The neurotrophin receptor TrkB is colocalized to mitochondrial membranes

The transmembrane tyrosine-specific protein kinase TrkB has been shown to serve as a receptor for the neurotrophic factors BDNF and NT-4. Neurotrophin binding to TrkB isoformes mediates many intracellular signaling pathways, including calcium signalling. Two truncated isoforms of the receptor, lacking the tyrosine kinase activity, signal through a yet unknown pathway. Specific signals modulate the surface expression of TrkB, which is localized in considerable amounts in intracellular pools. These intracellular pools has not been specified so far. We therefore investigated the intracellular distribution of TrkB by colocalisation studies. In contrast to the unspecific neurotrophin receptor NGFRp75, TrkB immunohistochemistry showed a staining pattern very similar to mitochondrial stainings in adult human skeletal muscle fibers. Immunofluorescence techniques revealed in different types of permeabilized cells that TrkB is bound to mitochondrial membranes. This observation was confirmed on isolated astrocyte mitoplasts. Colocalisation of the TrkB ligand NT-4 and the specific mitochondrial marker cytochrome c oxidase was also observed. Western blot analysis of isolated mitochondria from rat brain and skeletal muscle verified that a truncated isoform of TrkB is present in both, brain and muscle mitochondria, and full-length TrkB is additionally present in brain mitochondria. Our results imply that neurotrophins can be stored in mitochondria and possibly act as signalling molecules on mitochondria.     

Discovery of an isocoumarin analogue that modulates neuronal functions via neurotrophin receptor TrkB

Isocoumarins are lactone ring-containing natural products, are quite abundant in microbes and higher plants, and have been shown to exhibit a broad range of pharmacological properties. However, the molecular mechanism or target of this class of molecules is not known. In this study, we have synthesized 14 isocoumarin derivatives and evaluated for their activity at TrkB receptor in transiently transfected HEK293T cells. We identified 8-hydroxy-3-aryl isocoumarin (1) as a high-affinity agonist at the TrkB receptor. We also demonstrated that isocoumarin 1 activated endogenously TrkB receptor in primary cortical neurons and modulated various markers of synaptic plasticity, and increased dendritic arborization. These results indicate therapeutic potential and molecular target of 8-hydroxy-3-aryl isocoumarin 1 for the treatment of various CNS disorders.     

TTIP is a novel protein that interacts with the truncated T1 TrkB neurotrophin receptor

Alternative splicing of the TrkB gene produces a full length tyrosine kinase receptor as well as two truncated isoforms that contain extracellular and transmembrane domains but lack the kinase domain and have unique C terminal tails. The function of the truncated TrkB isoforms is unclear and to gain insights into their function, we have isolated a protein from 15N neuroblastoma cells that specifically binds the TrkB.T1 isoform. Pulldown experiments using a GST fusion protein containing the TrkB.T1 intracellular domain identified a 61 kDa protein from radiolabeled 15N lysates. Coimmunoprecipitation experiments showed that the 61 kDa protein interacted with epitope-tagged TrkB.T1 overexpressed in 15N cells as well as with TrkB.T1 which was endogenously expressed. Peptide competition experiments revealed that the protein, designated TTIP (for Truncated TrkB Interacting Protein), showed specific binding to the TrkB.T1 tail. MALDI MS and MS/MS analysis has revealed that TTIP is a novel protein not yet listed in the current databases.     

Down-regulation of the neurotrophin receptor TrkB following ligand binding. Evidence for an involvement of the proteasome and differential regulation of TrkA and TrkB

This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA.     

Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB

The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine‐binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α‐PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. Proteins 2014; 82:1534–1541. © 2014 Wiley Periodicals, Inc.     

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.     

On the presence of neurotrophin p75 receptor on rat sympathetic cerebrovascular nerves

Although the presence of neurotrophin p75 receptor on sympathetic nerves is a well-recognised feature, there is still a scarcity of details of the distribution of the receptor on cerebrovascular nerves. This study examined the distribution of p75 receptor on perivascular sympathetic nerves of the middle cerebral artery and the basilar artery of healthy young rats using immunohistochemical methods at the laser confocal microscope and transmission electron microscope levels. Immunofluorescence methods of detection of tyrosine hydroxylase (TH) in sympathetic nerves, p75 receptor associated with the nerves, and also S-100 protein in Schwann cells were applied in conjunction with confocal microscopy, while the pre-embedding single and double immunolabelling methods (ExtrAvidin and immuno-gold-silver) were applied for the electron microscopic examination. Immunofluorescence studies revealed “punctuate” distribution of the p75 receptor on sympathetic nerves including accompanying Schwann cells. Image analysis of the nerves showed that the level of co-localization of p75 receptor and TH was low. Immunolabelling applied at the electron microscope level also showed scarce co-localization of TH (which was intra-axonal) and p75. Immunoreactivity for p75 receptor was present on the cell membrane of perivascular axons and to a greater extent on the processes of accompanying Schwann cells. Some Schwann cell processes were adjacent to each other displaying strong immunoreactivity for p75 receptor; immunoreactivity was located on the extracellular sites of the adjacent cell membranes suggesting that the receptor was involved in cross talk between these. It is likely that variability of locations of p75 receptor detected in the study reflects diverse interactions of p75 receptor with axons and Schwann cells. It might also imply a diverse role for the receptor and/or the plasticity of sympathetic cerebrovascular nerves to neurotrophin signalling.     

Characterization of angiotensin II receptor subtypes in the rat spleen.

Quantitative autoradiography was used to determine the subtype of ANG receptors in the red pulp of the rat spleen. The AT1 antagonist DuP 753 competed for ANG binding with high affinity; binding was abolished by dithiothreitol. The AT2 competitor CGP 42112 A showed lower affinity, and the AT2 competitor PD 123177 did not affect binding at 10(-5) M. These data indicated the presence of only AT1 receptors. AT1 receptor number was similar in immature (2 weeks old) and adult (8 weeks old) rats. Binding was sensitive to guanine nucleotides, suggesting an association with G-proteins. Angiotensin II, at a dose of 10(-7) M, stimulated inositol phosphate formation 33% over control values in spleen from 8-week-old rats. This effect was significantly blocked by 10(-5) M DuP 753. We suggest a possible role of AT1 receptors in the regulation of splenic volume, blood flow, and lymphocyte function.     

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.     

The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.