Neuronal BC RNA Transport Impairments Caused by Systemic Lupus Erythematosus Autoantibodies.

The etiology of the autoimmune disorder systemic lupus erythematosus (SLE) remains poorly understood. In neuropsychiatric SLE (NPSLE), autoimmune responses against neural self-antigens find expression in neurological and cognitive alterations. SLE autoantibodies often target nucleic acids, including RNAs and specifically RNA domains with higher-order structural content. We report that autoantibodies directed against neuronal regulatory brain cytoplasmic (BC) RNAs were generated in a subset of SLE patients. By contrast, anti-BC RNA autoantibodies (anti-BC abs) were not detected in sera from patients with autoimmune diseases other than SLE (e.g., rheumatoid arthritis or multiple sclerosis) or in sera from healthy subjects with no evidence of disease. SLE anti-BC abs belong to the IgG class of immunoglobulins and target both primate BC200 RNA and rodent BC1 RNA. They are specifically directed at architectural motifs in BC RNA 5' stem-loop domains that serve as dendritic targeting elements (DTEs). SLE anti-BC abs effectively compete with RNA transport factor heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) for DTE access and significantly diminish BC RNA delivery to synapto-dendritic sites of function. In vivo experiments with male BALB/c mice indicate that, upon lipopolysaccharide-induced opening of the blood-brain barrier, SLE anti-BC abs are taken up by CNS neurons where they significantly impede localization of endogenous BC1 RNA to synapto-dendritic domains. Lack of BC1 RNA causes phenotypic abnormalities including epileptogenic responses and cognitive dysfunction. The combined data indicate a role for anti-BC RNA autoimmunity in SLE and its neuropsychiatric manifestations.SIGNIFICANCE STATEMENT Although clinical manifestations of neuropsychiatric lupus are well recognized, the underlying molecular-cellular alterations have been difficult to determine. We report that sera of a subset of lupus patients contain autoantibodies directed at regulatory brain cytoplasmic (BC) RNAs. These antibodies, which we call anti-BC abs, target the BC RNA 5' domain noncanonical motif structures that specify dendritic delivery. Lupus anti-BC abs effectively compete with RNA transport factor heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) for access to BC RNAs. As a result, hnRNP A2 is displaced, and BC RNAs are impaired in their ability to reach synapto-dendritic sites of function. The results reveal an unexpected link between BC RNA autoantibody recognition and dendritic RNA targeting. Cellular RNA dysregulation may thus be a contributing factor in the pathogenesis of neuropsychiatric lupus.

Regulatory BC1 RNA in cognitive control.

Dendritic regulatory BC1 RNA is a non-protein-coding (npc) RNA that operates in the translational control of gene expression. The absence of BC1 RNA in BC1 knockout (KO) animals causes translational dysregulation that entails neuronal phenotypic alterations including prolonged epileptiform discharges, audiogenic seizure activity in vivo, and excessive cortical oscillations in the γ frequency band. Here we asked whether BC1 RNA control is also required for higher brain functions such as learning, memory, or cognition. To address this question, we used odor/object attentional set shifting tasks in which prefrontal cortical performance was assessed in a series of discrimination and conflict learning sessions. Results obtained in these behavioral trials indicate that BC1 KO animals were significantly impaired in their cognitive flexibility. When faced with conflicting information sources, BC1 KO animals committed regressive errors as they were compromised in their ability to disengage from recently acquired memories even though recall of such memories was in conflict with new situational context. The observed cognitive deficits are reminiscent of those previously described in subtypes of human autism spectrum disorders.

Translational control at the synapse: role of RNA regulators.

Translational control of gene expression is instrumental in the regulation of eukaryotic cellular form and function. Neurons in particular rely on this form of control because their numerous synaptic connections need to be independently modulated in an input-specific manner. Brain cytoplasmic (BC) RNAs implement translational control at neuronal synapses. BC RNAs regulate protein synthesis by interacting with eIF4 translation initiation factors. Recent evidence suggests that such regulation is required to control synaptic strength, and that dysregulation of local protein synthesis precipitates neuronal hyperexcitability and a propensity for epileptogenic responses. A similar phenotype results from lack of fragile X mental retardation protein (FMRP), indicating that BC RNAs and FMRP use overlapping and convergent modes of action in neuronal translational regulation.

Autoimmune RNA dysregulation and seizures: therapeutic prospects in neuropsychiatric lupus.

Lupus autoimmunity frequently presents with neuropsychiatric manifestations, but underlying etiology remains poorly understood. Human brain cytoplasmic 200 RNA (BC200 RNA) is a translational regulator in neuronal synapto-dendritic domains. Here, we show that a BC200 guanosine-adenosine dendritic transport motif is recognized by autoantibodies from a subset of neuropsychiatric lupus patients. These autoantibodies impact BC200 functionality by quasi irreversibly displacing two RNA transport factors from the guanosine-adenosine transport motif. Such anti-BC autoantibodies, which can gain access to brains of neuropsychiatric lupus patients, give rise to clinical manifestations including seizures. To establish causality, naive mice with a permeabilized blood-brain barrier were injected with anti-BC autoantibodies from lupus patients with seizures. Animals so injected developed seizure susceptibility with high mortality. Seizure activity was entirely precluded when animals were injected with lupus anti-BC autoantibodies together with BC200 decoy autoantigen. Seizures are a common clinical manifestation in neuropsychiatric lupus, and our work identifies anti-BC autoantibody activity as a mechanistic cause. The results demonstrate potential utility of BC200 decoys for autoantibody-specific therapeutic interventions in neuropsychiatric lupus.

Spatial codes in dendritic BC1 RNA.

BC1 RNA is a dendritic untranslated RNA that has been implicated in local translational control mechanisms in neurons. Prerequisite for a functional role of the RNA in synaptodendritic domains is its targeted delivery along the dendritic extent. We report here that the targeting-competent 5' BC1 domain carries two dendritic targeting codes. One code, specifying somatic export, is located in the medial-basal region of the 5' BC1 stem-loop structure. It is defined by an export-determinant stem-bulge motif. The second code, specifying long-range dendritic delivery, is located in the apical part of the 5' stem-loop domain. This element features a GA kink-turn (KT) motif that is indispensable for distal targeting. It specifically interacts with heterogeneous nuclear ribonucleoprotein A2, a trans-acting targeting factor that has previously been implicated in the transport of MBP mRNA in oligodendrocytes and neurons. Our work suggests that a BC1 KT motif encodes distal targeting via the A2 pathway and that architectural RNA elements, such as KT motifs, may function as spatial codes in neural cells.

On BC1 RNA and the fragile X mental retardation protein.

The fragile X mental retardation protein (FMRP), the functional absence of which causes fragile X syndrome, is an RNA-binding protein that has been implicated in the regulation of local protein synthesis at the synapse. The mechanism of FMRP's interaction with its target mRNAs, however, has remained controversial. In one model, it has been proposed that BC1 RNA, a small non-protein-coding RNA that localizes to synaptodendritic domains, operates as a requisite adaptor by specifically binding to both FMRP and, via direct base-pairing, to FMRP target mRNAs. Other models posit that FMRP interacts with its target mRNAs directly, i.e., in a BC1-independent manner. Here five laboratories independently set out to test the BC1-FMRP model. We report that specific BC1-FMRP interactions could be documented neither in vitro nor in vivo. Interactions between BC1 RNA and FMRP target mRNAs were determined to be of a nonspecific nature. Significantly, the association of FMRP with bona fide target mRNAs was independent of the presence of BC1 RNA in vivo. The combined experimental evidence is discordant with a proposed scenario in which BC1 RNA acts as a bridge between FMRP and its target mRNAs and rather supports a model in which BC1 RNA and FMRP are translational repressors that operate independently.

BC1 regulation of metabotropic glutamate receptor-mediated neuronal excitability.

Regulatory RNAs have been suggested to contribute to the control of gene expression in eukaryotes. Brain cytoplasmic (BC) RNAs are regulatory RNAs that control translation initiation. We now report that neuronal BC1 RNA plays an instrumental role in the protein-synthesis-dependent implementation of neuronal excitation-repression equilibria. BC1 repression counter-regulates translational stimulation resulting from synaptic activation of group I metabotropic glutamate receptors (mGluRs). Absence of BC1 RNA precipitates plasticity dysregulation in the form of neuronal hyperexcitability, elicited by group I mGluR-stimulated translation and signaled through the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway. Dysregulation of group I mGluR function in the absence of BC1 RNA gives rise to abnormal brain function. Cortical EEG recordings from freely moving BC1(-/-) animals show that group I mGluR-mediated oscillations in the gamma frequency range are significantly elevated. When subjected to sensory stimulation, these animals display an acute group I mGluR-dependent propensity for convulsive seizures. Inadequate RNA control in neurons is thus causally linked to heightened group I mGluR-stimulated translation, neuronal hyperexcitability, heightened gamma band oscillations, and epileptogenesis. These data highlight the significance of small RNA control in neuronal plasticity.

Dendritic BC1 RNA in translational control mechanisms.

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3' domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3' BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.

RNA reigns in neurons.

A workshop entitled "RNA Control of Neuronal Function" was recently held in Kfar Blum, Israel. The main topics discussed at the meeting included neuronal RNA targeting mechanisms and the contributing codes and components, translational control mechanisms in dendrites and axons, and the relevance of these mechanisms for neuronal development, plasticity, and dysfunction.

Regulatory BC200 RNA in peripheral blood of patients with invasive breast cancer.

Regulatory brain cytoplasmic 200 RNA (BC200 RNA) is highly expressed in human mammary carcinoma cells. Here, we ask whether BC200 RNA becomes detectable in peripheral blood of patients with invasive breast cancer. Using quantitative reverse-transcription PCR (qRT-PCR) methodology, we observed that BC200 RNA blood levels were significantly elevated, in comparison with healthy subjects, in patients with invasive breast cancer prior to tumorectomy (p=0.001) and in patients with metastatic breast cancer (p=0.003). In patients with invasive breast cancer who had recently undergone tumorectomy, BC200 RNA blood levels were not distinguishable from levels in healthy subjects. However, normality analysis revealed a heterogeneous distribution of patients in this group, including a subgroup of individuals with high residual BC200 RNA blood levels. In blood from patients with invasive breast cancer, BC200 RNA was specifically detected in the mononuclear leukocyte fraction. The qRT-PCR approach is sensitive enough to detect as few as three BC200 RNA-expressing tumor cells. Our work establishes the potential of BC200 RNA detection in blood to serve as a molecular indicator of invasive breast malignancy.

BC RNA Mislocalization in the Fragile X Premutation.

Fragile X premutation disorder is caused by CGG triplet repeat expansions in the 5' untranslated region of FMR1 mRNA. The question of how expanded CGG repeats cause disease is a subject of continuing debate. Our work indicates that CGG-repeat structures compete with regulatory BC1 RNA for access to RNA transport factor hnRNP A2. As a result, BC1 RNA is mislocalized in vivo, as its synapto-dendritic presence is severely diminished in brains of CGG-repeat knock-in animals (a premutation mouse model). Lack of BC1 RNA is known to cause seizure activity and cognitive dysfunction. Our working hypothesis thus predicted that absence, or significantly reduced presence, of BC1 RNA in synapto-dendritic domains of premutation animal neurons would engender cognate phenotypic alterations. Testing this prediction, we established epileptogenic susceptibility and cognitive impairments as major phenotypic abnormalities of CGG premutation mice. In CA3 hippocampal neurons of such animals, synaptic release of glutamate elicits neuronal hyperexcitability in the form of group I metabotropic glutamate receptor-dependent prolonged epileptiform discharges. CGG-repeat knock-in animals are susceptible to sound-induced seizures and are cognitively impaired as revealed in the Attentional Set Shift Task. These phenotypic disturbances occur in young-adult premutation animals, indicating that a neurodevelopmental deficit is an early-initial manifestation of the disorder. The data are consistent with the notion that RNA mislocalization can contribute to pathogenesis.

Neuronal BC1 RNA: microtubule-dependent dendritic delivery.

RNA localization is an important means of post-transcriptional regulation of gene expression in many eukaryotic cell types. In neurons, select RNAs are delivered to postsynaptic dendritic microdomains, a mechanism that is considered a key underpinning in the administration of long-term synaptic plasticity. BC1 RNA is a small untranslated RNA that interacts with translation initiation factors and functions as a translational repressor by targeting assembly of 48S initiation complexes. BC1 RNA is specifically and rapidly transported to dendrites where it is found concentrated in postsynaptic microdomains. The cytoskeletal infrastructure underlying dendritic localization of BC1 RNA has not been investigated. We now report that the dendritic delivery of BC1 RNA is dependent on intact microtubules. In two neuronal cell types, hippocampal neurons and sympathetic neurons in primary culture, disruption of microtubules abolished dendritic localization of BC1 RNA. In contrast, disruption of actin filaments had no significant effect on the somatodendritic distribution of BC1 RNA. It is concluded that the long-range dendritic delivery of BC1 RNA is supported by microtubules. At the same time, a role for actin filaments, while unlikely for long-range BC1 delivery, is not ruled out for short-range local translocation and anchoring at dendritic destination sites.

Neuronal untranslated BC1 RNA: targeted gene elimination in mice.

Despite the potentially important roles of untranslated RNAs in cellular form or function, genes encoding such RNAs have until now received surprisingly little attention. One such gene encodes BC1 RNA, a small non-mRNA that is delivered to dendritic microdomains in neurons. We have now eliminated the BC1 RNA gene in mice. Three independent founder lines were established from separate embryonic stem cells. The mutant mice appeared to be healthy and showed no anatomical or neurological abnormalities. The gross brain morphology was unaltered in such mice, as were the subcellular distributions of two prototypical dendritic mRNAs (encoding MAP2 and CaMKIIalpha). Due to the relatively recent evolutionary origin of the gene, we expected molecular and behavioral consequences to be subtle. Behavioral analyses, to be reported separately, indicate that the lack of BC1 RNA appears to reduce exploratory activity.

K-turn motifs in spatial RNA coding.

Three-dimensional architectural motifs are increasingly recognized as determinants of RNA functionality. We submit that such motifs can encode spatial information. RNAs are targeted to subcellular localities in many eukaryotic cell types, and especially in neuronal and glial cells, RNAs can be transported over long distances to their final destination sites. Such RNAs contain cis-acting long-range targeting elements, and recent evidence suggests that kink-turn motifs within such elements may act as spatial codes to direct transport. Kink-turns are complex RNA motifs that feature double- and single-stranded components and introduce a signature three-dimensional structure into helical stems. We propose that the overall architectural design as well as the individual character--as specified by nucleotide identity and arrangement--of kink-turn motifs can serve as RNA targeting determinants.

Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

Dendritic BC1 RNA: functional role in regulation of translation initiation.

In neurons, local protein synthesis in synaptodendritic microdomains has been implicated in the growth and plasticity of synapses. Prerequisites for local translation are the targeted transport of RNAs to distal sites of synthesis in dendrites and translational control mechanisms to limit synthesis to times of demand. Here we identify dendritic BC1 RNA as a specific repressor of translation. Experimental use of internal ribosome entry mechanisms and sucrose density gradient centrifugation showed that BC1-mediated repression targets translation at the level of initiation. Specifically, BC1 RNA inhibited formation of the 48S preinitiation complex, i.e., recruitment of the small ribosomal subunit to the messenger RNA (mRNA). However, 48S complex formation that is independent of the eukaryotic initiation factor 4 (eIF4) family of initiation factors was found to be refractory to inhibition by BC1 RNA, a result that implicates at least one of these factors in the BC1 repression pathway. Biochemical experiments indicated a specific interaction of BC1 RNA with eIF4A, an RNA unwinding factor, and with poly(A)-binding protein. Both proteins were found enriched in synaptodendritic microdomains. Significantly, BC1-mediated repression was shown to be effective not only in cap-dependent translation initiation but also in eIF4-dependent internal initiation. The results suggest a functional role of BC1 RNA as a mediator of translational control in local protein synthesis in nerve cells.

Glia induce dendritic growth in cultured sympathetic neurons by modulating the balance between bone morphogenetic proteins (BMPs) and BMP antagonists.

Dendritic growth in cultured sympathetic neurons requires specific trophic interactions. Previous studies have demonstrated that either coculture with glia or exposure to recombinant bone morphogenetic proteins (BMPs) is both necessary and sufficient to induce dendrite formation. These observations led us to test the hypothesis that BMPs mediate glial-induced dendritic growth. In situ hybridization and immunocytochemical studies indicate that the spatiotemporal expression of BMP5, -6, and -7 in rat superior cervical ganglia (SCG) is consistent with their proposed role in dendritogenesis. In vitro, both SCG glia and neurons were found to express BMP mRNA and protein when grown in the presence or absence of the other cell type. However, addition of ganglionic glia to cultured sympathetic neurons causes a marked increase in BMP proteins coincident with a significant decrease in follistatin and noggin. Functional assays indicate that glial-induced dendritic growth is significantly reduced by BMP7 antibodies and completely inhibited by exogenous noggin and follistatin. These data suggest that glia influence the rapid perinatal expansion of the dendritic arbor in sympathetic neurons by increasing BMP activity via modulation of the balance between BMPs and their antagonists.

Neuronal MAP2 mRNA: species-dependent differential dendritic targeting competence.

Providing the basis for local protein synthesis in dendritic microdomains, RNA transport in dendrites is thought to be underlying long-term neuronal plasticity. Dendritic RNA targeting mechanisms can therefore be expected to confer selective advantages in the evolution of complex neural systems. The question thus arises as to when and how dendritically targeted transcripts first acquired their targeting competence. To address this question, the dendritic targeting competence of MAP2 transcripts was examined in chicken, mouse and rat. In one approach, we established the somato-dendritic distribution of MAP2 transcripts in vivo. We found that in contrast to rodent MAP2 mRNAs, which are highly enriched in dendritic regions of the retina, chicken MAP2 transcripts are virtually absent from such areas and are rather confined to neuronal somata. In an independent line of investigation, we determined that a dendritic targeting element (DTE) corresponding to the mammalian MAP2 DTE is not contained in the 3' untranslated region (UTR) of avian MAP2 mRNA. The combined results indicate that in contrast to mammalian MAP2 transcripts, avian MAP2 mRNA is lacking dendritic targeting competence. The data thus suggest that the acquisition of such competence has likely been a relatively recent event in evolution.

Poly(A)-binding protein is associated with neuronal BC1 and BC200 ribonucleoprotein particles.

BC1 RNA and BC200 RNA are two non-homologous, small non-messenger RNAs (snmRNAs) that were generated, evolutionarily, quite recently by retroposition. This process endowed the RNA polymerase III transcripts with central adenosine-rich regions. Both RNAs are expressed almost exclusively in neurons, where they are transported into dendritic processes as ribonucleoprotein particles (RNPs). Here, we demonstrate with a variety of experimental approaches that poly(A)-binding protein (PABP1), a regulator of translation initiation, binds to both RNAs in vitro and in vivo. We identified the association of PABP with BC200 RNA in a tri-hybrid screen and confirmed this binding in electrophoretic mobility-shift assays and via anti-PABP immunoprecipitation of BC1 and BC200 RNAs from crude extracts, immunodepleted extracts, partially purified RNPs and cells transfected with naked RNA. Furthermore, PABP immunoreactivity was localized to neuronal dendrites. Competition experiments using variants of BC1 and BC200 RNAs demonstrated that the central adenosine-rich region of both RNAs mediates binding to PABP. These findings lend support to the hypothesis that the BC1 and BC200 RNPs are involved in protein translation in neuronal dendrites.