UNASSIGNED

A novel astrocytic autoantibody has been identified as a biomarker of a relapsing autoimmune meningoencephalomyelitis that is immunotherapy responsive. Seropositivity distinguishes autoimmune glial fibrillary acidic protein (GFAP) meningoencephalomyelitis from disorders commonly considered in the differential diagnosis.

UNASSIGNED

To describe a novel IgG autoantibody found in serum or cerebrospinal fluid that is specific for a cytosolic intermediate filament protein of astrocytes.

UNASSIGNED

Retrospective review of the medical records of seropositive patients identified in the Mayo Clinic Neuroimmunology Laboratory from October 15, 1998, to April 1, 2016, in blinded comprehensive serologic evaluation for autoantibody profiles to aid the diagnosis of neurologic autoimmunity (and predict cancer likelihood).

UNASSIGNED

Frequency and definition of novel autoantibody, the autoantigen's immunochemical identification, clinical and magnetic resonance imaging correlations of the autoantibody, and immunotherapy responsiveness.

UNASSIGNED

Of 103 patients whose medical records were available for review, the 16 initial patients identified as seropositive were the subject of this study. Median age at neurologic symptom onset was 42 years (range, 21-73 years); there was no sex predominance. The novel neural autoantibody, which we discovered to be GFAP-specific, is disease spectrum restricted but not rare (frequency equivalent to Purkinje cell antibody type 1 [anti-Yo]). Its filamentous pial, subventricular, and perivascular immunostaining pattern on mouse tissue resembles the characteristic magnetic resonance imaging findings of linear perivascular enhancement in patients. Prominent clinical manifestations are headache, subacute encephalopathy, optic papillitis, inflammatory myelitis, postural tremor, and cerebellar ataxia. Cerebrospinal fluid was inflammatory in 13 of 14 patients (93%) with data available. Neoplasia was diagnosed within 3 years of neurologic onset in 6 of 16 patients (38%): prostate and gastroesophageal adenocarcinomas, myeloma, melanoma, colonic carcinoid, parotid pleomorphic adenoma, and teratoma. Neurologic improvement followed treatment with high-dose corticosteroids, with a tendency of patients to relapse without long-term immunosuppression.

UNASSIGNED

Glial fibrillary acidic protein-specific IgG identifies a distinctive, corticosteroid-responsive, sometimes paraneoplastic autoimmune meningoencephalomyelitis. It has a lethal canine equivalent: necrotizing meningoencephalitis. Expression of GFAP has been reported in some of the tumor types identified in paraneoplastic cases. Glial fibrillary acidic protein peptide-specific cytotoxic CD8+ T cells are implicated as effectors in a transgenic mouse model of autoimmune GFAP meningoencephalitis.

BACKGROUND

Frontotemporal dementia (FTD) is recognised as a clinically and morphologically heterogeneous group of interrelated neurodegenerative conditions. One of the subtypes within this disease spectrum is the behavioural variant FTD (bvFTD). This is known to be a varied disorder with a mixture of tau-positive and tau-negative underlying pathologies. The other subtypes include semantic dementia (SD), which generally exhibits tau-negative pathology, and progressive non-fluent aphasia (PNFA), which is usually tau-positive. As the clinical presentation of these subtypes may overlap, a specific diagnosis can be difficult to attain and today no specific biomarker can predict the underlying pathology. Neurofilament light chain protein (NFL), a cytoskeletal constituent of intermediate filaments, is thought to reflect neuronal and axonal death when appearing in the cerebrospinal fluid (CSF). NFL has been shown to be elevated in CSF in patients with FTD compared with AD and controls. Our hypothesis was that the levels of NFL also differ between the subtypes of FTD and may indicate the underlying pathological subtype.

METHODS

We retrospectively analysed data from previous CSF analyses in 34 FTD cases (23 bvFTD, seven SD, four PNFA), 20 AD cases, and 26 healthy controls. A separate group of 10 neuropathologically verified and subtyped FTD cases (seven tau-negative, three tau-positive) were also analysed.

RESULTS

NFL levels were significantly higher in FTD compared with both AD (p<0.001) and controls (p<0.001). The NFL levels of SD and bvFTD were significantly higher (p<0.001) compared with AD. The biomarker profiles of PNFA and AD were similar. In the neuropathologically verified FTD cases, NFL was higher in the tau-negative than in the tau-positive cases (exact p=0.017).

CONCLUSIONS

The marked NFL elevation in some but not all FTD cases is likely to reflect the different underlying pathologies. The highest NFL values found in the SD group as well as in the neuropathologically verified tau-negative cases may be of subtype diagnostic value, if corroborated in larger patient cohorts. In bvFTD, a mixture of tau-positive and tau-negative underlying pathologies could possibly explain the intermediate NFL values.

Three new, well growing cell lines (GLC-1, GLC-2, and GLC-3) have been established from small cell lung carcinoma (SCLC) and characterized. A subclone (GLC-1-M13) markedly different from its parent line GLC-1 was also isolated and characterized. Cytogenetic analysis of the cell lines revealed deletions in the short arm of chromosome 3 as a most consistent chromosomal aberration. The deleted region was not identical in all metaphases, 3p(21-23) being the shortest region of overlap. Despite their SCLC origin GLC-1, GLC-2, and GLC-3 do not show pronounced SCLC differentiation features. Neurosecretory granula were very rare (GLC-1) or completely absent (GLC-2 and GLC-3), whereas the SCLC-related enzyme and hormone markers L-3,4-dihydroxyphenylalanine decarboxylase, neuron-specific enolase, creatine kinase BB, and bombesin-like immunoreactivity were variably expressed. Although the subclone GLC-1-M13 was derived from the poorly differentiated GLC-1, it behaved according to the above criteria as a differentiated "classic" SCLC cell line. When assessed with specific monoclonal antibodies the different cell lines appeared to express different subsets of intermediate filament proteins, indicative for different stages and directions of differentiation: "undifferentiated" (GLC-1 and GLC-2); "neural tissue related" (GLC-2); "simple epithelium" related (GLC-1-M13); and a combination of simple and squamous epithelium related (GLC-3). We conclude that GLC-1, GLC-2, and GLC-3 represent dedifferentiated forms of SCLC, related to the recently described "variant" type of SCLC, whereas the clonal derivate GLC-1-M13 behaves like a differentiated "classic" SCLC cell line.

Keratin 18 is a type-I keratin that is found in a variety of simple epithelial tissues. In mice, the corresponding protein, called Endo B, is expressed at the 4- to 8-cell stage of mouse development and may be one of the first intermediate-filament proteins synthesized after fertilization. A cDNA clone for keratin 18, designated pK18, was isolated from a human placental cDNA library by hybridization with the mouse Endo-B probe. It was characterized by hybridization selection of RNA, translation, immunoprecipitation, Northern blotting, and sequence analysis. Synthetic T7 polymerase transcripts of the cDNA were indistinguishable in size from keratin-18 mRNA, suggesting that pK18 represents a full-length copy of the RNA. The cDNA insert is 1,428 nucleotides long and contains a single open reading frame of 1,342 nucleotides coding for 429 amino acids. The deduced amino acid sequence is 89.7% identical with that of Endo B. The only extensive difference between the two sequences is due to 9 additional amino acids being present in the last half of the N-terminal domain of keratin 18. The 38-nucleotide-long 3' noncoding region of the cDNA is 75% identical with the corresponding portion of Endo B. The 5' noncoding regions are 59% identical. The expression of keratin-18 mRNA was found to vary more than tenfold when HeLa cells and BeWo trophoblastic cells were compared.

Purified proteins have been used to reconstitute an in vitro system for the medial-to-late stages of recombination in E. coli. In this system, RecA protein formed recombination intermediates that were processed by the actions of the RuvA, RuvB, and RuvC proteins. RuvAB was found to promote branch migration, to dissociate the RecA filament, and to modulate the orientation of cleavage of Holliday junction resolution by RuvC. Monoclonal antibodies directed against RuvA, RuvB, or RuvC inhibited resolution in the reconstituted system. Specific protein-protein interactions between the branch migration motor (RuvB) and the resolvase (RuvC) were also observed. These results provide evidence for coordinated action during the late stages of recombination, possibly involving the assembly of a RuvABC branch migration/resolution complex.

Lamins are nucleoskeletal proteins which form intermediate type filaments in close association with the inner nuclear envelope membrane. Based on molecular and biochemical properties the lamins were grouped as type-A and type-B lamins, respectively. I have cloned the cDNA encoding lamin LIII of Xenopus which is the lamin protein present in oocyte nuclei and in cleavage nuclei. The data presented here indicate that a pool of maternal lamin LIII RNA is synthesized very early in oogenesis and that it continues to be present until gastrulation when the vast majority of the LIII RNA is degraded. Despite the similarities shared by all lamin proteins, the lamin LIII sequence neither possesses the features diagnostic for either type-A or type-B lamins nor does it show greater sequence similarity to one of the lamin types than to the other and thus it may represent a third type of lamin protein which may reflect its special function in oogenesis and early development.

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and Caenorhabditis elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.

Monospecific antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with rat skeletal myofibrils. After cloning 3 times on agarose, two stable clones were obtained and chosen for further characterization. The first clone, JLB1, produced an antibody that recognizing an antigen distributed in the M-line region and on either site of the Z line of myofibrils. The second clone, JLB7, produced an antibody reacting only with an antigen located at the M-line region of myofibrils. Both JLB1 and JLB7 antibodies decorate the typical intermediate filaments of a variety of cultured cells. Colcemid treatment of cells before reaction with both antibodies resulted in the coiling or capping (or both) of the fibers around the nucleus. Brief treatment of cells with cytochalasin B did not affect the integrity of the fibers stained by both antibodies whereas, under the same conditions, microfilament bundles visualized by another monoclonal antibody (JLA20) against actin were disassembled into many aggregates in the cytoplasm. Identical staining patterns of the intermediate filaments are obtained by double-label immunofluorescence microscopy of the same cell stained with these monoclonal antibodies and rabbit autoimmune serum (which has been shown to react with the components of the intermediate filaments). By using immunoprecipitation, protein bands at 210,000 and 95,000 daltons from chicken embryo fibroblasts were identified as the potential antigens recognized by JLB1 and JLB7 monoclonal antibodies, respectively. The widespread occurrence of these antigenic determinants in different cultured cells suggests the highly conservative property of these intermediate-filament components.

Intermediate filaments are built from one to several members of a multigene family encoding fibrous proteins that share a highly conserved hierarchic assembly plan for the formation of multistranded filaments from distinctly structured extended coiled coils. Despite the rather low primary sequence identity, intermediate filaments form apparently similar filaments with regard to their spatial dimensions and physical properties. Over the past few years, substantial progress has been made in the elucidation of the complex expression patterns and clinically relevant phenotypes of intermediate filaments. The key question of how these filaments assemble and what the molecular architecture of their distinct assembly intermediates comprises, however, has still not been answered to the extent that has been achieved for microfilaments and microtubules.

Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivo distribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation.

The developing tooth represents a suitable model for understanding the molecular mechanisms involved in induction, morphogenesis and differentiation of organs. It is conceivable that the developmental changes could be reflected in the distribution of different cytoskeletal components and in this report we analyze the expression of the intermediate filament nestin during rodent tooth development at the protein and mRNA levels (by immuno light and electron microscopy, and by in situ hybridization). Nestin is expressed at all stages of tooth development, but the expression levels increase after birth in both ectodermal and ectomesenchymal derivatives. The shift in nestin distribution, from the proliferating dental lamina to the dental mesenchyme, indicates that nestin may be involved in inductive phenomena. At early stages of mineralization, nestin is seen within the apical parts of the presecretory ameloblasts. Nestin is also expressed in odontoblasts, both during odontogenesis and after tooth eruption. The increase in nestin expression from early to late developmental stages and sustained expression in a differentiated cell type contrasts with previously observed patterns of nestin expression during nerve and muscle development. This suggests that nestin could be used as a specific marker for the odontoblast.

Monoclonal antibodies were used in indirect immunofluorescence and immunoblot studies to examine the expression of four different classes of intermediate filaments, namely, neurofilaments, glial filaments, cytokeratin, and vimentin, in NTERA-2 cl.D1 (NT2/D1) pluripotent human embryonal carcinoma (EC) cells, and in the neurons derived from these cells by differentiation induced with retinoic acid. In the EC cell cultures, grown in the absence of retinoic acid, cytokeratin was the predominant intermediate filament detected by immunofluorescence; only a few cells expressed vimentin, and none expressed glial filament protein or any of the three neurofilament proteins (NF195, NF170, and NF70). Immunoblot analyses of cytoskeletal extracts of these cells supported these data. Two days after exposure to retinoic acid, all three neurofilament subunits were detected in a few cells with a non-neuronal morphology and, by double indirect immunofluorescence, were observed to colocalize with cytokeratin. The number of neurofilament-positive cells increased with time after initial exposure to retinoic acid, and although 95% of these cells contained cytokeratin initially, less than 5% of the neurofilament-positive cells retained cytokeratin 2 weeks later. By this time, many of the cells expressing all three neurofilaments but no cytokeratin exhibited a neuronal morphology. Vimentin was evident in a large number of cells in the cultures, but it was not detected in the neurofilament-positive cells. Also, many of the neurofilament-negative cells continued to express cytokeratin. No cells expressing glial filament proteins were found. Immunoblot analysis of the differentiated cultures also revealed all three neurofilament subunits, and vimentin and cytokeratin, but no glial filament protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The surface distribution of several proteins (porin, hexokinase, and two proteins associated with microtubules or actin filaments) on the outer membrane of brain mitochondria was analyzed by immunogold labelling of purified mitochondria in vitro. The results suggest the existence of specialized domains for the distribution of porin in the outer mitochondrial membrane. Similarities between the distribution of porin and the distribution of microtubule-associated proteins bound in vitro to mitochondria suggested that mitochondria and microtubules interact by binding microtubule-associated proteins to porin-containing domains of the outer membrane. This hypothesis was supported by biochemical studies on outer mitochondrial proteins involved in in vitro binding of cytoskeleton elements. In vitro interactions between mitochondria and microtubules or neurofilaments were analyzed by electron microscopy. These studies revealed cross-bridging between the outer membrane of mitochondria and the two cytoskeleton elements. Cross-bridging was influenced by ATP hydrolysis and by several proteins associated with the surface of mitochondria or with microtubules. In addition, unidentified proteins which were recognized by antibodies to all intermediate filaments subunits were associated either with the mitochondrial surface or with microtubules. This data suggest the participation of additional cytoplasmic proteins in the interactions between cytoskeleton elements and mitochondria.

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.

Dystrophin is one of a number of large cytoskeleton associated proteins that connect between various cytoskeletal elements and often are tethered to the membrane through other transmembrane protein complexes. These cytolinker proteins often provide structure and support to the cells where they are expressed, and mutations in genes encoding these proteins frequently gives rise to disease. Dystrophin is no exception in any of these respects, providing connections between a transmembrane complex known as the dystrophin-glycoprotein complex and the underlying cytoskeleton. The most established connection and possibly the most important is that to F-actin, but more recently evidence has been forthcoming of connections to membrane phospholipids, intermediate filaments and microtubules. Moreover it is becoming increasingly clear that the multiple spectrin-like repeats in the centre of the molecule, that had hitherto been thought to be largely redundant, harbour binding activities that have a significant impact on dystrophin functionality. This functionality is particularly apparent when assessed by the ability to rescue the dystrophic phenotype in mdx mice. This review will focus on the relatively neglected but functionally vital coiled-coil region of dystrophin, highlighting the structural relationships and interactions of the coiled-coil region and providing new insights into the functional role of this region.

In this review we discuss some of the proteins for which a role in linking actin to the fibroblast plasma membrane has been suggested. We focus on the family of proteins related to erythrocyte spectrin, proteins that have generally been viewed as having an organization and a function in actin-membrane attachment similar to those of erythrocyte spectrin. Experiments in which we precipitated the nonerythrocyte spectrin within living fibroblasts have led us to question this supposed similarity of organization and function of the nonerythrocyte and erythrocyte spectrins. Intracellular precipitation of fibroblast spectrin does not affect the integrity of the major actin-containing structures, the stress fiber microfilament bundles. Unexpectedly, however, we found that the precipitation of spectrin results in a condensation and altered distribution of the vimentin class of intermediate filaments in most cells examined. Although fibroblast spectrin may have a role in the attachment of some of the cortical, submembranous actin, it is surprising how little the intracellular immunoprecipitation of the spectrin affects the cells. Several proteins have been found concentrated at the ends of stress fibers, where the actin filaments terminate at focal contacts. Two of these proteins, alpha-actinin and fimbrin, have properties that suggest that they are not involved in the attachment of the ends of the bundles to the membrane but are more probably involved in the organization and cross-linking of the filaments within the bundles. On the other hand, vinculin and talin are two proteins that interact with each other and may form part of a chain of attachments between the ends of the microfilament bundles and the focal contact membrane. Their role in this attachment, however, has not been established and further work is needed to examine their interaction with actin and to identify any other components with which they may interact, particularly in the plasma membrane.

Preparations of isolated brain postsynaptic densities (PSDs) contain a characteristic set of proteins among which the most prominent has a molecular weight of approximately 50,000. Following the suggestion that this major PSD protein might be related to a similarly sized component of neurofilaments (F. Blomberg et al., 1977, J. Cell Biol., 74:214-225), we searched for evidence of neurofilament proteins among the PSD polypeptides. This was done with a novel technique for detecting protein antigens in SDS-polyacrylamide gels (immunoblotting) and an antiserum that was selective for neurofilaments in immunohistochemical tests. As a control, an antiserum against glial filament protein (GFAP) was used because antisera against GFAP stain only glial cells in immunohistochemical tests. They would, therefore, not be expected to react with PSDs that occur only in neurons. The results of these experiments suggested that PSDs contain both neuronal and also glial filament proteins at higher concentrations than either synaptic plasma membranes, myelin, or myelinated axons. However, immunoperoxidase staining of histological sections with the same two antisera gave contradictory results, indicating that PSDs in intact brain tissue contain neither neuronal or glial filament proteins. This suggested that the intermediate filament proteins present in isolated PSD preparations were contaminants. To test this possibility, the proteins of isolated brain intermediate filaments were labeled with 125I and added to brain tissue at the start of a subcellular fractionation schedule. The results of this experiment confirmed that both neuronal and glial filament proteins stick selectively to PSDs during the isolation procedure. The stickiness of PSDs for brain cytoplasmic proteins indicates that biochemical analysis of subcellular fractions is insufficient to establish a given protein as a synaptic junctional component. An immunohistochemical localization of PSDs in intact tissue, which has now been achieved for tubulin, phosphoprotein I, and calmodulin, appears to be an essential accessory item of evidence. Our findings also corroborate recent evidence which suggests that isolated preparations of brain intermediate filaments contain both neuronal and glial filaments.

Previous results from our laboratory have indicated a requirement for CK intermediate filaments (IF) for the organization of the apical domain in polarized epithelial cells in culture. The results seemed to be challenged by the phenotype of cytokeratin (CK) 8-deficient mice, which comprises only colorectal hyperplasia, female sterility and a weaker hepatocyte integrity. In this work localization with anti-CK antibodies indicated that many Ck8-/- epithelia still form IF in CK8-deficient mice, perhaps because of the expression of the promiscuous CK7. In the small intestine, only villus enterocytes lacked IFs. These cells appeared to lose syntaxin 3, and three apical membrane proteins (alkaline phosphatase, sucrase isomaltase and cystic fibrosis transmembrane conductance regulator) as they progressed along the villus. At the distal third of the villi, gamma-tubulin was found scattered within the cytoplasm of enterocytes, in contrast to its normal sub-apical localization, and the microtubules were disorganized. These results could not be attributed to increased numbers of apoptotic or necrotic cells. The only other cell type we found without IFs in CK8 null mice, the hepatocyte, displayed increased basolateral levels of one apical marker (HA4), indicating a correlation between the lack of intermediate filaments and an apical domain phenotype. These data suggest a novel function for intermediate filaments organizing the apical pole of simple polarized epithelial cells.

Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.

To study the functional role of activated astrocytes in glutamate homeostasis in vivo, we used a model of sustained astrocytic activation in the rat striatum through lentiviral-mediated gene delivery of ciliary neurotrophic factor (CNTF). CNTF-activated astrocytes were hypertrophic, expressed immature intermediate filament proteins and highly glycosylated forms of their glutamate transporters GLAST and GLT-1. CNTF overexpression produced a redistribution of GLAST and GLT-1 into raft functional membrane microdomains, which are important for glutamate uptake. In contrast, CNTF had no detectable effect on the expression of a number of neuronal proteins and on the spontaneous glutamatergic transmission recorded from striatal medium spiny neurons. These results were replicated in vitro by application of recombinant CNTF on a mixed neuron/astrocyte striatal culture. Using microdialysis in the rat striatum, we found that the accumulation of extracellular glutamate induced by quinolinate (QA) was reduced threefold with CNTF. In line with this result, CNTF significantly increased QA-induced [(18)F]-fluoro-2-deoxyglucose uptake, an indirect index of glutamate uptake by astrocytes. Together, these data demonstrate that CNTF activation of astrocytes in vivo is associated with marked phenotypic and molecular changes leading to a better handling of increased levels of extracellular glutamate. Activated astrocytes may therefore be important prosurvival agents in pathological conditions involving defects in glutamate homeostasis.

Neuronal intranuclear inclusions (NIIs) are a histopathological hallmark of several neurodegenerative disorders. However, the role played by NIIs in neurodegenerative pathogenesis remains enigmatic. Defining their molecular composition represents an important step in understanding the pathophysiology of these disorders. Recently, a nuclear protein, "fused-in-sarcoma" (FUS) was identified as the pathological protein in two forms of frontotemporal lobar degeneration (FTLD-IF, formerly known as neuronal intermediate filament inclusion disease, and FTLD-UPS, formerly known as atypical FTLD-U), both of which are characterized by the presence of NII. The objective of the present study was to determine the range of neurodegenerative disorders characterized by FUS-positive NIIs. Immunostaining for FUS revealed intense reactivity of NIIs in FTLD-IF and FTLD-UPS as well as in Huntington's disease, spinocerebellar ataxias 1 and 3, and neuronal intranuclear inclusion body disease. In contrast, there was no FUS staining of NIIs in inherited forms of FTLD-TDP caused by GRN and VCP mutations, fragile-X-associated tremor ataxia syndrome, or oculopharyngeal muscular dystrophy. In a cell culture model of Huntington's disease, NIIs were intensely FUS-positive. NII-bearing cells displayed loss of the normal diffuse nuclear pattern of FUS staining. This suggests that sequestration of nuclear FUS by NIIs may interfere with its normal nuclear localization.

Neurofilaments (also called 10-nm filaments or intermediate filaments) from bovine brain were incubated with microtubule protein at 37 degrees C in the presence or absence of 1 mM ATP and in a buffer that allowed microtubule assembly. Falling-ball viscometry revealed that the (non-Newtonian) apparent viscosity of the ATP-containing mixtures is 5-20 times greater than that of the mixtures prepared without ATP. A larger ATP-dependent increase in viscosity (approximately 100-fold) was seen when purified tubulin replaced microtubule protein. The magnitude of the increase depended on the concentrations of both neurofilaments and tubulin. The presence of both neurofilaments and assembled microtubules was necessary for the increase to occur. The viscosity was drastically reduced by stirring or by cooling of the mixtures to 0 degrees C. Sedimentation velocity experiments, conducted at 35 degrees C on mixtures previously incubated at 35 degrees C, revealed the presence of a fraction of very rapidly sedimenting material (sedimentation coefficient greater than 1000 S) in the ATP-containing solutions but not in those prepared without ATP. It is concluded that an ATP-induced complex is formed between microtubules and neurofilaments. The observed complex may reflect interactions between microtubules and neurofilaments that are significant in vivo.