Proteins in unexpected locations.

Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer.

RNAi and brain function: was McConnell on the right track?

RNA interference (RNAi), one of the hottest topics of molecular biology research today, has unique features that are eerily reminiscent of the phenomenon of "RNA-mediated memory transfer," a controversial line of work that was investigated with great enthusiasm in the 1960s. If not a coincidence, then this suggests taking a new look at RNA-mediated modulation of neural function and raises the possibility that RNAi might be one of the physiologic mechanisms that regulate long-term gene expression in the brain.

Chronic corticosterone-mediated dysregulation of microRNA network in prefrontal cortex of rats: relevance to depression pathophysiology.

Stress plays a major role in inducing depression, which may arise from interplay between complex cascades of molecular and cellular events that influence gene expression leading to altered connectivity and neural plasticity. In recent years, microRNAs (miRNAs) have carved their own niche owing to their innate ability to induce disease phenotype by regulating expression of a large number of genes in a cohesive and coordinated manner. In this study, we examined whether miRNAs and associated gene networks have a role in chronic corticosterone (CORT; 50 mg kg(-1) × 21 days)-mediated depression in rats. Rats given chronic CORT showed key behavioral features that resembled depression phenotype. Expression analysis revealed differential regulation of 26 miRNAs (19 upregulated, 7 downregulated) in prefrontal cortex of CORT-treated rats. Interaction between altered miRNAs and target genes showed dense interconnected molecular network, in which multiple genes were predicated to be targeted by the same miRNA. A majority of altered miRNAs showed binding sites for glucocorticoid receptor element, suggesting that there may be a common regulatory mechanism of miRNA regulation by CORT. Functional clustering of predicated target genes yielded disorders such as developmental, inflammatory and psychological that could be relevant to depression. Prediction analysis of the two most prominently affected miRNAs miR-124 and miR-218 resulted into target genes that have been shown to be associated with depression and stress-related disorders. Altogether, our study suggests miRNA-mediated novel mechanism by which chronic CORT may be involved in depression pathophysiology.

Expression of a neurally related laminin binding protein by neural crest-derived cells that colonize the gut: relationship to the formation of enteric ganglia.

In order to give rise to the enteric nervous system (ENS), cells migrating from the neural crest must find the bowel and cease migrating at appropriate locations within the gut. Previous studies of the development of the ENS in a mutant mouse have led to the hypothesis that laminin in the enteric mesenchyme may act as a signal to crest-derived cells to cease migrating and extend neurites (or glial processes). Implied in this hypothesis is the idea that crest-derived cells, as a prelude to their participation in ganglion formation, acquire a neurally related laminin receptor, which they do not express at pre-enteric stages of migration. As a partial test of this hypothesis, single and double label immunocytochemistry at light and electron microscopic (EM) levels were used to study the expression of cell surface laminin binding proteins by crest-derived cells in the process of migrating to or within the developing chick gut. Two antibodies (called 3070 and alpha-110) raised against neuronal cell surface laminin binding proteins were employed for this purpose. Laminin binding protein immunoreactivity was found to be expressed within the bowel and ganglion of Remak by a subset of crest-derived cells (identified immunocytochemically with NC-1/HNK-1 antibodies) and by all of those developing as neurons (identified immunocytochemically with antibodies to neurofilament-associated proteins). Laminin binding protein immunoreactivity was also found to be expressed in fixed neural structures elsewhere in the embryos, including cranial and spinal roots, nerves, and ganglia. In contrast, laminin binding protein immunoreactivity was not expressed by migrating crest-derived cells in the vicinity of the vagal or sacral regions of the neuraxis (from which the precursors of the ENS take origin); nor was it expressed by juxta-pharyngeal vagal crest-derived cells migrating to the foregut through the caudal branchial arches or by the caudal stream of sacral crest-derived cells approaching the hindgut. EM immunocytochemistry confirmed that laminin binding protein immunoreactivity in the bowel was located on the surfaces of crest-derived cells, and was exhibited both by those cells that could only be distinguished from their neighbors by their NC-1/HNK-1 immunoreactivity and by cells developing as neurons or glia. EM immunocytochemistry also revealed that the surfaces of crest-derived cells migrating through the enteric mesenchyme were contacted by many small osmiophilic "puffs" of laminin-immunoreactive extracellular material. These puffs coincided in location with membrane sites that expressed the immunoreactivity of the laminin binding protein. These observations are consistent with the hypothesis that laminin plays a role in the formation of enteric ganglia.

Linking estrogen to Alzheimer's disease: an informatics approach.

Epidemiologic studies suggest that estrogen protects against AD. We employ ARROWSMITH, a novel computer-assisted approach, to identify possible links between estrogen and AD that are not explicit in the biomedical literature, by searching for substances or processes that are known targets of estrogen action and that have also been separately studied in relation to AD. Several links appear particularly promising (e.g., estrogen's antioxidant activity) and merit attention by neuroscientists.

Expression of Egr-1 in the brain of sleep deprived rats.

In previous research, rats subjected to prolonged sleep deprivation have shown disturbances of thermoregulation, hormonal and metabolic changes in apparent response to the thermoregulatory problems, lesions on the tail and paws, and eventual death. To search for alterations of functional activity in brain, the expression of the immediate early gene Egr-1 was examined by immunocytochemistry and Northern blotting in rats subjected to total sleep deprivation (TSD) for 10 days. Controls included yoked stimulus-control (TSC) rats, surgically implanted but otherwise undisturbed control rats, and unoperated control rats. Photographs of immunoreacted coronal sections from four sets of rats were ranked blindly for 25 brain regions. TSD rats showed tendencies for regionally specific increases in Egr-1-like immunoreactivity in dorsal raphe, lateral habenula, superior colliculus, and ventral periaqueductal grey. However, most regions showed no differences in Egr-1-like immunoreactivity between TSD and control rats. Neither was there a difference in whole brain Egr-1 mRNA by Northern blot in two additional sets of rats. Thus, this study, like previous studies of brain histology, amines, adrenoceptors, and glucose utilization, does not provide positive support for the hypothesis that sleep protects the central nervous system against massive global damage, fatigue, or dysfunction.

Positional specificity tests in co-cultures of retinal and tectal explants.

In previous studies we demonstrated that HRP-labeled ganglion-cell axons arising from fetal mouse retinal explants can distinguish appropriate (tectum) from inappropriate (spinal cord) CNS target regions in vitro. Retinal fibers preferentially invade the tectum where they ramify, develop terminal arbors, and make functional connections. The present study attempts to determine if fibers from a half-retina prefer the 'appropriate' half-region of a tectal explant. In one series, nasal or temporal halves were placed near the medial edge of the tectum, to test turning toward or away from the appropriate (posterior or anterior) half-tectal region. Several co-cultures showed apparent preferences, especially those with many ingrowing retinal fibers; but in other co-cultures the data were equivocal. A second paradigm placed the half-retinas near anterior or posterior tectal edges, and simply scored presence vs absence of retinal fiber ramifications within the nearest tectal half-region. This identified entry of retinal fibers into appropriate vs inappropriate half-tectal regions in 8 out of 8 blind scorings. These data encourage further, more critical analyses of retinotectal co-cultures to explore the basis of the specific neuritic connections which form in situ.

Radiosensitivity and differentiation of ganglion cells within fetal mouse retinal explants in vitro.

Fetal mouse retinas were explanted at 13-14 days of gestation, and exposed to gamma radiation in vitro. Not all regions of the retina were equally susceptible to radiation-induced necrosis; when exposed to 5000 rads soon after explanation, each explant had a single small radioresistant nubbin of apparently intact tissue, located near the optic nerve-head. This region of radioresistant tissue was larger when the dose of radiation was reduced and when the explants were exposed at later times in vitro, indicating the existence of a gradient of radioresistance across retinal explants which spread outward through at least the first week in vitro, the period examined. Based upon the extensive in situ literature which has correlated the emergence of radioresistance with the differentiation of retinal neurons, we conclude that the in situ central-to-peripheral sequence of cellular differentiation continues in vitro within our retinal explants. Whereas the ganglion cell axonal outgrowth from control retinas grown in isolation on collagen substrates underwent a gradual disintegration over 3 weeks in vitro, the sparse axonal outgrowth from explants exposed to 5000 rads disintegrated abruptly at 5-7 days in vitro. This did not appear to be due to direct damage from radiation, but instead reflected the fact that axons in irradiated cultures arose from central retinal regions only, while many axons in control cultures emerged from later-differentiating peripheral regions. We suggest that disintegration of individual axons in the outgrowth may occur rapidly and in a central-to-peripheral sequence. These findings should be useful in designing assays for trophic factors which may prevent ganglion cell axon disintegration in this in vitro model system.

Kinetic analysis of 'rapid onset' neurite formation in NG108-15 cells reveals a dual role for substratum-bound laminin.

Undifferentiated neural hybrid NG108-15 cells plated on laminin-coated, polylysine-treated plastic Petri dishes in minimal serum-free media formed long neurites within 1-4 h post-plating. Morphologic features and pharmacologic responses of these 'rapid onset' neurites were strikingly similar to those of neuronal growth cones. Cycloheximide (1-10 micrograms/ml) and forskolin (10(-7) to 10(-6) M) accelerated the initial formation of laminin-stimulated neurites, but did not cause rapid onset neurites to emerge upon Petri dishes coated with polylysine alone. Quantitative study of the substratum-dependent effect of cycloheximide showed that it was additive even with maximally effective amounts of laminin, independent of the magnitude of the laminin-stimulated baseline rate and not limited by an inherent ceiling in the rate at which neurites could form. The methylation inhibitor 5'-deoxy-5'-methyl thioadenosine (MTA) (3 X 10(-4) to 3 X 10(-3) M) did stimulate neurites to form on polylysine. MTA- and laminin-stimulated neurites were similar in their susceptibility to calmodulin antagonists and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, formation of MTA-stimulated neurites was not accelerated by cycloheximide. A simple two-compartment kinetic model of 'rapid onset' neurite formation is proposed: compartment A is common to both laminin- and MTA-stimulated neurites. Compartment B is affected by cycloheximide, and its access to the neurite formation machinery contained in compartment A is gated according to the nature of the substratum. In addition to its direct effects, laminin controls the relative dominance of the two kinetic compartments via modulating the effectiveness of other signals acting upon an endogenously active compartment B.

Coordinate enrichment of cranin (dystroglycan) subunits in synaptic membranes of sheep brain.

Cranin (dystroglycan), a mucin-like extracellular matrix receptor comprised of two subunits (alpha and beta), is involved in regulating cell-matrix interactions in a variety of tissues, including brain. A basic issue remains unresolved concerning the distribution of cranin in brain: are the alpha and beta subunits coordinately expressed at the synapse? We report here that cranin is indeed enriched progressively in synaptosomes and synaptic membranes of sheep brain, as assessed by immunoblotting and laminin-blotting assays, and that the extent of enrichment is similar for both alpha and beta subunits. These findings support the hypothesis that cranin (dystroglycan) contributes to synaptic function in the CNS.

Acute neurite retraction triggered by lysophosphatidic acid: timing of the inhibitory effects of genistein.

Acute neurite retraction, elicited by diverse agents in several neuronal cell types, has been reported to be inhibited by genistein, a kinase antagonist that is relatively (though not absolutely) selective for tyrosine kinases. It was hypothesized that genistein acts upon some final common pathway that integrates multiple extrinsic and intrinsic signals to regulate whether neurites will execute a retraction response (J. Neurochem., 61 (1993) 340-343). To define this pathway in more detail, a quantitative study of NG108-15 cell rapid-onset neurites was carried out as they retract in response to lysophosphatidic acid (LPA, 10 microM). Following the application of LPA, most neurites exhibited early morphologic changes between 0.5 and 1.5 min, followed by progressive shortening and eventual retraction, with 50% of neurites completely retracted by 5 min and 80% gone by 10 min. Genistein did not inhibit the formation of subcortical F-actin, nor its functional competence in several assays. Genistein protected neurites when added at any time prior to the onset of the earliest morphologic changes, but failed to block progression when added to neurites that were already undergoing retraction. These findings imply that the final common pathway (i.e. the critical target(s) for genistein) must be activated late, after the increase in F-actin levels has peaked and just before retraction is initiated.

Rapid regulation of neurite outgrowth and retraction by phospholipase A2-derived arachidonic acid and its metabolites.

Arachidonic acid and lipoxygenase metabolites have been proposed to act as retrograde synaptic messengers and as early mediators of neuronal injury, but few studies have analyzed their roles in controlling neurite behavior within a time window of minutes to hours. Phospholipase A2 inhibitors (BPB, ONO-RS-082, quinacrine and AACOCF3) and the lipoxygenase inhibitor AA861 delayed the initial outgrowth of NG108-15 cell neurites on laminin. Inhibitors of diacylglycerol lipase (RHC 80267), cyclooxygenase (indomethacin) and free radicals (N-acetyl cysteine and vitamin E) did not produce similar effects. Phospholipase A2 and lipoxygenase inhibitors also prevented acute neurite retraction in response to lysophosphatidic acid and eight other agents tested, and decreased F-actin staining at cell margins. Conversely, exogenous arachidonic acid (1 microM) enhanced the responses of neurites in outgrowth and retraction assays. Phospholipase A2 and lipoxygenase pathways appear to have a general role in maintaining the ability of neurites to respond rapidly to external stimuli, possibly via regulating the ability of the cytoskeleton to remodel.

Laminin as a substrate for retinal axons in vitro.

Fetal mouse retinal ganglion cell axons have been shown to ramify within co-cultured basement membrane secreting tumor explants and upon isolated basement membranes. Here we report that laminin, a glycoprotein found in basement membranes and adhesion sites of a variety of cell types, acted as a substrate for retinal explant attachment and axonal outgrowth. When axons were given a direct choice, laminin was preferred over collagen. Under suitable conditions (air-dried upon underlying collagen gels), laminin was more effective than fibronectin for promoting axon emergence from retinal explants. These findings have implications for the study of molecular mechanisms underlying CNS axonal outgrowth.

Development of ganglion cells and their axons in organized cultures of fetal mouse retinal explants.

Retinas from 13-15 day fetal mice were explanted alone, with adjacent eyeball tissue, or with nearby superior colliculus explants. The organotypic structure of the retina developed in situ, including photoreceptors, interneurons, plexiform layers, ganglion cells, and an optic fibre layer. Electrophysiologic recordings demonstrated that functional synaptic networks developed resembling bioelectric response patterns seen in situ. Within half-retinas, arrays of optic fibers converged to the optic nerve head; in co-cultures with tectum they could become myelinated. Large bundles of long, naked neurites--1 degree primary retinal fibers--emerged from the explant in the first few days in vitro; these could often be traced back to the optic nerve head and a detailed survey of their properties using horseradish peroxidase (HRP) tracing methods identified tham as ganglion cell axons. When growing upon collagen substrata, 1 degree fibers began to disintegrate during the second week in vitro; however, many 1 degree fibers that grew into superior colliculus explants were maintained for at least 5 weeks in vitro, where they formed elaborate, functional terminal arborizations. In a few cases, 1 degree fibers grew across skeletal muscle fibers and appeared to induce them to contract. A second type of neuritic outgrowth pattern appeared after the first week in vitro: 2 degrees retinal fibers. This was composed of a mixed population of interneuronal neurites; a small percentage was catecholaminergic. Our characterization of the morphologic properties of retinal ganglion cells and their axons in organotypic cultures provides the necessary background to interpret electrophysiologic mapping and neural-specificity analyses of retino-CNS co-cultures. This in vitro model system may have biological relevance to understanding the cues that control the development of the retinotectal projection in situ.

Neurites from mouse retina and dorsal root ganglion explants show specific behavior within co-cultured tectum or spinal cord.

We have utilized extracellular microiontophoretic injections of horseradish peroxidase into fetal mouse retinal explants to label retinal ganglion cell axons innervating co-cultured tectal explants in a solid Golgi-like manner. Using dorsal root ganglia-tectum and retina-spinal cord co-cultures as controls, our results indicate that retinal neurites show selective growth and arborizations within their appropriate tectal, target tissue. Retinotectal explant co-cultures may be a useful model system for studying aspects of neuronal specificity.

Specific neuritic pathways and arborizations formed by fetal mouse dorsal root ganglion cells within organized spinal cord explants in culture: a peroxidase-labeling study.

Extracellular microiontophoretic injections of horseradish peroxidase (HRP) into NGF-enhanced fetal mouse dorsal root ganglia (DRGs) produced an anterograde solid Golgi-like labeling of DRG neurites and their terminal arborizations within co-cultured spinal cord explants. In cultures of spinal cord transverse cross-sections with attached DRGs, the large NGF-enhanced DRGs remained in close proximity to the cord, often adjacent to both dorsal and ventral cord regions. Despite this, nearly all DRG neurites that entered the cord did so via dorsal root fascicles. They branched and ramified extensively within the dorsal region, taking on a wavy or kinky course and showed various types of arborizations. The density of cord innervation was much lower when isolated DRGs and cord explants were co-cultured 0.5-1 mm apart. Although fewer entering DRG fibers were labeled by our HRP injections the same qualitative growth and arborization patterns were seen within dorsal and ventral cord regions as in explants of cord with attached DRGs. When the facing edge contained both dorsal and ventral tissues, HRP-labeled DRG fibers entered dorsal regions selectively. DRG fibers readily entered, ramified and arborized within isolated strips of dorsal cord, whereas they sharply avoided isolated ventral cord explants. The avoidance of ventral cord cannot simply be due to the paucity of specific synaptic targets within the tissue, for larger numbers of DRG fibers entered completely inappropriate CNS target tissues, e.g. superior colliculus explants--though they did not ramify or arborize to any degree comparable to that seen within dorsal cord regions.

Antidepressants alter cell proliferation in the adult brain in vivo and in neural cultures in vitro.

The action of antidepressants on cell proliferation (bromodeoxyuridine (BrdU) or [3H]thymidine incorporation) was studied in the adult rat hippocampus in vivo and in neural precursors (immature rat cerebellar granule cells) in vitro. In vivo, prolonged (21 days) but not acute (single) intraperitoneal treatment with fluoxetine (5 mg/kg) resulted in a 3.4-fold increase of bromodeoxyuridine-positive cells in the subgranular zone of the dentate gyrus. In cell cultures, at 1 and 10 days in vitro, 48-h fluoxetine exposure (1 microM, which is comparable to therapeutic plasma concentrations) reduced thymidine incorporation when initiated at 1 day in vitro, but increased cell proliferation when initiated at 10 days in vitro. Clomipramine and imipramine produced similar action in vitro; desipramine was ineffective.

Morphologic plasticity of rapid-onset neurites in NG108-15 cells stimulated by substratum-bound laminin.

Undifferentiated NG108-15 cells, when replated onto laminin-coated substrata, extend multipolar, highly branched neurite-like extensions up to 200 microns in length within 4 h; morphologic and pharmacologic properties of these 'rapid-onset neurites' have been described recently. The present study has extended these observations, using time lapse video recordings of their dynamic behavior and additional pharmacologic studies. Rapid-onset neurites and neuronal growth cones were shown to be regulated in an identical manner in all respects examined, including inhibition of outgrowth by cytochalasin B. Of particular interest was the observation that rapid-onset neurites in contact with laminin exhibited an extremely high rate of turnover, which was inhibited by 5'-deoxy-5'-methylthioadenosine (MTA). This system provides a uniquely favorable in vitro preparation in which neuritic plasticity can be elicited, directly observed and experimentally modulated under controlled conditions.

Analysis of slow-onset neurite formation in NG108-15 cells: implications for a unified model of neurite elongation.

When undifferentiated NG108-15 cells are plated onto polylysine coated Petri dishes in serum-free medium, they form neurites within 1-4 h if plated in the presence of laminin or 5'-deoxy-5'-methylthioadenosine (rapid-onset neurites). In the absence of such agents, serum-deprived NG108-15 cells extend axon-like neurites onto polylysine over several days; here we characterize the dynamic behavior of this slow-onset outgrowth pattern in detail. Individual cells plated on laminin expressed a gradual multipolar-to-unipolar transition due to rapid-onset neurites becoming remodelled into the appearance of slow-onset neurites. This phenomenon reflected the selective stabilization of certain rapid-onset neurites, along with the restriction of motility to their distal tips. Based upon the properties and interactions of both rapid- and slow-onset neurites in NG108-15 cells, a unified model for neurite formation is presented.

Altered cell shapes in fibroblasts treated with 5'-deoxy-5'-methylthioadenosine: relation to morphogenesis of neural cells.

In the present study, mouse 3T3 fibroblasts and primary human foreskin fibroblasts exposed to MTA (5'-deoxy-5'-methylthioadenosine) were found to achieve neural-like cell shapes and to extend long, multipolar processes rapidly and reversibly. Time lapse recordings and pharmacologic studies revealed that process formation in MTA-treated fibroblasts was mechanistically related to the rapid-onset mode of neurite formation previously characterized in neural hybrid NG108-15 cells. These data, together with evidence presented elsewhere, indicate that MTA selectively reorganizes the mode of expression of a specific cytoplasmic machinery that is active in many types of cells, and which is involved in regulating cell shape and neurite formation in developing neurons.