Book of abstracts - British Neuroscience Association

8.05
Regulation of Neuronal Mitochondrial Transport by a GRIF-1 /
Miro1/ Kinesin Motor Protein Trafficking Complex
Macaskill A F, Kittler J T
Department of Physiology, University College London, Gower Street, ,
London, , WC1E 6BT
The transport of mitochondria to specific neuronal locations is
necessary to meet local cellular energy demands and for the buffering
of intracellular calcium. Although a critical role for kinesin motor
proteins in mitochondrial transport has been demonstrated, little is
known regarding the molecular mechanisms that regulate the specific
recruitment of motor proteins to mitochondrial cargo. Here we provide
evidence that the Mitochondrial Rho GTPase Miro1 acts as an
acceptor site for the recruitment of motor proteins to mitochondria in
nerve cells. Miro1 is highly expressed in hippocampal neurons and comigrates
with mitochondria along neuronal processes in live cells.
Furthermore, using immunofluorescence and co-immunoprecipitation
we demonstrate that Miro1 forms a protein complex with the adaptor
protein GRIF-1 (Trak2) and kinesin-1 in mammalian neurons. The
formation of this complex is dependent on a direct interaction between
Miro1 and the adaptor protein GRIF-1, which interacts directly with
kinesin family (KIF) 5 heavy chains. In agreement with this Miro1 can
recruit both GRIF-1 and kinesin-1 motors to mitochondria in both
neurons and HEK cells, dependent on its transmembrane domain. In
support of a critical role for this complex in regulating mitochondrial
transport, altering Miro1 function, or the Miro-dependent recruitment of
kinesin-1 motors, dramatically alters mitochondrial distribution along
neuronal processes. These results support an important role for Miro1
in regulating the kinesin motor protein dependent transport of
mitochondria in mammalian nerve cells.
9.01
Mitochondrial and plasma membrane potential of cultured cerebellar
neurons during glutamate induced necrosis, apoptosis and tolerance
Ward M W, Huber H J, Weisová P, Duessmann H, Prehn J H M
Department of Physiology and Medical Physics and RCSI Neuroscience
Research Centre, Royal College of Surgeons in Ireland, 123 St
Stephenâ€s Green, Dublin 2, Ireland. 2 Siemens Medical Division,
Siemens Ireland, Dublin 2, Ireland.
A failure of mitochondrial bioenergetics has been shown to be closely
associated with the onset of apoptotic and necrotic neuronal injury. Here,
we developed an automated computational model that interprets the single
cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a
consequence of changes in either the ΔΨm or ΔΨp, allowing for a
characterization of responses in populations of single cells and subsequent
statistical analysis. Necrotic injury triggered by prolonged glutamate
excitation resulted in a rapid monophasic or biphasic loss of ΔΨm that was
closely associated with a loss of ΔΨp, neuronal swelling and early
membrane lysis. Delayed apoptotic injury was induced by transient
glutamate excitation and resulted in a small, reversible decrease in TMRM
fluorescence followed by a sustained increase in TMRM fluorescence.
While the initial decrease was found to be caused by a ΔΨp depolarization,
the subsequent increase was due to a hyperpolarization of ΔΨm as
confirmed using the anionic probe DiBAC2(3). The hyperpolarization of
ΔΨm was sustained until a collapse of ΔΨm ensued (after 11.8 h ± 0.8h).
Statistical analysis identified two major correlations; neurons that displayed
a more pronounced depolarization of ΔΨp during the initial phase of
glutamate excitation entered apoptosis more rapidly, and those neurons
that displayed a more pronounced hyperpolarization of ΔΨm survived
longer. Indeed, neurons tolerant to transient glutamate excitation (18%)
showed the most significant increases in ΔΨm, indicating that mitochondrial
hyperpolarization is associated with survival responses during excitotoxic
injury.
9.02
Functional differences in GLT-1 splice variants
Peacey E, Rattray M, Lou Z, Kramer A, Dunlop J
Authors 1 & 2:, King`s College London, Wolfson Centre for Age-
Related Diseases, St. Thomas` St, London, SE1 1UL, , Authors 3, 4 &
5:, Wyeth Research, CN 8000, Princeton, NJ 08543
Glutamate transporter 1 (GLT-1) is expressed predominantly in
astrocytes and is responsible for up to 95% of glutamate uptake in the
mammalian CNS. Multiple N- and C-terminal splice variants of GLT-1
have been described, but are as yet functionally uncharacterised. It is
possible that the splice variants may show differences in expression,
multimerisation, uptake kinetics or degradation, and may play a
pathological role in conditions such as amyotrophic lateral sclerosis
(ALS), in which there is a significant down-regulation of GLT-1.
In order to address these questions, four GLT-1 splice variants
expressed in mouse cerebral cortex (termed MAST KREK, MAST
DIETCI, MPK KREK or MPK DIETCI according to amino acid
sequence) were cloned and epitope tagged. When transiently
expressed in COS-7 or HEK-293 cells, Western blots showed similar
expression of monomeric and trimeric bands for all splice variants. Coimmunoprecipitation
experiments using the tagged splice variants
indicated that they are capable of forming homomeric and heteromeric
trimers.
Pharmacological and kinetic characterisations of the splice variants
were carried out using stably transfected HEK-293 cells. The splice
variants were pharmacologically indistinguishable using any of the 12
glutamate transporter inhibitor compounds tested. [3H] glutamate
uptake studies showed that MPK DIETCI has significantly lower
affinity than the more common MAST KREK variant (Km = 46.5 ± 5.9
µM and 24.6 ± 0.6 µM respectively). This difference in affinity may be
of functional importance, especially if there is an alteration of
regulation of splice variant expression or degradation under
pathological conditions.
9.03
Histamine H4 receptor: the first evidence for CNS and PNS expression
and isoform hetero-oligomerisation
Shenton F C(1), Rijn Rv (2), Bakker R (2), Leurs R (2), Grandi D (3), Morini
G (3), Chazot P L (1)
1.Centre for Integrative Neuroscience, Durham University, UK;
2.Amsterdam/ Leiden University, The Netherlands; 3.Department of Human
Anatomy, Pharmacology and Forensic Medicine, University of Parma, Italy
The histamine H3R is found predominantly in the CNS. Herein, we provide
the first evidence that the H4R is also expressed on neuroendocrine cells,
and in selective structures within the rodent and human central and
autonomic nervous system. Both the H3R and H4R are GPCRs, which
undergo alternative splicing to yield shorter isoforms, with distinct
distribution patterns. The functional role of splicing is of great interest, and in
previous work, we have demonstrated the existence of rat and human H3
and human H4 homo-dimers using biophysical and immunobiochemical
techniques, with our novel selective anti-H3 and anti-H4 antibodies(1,2). We
have also reported the first evidence that the rat H3 isoforms act as activitydependent
dominant negative subunits in the brain(1). Using biophysical,
immunobiochemical and pharmacological experiments, we show that
human H4 isoforms homo- and hetero-dimerise in mammalian cells. The
hH4(302) and (67) isoforms alone did not bind [3H]histamine or transduce a
signal, and did not significantly affect the affinity of [3H]histamine binding to
the hH4(390), however both short isoforms reduced the level of binding by
55% and 30%, respectively. Surface biotinylation experiments showed that
all three isoforms can reach the cell surface when expressed alone, and
concur with the negative effects of the isoforms upon H4(390) surface
receptor number. Dominant negative action of splice isoforms appears to be
a general theme for both H3- and H4-histamine receptors, and may
represent a common regulatory system for GPCRs in the brain.
1. Mol Pharmacol. 69, 1194-1206.
2. Mol. Pharmacol. 70, 604-615
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