NOS1AP regulates dendrite patterning of hippocampal neurons through a carboxypeptidase E-mediated pathway.

During neuronal development, neurons form elaborate dendritic arbors that receive signals from axons. Additional studies are needed to elucidate the factors regulating the establishment of dendritic patterns. Our work explored possible roles played by nitric oxide synthase 1 adaptor protein (NOS1AP; also known as C-terminal PDZ ligand of neuronal nitric oxide synthase or CAPON) in dendritic patterning of cultured hippocampal neurons. Here we report that the long isoform of NOS1AP (NOS1AP-L) plays a novel role in regulating dendrite outgrowth and branching. NOS1AP-L decreases dendrite number when overexpressed at any interval between day in vitro (DIV) 0 and DIV 12, and knockdown of NOS1AP-L results in increased dendrite number. In contrast, the short isoform of NOS1AP (NOS1AP-S) decreases dendrite number only when overexpressed during DIV 5-7. Using mutants of NOS1AP-L, we show that neither the PDZ-binding domain nor the PTB domain is necessary for the effects of NOS1AP-L. We have functionally narrowed the region of NOS1AP-L that mediates this effect to the middle amino acids 181-307, a region that is not present in NOS1AP-S. Furthermore, we performed a yeast two-hybrid screen and identified carboxypeptidase E (CPE) as a binding partner for the middle region of NOS1AP-L. Biochemical and cellular studies reveal that CPE mediates the effects of NOS1AP on dendrite morphology. Together, our results suggest that NOS1AP-L plays an important role in the initiation, outgrowth, and maintenance of dendrites during development.

Brain-derived neurotrophic factor effects on oligodendrocyte progenitors of the basal forebrain are mediated through trkB and the MAP kinase pathway.

Previous work has indicated that BDNF increases the differentiation of basal forebrain (BF) oligodendrocytes (OLGs) in culture through the mediation of trkB and the MAPK pathway (Du et al. [ 2006a, b] Mol. Cell. Neurosci. 31:366-375; J. Neurosci. Res. 84:1692-1702). In the present work, effects of BDNF on BF OLG progenitor cells (OPCs) were examined. BDNF increased DNA synthesis of OPCs, as assessed by thymidine and bromodeoxyuridine incorporation. Effects of BDNF on DNA synthesis were mediated through the trkB receptor and not the p75 receptor, as shown by inhibitors that block neurotrophin binding to the receptors and by the phosphorylation of trkB. TrkB can activate the mitogen- activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3-K), and phospholipase C-gamma (PLC-gamma) pathways. BDNF elicited the phosphorylation of MAPK and Akt, a kinase downstream of PI3K, but not PLC-gamma in OPCs. Through the use of specific inhibitors to the MAPK and PI3-K pathways, it was found that the MAPK pathway was responsible for the effect of BDNF on DNA synthesis. These data indicate that BDNF affects OPC proliferation and development through the mediation of trkB and the MAPK pathway.

The trophic role of oligodendrocytes in the basal forebrain.

Traditionally, the primary function of oligodendrocytes (OLGs) in the CNS has been considered to be myelination. Here, we investigated whether OLGs may play a trophic role, particularly during development. Neurotrophin expression was assessed in postnatal day 7 basal forebrain (BF) OLGs, using in situ hybridization and detection of myelin basic protein. Nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs were revealed in OLGs in vivo and in culture. To determine whether OLGs support nearby neurons, we examined the influence of OLGs on BF cholinergic neurons. Neuronal function was enhanced by cocultured OLGs and OLG conditioned medium. Moreover, trophic effects of OLG conditioned medium were partially blocked by K252a, a trk tyrosine kinase inhibitor, and by neutralizing anti-BDNF or anti-NT-3 antisera, indicating that neurotrophins may mediate these effects, perhaps in concert with other signals. Our studies support a novel role for OLGs in providing local trophic support for neurons in the CNS.

Astroglia-mediated effects of uric acid to protect spinal cord neurons from glutamate toxicity.

Uric acid (UA) has been demonstrated to reduce damage to neurons elicited by oxidative stress. However, our studies utilizing cultures derived from embryonic rat spinal cord indicate that an astroglia-mediated mechanism is involved in the effects of UA to protect neurons from glutamate toxicity. The damage elicted by glutamate to neurons in a mixed culture of spinal cord cells can be reversed by UA. Furthermore, addition of UA after the termination of glutamate exposure suggests that UA plays an active role in mediating neuroprotection rather than purely binding peroxynitrite, as previously thought. Importantly, in pure neuron cultures from the same tissue, UA does not protect against glutamate toxicity. Addition of astroglia to the pure neuron cultures restores the ability of UA to protect the neurons from glutamate-induced toxicity. Our results also suggest that glia provide EAAT-1 and EAAT-2 glutamate transporters to protect neurons from glutamate, that functional EAATs may be necessary to mediate the effects of UA, and that treatment with UA results in upregulation of EAAT-1 protein. Taken together, our data strongly suggest that astroglia in mixed cultures are essential for mediating the effects of UA, revealing a novel mechanism by which UA, a naturally produced substance in the body, may act to protect neurons from damage during insults such as spinal cord injury.

Mitogen-activated protein kinase pathway mediates effects of brain-derived neurotrophic factor on differentiation of basal forebrain oligodendrocytes.

Previous studies indicate that brain-derived neurotrophic factor (BDNF), through the mediation of the trkB receptor, modulates the expression of differentiated traits in basal forebrain (BF) oligodendrocytes (OLGs). Specifically, BDNF up-regulates the expression of myelin basic protein (MBP), proteolipid protein (PLP), and myelin associated glycoprotein (MAG; Du et al. [2006] Mol. Cell. Neurosci. 31:366-375). However, the signaling cascades mediating the effects of BDNF have not been defined. The current study employs biochemical and molecular biological approaches to examine the involvements of the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3 kinase (PI3K) pathway, and the phospholipase C-gamma (PLC-gamma) pathway. Our results indicate that, in BF OLGs, BDNF activates the MAPK pathway and the PLC-gamma pathway but not the PI3K-Akt signaling cascade. By using specific inhibitors and mutated dominant negative or constitutively active forms of MAPK kinase, we demonstrate that the MAPK pathway is mediating the effects of BDNF on expression of differentiated traits in BF OLGs.

Distinct effects of p75 in mediating actions of neurotrophins on basal forebrain oligodendrocytes.

Previous studies indicate that brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) increase myelin basic protein, (MBP) in differentiating basal forebrain (BF) oligodendrocytes (OLGs) (Du, Y., Fischer, T.Z., Lee, L.N., Lercher, L.D., Dreyfus, C. F., 2003. Regionally specific effects of BDNF on oligodendrocytes. Dev. Neurosci. 25, 116-126). While receptors, trk and p75, are expressed by subsets of oligodendrocytes (Du, Y., Fischer, T.Z., Lee, L.N., Lercher, L.D., Dreyfus, C. F., 2003. Regionally specific effects of BDNF on oligodendrocytes. Dev. Neurosci. 25, 116-126), those responsible for affecting differentiation have not been defined. In contrast, studies of peripheral Schwann cells reported that myelination is enhanced by BDNF working through p75, and diminished by trkC mediated processes (Cosgaya, J.M., Chan, J.R., Shooter, E.M., 2002. The neurotrophin receptor p75NTR as a positive modulator of myelination. Science 298, 1245-1248). To define receptors affecting central oligodendrocyte MBP, p75 knockout animals, p75 blocking antibodies, and an inhibitor of neurotrophin binding to p75, PD90780, were utilized. While p75 was implicated in the actions of NGF and NT-3, it did not affect actions of BDNF. On the other hand, K252a, an inhibitor of trk receptors, abolished the effects of the neurotrophins, including BDNF. All neurotrophins activated their respective trk receptors.

Oligodendrocytes as providers of growth factors.

Glial cells recently are being appreciated as supporters of brain neurons. This review addresses their role as growth factor providers. While the function of astrocytes in this capacity is known, new data indicate that oligodendrocytes, the myelinating cells of the brain, exhibit similar abilities. Oligodendrocytes provide trophic signals to nearby neurons and synthesize defined growth factors. Expression of growth factors is influenced by neural signals. The review summarizes these roles and their implications in brain function.

Regionally specific effects of BDNF on oligodendrocytes.

To define the effects of neurotrophins on oligodendrocytes, we monitored NGF, BDNF and NT-3 actions on basal forebrain (BF) and cortical populations. NGF, BDNF and NT-3 applied to BF oligodendrocytes elicited increases in expression of myelin basic protein (MBP) and enhanced the numbers of MBP+ cells, without affecting total cell numbers. In the cortex, however, while NGF and NT-3 influenced MBP expression, BDNF was without effect. To explore this apparent regional difference in BDNF action, we compared expression of the neurotrophin receptors trkA, trkB and trkC. While BF cells expressed all three trks, cortical cells did not express the full-length BDNF receptor, trkB. Interestingly, in no case was any receptor expressed by all oligodendrocytes, indicating that oligodendrocytes may be heterogeneous within a brain region. The data suggest that BF oligodendrocytes are influenced by BDNF to express MBP and are distinct in this ability from cortical cells.