Axonal tubulin and axonal microtubules: biochemical evidence for cold stability.

Nerve extracts containing tubulin labeled by axonal transport were analyzed by electrophoresis and differential extraction. We found that a substantial fraction of the tubulin in the axons of the retinal ganglion cell of guinea pigs is not solubilized by conventional methods for preparation of microtubules from whole brain. In two-dimensional polyacrylamide gel electrophoresis this cold-insoluble tubulin was biochemically distinct from tubulin obtained from whole brain microtubules prepared by cold cycling. Cleveland peptide maps also indicated some differences between the cold-extractable and cold-insoluble tubulins. The demonstration of cold-insoluble tubulin that is specifically axonal in origin permits consideration of the physiological role of cold-insoluble tubulin in a specific cellular structure. It appears likely that much of this material is in the form of cold-stable microtubules. We propose that the physiological role of cold-insoluble tubulin in the axon may be associated with the regulation of the axonal microtubule complexes in neurons.

Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins.

Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000-dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations.

Fever: reciprocal shift in brain sodium to calcium ratio as the set-point temperature rises.

A bacterial pyrogen acts on the brain by disturbing the natural balance between two essential cations in the cerebral region involved in thermoregulation. After typhoid vaccine is administered to the unanesthetized cat, (45)Ca(2+) efflux into the third cerebral ventricle increases while (22)Na(+) is retained in hypothalamic tissue at the same time that the set-point temperature begins to rise. The subsequent rates of (22)Na(+) and (45)Ca(2+) efflux parallel the course of the bacterial fever but in a reciprocal fashion. This supports the theory that a change in the set-point temperature is determined by an alteration in the inherent ratio of Na(+) to Ca(2+) ions in the hypothalamus.

Hyperthermia protects against light damage in the rat retina.

An increase in the synthesis of heat shock proteins that is induced in cells in vitro by hyperthermia or other types of metabolic stress correlates with enhanced cell survival upon further stress. To determine if a similar increase in stress tolerance could be elicited in vivo, rats were made hyperthermic, and then their retinas were tested for sensitivity to light damage. This treatment resulted in a marked decrease in photoreceptor degeneration after exposure to bright light as compared to normothermic animals. Concomitant with such protection was an increase in retinal synthesis of three heat shock proteins. Thus, a physiological rise in body temperature enhances the stress tolerance of nerve tissue, perhaps by increasing heat shock protein production.

Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes.

The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.

Hsp25 and -90 immunoreactivity in the normal rat eye.

PURPOSE: The distributions of heat shock protein (Hsp)25 and -90 in various regions of the rat eye are described to provide a basis for understanding their roles in normal, damaged, and diseased ocular tissues. This work complements the earlier examination of Hsp70 and Hsc70 (the constitutive form). METHODS: Eyes of adult male Sprague-Dawley rats were fixed in methacarn and embedded in paraffin. Sagittal sections (10 microm) through the optic nerve were stained with hematoxylin and eosin, or incubated with anti-Hsp90, anti-Hsp25, or control IgG. Bound antibody was visualized using an avidin-biotin-horseradish peroxidase detection system. RESULTS: Hsp90 immunoreactivity was abundant in the retina, whereas only low levels of Hsp25 were detected there. In the optic nerve, the relative difference in immunoreactivity for the two Hsps was reversed, with Hsp25 being considerably greater than Hsp90. Both Hsps were detected at low levels in the retinal pigment epithelium (RPE), except for that portion within 250 microm of the optic disc, where Hsp25 and -90 immunoreactivities were increased. Similar to the optic nerve, the corneal epithelium showed greater staining for Hsp25 than for Hsp90, and basal cells contained the highest levels of immunoreactivity for both Hsps. In the ciliary body and iris, Hsp25 and -90 were abundant and similarly distributed in the epithelial and stromal layers. CONCLUSIONS: Each of the ocular tissues had distinctive patterns of Hsp25 and -90 immunostaining. These results suggest that the various structures of the eye have unique requirements for the particular chaperoning and supportive functions of these two Hsp families.

Constitutive and inducible heat shock protein 70 immunoreactivity in the normal rat eye.

PURPOSE: Distributions for the constitutive and inducible 70-kDa heat shock proteins, Hsc70 and Hsp70, in different parts of the rat eye are likely to be related to the metabolic demands required for absorption and detection of light. This study was conducted to better understand the functions of Hsc70 and Hsp70 in these tissues and to provide a basis for elucidating their contributions to the maintenance and repair of ocular structures subsequent to tissue injury or cellular degeneration. METHODS: Eyes from male Sprague-Dawley rats (200-300 g) were fixed in methacarn and embedded in paraffin. Sagittal sections (10 microm) through the optic nerve were stained with hematoxylin and eosin or incubated with heat shock protein antibody or control IgG. Bound antibody was visualized using an avidin- biotin- horseradish peroxidase detection system. RESULTS: Hsc70 immunoreactivity was detected in all layers of the retina, except the outer segments. In the retinal pigment epithelium, staining was restricted to cells near the optic nerve-retina junction. Intense staining was also observed in glial nuclei of the optic nerve, whereas weaker staining was observed in the basal and wing cells of the limbal and corneal epithelia. In contrast, Hsp70 immunoreactivity was restricted to the outer nuclear layer and inner segments of the retina. Hsp70 staining was also prominent in basal and wing cells of the limbal cornea and to a lesser extent in the central corneal epithelium. The optic nerve was Hsp70 negative. CONCLUSIONS: Hsc70 and Hsp70 have distinct distributions in the normal rat eye, which imply regional and cell-specific functions.

Retinal uptake of intravitreally injected Hsc/Hsp70 and its effect on susceptibility to light damage.

PURPOSE: To evaluate the uptake by the rat retina of an intravitreally injected mixture of the constitutive and inducible forms of the 70 kD heat shock protein (Hsc/Hsp70) and test its potential to protect photoreceptors from light damage. METHODS: Hsc/Hsp70 and actin (control protein) were labeled with fluorescein (referred to as fl-Hsc/Hsp70 and fl-actin). The labeled proteins were microinjected intravitreally into the normal or light damaged rat eye and each eye collected at three intervals after the injections. Retinal uptake of Hsc/Hsp70 or actin was studied in frozen sections using epifluorescence microscopy and in western blots of retinal homogenates using an anti-fluorescein antibody. Additionally, the cytoprotective effects of Hsc/Hsp70 were tested in rats that first were exposed to bright light (170 ft-c) for 24 h and then given an intravitreal injection of the protein immediately thereafter. Ten days later, photoreceptor damage was evaluated by measuring the area of the outer nuclear layer at fixed locations along the circumference of the retina. RESULTS: The fluorescein-labeled proteins were detected in the retina one h after administration and were retained there for more than 6 h. They were diffusely distributed, primarily in the nerve fiber layer, ganglion cell layer, and plexiform layers. Fl-Hsc/Hsp70 was also found in the outer nuclear layer (ONL) at 6 h after injection. At 24 h post-injection, the proteins were undetectable by epifluorescence microscopy of retinal sections, but could still be detected in western blots of retinal homogenates. The pattern of protein uptake was similar in light-damaged retinas. Ten days after light damage, the retinas in those eyes that received injections of Hsc/Hsp70 had greater ONL areas compared to either the light-damaged retinas of uninjected eyes or those that had received actin. The difference was statistically significant (p<0.05). CONCLUSIONS: Intravitreally injected Hsc/Hsp70 is taken up by retinal cells and, when administered after an acute injury like light damage, increased the number of surviving photoreceptors.

Exogenous heat shock cognate protein Hsc 70 prevents axotomy-induced death of spinal sensory neurons.

Elevation of intracellular heat shock protein (Hsp)70 increases resistance of cells to many physical and metabolic insults. We tested the hypothesis that treatment with Hsc70 can also produce that effect, using the model of axotomy-induced neuronal death in the neonatal mouse. The sciatic nerve was sectioned and in some animals purified bovine brain Hsc70 was applied to the proximal end of the nerve immediately thereafter and again 3 days later. Seven days postaxotomy, the surviving sensory neurons of the lumbar dorsal root ganglion (DRG) and motoneurons of the lumbar ventral spinal cord were counted to assess cell death. Axotomy induced the death of approximately 33% of DRG neurons and 50% of motoneurons, when examined 7 days postinjury. Application of exogenous Hsc70 prevented axotomy-induced death of virtually all sensory neurons, but did not significantly alter motoneuron death. Thus, Hsc70 may prove to be useful in the repair of peripheral sensory nerve damage.

Estrogen and the subcellular distribution of luteinizing hormone releasing hormone: rate sedimentation studies.

Rate sedimentation of the 900 X G supernatants (S1) of hypothalamic homogenates from untreated male rats or ovariectomized rats with or without 5 micrograms estradiol benzoate (EB) revealed two populations of LHRH particles: a minor, slowly sedimenting one (peak 1) and a major, more rapidly sedimenting one (peak 2). Some LHRH-containing material also sedimented to the bottom of the gradient. The ovariectomized rats displayed more heterogeneity of particulate LHRH than did the male rats. Furthermore, the administration of EB to ovariectomized rats altered the relative sedimentation pattern of LHRH. In ovariectomized rats, hypotonic shock of S1 prior to rate sedimentation eliminated peak 2 and post-peak 2 LHRH and increased free LHRH at the top of the gradient. Peak 1 LHRH was still present and was elevated after EB treatment. Also, EB treatment lowered the free LHRH at the top of the gradient. These data demonstrate that the administration of EB to an ovariectomized rat alters the subcellular distribution of LHRH.

Axonal transport of clathrin-associated proteins.

Clathrin, the main constituent of coated vesicles, is anterogradely transported exclusively in the slow component b (SCb) of axonal transport. However, it has not been shown whether the 30-36-kDa clathrin-associated proteins (CAPs), which may regulate assembly of clathrin into coated vesicles, are transported along with clathrin in SCb. Clarification of this point has implications for the functional state of anterogradely transported clathrin. To investigate CAPs transport, retinal ganglion cells of the guinea pig were labeled with 35S-methionine and the optic nerves harvested at 6 h, 4 days, and 30 days to collect radiolabeled proteins from each major rate component of axonal fast component (FC), slow component a (SCa), and SCb. The radiolabeled rate component proteins were analyzed by using two-dimensional polyacrylamide gel electrophoresis and fluorography. The results showed that CAPs, like clathrin, were transported exclusively with the proteins of SCb. In addition, a comparison of radiolabeled CAPs isolated from axons with whole-brain CAPs failed to demonstrate an appreciable difference in molecular weight or isoelectric point between the two, suggesting that CAPs did not undergo a major post translational modification upon passage into the synaptic terminal. It appears that the distinctive microenvironment within the synaptic terminal is likely to contribute to the ability of clathrin and CAPs to interact with membranes.

Glial polypeptides transferred into the squid giant axon.

The proteins synthesized by the glial sheath of an isolated segment of squid giant axon and by the cell bodies of the giant axon in the isolated stellate ganglion were labeled by incubation in the presence of [3H]leucine. The axoplasm, which contained labeled proteins transferred from the glial sheath, was separated from the sheath by mechanical extrusion. The labeled proteins in the axoplasm, the empty sheath and the stellate ganglion were analyzed and compared by one- and two-dimensional polyacrylamide gel electrophoresis. Over 80 glial polypeptides were found to be selectively transferred into the axoplasm and many of these were distinct from stellate ganglion polypeptides which presumably could be supplied to the axon via axonal transport. Three of the more highly labeled transferred glial polypeptides (TGPs) were actin, a fodrin-like polypeptide and a polypeptide we have named traversin. Our observations, considered in the context of other reports, suggest that the squid axon receives a large number of polypeptides from its surrounding glia either by phagocytozing glial cell process that project into it or via cytoplasmic channels between adaxonal glia and the axon. These TGPs may help the axon survive unfavorable conditions.

Heat shock-like protein is transferred from glia to axon.

Glia-axon protein transfer was examined in the squid giant axon. Proteins synthesized by the glial sheath surrounding the axon were labeled with [3H]leucine. Raising the temperature of the incubation medium from 20 degrees C to 30 degrees C increased the synthesis of glial proteins that resembled heat-shock proteins. These proteins were among the group known to be transferred into the axon. Thus, glia provide the axon with proteins that may be involved in the reaction to trauma.

Immunocytochemical and ultrastructural characterization of type 1 astrocytes and 0-2A lineage cells in long-term co-cultures.

We examined cultures of purified type 1 astrocytes and mixed glial co-cultures containing type 1 astrocytes and 0-2A lineage cells in media containing fetal calf serum at 5 days in vitro (DIV), 12 DIV, and 30 DIV, using cell-specific immunocytochemical markers and electron microscopy. At all three time points and in both culture systems, the polygonal-shaped type 1 astrocytes were A2B5-, GFAP+, and GalC-(specific markers for 0-2A lineage cells, and mature astrocytes and oligodendrocytes, respectively). From 5 to 30 DIV, the type 1 astrocytes increased markedly in size and the appearance of the cytoskeleton changed dramatically, with the amount of glial filaments increasing and microtubules decreasing. At 5, 12, and 30 DIV, the 0-2A lineage cells were multipolar, A2B5 +, HNK-1 +, GFAP-, and GalC-. The 0-2 lineage cells could not be distinguished as either astrocytes or oligodendrocytes on the basis of immunocytochemical or ultrastructural characteristics. These cells had dense cytoplasm, very few intermediate filaments, and a large number of vacuoles and dense bodies. The general characteristics of the cultured astrocytes at 12 DIV and 30 DIV were similar to mature and aged astrocytes in vivo, respectively. These findings suggest that the culture environment in this study accelerated aging of type 1 astrocytes. 0-2A lineage cells, on the other hand, appeared unable to differentiate into either type 2 astrocytes or oligodendrocytes when cultured in the presence of both type 1 astrocytes and fetal calf serum.

In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance.

Glial cells release a variety of molecules that support neuronal function. Because heat shock proteins (Hsps) are important in the survival of neurons subjected to metabolic stress, the possibility that glia can release the inducible form of the 70 kDa Hsp (Hsp70) was examined. Additionally, the ability of neuronal cells to show increased stress tolerance by taking up a mixture of constitutive and inducible forms of Hsp70 (Hsc/Hsp70) added to the extracellular fluid was tested. Human T98G glioma cells and differentiated LA-N-5 neuroblastoma cells were used as model glia and neurons to investigate these points. Hsp70 was analyzed using affinity chromatography, Western blotting, and immunofluorescence microscopy. The glioma cells were shown to export Hsp70 into the culture medium whether under normal conditions or subjected to heat shock. The amount of glial Hsp70 released ranged from 5 to 15 pg per 10(6) cells per day, being greater following heat shock. Neuroblastoma cells took up biotinylated Hsc/Hsp70 within 1 h after it was added to the culture medium and it made them more resistant to heat shock (44 degrees C) and to staurosporine-induced apoptosis. This increased stress tolerance was especially important in neuroblastoma cells induced to differentiate with phorbol ester because those 'mature neurons' showed a 10-fold decline in endogenous Hsp70, which was accompanied by increased susceptibility to heat shock and staurosporine-induced apoptosis. These results suggest that extracellular Hsp70 may provide a means by which glia can affect neuronal function, perhaps enhancing neuronal stress tolerance.

Water deprivation protects photoreceptors against light damage.

Photoreceptor cell death after light-damage and during aging in rats is associated with the hormonal status of the animal, as well as other environmental and intrinsic factors. Restricted caloric intake extends the life of rodents and is usually accompanied by a reduction in water consumption. In this study, male and female rats were placed on restricted water intake for either 3 or 7 days to induce dehydration. Following exposure to damaging visible light, the retinas were evaluated for severity of damage and photoreceptor survival, heat shock (stress) protein (HSP) and total protein synthesis, and plasma arginine vasopressin (AVP) levels. Photoreceptor cells of 7-day, dehydrated male and female rats survived light-damage significantly better than those allowed water ad libitum; however, after 3 days of water restriction, only the male rats demonstrated protection from photodamage. Severity of photoreceptor damage could not be correlated with retinal HSP synthesis and content, although the latter was significantly reduced in dehydrated animals. Total retinal protein content and synthesis were unchanged by restricted water intake. AVP increased by 350% during the 7-day period of dehydration. Protection of photoreceptors from light-damage in this study may be correlated with osmotically stimulated changes in the retinas of dehydrated animals.

Transplantation of cultured type 1 astrocyte cell suspensions into young, adult and aged rat cortex: cell migration and survival.

The present study examined the fate and migration of transplanted astrocytes in different host ages. Additionally, the effect of donor cell age was examined in relation to cell migration. Cultured astrocytes from 5, 12 and 30 days in vitro were transplanted into young (postnatal day 5 and 21), adult (4.5 month), and aged (21 month) animals. The transplanted cells were labeled with Fast Blue, Fluorogold or DiI. The results confirmed previous studies demonstrating that transplanted cells were able to migrate successfully through host central nervous system and extended those findings to show that the age of the host significantly influenced donor cell migration distance. Migration was most extensive in young animals, as conditions supporting cell migration appeared to be lacking in older animals. Donor cells preferentially migrated on myelinated fiber tracts, rather than on unmyelinated fiber tracts or gray matter. The donor cells were not glial fibrillary acidic protein positive, indicating that either the cultured type 1 astrocytes did not survive transplantation or underwent significant remodeling of the intermediate filament network. It is also possible that a subpopulation of cells, possibly immature astrocytes which are present in the transplanted cell suspensions, flourished and subsequently migrated in the host brains.

Slow axonal protein transport and axoplasmic organization.

A recently formulated structural theory of axonal transport suggests that each group of transported material is a distinct class of functionally related structures and that the proteins making up those structures, with few exceptions, are found in only one rate component, thus maintaining a close noncovalent association during transport. If such a relationship exists, one would postulate that the labelled proteins at the leading edge and trailing edge of a rate component following a pulse of radioactive amino acid would be present in similar proportions. After retinal ganglion cell proteins were labelled by intraocular injection of radioactive amino acid in the guinea pig eye, the optic nerve and tract were analyzed at several post-injection intervals. The data thus derived support the existence of a close relationship among the "soluble" proteins of slow component b and are consistent with the structural concept of axonal transport. If diffusion were the mechanism of transport, the larger proteins would be expected to move slower than the smaller proteins. Such insights into the process of axonal transport should help to identify the variables critical to survival of neurons following acute trauma and degenerative diseases.

Changes in glutamate receptor subunit composition in hippocampus and cortex in patients with refractory epilepsy.

An assessment of glutamate receptor subunit profiles was made in hippocampus and temporal lobe cortex of patients with refractory epilepsy. Molecular biological analyses using reverse transcription reaction (RT) followed by polymerase chain reaction (PCR) revealed changes in the distribution profile of the transcripts of AMPA/KA glutamate receptor subunits in hippocampal and cortical tissue from patients with refractory epilepsy when compared to similar tissue from six human and four non-human primate samples with no history of seizures or seizure medication. A severe mean decrease (38% of control) in mRNA for the GluR1 subunit was found in 400 mm cross-sections of hippocampus from patients with epilepsy. Less severe but significant reductions in that GluR1 subunit expression (54% of control) were exhibited in samples of excised temporal pole cortex from the same subjects. Message for the GluR4 subunit was also significantly decreased in hippocampus (68% of control), but in contrast to GluR1, GluR4 mRNA level was not decreased in temporal cortex. Levels of GluR2 mRNA were not significantly changed in epileptic hippocampal and cortical tissue relative to control samples. Protein levels of the GluR1 and GluR4 subunits quantified by Western blot analysis were also reduced in hippocampal and cortical tissue from epilepsy patients. Two other kainate subunit transcripts, GluR6 and KA1 also showed significant changes compared to non-epileptic tissue (136% and 71% of control, respectively). Results are discussed in terms of possible mechanisms by which protracted seizures could produce selective loss of certain AMPA/KA subunits.

In situ fixation of the spinal cord using microwave radiation.

Due to its investiture with bone, the spinal cord can be difficult to study anatomically and histologically. Tissue degradation during immersion fixation or mechanical trauma during extraction of unfixed tissue often produces confusing artifacts. Perfusion fixation eliminates many of these problems, but it is a slow, tedious, and technically demanding procedure. This report demonstrates that microwave irradiation of the spinal cord before its removal from the spine is a rapid and easy method of tissue fixation with an absence of artifacts comparable to that with perfusion fixation.