Distinct Roles of Different Presynaptic and Postsynaptic NCAM Isoforms in Early Motoneuron-Myotube Interactions Required for Functional Synapse Formation.

The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM-/- myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation.SIGNIFICANCE STATEMENT Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease motility are not well understood. Using dynamic imaging of motoneuron-myotube cocultures, we show that NCAM is required on both the growth cone and myotube and that different NCAM isoforms mediate initial adhesion and the downregulation of growth cone motility. The agrin receptor Lrp4 was also essential for initial adhesive contacts and was downregulated on NCAM-/- myotubes. Our identification of novel roles for NCAM and Lrp4 and possible interactions between them in transforming motile growth cones into stable contacts opens interesting new avenues for exploration.

Application of viral vectors to the study of neural connectivities and neural circuits in the marmoset brain.

It is important to study the neural connectivities and functions in primates. For this purpose, it is critical to be able to transfer genes to certain neurons in the primate brain so that we can image the neuronal signals and analyze the function of the transferred gene. Toward this end, our team has been developing gene transfer systems using viral vectors. In this review, we summarize our current achievements as follows. 1) We compared the features of gene transfer using five different AAV serotypes in combination with three different promoters, namely, CMV, mouse CaMKII (CaMKII), and human synapsin 1 (hSyn1), in the marmoset cortex with those in the mouse and macaque cortices. 2) We used target-specific double-infection techniques in combination with TET-ON and TET-OFF using lentiviral retrograde vectors for enhanced visualization of neural connections. 3) We used an AAV-mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex. We also established systems for shRNA mediated gene targeting in a neocortical region where a gene is significantly expressed and for expressing the gene using the CMV promoter for an unexpressed neocortical area in the primate cortex using AAV vectors to understand the regulation of downstream genes. Our findings have demonstrated the feasibility of using viral vector mediated gene transfer systems for the study of primate cortical circuits using the marmoset as an animal model. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 354-372, 2017.

DNA methylation and methyl-binding proteins control differential gene expression in distinct cortical areas of macaque monkey.

Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes both in vitro and in vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex.

Selective targeting of different neural cell adhesion molecule isoforms during motoneuron myotube synapse formation in culture and the switch from an immature to mature form of synaptic vesicle cycling.

Characterization of neuromuscular junction formation and function in mice lacking all neural cell adhesion molecule (NCAM) isoforms or only the 180 isoform demonstrated that the 180 isoform was required at adult synapses to maintain effective transmission with repetitive stimulation whereas the 140 and/or 120 isoform(s) were sufficient to mediate the downregulation of synaptic vesicle cycling along the axon after synapse formation. However, the expression and targeting of each isoform and its relationship to distinct forms of synaptic vesicle cycling before and after synapse formation was previously unknown. By transfecting chick motoneurons with fluorescently tagged mouse 180, 140 and 120 isoforms, we show that before myotube contact the 180 and 140 isoforms are expressed in distinct puncta along the axon which are sites of an immature form (Brefeldin A sensitive, L-type Ca2+ channel mediated) of vesicle cycling. After myotube contact the 140 and 180 isoforms are downregulated from the axon and selectively targeted to the presynaptic terminal. This coincided with the downregulation of vesicle cycling along the axon and the expression of the mature form (BFA insensitive, P/Q type Ca2+ channel mediated) of vesicle cycling at the terminal. The synaptic targeting of exogenously expressed 180 and 140 isoforms also occurred when chick motoneurons contacted +/+ mouse myotubes; however only the 180 but not the 140 isoform was targeted on contact with NCAM-/- myotubes. These observations indicate that postsynaptic NCAM is required for the synaptic targeting of presynaptic 140 NCAM but that the localization of presynaptic 180 NCAM occurs via a different mechanism.

Ciliary neurotrophic factor inhibits differentiation of photoreceptor-like cells in rat pineal glands in vitro.

Ciliary neurotrophic factor (CNTF) is a unique member of the interleukin-6 (IL-6) family, whose receptor subunit for ligand binding is exclusively expressed in the nervous system and muscle. The role of CNTF in mammalian development remains unknown. We recently reported the specific expression of CNTF in the pineal gland and eyes. To further examine the expression pattern and role of CNTF in development, we prepared a polyclonal antibody against rat CNTF, performed western blotting with this antibody, and confirmed a strong and specific expression of the CNTF protein in pineal glands and a moderate expression in the eyes among the various tissues examined in newborn rats. In pineal organ cultures of newborn rats, exogenously added recombinant rat CNTF potently inhibited the differentiation of photoreceptor-like cells in a dose-dependent manner, while CNTF did not influence the survival of pineal cells. Among several cell growth factors known to have a similar effect in retinal cultures examined, strong inhibitory effects were seen only with CNTF and the leukemia inhibitory factor (LIF), both of which belong to the IL-6 cytokine family. This inhibitory effect was the strongest during three to 6 days of culture when CNTF was added to these cultures. These results suggest that CNTF plays an inhibitory role in the development of photoreceptor-like cells in early postnatal rat pineal glands.

Inhibitory effect of BCG cell-wall skeletons (BCG-CWS) emulsified in squalane on tumor growth and metastasis in mice.

The antimetastatic effect of BCG-CWS, which was emulsified in an oil-in-water form with either Drakeol 6VR mineral oil (BCG-CWS/DK) or squalane (BCG-CWS/SQA), on lung metastasis produced by highly metastatic murine tumor cells, Colon26-M3.1 carcinoma cells and B16-BL6 melanoma cells, was investigated in syngeneic mice. An intravenous (i.v.) administration of BCG-CWS (100 mg/mouse) 1 day after tumor inoculation significantly inhibited tumor metastasis of both Colon26-M3.1 carcinoma and B16-BL6 melanoma cells in experimental lung metastasis models. No differences in the antitumor activity of the two oil-based formulations (BCG-CWS/DK and BCG-CWS/SQA) were obverved. However, BCG-CWS/SQA administered through subcutaneous (s.c.) route was shown to be effective only when it was consecutively injected (3 times) after tumor inoculation. An in vivo analysis for tumor-induced angiogenesis showed that a single i.v. administration of BCG-CWS/SQA inhibited the number of tumor-induced blood vessels and suppressed tumor growth. Furthermore, the multiple administration of BCG-CWS/SQA given at on week intervals led to a significant reduction in spontaneous lung metastasis of B16-BL6 melanoma cells in a spontaneous metastasis model. These results suggest that BCG-CWS emulsified with squalane is a potent inhibitory agent of lung metastasis, and that the antimetastatic effect of BCG-CWS is related to the suppression of tumor growth and the inhibition of tumor-induced angiogenesis.

CNTF is specifically expressed in developing rat pineal gland and eyes.

Ciliary neurotrophic factor (CNTF) attracts considerable attention because it supports survival and differentiation of various types of neurons and glial cells in vitro. Although CNTF functions as a moderate neurotrophic factor in mature motor neurons, its role in embryonic development remains unknown. Here, we found a specific CNTF expression in the rat pineal gland and eyes during embryonic development. In vitro, neonatal rat pineal extract including CNTF supported the survival of neonatal sympathetic neurons, which innervate pineal glands immediately after birth.