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., 2010; Watanebe et al., 2013; Austin 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).
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., 2013; Wang 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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