When RNA transcripts aren’t processed properly or don’t make it to their final destinations for translation into protein, the consequences could trigger neurodegenerative disease. At the 5th RNA Metabolism in Neurological Disease meeting, researchers reported how RNA-binding proteins tasked with keeping transcripts in order manage to fall out of line themselves—gelling with other proteins and RNA in cytoplasm. Scientists linked previously disparate mechanisms of C9ORF72 toxicity into cohesive feed-forward loops, and hatched plans for new therapeutic approaches.
It’s ‘And,’ Not ‘Either-Or’: C9ORF72 Mechanisms of Action are Linked
Hexanucleotide expansions in the first intron of the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Scientists entertain several possible explanations for what makes these run-on repeats toxic. C9ORF72 function is kaput, RNA foci form, dipeptide repeats coalesce into cytoplasmic aggregates. But which of these is it? At the 5th RNA Metabolism in Neurological Disease Conference, held November 1–2 in San Diego, researchers realized it might be all of the above.
Scientists drew pivotal connections, not distinctions, between these mechanisms. They saw that a cell’s inability to express C9ORF72 due to the cumbersome expansions taxes its ability to execute autophagy and this, in turn, allows dipeptide proteins (DPRs) translated from the repeat sequence to pile up. They described a scenario in which repeat-laden RNA transcripts tip off the cellular stress response, bolstering the process of repeat-associated non-AUG (RAN) translation that produces the toxic DPRs. In dissecting mechanisms of RAN translation, scientists discovered unexpected consequences. Therapeutic ideas for halting these vicious cycles emerged at the conference, as well.
Nicolas Charlet–Berguerand of the University of Strasbourg, France, had previously reported that the protein translated from C9ORF72 interacts with the Rab GTP/GDP exchange factor SMCR8; both together promote flux in autophagy. A dip in C9ORF72 expression—as has been observed recently in brain tissue of expansion carriers (Aug 2018 news)—therefore disrupts this flow. By itself, this dampening of autophagy does not wreak havoc on neurons. Instead, it is made worse by a second known genetic modifier of ALS—Ataxin-2 polyglutamine repeats—which build up when C9ORF72 expression is low (Sellier et al., 2016).
In San Diego, Charlet-Berguerand told fellow scientists that he also sees this harmful synergy with the very DPRs translated from the repeat-hobbled C9ORF72 gene. Using HEK293 cells transfected with different C9ORF72-hexanucleotide repeat expansion (HRE) constructs, he found that RAN translation of DPRs is a particularly inefficient process. However, when he deployed siRNA to silence C9ORF72 in those repeat-expressing cells, or when he inhibited autophagy, cellular DPR accumulation ramped up to become deleterious.
Charlet-Berguerand proposed a “double hit” mechanism, in which low levels of DPRs translated via RAN accumulate when autophagy wanes. In vivo evidence in support of this mechanism came from Qiang Zhu, a postdoc in Don Cleveland’s lab at the University of California, San Diego. Zhu and colleagues generated transgenic mice expressing a bacterial artificial chromosome carrying the human C9ORF72 gene strapped with 450 GGGGCC repeats. These repeats were expressed separately from the endogenous mouse gene, and did not produce a functional C9ORF72 protein, only products of the repeat sequence (RNA foci and DPRs). The researchers had previously reported that these animals had pathological and behavioral phenotypes, including gliosis, fewer neurons in the hippocampus, poor performance on memory tests, and weak motor skills (Apr 2016 news).
Zhu then crossed these mice to C9ORF72-deficient mice, which already had enlarged spleens, an indicator of inflammation. In San Diego, he reported that all of the repeat-associated disease phenotypes were more extreme in the crosses. The scientists got similar results when they used adeno-associated virus to express HRE sequences in the brains of C9ORF72-deficient mice. To Zhu, this implies that reduced autophagy due to an absence of C9ORF72 expression rendered the DPR accumulation more toxic. Zhu and Charlet-Berguerand agreed that their data complement each other in support of this idea.
Could boosting autophagy counteract damage by C9ORF72-HRE? This is an active area of preclinical drug development, and scientists at the conference took different views. Charlet-Berguerand considers the pursuit worthwhile but cautioned that despite considerable effort to develop autophagy-enhancing drugs, their toxicity remains a problem. Zhu favors the more direct approach of taking down expression of DPRs with antisense oligonucleotides, an approach he has employed to degrade repeat-containing C9ORF72 transcripts while sparing expression of the normal copy of the gene. Yet other researchers called autophagy a viable target that could have benefit in any disease marked by pathological protein accumulation.
Nathaniel Safren, a postdoc in Sami Barmada’s lab at the University of Michigan in Ann Arbor, described a new autophagy-screening system. He tags LC3 proteins, which associate with autophagosomes throughout their maturation process, with fluorescent dyes that change color with time. This allows him to gauge autophagic flux dynamically. Safren screened 24,000 compounds for hits that enhance autophagy in rat primary cortical neurons. One is NVP-BEZ235, aka dactolisib, an old PI3K-mTOR inhibitor and investigational cancer treatment. In Safren’s hands, NVP-BEZ235 extended survival of rat cortical neurons overexpressing TDP-43.
Besides waning autophagy, there is another way in which DPRs could end up accumulating in neurons. RAN translation itself is somehow revved up in cells bearing C9ORF72 HREs in their genome. Despite being tucked within an intron, with nary an AUG initiation codon upstream, this expansion somehow manages to be transcribed and translated. While scientists are still hammering out the particulars of this non-canonical process, they do know that translation starts at non-AUG codons—a process usually considered far less efficient than the canonical AUG start.
Or is it? In San Diego, Laura Ranum, University of Florida, Gainesville, shared her new discovery that the C9ORF72 repeat-laden transcripts trigger the cell’s integrated stress response. Ranum first described RAN translation from CAG repeat expansions seven years ago (Zu et al., 2011). The ISR starts up when the master kinase PKR phosphorylates elongation initiation factor 2α (eIF2α). It then shuts down canonical protein translation in favor of non-canonical pathways such as RAN. Ranum speculated that the repeat-containing transcripts trigger the stress response because they form hairpin structures akin to those formed by viral RNA.
At the conference, Ranum discussed initial data from two distinct strategies her lab pursued to take down DPRs. The scientists removed DPRs with a new poly-GA antibody found by screening the plasma of super-agers. They also shut down stress-induced DPR production by way of a dominant-negative form of PKR. Ranum tested both in the C9ORF72-BAC mouse model her group developed (May 2016 news). These mice recapitulate aspects of ALS/FTD, including development of RNA foci and DPRs, motor neuron degeneration, motor deficits, and early death. In San Diego, Ranum reported that both strategies substantially ameliorated these phenotypes. Surprisingly, although the antibody only targeted the poly-GA, it somehow triggered the clearance of other DPRs as well.
Peter Todd of the University of Michigan in Ann Arbor said that his lab’s preliminary data jibe with Ranum’s. RNA transcribed from the C9ORF72 repeat expansions instigated the eIF2α stress response, which, in turn, promoted RAN translation of DPRs.
Stalling—A Virtue in Biology?
Instead of presenting that data, though, Todd unveiled an unexpected consequence of RAN translation. It stalls production of FMRP. Located on the X chromosome, the FMR1 gene is implicated in both the neurodevelopmental disorder Fragile X Syndrome as well as the late-onset neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). People with FXTAS have a string of 55 to 200 CGG repeats located upstream of the open reading frame of the FMR1 gene, and RAN translation of this expansion occurs in both the sense and antisense directions (for review, see Glineburg et al., 2018). In San Diego, Todd reported a surprising finding. These RAN translation products are detectable even at the normal repeat size in people, and both the repeat and their translation are highly conserved in mammals. This suggests that RAN translation of this sequence is a normal phenomenon.
What purpose could it serve? Todd found that the CGG repeats and RAN translation snagged scanning ribosomes, preventing them from reaching and engaging the proper AUG start site for the FMR1 gene. This dampened expression of the downstream FMR1 protein, FMRP. Most interestingly, Todd found that certain forms of synaptic plasticity suppress this RAN-mediated stalling as a method of upregulating FMRP synthesis in neuronal dendrites, allowing for local regulation of translation of this synaptic protein.
In collaboration with Ionis Pharmaceuticals in Carlsbad, California, Todd and colleagues have developed antisense oligonucleotides to block the non-cognate codons where RAN translation initiates upstream of the FMR1 gene. As predicted, shutting down RAN with these ASOs led to a spike in FMRP, and also a blockade of translation of the toxic repeat protiens at expanded repeats. In turn, this treatment lengthened the survival time of neurons derived from patients with expanded CGG repeats, Todd showed.
More broadly, Todd proposed that RAN translation and nucleotide repeats could serve physiological roles in regulating gene expression throughout the genome. He noted that there more than two million tandem microsatellte repeat sequences in the human genome, most of which are unstudied. Future work is needed to understand the mechanisms by which these repeats might influence brain function and disease, Todd believes.
Translation Right Off Transcript’s Trash?
Getting back to C9ORF72-HRE, prior studies have ignored the elephant in the room: The repeat expansion resides within an intron, and introns are removed from mRNA before ribosomes get to work. How is the expansion translated? Most scientists favor the idea that the intron is aberrantly retained as the raw mRNA transcript is being spliced, leading to translation starting at non-cognate codons just upstream of the repeat sequence (Jan 2018 news on Green et al., 2017 and Tabet et al., 2018; Sznajder et al., 2018). However, Shuying Sun, Johns Hopkins University, Baltimore, has evidence for a different mechanism. She believes the intron is mostly spliced out, and RAN translation occurs using this excised bit as a transcript. Introns lack the 5' m7G caps that usually anchor ribosomes and their associated translational machinery. Even so, Sun recently reported, these excised C9ORF72 introns were able to yield DPRs, especially, once again, when the integrated stress response was switched on (Jan 2018 news on Cheng et al., 2018).
In San Diego, Sun bolstered this idea with additional data. She created a stir when she showed striking visuals of RAN translation in progress but, like many scientists intimidated by scientific journals, declined to share an image prior to formal publication. In collaboration with Bin Wu, also at Johns Hopkins, (Wu et al., 2016), who developed a technique to visualize RNA translation, Sun generated genetic constructs in which exons and the repeat-containing intron of C9ORF72 were tagged with either blue or red fluorescent dyes, respectively, after transcription. At the same time, she labeled the translating peptides with a SunTag (no relation to her name), a form of GFP that emits an amplified signal. In living cells expressing all these components, Sun was able to visualize and distinguish between translation occurring from the intron alone (red), exons alone (blue), or from sequences containing both.
What did she see? Mostly translating peptides whirring off either red or blue transcripts. Out of 380 cells Sun and Wu visualized, only 22 contained very few co-localized introns and exons in the cytoplasm. Importantly, Sun reported that the excised intron was exported only when it contained the repeat expansion.
Researchers complimented the beauty of Sun’s data. They also wondered how relevant these artificially tagged RNAs are to physiological transcripts in vivo. Susan Ackerman of the University of California, San Diego, asked whether nuclear export of excised introns was a broad phenomenon, or only occurred in specific cell types. Roy Parker of the University of Colorado, Denver, asked whether the secondary structure of the repeat-containing RNAs might somehow stabilize the excised intron. Does it form the characteristic, lasso-like lariat structure, as most excised introns do? Ranum asked whether other instances of RAN translation from excised introns have been reported. Sun is working on these aspects. Meanwhile, she said, her findings provide evidence that RAN translation from excised introns can happen, at least under certain conditions.—Jessica Shugart
Zu T, Gibbens B, Doty NS, Gomes-Pereira M, Huguet A, Stone MD, Margolis J, Peterson M, Markowski TW, Ingram MA, Nan Z, Forster C, Low WC, Schoser B, Somia NV, Clark HB, Schmechel S, Bitterman PB, Gourdon G, Swanson MS, Moseley M, Ranum LP.
Non-ATG-initiated translation directed by microsatellite expansions.
Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):260-5. Epub 2010 Dec 20
PubMed.
Beyond the Nucleus: TDP-43 Sticks Together, For Better or Worse
Similar to a newborn human, nascent RNA transcripts need a lot of coddling before they realize their purpose in life (that is, translation into protein). It takes a village of RNA-binding proteins to accomplish this. They see to the transcripts’ proper splicing, capping, polyadenylation, and export from the nucleus, and they send the finished RNAs off to sometimes far-flung locales such as axonal terminals for translation. When the RNA-binding proteins themselves get mixed up, the cell is in trouble. This goes for FUS (see Part 3 of this series) and for TDP-43. While most TDP-43 works from the neuronal nucleus in healthy people, the protein increasingly acts in the cytoplasm in ALS/FTD. At the 5th RNA Metabolism in Neurological Disease Conference, held November 1-2 in San Diego, researchers exchanged surprising new insights on how the protein gets trapped in the cytoplasm, and hatched plans for new therapeutic targets.
Sami Barmada of the University of Michigan, Ann Arbor, connected the dots between TDP-43’s exile in the cytoplasm and neuronal hyperactivity—common phenomena in ALS/FTD. When he stimulated human neurons derived from induced pluripotent stem cells (iPSC) with tetraethylammonium or other activators, he found that this shifted TDP-43 into the cytoplasm. Deploying a collection of N- and C-terminal antibodies against TDP-43, Barmada zeroed in on an unlikely cause. Somehow, neuronal activity led to the appearance of a C-terminally truncated version of TDP-43. It arose from alternative splicing and, strikingly, the truncation introduced a nuclear export signal at the C-terminus. This relegated the shortened protein to the cytoplasm, where it promptly interacted with full-length TDP-43. Barmada proposed that this stunted form of TDP-43 promotes cytoplasmic accumulation of full-length TDP-43. In support of this idea, more full-length TDP-43 piled up in the cytoplasm when Barmada co-expressed the short isoform in neurons.
The findings not only offer a new explanation for how TDP-43 finds itself in the cytoplasm, they could also explain discrepancies in past data depending on whether N- or C-terminal antibodies were used to pinpoint where in the cell TDP-43 was. The more commonly used C-terminal antibodies do not recognize truncated TDP-43, and therefore undercount cytoplasmic localization relative to N-terminal antibodies, which recognize full-length and truncated forms, Barmada said.
What might this short form do, researchers wanted to know? While Barmada has confirmed that it binds RNA, he does not know yet whether it is functional, how it affects full-length TDP-43, or how it interacts with cytoplasmic stress granules.
Previous studies have hinted at oligomerization as a prerequisite for TDP-43’s accumulation and potential entrapment in such granules. To nail down the consequences of this coupling, some investigators use light to force to protein to oligomerize. Christopher Donnelly of the University of Pittsburgh, Pennsylvania, built upon the “optodroplet” system previously developed by Clifford Brangwynne at Princeton University, New Jersey. He fused TDP-43 to cryptochrome 2 (Cry2), a photoreceptor that dimerizes fast when exposed to blue light (Jan 2017 news). In San Diego, Donnelly reported that when cells expressing this Cry2-TDP-43 hybrid were chronically exposed to blue light, the protein left the nucleus and built up in the cytoplasm. There, it formed inclusions that recruited endogenous TDP-43, a hallmark of proteinopathy. Interaction with RNA rendered TDP-43 resistant to this state, indicating that the nucleic acid antagonizes TDP-43’s propensity to form inclusions.
These TDP-43 “opto-inclusions” were more than experimental playthings. Donnelly told the audience that they are toxic, killing cells two days after induction of TDP-43 oligomerization. His team made an “opto-fly” that expresses the Cry2-TDP-43 hybrid, and are starting to see that TDP-43 dimerization triggers motor deficits in the insects. They also showed that treating cells with decoy oligonucleotides composed of TDP-43 binding sequences prevented these oligomerization events and delayed neurotoxicity.
Kazuhide Asakawa of the National Institute of Genetics in Mishima, Japan, expressed a similar Cry2-TDP-43 construct in zebrafish, saying that the see-through minnows make excellent models to study cellular consequences of TDP-43 aggregation. Flashing the fish with pulses of light coaxed TDP-43 out of the nucleus and into the cytoplasm of motor neurons. Echoing Barmada’s findings with short TDP-43, Asakawa reported that these flashes essentially transformed opto-TDP-43 into a dominant-negative protein as it coupled with normal TDP-43 in the cytoplasm. This cytoplasmic mislocalization somehow brought axonal outgrowth to a halt.
Don Cleveland of the University of California, San Diego was curious about Asakawa’s latter result, for reasons that became clear during his own presentation. TDP-43 influences myriad RNA processing steps for numerous transcripts, and its depletion from the nucleus changes the expression level of more than 1,500 genes. How to find the important ones? Cleveland suspects many of the toxic consequences of TDP-43’s nuclear exodus come down to changes in a single transcript. Why? The microtubule binding protein stathmin-2 was the most severely downregulated gene subsequent to TDP-43 deficiency. Cleveland reported that with loss of nuclear TDP-43 somehow came a premature polyadenylation site in the first exon of stathmin-2, resulting in a stunted transcript that produced no functional protein. Notably, he also found stathmin-2 expression to be deficient in neurons generated from ALS/FTD patients with mutations in C9ORF72 or TDP-43, though not SOD1. Neurons from sporadic cases had the defect, too.
Cleveland’s group used a microfluidic model of axonal regeneration to investigate what losing stathmin-2 would do. Motor neurons grew in a compartment that was connected to a neighboring chamber by a segment that allowed axons to project through. When these axons were severed, normal motor neurons rapidly grew new axons. However, antisense oligonucleotides (ASOs) that knocked down either TDP-43 or stathmin-2 completely blocked this regeneration. Notably, restoring stathmin-2 expression alone completely rescued this defect. Cleveland proposed using ASOs targeting the premature polyadenylation site in stathmin-2 as a therapeutic strategy for any form of ALS/FTD marked by TDP-43 pathology.
Researchers peppered Cleveland with questions. Fen Biao Gao, University of Massachusetts Medical School, Worcester, asked whether stathmin-2 expression was abnormal in tissue sections from ALS/FTD patients. Cleveland said he had not looked, and that such an analysis would be limited by the specificity of antibodies; moreover, many vulnerable neurons no longer exist in postmortem sections. Benjamin Wolozin, Boston University, suggested that this result be confirmed using TDP-43 sans its nuclear localization sequence, to be sure that the stathmin-2 loss was due to TDP-43 depletion from the nucleus. When asked what exactly spurs introduction of the polyadenylation site, Cleveland said it was still unclear.
Dieter Edbauer, German Center for Neurodegenerative Diseases, Munich, asked about stathmin-2 in TDP-43 mouse models. Cleveland responded that the polyadenylation site—which is modified from UU to AA in the human stathmin-2 gene—does not exist in mice; they have two Gs in that position instead. Perhaps this is why TDP-43 mouse models do not fully recapitulate disease, Cleveland said. Still, others wondered what explains the ALS/FTD phenotypes that TDP-43 mouse models do have, despite their apparent resistance to premature polyadenylation of stathmin-2.
Despite the unknowns, some researchers were cautiously optimistic about the approach. Gao told Alzforum that stathmin-2 need not explain all TDP-43 toxicity to be a viable target. Others were less convinced. Ke Zhang of Johns Hopkins University in Baltimore challenged Cleveland on his finding, noting that the axonal regeneration phenotype has little to do with neurodegeneration. He believes that without evaluating stathmin-2 loss in animal models more broadly first, pushing stathmin-2 as a therapeutic target is premature.
Myogranules to the Rescue?
TDP-43 (red) stays in the nucleus in uninjured myocytes (top), but moves into the cytoplasm five days post-injury (middle, injured myocytes: eMHC, green). By 10 days post-injury (bottom), TDP-43 repairs back to the nucleus. [Courtesy of Vogler et al., Nature, 2018.]
Damage to neurons from TDP-43’s extranuclear wanderings was a predominant theme at this meeting. And yet, Roy Parker of the University of Colorado, Boulder, offered a counternarrative, at least in muscle cells. As recently published, Parker showed that TDP-43 not only forms amyloid-like structures with RNA in the cytoplasm, but suggested that these little nuggets serve a purpose during development and regeneration (Vogler et al., 2018).
Parker reasoned that RNA’s notorious tendency toward self-assembly made it surprising, if anything, that granules chock-full of entangled RNAs do not continually choke the cytoplasm. RNA-binding proteins clearly maintain order, Parker said, and nowhere is there more of a need for this than in muscle cells. Myocytes boast the longest-known RNA message in the transcriptome, a 100 kb monster encoding the giant muscle protein titin, and they have to deliver such RNAs over long distances to form super structures such as the sarcomere, a highly organized collection of bundled fibers. What’s more, TDP-43 is known to play a pivotal role in muscle development and maintenance, and cytoplasmic accumulation is a feature of degenerating muscles.
Parker found that when muscle fibers develop in mice, TDP-43 moves from the nucleus of myocytes into the cytoplasm, where it binds numerous mRNAs. From within the tube-like myocytes, TDP-43 formed rings around growing myofibers. Once the sarcomere had formed, TDP-43 returned to the myocyte nucleus. The same was true in adult myocytes spurred to regenerate after an injury: TDP-43 sprang out of the nucleus, RNA in tow, until the sarcomere was repaired.
Closer inspection of these transient, high-molecular-weight TDP-43-RNA structures brought up a resemblance to amyloid oligomers. Diffraction patterns indicated β-sheet content on par with oligomers made of α-synuclein and Aβ, yet none of the steric zipper motifs found in amyloid fibrils. The A11 antibody, known to bind a range of oligomers but not fibrils, latched onto these so-called “myogranules” as well (Apr 2003 news). Notably, the TDP-43/RNA structures contained some, but not all, components of stress granules. Myogranules were also distinct from the stress granules that formed when the researchers subjected myofibers to stress conditions. Parker identified numerous TDP-43 binding sites within known myocyte mRNAs, including titin, suggesting several TDP-43 molecules might dot the surface of mature mRNAs in the cytoplasm.
Oligomers with a Purpose. Five days post-injury, oligomers recognized by the A11 amyloid antibody (red) co-localize with TDP-43 (green) in the cytoplasm of myocytes. [Courtesy of Vogler et al., Nature, 2018.]
Parker proposed that these transient structures might keep RNA from getting entangled during times of intense gene-expression changes, e.g., during development or regeneration. He envisions TDP-43 amyloid-like structures as protective cages that shuttle RNA to distant sites for local translation. In myopathies, when muscles are constantly trying to repair themselves, an overabundance of these structures, coupled with a failure to clear them, could seed a pathological aggregation. Parker found numerous A11-positive TDP-43 aggregates in muscle samples from people with necrotizing myopathy, a disease in which myocytes are in a perpetual state of repair.
Could similar mechanisms be at play in neurons? Following neuronal injury, or in the face of chronic neurodegeneration, neurons might require the services of TDP-43 to shuttle numerous mRNAs out to axons, dendrites, and other distant locations for translation. Could such myogranules jump across neuromuscular junctions, and seed pathological aggregation in motoneurons? No evidence exists to suggest this could be the case, Parker told Alzforum. Even so, he suggested that an interesting future experiment would be to see if TDP-43 containing myogranules can also initiate a trans-synaptic propagation of TDP-43 aggregates.
At the meeting, Virginia Lee of the University of Pennsylvania in Philadelphia showed data that ranked TDP-43 itself—not myogranules—among the proteins that can propagate via proteopathic spread. Alzforum covered her group’s paper, which reported that aggregates extracted from postmortem brains of people with FTD, when injected into TDP-43 transgenic mouse brain, seeded trans-synaptic propagation (Oct 2018 news).
Wolozin, for one, was fascinated by Parker’s findings. They mesh with his and others’ observation that except in cultured cell lines, cytoplasmic TDP-43 generally does not coalesce into stress granules under physiological conditions. It appears more diffusely distributed. Other researchers were intrigued by Parker’s discovery of a normal, physiological purpose for cytoplasmic TDP-43 aggregates. Paul Taylor of St. Jude’s Children’s Hospital in Memphis, Tennessee, noted that this role as a protector of RNA molecules could also be true of other RNA-binding proteins, such as FUS, which have been spotted accompanying RNA to distant reaches of neurons. “When conditions change, such as when cells become stressed, then these protective structures could join up with stress granules or other membraneless organelles,” Taylor suggested. If this continues unabated, toxic aggregation might ensue, he added. For more on FUS, see Part 3 in this series.—Jessica Shugart
Vogler TO, Wheeler JR, Nguyen ED, Hughes MP, Britson KA, Lester E, Rao B, Betta ND, Whitney ON, Ewachiw TE, Gomes E, Shorter J, Lloyd TE, Eisenberg DS, Taylor JP, Johnson AM, Olwin BB, Parker R.
TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.
Nature. 2018 Nov;563(7732):508-513. Epub 2018 Oct 31
PubMed.
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Going the Distance: FUS Travels to Terminals, Drops Off RNA
Following their transcription from the genome, RNA messages exit the nucleus and enter the cytoplasm, sometimes traveling vast distances before they are finally translated into protein. RNA-binding proteins are helping hands on this journey from start to finish. At the 5th RNA Metabolism in Neurological Disease Conference, held November 1‒2 in San Diego, scientists shared news about FUS. This RNA-binding protein not only accompanies RNA cargo on its trip to distant axons for translation, it also requires its own chaperones to avoid getting sidetracked into globs of liquid droplets called ribonucleoprotein granules along the way. This careful shepherding gets derailed when FUS carries mutations known to cause ALS/FTD. Finally, researchers raised the possibility that dalliances with chaperones and specialized ribosomes in the neuronal synapse might push FUS into releasing its precious RNA cargo for axonal translation.
Sandrine Da Cruz of the University of California, San Diego, showed recently published data suggesting that cellular stress derails axonal translation when FUS is mutated (Lopez-Erauskin et al., 2018). The researchers used a bacterial artificial chromosome to express human FUS under control of the mouse promoter. Mice lacking the FUS gene die before birth, but live when expressing the human gene. By all accounts, human wild-type FUS functionally replaced the mouse gene, but mice that expressed either of two ALS-linked FUS mutants developed an ALS-like disease marked by a weakening grip and loss of a third of their neuromuscular junctions by 18 months of age. By two years, the mice started slipping on memory tests, and neurons in their hippocampi had fewer synapses.
Da Cruz reported that in both wild-type and mutant mice, the majority of FUS protein was in the nucleus; she saw no cytoplasmic aggregates. What’s more, Da Cruz saw no obvious changes in expression or splicing of genes normally affected by loss of FUS expression. This means that FUS mutations cause neurodegeneration in ways other than altering FUS function in the nucleus.
What could it be? Da Cruz saw downregulation of genes encoding the translational machinery, including ribosomal proteins, in the spinal cords of mice expressing mutant FUS. Expression of genes encoding key synaptic proteins also took a plunge. The spinal cord was marked by increased phosphorylation of eIF2α, which shuts off translation in response to cellular stress. The twist: This stress response and translational shutdown happened only in axons. By labeling newly synthesized proteins with puromycin, Da Cruz found blocked protein synthesis in axons, not cell bodies, of neurons expressing mutant FUS. This was true in cultured hippocampal neurons and in sciatic nerves of mice. Furthermore, a higher proportion of FUS accumulated in axons of mutant FUS mice compared with wild-type FUS mice.
Axons Stopped in Translation. Newly synthesized proteins (green, puromycin) populate both cell bodies and axons of hippocampal neurons from non-transgenic mice (left). Only cell bodies have substantial puromycin in FUS mutant mice (right) [Courtesy of Lopez-Erauskin et al., Neuron, 2018.]
Da Cruz believes mutant FUS somehow triggers the integrated stress response in axons, halting translation locally. Some quibbled that the dearth of newly synthesized proteins in axons could be explained by a deficit in axonal transport, or by the general degeneration of axons. Da Cruz replied that she sees this deficit in axonal, not somal, translation almost immediately after injecting puromycin, suggesting the defect was localized and specific. Exactly how mutant FUS incites this response, and what physiological role FUS normally plays in axonal transport and translation, remains unclear. Fielding more questions after her talk, Da Cruz said her group searched for signs of stress granules or FUS aggregates in axons, but found none.
Dorothee Dormann of Ludwig-Maximilians-University, Munich, described a mechanism that could sidetrack FUS in the cytoplasm, possibly preventing the RNA-binding protein from delivering its cargo to sites of translation. As described in Alzforum’s recent coverage of Dormann’s paper, under normal conditions, FUS interacts with the nuclear import receptor TNPO1 (Apr 2018 news on Hofweber et al., 2018). This protein chaperones FUS in the cytoplasm and keeps it from coalescing into RNA granules. In this way, TNPO1 allows FUS to travel, unfettered, to sites of translation. ALS/FTD mutations hinder this interaction, rendering FUS prone to sequestration.
Dormann found that besides binding to FUS’s nuclear localization sequence, TNPO1 can also latch onto its RNA-binding domain, competing with RNA for attachment to FUS. In San Diego, Dormann said that she spotted TNPO1 lingering in RNA granules way out in the neurites of primary rat cortical neurons. She thinks TNPO1 might outcompete FUS-bound RNA there, thus releasing transcripts for local translation.
Roy Parker of the University of Colorado in Boulder told Alzforum that Dormann’s findings underscore the importance of maintaining a dynamic environment in the cytoplasm. “Just as RNA binding proteins like FUS and TDP-43 prevent RNA entanglement, it seems that chaperones like TNPO1 prevent these RNA-binding proteins from becoming ensnared in granules,” he said.
Peter St George-Hyslop’s data meshed with Dormann’s. His lab at the University of Toronto focused on the dynamics of FUS phase separation in axonal terminals. He found that both arginine hypomethylation and ALS/FTD mutations promote FUS phase separation and keep FUS enmeshed in granules. Similar to Dormann, he reported that TNPO1 counters this gelling tendency. If FUS remains stuck in granules, local translation at axonal terminals cannot happen (Apr 2018 news on Qamar et al., 2018). Likening granules to Borg assimilation in "Star Trek," St George-Hyslop said that occasionally, FUS granules merge, creating an ever-larger trap for FUS, RNA, and possibly translational machinery.
The translation machinery itself drew attention at this workshop. It includes a distinctive palette of proteins depending on where in the cell, and under what conditions, translation takes place, according to Maria Barna of Stanford University in Palo Alto, California. Barna described a startlingly diverse world of specialized ribosomes. Cast as blobs floating in a vast cytoplasmic sea, ribosomes have long been considered boring workhorses that translate any old transcript they come across. Barna’s work casts ribosomes as sites of exquisite specificity.
Using triple quadrupole mass spectrometry, Barna found that a fraction of the 80 core ribosomal proteins only appear in a subset of so-called specialized ribosomes. Strikingly, Barna reported that specialized ribosomes translated mRNAs involved in distinctive processes or pathways. For example, ribosomes containing the protein Rp10a tended to translate mRNAs involved in cell growth, but avoided mRNAs linked to opposing processes such as cell stress or apoptosis. Comparing specialized ribosomes with a bacterial operon system, Barna reported that certain ribosomes were responsible for translating proteins involved in entire pathways, such as vitamin B12 biosynthesis (Shi et al., 2017). The cadre of specialized ribosomes also shifted during cellular differentiation from stem cells to other cell types, according to preliminary data from her lab.
Barna also sees this specificity in the ribosomal interactome. She identified more than 200 ribosome-associated proteins, aka RAPs. They differed based on cell type and intracellular location. Barna has yet to investigate the landscape of specialized ribosomes or RAPs within neurons, but proposed that they could form the basis for specific translation in the axon or synapse. One tidbit she does know already: the ALS/FTD related proteins FUS, VCP, and FMRP1 are among the proteins that interacted with subsets of ribosomes (Simsek et al., 2017).
Da Cruz wondered whether these interactions might explain the specialized delivery of mRNAs by FUS or other RNA binding proteins out to the synapses. Dovetailing with Dormann’s idea that TNPO1-FUS interactions at the synapse help FUS shed its mRNA cargo there, perhaps associations with specialized ribosomes come into play as well, she speculated.
Benjamin Wolozin of Boston University told Alzforum that Barna’s findings about ribosomal complexity are an important factor the neurodegenerative disease field has yet to address. Specialized ribosomes could play a role in the selective vulnerability of neurons to disease.—Jessica Shugart
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