Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival-promoting effects of BDNF and IGF-1 on serum deprivation-induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF-1 in the neurons. We found that the survival-promoting effect of BDNF was much weaker than that of IGF-1 in serum deprivation-induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3-kinase (PtdIns3-K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF-1 in the cultured cortical neurons, although many reports suggest that PtdIns3-K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen-activated protein kinase (MAPK) and cAMP responsive element-binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF-1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3-K-Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival-promoting effects of BDNF and IGF-1.     

LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathways

The identities of receptor protein tyrosine phosphatases (PTPs) that associate with Trk protein tyrosine kinase (PTK) receptors and modulate neurotrophic signaling are unknown. The leukocyte common antigen-related (LAR) receptor PTP is present in neurons expressing TrkB, and like TrkB is associated with caveolae and regulates survival and neurite outgrowth. We tested the hypothesis that LAR associates with TrkB and regulates neurotrophic signaling in embryonic hippocampal neurons. Coimmunoprecipitation and coimmunostaining demonstrated LAR interaction with TrkB that is increased by BDNF exposure. BDNF neurotrophic activity was reduced in LAR-/- and LAR siRNA-treated LAR+/+ neurons and was augmented in LAR-transfected neurons. In LAR-/- neurons, BDNF-induced activation of TrkB, Shc, AKT, ERK, and CREB was significantly decreased; while in LAR-transfected neurons, BDNF-induced CREB activation was augmented. Similarly, LAR+/+ neurons treated with LAR siRNA demonstrated decreased activation of Trk and AKT. LAR is known to activate the Src PTK by dephosphorylation of its negative regulatory domain and Src transactivates Trk. In LAR-/- neurons, or neurons treated with LAR siRNA, phosphorylation of the Src regulatory domain was increased (indicating Src inactivation), consistent with a role for Src in mediating LAR's ability to up-regulate neurotrophic signaling. Interactions between LAR, TrkB, and Src were further confirmed by the findings that Src coimmunoprecipitated with LAR, that the Src inhibitor PP2 blocked the ability of LAR to augment TrkB signaling, and that siRNA-induced depletion of Src decreased LAR interaction with TrkB. These studies demonstrate that receptor PTPs can associate with Trk complexes and promote neurotrophic signaling and point to receptor PTP-based strategies as a novel approach for modulating neurotrophin function.     

Brain-Derived Neurotrophic Factor Stimulates Interactions of Shp2 with Phosphatidylinositol 3-Kinase and Grb2 in Cultured Cerebral Cortical Neurons

Shp2, a protein tyrosine phosphatase possessing SH2 domains, is utilized in the intracellular signaling of various growth factors. Shp2 is highly expressed in the CNS. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, which also shows high levels of expression in the CNS, exerts neurotrophic and neuromodulatory effects in CNS neurons. We examined how BDNF utilizes Shp2 in its signaling pathway in cultured cerebral cortical neurons. We found that BDNF stimulated coprecipitation of several tyrosine-phosphorylated proteins with anti-Shp2 antibody and that Grb2 and phosphatidylinositol 3-kinase (PI3-K) were coprecipitated with anti-Shp2 antibody in response to BDNF. In addition, both anti-Grb2 and anti-PI3-K antibodies coprecipitated Shp2 in response to BDNF. The BDNF-stimulated coprecipitation of the tyrosine-phosphorylated proteins, Grb2, and PI3-K with anti-Shp2 antibody was completely inhibited by K252a, an inhibitor of TrkB receptor tyrosine kinase. This BDNF-stimulated Shp2 signaling was markedly sustained as well as BDNF-induced phosphorylation of TrkB and mitogen-activated protein kinases. In PC12 cells stably expressing TrkB, both BDNF and nerve growth factor stimulated Shp2 signaling similarly to that by BDNF in cultured cortical neurons. These results indicated that Shp2 shows cross-talk with various signaling molecules including Grb2 and PI3-K in BDNF-induced signaling and that Shp2 may be involved in the regulation of various actions of BDNF in CNS neurons.     

Possible involvement of brain-derived neurotrophic factor (BDNF) in the innervation of dopaminergic neurons from the rat periventricular nucleus to the pars intermedia

Developing neurons are guided to their appropriate targets by specific guidance substances that have neurotrophic actions. The aim of the present study was to elucidate the mechanism by which hypothalamic neurons reach the pars intermedia (PI) by correlating the development of dopaminergic (DA) neurons arising in the periventricular nucleus (PeV) of fetal rats with the expression of brain-derived neurotrophic factor (BDNF) in the rat pituitary. The differentiation of DA neurons was observed by immunohistochemistry using an antibody against tyrosine hydroxylase (TH), whereas the ontogenesis of BDNF mRNA in the PI was examined by in situ hybridization and RT-PCR. Immunoreactive TH-neurons were first observed in the PeV at embryonic day (E) 16.5, following which time their axons elongated toward the pituitary. TH-positive reactions were observed in the connective tissue between the PI and the pars nervosa at E20.5. Innervation of the PI by TH-positive neurons was determined at postnatal day (P) 1.5; however, BDNF mRNA was first detected in the PI cells at E17.5, with an increase in its expression clearly visible at E21.5 and continuing high expression levels in the PI thereafter. These results suggest that BDNF is a specific guidance cue for DA neurons elongating from the PeV to the PI.     

Defective Self-Renewal and Differentiation of GBA-Deficient Neural Stem Cells Can Be Restored By Macrophage Colony-Stimulating Factor

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene (GBA), which encodes the lysosomal enzyme glucosylceramidase (GCase). Deficiency in GCase leads to characteristic visceral pathology and lethal neurological manifestations in some patients. Investigations into neurogenesis have suggested that neurodegenerative disorders, such as GD, could be overcome or at least ameliorated by the generation of new neurons. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are potential candidates for use in the treatment of neurodegenerative disorders because of their ability to promote neurogenesis. Our objective was to examine the mechanism of neurogenesis by BM-MSCs in GD. We found that neural stem cells (NSCs) derived from a neuronopathic GD model exhibited decreased ability for self-renewal and neuronal differentiation. Co-culture of GBA-deficient NSCs with BM-MSCs resulted in an enhanced capacity for self-renewal, and an increased ability for differentiation into neurons or oligodendrocytes. Enhanced proliferation and neuronal differentiation of GBA-deficient NSCs was associated with elevated release of macrophage colony-stimulating factor (M-CSF) from BM-MSCs. Our findings suggest that soluble M-CSF derived from BM-MSCs can modulate GBA-deficient NSCs, resulting in their improved proliferation and neuronal differentiation.     

In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: Inducing effects of brain-derived neurotrophic factor

Neural stem cells proliferate in vitro and form neurospheres in the presence of epidermal growth factor (EGF), and are capable of differentiating into both neurons and glia when exposed to a substrate. We hypothesize that specific neurotrophic factors induce differentiation of stem cells from different central nervous system (CNS) regions into particular fates. We investigated differentiation of stem cells from the postnatal mouse hippocampus in culture using the following trophic factors (20 ng/mL): brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF). Without trophic factors, 32% of stem cells differentiated into neurons by 4 days in vitro (DIV), decreasing to 10% by 14 DIV. Addition of BDNF (starting at either day 0 or day 3) significantly increased neuron survival (31–43% by 14 DIV) and differentiation. Morphologically, many well-differentiated neurons resembled hippocampal pyramidal neurons. 5′-Bromodeoxyuridine labeling demonstrated that the pyramidal-like neurons originated from stem cells which had proliferated in EGF-containing cultures. However, similar application of NT-3 and GDNF did not exert such a differentiating effect. Addition of BDNF to stem cells from the postnatal cerebellum, midbrain, and striatum did not induce these neuronal phenotypes, though similar application to cortical stem cells yielded pyramidal-like neurons. Thus, BDNF supports survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons. The presence of pyramidal neurons in BDNF-treated hippocampal and cortical stem cell cultures, but not in striatal, cerebellar, and midbrain stem cell cultures, suggests that stem cells from different CNS regions differentiate into region-specific phenotypic neurons when stimulated with an appropriate neurotrophic factor. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 395–425, 1998     

The effect of brain-derived neurotrophic factor on neuritogenesis and synaptic plasticity in Aplysia neurons and the hippocampal cell line HiB5

Brain-derived neurotrophic factor (BDNF) plays a key role in the differentiation and neuritogenesis of developing neurons, and in the synaptic plasticity of mature neurons, in the mammalian nervous system. BDNF binds to the receptor tyrosine kinase TrkB and transmits neurotrophic signals by activating neuron-specific tyrosine phosphorylation pathways. However, the neurotrophic function of BDNF in Aplysia neurons is poorly understood. We examined the specific effect of BDNF on neurite outgrowth and synaptic plasticity in cultured Aplysia neurons and a multipotent rat hippocampal stem cell line (HiB5). Our study indicates that mammalian BDNF has no significant effect on the neuritogenesis, neurotransmitter release, excitability, and synaptic plasticity of cultured Aplysia neurons in our experimental conditions. In contrast, BDNF in combination with platelet-derived growth factor (PDGF) increases the length of the neurites and the number of spine-like structures in cells of HiB5.     

Long-term gene therapy causes transgene-specific changes in the morphology of regenerating retinal ganglion cells

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.     

Dopamine suppresses osteoclast differentiation via cAMP/PKA/CREB pathway

How the nervous system regulates bone remodeling is an exciting area of emerging research in bone biology. Accumulating evidence suggest that neurotransmitter-mediated inputs from neurons may act directly on osteoclasts. Dopamine is a neurotransmitter that can be released by hypothalamic neurons to regulate bone metabolism through the hypothalamic-pituitary-gonadal axis. Dopamine is also present in sympathetic nerves that penetrate skeletal structures throughout the body. It has been shown that dopamine suppresses osteoclast differentiation via a D2-like receptors (D2R)-dependent manner, but the intracellular secondary signaling pathway has not been elucidated. In this study, we found that cAMP-response element binding protein (CREB) activity responds to dopamine treatment during osteoclastogenesis. Considering the critical role of CREB in osteoclastogenesis, we hypothesize that CREB may be a critical target in dopamine's regulation of osteoclast differentiation. We confirmed that D2R is also present in RAW cells and activated by dopamine. Binding of dopamine to D2R inhibits the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway which ultimately decreases CREB phosphorylation during osteoclastogenesis. This was also associated with diminished expression of osteoclast markers that are downstream of CREB. Pharmacological activation of adenylate cyclase (to increase cAMP production) and PKA reverses the effect of dopamine on CREB activity and osteoclastogenesis. Therefore, we have identified D2R/cAMP/PKA/CREB as a candidate pathway that mediates dopamine's inhibition of osteoclast differentiation. These findings will contribute to our understanding of how the nervous and skeletal systems interact to regulate bone remodeling. This will enable future work toward elucidating the role of the nervous system in bone development, repair, aging, and degenerative disease.     

Neutralization of BDNF Attenuates the in vitro Protective Effects of Olfactory Ensheathing Cell-Conditioned Medium on Scratch-Insulted Retinal Ganglion Cells

Transplantation of olfactory ensheathing cells (OECs) becomes one of the promising strategies in restoring lost functions of injured central nervous system. Elevated level of expressed brain-derived neurotrophic factor (BDNF) was revealed in the previous studies to be related to the protective effects of OECs on injured cortical and brain stem neurons as well as retinal ganglion cells (RGCs), but no evidence has been obtained to demonstrate whether transplanted OECs protect injured central neurons directly by their secreted BDNF. In the present study, the effects of BDNF neutralization on the neuroprotection of adult OEC-conditioned medium (OEC-CM) on scratch-insulted RGCs were examined. The results showed that OEC-CM protected cultured RGCs from scratch insult, and neutralization of BDNF by BDNF neutralizing antibody attenuated such neuroprotection of the medium. It is thus concluded that neurotrophic factors including BDNF secreted by OECs can protect injured OECs in vitro and BDNF plays a major role in such a protection of OECs.