Boosting autophagy helps cells dispose of toxic forms of TDP-43 and enhances survival in cellular models of amyotrophic lateral sclerosis, according to findings reported in the June 29 Nature Chemical Biology online. Researchers from the Gladstone Institute of Neurological Disease in San Francisco monitored TDP-43 degradation at a single-cell level and identified approved drugs that help cells dismantle the nefarious protein. Related compounds might treat Huntington’s and Parkinson’s diseases as well, suggested senior author Steven Finkbeiner.

In TDP-43 proteinopathies, including ALS and frontotemporal dementia, the protein accumulates in soluble and aggregated forms. Finkbeiner and colleagues, including first author Sami Barmada, who has moved to the University of Michigan in Ann Arbor, suspected that they could return TDP-43 levels to normal by ratcheting autophagy up a notch. While some previous research suggests more autophagy can help in ALS models (see Sep 2009 news story), other work implies it would be detrimental  (Zhang et al., 2011). The equivocal results stem partly from off-target effects of the molecules used to induce autophagy, as well as different cell types used in various studies.

Finkbeiner’s group looked for autophagy inducers that would be more specific. They had previously identified a compound, called NCP (10-(4’-(N-diethylamino)butyl)-2-chlorophenoxazine), that turns on autophagy in neurons (Tsvetkov et al., 2010). Based on the chemical structure of that molecule, study co-author Michael Pleiss screened a computer library of more than one million compounds. He found two with a similar shape, fluphenazine and methotrimeprazine, which are both FDA-approved antipsychotics. These, and NCP, amped up assembly of autophagosomes and shortened the half-life of an autophagy biomarker in the rodent cortical neurons, the researchers found.

Measuring Turnover with Precision
Barmada and colleagues wanted to focus on the degradation of TDP-43 in cells treated with the drugs. First they developed a method to accurately measure protein turnover. Traditionally, researchers have done this by radioactively labelling the protein, waiting a certain time, then measuring how much remained after capturing it with antibodies. However, there are some problems with that method, Barmada said. For one, the protein purification is only as good as the antibody, and the process might or might not pick up aggregated proteins. For another, TDP-43 can kill cells, which would remove their protein from the analysis, making the protein’s half-life look shorter than it is. Instead, Barmada and colleagues measured TDP-43 turnover in single cells by microscopy (Tsvetkov et al., 2013). “We think it is a little more accurate than traditional measures because we can account for the contributions of cell death and [protein] aggregation,” Barmada said. 

The researchers used an optical pulse chase method. They labeled TDP-43 with the fluorescent protein Dendra2 and expressed it in rat primary cortical neurons. Dendra2 starts out green, but switches to red if hit with blue light. By exposing the cells to a pulse of blue light, Barmada labeled all the TDP-43 at that time point. He then followed the disappearance of the red fluorescence in each cell over time with automated microscopy. Barmada calculated turnover of soluble TDP-43 only, by excluding cells with visible inclusions. He found that wild-type, soluble TDP-43 had a half-life of 68 hours, and a mutant version linked to disease lasted less time, with a half-life of 50 hours. To Barmada, this suggested the cells sensed the abnormal mutant protein and attempted to eliminate it.

These data conflict with several other studies that reported mutant TDP-43 hung around longer than the wild type (see Jul 2010 news story on Ling et al., 2010Watanebe et al., 2013Austin et al., 2014). Those studies measured turnover in cell populations that would have had protein aggregates. When Barmada calculated his optical pulse-chase results for all cells as a population, including those with aggregates, he obtained similar half-lives as the standard radioactive assay. “The stability of TDP-43 mutants is still being debated,” commented Jemeen Sreedharan of the University of Massachusetts Medical School in Worcester, who was not involved in the study (see full comment below).

Drug Effects
Having perfected his measurements for TDP-43 turnover, Barmada turned to the autophagy-inducing drugs. When he treated TDP-43-expressing cells, he found by the optical chase assay that all three molecules shortened the half-life of wild-type and mutant TDP-43. The latter formed fewer inclusions in cells treated with fluphenazine or methotrimeprazine. Moreover, rodent neurons overexpressing wild-type and mutant TDP-43 lived longer on the two medications, as did motor neurons derived from iPS cells from a person with ALS due to mutant TDP-43. 

“The article is technically elegant and conceptually impressive,” commented Robert Brown, also of the University of Massachusetts Medical School in Worchester, who was not involved in the work. “I think the critical next step will be to test this concept in TDP-43 ALS mice,” he wrote to Alzforum.

The study authors have not yet done so, Finkbeiner told Alzforum, in part because he doubts current TDP-43 mice are suitable for preclinical studies (see Sep 2012 news story). Other researchers have tested such mice with rapamycin, which turns up autophagy but also has other effects, and found it improved memory and motor function (Wang et al., 2013Wang et al., 2012). In addition, one small clinical trial in Poland reported that a compound related to fluphenazine, called pimozide, slowed ALS progression (Szczudlik et al., 1998). While Barmada does not put much stock in this 44-person study, he said it offered a hint that the autophagy-augmenting treatment might indeed help people with the disease. Sreedharan wrote that even those with sporadic ALS, not due to TDP-43 mutations, might benefit,  because that disease also involves TDP-43 pathology.

Fluphenazine and methotrimeprazine are known to cross the blood-brain barrier. However, they are also powerful dopamine receptor blockers, and might have undesirable side effects for ALS patients. Finkbeiner wrote that the researchers have already modified both molecules to improve the effects on autophagy and brain entry while removing chemical groups that might cause unwanted side effects. He has filed a patent application for the new, modified compounds and is in discussions with several companies to develop these medications to treat a variety of neurodegenerative diseases where correcting protein homeostasis could be beneficial.—Amber Dance

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Comments on News and Primary Papers

  1. This is quite an intricate paper, as is the authors' method for studying TDP-43-mediated toxicity, which speaks to the fact that TDP-43 levels must be regulated carefully (it does of course regulate its own expression). Overall, the study is well done. Perhaps the most significant observation is that inducing autophagy with compounds that could potentially be used in humans seemed to work in human cells in vitro.

    In terms of the biology of TDP-43, it is already known that too much of this protein is a bad thing. That the authors quantified this while taking into account cell death and aggregation, which are confounding factors in the study of protein turnover, is an important contribution and may help resolve some previous discrepancies in the literature on mutant TDP-43 half-life. The stability of TDP-43 mutants is still being debated.

    For sporadic, or non-familial, ALS (sALS), the paper is highly relevant, as it is based on TDP-43 biology. I don't think it is a coincidence that WT TDP-43 aggregates in sALS, and that TDP-43 gene mutations occur in ALS as well. Whatever results we get with TDP-43 mutations will be highly relevant to sALS, so the implication is that autophagy induction could be of value in sporadic disease. For that reason it is interesting that autophagy induction appeared to reduce levels of non-aggregated TDP-43 as well as aggregated TDP-43. I was not aware that autophagy could have such an effect.

    Testing the autophagy effects using in vivo models will be important and should be done in TDP-43 transgenic mice and rats, but negative results from these animal studies should not necessarily stop us from considering FDA approved compounds for use in humans (particularly as rodent models of TDP-43 toxicity are still very much a work in progress).

    View all comments by Jemeen Sreedharan

References

News Citations

  1. Research Brief: Mutant Cells Eat Mutant SOD1
  2. Toxic TDP-43 Too Tough to Degrade, Plays Prion?
  3. Are TDP-43 Mice Living Up to Expectations?

Paper Citations

  1. . Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Autophagy. 2011 Apr;7(4):412-25. PubMed.
  2. . A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model. Proc Natl Acad Sci U S A. 2010 Sep 28;107(39):16982-7. PubMed.
  3. . Proteostasis of polyglutamine varies among neurons and predicts neurodegeneration. Nat Chem Biol. 2013 Sep;9(9):586-92. PubMed.
  4. . ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A. 2010 Jul 27;107(30):13318-23. PubMed.
  5. . Accelerated Disease Onset with Stabilized Familial Amyotrophic Lateral Sclerosis (ALS)-linked Mutant TDP-43 Proteins. J Biol Chem. 2013 Feb 1;288(5):3641-54. PubMed.
  6. . Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life. Proc Natl Acad Sci U S A. 2014 Mar 18;111(11):4309-14. Epub 2014 Mar 3 PubMed.
  7. . Autophagy activation ameliorates neuronal pathogenesis of FTLD-U mice: A new light for treatment of TARDBP/TDP-43 proteinopathies. Autophagy. 2013 Feb 1;9(2):239-40. PubMed.
  8. . Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43. Proc Natl Acad Sci U S A. 2012 Sep 11;109(37):15024-9. Epub 2012 Aug 29 PubMed.
  9. . [Assessment of the efficacy of treatment with pimozide in patients with amyotrophic lateral sclerosis. Introductory notes]. Neurol Neurochir Pol. 1998 Jul-Sep;32(4):821-9. PubMed.

Further Reading

Papers

  1. . Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species. J Cell Sci. 2014 Mar 15;127(Pt 6):1263-78. Epub 2014 Jan 14 PubMed.
  2. . Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain. Mol Psychiatry. 2012 Aug 7; PubMed.
  3. . Cdc37/Hsp90 Protein Complex Disruption Triggers an Autophagic Clearance Cascade for TDP-43 Protein. J Biol Chem. 2012 Jul 13;287(29):24814-20. PubMed.
  4. . Autophagy dysregulation in amyotrophic lateral sclerosis. Brain Pathol. 2012 Jan;22(1):110-6. PubMed.
  5. . Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1. J Neurochem. 2011 Jan;116(2):248-59. PubMed.
  6. . The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS). Hum Mol Genet. 2010 Sep 1;19(17):3440-56. PubMed.

Primary Papers

  1. . Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models. Nat Chem Biol. 2014 Aug;10(8):677-85. Epub 2014 Jun 29 PubMed.