Increases in two truncated TrkB isoforms in the prefrontal cortex of people with schizophrenia.
ABSTRACT: The truncated brain-derived neurotrophic factor (BDNF) receptors (truncated TrkB [TrkB-TK-] and sarc homology containing TrkB [TrkB-Shc]) are alternative transcripts of the full-length TrkB receptor (TrkB-TK+) that produce isoforms capable of binding to BDNF but not being able to mediate the classic neurotrophic response via tyrosine kinase signaling. We hypothesized that in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia, truncated TrkB receptors (TK- and Shc) would be altered and may contribute to deficits in BDNF function. Using a large cohort of controls and schizophrenics (n = 72/72), we measured mRNA expression of the full-length TrkB receptor, TrkB-TK+ and the truncated TrkB receptors, TrkB-TK- and TrkB-Shc, by quantitative real-time polymerase chain reaction and protein expression by western blotting. We found highly significant increases in mRNA expression of both truncated TrkB receptor isoforms in people with schizophrenia. When we examined the full-length TrkB-TK+:truncated TrkB ratios, we observed significant decreases in schizophrenia both on the mRNA and protein level. We found a slight reduction in TrkB-TK+ mRNA and a significant reduction in TrkB-TK+ protein expression in schizophrenia, which was evident in females. No gender-specific changes were found for the truncated TrkB receptors. Diagnostic changes in TrkB-TK+ mRNA and protein may be subtle and/or gender-specific, whereas changes in TrkB-TK- and TrkB-Shc expression are robust and may generalize to both males and females with schizophrenia. Increased truncated TrkB receptors may contribute to reduced overall BDNF/tyrosine receptor kinase B (TrkB) signaling and lead to reduced neuronal plasticity in the DLPFC in schizophrenia suggesting that therapies aimed at ameliorating neurotrophin deficits may need to consider blocking excessive truncated TrkB function.
Project description:Abnormalities in brain-derived neurotrophic factor (BDNF)/trkB signaling have been implicated in the etiology of schizophrenia and mood disorders. Patients with schizophrenia, bipolar disorder (BPD) and major depression (MDD) have reduced levels of neurotrophins in their brains when compared with normal unaffected individuals; however, only a few brain areas have been examined to date. Owing to the broad range of symptoms manifested in these disorders, we hypothesized that multiple associative areas of the neocortex may be implicated and that the degree of change in BDNF and trkB-TK+ mRNA expression and the cortical region or layers involved may vary according to Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnosis. We compared BDNF and trkB-TK+ mRNA levels across all layers of the prefrontal cortex (dorsolateral prefrontal cortex, DLPFC), orbital frontal cortex (OFC), anterior cingulate cortex (ACC), inferior temporal gyrus (ITG) and superior temporal gyrus (STG) in four groups: schizophrenia, BPD, MDD and unaffected controls (n=60). BDNF mRNA levels were significantly decreased in layers IV and V of DLPFC in schizophrenia patients, in layer VI of ACC in schizophrenia and MDD and in layer VI of ITG in schizophrenia, BPD and MDD. BDNF mRNA levels were also significantly decreased in layer V and/or VI of STG in schizophrenia, BPD and MDD. TrkB-TK+ mRNA levels were only significantly decreased in the cortical layer VI of OFC in BPD. The shared and distinct patterns of neurotrophin transcript reductions, with some specific to each group, may compromise the function and plasticity of distinct cortical areas to various degrees in the different groups and contribute to the range and overlap of symptoms manifested across the diagnoses.
Project description:BACKGROUND: Brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor (trkB-TK+) and glutamic acid decarboxylase (GAD67) mRNA levels have previously been found to be reduced in the prefrontal cortex of patients with schizophrenia. To determine whether this reduction extends to other brain regions, we measured the expression levels of BDNF, trkB-TK+ and GAD67 mRNA in regions of the hippocampus, including the dentate gyrus (DG), cornu ammonis subfields (CA1-4), subiculum and entorhinal cortex (EC) of individuals with schizophrenia, bipolar disorder, major depression and unaffected controls. METHODS: In situ hybridization was performed on postmortem brain tissue obtained from the Stanley Foundation Consortium and analyzed using film-based quantification. RESULTS: Analyses of covariance comparing the expression of mRNA among all groups revealed a significant decrease in BDNF mRNA in CA4 in the bipolar disorder group compared with controls (33%). We found trkB-TK+ mRNA levels to be significantly reduced in CA4 in the schizophrenia group (36%) and in layer II of the EC in the bipolar disorder and major depression groups (28%, 21%, respectively) compared with controls. In addition, GAD67 mRNA levels were reduced in patients with schizophrenia in both the DG (23%) and CA4 (60%) compared with controls. Individuals with major depression also expressed significantly less GAD67 mRNA (44%) compared with controls in CA4 of the hippocampus. LIMITATIONS: It is necessary to account for factors that influence the molecular preservation in postmortem brain tissue, including pH, postmortem interval and tissue storage time. Moreover, there are limitations to the sensitivity of the film-based method of quantification. CONCLUSION: Our findings show abnormal BDNF, trkB-TK+ and GAD67 mRNA expression in the hippocampus of individuals with schizophrenia and mood disorders, indicating that fundamental properties of hippocampal signalling transmission, plasticity and circuitry may be affected in individuals with these major mental illnesses.
Project description:Signaling by brain-derived neurotrophic factor (BDNF) via the TrkB receptor, or by neurotrophin-3 (NT3) through the TrkC receptor support distinct populations of sensory neurons. The intracellular signaling pathways activated by Trk (tyrosine kinase) receptors, which in vivo promote neuronal survival and target innervation, are not well understood. Using mice with TrkB or TrkC receptors lacking the docking site for Shc adaptors (trkB(shc/shc) and trkC(shc/shc) mice), we show that TrkB and TrkC promote survival of sensory neurons mainly through Shc site-independent pathways, suggesting that these receptors use similar pathways to prevent apoptosis. In contrast, the regulation of target innervation appears different: in trkB(shc/shc) mice neurons lose target innervation, whereas in trkC(shc/shc) mice the surviving TrkC-dependent neurons maintain target innervation and function. Biochemical analysis indicates that phosphorylation at the Shc site positively regulates autophosphorylation of TrkB, but not of TrkC. Our findings show that although TrkB and TrkC signals mediating survival are largely similar, TrkB and TrkC signals required for maintenance of target innervation in vivo are regulated by distinct mechanisms.
Project description:Dysfunction of inhibitory neurons in the prefrontal cortex (PFC), represented by decreased expression of GABA-related genes such as the 67 kDa isoform of glutamate decarboxylase (GAD67) and parvalbumin (PV), appears to contribute to cognitive deficits in subjects with schizophrenia. We investigated the involvement of signaling mediated by brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB in producing the altered GABA-related gene expression in schizophrenia. In 15 pairs of subjects with schizophrenia and matched control subjects, both BDNF and TrkB mRNA levels, as assessed by in situ hybridization, were significantly decreased in the PFC of the subjects with schizophrenia, whereas the levels of mRNA encoding the receptor tyrosine kinase for neurotrophin-3, TrkC, were unchanged. In this cohort, within-pair changes in TrkB mRNA levels were significantly correlated with those in both GAD67 and PV mRNA levels. Decreased BDNF, TrkB, and GAD67 mRNA levels were replicated in a second cohort of 12 subject pairs. In the combined cohorts, the correlation between within-pair changes in TrkB and GAD67 mRNA levels was significantly stronger than the correlation between the changes in BDNF and GAD67 mRNA levels. Neither BDNF nor TrkB mRNA levels were changed in the PFC of monkeys after a long-term exposure to haloperidol. Genetically introduced decreases in TrkB expression, but not in BDNF expression, also resulted in decreased GAD67 and PV mRNA levels in the PFC of adult mice; in addition, the cellular pattern of altered GAD67 mRNA expression paralleled that present in schizophrenia. Decreased TrkB signaling appears to underlie the dysfunction of inhibitory neurons in the PFC of subjects with schizophrenia.
Project description:The brain-derived neurotrophic factor (BDNF) gene contains multiple 5' promoters which generate alternate transcripts. Previously, we found that pan-BDNF mRNA and protein are reduced in the dorsolateral prefrontal cortex (DLPFC) from patients with schizophrenia. In this study, we determined which of the four most abundant and best characterized BDNF alternate transcripts, I-IX, II-IX, IV-IX, and VI-IX are altered in schizophrenia. Using a cohort from the NIMH, USA, we found that BDNF II-IX mRNA was significantly reduced in the DLPFC of patients with schizophrenia, and we replicated this finding using a second cohort from Sydney, Australia. Moreover, we show that BDNF protein expression [including prepro ( approximately 32 kDa), pro ( approximately 28 kDa) and mature ( approximately 14 kDa) BDNF] is reduced in the DLPFC of patients with schizophrenia. We next determined the regional specificity of the BDNF mRNA reduction by measuring BDNF transcripts in the parietal cortex and hippocampus and found no significant changes. The effect of antipsychotics on BDNF alternate transcript expression was also examined and we found no relationship between BDNF mRNA expression and antipsychotic use. As schizophrenic patients are often prescribed antidepressants which can up-regulate expression of BDNF, we investigated the relationship between antidepressant treatment and BDNF transcript expression. All four BDNF transcripts were significantly up-regulated in schizophrenic patients treated with antidepressants. Moreover, we found significant reductions in BDNF transcripts II-IX and IV-IX in the parietal cortex and VI-IX in the hippocampus of patients with schizophrenia who did not have a history of treatment with antidepressants. This suggests that down-regulation of at least one out of four major BDNF transcripts occurs in various brain regions of patients with schizophrenia, particularly in the DLPFC which appears to have the most robust BDNF deficit in schizophrenia.
Project description:In PC12 cells, it has been previously reported that nerve growth factor stimulates neuropeptide Y (NPY) gene expression. In the current study we examined the signalling pathways involved in this effect by transiently expressing in PC12 cells the receptor (TrkB) for the related neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3-fold induction of luciferase expression from a transiently co-transfected plasmid possessing the firefly luciferase gene under the control of the NPY promoter. This effect of BDNF was completely blocked by either a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphorylation and activation of phospholipase Cgamma (PLCgamma)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC was also completely blocked by a naturally occurring kinase insert which prevents the high-affinity binding of Shc and PLCgamma. NPY promoter activation by BDNF was blocked by PD98059, suggesting a role for mitogen-activated protein kinase (MAP kinase). Stimulation of NPY gene expression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protein kinase in the effect of BDNF. In addition, BDNF did not cause an elevation in cytosolic Ca2+ concentration. Taken together, our results suggest that stimulation of the NPY promoter by BDNF requires the simultaneous activation of two distinct pathways; one involves Shc and MAP kinase, and the other appears to be PLCgamma-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified.
Project description:Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.
Project description:Neurotrophins are a family of growth factors playing key roles in the survival, development, and function of neurons. The neurotrophins brain-derived neurotrophic factor (BDNF) and NT4 both bind to and activate TrkB receptors, however, they mediate distinct neuronal functions. The molecular mechanism of how TrkB activation by BDNF and NT4 leads to diverse outcomes is unknown. Here, we report that BDNF and NT4 lead to differential endocytic sorting of TrkB receptors resulting in diverse biological functions in cultured cortical neurons. Fluorescent microscopy and surface biotinylation experiments showed that both neurotrophins stimulate internalization of TrkB with similar kinetics. Exposure to BDNF for 2-3 h reduced the surface pool of TrkB receptors to half, whereas a longer treatment (4-5 h) with NT4 was necessary to achieve a similar level of down-regulation. Although BDNF and NT4 induced TrkB phosphorylation with similar intensities, BDNF induced more rapid ubiquitination and degradation of TrkB than NT4. Interestingly, TrkB receptor ubiquitination by these ligands have substantially different pH sensitivities, resulting in varying degrees of receptor ubiquitination at lower pH levels. Consequently, NT4 was capable of maintaining longer sustained downstream signaling activation that correlated with reduced TrkB ubiquitination at endosomal pH. Thus, by leading to altered endocytic trafficking itineraries for TrkB receptors, BDNF and NT4 elicit differential TrkB signaling in terms of duration, intensity, and specificity, which may contribute to their functional differences in vivo. The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), both bind to and activate TrkB receptors, however, they mediate distinct neuronal functions. Here, we propose that BDNF and NT4 lead to differential endocytic sorting of TrkB receptors resulting in diverse biological functions. BDNF induces more rapid ubiquitination and degradation of TrkB than NT4. Consequently, NT4 is capable of maintaining more sustained signaling downstream of TrkB receptors.
Project description:BACKGROUND: Antidepressant drugs (ADs) have been shown to activate BDNF (brain-derived neurotrophic factor) receptor TrkB in the rodent brain but the mechanism underlying this phenomenon remains unclear. ADs act as monoamine reuptake inhibitors and after prolonged treatments regulate brain bdnf mRNA levels indicating that monoamine-BDNF signaling regulate AD-induced TrkB activation in vivo. However, recent findings demonstrate that Trk receptors can be transactivated independently of their neurotrophin ligands. METHODOLOGY: In this study we examined the role of BDNF, TrkB kinase activity and monoamine reuptake in the AD-induced TrkB activation in vivo and in vitro by employing several transgenic mouse models, cultured neurons and TrkB-expressing cell lines. PRINCIPAL FINDINGS: Using a chemical-genetic TrkB(F616A) mutant and TrkB overexpressing mice, we demonstrate that ADs specifically activate both the maturely and immaturely glycosylated forms of TrkB receptors in the brain in a TrkB kinase dependent manner. However, the tricyclic AD imipramine readily induced the phosphorylation of TrkB receptors in conditional bdnf?/? knock-out mice (132.4±8.5% of control; P?=?0.01), indicating that BDNF is not required for the TrkB activation. Moreover, using serotonin transporter (SERT) deficient mice and chemical lesions of monoaminergic neurons we show that neither a functional SERT nor monoamines are required for the TrkB phosphorylation response induced by the serotonin selective reuptake inhibitors fluoxetine or citalopram, or norepinephrine selective reuptake inhibitor reboxetine. However, neither ADs nor monoamine transmitters activated TrkB in cultured neurons or cell lines expressing TrkB receptors, arguing that ADs do not directly bind to TrkB. CONCLUSIONS: The present findings suggest that ADs transactivate brain TrkB receptors independently of BDNF and monoamine reuptake blockade and emphasize the need of an intact tissue context for the ability of ADs to induce TrkB activity in brain.
Project description:Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.