Mesoporous bioactive glass scaffolds for the delivery of bone marrow stem cell-derived osteoinductive extracellular vesicles lncRNA promote senescent bone defect repair by targeting the miR-1843a-5p/Mob3a/YAP axis.

Bone repair in elderly patients poses a huge challenge due to the age-related progressive decline in regenerative abilities attributed to the senescence of bone marrow stem cells (BMSCs). Bioactive scaffolds have been applied in bone regeneration due to their various biological functions. In this study, we aimed to fabricate functionalized bioactive scaffolds through loading osteoinductive extracellular vesicles (OI-EVs) based on mesoporous bioactive glass (MBG) scaffolds (1010 particles/scaffold) and to investigate its effects on osteogenesis and senescence of BMSCs. The results suggested that OI-EVs upregulate the proliferative and osteogenic capacities of senescent BMSCs. More importantly, The results showed that loading OI-EVs into MBG scaffolds achieved better bone regeneration. Furthermore, OI-EVs and BMSCs RNAs bioinformatics analysis indicated that OI-EVs play roles through transporting pivotal lncRNA acting as a "sponge" to compete with Mob3a for miR-1843a-5p to promote YAP dephosphorylation and nuclear translocation, ultimately resulting in elevated proliferation and osteogenic differentiation and reduced senescence-related phenotypes. Collectively, these results suggested that the OI-EVs lncRNA ceRNA regulatory networks might be the key point for senescent osteogenesis. More importantly, the study indicated the feasibility of loading OI-EVs into scaffolds and provided novel insights into biomaterial design for facilitating bone regeneration in the treatment of senescent bone defects. STATEMENT OF SIGNIFICANCE: Constructing OI-EVs/MBG delivering system and verification of its bone regeneration enhancement in senescent defect repair. Aging bone repair poses a huge challenge due to the age-related progressive degenerative decline in regenerative abilities attributed to the senescence of BMSCs. OI-EVs/MBG delivering system were expected as promising treatment for senescent bone repair, which could provide an effective strategy for bone regeneration in elderly patients. Clarification of potential OI-EVs lncRNA ceRNA regulatory mechanism in senescent bone regeneration OI-EVs play important roles through transferring lncRNA-ENSRNOG00000056625 sponging miR-1843a-5p that targeted Mob3a to activate YAP translocation into nucleus, ultimately alleviate senescence, promote proliferation and osteogenic differentiation in O-BMSCs, which provides theoretical basis for EVs-mediated therapy in future clinical works.

Mandible-First Sequencing Increase Surgical Accuracy for Patients With Skeletal Class II Malocclusion Concomitant With Unstable Condyle-Fossa Relation.

The aim of this study was to explore whether mandible-first sequencing increases the surgical accuracy in bimaxillary orthognathic surgery for patients with skeletal class II malocclusion concomitant with the unstable condyle-fossa relation. A retrospective evaluation of 19 patients who had undergone virtually planned double-splint orthognathic surgery with different operation sequences was performed: maxilla-first (n=9) or mandible-first (n=10) surgery. The centroid position, translational, and rotational differences in the maxilla were evaluated by comparing the virtual plans with actual results. The stability was assessed by comparing the actual results with the follow-up outcomes 6 months postoperatively. The accuracy of the maxilla centroid position was improved in mandible-first sequencing surgery: mandible-first 1.87±0.94 mm versus maxilla-first 2.70±0.75 mm (P<0.05). Moreover, no significant difference was detected in the translational and orientational discrepancies between the 2 groups. Neither sequencing procedure differed in the overall stability: maxilla-first (1.48±1.13 mm) versus mandible-first (1.57±0.90 mm). This study indicated that the mandible-first surgery leads to a more accurate maxilla position than the maxilla-first surgery for patients with skeletal class II malocclusion concomitant with the unstable condyle-fossa relation.

Evaluation of the transdentinal capability of the intrinsic antibacterial cetylpyridinium chloride/cholesterol sterosomes in vitro and in vivo.

AIM: Dentinal tubules serve as disease-causing channels for infiltration and penetration of bacteria and their by-products; which are regarded as the major driver of pathogenesis in pulpal inflammation and infection. In this study, we aimed to evaluate the transdentinal potential of nanoscale cetylpyridinium chloride/cholesterol (CPC/Chol) sterosomes, which are a recently developed type of cationic non-phospholipid liposomal nanocarrier; as well as their intrinsic and universal antibacterial activity. METHODOLOGY: Cetylpyridinium chloride/cholesterol sterosomes were formulated, with a hydrodynamic diameter of 134 ± 4 nm, a low polydisperse index of 0.161 ± 0.007, and a positive zeta potential of 41 ± 3 mV at pH 7.4. Transdentinal diffusion ability of sterosomes was evaluated using human dentine blocks in vitro, and Wistar rat molar teeth in vivo. The intrinsic antibacterial activities of CPC/Chol sterosomes against Enterococcus faecalis, Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis were further examined. RESULTS: Cetylpyridinium chloride/cholesterol sterosomes successfully penetrated through the dentinal tubules, and diffused into the pulp, which could be internalized by dental pulp cells with a high efficiency. In addition, they exhibited substantial levels of intrinsic antibacterial activity against these Gram-positive and Gram-negative endodontic bacteria and their biofilms. CONCLUSIONS: Given its high penetration and diffusion ability through the dentine and pulp, great potential for multi-drug delivery, and distinct intrinsic antibacterial activity; sterosome-based nanocarriers might serve as a promising therapeutic strategy aimed at targeting various specific pathways associated with pulpal diseases. This will help determine and characterize the most appropriate prophylactic and therapeutic targets for early intervention in our future dentistry practice.

Use of mesoporous polydopamine nanoparticles as a stable drug-release system alleviates inflammation in knee osteoarthritis.

Osteoarthritis drugs are often short-acting; therefore, to enhance their efficacy, long-term, stable-release, drug-delivery systems are urgently needed. Mesoporous polydopamine (MPDA), a natural nanoparticle with excellent biocompatibility and a high loading capacity, synthesized via a self-aggregation-based method, is frequently used in tumor photothermal therapy. Here, we evaluated its efficiency as a sustained and controlled-release drug carrier and investigated its effectiveness in retarding drug clearance. To this end, we used MPDA as a controlled-release vector to design a drug-loaded microsphere system (RCGD423@MPDA) for osteoarthritis treatment, and thereafter, tested the efficacy of the system in a rat model of osteoarthritis. The results indicated that at an intermediate drug-loading dose, MPDA showed high drug retention. Furthermore, the microsphere system maintained controlled drug release for over 28 days. Our in vitro experiments also showed that drug delivery using this microsphere system inhibited apoptosis-related cartilage degeneration, whereas MPDA-only administration did not show obvious cartilage degradation improvement effect. Results from an in vivo osteoarthritis model also confirmed that drug delivery via this microsphere system inhibited cartilage damage and proteoglycan loss more effectively than the non-vectored drug treatment. These findings suggest that MPDA may be effective as a controlled-release carrier for inhibiting the overall progression of osteoarthritis. Moreover, they provide insights into the selection of drug-clearance retarding vectors, highlighting the applicability of MPDA in this regard.

Evaluation of Related Factors of Maxillary Sinusitis After Le Fort I Osteotomy Based on Computed Tomography: A Retrospective Case-Control Study.

ABSTRACT: Maxillary sinusitis is 1 of the postoperative complications of the Le Fort I osteotomy, this study investigated the related factors of maxillary sinusitis after Le Fort I osteotomy. A total of 23 cases, 92 controls, and 11 related factors were included in this case-control study with a 1:4 case-control ratio. The risk factors for maxillary sinusitis after Le Fort I were examined by least absolute shrinkage and selection operator multivariate conditional logistic regression and least absolute shrinkage and selection operator multivariate linear regression. The patency of maxillary sinus ostium at 6 months after surgery was significantly associated with maxillary sinusitis after Le Fort I osteotomy. Compared with the obstructed maxillary sinus ostium, the percentage of the volume of the healthy air cavity in the complete sinus cavity increased 70.7% when the maxillary sinus ostium was unobstructed, and 95% confidence interval was 0.610 to 0.805. Similarly, when the maxillary sinus ostium was wide, the percentage increased 6.0% compared with the narrow 1, and 95% confidence interval was 0.013 to 0.107. This study indicated that the patency of maxillary sinus ostium has an important impact on maxillary sinusitis after Le Fort I osteotomy. Close attention should be paid to maintain the maxillary sinus ostium and the drainage of maxillary sinuses unobstructed in a clinical setting.

Compromised osteogenic effect of exosomes internalized by senescent bone marrow stem cells via endocytoses involving clathrin, macropinocytosis and caveolae.

Stem cell senescence leads to progressive functional declines and disrupts the physiological homeostasis of bone environment. Stem cell-derived exosomes are emerging as promising therapeutical approaches to treat diverse aging-related osseous diseases. Herein, a previously reported osteoinductive exosome (OI-exo) was applied as a therapeutic agent for bone repair in aging individuals and its internalization mechanisms in senescent bone marrow stem cells (BMSCs) were explored. The results demonstrated that OI-exos derived from young BMSCs could partially rescue the proliferation, osteogenic differentiation and alleviate aging phenotypes in vitro. OI-exo-delivered hierarchical mesoporous bioactive glass (MBG) scaffold effectively promote in vivo bone formation in aging rat cranial defect model. However, the osteogenic effects of OI-exo both in vitro and in vivo were compromised in senescent individuals and for aging BMSCs compared to younger ones. This study revealed that non-senescent BMSCs internalized exosomes exclusively via clathrin-mediated endocytosis, while senescent BMSCs additionally evoked macropinocytosis and caveolae-mediated endocytosis to mediate the internalization of exosomes. The alteration of endocytic manner of senescent BMSCs and the involvement of macropinocytosis might be responsible for the compromised effects of therapeutical exosomes. The phenomena discovered in this study could also be extended to other scenarios where drugs or treatments exerted compromised effects in aging individuals. The influence of endocytic manner, avoidance of macropinocytosis-related negative effects should be taken into considerations in future therapeutic design for aging populations.

Effects of autologous concentrated growth factor on gingival thickness in periodontal accelerated osteogenic orthodontics: a 6-month randomized controlled trial.

BACKGROUND: Earlier studies have not given clear results of concentrated growth factor (CGF) on gingival thickness (GT) in periodontal accelerated osteogenic orthodontics (PAOO). This randomized controlled trial aimed to evaluate the effects of CGF on GT in patients with thin gingival phenotype undergoing PAOO. METHODS: Forty four patients presenting 264 anterior mandibular teeth were recruited and randomly allocated to one of the groups: test-positioning of autologous CGF after PAOO or control-positioning of a collagen membrane after PAOO. GT, gingival height (GH), buccal alveolar bone thickness (BT), and buccal alveolar bone height (BH) were evaluated depending on cross-sectional CBCT images at t0 (before surgery) and t1(6 months after surgery). RESULTS: GT were increased in both groups at t1 compared to t0. Yet, higher values were observed in the test group (from 0.94 ± 0.23 to 1.31 ± 0.33 mm) compared to the control group (from 0.94 ± 0.19 to 1.02 ± 0.16 mm) (p < 0.05). Moreover, in the intergroup comparison, GT at t1 in the test group was significantly higher compared to the control group (p < 0.01). Furthermore, the GT of central incisors, lateral incisors and canine teeth all showed significantly changes compared with baseline and the test group showed higher increase (p < 0.01). No statistically significant difference were found in GH, BT, BH and all clinical parameters between two groups at t1 (p > 0.05). CONCLUSIONS: Within the limitation of this study, gingival thickness could be increased by using CGF in PAOO for the patients with thin gingival phenotype. Trial registration The study was registered in Chinese Clinical Trial Registry ( http://www.chictr.org.cn/index.aspx ) under the number ChiCTRINR17013346, Registered 11 November 2017.

A new method for individual condylar osteotomy and repositioning guides used in patients with severe deformity secondary to condylar osteochondroma.

BACKGROUND: Mandibular condylar osteochondroma (OC) could lead to facial morphologic and functional disturbances, such as facial asymmetry, malocclusion, and temporomandibular joint dysfunction. However, after condylar OC resection, the inaccurate reposition of the neocondyle still needs to be solved. The purpose of this study was to explore the feasibility of the condylar osteotomy and repositioning guide to reposition the neocondyle in the treatment of patients with severe deformity secondary to condylar OC. RESULTS: Three patients with severe deformity secondary to OC of the mandibular condyle were enrolled in this study. With the aid of condylar osteotomy and repositioning guide, condylar OC resection and repositioning were carried out, and the accuracy and stability of these guides were evaluated. All patients healed uneventfully, and no facial nerve injury and condylar ankylosis occurred. Compared with the computerized tomography scans in centric relation before surgery and 3 days after surgery, the results showed that the facial symmetry was greatly improved in all the patients. Also, after the superimposition of the condylar segments before surgery and 3 days after surgery, the postoperative reconstructed condyles had a high degree of similarity to the reconstruction of the virtual surgical planning. Observed from the sagittal and coronal directions, the measurements of condylar positions were very close to those of virtual surgical planning. Moreover, it also showed stable results after a 1-year follow-up. CONCLUSIONS: For patients with severe deformity secondary to condylar OC, condylar osteotomy, and repositioning guide was expected to provide a new option for the improvement of facial symmetry and occlusal relationship.

Amyotrophic lateral sclerosis: Protein chaperone dysfunction revealed by proteomic studies of animal models.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons and causes progressive muscle weakness and atrophy. The etiology and pathogenesis of ALS are largely unknown and no effective treatment is presently available. About 10% of patients have the familial or inherited form of the disease (fALS), among which 20% is linked to mutations with Cu(2+)/Zn(2+) superoxide dismutase (mSOD1). Transgenic animals expressing human mSOD1 are excellent models for understanding not only fALS but sporadic ALS as well. Pathological features in both ALS patients and mSOD1 transgenic animals' spinal cords share commonalties including the accumulation of misfolded protein inclusions. Recent proteomic investigations on ALS animal models have discovered alterations in protein expression, protein-protein interactions and post-translational modifications. These efforts have revealed aspects of potential pathogenic mechanisms and identified probable therapeutic targets. The present review summarizes the major findings of proteomics studies performed on the mSOD1 mice with particular emphasis on the spinal cord proteome. These results are compared with those reported using cell cultures or specimens obtained from ALS patients. The convergence of pathogenic processes on protein chaperone function, and its relationship to protein degradation, metabolic dysfunction and oxidative signaling events is discussed.

Immune modulation and tolerance induction by RelB-silenced dendritic cells through RNA interference.

Dendritic cells (DC), the most potent APCs, can initiate the immune response or help induce immune tolerance, depending upon their level of maturation. DC maturation is associated with activation of the NF-kappaB pathway, and the primary NF-kappaB protein involved in DC maturation is RelB, which coordinates RelA/p50-mediated DC differentiation. In this study, we show that silencing RelB using small interfering RNA results in arrest of DC maturation with reduced expression of the MHC class II, CD80, and CD86. Functionally, RelB-silenced DC inhibited MLR, and inhibitory effects on alloreactive immune responses were in an Ag-specific fashion. RelB-silenced DC also displayed strong in vivo immune regulation. An inhibited Ag-specific response was seen after immunization with keyhole limpet hemocyanin-pulsed and RelB-silenced DC, due to the expansion of T regulatory cells. Administration of donor-derived RelB-silenced DC significantly prevented allograft rejection in murine heart transplantation. This study demonstrates for the first time that transplant tolerance can be induced by means of RNA interference using in vitro-generated tolerogenic DC.

Calcium mediated excitotoxicity in neurofilament aggregate-bearing neurons in vitro is NMDA receptor dependant.

We have previously shown that the co-localization of neuronal nitric oxide synthase (nNOS) with neurofilament (NF) aggregates in motor neurons derived from transgenic mice over-expressing the human low molecular weight NF protein (hNFL+/+) is associated with a deregulation of calcium influx via the N-methyl-d-aspartate (NMDA) receptor, resulting in apoptosis. Because the absence of the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor confers calcium permeability and has been implicated in the process of excitotoxicity in ALS, we have examined the role of the AMPA receptor in this model. GluR2 protein expression and mRNA were examined in hNFL+/+ and wild-type motor neurons (wt). Live cell calcium imaging was performed using Oregon-Green Bapta and Fura-2 calcium dyes. For apoptotic studies, neurons were treated with glutamate, with or without glutamate receptor antagonists [6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) or (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)] and examined for active caspase-3 or phospholipid inversion. We observed that although both GluR2 mRNA and protein levels were decreased in hNFL+/+ motor neurons compared to wt, there was no appreciable calcium influx via the AMPA receptor. These studies demonstrate that calcium mediated excitotoxicity in NF aggregate-bearing neurons is NMDA receptor dependant.

14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

Neuronal tissue-specific ribonucleoprotein complex formation on SOD1 mRNA: alterations by ALS SOD1 mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal disease of unknown etiology. Mutations in copper/zinc superoxide dismutase (SOD1) are the most commonly associated genetic abnormality. Given that SOD1 is ubiquitously expressed, the exclusive vulnerability of motor neurons is one of the most puzzling issues in ALS research. We here report that wild-type SOD1 mRNA forms ribonucleoprotein (RNP) complexes with protein homogenates of neuronal tissue but not with homogenates of non-neuronal tissues. 3' Untranslated region of SOD1 mRNA-dependent RNP complexes functioned to stabilize SOD1 mRNA. Moreover, SOD1 mRNAs harboring ALS-associated mutations, including silent mutations, were deficient in forming RNP complexes. In contrast, SOD1 mRNAs harboring artificial mutations, not known to be associated with ALS, demonstrated preserved RNP complex formation. This paper reports RNP complex formation on SOD1 mRNA as a neuronal tissue-specific and ALS-associated mutation sensitive feature.

Activated microglial supernatant induced motor neuron cytotoxicity is associated with upregulation of the TNFR1 receptor.

We have previously reported that supernatant derived from LPS-activated BV-2 cells, an immortalized microglial cell line, induces death of NSC-34 cells (a motor neuron hybridoma) through a TNFalpha and nitric oxide synthase (NOS) dependant mechanism. In this study, we have observed that LPS-activated BV-2 supernatant induces NSC-34 cell death in association with an upregulation of the TNF receptor 1 (TNFR1) expression on NSC-34 cells, both at the transcription level and at the cell surface protein level. The upregulation of TNFR1 receptor was independent of TNFalpha, and could be partly inhibited by the inhibition of iNOS activation in the BV-2 cells. The TNFR2 receptor was not involved. These observations have important implications in understanding the mechanism by which microglial activation contributes to the motor neuron degeneration.

Mutant copper-zinc superoxide dismutase binds to and destabilizes human low molecular weight neurofilament mRNA.

The mechanism by which mutated copper-zinc superoxide dismutase (SOD1) causes familial amyotrophic lateral sclerosis is believed to involve an adverse gain of function, independent of the physiological antioxidant enzymatic properties of SOD1. In this study, we have observed that mutant SOD1 (G41S, G85A, and G93A) but not the wild type significantly reduced the stability of the low molecular weight neurofilament mRNA in a dosage-dependent manner. We have also demonstrated that mutant SOD1 but not the wild type bound directly to the neurofilament mRNA 3'-untranslated region and that the binding was necessary to induce mRNA destabilization. These observations provide an explanation for a novel gain of function in which mutant SOD1 expression in motor neurons alters an intermediate filament protein expression.

Induction of RNA interference in dendritic cells.

Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

Intermediate filament steady-state mRNA levels in amyotrophic lateral sclerosis.

We have examined the steady-state levels of intermediate filament mRNA in amyotrophic lateral sclerosis using the RNAse protection assay (NFL, NFM, NFH; corrected against GAPDH) or by PCR (peripherin, alpha-internexin, nestin, and vimentin; corrected against beta-actin). Significant elevations of NFL and peripherin mRNA levels were observed within the ALS cervical and lumbar spinal cord, with all other IF mRNA levels being comparable between control and ALS cases. These findings suggest that disturbances in both NFL and peripherin expression, independently known to contribute to the generation of motor neuron dysfunction in transgenic mice, are evident in ALS.

Cytoplasmic retention sites in p190RhoGEF confer anti-apoptotic activity to an EGFP-tagged protein.

p190RhoGEF is a large multi-functional protein with guanine nucleotide exchange (GEF) activity. The C-terminal region of p190RhoGEF is a highly interactive domain that binds multiple factors, including proteins with anti-apoptotic activities. We now report that transfection of EGFP-tagged p190RhoGEF protects Neuro 2a cells from stress-induced apoptosis and that anti-apoptotic activity is localized to cytoplasmic retention sequences (CRS-1 and CRS-2) in the C-terminal region of p190RhoGEF. Cytoplasmic retention is conferred to an EGFP fluorescent marker when fused to either CRS-1 or CRS-2. Both cytoplasmic retention and anti-apoptotic activity are lost by deleting CRS-1 and CRS-2 in the p190RhoGEF sequence and can be recovered by restoring either CRS-1 or CRS-2 to the EGFP-tagged sequence. Since the CRS-1 and CRS-2 contain the JIP-1 and 14-3-3 binding sites, we propose that anti-apoptotic activity may be conferred by the binding of p190RhoGEF to JIP-1 or 14-3-3, possibly by altering their interactive properties or nucleocytoplasmic movements. Taken together, our findings support a model whereby multiple interactions of p190RhoGEF confer homeostatic properties to differentiated neurons and may link neuronal homeostasis to the regulation of NF-L expression.

Selective loss of trans-acting instability determinants of neurofilament mRNA in amyotrophic lateral sclerosis spinal cord.

Neurofilament (NF) aggregates in motor neurons are a key neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously observed an alteration in the stoichiometry of NF subunit steady state mRNA levels in ALS spinal motor neurons using in situ hybridization and proposed that this led to aggregate formation. We have now examined the levels of NF mRNA in whole tissue homogenates of spinal cord using the RNase protection assay and real time reverse transcriptase-PCR and observed significant elevations of NF mRNA level in ALS. Compared with age-matched control, we observed a greater stability of heterogeneously expressed NFL mRNA in the presence of ALS spinal cord homogenates. Heat denaturing or protease K digestion of the control homogenates increased the stability of the NFL mRNA to levels observed in ALS homogenate. Increased NFL mRNA stability was also induced by increasing the percentage of ALS homogenate in an admixture of control and ALS homogenates. These observations suggest the presence of trans-acting NFL mRNA-destabilizing elements in control but not in ALS spinal cord homogenates. This was confirmed in gel retardation assays. We also observed that the destabilizing elements interact with the 3'-untranslated region of NFL mRNA. These findings suggest that the trans-acting NFL-destabilizing elements are selectively suppressed in ALS homogenates, resulting in an increased stability and level of NFL mRNA.

Binding of p190RhoGEF to a destabilizing element on the light neurofilament mRNA is competed by BC1 RNA.

The enhancement of RNA-mediated motor neuron degeneration in transgenic mice by mutating a major mRNA instability determinant in a light neurofilament (NF-L) transgene implicates cognate RNA binding factors in the pathogenesis of motor neuron degeneration. p190RhoGEF is a neuron-enriched guanine exchange factor (GEF) that binds to the NF-L-destabilizing element, to c-Jun N-terminal kinase-interactive protein-1 (JIP-1), and to 14-3-3 and may link neurofilament expression to pathways affecting neuronal homeostasis. This study was undertaken to identify additional RNA species that bind p190RhoGEF and could affect interactions of the exchange factor with NF-L transcripts. The C-terminal domain of p190RhoGEF, containing the RNA-binding site, was expressed as a glutathione S-transferase fusion protein and was used as an affinity probe to isolate interactive RNAs in rat brain extracts. As expected, NF-L mRNA was identified as an RNA specie eluted from the affinity column. In addition, BC1 RNA was also found enriched in the bound RNA fraction. BC1 is a 152-nucleotide RNA that is highly expressed but untranslated in differentiated neurons. We show that BC1 and NF-L mRNA bind to a similar site in the C-terminal domain of p190RhoGEF, and their bindings to p190RhoGEF are readily cross-competed. Moreover, we identify a novel binding site in BC1 to account for its interaction with p190RhoGEF. The findings suggest a novel role of BC1 in differentiated neurons involving RNA-protein interactions of p190RhoGEF.