OIP106 (TRAK1) and GRIF-1 (TRAK2) kinesin-associated adaptor proteins: a study of their role in mitochondrial trafficking processes in neurones
Lead Research Organisation:
University College London
Department Name: Pharmaceutical and Biological Chem
Abstract
Information in our brains is processed by a discontinuous network of nerve cells or neurones. Communication between these cells occurs at specialized regions called synapses that are found at axon terminals. Synapses are metabolically dynamic and energy sources and proteins need to be constantly replenished to ensure fidelity of brain function. Since the synapse is often distant from the neuronal cell body (it can be up to 1 metre away), newly synthesized proteins need to be transported to these active zones. This transport process is achieved by motor proteins that associate with their cargoes via adaptor proteins that travel along the microtubular network within the neurones. My research group discovered a protein, GABA-A Receptor Interacting Factor-1 (GRIF-1, also called TRAK2), a member of a new gene family that is integral to these transport processes. The name derives from GRIF-1's first identified functional role in the transport of inhibitory GABA-A neurotransmitter receptor proteins to synapses but, it is now thought to play a more general role in neuronal trafficking processes since it has been shown to associate with the motor protein, kinesin. In flies, often used as model organisms, researchers have identified a protein that is similar to GRIF-1. This protein is called Milton. The name derives from the blind poet, John Milton; flies that do not have Milton are blind. In these mutant flies, it was found that mitochondria, a subcomponent of the cell that supplies energy, are absent from synapses in the neurones in the eyes of flies. It was thus speculated that Milton plays an important role in the trafficking of these organelles to synapses to supply energy for the proper communication between adjacent neurones. In this research proposal, we wish to study the role of GRIF-1 (TRAK2) and the related protein OIP106 (TRAK1) in model cell systems and in neurones to test if they have a similar function to Milton; to identify which mitochondrial proteins GRIF-1 and OIP106 bind and to determine how their activities are regulated. A deficiency in trafficking mechanisms may contribute to the pathology of neurodegenerative disorders such as Alzheimer's disease and spasticity. Thus, if we understand these basic mechanisms, in the future it may be possible to contribute towards the development of innovative therapies for their treatment.
Technical Summary
An emerging feature of trafficking mechanisms in neurones is that the transport of proteins and/or organelles occurs via adaptor proteins that link kinesin motor proteins to their cargoes. My research group discovered and cloned a protein, GRIF-1 (TRAK2), that we have recently shown is an example of a kinesin associated adaptor protein. GRIF-1 and the homologue, OIP106 (TRAK1), are members of a novel coiled-coil gene family. Whilst we originally proposed that GRIF-1 was involved in trafficking inhibitory GABA-A neurotransmitter receptors, it appears that both proteins may play a more general role in trafficking in excitable tissues. In this proposal, we wish to study in more detail this family of adaptor proteins. The impetus for the experiments described herein has come from our previous work and also, from the recent work describing the Drosophila orthologue of GRIF-1 and OIP106, Milton. Milton is purported to play a role in the trafficking of mitochondria in neurones. We have shown that both GRIF-1 and OIP106, like Milton, aggregate mitochondria, that this aggregation is mediated via the C-terminal, non-KHC binding domain of GRIF-1 and that in heterologous systems, mitochondria are trafficked in a GRIF-1 mediated kinesin-1-dependent manner. Here we plan to investigate the role of both GRIF-1 and OIP106 in mitochondrial trafficking by identifying mitochondrial partners of GRIF-1 and OIP106; by studying the transport of mitochondria via siRNA gene knock-down experiments, how this may be regulated via post-translational modification mechanisms and determining the cargoes that the KHC/GRIF-1 or KHC/OIP106 complexes transport. Results from the proposal will yield fundamental information on trafficking processes in neurones that may impact in the long term on the understanding of neurodegenerative disorders.
Organisations
People |
ORCID iD |
| Frances Stephenson (Principal Investigator) |
Publications
Brickley K
(2011)
N-acetylglucosamine transferase is an integral component of a kinesin-directed mitochondrial trafficking complex.
in Biochimica et biophysica acta
MacAskill AF
(2009)
GTPase dependent recruitment of Grif-1 by Miro1 regulates mitochondrial trafficking in hippocampal neurons.
in Molecular and cellular neurosciences
Stephenson F
(2011)
Folding for the Synapse
Stephenson FA
(2010)
Activity-dependent immobilization of mitochondria: the role of miro.
in Frontiers in molecular neuroscience
Wyttenbach Andreas
(2011)
Folding for the Synapse
| Description | Information in our brains is processed by a discontinuous network of nerve cells or neurones. Communication between these cells occurs at specialized regions called synapses that are found at axon terminals. Synapses are metabolically dynamic and energy sources and proteins need to be constantly replenished to ensure fidelity of brain function. Since the synapse is often distant from the neuronal cell body (it can be up to 1 metre away), newly synthesized proteins need to be transported to these active zones. This transport process is achieved by motor proteins that associate with their cargoes via adaptor proteins that travel along the microtubular network within the neurones. My research group discovered a protein, GABA-A Receptor Interacting Factor-1 (GRIF-1, now called TRAK2), a member of a new gene family that is integral to these transport processes. The original name was derived from GRIF-1's first identified functional role in the transport of inhibitory GABA-A neurotransmitter receptor proteins to synapses. But subsequent work carried out in my research group showed that GRIF-1 binds to the motor protein, kinesin, hence the change in name to TRAK2 derived from Trafficking kinesin protein. TRAK2 belongs to a gene family of at least two members, hence TRAK1 and TRAK2. It is now thought that TRAK1 and TRAK2 have similar functions both playing a more general role in neuronal trafficking processes. In flies, often used as model organisms, researchers have identified a protein that is similar to TRAK1/2. This protein is called Milton. The name derives from the blind poet, John Milton; flies that do not have Milton are blind. In these mutant flies, it was found that mitochondria, a subcomponent of the cell that supplies energy, are absent from synapses in the neurons in the eyes of flies. It was thus speculated that Milton plays an important role in the trafficking of these organelles to synapses to supply energy for the proper communication between adjacent neurones. In this research proposal, we have investigated the role of TRAK1 and TRAK2 in neurones to test if they have a similar function to Milton. To do this we cultured hippocampal pyramidal neuronal cells in the laboratory. We selected this class of neuronal cell because it is readily grown in culture to yield a broadly homogeneous neuronal cell population. Importantly also, these neurones are relatively large with extensive processes so they are advantageous for imaging studies. We visualized the mitochondria in these neurones by introducing into them a vector, pDsRed1-Mito, a process termed transfection. This is an inefficient process, usually only approximately 1 in 1000 neurones take up the vector. But, it is a powerful technique since once the vector is taken up by the neurone, it results in the generation of a red fluorescent protein which has an attached mitochondrial targeting sequence. Thus all the protein encoded by the vector cDNA that is made in the transfected neurone is targeted to mitochondria. So, mitochondria can be visualized and the parameters of Page 6 of 8 Date printed: 21/07/2010 16:18:59 BB/E021549/1 Date saved: 21/07/2010 16:16:01 their mobility determined by live imaging using a confocal microscope. To determine the role of the TRAK proteins in mitochondrial transport, we then used specialized reagents to interfere with either the production of the TRAK proteins (shRNA approaches) or, alternatively, we prevented the binding of TRAKs to the motor protein kinesin. If the TRAKs cannot bind to the motor protein, they may still bind to mitochondria but mitochondrial movement should be impaired because the motor protein is detached. This is termed a dominant negative strategy. These reagents, i.e. the dominant negative and shRNAs are also introduced by the process of transfection. We need to identify which neurones contain these specialized reagents. Each is encoded by a vector which, like the mitochondrial vector, has a fluorescent reporter molecule so that transfected cells can be identified by microscopy. This time the fluorescent marker is green so that in our studies we identified neurones that contained both red (mitochondrial labelling) and green (shRNA or dominant negative test and control reagents) and carried out the live imaging of mitochondrial mobility. We found that introduction of the dominant negative resulted in an approximate 73% decrease in mitochondrial mobility demonstrating for the first time that indeed, endogenous TRAK2 is an important regulator of neuronal mitochondrial mobility analogous to the fly protein, Milton. This finding is important because deficiencies in neuronal trafficking mechanisms are known occur in neurodegenerative disorders such as Alzheimer's disease, hereditary spastic paraplegia and motor neurone disease. Deficits in mitochondrial trafficking may be early events compromising neuronal function eventually contributing or even being causal to these diseases. Thus, understanding these basic trafficking mechanisms may in the future contribute towards the development of innovative therapies for the treatment of these disorders. At any one time only approximately 13-30% of neuronal mitochondria are mobile. They are known to translocate to relevant sites in response to particular needs, so it is probable that there are intracellular signalling mechanisms that initiate this movement and that appropriate trafficking mechanisms exist to facilitate transport. We and others have demonstrated that TRAKs are part of a multi-component protein complex that contains the kinesin motor protein, TRAK (the kinesin adaptor), an enzyme, N-acetylglucosamine transferase, and the protein, Miro, which is the mitochondrial acceptor for TRAKs. Thus the transport processes that respond to these events may be regulated by the formation of the kinesin/adaptor, the kinesin/adaptor/cargo mitochondrial complex, the dissociation of the kinesin/adaptor/mitochondrial complex i.e. the delivery of cargo and the docking of the mitochondria. We investigated the role of the enzyme, Nacetylglucosamine transferase in the formation of this complex. This enzyme can modify proteins by the addition of the sugar molecule, N-acetylglucosamine, to proteins. When proteins are modified in this way, their activity can be regulated so that they may have reduced/increased activity or it may inhibit their association with other proteins in protein complex such as the kinesin/TRAK/Miro complex. We found that TRAK2 can be glycosylated by this enzyme. We identified a site where the sugar residue is attached and furthermore, we showed that over-expression of the enzyme with TRAK1/2 and kinesin resulted in aberrant distribution of mitochondria suggesting that indeed sugar modification may play a role regulating the formation of motor protein/cargo complexes. Generally, there is little information available regarding the mechanisms that govern the binding and the delivery of cargoes which is especially important in neuronal cells. Thus we have identified a possible regulatory mechanism which we hope to study further in future studies. |
| Exploitation Route | The training of research personnel. Overall, we have provided new information on mechanisms of mitochondrial transport in neurones. Understanding these basic trafficking mechanisms may in the future contribute towards the development of innovative therapies for the treatment of neurodegenerative disorders in which mitochondrial dysfunction is implicated. |
| Sectors | Other |
| Description | TRAK-mediated neuronal mitochondrial trafficking mechanisms: regulation and impact on neuronal function |
| Amount | £700,000 (GBP) |
| Funding ID | BB/K014285/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2013 |
| End | 08/2016 |