Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body.

Intracellular trafficking of hnRNP A2 in oligodendrocytes.

Heterogeneous ribonucleoprotein (hnRNP) A2 is a trans-acting factor that mediates intracellular trafficking of specific RNAs containing the A2 response element. HnRNP A2 is localized in the nucleus and also in granules in the perikaryon and processes in oligodendrocytes. The distribution of the cytoplasmic pool of hnRNP A2 is microtubule-dependent. HnRNP A2 is composed of two sequential RNA binding domains (RBDI and RBDII), a glycine-rich domain, and a nuclear import domain (M9). In order to analyze the roles of individual domains in determining the intracellular distribution of hnRNP A2, chimeric mRNAs encoding various domains fused with green fluorescent protein (GFP) were injected into oligodendrocytes, and the subcellular distribution of the GFP hybrid proteins was analyzed by fluorescence microscopy. Chimeric GFP proteins containing the M9 domain were localized to the nucleus. In the absence of the M9 domain, proteins containing the RBDII domain were preferentially concentrated in the distal processes of the cells. Localization of RBDII-containing proteins in the periphery was dependent on the presence of intact microtubules. These data suggest that the RBDII domain of hnRNP A2 targets hnRNP A2 to the periphery of the cell in a microtubule-dependent manner.

The balance of power in RNA trafficking.

In the past two years, several different RNA trafficking pathways have been characterized in oligodendrocytes; similar trafficking pathways have been discovered in neuronal and retroviral systems; co-assembly of multiple different RNAs into the same granules has been analyzed as a mechanism for coordinating gene expression; and a new hypothesis for RNA trafficking, based on the balance of power between kinesin and dynein in individual RNA granules, has been proposed.

Structural dynamics of oligodendrocyte lysis by perforin in culture: relevance to multiple sclerosis.

The mechanism by which oligodendrocytes are depleted from active lesions in multiple sclerosis (MS) is not clear but many reports implicate a cytolytic process. The most applied animal model for MS, chronic relapsing experimental autoimmune encephalomyelitis (EAE), has been established in inbred strains of mice, especially SJL and PL. Studies on oligodendrocytes from these strains in vitro have been hampered to date by an inability to grow these cells from mouse CNS tissue. We report here a successful method to culture SJL mouse oligodendrocytes and have analyzed lysis of these cells in vitro mediated by the pore-forming protein, perforin, a candidate effector molecule in inflammatory demyelination. Cultures were exposed to murine perforin, 36-72 hemolytic U, for up to 2.5 hr and examined using the oligodendrocyte phenotypic markers O4, galactocerebroside and myelin basic protein (MBP), in addition to a membrane dye (DiI) and a marker of necrosis, propidium iodide, (PI). Cultures were imaged chronologically by phase contrast, immunofluorescence, digital, light and electron microscopy. Findings showed that the majority of oligodendrocytes were killed within 60-90 min via pore expansion and ultimately, membrane disruption. The structural features of the cellular damage comprised swelling of the cell body, fenestration and fragmentation of membranes and processes, cytoplasmic vacuolation and breakdown of the nuclear envelope. Astrocytes in the same system were relatively resistant to cell lysis. The above patterns of oligodendrocyte damage in SJL oligodendrocytes were reminiscent of patterns in the MS lesion, leaving us to conclude that perforin may play an important role in the human disease.

RNA trafficking in oligodendrocytes.

A2RE and hnRNP A2 have been identified as important cis/trans determinants for MBP RNA trafficking in oligodendrocytes. Since A2RE-like sequences are found in several different transported RNAs, and since hnRNP A2 is expressed in most cell types, this may represent a general RNA trafficking pathway shared by a variety of different RNAs in different cell types. In oligodendrocytes, A2RE/hnRNP A2 determinants are involved in at least four steps in the RNA trafficking pathway: (1) export from the nucleus to the cytoplasm, (2) granule assembly in the perikaryon, (3) transport along microtubules in the processes, and (4) translation activation in the myelin compartment. The components of the cellular machinery mediating each of these steps are known. How A2RE/hnRNP A2 determinants interact with these components to mediate RNA trafficking is being investigated by a combination of: biochemistry to analyze molecular interactions in vitro, imaging to visualize molecular interactions in living cells, and computational modeling to simulate molecular interactions in the Virtual Cell.

RNA trafficking signals in human immunodeficiency virus type 1.

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Myelin basic protein gene dosage effects in the PNS.

Myelin basic protein (MBP) plays an essential adhesive role in the formation of compact myelin in the central nervous system (CNS), but not in the peripheral nervous system (PNS). Morphologic data suggest that MBP controls the number of cytoplasmic channels or Schmidt-Lanterman incisures (SLI) present in PNS myelin. The levels of connexin-32 (Cx32) and myelin-associated glycoprotein (MAG), two components of the incisures, are inversely proportional to the levels of MBP in sciatic nerves of mice affected by the shiverer (shi) mutation, while protein zero (P0) and peripheral membrane protein 22 (PMP22), two structural components of compact myelin, remain constant. The levels of P0, PMP22, Cx32, and MAG mRNA do not vary in relationship to the levels of MBP. This indicates that MBP exerts its effect on Cx32 and MAG at a posttranscriptional level and suggests a new function for MBP in regulating gene expression in the PNS.

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking.

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.

The cis-acting RNA trafficking signal from myelin basic protein mRNA and its cognate trans-acting ligand hnRNP A2 enhance cap-dependent translation.

The 21 nucleotide RNA trafficking signal (RTS), originally identified in myelin basic protein mRNA, but also found in a variety of other localized RNAs, is necessary and sufficient for transport of RNA along microtubules in oligodendrocytes. The RTS binds specifically to the RNA binding protein, hnRNP A2. Together, the RTS and hnRNP A2 comprise cis/trans determinants for several steps in the RNA trafficking pathway. Here we show that insertion of the RTS into green fluorescent protein (GFP) RNA enhances translation without affecting stability of microinjected RNA. In dicistronic RNA, the RTS enhances cap-dependent translation without affecting internal ribosome entry site (IRES)-dependent translation. The translation enhancer function of the RTS is position, copy number, and cell type independent, hnRNP A2 dependent, and saturable with increasing amounts of injected RNA. This represents one of the first specific translation enhancer elements identified in a mammalian system.

Myelin-associated oligodendrocytic basic protein mRNAs reside at different subcellular locations.

The mRNAs for two myelin proteins, myelin basic protein (MBP) and myelin-associated oligodendrocytic basic protein (MOBP)-81A, are uniquely located at sites where myelin sheaths are assembled. Here, we use subcellular fractionation to show that four MOBP mRNAs, like MBP mRNA, are located at sites of myelin sheath assembly, and that three other MOBP mRNAs are located in oligodendrocyte soma. The MOBP-81 protein is found in myelin and in another subcellular fraction, whereas other myelin proteins, including MBP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin-associated glycoprotein, are largely restricted to myelin. Different MBP mRNAs are generated by alternative splicing. All of them contain an RNA transport sequence (RTS) that directs them to sites in oligodendrocytes, where myelin sheaths are assembled. Consequently, all are enriched in myelin. After fractionation, four MOBP mRNAs, MOBP-71, MOBP-81A, MOBP-99, and MOBP-169 (identified in this study), are enriched in myelin. These mRNAs contain a common exon, exon 8b, which has a nucleotide sequence that is similar to MBP mRNA RTS. This sequence likely directs these mRNAs to sites of myelin sheath assembly. Three other MOBP mRNAs, MOBP-69, MOBP-81B, and MOBP-170, lack this exon. Their subcellular distribution indicates that they are largely retained in oligodendrocyte soma. We conclude that the distribution of MOBPs in oligodendrocytes is strongly influenced by alternative splicing of the corresponding mRNAs.

Concurrent loss and proliferation of astrocytes following lateral fluid percussion brain injury in the adult rat.

Astrocyte populations were analyzed over a period of 1 month in the hippocampus following lateral fluid percussion (FP) brain injury. Rats (n = 23) were subjected either to a brain injury of moderate severity, or to anesthesia and surgery without injury (n = 7). At 3 days, 1, 2, or 4 weeks postinjury, subgroups of animals were sacrificed and the brains removed and sectioned for histochemical analysis. The density of astrocytes, identified with gold sublimate staining, decreased significantly in the ipsilateral hippocampus of injured rats 3 days following injury, eventually falling to 64% of the total astrocyte population present in uninjured animals by 1 week postinjury. One month postinjury, the density of hippocampal astrocytes had returned to 85% of the total number of astrocytes observed in the hippocampus of uninjured animals. In order to characterize the post-traumatic formation of new astrocytes, immunohistochemistry was performed using antibodies to proliferating cell nuclear antigen (PCNA) and to glial fibriallary acidic protein (GFAP). Positive immunolabeling for both PCNA and GFAP was most abundant at 3 days following FP brain injury in regions where the blood brain barrier was compromised, and was not detectable by 1 month postinjury. These results indicate that astrocyte proliferation after injury may be evoked by mitogens released from vascular sources, and may be an attempt to compensate for some of the astrocytic cell loss observed after injury.

RNA on the road to myelin.

In oligodendrocytes some mRNAs are transported from the perikaryon to the distal processes and localized in the myelin compartment where they are translated. This review describes the cis-acting signals and trans-acting factors that mediate intracellular trafficking of myelin basic protein (MBP) RNA, the prototype for such mRNAs in myelinating glia.

RNA trafficking in myelinating cells.

In the past year, several key molecular components of the RNA trafficking pathway in myelinating cells have been identified: distinct cis-acting elements for RNA transport and localization have been characterized in myelin basic protein mRNA; hnRNP A2 has been identified as a trans-acting factor in oligodendrocytes that binds specifically to the RNA transport sequence; and microtubules and kinesin have been identified as cytoskeletal elements required for RNA transport in oligodendrocytes.

Cytoplasmic dynein contains a family of differentially expressed light chains.

Cytoplasmic dynein contains a series of accessory proteins associated with the motor containing heavy chains.1 These include three distinct classes of light chains (Mr < approximately 22 000). Here we demonstrate that a previously cloned protein termed rp3 is a bona fide Mr 14 000 light chain component of this microtubule motor complex. The rp3 polypeptide is approximately 55% identical to the Tctex1 dynein light chain, and together, these two proteins define one branch of a diverse family of Mr 14 000 light chains associated with both cytoplasmic and flagellar dyneins. The Tctex1 and rp3 light chains are differentially expressed in various tissues: rp3 is most prevalent in liver and brain cytoplasmic dynein, whereas those tissues contain the least amounts of Tctex1. Immunofluorescence analysis was consistent with the tissue-specific distribution of these proteins and revealed that both rp3 and Tctex1 are present in multiple perinuclear punctate particles. Furthermore, in two cell lines, rp3 was found associated with an elongated structure located in the layer of cytoplasm above the nucleus. Electrophoretic/immunological analysis indicates that there are only single isoforms for these proteins in brain and PC-12 cells, suggesting that alterations in the Mr 14 000 light chains of dynein are achieved at the level of the individual proteins and not by posttranslational modification. Dissection of the cytoplasmic dynein complex revealed that Tctex1, an Mr 8000 LC dimer, and IC74 associate to define a basal-located intermediate chain/light chain complex analogous to that found in flagellar outer arm dynein.

Transport and localization elements in myelin basic protein mRNA.

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3' UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1, 473 in the 3' UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.

Evaluation of lipid-coated microbubbles as a delivery vehicle for Taxol in brain tumor therapy.

OBJECTIVE: This work evaluates the potential of lipid-coated microbubbles (LCM) as a delivery vehicle for lipid-soluble antineoplastic agents. We have shown, in rats, the selective affinity of intravenously administered LCM for tumor cells. They are internalized by the tumor cells both in vitro and in vivo. The specificity of LCM for tumors and subsequent incorporation into the cytoplasm could significantly reduce the systemic effects of an agent incorporated into the bubbles, such as Taxol. METHODS: The in vitro methods were as follows. C6 cells (10(5) cells) were treated with Taxol-LCM (6 micrograms/ml), Taxol-Cremophore (6 micrograms/ml), or LCM alone for 8 or 24 hours. Cell death was determined by staining the cells with nuclear staining. Abnormalities of microtubule structures were ascertained by confocal microscopy. The in vivo methods were as follows. Two rat tumor models (C6 and 9L) were used. Rats were treated with single bolus injections or with repetitive (two or three) treatment courses, with respective control animals. Each course consisted of one daily tail vein injection for 5 consecutive days and then 2 days of rest. RESULTS: When compared with either a saline control group or a group receiving Taxol in an oil vehicle, Taxol-LCM reduced tumor progression in Fischer 344 rats inoculated with 9L glioma. The most profound effect was observed with rats treated with three treatment cycles (five daily injections/cycle) separated by two rest periods (2 d/period). CONCLUSION: Both in vitro and in vivo data indicate that Taxol can be incorporated into LCM, can be delivered to the tumor site, and can exert a measurable antitumor biological effect.

Rearrangement and reactivation of the myelin basic protein locus in myelin-deficient (mld) mouse brain.

Myelin-deficient (mld) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in approximately 5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of approximately 1.4 x 10(-6) per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant (mld/mld, scid/scid) and single mutant (mld/mld, +scld/+scld) mice, indicating that the scid factor is not required for MBP gene reactivation in mld. The significance of DNA rearrangement in mammalian development is discussed.

The affinity of lipid-coated microbubbles for maturing brain injury sites.

The availability of a vehicle to deliver lipid soluble agents to a brain injury site may be of potential value in management of brain injury. This work describes the aggregation of intravenously administered Lipid-Coated Microbubbles (LCM) in the injury site following an experimental radiofrequency rat brain lesion. The bubbles can be identified around the region of the injury after the lesion has matured at least 48 h. The greatest bubbles density is evident after the lesion has matured for 10 days. This bubble density, reflecting "affinity," decreases to a plateau level from the second to the third week after injury. In order to investigate the potential relationship of bubble influx to posttraumatic astrocytosis and to cell turnover in the region, we utilized dual-channel laser-scanning confocal microscopy to track both bubble influx into the region and concomitant Glial Fibrillary Acidic Protein (GFAP) expressing astroctyte cell distribution. Cell turnover was assayed in separate sections using immunohistochemical staining of Proliferating Cell Nuclear Antigen (PCNA). We suggest a relationship between the LCM affinity and reactive astrocytes, but found no affinity of LCM for cells which stained positive with PCNA.

Translocation of myelin basic protein mRNA in oligodendrocytes requires microtubules and kinesin.

Myelin basic protein (MBP) mRNA is localized to the myelin membranes of oligodendrocytes. When exogenous MBP mRNA is microinjected into oligodendrocytes in culture, it is transported along the processes and localized to the myelin compartment in a multistep intracellular RNA trafficking pathway. In the work described here, oligodendrocytes were treated with agents that affect the cytoskeleton including: nocodazole, to disrupt microtubules; taxol, to stabilize microtubules; cytochalasin, to disrupt microfilaments; and kinesin anti-sense oligonucleotide, to suppress kinesin expression. Digoxigenin-labeled MBP mRNA was microinjected into the treated cells and the extent of translocation of the microinjected RNA was determined by confocal microscopy. Nocodazole, taxol, and kinesin anti-sense oligonucleotide inhibited translocation of microinjected MBP mRNA, while cytochalasin B and kinesin sense oligonucleotide did not. These results indicate that translocation of MBP mRNA in oligodendrocytes requires intact microtubules and kinesin but does not require intact microfilaments. The results are discussed in relation to the current multistep model for intracellular RNA trafficking in oligodendrocytes.

Regional heterogeneity in the response of astrocytes following traumatic brain injury in the adult rat.

The regional distribution and temporal appearance of astrocytes expressing glial fibrillary acidic protein (GFAP), S100 protein, and vimentin were determined in a nonpenetrating lateral fluid percussion (LFP) brain injury model. Following injury, reactive astrocytes were observed in the subcortical white matter tracts as early as 1 day, in the hippocampus and injured cortex by 3 days, and in the thalamus by 1 week. Reactive astrocytes in the injured cortex, subcortical white matter tracts, and CA3 region of the hippocampus were all vimentin positive at 1 month post-injury. These astrocytes had a distinct morphology characterized by an enlarged cell body and long intertwined processes. In contrast, reactive astrocytes in the thalamic nuclei never expressed vimentin, and displayed an enlarged cell body with thick shortened processes. An increase in S100 protein was detected in all reactive astrocytes following LFP brain injury. Quantitative assessment of GFAP, S100, and vimentin polypeptides confirmed the immunohistochemical evaluation. Our data indicate that although astrogliosis mirrors the spatial pattern of post-traumatic neuronal cell loss, the expression of vimentin and the cellular morphology of the cells were regionally distinct, suggesting that astrogliosis may be modulated by factors present in the post-traumatic brain.