Rethinking Unconventional Translation in Neurodegeneration.

Eukaryotic translation is tightly regulated to ensure that protein production occurs at the right time and place. Recent studies on abnormal repeat proteins, especially in age-dependent neurodegenerative diseases caused by nucleotide repeat expansion, have highlighted or identified two forms of unconventional translation initiation: usage of AUG-like sites (near cognates) or repeat-associated non-AUG (RAN) translation. We discuss how repeat proteins may differ due to not just unconventional initiation, but also ribosomal frameshifting and/or imperfect repeat DNA replication, expansion, and repair, and we highlight how research on translation of repeats may uncover insights into the biology of translation and its contribution to disease.

Pausing on Polyribosomes: Make Way for Elongation in Translational Control.

Among the three phases of mRNA translation-initiation, elongation, and termination-initiation has traditionally been considered to be rate limiting and thus the focus of regulation. Emerging evidence, however, demonstrates that control of ribosome translocation (polypeptide elongation) can also be regulatory and indeed exerts a profound influence on development, neurologic disease, and cell stress. The correspondence of mRNA codon usage and the relative abundance of their cognate tRNAs is equally important for mediating the rate of polypeptide elongation. Here, we discuss recent results showing that ribosome pausing is a widely used mechanism for controlling translation and, as a result, biological transitions in health and disease.

Cytoplasmic polyadenylation element binding proteins in development, health, and disease.

The cytoplasmic polyadenylation element binding (CPEB) proteins are sequence-specific mRNA binding proteins that control translation in development, health, and disease. CPEB1, the founding member of this family, has become an important model for illustrating general principles of translational control by cytoplasmic polyadenylation in gametogenesis, cancer etiology, synaptic plasticity, learning, and memory. Although the biological functions of the other members of this protein family in vertebrates are just beginning to emerge, it is already evident that they, too, mediate important processes, such as cancer etiology and higher cognitive function. In Drosophila, the CPEB proteins Orb and Orb2 play key roles in oogenesis and in neuronal function, as do related proteins in Caenorhabditis elegans and Aplysia. We review the biochemical features of the CPEB proteins, discuss their activities in several biological systems, and illustrate how understanding CPEB activity in model organisms has an important impact on neurological disease.

The molecular biology of FMRP: new insights into fragile X syndrome.

Fragile X mental retardation protein (FMRP) is the product of the fragile X mental retardation 1 gene (FMR1), a gene that - when epigenetically inactivated by a triplet nucleotide repeat expansion - causes the neurodevelopmental disorder fragile X syndrome (FXS). FMRP is a widely expressed RNA-binding protein with activity that is essential for proper synaptic plasticity and architecture, aspects of neural function that are known to go awry in FXS. Although the neurophysiology of FXS has been described in remarkable detail, research focusing on the molecular biology of FMRP has only scratched the surface. For more than two decades, FMRP has been well established as a translational repressor; however, recent whole transcriptome and translatome analyses in mouse and human models of FXS have shown that FMRP is involved in the regulation of nearly all aspects of gene expression. The emerging mechanistic details of the mechanisms by which FMRP regulates gene expression may offer ways to design new therapies for FXS.

FMRP deficiency leads to multifactorial dysregulation of splicing and mislocalization of MBNL1 to the cytoplasm.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that is often modeled in Fmr1 knockout mice where the RNA-binding protein FMRP is absent. Here, we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, PTBP1, and MBNL1. FMRP regulates the translation of Mbnl1 mRNA as well as Mbnl1 RNA auto-splicing. Elevated Mbnl1 auto-splicing in FMRP-deficient cells results in the loss of a nuclear localization signal (NLS)-containing exon. This in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This redistribution of MBNL1 isoforms in Fmr1-deficient cells could result in downstream splicing changes in other RNAs. Indeed, further investigation revealed that splicing disruptions resulting from Fmr1 depletion could be rescued by overexpression of nuclear MBNL1. Altered Mbnl1 auto-splicing also occurs in human FXS postmortem brain. These data suggest that FMRP-controlled translation and RNA processing may cascade into a general dys-regulation of splicing in Fmr1-deficient cells.

FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.

FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.

Antisense oligonucleotide rescue of CGG expansion-dependent FMR1 mis-splicing in fragile X syndrome restores FMRP.

Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1-expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced FMR1, antisense oligonucleotide (ASO) treatment reduces FMR1-217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217, ASO treatment may offer a therapeutic approach to mitigate the disorder.

FMRP regulation of aggrecan mRNA translation controls perineuronal net development.

Perineuronal nets (PNNs) are mesh-like structures on the surfaces of parvalbumin-expressing inhibitory and other neurons, and consist of proteoglycans such as aggrecan, brevican, and neurocan. PNNs regulate the Excitatory/Inhibitory (E/I) balance in the brain and are formed at the closure of critical periods of plasticity during development. PNN formation is disrupted in Fragile X Syndrome, which is caused by silencing of the fragile X messenger ribonucleoprotein 1 (Fmr1) gene and loss of its protein product FMRP. FXS is characterized by impaired synaptic plasticity resulting in neuronal hyperexcitability and E/I imbalance. Here, we investigate how PNN formation is altered in FXS. PNNs are reduced in Fmr1 KO mouse brain when examined by staining for the lectin Wisteria floribunda agglutin (WFA) and aggrecan. Examination of PNNs by WFA staining at P14 and P42 in the hippocampus, somatosensory cortex, and retrosplenial cortex shows that they were reduced in these brain regions at P14 but mostly less so at P42 in Fmr1 KO mice. However, some differential FMRP regulation of PNN development in these brain regions persists, perhaps caused by asynchrony in PNN development between brain regions in wild-type animals. During development, aggrecan PNN levels in the brain were reduced in all brain regions in Fmr1 KO mice. Aggrecan mRNA levels were unchanged at these times, suggesting that FMRP is normally an activator of aggrecan mRNA translation. This hypothesis is buttressed by the observations that FMRP binds aggrecan mRNA and that ribosome profiling data show that aggrecan mRNA is associated with reduced numbers of ribosomes in Fmr1 KO mouse brain, indicating reduced translational efficiency. Moreover, aggrecan mRNA poly(A) tail length is also reduced in Fmr1 KO mouse brain, suggesting a relationship between polyadenylation and translational control. We propose a model where FMRP modulates PNN formation through translational up-regulation of aggrecan mRNA polyadenylation and translation.

Oppositional poly(A) tail length regulation by FMRP and CPEB1.

Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3'UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases GLD2 (TENT2, PAPD4, Wispy) and GLD4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor GLD4 to RNA, we expressed double tagged GLD4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that coprecipitated with GLD4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA nanopore sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation.

Ribosome profiling reveals novel regulation of C9ORF72 GGGGCC repeat-containing RNA translation.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5' cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

CPEB and translational control by cytoplasmic polyadenylation: impact on synaptic plasticity, learning, and memory.

The late 1990s were banner years in molecular neuroscience; seminal studies demonstrated that local protein synthesis, at or near synapses, was necessary for synaptic plasticity, the underlying cellular basis of learning and memory [1, 2]. The newly made proteins were proposed to "tag" the stimulated synapse, distinguishing it from naive synapses, thereby forming a cellular memory [3]. Subsequent studies demonstrated that the transport of mRNAs from soma to dendrite was linked with translational unmasking at synapses upon synaptic stimulation. It soon became apparent that one prevalent mechanism governing these events is cytoplasmic polyadenylation, and that among the proteins that control this process, CPEB, plays a central role in synaptic plasticity, and learning and memory. In vertebrates, CPEB is a family of four proteins, all of which regulate translation in the brain, that have partially overlapping functions, but also have unique characteristics and RNA binding properties that make them control different aspects of higher cognitive function. Biochemical analysis of the vertebrate CPEBs demonstrate them to respond to different signaling pathways whose output leads to specific cellular responses. In addition, the different CPEBs, when their functions go awry, result in pathophysiological phenotypes resembling specific human neurological disorders. In this essay, we review key aspects of the vertebrate CPEB proteins and cytoplasmic polyadenylation within the context of brain function.

Breaking the code of polyadenylation-induced translation.

The translation of many maternal mRNAs is regulated by dynamic changes in poly(A) tail length. During maturation of Xenopus oocytes, polyadenylation is mediated by three different cis elements in the 3' untranslated region (UTR) of maternal mRNAs. In this issue, Piqué et al. (2008) explore the interplay of these elements to elucidate a combinatorial code that predicts the timing of polyadenylation and translation of maternal mRNAs.

FMRP links optimal codons to mRNA stability in neurons.

Fragile X syndrome (FXS) is caused by inactivation of the FMR1 gene and loss of encoded FMRP, an RNA binding protein that represses translation of some of its target transcripts. Here we use ribosome profiling and RNA sequencing to investigate the dysregulation of translation in the mouse brain cortex. We find that most changes in ribosome occupancy on hundreds of mRNAs are largely driven by dysregulation in transcript abundance. Many down-regulated mRNAs, which are mostly responsible for neuronal and synaptic functions, are highly enriched for FMRP binding targets. RNA metabolic labeling demonstrates that, in FMRP-deficient cortical neurons, mRNA down-regulation is caused by elevated degradation and is correlated with codon optimality. Moreover, FMRP preferentially binds mRNAs with optimal codons, suggesting that it stabilizes such transcripts through direct interactions via the translational machinery. Finally, we show that the paradigm of genetic rescue of FXS-like phenotypes in FMRP-deficient mice by deletion of the Cpeb1 gene is mediated by restoration of steady-state RNA levels and consequent rebalancing of translational homeostasis. Our data establish an essential role of FMRP in codon optimality-dependent mRNA stability as an important factor in FXS.

Update on the Diagnosis and Treatment of Achalasia.

Achalasia is a rare disease of the esophagus with impaired relaxation of the lower esophageal sphincter and aperistalsis. The etiology is unknown but speculations include a viral or autoimmune etiology. All specialists dealing with swallowing and esophageal diseases should recognize the classic symptoms of dysphagia for solids/liquids, regurgitation, and choking, especially at night. High-resolution manometry is critical for the diagnosis with endoscopy and barium esophagram having a supportive role. The disease cannot be cured but most can return to near normal swallowing and a regular diet with appropriate therapy. Treatment includes smooth muscle relaxants, botulinum toxin injections to the lower sphincter, pneumatic dilation, Heller myotomy, and peroral endoscopic myotomy. One treatment does not fit all and a tailored approach through a multidiscipline team will give the best long-term outcomes.

CPEB1 regulates the inflammatory immune response, phagocytosis, and alternative polyadenylation in microglia.

Microglia are myeloid cells of the central nervous system that perform tasks essential for brain development, neural circuit homeostasis, and neural disease. Microglia react to inflammatory stimuli by upregulating inflammatory signaling through several different immune cell receptors such as the Toll-like receptor 4 (TLR4), which signals to several downstream effectors including transforming growth factor beta-activated kinase 1 (TAK1). Here, we show that TAK1 levels are regulated by CPEB1, a sequence-specific RNA binding protein that controls translation as well as RNA splicing and alternative poly(A) site selection in microglia. Lipopolysaccharide (LPS) binds the TLR4 receptor, which in CPEB1-deficient mice leads to elevated expression of ionized calcium binding adaptor molecule 1 (Iba1), a microglial protein that increases with inflammation, and increased levels of the cytokine IL6. This LPS-induced IL6 response is blocked by inhibitors of JNK, p38, ERK, NFκB, and TAK1. In contrast, phagocytosis, which is elevated in CPEB1-deficient microglia, is unaffected by LPS treatment or ERK inhibition, but is blocked by TAK1 inhibition. These data indicate that CPEB1 regulates microglial inflammatory responses and phagocytosis. RNA-seq indicates that these changes in inflammation and phagocytosis are accompanied by changes in RNA levels, splicing, and alternative poly(A) site selection. Thus, CPEB1 regulation of RNA expression plays a role in microglial function.

Demystifying Esophageal Lichen Planus: A Comprehensive Review of a Rare Disease You Will See in Practice.

Lichen planus (LP) is a chronic inflammatory disorder that often affects the skin, hair, nails, and mucus membranes. Although esophageal involvement has traditionally been felt to be rare, recent reports suggest that it is often unrecognized or misdiagnosed. The diagnoses of esophageal lichen planus can be challenging and is suspected based on patients' endoscopic and histologic findings and in the context of their clinical history and physical examination. Physicians must have an index of suspicion, particularly in older white women and in those patients with an atypical esophagitis or stricturing disease, which do not respond to traditional treatment. Currently, there are limited data on esophageal lichen planus patients, and no formal management guidelines for this disease, which all gastroenterologists will see in practice. This article reviews the etiology and histopathology of LP and provides a comprehensive discussion of the clinical features, diagnosis, and management of esophageal disease from the gastroenterologist's perspective. Finally, we address the esophageal complications of LP.

Endoscopic approach to eosinophilic esophagitis: American Society for Gastrointestinal Endoscopy Consensus Conference.

Endoscopy plays a critical role in caring for and evaluating the patient with eosinophilic esophagitis (EoE). Endoscopy is essential for diagnosis, assessment of response to therapy, treatment of esophageal strictures, and ongoing monitoring of patients in histologic remission. To date, less-invasive testing for identifying or grading EoE severity has not been established, whereas diagnostic endoscopy as integral to both remains the criterion standard. Therapeutic endoscopy in patients with adverse events of EoE may also be required. In particular, dilation may be essential to treat and attenuate progression of the disease in select patients to minimize further fibrosis and stricture formation. Using a modified Delphi consensus process, a group of 20 expert clinicians and investigators in EoE were assembled to provide guidance for the use of endoscopy in EoE. Through an iterative process, the group achieved consensus on 20 statements yielding comprehensive advice on tissue-sampling standards, gross assessment of disease activity, use and performance of endoscopic dilation, and monitoring of disease, despite an absence of high-quality evidence. Key areas of controversy were identified when discussions yielded an inability to reach agreement on the merit of a statement. We expect that with ongoing research, higher-quality evidence will be obtained to enable creation of a guideline for these issues. We further anticipate that forthcoming expert-generated and agreed-on statements will provide valuable practice advice on the role and use of endoscopy in patients with EoE.

Noncanonical cytoplasmic poly(A) polymerases regulate RNA levels, alternative RNA processing, and synaptic plasticity but not hippocampal-dependent behaviours.

Noncanonical poly(A) polymerases are frequently tethered to mRNA 3' untranslated regions and regulate poly(A) tail length and resulting translation. In the brain, one such poly(A) polymerase is Gld2, which is anchored to mRNA by the RNA-binding protein CPEB1 to control local translation at postsynaptic regions. Depletion of CPEB1 or Gld2 from the mouse hippocampus results in a deficit in long-term potentiation (LTP), but only depletion of CPEB1 alters animal behaviour. To test whether a related enzyme, Gld4, compensates for the lack of Gld2, we separately or simultaneously depleted both proteins from hippocampal area CA1 and again found little change in animal behaviour, but observed a deficit in LTP as well as an increase in long-term depression (LTD), two forms of protein synthesis-dependent synaptic plasticity. RNA-seq data from Gld2, Gld4, and Gld2/Gld4-depleted hippocampus show widespread changes in steady state RNA levels, alternative splicing, and alternative poly(A) site selection. Many of the RNAs subject to these alterations encode proteins that mediate synaptic function, suggesting a molecular foundation for impaired synaptic plasticity.

Discordance between high-resolution manometry, esophagoscopy and contrast esophagogram in determining landmarks for per-oral endoscopic myotomy in spastic esophageal disorders: a word of caution.

BACKGROUND: Myotomy length for per-oral endoscopic myotomy (POEM) is standardized for type I and II achalasia. However, for type III achalasia, jackhammer esophagus, diffuse esophageal spasms and esophagogastric junction outflow obstruction, there is no standard. Determining myotomy length based on the high-pressure zone found during high-resolution manometry (HRM) and spastic length found during esophagography may be used to determine adequate myotomy length without excess muscle destruction. METHODS: The records of patients who have undergone POEM procedures at our institution had the following data gleaned: age, sex, esophageal spastic diagnosis, length of high-pressure zone and lower esophageal sphincter (LES) position by HRM, length of spastic esophagus by esophagography, position of the z-line by esophagoscopy and length of myotomy performed. Outcomes were assessed based on patient symptomatic improvement and need for re-intervention. RESULTS: 71 patients were evaluated for POEM, with 67 completing POEM. There was an average difference in LES position by HRM and z-line position by esophagoscopy of 3.9 ± 3.0 cm. There was an average difference in high-pressure zone by HRM and spastic length by esophagography of 4.9 ± 3.2 cm. Overall, with a median of 20 months follow-up, 74% achieved long-term symptomatic improvement, with 17 patients requiring re-intervention. CONCLUSIONS: Discordance among HRM, esophagography and esophagoscopy can be significant. Caution should be employed with using these methods to determine myotomy length in POEM.

Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice.

Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases including fragile X syndrome. In order to capture subtle but biologically significant differences in translation in these disorders, a robust technique is required. One powerful tool to study translational control is ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease (RNase) digestion by ribosomes. However, this approach has been mainly applied to rapidly dividing cells where translation is active and large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for RNase digestion. We performed RNase titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling for low-input brain tissue.