F. Anne Stephenson

Mapping the GRIF-1 Binding Domain of the Kinesin, KIF5C, Substantiates a Role for GRIF-1 as an Adaptor Protein in the Anterograde Trafficking of Cargoes

γ-Aminobutyric acid, type A (GABAA) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating β2 subunit-containing GABAA receptors, in the brain.

Neto1 associates with the NMDA receptor/amyloid precursor protein complex.

Neuropilin tolloid‐like 1 (Neto1), is a CUB domain‐containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein (APP)695/GluN1/GluN2A and APP695/GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1HA was shown to co‐immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1HA did not co‐immunoprecipitate APP695FLAG. Co‐immunoprecipitations from mammalian cells co‐transfected with APP695FLAG, Neto1HA and GluN1/GluN2A or GluN1/GluN2B revealed that all four proteins co‐exist within one macromolecular complex. Immunoprecipitations from native brain tissue similarly revealed the existence of a GluN1/GluN2A or GluN2B/APP/Neto1 complex. Neto1HA caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695FLAG‐ or PSD‐95αc‐Myc enhanced surface receptor expression. The Neto1 binding domain of GluN2A was mapped using GluN1/GluN2A chimeras and GluN2A truncation constructs. The extracellular GluN2A domain does not contribute to association with Neto1HA but deletion of the intracellular tail resulted in a loss of Neto‐1HA co‐immunoprecipitation which was paralleled by a loss of association between GluN2A and SAP102. Thus, Neto1 is concluded to be a component of APP/NMDA receptor trafficking complexes.

GABAA receptors can initiate the formation of functional inhibitory GABAergic synapses

The mechanisms that underlie the selection of an inhibitory GABAergic axons postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABAA receptors (GABAARs) themselves – the essential functional postsynaptic components of GABAergic synapses – can be sufficient to initiate formation of synaptic contacts, a novel co‐culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2‐GABAARs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse‐like contacts in these co‐cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle‐luminal domain‐specific antibody; and they supported spontaneous and action potential‐driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N‐methyl‐d‐aspartate receptor‐expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin‐2 was co‐expressed with GABAARs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABAARs can promote the adhesion of inhibitory axons and the development of functional synapses.

APLP1 and APLP2, members of the APP family of proteins, behave similarly to APP in that they associate with NMDA receptors and enhance NMDA receptor surface expression.

The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N‐methyl‐d‐aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor‐like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co‐immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co‐immunoprecipitation of APLP1 and APLP2 with GluN2A‐ and GluN2B‐containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus, all the three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homoeostasis.

Localization of the kinesin adaptor proteins trafficking kinesin proteins 1 and 2 in primary cultures of hippocampal pyramidal and cortical neurons

Neuronal function requires regulated anterograde and retrograde trafficking of mitochondria along microtubules by using the molecular motors kinesin and dynein. Previous work has established that trafficking kinesin proteins (TRAKs),TRAK1 and TRAK2, are kinesin adaptor proteins that link mitochondria to kinesin motor proteins via an acceptor protein in the mitochondrial outer membrane, etc. the Rho GTPase Miro. Recent studies have shown that TRAK1 preferentially controls mitochondrial transport in axons of hippocampal neurons by virtue of its binding to both kinesin and dynein motor proteins, whereas TRAK2 controls mitochondrial transport in dendrites resulting from its binding to dynein. This study further investigates the subcellular localization of TRAK1 and TRAK2 in primary cultures of hippocampal and cortical neurons by using both commercial antibodies and anti‐TRAK1 and anti‐TRAK2 antibodies raised in our own laboratory (in‐house). Whereas TRAK1 was prevalently localized in axons of hippocampal and cortical neurons, TRAK2 was more prevalent in dendrites of hippocampal neurons. In cortical neurons, TRAK2 was equally distributed between axons and dendrites. Some qualitative differences were observed between commercial and in‐house‐generated antibody immunostaining.

Revisiting the TRAK Family of Proteins as Mediators of GABAA Receptor Trafficking

Abstract γ-Aminobutyric acid type A (GABAA) receptor interacting factor-1 (GRIF-1) was originally discovered as a result of studies aiming to find the elusive GABAA receptor clustering protein. It was identified as a GABAA receptor associated protein by virtue of its specific interaction with the GABAA receptor β2 subunit intracellular loop in a yeast two-hybrid screen of a rat brain cDNA library. Further work however, established that GRIF-1, now known as trafficking kinesin protein 2 (TRAK2), is a member of the TRAK family of kinesin adaptor proteins. A pivotal role for TRAK1 and TRAK2 in the transport of mitochondria is well recognized. Notwithstanding this progress, there is a body of evidence that still supports a role for TRAKs in the intracellular transport of GABAA receptors. This is critically reviewed in this article.

NMDA receptor/amyloid precursor protein interactions: A comparison between wild-type and amyloid precursor protein mutations associated with familial Alzheimer's disease

Two recent reports showed that amyloid precursor protein (APP) may contribute to postsynaptic mechanisms via the regulation of the surface trafficking of excitatory N-methyl-D-aspartate (NMDA) receptors. Here we have investigated the interactions and surface trafficking of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptor subtypes with three APP mutations linked to familial Alzheimers disease, APP695(Indiana), APP695(London) and APP695(Swedish). Flag-tagged mutated APP695s were generated and shown to be expressed at equivalent levels to wild-type APP695 in mammalian cells. Each APP mutant co-precipitated with NR1-1a/NR2A and NR1-1a/NR2B receptors following co-expression in mammalian cells. Further, as found for wild-type APP695, each enhanced NMDA receptor surface expression with no concomitant increase in total NR1-1a, NR2A or NR2B subunit expression. Thus these three familial APP mutations behave as wild-type APP695 with respect to their association with assembled NMDA receptors and their APP695-enhanced receptor cell surface trafficking.

Delineation of the TRAK binding regions of the kinesin-1 motor proteins

Understanding specific cargo distribution in differentiated cells is a major challenge. Trafficking kinesin proteins (TRAKs) are kinesin adaptors. They bind the cargo binding domain of kinesin‐1 motor proteins forming a link between the motor and their cargoes. To refine the TRAK1/2 binding sites within the kinesin‐1 cargo domain, rationally designed C‐terminal truncations of KIF5A and KIF5C were generated and their co‐association with TRAK1/2 determined by quantitative co‐immunoprecipitations following co‐expression in mammalian cells. Three contributory regions forming the TRAK2 binding site within KIF5A and KIF5C cargo binding domains were delineated. Differences were found between TRAK1/2 with respect to association with KIF5A.

Developmental changes in trak-mediated mitochondrial transport in neurons

Abstract Previous studies established that the kinesin adaptor proteins, TRAK1 and TRAK2, play an important role in mitochondrial transport in neurons. They link mitochondria to kinesin motor proteins via a TRAK acceptor protein in the mitochondrial outer membrane, the Rho GTPase, Miro. TRAKs also associate with enzyme, O‐linked N‐acetylglucosamine transferase (OGT), to form a quaternary, mitochondrial trafficking complex. A recent report suggested that TRAK1 preferentially controls mitochondrial transport in axons of hippocampal neurons whereas TRAK2 controls mitochondrial transport in dendrites. However, it is not clear whether the function of any of these proteins is exclusive to axons or dendrites and if their mechanisms of action are conserved between different neuronal populations and also, during maturation. Here, a comparative study was carried out into TRAK‐mediated mitochondrial mobility in axons and dendrites of hippocampal and cortical neurons during maturation in vitro using a shRNA gene knockdown approach. It was found that in mature hippocampal and cortical neurons, TRAK1 predominantly mediates axonal mitochondrial transport whereas dendritic transport is mediated via TRAK2. In young, maturing neurons, TRAK1 and TRAK2 contribute similarly in mitochondrial transport in both axons and dendrites in both neuronal types. These findings demonstrate maturation regulation of mitochondrial transport which is conserved between at least two distinct neuronal subtypes. Graphical abstract Figure. No caption available. HighlightsMitochondrial transport and velocity changes during neuronal maturation.TRAK1 and TRAK2 contribute to transport in axons and dendrites of immature neurons.In mature neurons TRAK1 controls axonal mitochondrial transport.In mature neurons TRAK2 controls dendritic mitochondrial transport.

Marie T. Filbin

Distinguished Professor Marie T. Filbin, late of Hunter College of The City University of New York, died in January this year at the height of her scientific career at the age of just 58, following a long illness. Marie and I were PhD students together at the University of Bath, UK, in the laboratory of George Lunt, one time Editor in Chief of this journal. We also shared a flat that was in an elegant Georgian terrace, typical of the architecture of the historic city. We worked hard but we also enjoyed ourselves. Life with Marie was never boring. She knew everyone and all the right places to go for fun. If at that time, she was asked to reflect I am sure that she would have agreed that she could never have envisaged the successful scientific career that would come her way. Indeed in her own words on receiving the Presidential Award for Excellence in Scholarship from Hunter College after 20 years of service she said, ‘. . . I have excelled and succeeded beyond my wildest dreams. . ..’. It is thus with great sadness that I write this tribute to her. Marie was from Northern Ireland. She and her great friend, Nuala Mooney, both came to England from their home town, Lurgan, to study for a BSc degree in biochemistry at the University of Bath, ostensibly to escape the troubles. Marie thrived at Bath. She made many lifelong friends and succeeded in her academic work despite both of her parents dying within a short time of each other during her undergraduate studies. After graduating, she joined George Lunt’s laboratory to undertake PhD studies. Her PhD was sponsored by Shell Research and was supervised by George in collaboration with John Donnellan at Shell Research Laboratories in Sittingbourne, Kent. Marie’s thesis work involved the biochemical characterization of neurotransmitter receptors in invertebrates. It was becoming clear from electrophysiological studies that neurotransmission at the insect neuromuscular junction was mediated by glutamate rather than by nicotinic cholinergic transmission as in vertebrates. Thus, Marie started out attempting to measure glutamate receptors in skeletal muscle of the locust, Schistocerca Gregaria, using radioligand binding techniques. This was exceptionally challenging since good pharmacological tools to study glutamate receptors were not yet available. Indeed, there were few if any, published works on the molecular properties of glutamate receptors. After 2 years, Marie switched her attention to the locust supraesophageal ganglion and succeeded in using a-bungarotoxin to identify, again by radioliogand binding, a nicotinic acetylcholine receptor in the locust central nervous system. This work was published in the European Journal of Biochemistry, her first paper (Filbin et al. 1983). It was the interest in invertebrate neurotransmitter receptors that took Marie to the USA to work in the laboratory of Mo and Molly Eldefrawi in Edson Albuquerque’s Department at the University of Maryland in Baltimore to continue her studies on the characterization of the insect central nicotinic cholinergic system. She soon moved, however, from invertebrates to vertebrates, crossed the city and joined Dr Marie T. Filbin