Autophagic dysfunction and autophagosome escape in the mdx mus musculus model of Duchenne muscular dystrophy.

AIM: Duchenne muscular dystrophy is caused by the absence of functional dystrophin protein and results in a host of secondary effects. Emerging evidence suggests that dystrophic pathology includes decreased pro-autophagic signalling and suppressed autophagic flux in skeletal muscle, but the relationship between autophagy and disease progression is unknown. The purpose of this investigation was to determine the extent to which basal autophagy changes with disease progression. We hypothesized that autophagy impairment would increase with advanced disease. METHODS: To test this hypothesis, 7-week-old and 17-month-old dystrophic diaphragms were compared to each other and age-matched controls. RESULTS: Changes in protein markers of autophagy indicate impaired autophagic stimulation through AMPK, however, robust pathway activation in dystrophic muscle, independent of disease severity. Relative protein abundance of p62, an inverse correlate of autophagic degradation, was dramatically elevated with disease regardless of age. Likewise, relative protein abundance of Lamp2, a lysosome marker, was decreased twofold at 17 months of age in dystrophic muscle and was confirmed, along with mislocalization, in histological samples, implicating lysosomal dysregulation in this process. In dystrophic muscle, autophagosome-sized p62-positive foci were observed in the extracellular space. Moreover, we found that autophagosomes were released from both healthy and dystrophic diaphragms into the extracellular environment, and the occurrence of autophagosome escape was more frequent in dystrophic muscle. CONCLUSION: These findings suggest autophagic dysfunction proceeds independent of disease progression and blunted degradation of autophagosomes is due in part to decreased lysosome abundance, and contributes to autophagosomal escape to the extracellular space.

The use of avidin as a probe for the distribution of mitochondrial carboxylases in developing chick retina.

During development, the inner chick retina progresses from an aerobic to an anaerobic metabolic basis because of the lack of a vascular system. To investigate this process further, we have examined the expression and distribution of mitochondrial carboxylases. Because these enzymes use covalently bound biotin as a co-enzyme, we were able to develop a new detection protocol for mitochondria using avidin as a probe for the biotin. Chemiluminescent detection of bound avidin-peroxidase was used to examine a developmental series of extracts of retinas that had been separated by electrophoresis and blotted to nitrocellulose. Avidin-peroxidase, visualized with the sensitive peroxidase substrate True Blue, permitted detection in epoxy-embedded tissue sections. In the extracts, specific bands of approximate molecular weights 130 and 70 kD were found, corresponding to biotinylated subunits of several mitochondrial carboxylases. During development, the intensity of the bands decreases, although at different rates. In tissue sections, 8-day embryonic retinas display reaction product throughout the tissue, with higher local concentrations in the vitread and sclerad regions. During further development, the reaction product becomes segregated into bands at the borders of the plexiform layers. As the photoreceptors mature, stain becomes concentrated in the developing ellipsoids and the sclerad ends of Müller cells.

Inhibition of retinal growth cone activity by specific metalloproteinase inhibitors in vitro.

The developing neural retina expresses a set of extracellular proteases including plasminogen activator and gelatinases. Since neurites of retina cells cultured on fluorescent gelatin digest the substrate in their paths, we have suggested that the proteases are used by the tips of growing fibers to allow them to migrate within the mass of the tissue in vivo. In order to obtain further information about relationships between extracellular proteases and fiber growth, we have examined the effects of the specific inhibitors HS-LFA (HS-Leu-Phenylala-Ala, enantiomeric forms 1 and 2), bathophenanthroline sulfonate (BPS), phenylmethyl sulfonyl fluoride (PMSF), and relevant controls on the activity of retinal growth cones in vitro, monitored by time lapse video microscopy. Of the inhibitors tested, only the two enantiomeric forms of HS-LFA caused a reproducible cessation of both spike extension and filopodial processes at the growth cone ruffling, while control media had no effect. In some cases, the growth cone swelled and exhibited small protrusions. The behavior of growth cones was in sharp distinction to that of the cytoplasm of neural cells, and membrane ruffling of flat cells, which continued in activity throughout. Growth cone activity returned after several hours in the presence of the agent. BPS was toxic at concentrations above 2.5 mM. Below that, it had no effect. L-cysteine, PMSF, and control media had no effect. The relevance of these results to the possible role of proteases in fiber outgrowth from retinal cells is discussed.

Changes in expression and distribution of lactate dehydrogenase isoenzymes in the developing chick retina.

We have investigated the expression and distribution of lactate dehydrogenase (LDH) isoenzymes in the developing and adult chicken retina. Non-denaturing polyacrylamide gel electrophoresis was used to follow the appearance and expression of the five main LDH isoenzymes in tissue homogenates. Immunohistochemistry was used to define the distribution of the aerobic heart type LDH and the anaerobic muscle type LDH in paraffin sections of embryonic and adult chick retina. The electrophoretic results show that the expression of the anaerobic isoenzyme increases in the retina as development proceeds. Immunoreactivity against the anaerobic isoenzyme localizes to the inner plexiform layer and other regions of the inner chick retina. The aerobic isoenzyme immunoreactivity is localized to the inner segments of the photoreceptor cells as well as the ganglion cell layer. Both antibodies bind weakly in other layers of the retina with the notable exception that the anaerobic protein does not localize in the photoreceptor cell inner segments. These results provide further evidence for the anaerobic nature of the adult inner retina. As retinal development proceeds, the expression of the anaerobic isoenzyme in the retina increases.

Changes in distribution of mitochondria in the developing chick retina.

The distribution of mitochondria in the developing chick retina was examined by enzyme histochemistry, immunohistochemistry, and electron microscopy. Two distinct phenomena were observed: (1) progressive segregation of mitochondria in specific locations in the developing tissue; and (2) progressive loss of mitochondrial activity from the inner retina as it matures. Densitometric scans of stained tissue sections were used to quantitate the relative amounts of mitochondrial activity in the retinal layers. Mitochondria were localized to tissue regions by transmission electron microscopy, enzyme histochemistry for the inner membrane bound mitochondrial enzymes succinic dehydrogenase and cytochrome oxidase, a combined histochemical and ultrastructural method for cytochrome oxidase, and immunolocalization of the mitochondrial matrix enzyme glutamate dehydrogenase. Seven-day embryonic chick retina has a high number of mitochondrial structures and a high level of activity. As development proceeds, the mitochondria organize into layers within the tissue. However, the relative activity of mitochondria in much of the inner retina decreases. In the post-hatch retina, 50% of the mitochondrial activity is found in less than 10% of the tissue area, in the inner segments of the photoreceptor cells.

Extracellular proteases in developing chick neural retina.

During retinal histogenesis, cells and their extensions migrate within the tissue to final positions. In order for the cells to move through the matrix of tissue, space must be made available. We report evidence that extracellular proteolytic activity might be associated with this process. (1) When embryonic chick neural retinal cells are seeded onto a substrate of rhodamine conjugated fluorescent gelatin, the tips of growing neurites remove the fluorescence from the substrate. (2) Latent gelatinolytic activity can be identified with soluble assays of homogenates of embryonic chick neural retina. (3) Zymogram analysis demonstrates the presence of high molecular weight bands of proteolytic activity. The activity is inhibited by 1.10 phenanthroline, suggesting that it is due to a metalloproteinase. Activity can be detected in supernatants of retinal cells grown in vitro. Gelatinolysis is not the only proteolytic activity detected in the retina. Addition of plasminogen to zymograms results in an additional band of activity.

Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina.

When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish. We have carried out a morphologic and immunologic study of the emergence of this population in vitro. Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein. SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture. Not all of the cells that attach flatten. As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them. The flattened cells are positive for Vimentin by IF within 10 min of attachment. The percent of fluorescent cells found on the substrate is constant during the time in culture. This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate. When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate. Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized. Thus, the same cells that contained Vimentin in the retina now express it as flat cells. This supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Müller cells. We have also observed the emergence of a population of cells with short (0.5 micron) microvilli that appear within 8 h of culture. They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters.

Purkinje cell number related to rate of classical conditioning.

The cerebellar cortex is normally involved (but not essential) in acquisition of the classically conditioned nictitating membrane response. Cerebellar Purkinje cells probably play a role in acquisition, but these cells are vulnerable to hypoxia, ischemia, and aging. Since large age differences exist in rate of conditioning, we suspected that the number of Purkinje cells in individual rabbits would correlate with learning rate. Purkinje cell counts were undertaken in 12 rabbits aged 3-50 months. The correlation between trials to learning criterion and Purkinje cells was -0.79. A second study used 18 3-month-old rabbits. Even with age held constant, the correlation was -0.60. Individual variation in Purkinje cell number accounts for significant variability in learning rate.

Patterns of cell movement in early organ primordia of the chick embryo.

Purse-string constriction of the cytoskeleton at cell poles is generally accepted as the causal mechanism for invagination during early stages of organ formation. However, it is known that other cell movements, including intercalation, play a role in the organotypic shape changes that occur during gastrulation and neurulation. Such cell movements have not been investigated in pouching and branching epithelial primordia. There is reason to suspect that cells within these organ primordia might exchange their neighbors for others, that is, intercalate or translocate, at sites of sharp folding such as borders with the surrounding epithelial sheet or where a bend occurs within the primordium. The greatest difficulty in identifying these movements has been the need to use intact embryos so that the processes are not distorted. This study explores the possibility of using time-lapse video recording to identify cell movement at these locations. Three organ primordia were tested: otic and thyroid placodes, which had not been tested previously, and neural plate as a control, where movements of this sort have been documented. Embryos or parts containing the primordia were immobilized and cell apices visualized with Hoffman modulation contrast optics. Recordings to an optical memory disc recorder were transferred to a microcomputer for image analysis. The viewing procedure allows reasonably clear visualization of cell apices, and image analysis permits tracking of a number of adjacent cell apices over an extended time period. Several types of movement were found to occur within cell sheets, and the relative abundance of each type depends on the specific primordium. In the neural plate, some cells move many cell diameters from their neighbors. In the other two primordia, most cells show limited shifts in position relative to their neighbors except at regions where folds are formed. In other regions, adjacent cells move as a unit. Knowledge of the movements which occur in any particular primordium is essential to an understanding of the mechanisms controlling its formation.

Use of a touch sensitive screen and computer assisted image analysis for quantitation of developmental changes in pulmonary structure.

The extensive changes in pulmonary function occurring during early development may reflect variations in the anatomic structure of the respiratory apparatus during this period. Accurate definition of these alterations could yield important information concerning the structure-function correlations of the respiratory system. To facilitate the acquisition of morphometric data from histologic sections of pulmonary tissues, we propose the use of a computer assisted image analysis system with a touch sensitive screen as an interactive peripheral. This allows planimetric measurements and computation of the dimensions of areas of selected light intensities within an image. We present the description, design, and applications of such an image analysis system and report representative results regarding developmental changes in pulmonary structure. In addition, we correlate these results with previously published information regarding pulmonary mechanics during early development to help clarify the maturational changes in pulmonary structure-function relationships.

A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances.

We describe a simple assay for small amounts of protein that is insensitive to sodium dodecyl sulfate (SDS) or many common interfering substances including Tris and reducing sugars. For this reason, it is particularly useful in the analysis of protein content of samples prior to SDS electrophoresis. The assay consists of the following steps: (i) absorption of protein to nitrocellulose; (ii) fixation of the bound protein with methanol; (iii) staining of the bound protein with amido black; and (iv) elution and spectrophotometric measurement of the bound dye. The assay is sensitive to as little as 0.5 microgram of protein in 1 microliter of solution. Although SDS does not interfere appreciably with measurement, Nonidet-P40 does.

Retinal flat cells are a substrate that facilitates retinal neuron growth and fiber formation.

When embryonic chick neural retinas are dissociated into a suspension of single cells and plated in stationary cultures, "flat cells" spread out and form a monolayer to which the neuronal cells attach. It has been shown previously that the flat cells are related to the Müller cell population of the retina. The neuronal cells form aggregates interconnected by bundles of axon-like fibers. The authors have been able to isolate relatively pure flat cells by shaking off the neuronal aggregates after 5 or 6 days of culture. In order to determine if the flat cells have a unique relationship with the neuronal cells, freshly dissociated neural retina cells were added to monolayers of flat cells and their behavior compared to that on chick embryo mesodermal cells. It has been observed by phase contrast and scanning electron microscopy that the growth behavior of the retina cells on flat cells is significantly different from that on mesodermal cells. On flat cells, neuronal retina cells form flat patches in which new growing flat cells fuse with the monolayer, and neuronal cells attach as single cells or small clusters. Axon-like fibers are present several hours after plating, and by day 4 an extensive network of fibers connects single cells and clusters on the surface of the monolayer. When retina cells are plated onto mesodermal cells, the cells form aggregates which are organized along the long axis of the mesodermal cells. The flat cells provide a unique substrate for the differentiation and neurite extension of neuronal cells from embryonic chick retina.

Retinal flat cells participate in the formation of fibers by retinal neuroblasts in vitro. Time lapse video studies.

Freshly dissociated cells from embryonic chick neural retinas grow in characteristic patterns on flat cells or on chick embryo mesodermal cells. A striking difference between the two patterns is that the cells grown on flat cells are interconnected by a complex network of fibers, whereas those grown on mesodermal cells are aggregated into clusters that remain relatively isolated within the mesodermal monolayer. Analysis by time-lapse video microscopy indicates that two processes produce the fibers. (1) Fibers grow out by the extension of growth cones from cells within aggregates. (2) Neuronal cell aggregates that attach to two flat cells are pulled apart by the movement of the cells beneath them. As the aggregate is pulled apart, portions of the cells remain attached to the two halves, and their cytoplasm is drawn into thin fibers. The lack of fibers on a mesodermal substrate is due to two factors: (1) Aggregates are widely spaced on the substrate surface and do not come into contact often. (2) On those occasions when they do come into contact, the movement of the monolayer is so vigorous that emerging fibers are torn.

Isolation and characterization of flat cells, a subpopulation of the embryonic chick retina.

When the embryonic neutral retina is dissociated into single cells which are maintained in stationary culture, the neuronal cells associate on the surfaces of a second population which we refer to as flat cells. The flat cells appear in the culture in significant numbers after 2 days and are required for neuronal cell attachment. We have been able to isolate pure flat cells from early cultures of mixed retina cells and have identified several antigens which support the concept that these cells are related to the glia. The cells have been tested by immunofluorescence for glial fibrillary acidic protein and have been found positive. Cell surfaces were labeled by transfer of tritiated galactose from UDP-galactose to endogenous acceptors in the presence of exogenous galactosyl transferase. After SDS-PAGE and fluorography, the surface glycoproteins of flat cells were seen to be significantly different from those of the original retina, and from chick fibroblasts. Immunoelectron microscope studies of detergent-extracted flat cells have demonstrated a complex network of intermediate filaments and actin fibers. We conclude that the flat cells are derived from the glia subpopulation of the retina and have adapted to the tissue culture environment by assuming this configuration. The unique surface properties of flat cells may be related to their role as an intermediate substrate between the neuronal cells and the tissue culture dish.

Alterations of the cytoplasmic organization of WIRL cells induced by trifluoperazine.

The antipsychotic drug trifluoperazine (TFP) causes a reversible rounding of cells of the rat liver epithelial cell line, WIRL. We have investigated the cytoplasmic organization of these cells after TFP treatment using SEM, TEM and immunofluorescence and have observed significant differences between the control and treated cells. Mitochondria are converted to the condensed configuration with distended cristae and the endoplasmic reticulum becomes tubular with distended cisternae. Intermediate filaments, visualized with a monoclonal antibody, are aggregated to a cap on the nucleus in an arrangement different from that induced by colcemid.

Glycoprotein differences among cells of the 14-day embryonic chick neural retina.

In order to test the hypothesis that the progressive layering and differentiation of cell types during the development of the neural retina are associated with cell surface alterations we have separated distinct cell populations from the 14-day embryonic chick retina. Cells of these populations have been shown to differ in associative behavior and intramembrane particle content. We now report that these cells differ in cell surface glycoproteins. Proteins were labeled with two different extrinsic labels and one metabolic label. We used enzymatic transfer to galactose from UDP-gal to cellular acceptors, and borotritide reduction after galactose oxidation as extrinsic labels. Glucosamine incorporation was used as the metabolic label. In all these cases, we were able to identify bands on electrophoretic gels which were unique to individual populations.

Cells isolated from the embryonic neural retina differ in behavior in vitro and membrane structure.

Several subpopulations of cells were isolated from trypsin-dissociated embryonic (14 days) chick retinas. The cells of each subpopulation differed in associative behavior measured by cell aggregation and stationary culture assays and in glycoproteins that contain glucosamine. Freeze-fracture analysis showed that these populations also differed in intramembrane particle content.