Regulation of epithelial transitional states in murine and human pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (KRT8) genetic variants were associated with IPF. Krt8-/- mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.

Identification of PSMB5 as a genetic modifier of fragile X-associated tremor/ataxia syndrome.

Fragile X­associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58­70 (2015); S. Jacquemont et al., JAMA 291, 460­469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55­200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.

DDX3X and specific initiation factors modulate FMR1 repeat-associated non-AUG-initiated translation.

A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.

RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response.

Repeat-associated non-AUG (RAN) translation allows for unconventional initiation at disease-causing repeat expansions. As RAN translation contributes to pathogenesis in multiple neurodegenerative disorders, determining its mechanistic underpinnings may inform therapeutic development. Here we analyze RAN translation at G4C2 repeat expansions that cause C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9RAN) and at CGG repeats that cause fragile X-associated tremor/ataxia syndrome. We find that C9RAN translation initiates through a cap- and eIF4A-dependent mechanism that utilizes a CUG start codon. C9RAN and CGG RAN are both selectively enhanced by integrated stress response (ISR) activation. ISR-enhanced RAN translation requires an eIF2α phosphorylation-dependent alteration in start codon fidelity. In parallel, both CGG and G4C2 repeats trigger phosphorylated-eIF2α-dependent stress granule formation and global translational suppression. These findings support a model whereby repeat expansions elicit cellular stress conditions that favor RAN translation of toxic proteins, creating a potential feed-forward loop that contributes to neurodegeneration.

RAN translation-What makes it run?

Nucleotide-repeat expansions underlie a heterogeneous group of neurodegenerative and neuromuscular disorders for which there are currently no effective therapies. Recently, it was discovered that such repetitive RNA motifs can support translation initiation in the absence of an AUG start codon across a wide variety of sequence contexts, and that the products of these atypical translation initiation events contribute to neuronal toxicity. This review examines what we currently know and do not know about repeat associated non-AUG (RAN) translation in the context of established canonical and non-canonical mechanisms of translation initiation. We highlight recent findings related to RAN translation in three repeat expansion disorders: CGG repeats in fragile X-associated tremor ataxia syndrome (FXTAS), GGGGCC repeats in C9orf72 associated amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and CAG repeats in Huntington disease. These studies suggest that mechanistic differences may exist for RAN translation dependent on repeat type, repeat reading frame, and the surrounding sequence context, but that for at least some repeats, RAN translation retains a dependence on some of the canonical translational initiation machinery. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

CGG Repeat-Associated Non-AUG Translation Utilizes a Cap-Dependent Scanning Mechanism of Initiation to Produce Toxic Proteins.

Repeat-associated non-AUG (RAN) translation produces toxic polypeptides from nucleotide repeat expansions in the absence of an AUG start codon and contributes to neurodegenerative disorders such as ALS and fragile X-associated tremor/ataxia syndrome. How RAN translation occurs is unknown. Here we define the critical sequence and initiation factors that mediate CGG repeat RAN translation in the 5' leader of fragile X mRNA, FMR1. Our results reveal that CGG RAN translation is 30%-40% as efficient as AUG-initiated translation, is m(7)G cap and eIF4E dependent, requires the eIF4A helicase, and is strongly influenced by repeat length. However, it displays a dichotomous requirement for initiation site selection between reading frames, with initiation in the +1 frame, but not the +2 frame, occurring at near-cognate start codons upstream of the repeat. These data support a model in which RAN translation at CGG repeats uses cap-dependent ribosomal scanning, yet bypasses normal requirements for start codon selection.

Electron tomography on γ-aminobutyric acid-ergic synapses reveals a discontinuous postsynaptic network of filaments.

The regulation of synaptic strength at γ-aminobutyric acid (GABA)-ergic synapses is dependent on the dynamic capture, retention, and modulation of GABA A-type receptors by cytoplasmic proteins at GABAergic postsynaptic sites. How these proteins are oriented and organized in the postsynaptic cytoplasm is not yet established. To better understand these structures and gain further insight into the mechanisms by which they regulate receptor populations at postsynaptic sites, we utilized electron tomography to examine GABAergic synapses in dissociated rat hippocampal cultures. GABAergic synapses were identified and selected for tomography by using a set of criteria derived from the structure of immunogold-labeled GABAergic synapses. Tomography revealed a complex postsynaptic network composed of filaments that extend ∼ 100 nm into the cytoplasm from the postsynaptic membrane. The distribution of these postsynaptic filaments was strikingly similar to that of the immunogold label for gephyrin. Filaments were interconnected through uniform patterns of contact, forming complexes composed of 2-12 filaments each. Complexes did not link to form an integrated, continuous scaffold, suggesting that GABAergic postsynaptic specializations are less rigidly organized than glutamatergic postsynaptic densities.