Cell regulation

Expression and localization of endogenous BDNF and BDNF-EGFP/pHluorin in cultured hippocampal neurons EGFP has the ability to increase GFP fluorescence, while pHlourin is a protein that consists of a pH-sensitive form of green fluorescent protein (GFP) fused to a vesicles-associated membrane protein (VAMP). Because the vesicle has an acidic pH, when the pHlourin fuse with BDNF there is low fluorescence emitted. But when BDNF-pHlourin goes out of the vesicle (cell medium has a neutral pH) the protein becomes more fluorescent. This technique allows the following of the BDNF release, and we can see that BDNF has a higher concentration in the dendrites besides the axons. Different release sites for BDNF 1. BDNF release from the soma and dendrites – BDNF release is dependent on extracellular Ca2+ -influx (mediated via VGCC and NMDAR) and/or intracellular Ca2+ -release from internal stores (Endoplasmatic reticulum) Ca2+ -influx. Ca2+ -release from ER is mediated via IP3R or RyR. Increased burst firing activity, glutamate, or other ligands of GPCR mediate transient intracellular Ca2+ -increase important for vesicle exocytosis. 2. BDNF release from axons – BDNF release is dependent on Ca2+ -influx from extracellular space via presynaptic NMDAR and intracellular Ca2+ -release from internal Ca2+ -stores. Transport and secretion of neurotrophins BDNF is released from dendrites (but also axons and soma) into the extracellular space, in a process like the release of neurotransmitters, but in this case, they’re released by the dendrites instead of axon terminals. Neurotrophins are synthesized in the cell body (soma). Later, neurotrophins and receptors are transported in vesicles to the dendrites, where they will be released. After released, when they bind to the receptors, in both pre- and post-synaptic membrane, they activate receptors/induce responses: ▪ The activation of post-synaptic receptors regulates the release of neurotrophins (originates the signalling endosome that migrates to the soma, promoting gene expression and survivance); ▪ The activation of pre-synaptic receptors leads to the internalization of the receptor bound to BDNF and the regulation of neurotransmitters release; internalized receptors and neurotrophins are driven to the nucleus where they modulate transcription and growth. Trk signalling endosomes regulate diverse developmental events in multiple neuronal compartments, namely: local signalling, which leads to axon growth; retrograde transport; local recycling; transcription; synapse assembly. Mechanisms of neurotrophin internalization It has been proposed that internalization of TRKA following binding to nerve growth factor (NGF) occurs through clathrin-dependent and clathrin-independent mechanisms. Although clathrin-mediated endocytosis results in the formation of early endosomes, internalization via macropinocytosis leads to the generation of multivesicular bodies. Newly internalized TRKA endosomes must overcome an F-actin barrier before docking with microtubules for long distance transport. This is achieved by activation of RAC1, which leads to actin depolymerization through recruitment of cofilin to the signalling endosome. Signalling endosomes Target-derived neurotrophins are required for the growth and survival of innervating neurons. The vesicle that has the complex neurotrophin-receptor, called signalling endosomes, can be transported along the axon, remaining the interaction between them. Signalling endosomes – contain both ligand and receptor and are essential carriers of retrograde neurotrophin signal. The vesicle is transported along the axon by the microtubules that are associated with a motor protein (dyneindynactin complex). They also have proteins around them that participate in the receptor’s signalling activity, namely Rap1 and Rab5, for instance. When the signalling endosomes reaches the nucleus, it may occur transcription regulation of the TrkA gene: its transcription will originate new endosomes with new TrkA. The signalling of the endosomes with NGF-TrkA can lead to activation of Rab11 which promotes the recycling of TrkA, leading them to the distal part of the axon. They will be transported to the terminal increasing the number of receptors in the membrane of the axon, to increase the response to NGF. There is also the release of signals that will lead to the death of neighbouring neurons that do not have sufficient support for the NGF. In the other hand, retrograde NGF signalling, likely via axonal transport of TrkA-signalling endosomes, activates transcription factors and the expression of downstream target genes essential for long-term axon growth. BDNF and proBDNF are found at presynaptic terminals Cells have, in their membranes, receptors for both the mature form of BDNF but also for the proBDNF and both molecules have different roles in the synapse. ▪ proBDNF – increases the number of some receptors and the internalization of others; it has an effect in the communication between two neurons (or more). Synthesis and soring of neurotrophins All neurotrophins are produced as pro-proteins (eg. proBDNF). The mRNA of neurotrophins can also be transported to the dendrites and be produced there. Neurotrophins are synthetized in the ER, then goes to the lumen of the ER and after that travel to the Golgi, by vesicles, where they will become mature. When the vesicles arrive to the Golgi compartment they interact with proteins on the surface of the membrane of Golgi. In the Golgi the pro domain interacts with sortilin (intracellular receptor), which will facilitate the proper folding of the mature domain. The sequence of the proprotein that corresponds to the mature form of BDNF have specific aminoacids that interact with CPE (enzyme). This interaction determines the fate of the protein, by sorting the proteins and delivering them to different types of vesicles: BDNF goes to the regulatory secretory pathway vesicles (regulated by calcium); NGF goes manly to the constitutive secretory pathway vesicles. CPE-BDNF interaction also determines where the neurotrophins will be delivered: axons, dendrites or cell bodies. Pro-neurotrophins can be cleaved into their mature forms by either furin or PC1, but a significant fraction of BDNF remains in the precursor form until being released. Neurotrophins containing vesicles can be delivered to axons or dendrites and sorted to different branches. BDNF containing vesicles fuse with the pre- or post-synaptic membrane in response to depolarization stimuli that raise the intracellular Ca2+ concentration. There are receptors only activated by the mature form of BDNF. In addition to the effects of mature BDNF, the prodomain may also have effects on the cell. As seen before, pro-domain of BDNF may also have a function: control the number of receptors on synaptic membrane, for example. ▪ Regulated secretory pathway – soluble proteins and other substances are initially stored in secretory vesicles for later release. It needs some kind of signal to be released (eg. BDNF). ▪ Constitutive secretory pathway – operates continuously in all cells and supply a continuous stream of vesicles containing lipids and proteins for plasma membrane. Don’t need a signal (eg. NGF). Note(s): Pro-neurotrophins that are release in this form are cleaved by extracellular enzymes, such as MMP and Plasmin. tPA is a protease responsible for the formation of the plasmin. The mature neurotrophins exist in solution as dimmers and bind to the Tkr receptors activating intracellular signalling pathways in the target cell. Note: Huntington protein (Huntington’s disease) is related with the transport of BDNF in vesicles along the axon; the mutant protein inhibits the interaction of the motor proteins with the microtubules (vesicle immobilization). Neurotrophins: structure and function of the Trk neurotrophin receptors proNGF protein structure All neurotrophins are initially synthesized as large pro-peptides of similar size. After translation, proneurotrophins dimerize and can follow distinct routes. They can either be secreted to the extracellular space or intracellularly cleaved to originate mature neurotrophins. The interaction is not covalent, and terminals of each one of the monomers are directed to the same side of the dimer. Besides the pro-domain, the proneurotrophins also have a pre-domain (short sequence in the terminals) that works as a signal and is also important for the trafficking of the protein in between the cell. The domains L4 and L2 are also very important for the interaction of the protein with the receptor. Neurotrophin receptors Neurotrophins bind to and activate a family of receptor tyrosine kinases called Trk. All neurotrophins have a similar structure, but with little differences in extracellular domains. These differences are responsible for different signals, and so they bind to different receptors: ▪ NGF binds to TrkA; ▪ BDNF binds to TrkB; ▪ NT-3 binds to TrkC (but also sometimes can bind TRkA and TrkB). The RTK receptor family All Trks constitute an extracellular domain containing a ligand-binding site (different extracellular domains for different ligands), a single hydrophobic transmembrane α-helix, and a cytosolic domain that includes a region with protein tyrosine kinase activity. When we look at all the receptors in this family, they all have a similar intracellular and intramembrane domain, and the differences are seen in the extracellular domains, providing specificity. All have similar signalling mechanisms. Whenever neurotrophins binds to the receptor, we have dimerization. Thus, it’s important to have domains (ligand-binding region) that prevents spontaneous dimerization, otherwise the neurotrophins wouldn’t be able to bind to the receptor itself. Note: the NGF molecules exists as a dimer, so when a cell is stimulated with NGF this leads to the dimerization of the receptors: NGF binds to 2 receptors that are put close together, each one of the re