This is Part 1 of a three-part series. See also Part 2, Part 3. Read a PDF of the entire series.
10 May 2013. Unlike scientific meetings that focus on a particular protein, pathway, or disease, the one that drew some 150 researchers to San Francisco 15-17 April 2013 featured an unusually broad array of topics from nitty-gritty molecular to bird’s-eye conceptual. “From Science to Therapeutics: The Best Way Forward” was the catch-all theme for this annual meeting sponsored jointly by the Gladstone Institute of Neurological Disease, San Francisco, and the German Center for Neurodegenerative Diseases (DZNE), Bonn. Their annual meetings alternate between sites. Last year’s workshop in Germany explored the role of synapses in neurodegenerative disease. The inaugural GIND/DZNE event in 2011 covered tau and tauopathies (see ARF conference series).
Lennart Mucke at the Gladstone Institute and Pierluigi Nicotera of the DZNE organized this year’s meeting with the complexity of AD in mind. “We deliberately brought together investigators pursuing diverse leads and strategies to comprehensively address the challenges posed by Alzheimer's disease,” Mucke told Alzforum. In addition, the organizers chose to separate presentations on similar themes and approaches, rather than cluster them together. “We did this to keep the audience engaged at all times and to prevent different groups from ‘tuning out’ during talks that fall outside their immediate areas of interest,” Mucke said.
In addition to new data on the physiological function of β-secretases and novel efforts to co-opt a longevity gene, attendees heard a handful of talks on therapeutic approaches that mobilize protein quality control mechanisms to keep amyloidosis in check. One of those promotes ubiquitin-proteasome degradation to counteract pathogenesis. Several others presented at the meeting expand or tweak proteostasis mechanisms to help rid cells of misfolded proteins.
Li Gan of the Gladstone Institute explores how acetylation and ubiquitination contribute to tauopathies by controlling tau degradation. Gan and colleagues previously reported that tau can be acetylated in cultured cells and mice that model tauopathies. They detected acetylation in AD brains at early Braak stages and showed that this post-translational modification keeps protein degradation machinery from clearing tau out of cells (see ARF related news story on Min et al., 2010). More recently, Virginia Lee and John Trojanowski at the University of Pennsylvania School of Medicine, Philadelphia, as well as Gan, reported finding acetylated tau in human tauopathies (see ARF related news story and Irwin et al., 2012).
In San Francisco, Gan discussed her lab’s latest work, showing that acetylated tau can impair dendritic sorting and microtubule dynamics. Her lab generated antibodies to acetylated tau and used them to map three acetylation sites—two in tau’s microtubule-binding region and one in its amino-terminal “projection domain,” which seems to determine microtubule spacing. The researchers identified the acetylation sites from AD brain extract using mass spectrometry. One of them—but not other residues in tau’s microtubule-binding domain—drives proteasome-mediated tau degradation, Gan reported. In collaboration with Dan Finley at Harvard Medical School in Boston, Gan found that a small molecule inhibitor of the deubiquitinase USP14 enhanced proteasome activity and degradation of tau in primary rat neurons expressing human tau. Conversely, the researchers found no such protection in neurons expressing an acetyl-mimic tau that cannot be ubiquitinated at the critical site. Furthermore, transgenic mice expressing this tau mutant in the hippocampus were hyperactive and did not adjust properly to their surroundings in an open field test that measures anxiety, Gan said.
To explore mechanisms underlying the behavioral impairment of this acetyl mimic, the researchers used fluorophores to label and track tau’s movement on microtubules in primary neurons. They found that the acetyl-mimic tau crossed the axon initial segment—a specialized membrane region where neurons initiate axon potentials—more readily than wild-type tau. “We think acetylation at the microtubule-binding domain makes tau hyperdynamic, missorting it to the somatodendritic compartment,” Gan said.
With an emerging picture of how tau acetylation endangers neurons, the Gladstone scientists are also exploring therapeutic approaches that block the histone acetyltransferase p300, an enzyme they determined to acetylate tau in their 2010 study. They are screening for additional p300-blocking molecules, and plan to generate conditional p300 knockouts and cross those with AD and tauopathy mouse models, Gan said.
However, p300 may not be the only tau acetylase. In an intriguing twist, Lee and colleagues at UPenn reported last month that tau can acetylate itself, identifying key cysteine residues in the microtubule-binding domain that are involved in the catalytic activity (Cohen et al., 2013). Gan and others found the data interesting, but said future work is needed to validate the in-vivo relevance of the auto-acetylation, and determine if it correlates with human disease progression.
Exactly what tau’s own acetylase activity contributes is still nebulous, but recent work by Gan and William Seeley at UCSF raises the possibility that tau might require acetylation to become toxic. Using a monoclonal antibody specific for tau acetylated at K274, the researchers probed 22 brain samples from people with AD and eight other tauopathies. They detected acetylated tau ac-K274 in all cases except argyrophilic grain disease (AGD)—a rare condition most often seen in people with long-lasting amnestic mild cognitive impairment that does not progress clinically. “The fact that [the AGD cases] are negative for tau ac-K274 is consistent with the notion that that tau acetylation may be required to accelerate tau toxicity,” Gan noted in an e-mail to Alzforum.
Proteostasis to the Rescue?
The theme of protein degradation also figured prominently in a presentation by Jeff Kelly of Scripps Research Institute, La Jolla, California, who described enlisting the unfolded protein response (UPR), a signaling pathway activated when misfolded proteins accumulate in the endoplasmic reticulum. The concept is simple, in theory: Get the system to degrade mutant proteins while still properly folding the wild-type. In practice, this may be hard to achieve, said Kelly, because the UPR activation turns on three transcription factors that each drive expression of distinct but overlapping sets of regulators that control protein degradation. Researchers are unsure which transcriptional program targets amyloidogenic proteins, such as those implicated in neurodegenerative diseases.
Kelly, together with Scripps colleague Luke Wiseman, devised a way to produce two UPR-associated transcription factors—X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6)—at physiological levels within the same cell. They turned on XBP1 using a conventional tetracycline-based promoter. To raise ATF6 levels in the cell, the scientists attached the transcription factor to a mutated variant of E. coli dihydrofolate reductase (DHFR), which does not fold properly. Normally, the cell’s proteosomal degradation system makes quick work of ATF6-DHFR fusion proteins because they are highly unstable. However, add some trimethoprim, a molecule that stabilizes the DHFR domain, and the chimera accumulates to high enough levels to trigger transcription of ATF-6 target genes.
Using whole-genome arrays and proteomics, the scientists looked in HEK293 cells for genes upregulated after activating XBP1, ATF6, or both transcription factors. XBP1 turned on 180 genes in a variety of ER proteostasis pathways, whereas ATF6 upregulated a smaller subset. Specifically, they found that transthyretin (TTR)—the amyloidogenic protein that causes familial amyloid polyneuropathy (FAP) and related diseases—was controlled principally by the ATF6 arm. Normal cellular secretion of misfolded, toxic TTR dropped 40 percent when ATF6 target genes were activated but held steady when XBP1-driven transcription was turned on. Wild-type TTR and other endogenous proteins were unaffected by activation of either transcriptional program. “Activating ATF6 target genes enhanced the cell’s ability to maintain quality control,” Kelly said.
Kelly hopes the findings will energize drug development by offering a way “to discern whether a given stress response pathway will be useful for ameliorating a given disease,” he said. The system “allows you to express a transcriptional factor at physiological levels and ask if this is something you would like to go after with a drug-like molecule.”
Switching gears from proteasomal degradation of aggregation-prone proteins, Kelly updated the audience on the long-term efficacy of tafamidis—a drug that stabilizes the normal tetramer adopted by transthyretin. Marketed by Pfizer as a treatment for FAP, tafamidis (trade name Vyndaqel®) prevents transthyretin from breaking into monomers, which can misfold and then misassemble into amyloid (see ARF related news story on Alhamadsheh et al., 2011). Others are using similar approaches to develop therapeutic compounds that target superoxide dismutase 1 (SOD1) in amyotrophic lateral sclerosis (ALS) and apolipoprotein E4 (ApoE4) in AD, Kelly told Alzforum. However, the approach is less likely to work for amyloid-β or tau. “Generally speaking, you need a protein that adopts a well-defined, folded structure to fashion high-affinity stabilizing ligands,” Kelly said.
The European Medicines Agency approved tafamidis in 2011, making it the first therapy to successfully treat a disease by blocking amyloid formation (see ARF related news story). In the U.S., Pfizer submitted a new drug application to the Food and Drug Administration that same year but the agency's advisory committee did not issue an approvable letter after its meeting in May 2012. Under the U.S. Orphan Drug Act, treatments for rare diseases in principle can gain approval based on positive results in a single trial, if the trial uses a surrogate biomarker that is likely to predict clinical efficacy. This latter point is still being disputed.
Kelly and colleagues have developed and patented an ELISA that they say specifically recognizes transthyretin oligomers, the presumed molecular culprit in FAP. Using this assay to screen human blood samples, the researchers distinguished FAP patients from their spouse controls (100 people total) with 100 percent accuracy. In addition, oligomer levels fell more than 50 percent after six months of treatment with tafamidis, Kelly said. He hopes the new data will enable the FDA to approve tafamidis without requiring a second FAP trial.
Jason Gestwicki, who moved last month from the University of Michigan to the University of California, San Francisco, presented his group’s latest work on small molecules targeting the molecular chaperone HSP70. The ADP-bound form of HSP70 binds tightly to misfolded proteins, preventing them from interacting with, and potentially corrupting, properly folded forms. Gestwicki worked with Chad Dickey at the University of South Florida, Tampa, to see if this strategy might work for tauopathies, which are marked by buildup of pathological tau aggregates. The researchers screened for small molecules that trap HSP70 in its ADP-bound state and, in doing so, promote tau degradation (see Evans et al., 2010).
From those screens came MKT-077, an anti-cancer compound that entered Phase 1 testing in the 1960s but was later abandoned because it caused kidney damage. MKT-077 potently reduced tau levels in neuronal cultures from Tg4510 tauopathy mice. Furthermore, the compound seemed to improve synaptic function, driving up long-term potentiation when administered to brain slices from these animals, said Gestwicki. He reported these data in the April 19 Biological Psychiatry (Abisambra et al., 2013; see also Rousaki et al., 2011).
In a separate study done in collaboration with University of Michigan colleague Andrew Lieberman, Gestwicki reported that an MKT-077 analogue curbs neurotoxicity in a fly model of spinobulbar muscular atrophy, also known as Kennedy’s disease (Wang et al., 2013). In this inherited motor neuron disorder, expanded polyglutamine repeats in the androgen receptor (AR) cause the protein to misfold and aggregate. Similar to MKT-077’s tau-reducing effects, this compound appears to relieve toxicity by stabilizing AR binding to HSP90 and HSP70, thereby promoting the receptor’s degradation.
While these data demonstrate proof of principle, MKT-077 and its analogues have a major shortcoming—they do not cross the mammalian blood-brain barrier. However, last month Gestwicki and colleagues reported their first brain-penetrant analogue and showed that it reduced levels of phosphorylated tau in cultured brain slices (Miyata et al., 2013). “We are changing the way tau is recognized by the system,” Gestwicki said.
“The big question we need to address next is whether this approach is safe,” Gestwicki noted. “Do other proteins (besides tau and androgen receptor) get degraded by these molecules? And what are the potential side effects?” His lab is working to address these issues.—Esther Landhuis.
This is Part 1 of a three-part series. See also Part 2, Part 3. Read a PDF of the entire series.