Book of abstracts - British Neuroscience Association

16.03
Quantitative analysis of membrane microdomains-associated
proteins in the DLPFC in the schizophrenic and bipolar disorder
brain
Áine T. Behan*, Kieran Wynne#, Connie Byrne#, Patricia Maguire#,
Niaobh O`Donoghue$, Michael J. Dunn$, Gerard Cagney#, David R.
Cotter*
*Department of Psychiatry, Royal College of Surgeons in Ireland ERC,
Beaumont Hospital, Dublin 9., $Proteome Research Centre, UCD
Conway Institute of Biomolecular and Biomedical Research, UCD,
#School of Biomolecular, and Biomedical Science, Conway Institute,
UCD.
Membrane microdomains (MM) are defined by their cholesterol and
sphingolipid-rich nature with numerous roles been ascribed them such
as their involvement as vehicles of endocytosis and trafficking during
signaling. Studies analysing protein changes potentially contributing to
human brain diseases have identified numerous MM associated
proteins as candidate players. We reason that by identifying and
quantifying MMs in the dorsolateral prefrontal cortex (DLPFC), an area
implicated in schizophrenia (SCZ), we may better understand the
molecular mechanisms underlying dysfunction in the SCZ and bipolar
disorder (BPD) brain.
A 10/10/10 series of the best matched brains (pH. PMI etc.) were
chosen from the 35/35/35 Stanley Foundation frozen post-mortem
brain tissue from DLPFC grey matter. Ten samples were pooled for
each of the groups (control, SCZ, BPD) and MMs were isolated and
analysed using two proteomic methods, (i) 2D-DIGE and MALDI-TOF
and (ii) 1D SDS PAGE and ion-trap MS. Proteins of interest were
validated in whole cell lysates (10/10/10 series – not pooled) using
Western blotting.
21 proteins were found to be differentially expressed by both proteomic
methods in our pooled samples. Further validation by Western blotting
demonstrated this differential expression for 3 synaptic/neuroplasticity
proteins, namely brain acid soluble protein 1(BASP1), limbic system
associated membrane protein(LAMP) and syntaxin binding protein
1(STXBP1).
BASP1, STXBP1 and LAMP are increased in the SCZ and BPD brain.
This suggests a dysfunction in synaptic function and/or neuroplasticity
in these psychiatric disorders.
16.04
Cellular and proteomics approaches to protein synthesis in axons.
Minnen Jv
Department of Molecular and Cellular Neurobiology, Center for
Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit, De
Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Recent studies have begun to focus on axonal protein synthesis and the
functional significance of localized protein synthesis. However, identification
of proteins that are synthesized in mammalian axons has not been
rigorously addressed. Here, we used axons purified from cultures of injuryconditioned
adult dorsal root ganglion (DRG) neurons and proteomics
methodology to identify axonally synthesized proteins. Proteins and the
encoding mRNAs for cytoskeletal proteins were identified in glial cell free
axonal preparations. In addition to the cytoskeletal elements, several heat
shock proteins, resident endoplasmic reticulum (ER) proteins, proteins
associated with neurodegenerative diseases (ubiquitin C-terminal
hydrolase L1, rat ortholog of human DJ-1/Park7, -synuclein, superoxide
dismutase 1), anti-oxidant proteins and metabolic proteins were identified
among the axonally synthesized proteins. Detection of the mRNAs
encoding each of the axonally synthesized proteins identified by mass
spectrometry in the axonal compartment indicates that the DRG axons
have the potential to synthesize a complex population of proteins.
Ultrastructural studies on in vivo injured and regenerating axons
demonstrated that these axons harbor high numbers of polyribosomes,
indicating active protein synthesis. Surprisingly, by creating GFP-tagged
ribosomes in Schwann cells, we demonstrated that the axonal
polyribosomes have originated in the Schwann cells. These data indicate
that mammalian axons have the ability to synthesize a large variety of
proteins, of which part of the encoding mRNAs and synthetic machinery
have originated in Schwann cells.
17.01
Molecular regulation of thalamocortical axonal navigation
Price D
Centres for Integrative Physiology and Neuroscience Research,
Edinburgh University, Hugh Robson Building, George Square,
Edinburgh EH8 9XD
The cerebral cortex receives most of its sensory innervation via
thalamocortical axons, whose development requires interactions
between advancing thalamic growth cones and guidance cues that
they encounter, attracting them towards or repelling them from specific
regions. We have been studying the regulation of guidance molecules
in thalamic neurons and in the environment through which their growth
cones navigate. Regarding control of navigation in thalamic cells, we
found that thalamic growth cones and neurons contain β-catenin
mRNA and that manipulations predicted to affect β-catenin levels
cause major disruption to thalamic axonal navigation. (1) In vivo, loss
of adenomatous polyposis coli (APC) in the forebrain blocks
completely thalamic axonal navigation from ventral to dorsal
telencephalon. (2) In vitro, application of lithium blocks thalamic axonal
growth in thalamocortical co-cultures. Regarding control of the
environment that thalamic axons navigate, we found direct evidence
that telencephalic expression of the transcription factor Pax6 is
required for thalamic axon guidance. We made mice with loxP sites
flanking exons encoding Pax6’s paired DNA binding domain
(Pax6loxP), so that exposure to Cre recombinase excises this crucial
domain and knocks the remainder of the protein out of frame, creating
a null-allele. We removed Pax6 from some cells in ventral
telencephalon using a Six3Cre allele that drives Cre recombinase
expression in cells around the developing internal capsule but neither
in diencephalon nor in cortex. In Pax6loxP/loxP;Six3Cre embryos, a
60-70% depletion of Pax6-expressing cells around the internal capsule
correlates with a ventral misrouting of a significant proportion of
thalamic axons.
17.02
Tangential neuronal migration controls axon guidance: a role for
neuregulin-1 in thalamocortical axon navigation
López-Bendito G
Instituto de Neurociencias de Alicante, CSIC & Universidad Miguel
Hernández, 03550 San Joan d’Alacant, Spain
Neuronal migration and axon guidance constitute fundamental processes in
brain development that are generally studied independently. Although both
share common mechanisms of cell biology and biochemistry, little is known
about their coordinated integration in the formation of neural circuits. Using
several in vitro coculture assays, in situ hybridation and
immunohistochemistry, here we show that the development of the
thalamocortical projection, one of the most prominent tracts in the
mammalian brain, depends on the early tangential migration of a population
of neurons derived from the ventral telencephalon. This tangential migration
contributes to the establishment of a permissive corridor that is essential for
thalamocortical axon pathfinding. Our results also demonstrate that in this
process two different products of the Neuregulin-1 gene, CRD-NRG1 and
Ig-NRG1, mediate the guidance of thalamocortical axons. These results
have been demonstrated in part by the analysis of specific mutant mice
deficient on either or all isoforms of Ngr1. TCAs fail to extend normally
through the telencephalon in mice with a loss of function mutation in the
Nrg1 gene. We have also demonstrated that this function seems to be
mediated by the tyrosine kinase receptor ErbB4. In sum, these results show
that neuronal tangential migration constitutes a novel mechanism to control
the timely arrangement of guidance cues required for axonal tract formation
in the mammalian brain
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