The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin.

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.

Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons.

Sympathetic neurons from perinatal rat pups extend only a single axon when maintained in culture in the absence of glia and serum. Exposure to recombinant osteogenic protein-1 (OP-1) selectively induces the formation of dendrites that correctly segregate and modify cytoskeletal and membrane proteins and form synaptic contacts of appropriate polarity. OP-1 requires nerve growth factor (NGF) as a cofactor, and, in the presence of optimal concentrations of NGF, OP-1-induced dendritic growth from cultured perinatal neurons is comparable to that observed in situ. Sympathetic neuroblasts that had not formed dendrites in situ also responded to OP-1 in culture, indicating that OP-1 can cause de novo formation as well as regeneration of dendrites. These data imply that specific signals can regulate the development of neuronal shape and polarity.

Distinct spatial localization of specific mRNAs in cultured sympathetic neurons.

We examined the subcellular distribution of specific mRNAs in cultured sympathetic neurons. Under appropriate conditions, sympathetic neurons extend both axons and dendrites that are distinguishable by light microscopic and immunocytochemical criteria. In situ hybridization revealed a differential localization of mRNA within dendrites. mRNA encoding MAP2 was abundant in cell bodies and distributed nonhomogeneously throughout the dendritic compartment, but was not detected in axons. In contrast, mRNAs encoding GAP-43 and alpha-tubulin were restricted to the cell body and largely excluded from dendrites as well as axons. Detergent extraction revealed that most dendrite-associated mRNA encoding MAP2 was associated with the Triton X-100 insoluble fraction of the cell. The subset of mRNAs present in the dendritic compartment may encode proteins involved in the morphogenesis and remodeling of dendrites.

Mechanisms of neuronal polarity.

The mechanisms that permit neurons to establish axons and dendrites involve an interplay between a cell's genetic program and signals in its environment. Recent experiments have identified some of the important extracellular molecules that regulate dendritic development and have furthered our understanding of the endogenous cell biological mechanisms that underlie protein sorting. Some of the signaling pathways that allow extracellular cues to regulate neuronal morphogenesis are also being elucidated.

Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons.

BACKGROUND: BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons. RESULTS: Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG) during times of initial growth and rapid expansion of the dendritic arbor. CONCLUSIONS: These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

Effect of leukemia inhibitory factor (LIF) on the morphology and survival of cultured hippocampal neurons and glial cells.

Leukemia inhibitory factor (LIF) is a cytokine involved in the survival, differentiation and regeneration of sympathetic, sensory and motor neurons. Its effects in the brain are less well characterized. In a previous study, we found LIF transcripts to be predominantly expressed in neurons of the adult rat brain. Highest levels were observed in the hippocampus, particularly in granular neurons of the dentate gyrus and in hilar interneurons. Here we report the effects of LIF on survival and differentiation of postnatal rat hippocampal cells in vitro. We find that LIF minimally influences the survival and differentiation of dentate gyrus neurons, causing a slight reduction of the number of dendrites per neuron. In contrast, LIF induces a pronounced increase in the number of astrocytes. This increase does not appear to be due to enhanced proliferation but rather to increased cell survival. On the other hand, epidermal growth factor (EGF) induces astrocyte proliferation, and addition of LIF inhibits the EGF effect. In summary, LIF does not appear to be crucial for the survival or differentiation of cultured dentate gyrus neurons. This cytokine increases astrocyte survival but does not enhance astrocyte proliferation, and LIF is able to counteract the growth stimulation elicited by EGF.

Manganese induces neurite outgrowth in PC12 cells via upregulation of alpha(v) integrins.

Previous studies have demonstrated that the divalent cation manganese (Mn) causes PC12 cells to form neurites in the absence of NGF. Since divalent cations modulate the binding affinity and specificity of integrins, and integrin function affects neurite outgrowth, we tested the hypothesis that Mn induces neurite outgrowth through an integrin-dependent signaling pathway. Our studies support this hypothesis. Function-blocking antisera specific for beta(1) integrins block the neurite-promoting activity of Mn by 90-95%. Bioassays and biochemical studies with antisera specific for the alpha(v), alpha(5), or alpha(8) integrin subunit suggest that the alpha(v)beta(1) heterodimer is one of the principal beta(1) integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express alpha(v) at levels detectable by immunoprecipitation or immunocytochemistry. SDS-PAGE analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells, as well as confocal laser microscopy of PC12 immunostained for surface alpha(v) indicate that Mn increases the surface expression of alpha(v) integrins. This increase appears to be due in part to synthesis of alpha(v) since specific inhibitors of RNA and protein synthesis block the neurite-promoting activity of Mn. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating alpha(v) integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.

The effects of extracellular matrix and osteogenic protein-1 on the morphological differentiation of rat sympathetic neurons.

The growth patterns of axons and dendrites differ with respect to their number, length, branching, and spatial orientation; therefore, it is likely that these processes differ in their growth requirements. To examine this hypothesis, we have been analyzing the responses of cultured rat sympathetic neurons to three types of stimuli: large structural proteins of the extracellular matrix, matrix-associated growth factors, and neurotrophins. Purified structural proteins such as laminin and collagen IV have been found to promote only axonal growth; whereas the matrix associated growth factor, osteogenic protein-1, selectively stimulates dendritic growth. In contrast, nerve growth factor modulates the growth of both types of processes. These data suggest that process-specific interactions with the extracellular environment may be critical determinants of cell shape in neurons. Perinatal rat sympathetic neurons grown in culture in the absence of serum or glial cells extend a single process which is axonal in nature. Exposure to osteogenic protein-1 causes the formation of additional processes which express the morphological, cytoskeletal, and ultrastructural characteristics of dendrites. Consistent with observations on the regulation of dendritic growth in sympathetic neurons in situ, the dendrite-promoting activity of osteogenic protein-1 is independent of synaptic or electrical activity, but is modulated by nerve growth factor. In the presence of optimal concentrations of osteogenic protein-1 and nerve growth factor, the size of the dendritic arbor extended by cultured sympathetic neurons approximates that seen in situ at comparable developmental stages. Osteogenic protein-1 does not promote dendritic growth in cultured neurons obtained from embryonic ciliary, dorsal root, trigeminal or nodose ganglia, suggesting that its morphogenetic effects are cell selective. Since mRNA for osteogenic protein-1 is expressed in mature as well as embryonic target tissues of the sympathetic nervous system, we also examined the effects of osteogenic protein-1 on cultures of sympathetic neurons derived from adult rats. Consistent with results obtained with perinatal neurons, osteogenic protein-1 selectively promoted dendritic growth in adult neurons. These data suggest that this matrix-associated growth factor could play a role not only in the morphogenesis of the developing nervous system, but also in the maintenance and remodeling of dendritic structures in the mature animal.

Protein synthesis is required for the initiation of dendritic growth in embryonic rat sympathetic neurons in vitro.

We have utilized an experimental paradigm which allows the manipulation of dendritic growth in sympathetic neurons in culture to examine the effects of inhibitors of protein synthesis and RNA synthesis on the development of dendrites. Embryonic rat sympathetic neurons extend only axons when they are grown in serum-free medium on a polylysine substrate. The addition of an extract of basement membrane proteins (BME) to this culture system elicits dendritic growth within 48 h. Both cycloheximide and actinomycin-D inhibited BME-induced dendritic growth in greater than 80% of the neuronal population and reduced the number of dendrites extended by greater than or equal to 97%. In contrast, cycloheximide was found to have minimal effects on axonal growth in short-term (less than or equal to 18 h) cultures as measured with respect to the percentage of the population with axons and the number of axons per neuron. However, this inhibitor did significantly reduce (84%) the length of the axonal plexus extended. These results indicate that dendritic and axonal growth in sympathetic neurons are differentially dependent on protein synthesis such that the formation of dendrites requires protein synthesis whereas the initiation, but not the elongation, of axons is relatively independent of protein synthesis.

Laminin selectively enhances axonal growth and accelerates the development of polarity by hippocampal neurons in culture.

We have examined the effects of laminin on the morphological development of embryonic rat hippocampal neurons maintained in tissue culture. Forty-eight hours after plating, neurons grown on a polylysine-coated substrate had become polarized, typically having one long axon and 4 or 5 minor processes. Adsorption of laminin to the substrate did not cause changes in the number of axons extended by hippocampal neurons but did cause significant increases in the length of the axonal plexus and in axonal branching. In contrast to its effects on axons, laminin did not influence the number, length, or branching of the minor processes that eventually become dendrites or the morphology of definite dendrites as assessed after 7 days in culture. In addition to selectively enhancing axonal growth, laminin greatly increased the rate of polarization of hippocampal neurons such that most became polarized within 18 h. Analysis of the time course of laminin's effects revealed that the acceleration of polarization was not associated with a change in the time of initial process formation, but rather with a selective stimulation of the growth of the longest process at all times from the 12th through the 48th h in vitro. These data suggest that even though the basic shape of hippocampal neurons may be intrinsically programmed, critical aspects of their morphological development may be modulated by extracellular matrix molecules such as laminin.

Leukemia inhibitory factor and ciliary neurotrophic factor regulate dendritic growth in cultures of rat sympathetic neurons.

Cytokines such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neurotransmitter and neuropeptide synthesis in sympathetic neurons [P.H. Patterson, Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology, Proc. Natl. Acad. Sci. USA 91 (1994) 7833-7835]. We considered the possibility that these agents may also affect the development of neuronal cell shape. Intracellular dye injection and immunocytochemistry were used to assess dendritic growth in cultures of perinatal rat sympathetic neurons and the effects of LIF and CNTF were compared to those of osteogenic protein-1 (OP-1), a growth factor that induces profuse dendritic growth in these neurons [P. Lein, M. Johnson, X. Guo, D. Rueger, D. Higgins, Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons, Neuron 15 (1995) 597-605]. Under control conditions, sympathetic neurons formed only axons. Exposure to either LIF or OP-1 stimulated dendritic growth, but the magnitude of the response to LIF was much less than that obtained with OP-1 with respect to both dendritic number and length. Simultaneous exposure to LIF and OP-1 resulted in dendritic growth equivalent to that observed in the presence of LIF alone, suggesting that LIF inhibits the response of neurons to OP-1. Both the stimulatory and inhibitory effects of LIF were mimicked by CNTF, but not by other growth factors. These data suggest that LIF and CNTF regulate dendritic development in a complex manner that is dependent on both the morphological state of the neuron and the presence of other growth factors. However, the net effect of exposure to these cytokines appears to be the production of a population of neurons with rudimentary arbors consisting of only one or two short dendrites.

Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro.

We have characterized the effects of laminin and a basement membrane extract (BME) on the morphology of embryonic rat sympathetic neurons maintained in tissue culture in the absence of nonneuronal cells. Neurons were grown on polylysine-coated coverslips in the presence or absence of laminin or BME in serum-free medium. Axons were distinguished from dendrites using intracellular dye injections, immunocytochemistry, and [3H]uridine autoradiography. In short-term (less than or equal to 24 hr) culture, laminin had a potent neurite-promoting effect, causing increases in the number of processes, total neuritic length, and neuritic branching. In long-term (3-35 days) cultures chronically exposed to laminin, most (greater than 75%) neurons maintained supernumerary axons but failed to form dendrites. In contrast, most neurons (greater than 70%) grown in long-term culture on polylysine in the absence of laminin were unipolar, extending a single axon. BME caused sympathetic neurons to extend multiple (range, 1-15) dendrites. Morphometric measurements made after 1 month of exposure to BME indicated that the amount of dendritic growth that occurred in vitro was similar to that normally occurring during a comparable period in situ. BME did not cause changes in the number of axons per neuron or in the uptake of neurotransmitter. Preliminary characterization of the dendrite-promoting activity of BME suggests that it resides in extracellular matrix (ECM) molecules and not in low-molecular weight contaminants. These observations indicate that (1) axonal and dendritic growth may be differentially regulated by various constituents of the ECM, and (2) such process-specific interactions can significantly affect the morphological development of sympathetic neurons.

Leukemia inhibitory factor and ciliary neurotrophic factor cause dendritic retraction in cultured rat sympathetic neurons.

Dendritic retraction occurs in many regions of the developing brain and also after neural injury. However, the molecules that regulate this important regressive process remain largely unknown. Our data indicate that leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) cause sympathetic neurons to retract their dendrites in vitro, ultimately leading to an approximately 80% reduction in the size of the arbor. The dendritic retraction induced by LIF exhibited substantial specificity because it was not accompanied by changes in cell number, in the rate of axonal growth, or in the expression of axonal cytoskeletal elements. An antibody to gp130 blocked the effects of LIF and CNTF, and both cytokines induced phosphorylation and nuclear translocation of stat3. Moreover, addition of soluble interleukin-6 (IL-6) receptor to the medium endowed IL-6 with the ability to cause dendritic regression. These data indicate that ligands activating the gp130 pathway have the ability to profoundly alter neuronal cell shape and polarity by selectively causing the retraction of dendrites.

Effect of short-term exposure to hexachlorophene on rat brain cell specific marker enzymes.

Seven cell specific marker enzymes in brain and optic nerve and morphological evaluation by light microscopy were used to characterize the neurotoxicity associated with exposure of rats to hexachlorophene (HCP; 40 mg/kg/day, po, for 9 days). In vitro exposure to HCP at concentrations up to 100 microM had no direct inhibitory effect on the marker enzymes, validating their use in evaluating brain function in vivo. Rats exhibited a reduction in body weight gain, weakness, and ataxia of the hind limbs by the ninth day of HCP exposure. At 24 hr following the last day of exposure to HCP, the activities of the three neuron specific enzymes, glutamic acid decarboxylase, tyrosine hydroxylase, and choline acetyltransferase, in rat brain were unchanged from those of the vehicle-treated control group. Of the two astroglial enzyme markers measured, a small but significant increase was observed in the activity of nonneuronal enolase in the cerebellum and glutamine synthetase in the hippocampus of HCP-treated rats. The optic nerve appeared to be the most sensitive tissue in that the activity of both the astroglial marker, nonneuronal enolase, and the myelin marker, 2',3'-cyclic nucleotide phosphohydrolase, was significantly decreased following HCP exposure. This decrease in enzyme activity is consistent with the histological observations demonstrating extensive vacuolization and edema in the optic nerve after exposure to HCP.

Dendritic growth induced by BMP-7 requires Smad1 and proteasome activity.

Bone morphogenetic proteins (BMPs) induce dendritic growth in cultured sympathetic neurons; however, the signaling pathways that mediate this dendrite-promoting activity have not been previously characterized. Here we report studies of the signaling events that regulate the growth of these afferent processes. We find that Smad1 is expressed in sympathetic neurons and that BMPs rapidly induce its phosphorylation and translocation from the cytoplasm to the nucleus. Furthermore, a dominant negative form of Smad1 inhibits BMP-7-induced dendritic growth, suggesting a requirement for Smad1 activation in this biological activity of BMP-7. A physical interaction between Smad1 and components involved in the proteasome-mediated degradation system was detected with a yeast two-hybrid screen, thereby prompting an examination of the effects of proteasome inhibitors on dendritic growth. Lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth without adversely affecting either cell viability or axonal growth. Moreover, studies of transfected P19 cells suggest that the proteasome inhibitors directly block the effects of Smad1 on the transcriptional activity of the Tlx-2 promoter. These data indicate that BMP-induced dendritic growth requires Smad1 activation and involves proteasome-mediated degradation events.

Cerebrospinal fluid contains biologically active bone morphogenetic protein-7.

Bone morphogenetic proteins (BMPs) regulate the development and function of many types of neurons. However, little is known of the actual concentrations of BMPs in the various parts of the brain. In this study, we considered the possibility that BMPs might be present in cerebrospinal fluid (CSF). Western blot analysis of normal adult bovine CSF revealed the presence of dimeric and monomeric forms of BMP-7, and the concentration of this molecule was found to be approximately 12 ng/ml in a radioimmunoassay. Since BMP-7 is known to induce dendritic growth in rat sympathetic neurons, this was used as a bioassay to examine the biological activity of the BMP-7 present in CSF. Addition of normal bovine CSF to cultures of sympathetic neurons produced a dose-dependent increase in dendritic growth and the magnitude of this response approximated that obtained with maximally effective concentrations of exogenous BMP-7. Moreover, CSF-induced dendritic growth was inhibited by follistatin, a protein that can sequester BMPs, and by either of two monoclonal antibodies that react with BMP-7. These results show that, unlike most other neurotrophic factors, BMP-7 is a constituent of normal CSF and is present at concentrations sufficient to elicit a near maximal biological response.