC article.
9. Prasad A, Bharathi V, Sivalingam V, Girdhar A, Patel BK.(2019) Molecular Mechanisms of TDP-43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis. Front Mol Neurosci. 2019 Feb 14;12:25. doi: 10.3389/fnmol.2019.00025. eCollection 2019. PMID: 30837838 Free PMC article. Review.
10. Oberstadt M, Stieler J, Simpong DL, Römuß U, Urban N, Schaefer M, Arendt T, Holzer M.(2018)TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole. Sci Rep. 2018 Feb 2;8(1):2248. Free PMC article.
11. Parakh S, Shadfar S, Perri ER, Ragagnin AMG, Piattoni CV, Fogolín MB, Yuan KC, Shahheydari H, Don EK, Thomas CJ, Hong Y, Comini MA, Laird AS, Spencer DM, Atkin JD. (2020) The Redox Activity of Protein Disulfide Isomerase Inhibits ALS Phenotypes in Cellular and Zebrafish Models.Science. 2020 May 22;23(5):101097. Free PMC article.
12. Mitsugi H, Niki T, Takahashi-Niki K, Tanimura K, Yoshizawa-Kumagaye K, Tsunemi M, Iguchi-Ariga SM, Ariga H.(2013) Identification of the recognition sequence and target proteins for DJ-1 protease. FEBS Lett. 2013 Aug 19;587(16):2493-9. doi: 10.1016/j.febslet.2013.06.032. Epub 2013 Jul 2. PMID: 23831022 Free article.
13. Girotto S, Cendron L, Bisaglia M, Tessari I, Mammi S, Zanotti G, Bubacco L.(2014) DJ-1 is a copper chaperone acting on SOD1 activation. J Biol Chem. 2014 Apr 11;289(15):10887-10899. Free PMC article.