Scientists Pore Over New Strategies to Tackle ALS

Gatecrasher? The nuclear pore, comprising more than 400 proteins, manages the constant flow of RNAs and proteins in and out of the nucleus. But in ALS, this intracellular gate may become compromised, contributing to the disease. [Image: Courtesy of Mohammad Soheilypour, Lawrence Berkeley National Laboratory].

The nucleoporins making up the nuclear pore are some of the longest-lived proteins in the human body. They stay put for decades, managing the constant flow of RNAs and proteins across the nuclear envelope at a rate of hundreds of molecules per minute. In so doing, they maintain a delicate balance between nuclear and cytosolic composition. But according to recent research, in ALS, that balance is upended in neurons. This leads certain RNAs and proteins to accumulate, inappropriately, in the cytoplasm, where they can aggregate and wreak havoc. Could this nucleocytoplasmic transport process be a suitable target for ALS therapeutics?

“Yes, I think so,” said Tom Lloyd of Johns Hopkins University School of Medicine in Baltimore. Lloyd is part of a consortium testing selective inhibitors of nuclear export (SINEs), developed by Karyopharm Therapeutics in Newton, Massachusetts, in a wide-range of ALS models. One of these inhibitors, called KPT-350, is now being further evaluated by Biogen, Inc., in Cambridge, Massachusetts, as a potential therapy for neurodegenerative diseases including ALS (see Feb 2018 news).

Problems at the Pore

There’s plenty of reason to focus on nucleocytoplasmic transport in ALS. Lloyd and others reported in 2015 that C9orf72 repeat expansions somehow obstruct nuclear traffic, which may play a role in the most common genetic form of the disease (see Aug 2015 news; Friebaum et al., 2015; Zhang et al., 2015; Jovičić et al., 2015). In addition, a mutation in the nuclear pore component Gle1 has been associated with certain rare cases of ALS, scientists reported in 2015 (Kaneb et al., 2015).

Clogged Pores. A proline-arginine poly-dipeptide encoded by C9orf72 expansions (green) fills up the nuclear pore (red) in Xenopus oocytes, potentially tying up nuclear traffic. [Courtesy of Shi et al., 2017, Proceedings of the National Academy of Sciences].

These nuclear traffic jams may contribute to sporadic forms of the disease, too. Nuclear proteins such as TDP-43 are frequently mislocalized to the cytoplasm in both genetic and sporadic ALS (for review, see Ederle and Dormann, 2017). Moreover, mislocalized, aggregated TDP-43 may itself disrupt nucleocytoplasmic trafficking, reported Wilfried Rossoll, of the Mayo Clinic in Jacksonville, Florida, in January (see Jan 2018 news; Chou et al., 2018).

In fact, these traffic jams are emerging as a general hallmark of neurodegenerative disease. The poly-dipeptides encoded by C9orf72 repeat expansions in ALS and FTD, polyglutamine peptides associated with Huntington’s disease and tau linked to Alzheimer’s all can affect transport (Shi et al., 2017; Grima et al., 2017; Eftekharzadeh et al., 2017; Dec 2015 News; Woerner et al., 2016).

Aging, too, leads to transport defects, Rossoll pointed out, potentially explaining why these traffic tie-ups occur in motor neurons in late-onset diseases like ALS. Since neurons in adults do not divide, they never dissolve the nuclear envelope and therefore are unable to replace worn-out nuclear pore components (D’Angelo et al., 2009). Therefore, as neurons grow older, speculated Rossoll, these pores may be more likely to become dysfunctional, making them vulnerable to disease.

In and Out. Importins, exportins, and Ran mediate the traffic across the nuclear pore. [Courtesy of Flather and Semler, 2015 under a CC BY 4.0 license.]

The nuclear pores are part of a crucial intracellular gatekeeping system. Many proteins, such as TDP-43, possess both a nuclear export signal (NES) and nuclear import signal (NLS). To cross the nuclear envelope, they bind to importins or exportins to help them get in or out of the nucleus. But according to a post-mortem analysis by Rossoll’s group, these pores become damaged in ALS (see Jan 2018 news; Chou et al., 2018). That might lead to certain nuclear proteins, such as TDP-43, mistakenly accumulating in the cytoplasm.

One way to cut down on neurotoxicity in ALS may be to block nuclear export. For example, Guillaume Hautbergue and colleagues at the University of Sheffield in England took aim at C9orf72 ALS by targeting the export of C9orf72 repeat-rich RNAs. These repeat expansions are intronic, so they should be spliced out before the mRNAs leave the nucleus. The faulty transcripts escape, however, the researchers found, thanks to a nuclear protein called SRSF1. Eliminating SRSF1, or its binding to the nuclear export factor NXF1, protected neurons in culture and in fruit flies (Hautbergue et al., 2017). Efforts to develop potential therapies that reduce SRSF1, including antisense oligonucleotides, are now underway (see Jul 2017 news; Jul 2017 news).

X-ing Out Exportin-1

Karyopharm Therapeutics is developing another approach, targeting the exportin XPO-1, to treat cancer. That’s because XPO1 handles export of many tumor suppressor proteins. Karyopharm’s compounds, it is hoped, will keep those suppressors in the nucleus, where they can fight tumor proliferation by silencing cancer-causing genes.

Could the same approach reduce neurotoxicity in ALS? So far, studies offer reason to hope. Lloyd and colleagues found that an early Karyopharm molecule, KPT-276, fixed nucleocytoplasmic transport and reduced neuron loss in a fly eye model of the disease (Zhang et al., 2015; Tamir et al., 2017). He and his collaborators are now evaluating a different XPO-1 inhibitor, KPT-350, as a potential ALS therapy in fly, mouse, and stem cell-based models. KPT-350 is a better option for ALS than Karyopharm’s other drug candidates because it crosses the blood-brain barrier more effectively. It’s already been shown to protect neurons expressing mutant huntingtin (Grima et al., 2017).

Rossoll’s team is also seeing neuroprotective benefits from some of Karyopharm’s drug candidates in ALS models (see Jan 2018 news; Chou et al., 2018). “What is really promising is that in several of these preclinical models, different groups have observed a beneficial effect,” said Rossoll.

Reduced neurotoxicity? Treatment with KPT-350 improves muscle strength in a rat model of TDP-43. But according to subsequent cell culture analysis, this partial benefit does not appear to be due to increased levels of nuclear TDP-43 (see Archbold et al., 2018).[Courtesy of Archbold et al., 2018, Scientific Reports. CC BY 4.0]

But according to recent results from Sami Barmada’s group at the University of Michigan in Ann Arbor, this potential benefit is likely not due to redistribution of TDP-43. At least three exportins help shuttle TDP-43 out of the nucleus, suggesting that its mislocalization is mediated by multiple mechanisms (Archbold et al., 2018).

“Future in vitro and in vivo studies are warranted to more closely examine the effect of SINE compounds in models of ALS, FTD and related disorders,” wrote Barmada and colleagues. “Identifying the downstream effectors of SINE compound-mediated neuroprotection is an important next step.”

One possible explanation may be lowering of inflammation, noted Lloyd. XPO1 shepherds the inhibitor of the transcription factor NF-kB, IkB, out of the nucleus, enabling the expression of pro-inflammatory cytokines (see December 2011 news; Kim et al., 2016; Frakes et al., 2014). By blocking this transport, KPT-350 ought to dampen neuroinflammation.

Plus, Lloyd adds, proteins like Sirt1 and FOXO, activators of neuroprotective genes, shuttle across the nuclear envelope. Blocking export should raise their nuclear concentrations, too.

The fact that XPO1 shuttles so many proteins is also a reason to be careful with any therapeutic acting on it, according to experts.

“If you escape a disease by breaking the nuclear pore complex, that creates a challenge,” points out André Hoelz, who studies nuclear pore biology at the California Institute of Technology in Pasadena. “This is definitely not going to be fine in the rest of the body.” In fact, phase 1 trials of one of Karyopharm’s drug candidates, selinexor (KPT-330), found side effects including nausea, fatigue, weight loss and low platelet count (Garzon et al., 2017; Chen et al., 2017).

The key is not to slam the nuclear exit completely shut. That’s what Karyopharm’s approach is designed to do, explains Lloyd. “It’s not a full blockage of export, it’s actually done in such a way that it only partially inhibits export,” explains Lloyd. That way, needed proteins can still travel, but hopefully those that cause problems in the cytoplasm, like TDP-43, will stay put.

To target or not to target, that is the question. Many mechanisms contribute to ALS. But which of these mechanisms primarily contributes to neurotoxicity and therefore targeted in the disease remains hotly debated. [Courtesy of Van Damme et al., 2017, Disease Models and Mechanisms. CC BY 4.0 License.]

“It will be very difficult to find the right balance,” Rossoll cautioned.

Karyopharm’s drug candidates appear to have a narrow therapeutic index, exhibiting significant neurotoxicity at higher levels in preclinical models of ALS (Chen et al., 2017; Archbold et al., 2018). However, the company’s early clinical trials indicate it’s possible to find a tolerable dose (Garzon et al., 2017; Chen et al., 2018).

While it’s true that pore defects may be a general contributor to neurodegenerative disease, said Hoelz, he cautioned that targeting XPO1 or other pore components is far from a sure route to therapeutics. “There is a lot of research that is missing,” Hoelz said. For one, it’s not clear what causes these defects in ALS: C9orf72 RNAs? The repetitive proteins translated from them? Mislocalized TDP-43?

The normal function of the nuclear pore is poorly understood, added Hoelz. In addition, the nuclear pore also has other roles such as anchoring chromatin and organizing the nucleus. Might those jobs, too, be impacted in ALS? With so many unanswered questions, Hoelz suspects that any treatment aiming to modulate nuclear pore function is still “some time away.”

It also remains unlikely that blocking this process will target the root cause of the disease, which may be the toxic, aggregating proteins themselves, said Rossoll. If scientists could identify and reverse the problem that kicks off neurodegeneration, they might not need to meddle with transport.

“On the positive side, I think this nucleocytoplasmic defect is probably present in, potentially, a very large patient population,” Rossoll added. “That makes it a good target.”

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Biogen blood brain barrier c9orf72 disease-ad disease-als disease-ftd karyopharm KPT-350 nuclear export nuclear pore nucleocytoplasmic transport SINE tdp-43 topic-preclinical topic-randd
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