Rosemary Kraemer

ProBDNF Induces Neuronal Apoptosis via Activation of a Receptor Complex of p75NTR and Sortilin

Brain-derived neurotrophic factor (BDNF) is best characterized for critical roles in neuronal survival, differentiation, and synaptic modulation mediated by the TrkB receptor tyrosine kinase. Developmentally regulated death signaling by BDNF has also been demonstrated via activation of p75NTR. Because recent studies suggest that proNGF, the precursor form of NGF, is more active than mature NGF in inducing apoptosis after binding to p75NTR and a coreceptor, sortilin, we asked whether the precursor of BDNF (proBDNF) is also a proapoptotic ligand in the nervous system. proBDNF is secreted by cultured neurons, and recombinant proBDNF binds to sortilin. In sympathetic neurons coexpressing sortilin and p75NTR, we found that proBDNF is an apoptotic ligand that induces death at subnanomolar concentrations. In contrast, mature BDNF, but not proBDNF, is effective in inducing TrkB phosphorylation. proBDNF effects are dependent on cellular coexpression of both p75NTR and sortilin, because neurons deficient in p75NTR are resistant to proBDNF-induced apoptosis, and competitive antagonists of sortilin block sympathetic neuron death. Moreover, addition of preformed complexes of soluble sortilin and proBDNF failed to induce apoptosis of cells coexpressing both sortilin and p75NTR, suggesting that interaction of proBDNF with both receptors on the cell surface is required to initiate cell death. Together with our past findings, these data suggest that the neurotrophin family is capable of modulating diverse biological processes via differential processing of the proneurotrophins.

Variant Brain-Derived Neurotrophic Factor (Val66Met) Alters Adult Olfactory Bulb Neurogenesis and Spontaneous Olfactory Discrimination

Neurogenesis, the division, migration, and differentiation of new neurons, occurs throughout life. Brain derived neurotrophic factor (BDNF) has been identified as a potential signaling molecule regulating neurogenesis in the subventricular zone (SVZ), but its functional consequences in vivo have not been well defined. We report marked and unexpected deficits in survival but not proliferation of newly born cells of adult knock-in mice containing a variant form of BDNF [a valine (Val) to methionine (Met) substitution at position 66 in the prodomain of BDNF (Val66Met)], a genetic mutation shown to lead to a selective impairment in activity-dependent BDNF secretion. Utilizing knock-out mouse lines, we identified BDNF and tyrosine receptor kinase B (TrkB) as the critical molecules for the observed impairments in neurogenesis, with p75 knock-out mice showing no effect on cell proliferation or survival. We then localized the activated form of TrkB to a discrete population of cells, type A migrating neuroblasts, and demonstrate a decrease in TrkB phosphorylation in the SVZ of Val66Met mutant mice. With these findings, we identify TrkB signaling, potentially through activity dependent release of BDNF, as a critical step in the survival of migrating neuroblasts. Utilizing a behavioral task shown to be sensitive to disruptions in olfactory bulb neurogenesis, we identified specific impairments in spontaneous olfactory discrimination, but not general olfactory sensitivity or habituation to olfactory stimuli in BDNF mutant mice. Through these observations, we have identified novel links between genetic variant BDNF and adult neurogenesis in vivo, which may contribute to significant impairments in olfactory function.

GGF/Neuregulin Induces a Phenotypic Reversion of Oligodendrocytes ☆

We have previously shown that glial growth factor (GGF), a member of the neuregulin (NRG) family of growth factors, is a mitogen and survival factor for oligodendrocyte progenitors in cell culture and blocks their differentiation at the pro-oligodendrocyte stage (P. D. Canoll et al., 1996, Neuron 17, 229-243). We now show that GGF is able to induce differentiated oligodendrocytes to undergo a phenotypic reversion characterized by loss of MBP expression, reexpression of the intermediate filament protein nestin, reorganization of the actin cytoskeleton, and a dramatic reduction in the number of processes per cell. TUNEL analysis demonstrates that GGF is not cytotoxic for mature oligodendrocytes, but rather enhances their survival. GGF also induces the rapid activation of the PI 3-kinase and MAP kinase signaling pathways. These results further support a role for the NRGs in promoting the proliferation and survival of and inhibiting the differentiation of cells in the oligodendrocyte lineage and demonstrate that oligodendrocytes that differentiate in culture retain a substantial degree of phenotypic plasticity.

Small, Nonpeptide p75NTR Ligands Induce Survival Signaling and Inhibit proNGF-Induced Death

Studies showing that neurotrophin binding to p75NTR can promote cell survival in the absence of Trk (tropomyosin-related kinase) receptors, together with recent structural data indicating that NGF may bind to p75NTR in a monovalent manner, raise the possibility that small molecule p75NTR ligands that positively regulate survival might be found. A pharmacophore designed to capture selected structural and physical chemical features of a neurotrophin domain known to interact with p75NTR was applied to in silico screening of small molecule libraries. Small, nonpeptide, monomeric compounds were identified that interact with p75NTR. In cells showing trophic responses to neurotrophins, the compounds promoted survival signaling through p75NTR-dependent mechanisms. In cells susceptible to proneurotrophin-induced death, compounds did not induce apoptosis but inhibited proneurotrophin-mediated death. These studies identify a unique range of p75NTR behaviors that can result from isolated receptor liganding and establish several novel therapeutic leads.

p75NTR Mediates Neurotrophin-Induced Apoptosis of Vascular Smooth Muscle Cells

The development of atherosclerotic lesions results from aberrant cell migration, proliferation, and extracellular matrix production. In advanced lesions, however, cellular apoptosis, leading to lesion remodeling, predominates. During lesion formation, the neurotrophins and the neurotrophin receptor tyrosine kinases, trks B and C, are induced and mediate smooth muscle cell migration. Here we demonstrate that a second neurotrophin receptor, p75NTR, is expressed by established human atherosclerotic lesions and late lesions that develop after balloon injury of the rat thoracic aorta. The p75NTR, a member of the tumor necrosis factor/FAS receptor family, can modulate trk receptor function as well as initiate cell death when expressed in cells of the nervous system that lack kinase-active trk receptors. p75NTR expression colocalizes to neointimal cells, which express smooth muscle cell α-actin and are expressed by cultured human endarterectomy-derived cells (HEDC). Areas of the plaque expressing p75NTR demonstrate increased TUNEL positivity, and HEDC undergo apoptosis in response to the neurotrophins. Finally, neurotrophins also induced apoptosis of a smooth muscle cell line genetically manipulated to express p75NTR, but lacking trk receptor expression. These studies identify the regulated expression of neurotrophins and p75NTR as an inducer of smooth muscle cell apoptosis in atherosclerotic lesions.

Neurotrophin Receptor Interacting Factor (NRIF) Is an Essential Mediator of Apoptotic Signaling by the p75 Neurotrophin Receptor

Activation of the p75 neurotrophin receptor leads to a variety of effects within the nervous system, including neuronal apoptosis. Both c-Jun N-terminal kinase (JNK) and the tumor suppressor p53 have been reported to be critical for this receptor to induce cell death; however, the mechanisms by which p75 activates these pathways is undetermined. Here we report that the neurotrophin receptor interacting factor (NRIF) is necessary for p75-dependent JNK activation and apoptosis. Upon nerve growth factor withdrawal, nrif–/– sympathetic neurons underwent apoptosis, whereas p75-mediated death was completely abrogated. The lack of cell death correlated with a lack of JNK activation in the nrif–/– neurons, suggesting that NRIF is a selective mediator for p75-dependent JNK activation and apoptosis. Moreover, we document that NRIF expression is sufficient to induce cell death through a mechanism that requires p53. Taken together, these results establish NRIF as an essential component of the p75 apoptotic pathway.

The annexin A2/S100A10 system in health and disease: emerging paradigms.

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

Reduced Apoptosis and Increased Lesion Development in the Flow-Restricted Carotid Artery of p75NTR-Null Mutant Mice

Abstract— Apoptosis of neointimal smooth muscle cells is a well-recognized component of the pathogenesis of vascular lesions. In recent studies, we have identified the neurotrophin receptor, p75NTR, as a mediator of apoptosis of neointimal smooth muscle cells. Neurotrophin ligands and p75NTR are selectively expressed in areas of atherosclerotic lesions with increased smooth muscle cell apoptosis and the neurotrophins are potent apoptotic agents for p75NTR-expressing smooth muscle cells in vitro. In the present study, we directly assess the role of p75NTR in lesion development in the flow-restricted carotid artery, a model of murine vascular injury. Ligation of the left carotid artery resulted in a 3- to 4-fold increase in lesion development in p75NTR-null mutant mice as compared with wild-type mice. The increase in lesion size was associated with a 70% decrease in apoptosis of neointimal smooth muscle cells, as assessed by in situ TUNEL analysis. These data suggest that under conditions of flow restriction, p75NTR activation impairs lesion formation by promoting smooth muscle cell apoptosis. These results further implicate p75NTR as an important regulator of smooth muscle cell apoptosis and lesion development after vascular injury.

Decreased Neurotrophin TrkB Receptor Expression Reduces Lesion Size in the Apolipoprotein E–Null Mutant Mouse

Background— Accumulation of macrophages and smooth muscle cells in the vascular wall is critical for the development of atherosclerotic lesions. Although much is known about the factors that regulate macrophage recruitment to the vascular wall, the ability of growth factors to regulate smooth muscle cell recruitment in lesion development in vivo is unclear. Our previous studies demonstrated that neurotrophins and their receptors, the Trk receptor tyrosine kinases, are potent chemotactic factors for smooth muscle cells, and the expression of brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB, is upregulated in human atherosclerotic lesions. Methods and Results— TrkB+/− mice on a 129/B6 background were backcrossed to apolipoprotein E (ApoE)–null (ApoE−/−) mice on the C57Bl/6 background for 6 to 8 generations. Immunohistochemical analysis demonstrated BDNF immunoreactivity in areas of macrophage and smooth muscle cell infiltration, whereas TrkB immunoreactivity was predominant in areas of neointimal smooth muscle cells. Moreover, haplodeficient expression of TrkB in ApoE−/− mice was associated with a 30% to 40% reduction in lesion size compared with ApoE−/− mice with normal expression of TrkB and a 45% decrease in smooth muscle cell accumulation in the lesions. Finally, reconstitution with bone marrow from ApoE−/− mice with normal TrkB expression did not restore lesion development in TrKB+/−/ApoE−/− mice. Conclusions— These results suggest that TrkB expression on smooth muscle cells contributes to lesion development in the cholesterol-fed ApoE–null mutant mouse. These data demonstrate, for the first time, a role for the neurotrophin TrkB receptor in atherosclerotic lesion development.

Human SolCD39 Inhibits Injury-induced Development of Neointimal Hyperplasia

Blood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolise nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADPase activity in plasma peaked 1 hour (h) post-injection, with an elimination half-life of 43 h. Accordingly, mice were administered solCD39 or saline 1 h prior to vessel injury, then either sacrificed 24 h post-injury or treated with solCD39 or saline (three times weekly) for an additional 18 days. Twenty-four hours post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and smooth muscle cell (SMC) proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56 +/- 0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimising platelet deposition and leukocyte recruitment, and suppressing neointimal hyperplasia.