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.

Reelin function in neural stem cell biology.

In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, alpha3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients.

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.

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.

Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells.

Reelin regulates telencephalic and cerebellar lamination during mammalian development and is expressed in several structures of the adult brain; however, only traces of reelin were believed to be in peripheral tissues. Because reelin structurally resembles extracellular matrix proteins, and because many of these proteins are expressed in blood, we hypothesized that reelin also might be detectable in the circulation. Reelin (420 kDa) and two reelin-like immunoreactive bands (310 and 160 kDa) are expressed in serum and platelet-poor plasma of rats, mice, and humans, but these three bands were not detectable in serum of homozygous reeler (rl/rl) mice. Reelin plasma levels in heterozygous (rl/+) mice were half of those in wild-type littermates. Western blotting and immunocytochemistry using antireelin mAbs indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the rat adrenal medulla and in a subset of cells coexpressing alpha-melanocyte-stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary failed to decrease the amount of reelin (420-kDa band) expressed in serum. Adult liver expressed one-third of the reelin mRNA concentration expressed in adult mouse cerebral cortex. Full-length reelin protein was detectable in liver extracts in situ; acutely isolated liver cells also secreted full-length reelin in vitro. Liver appears to be a prime candidate to produce and maintain the circulating reelin pool. It now becomes relevant to ask whether circulating reelin has a physiologic role on one or more peripheral target tissues.

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.

A decrease of reelin expression as a putative vulnerability factor in schizophrenia.

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.

Using ARROWSMITH: a computer-assisted approach to formulating and assessing scientific hypotheses.

Conventional computer searches of the biomedical literature (e.g. MEDLINE) allow investigators to retrieve much of the information that has already been published on a given topic. However, these searches are of limited utility at the frontier of scientific discovery, when one wishes to identify and assess new, untested scientific hypotheses, or to uncover biologically significant relations between two previously disparate fields of inquiry. We have designed a set of interactive software and database search strategies, collectively called ARROWSMITH, that facilitate the discovery of plausible hypotheses linking findings across specialties (Artif. Intell. 91 (1997) 183-203). In the simplest implementation of ARROWSMITH, the user begins with an experimental finding or hypothesis that two items A and C are related in some way. The titles of papers indexed in MEDLINE which contain the word 'A' (or synonyms) are downloaded into a file A, and similarly a file C is created. The software constructs a list of words and phrases B common to files A and C; automatic and manual editing are used to filter out uninteresting B-terms. For each B-term, the software generates an AB file of titles containing both 'A' and 'B', and a BC file of titles containing both 'B' and 'C'; these titles are juxtaposed to facilitate the user judging whether there is likely to be a biologically significant relation among A, B and C. ARROWSMITH has been employed to analyze research problems relating to oxidative stress, brain damage, Alzheimer's disease and schizophrenia. Applications of ARROWSMITH include: anticipating adverse drug reactions, identifying mechanisms by which agents modulate cellular or organismal responses, suggesting new therapeutic approaches, identifying possible risk factors for diseases, and identifying potential animal models for human conditions. A simplified experimental version of ARROWSMITH is now freely accessible on the World Wide Web (http:@kiwi.uchicago.edu).

The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction.

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.

Structural analysis of sequences O-linked to mannose reveals a novel Lewis X structure in cranin (dystroglycan) purified from sheep brain.

The Lewis X epitope, Galbeta1-4(Fucalpha1-3)GlcNAc-R, has been implicated in cell-cell recognition events in a number of systems including the central nervous system and is expressed on diverse glycoconjugates including cell adhesion molecules, glycolipids, and the proteoglycan phosphacan. Although Lewis X sequences 3-linked to mannose have been described within proteoglycan fractions of mammalian brain, these have not been reported in other contexts and have been widely believed to be peculiar constituents of brain proteoglycans. In the present paper, we confirm the existence of Lewis X structures O-linked to mannose within the mammalian brain, demonstrate that these structures are present on a well defined mucin-like glycoprotein, cranin (dystroglycan), and report studies suggesting that the linkages involved may be predominantly 2-linked to mannose. Mannose-linked Lewis X is the latest in an increasing list of oligosaccharide recognition "tags" that have been shown to be expressed on cranin (dystroglycan) purified from brain.

Conserved amphipathic helices near the N-terminus and C-terminus of the alpha subunit of cranin (dystroglycan).

Cranin (dystroglycan) is a ubiquitously expressed extracellular matrix receptor, synthesized as a single precursor, which is cleaved into an extracellular subunit (alpha) and a transmembrane subunit (beta). The primary sequence of cranin (dystroglycan) is known from cDNA cloning, and the protein has been strongly implicated in morphogenesis, cell adhesion and human disease. Nevertheless, the domain structure of the alpha subunit has not been well studied; although the protein binds to matrix proteins, to the beta subunit, to cell surfaces, and possibly to other membrane proteins such as sarcoglycans, the domains responsible for mediating these interactions remain unknown. Here I report computer analyses that identify two distinctive amphipathic alpha-helical regions near the N-terminus and C-terminus of the alpha subunit, which are conserved in all species for which sequence information is currently available. This finding should stimulate and guide experimental studies designed to understand how the alpha subunit is associated with the cell surface and with its various ligands.

Localization of cranin (dystroglycan) at sites of cell-matrix and cell-cell contact: recruitment to focal adhesions is dependent upon extracellular ligands.

We report that cranin (dystroglycan) can become recruited to focal adhesions of cultured rat REF 52 fibroblasts and human aortic smooth muscle cells. Within mature focal adhesions, cranin was present within the plaque region defined by beta 1 integrin, vinculin and phosphotyrosine staining, but occupied a larger domain corresponding to the terminal segments of stress fibers that was more precisely co-extensive with the cytoskeletal proteins alpha-actinin, utrophin and aciculin. When REF 52 fibroblasts were plated on different substrata in the absence of protein synthesis and secretion in serum-free medium, focal clusters of cranin readily formed within 2 hours on matrix proteins that bind cranin directly (laminin or agrin) which were maintained as the focal adhesions became mature. In contrast, cranin failed to become targeted to cell-substratum attachment sites, either at early or later times, when cells were plated on a variety of other substrata that elicit formation of focal adhesions but do not bind cranin directly (fibronectin, vitronectin, collagen type IV, or anti-beta 1 integrin antibody TS2/16). These data strongly suggest that targeting of cranin to focal adhesions was dependent upon the presence of an extracellular ligand capable of binding cranin directly. However, some cultured nonmuscle cell lines (e.g., human umbilical vein endothelial cells, NIH 3T3 and CHO cells) failed to localize cranin to focal adhesions, even when plated on laminin. Cranin was also enriched at cell-cell adherens-type junctions of human normal breast MCF-10 epithelial cells, and at growth cones of E17 rat hippocampal axons. That cranin can become targeted to sites of cell-cell and cell-substratum contact in diverse cell types supports the hypothesis that cranin may be involved in mediating or regulating cell adhesion. The absence of muscle-specific and synapse-specific proteins within fibroblasts and epithelial cells provides a different context for thinking about cranin (dystroglycan) that may aid in discerning general principles of its structure and function.

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.

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.

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.

Purification of cranin, a laminin binding membrane protein. Identity with dystroglycan and reassessment of its carbohydrate moieties.

Cranin was described in 1987 as a membrane glycoprotein expressed in brain and many other tissues, which binds laminin with high affinity in a calcium-dependent manner. Dystrophin-associated glycoprotein ("dystroglycan") is a laminin-binding protein cloned in 1992 whose relation to cranin has remained uncertain. Here we describe the purification of cranin to homogeneity from sheep brain, show cranin to be a form of dystroglycan, and localize the N terminus of beta-dystroglycan to amino acid residue 654. We find that brain alpha-dystroglycan is tightly associated with membranes, and localizes to regions of synaptic contact as assessed by immunocytochemistry of rat cerebellum. Brain alpha-dystroglycan expresses high mannose/hybrid N-linked saccharides, terminal GalNAc residues, and the HNK-1 epitope. Although dystroglycan has previously been presumed to be a proteoglycan, the amino acid sequence, pI, O-sialoglycoprotease susceptibility, lectin-binding profile, and laminin-binding properties of brain dystroglycan are more typical of mucin-like proteins. Furthermore, using CHO mutant cell lines deficient in xylosyltransferase and galactosyltransferase I, which are required for glycosaminoglycan biosynthesis, it is shown that chondroitin sulfate and heparan sulfate are not critical for laminin binding, and indeed are apparently not expressed at all in dystroglycan from CHO cells.