Reticulon Research Articles

Cell autonomous function of nogo and reticulons: The emerging story at the endoplasmic reticulum

The myelin-associated membrane protein reticulon-4 (RTN4)/Nogo has been extensively studied with regards to its neurite outgrowth inhibitory function, both in limiting plasticity in the healthy adult brain and regeneration during central nervous system injury. These activities are presumably associated with Nogo splice isoforms expressed on the cell surface and function largely in trans, exerting an influence as an intercellular membrane-bound ligand. Nogo, and other reticulon paralogues and orthologues, are however mainly localized to the endoplasmic reticulum (ER), and are likely to have cell autonomous functions that are not yet clear. Emerging evidence suggests that Nogo may have a role in modulating the morphology and functions of the ER. This role is apparently not essential for cell viability under normal growth conditions, but may be manifested under certain stress conditions.

Phosphorylation‐regulated cleavage of the reticulon protein Nogo‐B by caspase‐7 at a noncanonical recognition site

Reticulons (RTNs) are a large family of transmembrane proteins present throughout the eukaryotic domain in virtually every cell type. Despite their wide distribution, their function is still mostly unknown. RTN4, also termed Nogo, comes in three isoforms, Nogo-A, -B, and -C. While Nogo-A has been described as potent inhibitor of nerve growth, Nogo-B has been implicated in vascular remodeling and regulation of apoptosis. We show here that Nogo-B gets cleaved by caspase-7, but not caspase-3, during apoptosis at a caspase nonconsensus site. By a combination of MS and site-directed mutagenesis we demonstrate that proteolytic processing of Nogo-B is regulated by phosphorylation of Ser(16) within the cleavage site. We present cyclin-dependent kinase (Cdk)1 and Cdk2 as kinases that phosphorylate Nogo-B at Ser(16) in vitro. In vivo, cleavage of Nogo-B is markedly increased in Schwann cells in a lesion model of the rat sciatic nerve. Taken together, we identified an RTN protein as one out of a selected number of caspase targets during apoptosis and as a novel substrate for Cdk1 and 2. Furthermore, our data support a functionality of caspase-7 that is distinct from closely related caspase-3.

Overexpression of a Plant Reticulon Remodels the Lumen of the Cortical Endoplasmic Reticulum but Does not Perturb Protein Transport

We have cloned a member of the reticulon (RTN) family of Arabidopsis thaliana (RTNLB13). When fused to yellow fluorescent protein (YFP) and expressed in tobacco leaf epidermal cells, RTNLB13 is localized in the endoplasmic reticulum (ER). Coexpression of a soluble ER luminal marker reveals that YFP-tagged, myc-tagged or untagged RTNLB13 induces severe morphological changes to the lumen of the ER. We show, using fluorescence recovery after photobleaching (FRAP) analysis, that RTNLB13 overexpression greatly reduces diffusion of soluble proteins within the ER lumen, possibly by introducing constrictions into the membrane. In spite of this severe phenotype, Golgi shape, number and dynamics appear unperturbed and secretion of a reporter protein remains unaffected.

Identification and expression of XRTN1‐A and XRTN1‐C in Xenopus laevis

Reticulon1, also known as neuroendocrine-specific protein, belongs to the reticulon (RTN) family, whose members possess a conserved reticulon domain and are associated with the endoplasmic reticulum (ER) membrane. Here, we report cloning and expression of Xenopus homologues of Reticulon1-A (XRTN1-A) and -C (XRTN1-C). XRTN1-A and -C contain an open reading frame of 752 and 207 amino acids, respectively, each containing a conserved reticulon domain. Sequence analysis shows that XRTN1 proteins have an ER membrane retention signal and four putative membrane-spanning domains. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization showed that XRTN1-A is expressed in early neural precursors and differentiating neuronal populations, including the trigeminal placode, olfactory placode, lateral line placode, and otic vesicle. XRTN1-C is expressed in the developing brain and spinal cord. We found that XRTN1-C protein is localized to the ER of Xenopus and mammalian cells and the granules in neurites of primary neurons of the Xenopus spinal cord and rat hippocampus. We also showed that XRTN1-C protein is detected in the heavy membrane fraction, which contains lysosomal and ER-resident proteins, as well as in the nucleus and polysomal fractions of the Xenopus embryo. Finally, we showed that thyroid hormone specifically down-regulates XRTN1-A mRNA in the head of premetamorphic Xenopus tadpoles. Our work characterizes the intracellular roles of XRTN1 during Xenopus neural development.

The Membrane Topology of RTN3 and Its Effect on Binding of RTN3 to BACE1

Reticulon 3 (RTN3) has recently been shown to modulate Alzheimer BACE1 activity and to play a role in the formation of dystrophic neurites present in Alzheimer brains. Despite the functional importance of this protein in Alzheimer disease pathogenesis, the functional correlation to the structural domain of RTN3 remained unclear. RTN3 has two long transmembrane domains, but its membrane topology was not known. We report here that the first transmembrane domain dictates membrane integration and its membrane topology. RTN3 adopts a omega-shape structure with two ends facing the cytosolic side. Subtle changes in RTN3 membrane topology can disrupt its binding to BACE1 and its inhibitory effects on BACE1 activity. Thus, the determination of RTN3 membrane topology may provide an important structural basis for our understanding of its cellular functions.

Anti‐apoptotic activity of Bcl‐2 is enhanced by its interaction with RTN3

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.

Transgenic mice overexpressing reticulon 3 develop neuritic abnormalities

Dystrophic neurites are swollen dendrites or axons recognizable near amyloid plaques as a part of important pathological feature of Alzheimer's disease (AD). We report herein that reticulon 3 (RTN3) is accumulated in a distinct population of dystrophic neurites named as RTN3 immunoreactive dystrophic neurites (RIDNs). The occurrence of RIDNs is concomitant with the formation of high-molecular-weight RTN3 aggregates in brains of AD cases and mice expressing mutant APP. Ultrastructural analysis confirms accumulation of RTN3-containing aggregates in RIDNs. It appears that the protein level of RTN3 governs the formation of RIDNs because transgenic mice expressing RTN3 will develop RIDNs, initially in the hippocampal CA1 region, and later in other hippocampal and cortical regions. Importantly, we show that the presence of dystrophic neurites in Tg-RTN3 mice causes impairments in spatial learning and memory, as well as synaptic plasticity, implying that RIDNs potentially contribute to AD cognitive dysfunction. Together, we demonstrate that aggregation of RTN3 contributes to AD pathogenesis by inducing neuritic dystrophy. Inhibition of RTN3 aggregation is likely a therapeutic approach for reducing neuritic dystrophy.

Two hydrophobic segments of the RTN1 family determine the ER localization and retention

Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer’s disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.

Selectively oncolytic mutant of HSV-1 lyses HeLa cells mediated by Ras/RTN3

The selectively oncolytic mtHSV, a HSV icp34.5 mutant with lacz gene insertion, was proved that it was targeted for treating tumors but not other organs, however, its oncolytic mechanism is under confirmation. The results showed that HeLa cells could be lysed efficiently by mtHSV in vitro. In the flow cytometry and Western blot experiment, Ras protein was obviously down-regulated on plasma membrane (PM) while the whole Ras protein didn’t change along with up-regulation of reticulon 3(RTN3) protein at 48h post infection of mtHSV in HeLa cells . Expression of Ras protein on PM and whole Ras protein in HeLa cells was down-regulated by siFTa（inhibitor ofαsubunits of human farnesyltransferase with siRNA）and siRTN3(inhibitor of RTN3 with siRNA) respectively, and HeLa cells could be killed effectively by siFTa and siRTN3 at 48h post transfection. So siFTa and siRTN3 effectively suppressed mtHSV infection of HeLa cells. Further, experiments were made to study the relationship between Ras and RTN3 using confocal colocalization and co-immunoprecipitation. The results exhibited that Ras could interact with RTN3 at endoplasmic reticulum. The data put forward that Ras/RTN3 is an important access to HeLa cells for mtHSV. The molecular interaction between Ras and RTN3 may further improve the understanding of the function of Ras and RTN3 in mtHSV infection. The results provide further theoretical evidence that mtHSV may be used as an oncolytic agent for cancer therapy.

Reticulon 3 mediates Bcl-2 accumulation in mitochondria in response to endoplasmic reticulum stress

Reticulon3 (RTN3), firstly isolated from the retina and widely expressed in human tissues with the highest expression in the brain, is presumed to play an important role in the developing axons through the transport of liquids and proteins. We have identified and characterized RTN3 as a RTN4B/ASY interaction protein. Here we demonstrated that ER-stress activated RTN3 expression. CHOP and ATF6 were sufficient to up-regulate the expression of RTN3. The down-regulation of RTN3 would induce apoptosis and attenuate the anti-apoptotic activity of Bcl-2, indicating RTN3 was required for the cellular survival and optimal anti-apoptotic activity of Bcl-2. Our present studies also indicated ER-stress induced RTN3 up-regulation could trigger Bcl-2 translocation from ER to mitochondria. Moreover, the previous studies showed that RTN4B was also a Bcl-2-interacted protein. We found that RTN3 and RTN4B could block the access of Bcl-2 to each other and thereafter determined the Bcl-2 subcellular distribution. Taken together, our findings indicate that RTN3 is directly involved in the ER-constituents trafficking events through dually acting as an essential and important ER-stress sensor, and a trigger for the Bcl-2 translocation.

Reticulons RTN3 and RTN4‐B/C interact with BACE1 and inhibit its ability to produce amyloid β‐protein

Beta-secretase beta-site APP cleaving enzyme 1 (BACE1), is a membrane-bound aspartyl protease necessary for the generation of amyloid beta-protein (Abeta), which accumulates in the brains of individuals with Alzheimer's disease (AD). To gain insight into the mechanisms by which BACE1 activity is regulated, we used proteomic methods to search for BACE1-interacting proteins in human neuroblastoma SH-SY5Y cells, which overexpress BACE1. We identified reticulon 4-B (RTN4-B; Nogo-B) as a BACE1-associated membrane protein. Co-immunoprecipitation experiments confirmed a physical association between BACE1 and RTN4-B, RTN4-C (the shortest isoform of RTN-4), and their homologue reticulon 3 (RTN3), both in SH-SY5Y cells and in transfected human embryonic kidney (HEK) 293 cells. Overexpression of these reticulons (RTNs) resulted in a 30-50% reduction in the secretion of both Abeta40 and Abeta42 from HEK293 cells expressing the AD-associated Swedish mutant amyloid precursor protein (APP), but did not affect Abeta secretion from cells expressing the APP beta-C-terminal fragment (beta-CTF), indicating that these RTNs can inhibit BACE1 activity. Furthermore, a BACE1 mutant lacking most of the N-terminal ectodomain also interacted with these RTNs, suggesting that the transmembrane region of BACE1 is critical for the interaction. We also observed a similar interaction between these RTNs and the BACE1 homologue BACE2. Because RTN3 and RTN4-B/C are substantially expressed in neural tissues, our findings suggest that they play important roles in the regulation of BACE1 function and Abeta production in the brain.

Mapping of Interaction Domains Mediating Binding between BACE1 and RTN/Nogo Proteins

BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the β-secretase site. Membrane bound reticulon (RTN) family proteins interact with BACE1 and negatively modulate BACE1 activity through preventing access of BACE1 to its cellular APP substrate. Here, we focused our study on RTN3 and further show that a C-terminal QID triplet conserved among mammalian RTN members is required for the binding of RTN to BACE1. Although RTN3 can form homo- or heterodimers in cells, BACE1 mainly binds to the RTN monomer and disruption of the QID triplet does not interfere with the dimerization. Correspondingly, the C-terminal region of BACE1 is required for the binding of BACE1 to RTNs. Furthermore, we show that the negative modulation of BACE1 by RTN3 relies on the binding of RTN3 to BACE1. The knowledge from this study may potentially guide discovery of small molecules that can mimic the effect of RTN3 on the inhibition of BACE1 activity.

Spastin, the most commonly mutated protein in hereditary spastic paraplegia interacts with Reticulon 1 an endoplasmic reticulum protein

Spastin, an ATPase belonging to the AAA family of proteins is most commonly mutated in autosomal dominant hereditary spastic paraplegias (HSP). Spastin is a multifaceted protein with versatile role in cellular events, principally involved in microtubule dynamics. To gain further insight into the molecular function of spastin, we used the yeast two-hybrid approach to identify novel interacting partners of spastin. Using spastin as bait, we identified reticulon 1 (RTN1) and reticulon 3 (RTN3) as potential spastin interacting proteins. RTN1 and RTN3 belong to the reticulon (RTN) gene family, which are primarily expressed in the endoplasmic reticulum. Moreover, RTN1 is known to play a role in vesicular transport processes. Using in vitro and in vivo immunoprecipitation experiments, we were able to demonstrate that RTN1 interacts specifically with spastin. Intracellular distribution studies using immunostaining and overexpression of epitope-tagged protein revealed an obvious colocalization of spastin and RTN1 in discrete vesicles in the cytoplasm. Spastin mediates its interaction with RTN1 through its N-terminal region containing a microtubule-interacting and trafficking domain. It is interesting to note that the aberrant intracellular distribution of a truncated spastin protein was rescued by coexpression with RTN1, which highlights the physiological significance of this interaction. Our findings strengthen the hypothesis that disruption of intracellular vesicular transport processes could cause HSP. It is interesting to note that RTN1 is localized to 14q23.1 where SPG15 locus was mapped. Therefore, we considered RTN1 as a candidate gene for the SPG15 locus, but our mutational analysis possibly excludes RTN1 as causative gene.

Reticulon proteins: emerging players in neurodegenerative diseases.

Reticulons (RTNs) are a group of integral membrane proteins that have a uniquely conserved C-terminal domain named RHD. In mammalian genomes, transcripts are produced from four genes, rtn1 to rtn4, under the regulation of tissue or cell-type-specific expression. The presence of alternative promoters for gene expression and multiple cryptic splicing sites have resulted in large numbers of genes/proteins that are classified among the reticulon family. Although this family exists in almost all eukaryotes, only the rtn4 gene product, Nogo (RTN4), has gained relatively more in-depth attention. Despite predominant localization in the endoplasmic reticulum, Nogo on the cell surface appears to play a critical role as an inhibitory molecule for axonal growth and regeneration in humans and rodents. Recently, studies have expanded the biological functions of RTNs to other facets including modulating the enzymatic activity of beta-secretase in Alzheimer's disease. In this review, we summarize the accumulated findings concerning the structural and functional aspects of RTNs and speculate on their linkage to the pathogenesis of neurodegenerative diseases.

Cloning, expression, and preliminary structural characterization of RTN-1C

Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.

Screening and cloning of hepatitis C virus non-structural protein 4B interacting protein gene in hepatocytes

To investigate biological functions of non-structural protein 4B (NS4B) of hepatitis C virus (HCV), yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4B. HCV NS4B bait plasmid was constructed by ligating the NS4B gene with carrier plasmid pGBKT7 and transformed into yeast cells AH109 (type alpha). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent Escherichia coli and analysed by DNA sequencing and bioinformatics. Five genes in eight positive colonies were obtained. There were one NADH dehydrogenase subunit 3, one cytochrome c oxidase subunit III, one retinol binding protein 4, one reticulon 3-A (RTN3) and one fibrinogen gamma polypeptide (FGG). Genes of HCV NS4B interacting proteins in hepatocytes were successfully cloned and the results paved the way for studying the biological functions of NS4B and associated proteins.

Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.

Identification of a new RTN3 transcript, RTN3-A1, and its distribution in adult mouse brain

The Reticulon (RTN) family of proteins is thought to play important roles in the regulation of neuronal regeneration. In this study, we have identified a novel alternative splicing isoform of the RTN gene family, RTN3-A1, which contains an additional 2.3-kb exon. The transcripts of human and mouse RTN3-A1 (about 5.0 kb) were first discovered by database sequence mining and analysis, and verified by cloning and sequencing. Northern blot analysis of 16 human tissues with a common probe of RTN3 transcripts and a specific probe for RTN3-A1 demonstrated that human RTN3-A1 is expressed mainly in brain tissues with a weak expression in the skeletal muscle. With Western blot analysis, the expected 100-kDa RTN3-A1 protein was detected in mouse brain. In situ hybridization with a mouse RTN3-A1-specific cRNA probe revealed that the mouse RTN3-A1 mRNA was regionally expressed in the neurons of the cerebral cortex, hippocampus, hypothalamus, and cerebellum of the adult mouse brain. In contrast to the transcripts of RTN1 and RTN2, RTN3-A1 shares some significant similarity with RTN4-A in exon structure, tissue distribution, and brain expression profile. Since other reports have shown that RTN4-A inhibits neuronal outgrowth and restricts the plasticity of the central nervous system, we speculate that RTN3-A1 might play certain roles in the central nervous system.

Analysis of the Reticulon Gene Family Demonstrates the Absence of the Neurite Growth Inhibitor Nogo-A in Fish

Reticulons (RTNs) are a family of evolutionary conserved proteins with four RTN paralogs (RTN1, RTN2, RTN3, and RTN4) present in land vertebrates. While the exact functions of RTN1 to RTN3 are unknown, mammalian RTN4-A/Nogo-A was shown to inhibit the regeneration of severed axons in the mammalian central nervous system (CNS). This inhibitory function is exerted via two distinct regions, one within the Nogo-A-specific N-terminus and the other in the conserved reticulon homology domain (RHD). In contrast to mammals, fish are capable of CNS axon regeneration. We performed detailed analyses of the fish rtn gene family to determine whether this regeneration ability correlates with the absence of the neurite growth inhibitory protein Nogo-A. A total of 7 rtn genes were identified in zebrafish, 6 in pufferfish, and 30 in eight additional fish species. Phylogenetic and syntenic relationships indicate that the identified fish rtn genes are orthologs of mammalian RTN1, RTN2, RTN3, and RTN4 and that several paralogous fish genes (e.g., rtn4 and rtn6) resulted from genome duplication events early in actinopterygian evolution. Accordingly, sequences homologous to the conserved RTN4/Nogo RHD are present in two fish genes, rtn4 and rtn6. However, sequences comparable to the first approximately 1,000 amino acids of mammalian Nogo-A including a major neurite growth inhibitory region are absent in zebrafish. This result is in accordance with functional data showing that axon growth inhibitory molecules are less prominent in fish oligodendrocytes and CNS myelin compared to mammals.

Identification and expression of XRTN2 and XRTN3 duringXenopus development

The reticulon (RTN) family of proteins has been described as a new eukaryotic protein family. We have isolated Xenopus cDNA homologues of RTN2 and RTN3 and examined their expression patterns during Xenopus development. XRTN2 has two transcripts, XRTN2-B and XRTN2-C, which encode 321 and 191 amino acids, respectively. XRTN3 has only one transcript that encodes 214 amino acids. We detected the XRTN2-B transcript in the neural tissues and brain from the early neurula stage. XRTN2-C is strongly expressed in the myotome, future skeletal muscle. The XRTN3 mRNA is localized in the animal hemisphere of the egg and blastula stage embryos and then subsequently restricted, mainly in the neural tissues. At the subcellular level, The XRTN proteins are expressed in the endoplasmic reticulum network structure of the animal cap cells as well as COS-7 cells. Our results suggest the potential roles of XRTN2s and XRTN3 during Xenopus embryogenesis.