Detection of TTR Amyloid in the Conjunctiva Using a Novel Fluorescent Ocular Tracer.

Background: Transthyretin amyloidosis (ATTR) is a significant cause of cardiomyopathy and other morbidities in the elderly and Black Americans. ATTR can be treated with new disease-modifying therapies, but large shortfalls exist in its diagnosis. The objective of this study was to test whether TTR amyloid can be detected and imaged in the conjunctiva using a novel small-molecule fluorescent ocular tracer, with the implication that ATTR might be diagnosable by a simple eye examination. Methods: Three approaches were used in this study. First, AMDX-9101 was incubated with in vitro aggregated TTR protein, and changes in its excitation and emission spectra were quantified. Second, a cadaver eye from a patient with familial amyloid polyneuropathy type II TTR mutation and a vitrectomy sample from an hATTR patient were incubated with AMDX-9101 and counterstained with Congo Red and antibodies to TTR to determine whether AMDX-9101 labels disease-related TTR amyloid deposits in human conjunctiva and eye. Last, imaging of in vitro aggregated TTR amyloid labeled with AMDX-9101 was tested in a porcine ex vivo model, using a widely available clinical ophthalmic imaging device. Results: AMDX-9101 hyper-fluoresced in the presence of TTR amyloid in vitro, labeled TTR amyloid deposits in postmortem human conjunctiva and other ocular tissues and could be detected under the conjunctiva of a porcine eye using commercially available ophthalmic imaging equipment. Conclusions: AMDX-9101 enabled detection of TTR amyloid in the conjunctiva, and the fluorescent binding signal can be visualized using commercially available ophthalmic imaging equipment. Translational Relevance: AMDX-9101 detection of TTR amyloid may provide a potential new and noninvasive test for ATTR that could lead to earlier ATTR diagnosis, as well as facilitate development of new therapeutics.

RNA-binding protein RBM3 intrinsically suppresses lung innate lymphoid cell activation and inflammation partially through CysLT1R.

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3-/- and Rbm3-/-Rag2-/- mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3-/- ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3-/- lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3-/-Cyslt1r-/- mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.

A heterogeneous tRNA granule structure exhibiting rapid, bi-directional neuritic transport.

mRNA translation is regulated by diverse mechanisms that converge at the initiation and elongation steps to determine the rate, profile, and localization of proteins synthesized. A consistently relevant feature of these mechanisms is the spatial re-distribution of translation machinery, a process of particular importance in neural cells. This process has, however, been largely overlooked with respect to its potential role in regulating the local concentration of cytoplasmic tRNAs, even as a multitude of data suggest that spatial regulation of the tRNA pool may help explain the remarkably high rates of peptide elongation. Here, we report that Cy3/Cy5-labeled bulk tRNAs transfected into neural cells distribute into granule-like structures - "tRNA granules" - that exhibit dynamic mixing of tRNAs between granules and rapid, bi-directional vectorial movement within neurites. Imaging of endogenous tRNAgly and tRNAlys by fluorescent in situ hybridization revealed a similar granular distribution of tRNAs in somata and neurites; this distribution was highly overlapping with granules imaged by introduction of exogenous Cy5-tRNAthr and Cy3-tRNAval. A subset of tRNA granules located in the cell body, neurite branch points and growth cones displayed fluorescence resonance energy transfer (FRET) between Cy3 and Cy5-labeled tRNAs indicative of translation, and co-localization with elongation machinery. A population of smaller, rapidly trafficked granules in neurites lacked FRET and showed poor colocalization with translation initiation and elongation factors, suggesting that they are a translationally inactive tRNA transport particle. Our data suggest that tRNAs are packaged into granules that are rapidly transported to loci where translation is needed, where they may greatly increase the local concentration of tRNAs in support of efficient elongation. The potential implications of this newly described structure for channeling of elongation, local translation, and diseases associated with altered tRNA levels or function are discussed.

Antigen delivery to macrophages using liposomal nanoparticles targeting sialoadhesin/CD169.

Sialoadhesin (Sn, Siglec-1, CD169) is a member of the sialic acid binding Ig-like lectin (siglec) family expressed on macrophages. Its macrophage specific expression makes it an attractive target for delivering antigens to tissue macrophages via Sn-mediated endocytosis. Here we describe a novel approach for delivering antigens to macrophages using liposomal nanoparticles displaying high affinity glycan ligands of Sn. The Sn-targeted liposomes selectively bind to and are internalized by Sn-expressing cells, and accumulate intracellularly over time. Our results show that ligand decorated liposomes are specific for Sn, since they are taken up by bone marrow derived macrophages that are derived from wild type but not Sn(-/-) mice. Importantly, the Sn-targeted liposomes dramatically enhance the delivery of antigens to macrophages for presentation to and proliferation of antigen-specific T cells. Together, these data provide insights into the potential of cell-specific targeting and delivery of antigens to intracellular organelles of macrophages using Sn-ligand decorated liposomal nanoparticles.

Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3.

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ~70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ~70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events.

Developmentally regulated expression of the cold-inducible RNA-binding motif protein 3 in euthermic rat brain.

mRNA-binding proteins are critical regulators of protein synthesis during neural development. We demonstrated previously that the cold-inducible mRNA-binding protein 3 (RBM3) is present within euthermic neurons and that it enhances translation. Other studies have attributed anti-apoptotic and proliferative functions to RBM3. Here we characterize the developmental expression of RBM3 in rat brain. RBM3 is expressed widely during early brain development, peaking in the first to second postnatal weeks. This is followed by a decline in most brain regions and a shift from a nuclear to a more somatodendritic distribution by approximately P13. The highest levels of RBM3 in adult brain were observed in the cerebellum, olfactory bulb, proliferating cell fields and other regions reported to have high translation rates. RBM3 was expressed in glutamatergic and GABAergic cells, subtypes of which exhibited strong dendritic labeling for RBM3 mRNA and protein. Expression of RBM3 was also high in newly formed and migrating neurons marked by Ki67, nestin, and doublecortin, such as those in the subventricular zone and rostral migratory stream. These results indicate that expression of RBM3, a cold stress-responsive mRNA-binding protein, is dynamically regulated in the developing brain and suggest that it contributes to translation-dependent processes underlying proliferation, differentiation, and plasticity.

Two isoforms of the cold-inducible mRNA-binding protein RBM3 localize to dendrites and promote translation.

A diverse set of mRNA-binding proteins (BPs) regulate local translation in neurons. However, little is known about the role(s) played by a family of cold-inducible, glycine-rich mRNA-BPs. Unlike neuronal mRNA-BPs characterized thus far, these proteins are induced by hypothermia and are comprised of one RNA recognition motif and an adjacent arginine- and glycine-rich domain. We studied the expression and function of the RNA-binding motif protein 3 (RBM3), a member of this family, in neurons. RBM3 was expressed in multiple brain regions, with the highest levels in cerebellum and olfactory bulb. In dissociated neurons, RBM3 was observed in nuclei and in a heterogeneous population of granules within dendrites. In sucrose gradient assays, RBM3 cofractionated with heavy mRNA granules and multiple components of the translation machinery. Two alternatively spliced RBM3 isoforms that differed by a single arginine residue were identified in neurons; both were post-translationally modified. The variant lacking the spliced arginine exhibited a higher dendritic localization and was the only isoform present in astrocytes. When overexpressed in neuronal cell lines, RBM3 isoforms-enhanced global translation, the formation of active polysomes, and the activation of initiation factors. These data suggest that RBM3 plays a distinctive role in enhancing translation in neurons.

BDNF induces widespread changes in synaptic protein content and up-regulates components of the translation machinery: an analysis using high-throughput proteomics.

The brain-derived neurotrophic factor (BDNF) plays an important role in neuronal development, and in the formation and plasticity of synaptic connections. These effects of BDNF are at least partially due to the ability of the neurotrophin to increase protein synthesis both globally and locally. However, only a few proteins have been shown to be up-regulated at the synapse by BDNF. Using multidimensional protein identification technology (MudPIT) and relative quantification by spectra counting, we found that several hundred proteins are up-regulated in a synaptoneurosome preparation derived from cultured cortical neurons that were treated with BDNF. These proteins fall into diverse functional categories, including those involved in synaptic vesicle formation and movement, maintenance or remodeling of synaptic structure, mRNA processing, transcription, and translation. A number of translation factors, ribosomal proteins, and tRNA synthetases were rapidly up-regulated by BDNF. This up-regulation of translation components was sensitive to protein synthesis inhibitors and dependent on the activation of the mammalian target of rapamycin (mTOR), a regulator of cap-dependent mRNA translation. The presence of a subset of these proteins and their mRNAs in neuronal processes was corroborated by immunocytochemistry and in situ hybridization, and their up-regulation was confirmed by Western blotting. The data demonstrate that BDNF increases the synthesis of a wide variety of synaptic proteins and suggest that the neurotrophin may enhance the translational capacity of synapses.

Nuclear retention of MBP mRNAs in the quaking viable mice.

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.