The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment.

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF.

The loop diuretic bumetanide blocks posttraumatic p75NTR upregulation and rescues injured neurons.

Injured neurons become dependent on trophic factors for survival. However, application of trophic factors to the site of injury is technically extremely challenging. Novel approaches are needed to circumvent this problem. Here, we unravel the mechanism of the emergence of dependency of injured neurons on brain-derived neurotrophic factor (BDNF) for survival. Based on this mechanism, we propose the use of the diuretic bumetanide to prevent the requirement for BDNF and consequent neuronal death in the injured areas. Responses to the neurotransmitter GABA change from hyperpolarizing in intact neurons to depolarizing in injured neurons. We show in vivo in rats and ex vivo in mouse organotypic slice cultures that posttraumatic GABA(A)-mediated depolarization is a cause for the well known phenomenon of pathological upregulation of pan-neurotrophin receptor p75(NTR). The increase in intracellular Ca(2+) triggered by GABA-mediated depolarization activates ROCK (Rho kinase), which in turn leads to the upregulation of p75(NTR). We further show that high levels of p75(NTR) and its interaction with sortilin and proNGF set the dependency on BDNF for survival. Thus, application of bumetanide prevents p75(NTR) upregulation and neuronal death in the injured areas with reduced levels of endogenous BDNF.

Glia cell line-derived neurotrophic factor mediates survival of murine sympathetic precursors.

During embryonic development, neurons are first produced in excess, and final numbers are adjusted by apoptosis at later stages. Crucial to this end is the amount of target-derived growth factor available for the neurons. By this means, the target size correctly matches the innervating neuron number. This target-derived survival has been well studied for sympathetic neurons, and nerve growth factor (NGF) was identified to be the crucial factor for maintaining sympathetic neurons at late embryonic and early postnatal stages, with a virtual complete loss of sympathetic neurons in NGF knockout (KO) mice. This indicates that all sympathetic neurons are dependent on NGF. However, also different glia cell line-derived neurotrophic factor (GDNF) KO mice consistently presented a loss of sympathetic neurons. This was the rationale for investigating the role of GDNF for sympathetic precursor/neuron survival. Here we show that GDNF is capable of promoting survival of 30% sympathetic precursors dissociated at E13. This is in line with data from GDNF KOs in which a comparable sympathetic neuron loss was observed at late embryonic stages, although the onset of the phenotype was unclear. We further present data showing that GDNF ligand and canonical receptors are expressed in sympathetic neurons especially at embryonic stages, raising the possibility of an autocrine/paracrine GDNF action. Finally, we show that GDNF also maintained neonatal sympathetic neurons (40%) cultured for 2 days. However, the GDNF responsiveness was lost at 5 days in vitro.

PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signaling.

The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAi-mediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporine-induced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects neurons and repairs the Parkinson disease-like symptoms in a rat 6-hydroxydopamine model. We show a three-dimensional solution structure of human MANF that differs drastically from other neurotrophic factors. Remarkably, the C-terminal domain of MANF (C-MANF) is homologous to the SAP domain of Ku70, a well known inhibitor of proapoptotic Bax (Bcl-2-associated X protein). Cellular studies confirm that MANF and C-MANF protect neurons intracellularly as efficiently as Ku70.

Persephin signaling through GFRalpha1: the potential for the treatment of Parkinson's disease.

Neurotrophic factors promote survival, proliferation and differentiation of neurons inducing intracellular signaling via specific receptors. The conventional biochemical methods often fail to reveal full repertoire of neurotrophic factor-receptor interactions because of their limited sensitivity. We evaluated several approaches to study signaling of Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands and found that reporter-gene systems possess exceptionally high sensitivity and a heuristic power to identify novel biologically relevant growth factor-receptor interactions. We identified persephin, a GDNF family member, as a novel ligand for GFRalpha1/RET receptor complex. We confirmed this finding by several independent methods, including neurite outgrowth assay from the explants of sympathetic ganglia expressing Gfralpha1 and Ret mRNA but not persephin's conventional receptor GFRalpha4. As the activation of GFRalpha1/RET was shown to rescue dopaminergic neurons, our results suggest the potential of persephin for the treatment of Parkinson's disease.

Death receptors and caspases but not mitochondria are activated in the GDNF- or BDNF-deprived dopaminergic neurons.

Neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote survival of midbrain dopaminergic neurons, but the death pathways activated in the dopaminergic neurons by deprivation of these factors are poorly studied. We show here that deprivation of GDNF or BDNF triggers a novel mitochondria-independent death pathway in the cultured embryonic dopaminergic neurons: cytochrome c was not released from the mitochondria to cytosol, proapoptotic protein Bax was not activated, and overexpressed Bcl-xL did not block the death. Caspases were critically required, because the death was completely blocked by caspase inhibitor BAF [boc-aspartyl(OMe)-fluoromethylketone] and overexpression of dominant-negative mutants of caspase-9, -3, and -7 significantly blocked the death. Also, the death receptor pathway was involved, because blockage of caspase-8 or FADD (Fas-associated protein with death domain), an adapter required for caspase-8 activation, inhibited death induced by GDNF or BDNF deprivation. Ligation of Fas by agonistic anti-Fas antibody induced apoptosis in the GDNF- or BDNF-maintained neurons, and inhibition of Fas by Fas-Fc chimera blocked the death of GDNF- or BDNF-deprived neurons, whereas FAIM(L) (long isoform of Fas apoptosis inhibitory molecule) could control the activity of Fas in the dopaminergic neurons.

Cell differentiation: reciprocal regulation of Apaf-1 and the inhibitor of apoptosis proteins.

The molecular mechanisms by which differentiated cells combat cell death and injury have remained unclear. In the current issue, it has been shown in neurons that cell differentiation is accompanied by a decrease in Apaf-1 and the activity of the apoptosome with an increased ability of the inhibitor of apoptosis proteins (IAPs) to sustain survival (Wright et al., 2004). These results, together with earlier ones, deepen our understanding of how cell death and the apoptosome are regulated during differentiation and in tumor cells.

GDNF-deprived sympathetic neurons die via a novel nonmitochondrial pathway.

The mitochondrial death pathway is triggered in cultured sympathetic neurons by deprivation of nerve growth factor (NGF), but the death mechanisms activated by deprivation of other neurotrophic factors are poorly studied. We compared sympathetic neurons deprived of NGF to those deprived of glial cell line-derived neurotrophic factor (GDNF). In contrast to NGF-deprived neurons, GDNF-deprived neurons did not die via the mitochondrial pathway. Indeed, cytochrome c was not released to the cytosol; Bax and caspase-9 and -3 were not involved; overexpressed Bcl-xL did not block the death; and the mitochondrial ultrastructure was not changed. Similarly to NGF-deprived neurons, the death induced by GDNF removal is associated with increased autophagy and requires multiple lineage kinases, c-Jun and caspase-2 and -7. Serine 73 of c-Jun was phosphorylated in both NGF- and GDNF-deprived neurons, whereas serine 63 was phosphorylated only in NGF-deprived neurons. In many NGF-deprived neurons, the ultrastructure of the mitochondria was changed. Thus, a novel nonmitochondrial caspase-dependent death pathway is activated in GDNF-deprived sympathetic neurons.

Survival assay of transiently transfected dopaminergic neurons.

Death pathways in the apoptotic neurons are mostly studied by manipulating the levels of apoptosis-related proteins and counting the survival/death of affected neurons. Such assays are, however, technically complicated. We developed a transfection-survival assay for cultured embryonic dopaminergic (DA) neurons induced to die by deprivation of glial cell line-derived neurotrophic factor (GDNF). The calcium phosphate co-precipitation technique was used to transfect DA neurons. Microisland cultures and co-transfected enhanced green fluorescent protein allowed direct counting of transfected neurons from the same cultures at the beginning and the end of GDNF deprivation, whereas post hoc subtraction of tyrosine hydroxylase-negative neurons allowed exclusion of transfected non-DA neurons. Overexpression of dominant-negative mutant of caspase-6 significantly blocked the death of GDNF-deprived DA neurons. Thus, we have found a tool not only to transfect the neurons dissociated from midbrain, but also to analyze the apoptotic proteins particularly in DA neurons.

Antioxidative CXXC Peptide Motif From Mesencephalic Astrocyte-Derived Neurotrophic Factor Antagonizes Programmed Cell Death.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a potent survival-promoting protein with neurorestorative effect for neurodegenerative diseases. Its mechanism of action, albeit poorly known, depends strongly on the CXXC motif (CKGC). Here we studied the survival-promoting properties of the CKGC tetrapeptide from MANF. In the Jurkat T lymphocytic cell line, CKGC potently inhibits death receptor Fas-induced apoptosis and mildly counteracts mitochondrial apoptosis and necroptosis. The peptide with serines instead of cysteines (SKGS) has no survival-promoting activity. The cytoprotective efficiency of the peptide against Fas-induced apoptosis is significantly improved by reduction of its cysteines by dithiotreitol, suggesting that it protects the cells via cysteine thiol groups, partially as an antioxidant. CKGC neutralizes the reactive oxygen species, maintains the mitochondrial membrane potential and prevents activation of the effector caspases in the Jurkat cells with activated Fas. The peptide does not require intracellular administration, as it is endocytosed and resides mainly in the Golgi. Finally, the peptide also potently promotes survival of cultured primary dopaminergic neurons.

Poststroke delivery of MANF promotes functional recovery in rats.

Stroke is the most common cause of adult disability in developed countries, largely because spontaneous recovery is often incomplete, and no pharmacological means to hasten the recovery exist. It was recently shown that mesencephalic astrocyte-derived neurotrophic factor (MANF) induces alternative or M2 activation of immune cells after retinal damage in both fruit fly and mouse and mediates retinal repair. Therefore, we set out to study whether poststroke MANF administration would enhance brain tissue repair and affect behavioral recovery of rats after cerebral ischemic injury. We used the distal middle cerebral artery occlusion (dMCAo) model of ischemia-reperfusion injury and administered MANF either as a recombinant protein or via adeno-associated viral (AAV) vector. We discovered that, when MANF was administered to the peri-infarct region 2 or 3 days after stroke, it promoted functional recovery of the animals without affecting the lesion volume. Further, AAV7-MANF treatment transiently increased the number of phagocytic macrophages in the subcortical peri-infarct regions. In addition, the analysis of knockout mice revealed the neuroprotective effects of endogenous MANF against ischemic injury, although endogenous MANF had no effect on immune cell-related gene expression. The beneficial effect of MANF treatment on the reversal of stroke-induced behavioral deficits implies that MANF-based therapies could be used for the repair of brain tissue after stroke.

Intrastriatally Infused Exogenous CDNF Is Endocytosed and Retrogradely Transported to Substantia Nigra.

Cerebral dopamine neurotrophic factor (CDNF) protects the nigrostriatal dopaminergic (DA) neurons in rodent models of Parkinson's disease and restores DA circuitry when delivered after these neurons have begun to degenerate. These DA neurons have been suggested to transport striatal CDNF retrogradely to the substantia nigra (SN). However, in cultured cells the binding and internalization of extracellular CDNF has not been reported. The first aim of this study was to examine the cellular localization and pharmacokinetic properties of recombinant human CDNF (rhCDNF) protein after its infusion into rat brain parenchyma. Second, we aimed to study whether the transport of rhCDNF from the striatum to the SN results from its retrograde transport via DA neurons or from its anterograde transport via striatal GABAergic projection neurons. We show that after intrastriatal infusion, rhCDNF diffuses rapidly and broadly, and is cleared with a half-life of 5.5 h. Confocal microscopy analysis of brain sections at 2 and 6 h after infusion of rhCDNF revealed its widespread unspecific internalization by cortical and striatal neurons, exhibiting different patterns of subcellular rhCDNF distribution. Electron microscopy analysis showed that rhCDNF is present inside the endosomes and multivesicular bodies. In addition, we present data that after intrastriatal infusion the rhCDNF found in the SN is almost exclusively localized to the DA neurons, thus showing that it is retrogradely transported.

Microarray Analysis Reveals Increased Transcriptional Repression and Reduced Metabolic Activity but Not Major Changes in the Core Apoptotic Machinery during Maturation of Sympathetic Neurons.

Postnatal maturation of the neurons whose main phenotype and basic synaptic contacts are already established includes neuronal growth, refinement of synaptic contacts, final steps of differentiation, programmed cell death period (PCD) etc. In the sympathetic neurons, postnatal maturation includes permanent end of the PCD that occurs with the same time schedule in vivo and in vitro suggesting that the process could be genetically determined. Also many other changes in the neuronal maturation could be permanent and thus based on stable changes in the genome expression. However, postnatal maturation of the neurons is poorly studied. Here we compared the gene expression profiles of immature and mature sympathetic neurons using Affymetrix microarray assay. We found 1310 significantly up-regulated and 1151 significantly down-regulated genes in the mature neurons. Gene ontology analysis reveals up-regulation of genes related to neuronal differentiation, chromatin and epigenetic changes, extracellular factors and their receptors, and cell adhesion, whereas many down-regulated genes were related to metabolic and biosynthetic processes. We show that termination of PCD is not related to major changes in the expression of classical genes for apoptosis or cell survival. Our dataset is deposited to the ArrayExpress database and is a valuable source to select candidate genes in the studies of neuronal maturation. As an example, we studied the changes in the expression of selected genes Igf2bp3, Coro1A, Zfp57, Dcx, and Apaf1 in the young and mature sympathetic ganglia by quantitative PCR and show that these were strongly downregulated in the mature ganglia.

Neuronal apoptosis inhibitory protein: Structural requirements for hippocalcin binding and effects on survival of NGF-dependent sympathetic neurons.

Neuronal apoptosis inhibitory protein (NAIP) has been linked to the inherited disease, spinal muscular atrophy (SMA), which occurs in children with degeneration of the motorneurons. In the nervous system, NAIP is expressed by specific classes of neurons including spinal motorneurons. Recently, NAIP was shown to interact with hippocalcin, which belongs to the neuronal calcium sensor (NCS) protein family. Here we have studied this interaction in more detail, using deletions and a mutagenesis of the third baculovirus inhibitory repeat (BIR) motif in NAIP, and functional assays for neuronal death. The results showed that specific amino acids and the zinc finger domain in BIR3 are needed for efficient interaction of NAIP with hippocalcin. Cotransfections of NAIP-BIR3 and hippocalcin resulted in translocation and colocalisation of the two proteins in neuroblastoma cells. This was accompanied by an enhanced resistance towards cell death induced by high levels of calcium. In contrast, expression of NAIP-BIR3 and hippocalcin in sympathetic neurons did not protect against death induced by nerve growth factor (NGF) withdrawal. The results demonstrate a functional interaction of hippocalcin with NAIP-BIR3, which in neuroblastoma cells leads to rescue of cells after high intracellular calcium, but which in sympathetic neurons had no significant effect. The results indicate that NAIP in conjunction with hippocalcin can affect the survival of some, but not all neural cells, and this interaction may play a role in the neurodegenerative processes in SMA, and possible other human disorders.

Therapeutic potential of the endoplasmic reticulum located and secreted CDNF/MANF family of neurotrophic factors in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder where dopamine (DA) neurons in the substantia nigra degenerate and die. Since no cure for PD exists, there is a need for disease-modifying drugs. Glial cell line-derived neurotrophic factor (GDNF) and related neurturin (NRTN) can protect and repair DA neurons in neurotoxin animal models of PD. However, GDNF was unable to rescue DA neurons in an α-synuclein model of PD, and both factors have shown modest effects in phase two clinical trials. Neurotrophic factors (NTFs), cerebral DA NTF (CDNF) and mesencephalic astrocyte-derived NTF (MANF) form a novel family of evolutionarily conserved, endoplasmic reticulum (ER) located and secreted NTFs. CDNF and MANF have a unique structure and an unparalleled dual mode of action that differs from other known NTFs. Both protect cells from ER stress, and regulate the unfolded protein response via interacting with chaperons, and CDNF dissolves intracellular α-synuclein aggregates. By binding to putative plasma membrane receptors, they promote the survival of DA neurons similarly to conventional NTFs. In animal models of PD, CDNF protects and repairs DA neurons, regulates ER stress, and improves motor function more efficiently than other NTFs.

AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

BACKGROUND: For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. NEW METHOD: We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). RESULTS: We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. COMPARISON WITH EXISTING METHODS: Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. CONCLUSIONS: We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process.

Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.

Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo.

Acetylcholinesterase inhibitors rapidly activate Trk neurotrophin receptors in the mouse hippocampus.

Acetylcholinesterase inhibitors are first-line therapies for Alzheimer's disease. These drugs increase cholinergic tone in the target areas of the cholinergic neurons of the basal forebrain. Basal forebrain cholinergic neurons are dependent upon trophic support by nerve growth factor (NGF) through its neurotrophin receptor, TrkA. In the present study, we investigated whether the acetylcholinesterase inhibitors donepezil and galantamine could influence neurotrophin receptor signaling in the brain. Acute administration of donepezil (3 mg/kg, i.p.) led to the rapid autophosphorylation of TrkA and TrkB neurotrophin receptors in the adult mouse hippocampus. Similarly, galantamine dose-dependently (3, 9 mg/kg, i.p.) increased TrkA and TrkB phosphorylation in the mouse hippocampus. Both treatments also increased the phosphorylation of transcription factor CREB and tended to increase the phosphorylation of AKT kinase but did not alter the activity of MAPK42/44. Chronic treatment with galantamine (3 mg/kg, i.p., 14 days), did not induce changes in hippocampal NGF and BDNF synthesis or protein levels. Our findings show that acetylcholinesterase inhibitors are capable of rapidly activating hippocampal neurotrophin signaling and thus suggest that therapies targeting Trk signaling may already be in clinical use in the treatment of AD.