Neurons and glial cells of the embryonic human brain and spinal cord express multiple and distinct isoforms of laminin

We have identified by immunocytochemistry, Western blotting, and RT-PCR the isoforms of laminin expressed by glial cells and neurons cultured from human embryonic brain and spinal cord. We show that most of the known laminins are present in human neurons and glial cells. Importantly, Western analysis demonstrates that the isoforms of laminin present in embryonic human brain differ from those expressed in human spinal cord. Neurons of the brain and spinal cord also express their distinct and characteristic isoforms of laminin compared to the glial cells of the same CNS regions. These results suggest that, in addition to the known laminins, several novel isoforms may exist in the human embryonic CNS. The observed differences between the isoforms of laminin in brain and spinal cord neurons and glial cells may result from primary structural changes or from posttranslational modifications, e.g., variations in glycosylation. Thus, identification of these novel laminins and determination of their function(s) should further our understanding of the mechanisms of aging, disease, and trauma in the human CNS.     

Fetal rat septal cells adhere to and extend processes on basement membrane, laminin, and a synthetic peptide from the laminin A chain sequence

Responses of rat embryonic septal cells to reconstituted basement membrane, laminin, and laminin A chain-derived synthetic peptides were studied in culture. Dissociated fetal E16/17 septal cells were grown for three days on differently coated plastic substrata. Reconstituted basement membrane (Matrigel), laminin, and a 19-amino acid synthetic peptide CSRARKQAASIKVAVSADR-NH2 (PA22-2) from the laminin A chain sequence mediated cell-substratum adhesion and promoted neurite outgrowth. In contrast, cells did not attach to or form processes on uncoated plastic or on plastic substrata coated with synthetic, laminin-derived control peptides. Polyethylenimine (PEI) supported the adhesion and survival of fetal septal cells; however, when laminin was added to the medium during cell plating or 18 hr afterward, a dose-dependent increase was observed in neurite outgrowth of cells attached to this substratum. Cells grown for 6 days on PEI in the presence of laminin showed a determined increase in the number of cholinergic neurons as marked by acetylcholinesterase staining. These data suggest that the subpopulation of cholinergic septal neurons present in the septal cells studied here were also responding to laminin. The results of this in vitro study suggest potential uses for basement membrane, laminin, or synthetic peptides, such as PA22-2, in fetal septal grafts to enhance regeneration in the damaged septo-hippocampal system.     

Short exposure to methylazoxymethanol causes a long-term inhibition of axonal outgrowth from cultured embryonic rat hippocampal neurons

Methylazoxymethanol (MAM) is an alkylating agent that is used to induce microencephaly by killing mitotically active neuroblasts. We found that at later developmental times, MAM exposure can result in abnormal fiber growth in vivo. However, there have not been any previous studies on the effects of MAM on differentiating neurons. We examined the outcome of short exposure to MAM on postmitotic embryonic hippocampal cultures during the establishment of axonal polarity. At 0, 1, or 2 days in vitro (DIV), neurons were treated with 0.1 nM-1 μM MAM for 3 hr and then transferred to glial conditioned media. At 3 DIV, the cells were fixed and analyzed by immuno-fluorescent staining for neuron viability and differentiation. Control cells initiate several minor processes; one process elongates rapidly at about 1 DIV eventually becoming an axon, while extensive dendritic growth occurs after 3–4 DIV. Neurons treated with 1 μM MAM at 0 or 1 DIV showed a marked inhibition of neurite growth and withdrawal of axons without affecting cell viability. These cells continued to show minimal neurite outgrowth at 7 DIV, even when transferred to a glial coculture. In contrast, cells treated initially with MAM, after neuronal polarity is established at 2 DIV, showed no effect on axonal growth. To determine the effects of MAM on the neuronal cytoskeleton, we examined the in vitro assembly of brain microtubules in a one cycle assay. Exposure to MAM depleted the soluble pool of proteins, including microtubule-associated protein 1B (MAP1B) and MAP2, which are required for neurite outgrowth, through a nonspecific process. Under non-saturating conditions, there were no changes in the total amount of microtubules assembled or the coassembly of MAP1B and MAP2 in the presence of MAM. These results demonstrate that MAM can directly affect differentiating neurons, indicating that an early disruption of axonal outgrowth may have long-term effects. © 1996 Wiley-Liss, Inc.     

Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin.

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab')2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab')2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal-glial interactions.     

Cell surface changes in the developing optic nerve of mice

A recently defined antibody to a cell surface protein, M6, inhibits neurite outgrowth in culture (Lagenaur, Fushiki, and Schachner: Soc. Neurosci. Abstr. 10:739, '84). In the developing mouse, the antibody stains all parts of the primary optic pathway at birth. Over the next week, staining is lost from the proximal segment of the optic nerve and a week later from the more central part of the nerve. By contrast staining persists through adulthood in the optic fiber layer of the retina. This means that single axons in the mature optic nerve express the antigen over only the proximal few millimeters of their course and over their terminal region. The results are discussed in relation to the overall maturation of the optic pathway and to the processes of membrane maturation and myelination.     

Rhotekin modulates differentiation of cultured neural stem cells to neurons

Rhotekin is a downstream signal of Rho and is expressed in the central nervous system. However, the physiological role of rhotekin in the development of neural stem cells (NSCs) into neurons is unknown. In this study, we knocked down the expression of rhotekin protein with small interfering RNA (siRNA) in the NSCs and in neural differentiated cells and measured cell proliferation, differentiation, neurite length, and survival. By using immunocytochemistry and Western blot, the production of rhotekin was observed in NSCs and neuronal cells. Furthermore, rhotekin production was increased in accordance with neural differentiation. Rhotekin knock-down reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity and increased the cell death 72 hr after transfection in neurons. On the other hand, in NSCs, rhotekin knock-down increased MTT activity and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells. In the present study, we demonstrated that rhotekin is required for maintenance and survival of neurons and positively regulates differentiation and neurite outgrowth. Moreover, we found that rhotekin is produced in NSCs and that the role of rhotekin is to regulate cell proliferation negatively. In conclusion, these results suggest that rhotekin is one of the key molecules in the differentiation of NSCs into neurons.     

Laminin and its neurite outgrowth-promoting domain in the brain in Alzheimer's disease and Down's syndrome patients.

Immunocytochemistry, radioimmunoassay, immunoblotting, Northern analysis, and polymerase chain reaction (PCR) technique were applied to investigate the distribution of laminin and its neurite outgrowth domain in brains of neuropathologically verified cases of Alzheimer's disease and Down's syndrome. New antibodies against a neurite outgrowth domain of laminin were characterized and were used in localization of this peptide antigen in the human brain. Laminin was found as large punctate deposits in all plaques in the affected brains. Laminin synthesis was increased as assessed by RNA blotting and immunoblotting, and glial cells were heavily immunoreactive with antibodies for a neurite outgrowth-promoting peptide antigen of the B2 chain of laminin. This peptide antigen not only was produced by glial cells but also was deposited in the brain tissue. As this peptide antigen promotes neurite outgrowth at low concentrations, and is specifically neurotoxic at high concentrations, it may play a synergistic role with other molecules in inducing the sprouting and neurodegeneration occurring in brains of patients with either Alzheimer's disease or Down's syndrome.     

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett.308, 179–182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using ¹²⁵I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.     

Fibronectin and laminin elicit differential behaviors from SH-SY5Y growth cones contacting inhibitory chondroitin sulfate proteoglycans.

Neuronal growth cones integrate signals from outgrowth-promoting molecules, e.g., laminin (LN) or fibronectin (FN), and outgrowth-inhibiting molecules, e.g., chondroitin sulfate proteoglycans (CSPGs), to navigate through extracellular matrix (ECM). Sensory neurons on LN typically turn to avoid areas rich in inhibitory CSPGs, whereas neuron-like cells of human origin (SH-SY5Y) preferentially stop/stall. These different behaviors may reflect differences in neuron type, response to outgrowth-promoters, or the mechanisms involved in outgrowth vs. inhibition. We used image analysis to determine the effects of different outgrowth promoters on the response of SH-SY5Y cells to inhibitory CSPGs. LN increased neurite initiation and elongation compared to cells plated either on endogenous matrix or FN. On a patterned substratum consisting of alternating stripes of FN and CSPGs, 59.6 +/- 9.3% of SH-SY5Y growth cones turned upon CSPG contact, whereas only 31.9 +/- 8.2% of growth cones turned at a LN/CSPG border. Growth cones on LN spread more upon contact with CSPG than growth cones on FN, whereas growth cones on LN or FN not contacting CSPGs were morphologically similar. Because it is known that integrins are involved in outgrowth on promoters, we analyzed integrin expression in response to inhibitory CSPGs in a choice assay. CSPGs did not induce increases or redistribution of several integrin subunits in SH-SY5Y cells. Furthermore, an anti-beta1 integrin function-blocking antibody did not alter growth cone behavior at a CSPG border. These results indicate that significant mechanistic differences may exist between outgrowth on homogenous outgrowth promoters and growth cone turning at inhibitory molecules.     

Preferential adhesion of brain astrocytes to laminin and central neurites to astrocytes

Astrocytes from either fetal or newborn rat brain adhered preferentially to surfaces coated with active laminin. Neurites from septal neurons did not show a preference for active over inactive laminin. However, when given a choice between laminin and an astrocytic surface, septal cells preferred to extend neurities over astroglial processes. Hence, laminin paths can guide astrocyte migration, which, in turn, can guide neurite elongation.     

Ethanol-exposed central neurons fail to migrate and undergo apoptosis

Prenatal exposure of human brain to ethanol impairs neuronal migration and differentiation and causes mental retardation. The present results indicate that the adverse effects of ethanol on brain development may be partly due to the ethanol-induced disturbance of neuronal interaction with laminin, a protein involved in neuronal migration and axon guidance. This report shows that physiological concentrations (IC₅₀ = 28 mM) of ethanol inhibit neurite outgrowth and neuronal migration of the rat cerebellar granule neurons on a laminin substratum. The ethanol-treated granule neurons undergo apoptosis, degrade their laminin substratum, and appear to release and bind increased amounts of the B2-chain-derived peptides along their surfaces. A protease inhibitor aprotinin, and the NMDA receptor channel, and voltage-gated calcium channel antagonist MK801 partially protect cerebellar granule neurons from ethanol-induced neurotoxicity. These results imply that ethanol-treated granule neurons resemble the granule neurons of the homozygous weaver mouse cerebellum with respect to their apoptosis, laminin expression, and partial rescue by approtinin and MK-801. Thus, ethanol may influence neuronal survival and neurite outgrowth via molecular pathways similar to those involved in neuronal death in other neurodegenerative processes of the central nervous system. J. Neurosci. Res. 48:439–448, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of human neural precursor cells by laminin and integrins

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.     

Gamma 1 laminin and its biologically active KDI-domain may guide axons in the floor plate of human embryonic spinal cord

Immunocytochemistry, in situ hybridization and Matrigel-embedded cultures were used to investigate the distribution of laminins during development of the human embryonic spinal cord (7-11 weeks). Our results indicate that alpha 1, beta 1, beta 3 and gamma 1 laminins localize as punctate deposits in the floor plate region in association with commissural fibers crossing the ventral midline. In addition, the neurite outgrowth domain of gamma 1 laminin accumulates heavily in the floor plate region, in the notochord and in GFAP-immunoreactive glial fibers of the embryonic spinal cord. In culture experiments, the biologically active KDI-domain of gamma 1 laminin selectively attracted directional outgrowth of neurites from explants of the dorsal spinal cord. The spatial and temporal colocalization of punctate deposits of laminins with nerve fibers crossing the ventral midline, and the guidance of neurites by the KDI-peptide domain, indicate that laminins, specifically the gamma 1 laminin, may be involved in guidance of axons during embryonic development of the human spinal cord.     

Effects of Cerebrolysin on the outgrowth and protection of processes of cultured brain neurons

Summary. Cerebrolysin (Cere, EBEWE Arzneimittel, Austria), a peptidergic drug produced by a standardised enzymatic breakdown of porcine brain proteins, consists of a mixture of 75% free amino acids and 25% low molecular weight peptides (<10 k DA). Cerebrolysin was shown to protect against MAP2 loss in primary embryonic chick neuronal cultures after brief histotoxic hypoxia and in a rat model of acute brain ischemia. Since MAP2 is involved in processes like neuronal growth, plasticity and dendritic branching, we address the question whether Cere is protecting processes against degeneration in a chronic low serum (2% FCS) cell stress model and whether the spontaneous outgrowth of axon-like processes is influenced. This was accomplished by quantification of the neurite lengths of embryonic chicken telencephalon neurons after 4 and 8 days. Additionally, time-laps video microscopy was performed to study a possible influence of Cere on the growth cone behaviour of axon-like processes. To distinguish between effects caused by the peptide fraction and the effects related to free amino acids, we used an artificial amino acid solution (AA-mix). Results demonstrate a process outgrowth promoting effect of the AA-mix and Cere after 4 DIV. After 8 days neuronal network degeneration occurred in the AA-mix treated cultures, whereas Cere treated cultures still presented a well differentiated neuronal network. Dying neurons could release factors possibly impeding neurite outgrowth and Cere was shown to increase the viability of chicken cortical neurons. Neither the addition of BDNF nor serum supplementation (5% and 10% FCS) could protect the neuronal network against degeneration after 8 DIV, although these treatments were shown to ameliorate the viability of chicken telencephalon neurons. This result together with the finding obtained using the artificial amino acid solution points to the peptide fraction of Cere to be responsible for the protection of processes against degeneration. Time-laps studies of Cere treated cultures revealed a significant decrease of the velocities characterising random growth cone movements, which is thought to be responsible for an increase in the length of axon-like processes after 4 DIV. Received September 21, 2000; accepted November 22, 2000     

Secretion of amyloid precursor protein and laminin by cultured astrocytes is influenced by culture conditions

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as “astrogliosis.” Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neuritepromoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and longterm confluent (“aged”) neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, “Lesioned” (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite-promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite-promoting factors(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz-type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these protiens, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP-and laminin-enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti-APP and antilaminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non-conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM-induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ. © 1994 Wiley-Liss, Inc.     

Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells

Mn²⁺ has been shown to promote cell–substrate adhesion and cell spreading in many cell culture systems. In this study, we present data demonstrating that Mn²⁺ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells. In the presence of 1.0 mM MnCl₂, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes. These morphological effects of Mn²⁺ were both time- and dose-dependent. In the presence of cycloheximide, a protein synthesis inhibitor, both Mn²⁺-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn²⁺ to take place. Of the other divalent cations tested, Mg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, and Zn²⁺ were ineffective, and only Co²⁺ partially mimicked the effects of Mn²⁺. Although Mn²⁺-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth. The neurite outgrowth-promoting effects of Mn²⁺ were distinct from those of nerve growth factor in that the response to Mn²⁺ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites. Mn²⁺ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells. In cells grown in serum-free defined medium, Mn²⁺ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata. Similarly, Mn²⁺ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only. The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn²⁺ on PC12 cells. These findings are consistent with a hypothesis that Mn²⁺-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects. © 1993 Wiley-Liss, Inc.     

Fibroblast Growth Factor Treatment Produces Differential Effects on Survival and Neurite Outgrowth from Identified Bulbospinal Neurons in Vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

Heparan sulphate and HB-GAM (heparin-binding growth-associated molecule) in the development of the thalamocortical pathway of rat brain

Extracellular matrix (ECM) molecules, such as laminin, tenascin, chondroitin sulphate proteoglycans and heparan sulphate proteoglycans have been suggested to have 'signpost' and directing roles in the formation of axonal projections in cortical development. We show here that the expression of the neurite outgrowth-promoting protein heparin-binding growth-associated molecule (HB-GAM) and N-syndecan, a transmembrane heparan sulphate proteoglycan previously isolated as a receptor for HB-GAM, is spatiotemporally associated with the developing thalamocortical pathway in the rat brain. Using in situ hybridization, thalamic neurons were shown to express mRNA for N-syndecan, and in vitro, thalamic neurons grew more neurites on HB-GAM than on laminin. The HB-GAM-induced neurite outgrowth in thalamic neurons was inhibited by heparitinase, heparin, soluble N-syndecan and by an excess of soluble HB-GAM in the culture medium. In a pathway assay, thalamic neurons selectively preferred attaching and growing neurites on matrices containing HB-GAM than on those containing fibronectin or laminin alone, suggesting that HB-GAM may modulate the effect of other ECM proteins. On an unfixed brain slice preparation, thalamic neurons repeatedly showed a typical neurite outgrowth and attachment pattern resembling the expression pattern of HB-GAM. On the brain slices, the neurite outgrowth was significantly inhibited by heparitinase, heparin and soluble HB-GAM, thus displaying features of neurite outgrowth on matrix-bound HB-GAM. Our results suggest that HB-GAM is important for the neurite outgrowth of thalamic neurons and it may function as an ECM-bound guidance cue for thalamic neurons that possess HB-GAM-binding heparan sulphates on their cell membrane.