Dendrite-associated cell adhesion molecule, telencephalin, promotes neurite outgrowth in mouse embryo

Neuroscience Letters 240 (1998) 163–166

Dendrite-associated cell adhesion molecule, telencephalin, promotes neurite outgrowth in mouse embryo Atsushi Tamada a , b, Yoshihiro Yoshihara a , b , c, Kensaku Mori a , b ,* a

Laboratory for Neuronal Recognition Molecules, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-01, Japan b Department of Neuroscience, Osaka Bioscience Institute, Suita, Osaka 565, Japan c Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569, Japan Received 30 October 1997; received in revised form 11 December 1997; accepted 11 December 1997

Abstract Telencephalin (TLCN) is a dendrite-associated cell adhesion molecule expressed by neurons within the telencephalon. It belongs to the intercellular adhesion molecule subgroup of the immunoglobulin superfamily. To examine a neurite outgrowthpromoting activity, neurons dissociated from mouse embryos were cultured on the substrate of recombinant mouse TLCN protein. Hippocampal neurons extended multiple neurites on TLCN. The neurite outgrowth on TLCN was suppressed by an anti-TLCN antibody. Non-telencephalic neurons also extended neurites on TLCN. These results demonstrate a neurite outgrowth-promoting activity of TLCN and suggest that both telencephalic and non-telencephalic neurons express TLCN counterreceptor(s) which is coupled to the neurite outgrowth.  1998 Elsevier Science Ireland Ltd.

Keywords: Telencephalon; Cell adhesion molecule; Dendrite-associated cell adhesion molecules; Immunoglobulin superfamily; Intercellular adhesion molecules; Hippocampus; Mouse

Cell adhesion molecules expressed on the surface of neurons are involved in many aspects of neuron-neuron interactions that play critical roles in the development, maintenance and reorganization of neuronal circuits [4, 15]. Since neurons are polarized, extending an axon and dendrites from the soma [3], cell adhesion molecules can be classified by their subcellular localization into axon-associated cell adhesion molecules (A×CAMs), dendrite-associated cell adhesion molecules (DenCAMs), and cell adhesion molecules with distribution on both axons and dendrites [12,18,20]. While a large number of AxCAMs have been reported [2,11,12,14,19], studies on DenCAMs are limited to one molecule, telencephalin (TLCN) [7– 10,16,20]. TLCN is a type I integral membrane glycoprotein that belongs to the immunoglobulin (Ig) superfamily [2,12,19] with nine Ig-like domains in its extracellular region * Corresponding author. Tel.: +81 48 4671390; fax: +81 48 4672539; e-mail: [email protected]

[8,10,20]. Structure of TLCN is closely related to the intercellular adhesion molecules (ICAMs) [20], which play important roles in the immune system [13]. TLCN is expressed by subsets of neurons within the telencephalon, the most rostral segment of the brain [8–10]. TLCN is localized to soma-dendritic membranes, but not to axonal membranes [8,9]. Although TLCN has recently been shown to bind to lymphocyte function-associated antigen-1 (LFA-1) [7,16], a member of the integrin family [5] expressed on leukocytes and microglia [1], a neuronal counter-receptor(s) for TLCN and functional roles for TLCN in neuron-neuron interactions remain unknown. In the present study, we investigated whether or not TLCN has a neurite outgrowth-promoting activity to cultured embryonic neurons. Recombinant fusion proteins composed of the extracellular region of mouse TLCN or ICAM-1 and the Fc region of human IgG1 (TLCN/Fc or ICAM-1/Fc) were prepared as previously described [17]. An antibody against TLCN/Fc was raised by immunization of a hamster with TLCN/Fc. Purified mouse TLCN/Fc (25, 50, 100 mg/ml), mouse

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ICAM-1/Fc (100 mg/ml), human IgG (100 mg/ml; Cappel), mouse laminin (50 and 100 mg/ml; Koken) or poly-L-lysine (25 mg/ml; Sigma) in a volume of 50 ml were adsorbed onto nitrocellulose-coated 96-well culture plates [6]. The nitrocellulose was then blocked with 200 ml of bovine serum albumin (BSA; 10 mg/ml; Sigma). For antibody blockade experiments, TLCN/Fc- and laminin-coated wells were incubated with 50 ml of the anti-TLCN/Fc antibody (250

mg/ml) overnight at 4°C, and then added with 50 ml of cell suspension at two times the final density. The hippocampal region from Balb/c mice at embryonic day (E) 16 was used in all neurite outgrowth assays except those for checking the regional specificity. Pregnant mice were deeply anesthesized with sodium pentobarbital. Embryos were then removed, and brain was dissected out in cold Hanks’ solution. Cells were dissociated with 0.25%

Fig. 1. Neurite outgrowth of dissociated E16 hippocampal neurons on different substrates after 24 h in culture. (A–F) Photomicrographs of cultured neurons on plates coated with TLCN/Fc at 50 (A) and 100 mg/ml (B), IgG at 100 mg/ml (C), ICAM-1/Fc at 100 mg/ml (D), TLCN/Fc at 50 mg/ml incubated with the anti-TLCN/Fc antibody (E) and laminin at 50 mg/ml (F). Neurons were stained with anti-HNK-1 antibody. (G) A graph showing the mean length of neurites of hippocampal neurons. The lengths on TLCN/Fc were significantly larger than those on BSA and IgG (Mann–Whitney test, P , 0.0001), and increased in order of protein concentration (P , 0.0001). The length on ICAM-1/Fc was slightly larger than those on BSA (P , 0.05) and IgG (P , 0.01). Incubation with the anti-TLCN/Fc antibody caused a reduction of the neurite length on TLCN/ Fc (P , 0.0001), but did not change the length on laminin. Error bars indicate SEM. (H,I) Graphs showing the percentage of neurons (ordinate) with neurites longer than a given length (abscissa).

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trypsin, plated on the substrates at a density of 1 × 103 cells/ well, grown in 100 ml of Dulbecco’s modified Eagle’s medium (Nissui) and fixed after 24 h by addition of an equal volume of 6% formaldehyde. Neurons were stained with anti-HNK-1 monoclonal antibody (ATCC TIB200 cells), which labels the surface glycoconjugates of neuronal cell bodies and neurites, incubated with alkaline phosphataseconjugated anti-mouse IgG (Promega), and visualized by a chromogenic reaction with nitroblue tetrazolium and 5bromo-4-chloro-3-indolyl phosphate. Length of neurites was measured as the distance between the soma and the tip of its longest neurite. When a neurite interconnects with another one, it was excluded from the analysis. When length was less than the diameter of cell body, it was considered as zero. For quantification of morphology of neurites, the number of processes emerging directly from the soma on TLCN/Fc and laminin substrates was counted. Similar procedures were used for checking the regional specificity except in the following points. Cells from the hippocampus, cerebral neocortex, thalamus and cerebellum at E16 were plated at the density of 3 × 103 cells/well. Length of neurites was measured from phase-contrast photomicrographs taken from three randomly sampled fields of the plates. We first tested whether TLCN has a neurite outgrowthpromoting activity, using a culture of hippocampal cells dissociated from E16 mouse. After 24 h in culture, many neurons extended neurites on the plates coated with recombinant TLCN/Fc chimeric protein which was composed of the extracellular region of mouse TLCN and the Fc region of human IgG1 (Fig. 1A,B,G,H). In contrast, only a few neurons extended neurites on the control plates without TLCN/Fc (Fig. 1G). The length of neurite showed non-linear dose-dependent relationship with the concentration of TLCN/Fc (Fig. 1G,H). Since human IgG did not show any significant neurite outgrowth-promoting activity (Fig. 1C,G,H), the neurite outgrowth on TLCN/Fc appears to be caused by the extracellular region of TLCN, but not by the Fc region of IgG. Although ICAM-1 has a structure closely related to TLCN, only a negligible neurite outgrowth was found on ICAM-1/Fc even at the highest concentration (100 mg/ml) (Fig. 1D,G,H). Neurite outgrowth was greatly reduced when TLCN/Fc substrates were blocked by the incubation with the antiTLCN antibody (Fig. 1E,G,I). On the other hand, neurite

outgrowth on laminin substrates (Fig. 1F,G,I) was not significantly changed by the incubation with the same antibody (Fig. 1G,I). These results confirm that TLCN has a neurite outgrowth-promoting activity. Next, TLCN-mediated neurite outgrowth was tested for neurons of several brain regions of E16 mouse: hippocampus and cerebral neocortex from the telencephalon, and thalamus and cerebellum from non-telencephalic regions. Table 1 shows the mean length of neurites on TLCN/Fc, IgG, poly-L-lysine and laminin. In all regions tested, neurites on TLCN/Fc were significantly longer than those on IgG. These results indicate that TLCN enhances a neurite outgrowth of not only telencephalic neurons but also nontelencephalic neurons. Finally, we compared the morphology of hippocampal neurons cultured for 24 h on TLCN/Fc with that on laminin. While neurons on laminin had compact and round soma, extending one or two straight neurites with constant diameters (Fig. 1F), those on TLCN/Fc had large and flattened soma, extending multiple curved and tapered neurites with many bifurcations (Fig. 1A,B). The number of processes emerging from the soma on TLCN/Fc was larger than that on laminin (Fig. 2), and neurites on TLCN/Fc showed dendrite-like morphology. This study demonstrates that dissociated embryonic neurons extend neurites on TLCN/Fc, but not on IgG, showing that TLCN has a neurite outgrowth-promoting activity to these neurons. Although ICAM-1 has a structure closely related to TLCN [20], the neurite outgrowth-promoting activity of ICAM-1 was negligible when compared with that of TLCN (Fig. 1G,H). The present findings suggest the existence of a counterreceptor(s) for TLCN in neurons. The counter-receptor(s) on the neuronal surface may interact with TLCN, but not with ICAM-1, and mediate signals that lead to the promotion of neurite outgrowth. Since TLCN does not show homophilic binding activity [20], heterophilic interactions with a yet unknown counter-receptor(s) may mediate the neurite outgrowth. Existence of heterophilic interactions is also supported by the finding that non-telencephalic neurons, which do not express TLCN, also received neurite outgrowth-promoting activity by TLCN. A variety of Ig superfamily molecules have been shown to interact heterophilically with other Ig superfamily molecules or members in other superfamilies such as integrin family [2]. TLCN has

Table 1 Neurite outgrowth of various brain neurons Brain region

TLCN/Fc

Hippocampus Neocortex Thalamus Cerebellum

24 59 21 33

± ± ± ±

4 9 3 6

(n (n (n (n

IgG = = = =

72) 61) 82) 68)

0 0 0 0

± ± ± ±

0 0 0 0

(n (n (n (n

= = = =

54) 51) 74) 60)

Poly-L-lysine

Laminin

8 ± 1 (n = 91) 21 ± 4 (n = 77) 1 ± 0 (n = 109) 23 ± 3 (n = 92)

35 ± 8 (n = 46) 3 ± 2 (n = 62) 17 ± 3 (n = 74) 2 ± 1 (n = 76)

Data are the mean ± SEM lengths of neurites (mm). Concentration of protein was 100 mg/ml. Poly-L-lysine was 25 mg/ml. Note that lengths on TLCN/Fc and laminin were different among regions.

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We thank M. Kawasaki for help in preparing recombinant fusion proteins. This work was supported by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan.

Fig. 2. Percent histogram of the number of processes emitted directly from the soma of dissociated hippocampal neurons on laminin at 50 mg/ml (n = 229, open columns) and on TLCN/Fc at 50 mg/ml (n = 214, filled columns).

recently been shown to bind with LFA-1 integrin [7,16], which is expressed by leucocytes and microglia [1]. However, TLCN/LFA-1 interaction is not responsible for TLCNmediated neurite outgrowth, because neurons do not express LFA-1 [1]. Thus TLCN presumably interacts with a yet unknown counter-receptor(s) on the neuronal surface. This neuronal TLCN counter-receptor(s) may exist on both telencephalic and non-telencephalic neurons. The present neurite outgrowth assay may provide a good system for searching the unknown counter-receptor(s). TLCN is exclusively expressed on the soma-dendritic membrane of neuronal subsets within the telencephalon [8–10,20]. Such a specific expression pattern suggests a hypothesis that TLCN-mediated neurite outgrowth may play an important role in the formation or reorganization of specific neuronal circuits. Dendrites that express TLCN might interact in vivo with other axons or dendrites that express TLCN counter-receptor(s). For axon/dendrite interactions, TLCN might play roles in the formation or reorganization of axonal projections in the telencephalon, including afferent projections from non-telencephalic regions such as thalamocortical projections. Although the present neurite outgrowth assay could not be performed for a long period enough for a neurite to differentiate into either an axon or a dendrite [3], the present observation that neurons on TLCN had flattened somata and multiple and tapered neurites implies that TLCN mediates dendritic growth. TLCN may be involved in dendrite/ dendrite interactions such as fasciculation of dendrites or control of dendritic elongation of neighboring neurons. TLCN might function in dendro-dendritic synapses in the telencephalon, especially in those between granule cells and mitral cells of the olfactory bulb, where TLCN is expressed by granule cell dendrites [8,9].

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