ALS, PARP Diem?

Could the next ALS drug be…a cancer drug?

James Shorter thinks it just might. In three recent papers, researchers in Shorter’s group and that of Nancy Bonini, both at the University of Pennsylvania in Philadelphia, reported that inhibitors of poly(ADP-ribose) polymerases (PARPs) keep the ALS-linked protein TDP-43 in the nucleus where it belongs, and protect neurons from its toxic effects (McGurk et al., 2018; McGurk et al., 2018). Moreover, their work helps explain why cytoplasmic TDP-43 forms aggregates (McGurk et al., 2018).

A spoonful less of sugar? Inhibiting tankyrase may help protect neurons in ALS by reducing the accumulation of TDP-43 in cytoplasmic aggregates, enabling the RNA regulator to return to the nucleus. [Courtesy of McGurk et al., 2018, Molecular Cell.]

PARPs are a family of enzymes that add PAR groups to other proteins. There are 17 known PARPs in mammals, and they have many functions. One is to localize to DNA breaks, recruiting the DNA repair machinery, which includes the ALS-linked protein FUS (Mar 2018 news, Naumann et al., 2018). Cancer cells often already have DNA repair defects, making them especially sensitive to PARP inhibition. Blocking PARP1, and sometimes PARP2, can selectively kill cancer cells because they run out of options to fix their genomes.

PARPs promote cell health in multiple ways. They help regulate transcription, form the mitotic spindle, and manage telomere length. PARylation can also seed liquid-liquid phase separation of proteins, a phase transition critical to protect neurons under stress and to regulate, localize and stabilize essential RNAs (Oct 2015 news; Altmeyer et al., 2015; for review, see Langdon and Gladfelter, 2018; Boeynaems et al., 2018).

Overactivation of PARPs, however can incite inflammation, and may even contribute to neurodegenerative disease. Thus, scientists are increasingly repurposing PARP inhibitors as potential medicines for a variety of conditions, including stroke, traumatic brain injury and cardiovascular disease (Berger et al., 2017).

Two Pathways for TDP-43 Aggregation

Bonini and colleagues first discovered a potential link between PARPs and ALS studying how TDP-43 mediates neurotoxicity. In ALS, this normally nuclear protein accumulates and aggregates in the cytoplasm of neurons. Their approach: identify genes that, when downregulated, would mitigate the toxicity of TDP-43 in the fly eye (McGurk et al., 2018).

“One of the most potent modifiers from that screen was the knockdown of this PAR polymerase or PARP, tankyrase,” said Shorter. “It really gave almost a complete rescue.” Tankyrase is also known as PARP5; it promotes cell cycle progression, telomere elongation and exocytosis, and has also been considered as a cancer drug target.

Tankyrase downregulation also restored the normal lifespan of neuronal fruit fly model of ALS. It promoted nuclear localization of TDP-43 in neurons while diminishing cytoplasmic accumulation.

How could PARP downregulation reduce this neurotoxicity? It’s possible TDP-43 is PARylated, though the researchers saw no evidence of this in culture. What they did see is that TDP-43 binds PAR within its nuclear localization sequence. In addition, TDP-43 co-localized with this sugar in stress granules, membrane-less organelles that store proteins and RNAs to ensure only essential proteins are synthesized under stressful conditions.

All roads that lead to TDP-43 inclusions. In ALS, TDP-43 could aggregate in neurons by multiple mechanisms [Courtesy of Boeynaems and Gitler, 2018, Molecular Cell].

Short-term stress granules could act as a sort of “safe harbor,” preventing the TDP-43 from forming more toxic aggregates, wrote Aaron Gitler and Steven Boeynaems, of Stanford University School of Medicine in California, in a Molecular Cell commentary accompanying the study (Boeynaems and Gitler, 2018). But long-term stress — as in a neuron of a person with ALS — could force TDP into a permanently aggregated, and potentially more dangerous, conformation contributing to the disease.

Normally, stress granules disassemble when no longer needed. But under conditions of prolonged stress, akin to that in ALS, TDP-43 localized to inclusions distinct from stress granules in cultured cells. It also became phosphorylated, a sign it had turned toxic.

The researchers also noted that fragments of TDP-43 that lacked PAR-binding sequences did not accumulate in stress granules under stressful conditions. Instead, they localized to other inclusions.

The results suggest “there are two separate pathways: that you could go the stress granule route, or you could go a fibrillar pathology route,” commented Ben Wolozin of Boston University School of Medicine, who was not involved in the research. “That needs to be investigated more, but I think that could be important.” He speculated that during ALS, some signal—perhaps phosphorylation—shunts TDP-43 away from stress granules and into other aggregates which contribute to neurotoxicity.

In a subsequent study, the researchers found further reason to suspect PARP activity goes awry in ALS (McGurk et al., 2018). People who had ALS had elevated levels of nuclear PAR in their spinal cord motor neurons. That suggests abnormally high PARP activity in those cells.

Alternatively, the high PAR could also be explained by the presence of DNA damage, noted Ted Dawson, a neurologist at the Johns Hopkins University School of Medicine in Baltimore who was not involved in the studies. Other studies have also observed signs of DNA damage and inefficient repair in ALS models and human brain tissue (Aguirre et al., 2005; Kisby et al., 1997).

Repurposing PARP inhibitors?

Could overactivation of PARPs contribute to ALS? The results in fruit fly models of ALS suggest this is a possibility. But if PAR helps promote the recruitment of TDP-43 to stress granules, how does reducing levels of PAR mitigate its toxicity?

TDP-43 aggregates, a toxic sludge? The cytoplasmic buildup of TDP-43 may contribute to ALS by multiple mechanisms. This may include a power drop in neurons due to the inability to replenish mature oligodendroglial populations (see Wang et al., 2018). [Courtesy of Janssens and Broeckhoven, 2013, Human Molecular Genetics. CC BY 4.0.]

Perhaps, Shorter suggested, nuclear import receptors compete with PAR for TDP-43 binding. With less of a PARP around, there might be less PAR, giving the nuclear import system more opportunities to pull TDP-43 back into the nucleus.

To test the idea, they first treated cultures of stressed mammalian cells with three different tankyrase inhibitors. All three diminished the number of TDP-43 aggregates. Then the scientists experimented with cancer drug veliparib, a PARP1/2 inhibitor. In fibroblasts, it too reduced the fraction of cells with TDP-43 aggregates in the cytoplasm.

Next, the researchers tested veliparib in an ALS model. TDP-43 overexpression in rat primary spinal cord cultures cut neuron numbers by a quarter or more, compared to control cultures, but treatment with veliparib restored normal neuron counts.

The results suggest that blocking key PARPs might be beneficial for most cases of ALS, in which TDP-43 aggregates and likely contributes to motor neuron toxicity. This idea joins several other possible ways to manage these inclusions in ALS. To name a few: Shorter and colleagues are zeroing in on nuclear import receptors that can disaggregate TDP-43 (April 2018 news; Guo et al., 2018). Todd Cohen at the University of North Carolina in Chapel Hill is taking aim at an “acetylation switch” in TDP-43 to identify small molecules that can dissolve TDP-43 aggregates, too (September 2017 news, Wang et al., 2017). And, researchers in Japan are developing antibodies that specifically target and destroy these potentially toxic inclusions (July 2018 news; Tamaki et al., 2018).

The big advantage of PARP inhibitors is that a few are already approved for clinical use. “There are a lot of really great compounds out there,” said Shorter. The FDA has approved three PARP1/2 inhibitors for ovarian cancer: olaparib (Astrazeneca), rucaparib (Clovis Oncology), and niraparib (Tesaro). Olaparib and talazoparib (Pfizer) are also used for breast cancer. Rucaparib also inhibits PARP5, as does olaparib at high doses (Haikarainen et al., 2014; Narwal et al., 2012).

Most cancer patients using these medicines tolerate them well in comparison to chemotherapy, at least short-term; there hasn’t been time to collect long-term data (for review, see Mirza et al., 2018). Using pre-approved drugs could speed development of an ALS treatment, Shorter said.

Of course, to treat ALS, the drugs would have to cross the blood-brain barrier. Veliparib and niraparib are known to do so, said Nicola Curtin, who develops cancer treatments at Newcastle University in England, though olaparib and rucaparib don’t (Donawho et al., 2007; Durmus et al., 2015; Mikule and Wilcoxen, 2015; Chalmers et al., 2016).

Questions and Concerns

Many questions remain. For one, Shorter said, it is not clear whether PARP inhibitors break up existing TDP-43 aggregates in neurons, in addition to preventing new ones from forming. That’s important because neurons in people diagnosed with ALS may already have TDP-43 pathology.

Engage. Make It So. There are currently 5 PARP inhibitors available for clinical use to manage certain types of advanced-stage cancer in the US. Imaging tracers are now being developed to facilitate the clinical testing of these drugs for other indications by enabling measurement of target engagement [Courtesy of Carney et al., 2018, Nature Communications. CC BY 4.0].

Dawson noted that it will also be important to determine whether this strategy is of benefit in vertebrate models of the disease.

Moreover, the jury is still out whether this strategy could only be of benefit to TDP-43-positive forms of the disease. For example, some scientists have suggested that it would be beneficial to boost, not inhibit, PARP activity, in FUS ALS. The idea is to give these enzymes extra time to repair DNA breaks in ALS neurons (Mar 2018 news). Indeed, PARP 1/2 inhibitor veliparib treatment can cause FUS to mislocalize and aggregate in the cytosol of cultured cells, further contributing to cytotoxicity (Naumann et al., 2018).

What’s more, the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone treatment resulted in no benefit in a mouse model of SOD1 ALS (Andreassen et al., 2001).

Meanwhile, neurologists including Dawson worry about the long-term use of these drugs in treating chronic neurodegenerative diseases such as ALS.

“If you’re going to be using PARP inhibitors in humans, you’re going to be interfering with the DNA repair process,” he cautioned. “In these chronic diseases, that’ll be one of the things that will need to be taken under consideration.” However, he remains open to this treatment strategy for certain conditions with limited therapeutic options. Dawson and colleagues, in fact, are investigating their use as a potential treatment for Parkinson’s disease (Kam et al., 2018).

“I think in ALS, you’d want to have tankyrase inhibitors — [inhibitors] for PARP5 instead of PARP1/2,” Dawson suggested. These enzymes probably wouldn’t be involved in the DNA repair process, he said, making them potentially a safer option. Several highly selective PARP5 inhibitors are being developed for cancer (Haikarainen et al., 2014), though none have been clinically tested.

“[PARP inhibitors] could be interesting therapeutic options for ALS/FTD,” concluded Gitler and Boeynaems. “They potentially prevent cytoplasmic aggregation and additionally promote nuclear import—preventing, in one fell swoop, the two key steps on the path to pathological TDP-43 aggregation.”

Featured Papers

McGurk L, Gomes E, Guo L, Mojsilovic-Petrovic J, Tran V, Kalb RG, Shorter J, Bonini M. Poly(ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress granule localization. Mol Cell. 2018 Sep 6;71(5):703-717.e9. [PubMed]

McGurk L, Mojsilovic-Petrovic J, Van Deerlin VM, Shorter J, Kalb RG, Lee VM, Trojanowski JQ, Lee EB, Bonini NM. Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis. Acta Neuropathol Commun. 2018 Aug 29;6(1):84. [PubMed]

McGurk L, Gomes E, Guo L, Shorter J, Bonini N. Poly(ADP-ribose) engages the TDP-43 nuclear-localization sequence to regulate granulo-filamentous aggregation. Biochemistry. 2018 Dec 12. [PubMed]

Boeynaems S, Gitler AD. Pour some sugar on TDP(-43). Mol Cell. 2018 Sep 6;71(5):649-651. [PubMed]

References

Naumann M, Pal A, Goswami A, Lojewski X, Japtok J, Vehlow A, Naujock M, Günther R, Jim M, Stanslowsky N, Reinhardt P, Sterneckert J, Frickenhaus M, Pan-Montojo F, Storkebaum E, Poser I, Freischmidt A, Weishaupt JH, Holzmann K, Troost D, Ludolph AC, Boeckers TM, Liebau S, Petri S, Cordes N, Hyman AA, Wegner F, Grill SW, Weis J, Storch A, Hermann A. Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation. Nat Commun. 2018 Jan 23;9(1):335. [PubMed]

Altmeyer M, Neelsen KJ, Teloni F, Pozdnyakova I, Pellegrino S, Grofte M, Rask MB, Streicher W, Jungmichel S, Nielsen ML, Lukas J. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat Commun. 2015 Aug 18;6:8088. [PubMed]

Boeynaems S, Alberti S, Fawzi NL, Mittag T, Pollymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, Van Den Bosch L, Tompa P, Fuxreiter M. Protein phase separation: A new phase in cell biology. Trends Cell Biol. 2018 Jun;28(6):420-435. [PubMed]

Molliex A, Temirov J, Lee J, Coughlin M, Kanagaraj AP, Kim HJ, Mittag T, Taylor JP. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell. 2015 Sep 24;163(1):123-33. [PubMed]

Burke KA, Janke AM, Rhine CL, Fawzi NL. Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II. Mol Cell. 2015 Oct 15;60(2):231-41. [PubMed]

Berger NA, Besson VC, Boulares AH, Bürkle A, Chiarugi A, Clark RS, Curtin NJ, Cuzzocrea S, Dawson TM, Dawson VL, Haskó G, Liaudet L, Moroni F, Pacher P, Radermacher P, Salzman AL, Snyder SH, Soriano FG, Strosznajder RP, Sümegi B, Swanson RA, Szabo C. Opportunities for the repurposing of PARP inhibitors for the therapy of non-oncological diseases. Br J Pharmacol. 2018 Jan; 175(2):192-222. [PubMed]

Aguirre N, Beal MF, Matson WR, Bogdanov MB. Increased oxidative damage to DNA in an animal model of amyotrophic lateral sclerosis. Free Radic Res. 2005 Apr;39(4):383-8. [PubMed]

Kisby GE, Milne J, Sweatt C. Evidence of reduced DNA repair in amyotrophic lateral sclerosis brain tissue. Neuroreport. 1997 Apr 14;8(6):1337-40. [PubMed]

Wang P, Wander CM, Yuan CX, Bereman MS, Cohen TJ. Acetylation-induced TDP-43 pathology is suppressed by an HSF1-dependent chaperone program. Nat Commun. 2017 Jul 19;8(1):82. [PubMed]

Ciryam P, Lambert-Smith IA, Bean DM, Freer R, Cid F, Tartaglia GG, Saunders DN, Wilson MR, Oliver SG, Morimoto RI, Dobson CM, Vendruscolo M, Favrin G, Yerbury JJ. Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS. Proc Natl Acad Sci U S A. 2017 May 16;114(20):E3935-E3943. [PubMed]

Jackrel ME, DeSantis ME, Martinez BA, Castellano LM, Stewart RM, Caldwell KA, Caldwell GA, Shorter J. Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events. Cell. 2014 Jan 16;156(1-2):170-82. [PubMed]

Guo L, Kim HJ, Wang H, Monaghan J, Freyermuth F, Sung JC, O’Donovan K, Fare CM, Diaz Z, Singh N, Zhang ZC, Coughlin M, Sweeny EA, DeSantis ME, Jackrel ME, Rodell CB, Burdick JA, King OD, Gitler AD, Lagier-Tourenne C, Pandey UB, Chook YM, Taylor JP, Shorter J. Nuclear-import receptors reverse aberrant phase transitions of RNA-binding proteins with prion-like domains. Cell. 2018 Apr 19;173(3):677-692.e20. [PubMed]

Haikarainen T, Narwal M, Joensuu P, Lehtiö L. Evaluation and structural basis for the inhibition of tankyrases by PARP inhibitors. ACS Med Chem Lett. 2013 Nov 20;5(1):18-22. [PubMed]

Donawho CK, Luo Y, Luo Y, Penning TD, Bauch JL, Bouska JJ, Bontcheva-Diaz VD, Cox BF, DeWeese TL, Dillehay LE, Ferguson DC, Ghoreishi-Haack NS, Grimm DR, Guan R, Han EK, Holley-Shanks RR, Hristov B, Idler KB, Jarvis K, Johnson EF, Kleinberg LR, Klinghofer V, Lasko LM, Liu X, Marsh KC, McGonical TP, Meulbroek JA, Olson AM, Palma JP, Rodriguez LE, Shi Y, Stavropoulos JA, Tsurutani AC, Zhu GD, Rosenberg SH, Giranda VL, Frost DJ. ABT-888, an orally active poly(ADP-ribose) polymerase inhibitor that potentiates DNA-damaging agents in preclinical tumor models. Clin Cancer Res. 2007 May 1;13(9):2728-37. [PubMed]

Durmus S, Sparidans RW, van Esch A, Wagenaar E, Beijnen JH, Schinkel AH. Breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-GP/ABC1) restrict oral availability and brain accumulation of the PARP inhibitor rucaparib (AG-014699). Pharm Res. 2015 Jan;32(1):37-46. [PubMed]

Andreassen OA, Dedeoglu A, Friedlich A, Ferrante KL, hughes D, Szabo C, Beal MF. Effects of an inhibitor of poly(ADP-ribose) polymerase, desmethylselegiline, trientine, and lipoic acid in transgenic ALS mice. Exp Neurol. 2001 Apr;168(2):419-24. [PubMed]

Kam TI, Mao X, Park H, Chou SC, Karuppagounder SS, Umanah GE, Yun SP, Brahmachari S, Panicker N, Chen R, Andrabi SA, Qi C, Poirier GG, Pletnikova O, Troncoso JC, Bekris LM, Leverenz JB, Pantelyat A, Ko HS, Rosenthal LS, Dawson TM, Dawson VL. Poly(ADP-ribose) drives pathologic a-synuclein neurodegeneration in Parkinson’s disease. Science. 2018 Nov 2;362(6414). [PubMed]

Further Reading

Langdon EM, Gladfelter AS. A new lens for RNA localization: Liquid-liquid phase separation. Annu Rev Microbiol. 2018 Sep 8;72:255-271. [PubMed]

Narwal M, Venkannagari H, Lehtiö L. Structural basis of selective inhibition of human tankyrases. J Med Chem. 2012 Feb 9;55(3):1360-7. [PubMed]

Mirza MR, Pignata S, Ledermann JA. Latest clinical evidence and further development of PARP inhibitors in ovarian cancer. Ann Oncol. 2018 Jun 1;29(6):1366-1376. [PubMed]

Haikarainen T, Krauss S, Lehtio L. Tankyrases: Structure, function and therapeutic implications in cancer. Curr Pharm Des. 2014;20(41):6472-88. [PubMed]

disease-als DNA breaks DNA damage DNA repair neuroprotection neurotoxicity niraparib olaparib PARP poly ADP-ribose polymerase rucaparib talazoparib tdp-43 topic-preclinical
Share this:
Facebooktwittergoogle_plusmailFacebooktwittergoogle_plusmail