CD9, a Major Platelet Cell Surface Glycoprotein, is a ROCA Antigen and Is Expressed in the Nervous System

We previously generated a monoclonal antibody (mAb), ROCA1, which binds preferentially to rostral versus caudal sympathetic ganglia and intercostal nerves. Two other mAbs, ROCA2 and B2C11, bind to the same structures but not in rostrocaudal gradients. All three mAbs recognize a 26 kDa cell surface protein. Amino acid sequence data obtained from the affinity purified 26 kDa protein showed some homology with human CD9, a tetraspan protein implicated in intercellular signaling in hematopoietic cells. Using the PCR, we obtained cDNA clones representing the entire rat CD9 coding sequence from sciatic nerve and sympathetic ganglia. ROCA1, ROCA2, and B2C11 each immunoprecipitate a 26 kDa protein from CHO cells stably transfected with one of the clones, demonstrating that the ROCA cell surface antigen is indeed rat CD9. We find that CD9 mRNA is widely expressed, with particularly high levels present in a number of neural tissues. In situ hybridization demonstrates that peripheral neurons and Schwann cells, as well as adrenal chromaffin cells express CD9 mRNA. Consistent with immunoblot analyses showing that, unlike the ROCA1 epitope, the 26 kDa protein is not expressed in a rostrocaudal gradient, we find similar levels of rat CD9 mRNA in rostral and caudal intercostal nerves. In developing postnatal rat sciatic nerve, CD9 mRNA levels are coordinately regulated with the expression of myelin genes. These results provide another example of a cell surface protein expressed by both hematopoietic and neural cells, and suggest a role for CD9 in intercellular signaling in the nervous system

Characterizing DNA Condensation and Conformational Changes in Organic Solvents

Organic solvents offer a new approach to formulate DNA into novel structures suitable for gene delivery. In this study, we examined the in situ behavior of DNA in N, N-dimethylformamide (DMF) at low concentration via laser light scattering (LLS), TEM, UV absorbance and Zeta potential analysis. Results revealed that, in DMF, a 21bp oligonucleotide remained intact, while calf thymus DNA and supercoiled plasmid DNA were condensed and denatured. During condensation and denaturation, the size was decreased by a factor of 8–10, with calf thymus DNA forming spherical globules while plasmid DNA exhibited a toroid-like conformation. In the condensed state, DNA molecules were still able to release the counterions to be negatively charged, indicating that the condensation was mainly driven by the excluded volume interactions. The condensation induced by DMF was reversible for plasmid DNA but not for calf thymus DNA. When plasmid DNA was removed from DMF and resuspended in an aqueous solution, the DNA was quickly regained a double stranded configuration. These findings provide further insight into the behavior and condensation mechanism of DNA in an organic solvent and may aid in developing more efficient non-viral gene delivery systems

CD9 Plays a Role in Schwann Cell Migration in Vitro

To identify molecules that regulate Schwann cell migration, we have generated a panel of monoclonal antibodies against Schwann cell surface antigens that modulate Schwann cell migration in in vitro bioassays. One of these antibodies, SMRA1, recognizes a 26 kDa Schwann cell surface membrane protein identified here as CD9. SMRA1 enhances Schwann cell migration on two biologically relevant substrates: living axons of cultured dorsal root ganglion neurons, and cryostat sections of sciatic nerve. This CD9-induced regulation of Schwann cell motility is correlated with a rise in cytosolic calcium and enhanced tyrosine phosphorylation of several Schwann cell proteins. These results, together with the findings of Hadjiargyrou and Patterson (1994), implicate CD9 as an important regulator of Schwann cell behavior in peripheral nerve

An Antibody to the Tetraspan Membrane Protein CD9 Promotes Neurite Formation in a Partially α3β1 Integrin-Dependent Manner

The tetraspan cell surface glycoprotein, CD9, has been implicated in cellular signaling during growth and differentiation in the hematopoietic and nervous systems. Because CD9 expression is induced early in development in sensory and sympathetic neuroblasts, we investigated the role of CD9 in neurite outgrowth. We plated dissociated cells from neonatal sympathetic ganglia on immobilized anti-CD9 antibodies or antibodies against other cell surface molecules. We show here that B2C11, an anti-CD9 antibody that has been shown previously to activate Schwann cells in vitro, promotes robust neurite outgrowth from sympathetic neurons that is greater than that on other antibody surfaces and is comparable to neurite outgrowth on a collagen substratum. In addition, B2C11 causes dramatic morphological changes in neurons and glia from dissociated ganglia, including a flattening of these cells. Because CD9 interacts with integrins in many cell types including Schwann cells, and specifically with the α3β1 integrin in some cells, we tested whether the effect of B2C11 on neurite outgrowth is mediated by this integrin. An anti-α3β1 antibody, Ralph 3–1, attenuates the extent of neurite outgrowth on B2C11 and collagen, but not on laminin. Because the α3β1 integrin has been shown to mediate neurite outgrowth on different substrates, these results provide a functional significance for the CD9-α3β1 interaction; downstream signaling may be activated by this cis interaction on the cell surface in response to external cues that promote neurite outgrowth

The Extracellular Matrix and Blood Vessel Formation: Not Just a Scaffold

The extracellular matrix plays a number of important roles, among them providing structural support and information to cellular structures such as blood vessels imbedded within it. As more complex organisms have evolved, the matrix ability to direct signalling towards the vasculature and remodel in response to signalling from the vasculature has assumed progressively greater importance. This review will focus on the molecules of the extracellular matrix, specifically relating to vessel formation and their ability to signal to the surrounding cells to initiate or terminate processes involved in blood vessel formation

Mustn1: A Developmentally Regulated Pan-Musculoskeletal Cell Marker and Regulatory Gene

The Mustn1 gene encodes a small nuclear protein (~9.6 kDa) that does not belong to any known family. Its genomic organization consists of three exons interspersed by two introns and it is highly homologous across vertebrate species. Promoter analyses revealed that its expression is regulated by the AP family of transcription factors, especially c-Fos, Fra-2 and JunD. Mustn1 is predominantly expressed in the major tissues of the musculoskeletal system: bone, cartilage, skeletal muscle and tendon. Its expression has been associated with normal embryonic development, postnatal growth, exercise, and regeneration of bone and skeletal muscle. Moreover, its expression has also been detected in various musculoskeletal pathologies, including arthritis, Duchenne muscular dystrophy, other skeletal muscle myopathies, clubfoot and diabetes associated muscle pathology. In vitro and in vivo functional perturbation revealed that Mustn1 is a key regulatory molecule in myogenic and chondrogenic lineages. This comprehensive review summarizes our current knowledge of Mustn1 and proposes that it is a new developmentally regulated pan-musculoskeletal marker as well as a key regulatory protein for cell differentiation and tissue growth

The Intertwining of Transposable Elements and Non-Coding RNAs

Growing evidence shows a close association of transposable elements (TE) with non-coding RNAs (ncRNA), and a significant number of small ncRNAs originate from TEs. Further, ncRNAs linked with TE sequences participate in a wide-range of regulatory functions. Alu elements in particular are critical players in gene regulation and molecular pathways. Alu sequences embedded in both long non-coding RNAs (lncRNA) and mRNAs form the basis of targeted mRNA decay via short imperfect base-pairing. Imperfect pairing is prominent in most ncRNA/target RNA interactions and found throughout all biological kingdoms. The piRNA-Piwi complex is multifunctional, but plays a major role in protection against invasion by transposons. This is an RNA-based genetic immune system similar to the one found in prokaryotes, the CRISPR system. Thousands of long intergenic non-coding RNAs (lincRNAs) are associated with endogenous retrovirus LTR transposable elements in human cells. These TEs can provide regulatory signals for lincRNA genes. A surprisingly large number of long circular ncRNAs have been discovered in human fibroblasts. These serve as “sponges” for miRNAs. Alu sequences, encoded in introns that flank exons are proposed to participate in RNA circularization via Alu/Alu base-pairing. Diseases are increasingly found to have a TE/ncRNA etiology. A single point mutation in a SINE/Alu sequence in a human long non-coding RNA leads to brainstem atrophy and death. On the other hand, genomic clusters of repeat sequences as well as lncRNAs function in epigenetic regulation. Some clusters are unstable, which can lead to formation of diseases such as facioscapulohumeral muscular dystrophy. The future may hold more surprises regarding diseases associated with ncRNAs andTEs