Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice. We found that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also found that one of the arginine-rich DPRs (PR) can directly contribute to glucose metabolism and metabolic stress. These findings provide a mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and support a feedforward loop model that opens several opportunities for therapeutic intervention.

Mouse MCT3 gene is expressed preferentially in retinal pigment and choroid plexus epithelia.

Monocarboxylate transporters (MCTs) are a family of highly homologous membrane proteins that mediate the 1:1 transport of a proton and a lactate ion. In chicken, MCT3 is preferentially expressed in the retinal pigment epithelium (RPE). We have isolated the mouse MCT3 cDNA and gene and characterized the pattern of tissue expression. MCT3 is a single copy gene with a 1.8-kb transcript that encodes a protein with a predicted molecular mass of 51.5 kDa. Based on Northern hybridization analysis, MCT3 transcript was expressed in only two tissues: RPE and choroid plexus epithelium (CPE). The choroid plexus forms a barrier between the cerebrospinal fluid and fenestrated capillaries, similar to the organization of the RPE and choroidal vessels. Immunohistochemical staining demonstrated that MCT3 was restricted to the basolateral membranes of both epithelia but was more abundant in RPE than CPE. Differences in the level of protein expression were confirmed by Western blot analysis. The cloning of MCT3 identifies a specific transporter that could regulate lactate levels in fluid-bathing neuronal tissues.

Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast.

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.

Cloning of the human monocarboxylate transporter MCT3 gene: localization to chromosome 22q12.3-q13.2.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.

Genomic structure and developmental expression of the chicken nonocarboxylate transporter MCT3 gene.

MCT3 is a monocarboxylate transporter that is specifically expressed on the basolateral membrane of retinal pigment epithelial cells (RPE). In these studies the temporal expression of MCT3 during ocular development was examined using Northern blot analysis. A 2.2 kb transcript (MCT3b) was detected in RPE by embryonic day 7 (E7) and was present throughout embryonic development. A 2.45 kb transcript (MCT3a) was expressed at low levels before E11 but its expression increased between E11 and E17. Using 5'-RACE (rapid amplification of cDNA ends) it has demonstrated that MCT3a and MCT3b mRNA had distinct 5'-untranslated sequences but shared the same translation start site. To determine the exon-intron structure and to understand the elements that control the tissue specific and developmental expression of MCT3, the MCT3 gene was cloned and sequenced from a chicken genomic library. The MCT3 gene is distributed over 8 kb of DNA and is composed of 6 exons. Coding sequences for MCT3 are found on exon 2 through exon 5. Comparison of the 5'-RACE sequence with the genomic sequence reveals that the two 5'-untranslated regions of the mRNAs are encoded by distinct exons, 1a and 1b, which are alternatively spliced to exon 2. These data suggest that two forms of MCT3 mRNAs could be generated by two distinct promoters that may be regulated in response to changes in the metabolic activity of the retina during development.

Monocarboxylate transporter MCT1 is located in the apical membrane and MCT3 in the basal membrane of rat RPE.

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier and regulates the movement of nutrients, water, and ions between the choroidal blood supply and the retina. The transport properties of the RPE maintain retinal adhesion and regulate the pH and osmolarity in the space surrounding the photoreceptor cell outer segments. In this report we identify two monocarboxylate transporters, MCT1 and MCT3, expressed in rat RPE. On the basis of Northern and Western blot analyses, MCT1 is expressed in both the neural retina and the RPE, whereas the expression of MCT3 is restricted to the RPE. Using indirect immunolocalization we show that the two transporters are polarized to distinct membrane domains. MCT1 antibody labels the apical surface and the apical processes of the RPE. A polyclonal antibody produced against the carboxy terminus of rat MCT3 labels only the basolateral membrane of the RPE. The demonstration of MCT1 on the apical membrane and MCT3 on the basal membrane identifies specific proteins involved in the discriminate and critical regulation of water and lactate transport from the retina to the choroid.

Integrins and development: how might these receptors regulate differentiation of the lens.

Integrins transduce both internal signals and signals from the matrix. These interactions between integrins, their extracellular matrix ligands, and their cytoskeletal partners play an important role in the regulation of cellular differentiation. We have shown them to be important in lens cell differentiation. In the lens capsule there is a compartmentalization of matrix components with fibronectin, primarily localized to the anterior capsule, and tenascin in the posterior capsule. Integrins are developmentally regulated in the lens. alpha 5 beta 1 integrin, like fibronectin, is primarily associated with the lens epithelial cells, where together they are likely to be important in regulation of adhesion and proliferation. alpha 6A beta 1, the integrin laminin receptor, is expressed at its highest levels in the equatorial epithelium and the peripheral fiber cells, both migratory populations. Because laminin is uniformly distributed in the lens capsule, such changes in alpha 6A integrin expression are likely critical to the cell's ability to regulate its response to laminin in the matrix. The organization of cytoskeletal molecules associated with the integrin cytoplasmic face also changes with development. In the epithelial regions of the lens, where the initiation of lens cell differentiation occurs, expression of the cytoskeletal proteins involved in cell-substrate interactions, talin, alpha-actinin, and the signaling proteins, are high. In the fiber cell region of the lens, where the cells establish stable cell-cell contacts, vinculin predominates and becomes highly associated with the cytoskeletal fraction. The role of integrins in lens development is not only regulated by changes in the expression of different integrin receptors but is also closely correlated with the expression and organization of the molecules with which they associate.

Identification of a unique monocarboxylate transporter (MCT3) in retinal pigment epithelium.

The retinal pigment epithelium transports lactate between two tissue compartments, the interphotoreceptor matrix and the choriocapillaris. In this report we describe a 2.45-kb cDNA isolated from a chick cDNA RPE library that encodes a membrane protein found only in RPE cells. The deduced protein has 542 amino acids with twelve putative membrane spanning domains. The cDNA has been designated MCT3 based on its 45% identity in amino acid sequence and structural similarity with the monocarboxylate transporters MCT1 and MCT2. Stable transfectants (pCl-neo/MCT3), made in a rat thyroid epithelial cell line (FRTL-5), express MCT3 RNA. Transfectants had enhanced pyruvate uptake (used as a measure of lactate uptake) which was proton-dependent and inhibited by alpha-cyano-4-hydroxycinnamate. In summary, MCT3's unique expression in RPE cells, multiple potential phosphorylation sites, and basolateral distribution suggest that MCT3 may regulate lactate levels in the interphotoreceptor space.

Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of developing muscle cells.

Left-right asymmetric localization of flectin in the extracellular matrix during heart looping.

The early embryo is initially bilaterally symmetrical. One of the first distinct indications of asymmetry in the embryo occurs during heart looping. The midline tubular heart begins to bend to the right to form a C-shaped structure around 30 hr of development in the avian model. A molecular basis for heart asymmetry and direction of looping is not known, although factors inherent to the myocardium are believed to underlie looping. A left-right asymmetric localization of a specific molecule in the bilateral heart forming regions has not been reported previously. One molecule that we are calling flectin (flectere, in L., to bend or to loop) shows a bilateral asymmetric localization early in the heart forming mesoderm and continues to be expressed asymmetrically in a highly organized manner in the cardiac jelly during heart looping. This large extracellular matrix molecule has been identified using a monoclonal antibody F-22 (Mieziewska et al., 1994a,b). Flectin shows a discrete spatiotemporal pattern of extracellular matrix expression during avian heart development. An asymmetric expression of flectin is observed during heart development at stage 7+/8- (approximately at 24 hr of development around the 3-somite stage). It is predominantly expressed in the left precardiac mesoderm at this developmental period. Between stages 12 and 14, flectin continues to be asymmetrically expressed in the myocardium and is localized at high levels on the basal side of the myocardium and within the cardiac jelly extending to the endocardial cell surfaces. In the same plane of the looping part of the heart it is differentially organized within the cardiac jelly on the convex side and in the outer loop areas. A reduced expression is apparent anteriorly and posteriorly along the tubular heart. The initial asymmetry of localization is maintained throughout the tubular heart. At stage 22 (Embryonic Day 3.5), intensity of immunolocalization of flectin is significantly decreased, with left-right asymmetry becoming less discernible or absent. It again is expressed in Day 10 embryonic hearts. Flectin expression appears to be modulated by retinoids. In vitamin A-deficient quail embryonic hearts that do not loop (Dersch and Zile, 1993; Twal et al., 1995), flectin protein expression is decreased and disorganized, as are other extracellular matrix components comprising the cardiac jelly.

Developmental expression and molecular cloning of REMP, a novel retinal epithelial membrane protein.

The retinal pigment epithelium (RPE), like other transport epithelia, has a polarized distribution of membrane and cytoskeletal proteins. The establishment of a polarized phenotype is an essential step in the differentiation of the RPE and the development and maintenance of visual function. Using a monoclonal antibody (MAb 3C4) we have identified a novel membrane protein that is uniquely expressed in chick RPE. We have referred to this protein as REMP for retinal epithelial membrane protein. In these studies we characterized the expression and distribution of this protein during embryonic development and determined its primary structure by cDNA cloning. The developmental expression of REMP was examined by immunocytochemical localization. REMP was first detected in the chick RPE at Embryonic Day 5 (E5) in both apical and basolateral membranes. By E14 the distribution of REMP was restricted to the basolateral surface of the RPE cells. Biochemical fractionation and surface labeling of RPE cells suggested that REMP was an integral protein. The gene encoding REMP was isolated from an E15 chick RPE cDNA library, cloned into lambda gt11, and screened with MAb 3C4. The cDNA was sequenced and found to contain one 1350-bp open reading frame encoding for a 450-amino-acid protein. The deduced amino-acid sequence of REMP shares 32.9% identity with MCT1, a monocarboxylate transporter (Garcia, Goldstein, Pathak, Anderson, and Brown, Cell, 76, 865-873, 1994). By Northern blot analysis, REMP mRNA was detected only in RPE cells. There was an increase in the expression REMP transcript during development but when RPE cells were grown in primary culture the expression of REMP was turned off. The unique expression of REMP in the RPE in vivo would suggest a role for this protein in development and maintenance of normal retinal function.

Redistribution of insoluble interphotoreceptor matrix components during photoreceptor differentiation in the mouse retina.

The development of the nervous system is largely influenced by the extracellular matrix (ECM). In the neural retina, the photoreceptors are surrounded by a unique ECM, the interphotoreceptor matrix (IPM). The IPM plays a central and possibly crucial role in the development, maintenance and specific function of the photoreceptors. Therefore, the characterization of IPM components is necessary to understand the mechanisms regulating photoreceptor differentiation. The IPM in the mouse retina was examined during photoreceptor morphogenesis with the monoclonal antibody (MAb) F22, which recognizes a 250 kDa component of the interphotoreceptor matrix. The binding pattern of MAb F22 revealed a striking redistribution in the expression of the 250 kDa F22 antigen in late stage of postnatal photoreceptor differentiation in the mouse retina. The F22 staining was detectable in the IPM around the inner segments on the third postnatal day (P3). The MAb F22 initially labeled the region around inner segments, but as the outer segments elongated, the F22 distribution became concentrated to the matrix around the rod and cone outer segments until P16-17. At P17, the F22 label around rods began to disappear, while the label around cones became more defined. The shift in label distribution was largely completed by P20. Residual rod-associated label disappeared within a few days. In the adult animal, the F22 antibody labeled the cone-associated matrix only, and this labeling pattern remained stationary. The change in the distribution of MAb F22 demonstrated by immunolabeling was not accompanied by changes in the size of the molecule; F22 antigen isolated from the IPM of P13-15, and from adult IPM migrated with the same molecular weight on SDS gels. The distribution of MAb F22 was compared to that of chondroitin sulfate proteoglycans which are abundant in the IPM. The labeling patterns of MAbs CS-56, C6-S and C4-S were distinct from that of MAb F22. A general decrease of the label intensity was seen with two chondroitin sulfate MAbs (CS-56 and C4-S) between 16 days and 4 months, but a total loss of rod-associated label was not observed. All three chondroitin sulfate MAbs labeled the retina at embryonic day (E) 11.5-13.5, a time of outgrowth of ganglion cell axons, but the F22 antigen was not detected in the retina at this stage of development. The results demonstrate that the F22 and the chondroitin sulfate antibodies are recognizing different molecules that have distinct roles in retinal morphogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of integrin molecules involved in substrate adhesion.

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.

Diethylene glycol distearate (DGD): a versatile embedding medium for retinal cytochemistry.

Embedment in diethylene glycol distearate (DGD) was shown to be highly desirable and versatile for retinal cytochemical studies, including in situ hybridization, immuno- and lectin cytochemistry. This method allows for preservation of fine tissue detail as well as good reaction sensitivity. It appears to be more suitable than most other methods currently used for light microscopic retinal cytochemistry.

Tubulin in bovine retinal rod outer segments.

Bovine rod outer segment (ROS) preparations contain a major 58 kDa protein doublet that was identified by immunoblot as tubulin. Quantification by gel densitometry showed that the total amount of tubulin was 5- to 10-fold higher than that attributable to the rod axoneme, suggesting additional role(s) for tubulin in photoreceptor cells. Approximately 20% of this nonaxonemal tubulin (15% of total tubulin) is tightly associated with outer segment membranes. This fraction remains membrane-associated after extensive low- or high-salt washing, requiring detergents or protein denaturants for release from ROS membranes. Unlike ROS soluble tubulin it associates tightly with liposomes upon detergent solubilization and reconstitution. The ROS membrane-associated tubulin is highly enriched in isolated ROS plasma membrane fractions compared to the total outer segment membrane pool and can be localized to the plasma membrane but not to disks by immunofluorescent staining, suggesting a possible role in the structure or electrophysiology of the rod outer segment plasma membrane.

G-actin pool and actin messenger RNA during development of the apical processes of the retinal pigment epithelial cells of the chick.

It has been suggested that during development an increase in the pool of G-actin may drive the elongation of actin-containing processes which occur in several types of epithelial cells. The apical processes of chick retinal pigment epithelial (RPE) cells elongate during the last 7 days of embryonic life (E15-E21) reaching lengths of 20 microns or more by hatching (E21). F-actin bundles form the cores of these processes. We followed the elongation by measuring F-actin in the cells and cytoskeletons. In correlation with this, we studied by DNAse assay the levels of monomeric actin in supernatants of cell extracts from E13, before elongation starts, to E17, when elongation is well underway. Total F-actin increased 1.9-fold over this time period and cytoskeletal actin increased 2.5-fold. In supernatants from extracts of E13 RPE the monomeric actin concentration was 51 +/- 0.5 micrograms/ml. From estimates of cell volume we calculated the cellular monomeric actin concentration at E13 as at least 510 micrograms/ml (13 microM). We compared this with monomeric actin levels in extracts from RPE at E15 and E17. Allowing for the estimated increase in cell volume, our data show little overall change in cellular monomeric actin concentration at these times. Changes in the level of actin mRNA were measured over the same time period. Normalized to equal RNA, we found a twofold increase in beta actin mRNA and a four- to fivefold increase in message for gamma actin at E17 as compared to E13. In summary, we show that (1) there is a substantial pool of monomeric actin in these epithelial cells before elongation starts; (2) process elongation is not associated with a significant change in the size of this pool; and (3) process elongation is associated with a significant increase in actin mRNA.

Identification and localization of talin in chick retinal pigment epithelial cells.

Retinal pigmented epithelial cells are adherent at their basal surface to Bruch's membrane and at their apical surface to the neural retina. We examined the expression and distribution of two proteins that are found in regions of cell-matrix interaction, talin and integrin. Talin is a 235-kDa cytoplasmic protein that has been localized to regions of cell-substrate adhesion. It binds to both integrin, a transmembrane glycoprotein complex, and to vinculin, a cytoskeletal protein. In the present study, we produced a polyclonal antibody to chicken gizzard talin. Using this antibody we showed by western blot analysis that talin is expressed by RPE cells and is found in the triton-soluble fraction. Talin was shown to co-localize with integrin and vinculin in the basal region of chick RPE cells isolated from 18-day-old chick embryos. Neither talin nor integrin was found in the apical processes or in the zonula adherens. Antibodies to vinculin showed staining both in the apical and basal regions of the RPE cells. The localization of integrin, talin and vinculin along the basal membrane suggests that this complex is important in the attachment of the RPE cells to the basement membrane. The distribution of integrin and talin was examined in primary cultures of RPE cells grown on permeable filters. In these cells, a polarized distribution of integrin and talin was not observed. This may suggest that the neural retina may be important for maintaining the differentiated state of the RPE cells.

A double-blind single dose comparison of intramuscular ketorolac tromethamine and pethidine in the treatment of renal colic.

The efficacy of a single dose of intramuscular ketorolac 10 mg or 90 mg was compared with pethidine 100 mg in a randomized double-blind study in 121 patients reporting at least moderate pain due to renal colic. Pain was assessed before drug administration, and then at 1 hour and 12 hours after the dose. Sedation was also assessed at these times, and additionally at the 12 hour assessment the time of the next analgesic dose was recorded. At 1 hour after dosing, pain scores had decreased in all groups; the largest decrease was seen in the ketorolac 90 mg group. The difference in the decrease was significant between the two ketorolac groups, but the differences between ketorolac and pethidine were not significant. Fewer patients in the ketorolac 90 mg group (17%) required a further dose of analgesic within 10 hours than in either the ketorolac 10 mg group (39%) or the pethidine 100 mg group (47%). The difference between ketorolac 90 mg and pethidine 100 mg was statistically significant. At both assessment times the proportion of patients with no sedation was higher in the two ketorolac groups than in the pethidine group. The overall incidence of adverse events was low with all drugs, notably so for the occurrence of vomiting after ketorolac. The results of the study show that intramuscular ketorolac is efficacious in the treatment of renal colic.

S-antigen in a hereditary visual cell disease. Immunocytochemical and immunological studies.

S-antigen is a photoreceptor-specific and potentially autoantigenic protein. Using light microscopic immunocytochemistry, the localization of S-antigen was studied in the retinas of normal dogs and Irish setters affected with rod-cone dysplasia, a hereditary retinal degeneration characterized by abnormal cGMP metabolism and arrested outer segment differentiation. Normal and affected dogs were also tested for the presence of humoral and cellular immunity to S-antigen. S-antigen was present in both rods and cones during inner and outer segment differentiation, but there was an apparent loss of immunoreactivity in cones as the retina matured. The developmental appearance and localization of S-antigen in affected retinas was similar to that of normals. S-antigen immunoreactivity decreased during the early stages of rod loss (39-57 days), but was still present in photoreceptor somata in the late stages of retinal degeneration. No significant difference was found between normal and affected setters in humoral immunity to S-antigen, indicating that it probably does not stimulate autoimmunity in red 1. Because normal dog lymphocytes failed to respond when sensitized to bovine S-antigen, cellular immunity to S-antigen in this disease cannot be ruled out.