Download Conclusions Synapsin IIa is expressed in the brain of adult zebrafish

(Cesca et al. 2010)
The Identification and Expression of Synapsin IIa in Zebrafish
Kelly Welsh and Wendy Boehmler,
Department of Biological Sciences York College of Pennsylvania
addiction-dirkh.blogspot.com
Introduction
ZF 2a
•Synapsins (I, II, and III) are a family of neuronal phoshoproteins that play
an important role in neurotransmitter release by releasing synaptic vesicles
in the presynaptic terminal to move towards the plasma membrane for
exocytosis.
Human2b2
ZF 2a
ZF 2a
ZF 2a
ZF 2a
PCR Purification of
Synapsin IIa
Transformation
Ligation
Used tissues from
zebrafish brain, eye,
gut, heart & muscle
and 24, 42, and 72
hours post fertilization
(hpf) embryos
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Human2b302
ZF 2a
Results
349
Human2b362
ZF 2a
408
Ladder
Ladder
Human2b422
2. Determine if there is expression of synapsin IIa gene during
different developmental timepoints in zebrafish embryos.
•RT-PCR was conducted using RNA collected from adult zebrafish and
embryos.
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Human2b242
ZF 2a
1. Determine if there is expression of the synapsin IIa gene in adult
zebrafish tissues.
•Searches of expressed sequence tag databases
(http://www.ncbi.nlm.nih.gov/) led to the identification of a potential
zebrafish Synapsin IIa homolog (Gene Accession number NM001002597).
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Human2b182
•Because zebrafish are now being increasingly used as a genetic model and
to model a variety of human diseases, we focused our studies on the
cloning and characterization of zebrafish synapsin IIa.
Methods
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Human2b122
•Synapsins and synapsin-like genes have been characterized in a variety of
invertebrates and vertebrates, however the synapsin genes have yet to be
characterized in zebrafish.
Objectives
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•Synapsins have also been implicated in neurite outgrowth, the stabilization
of synapses, and synaptic vesicle docking, fusion, and recycling.
•It has been reported that synapsin II expression levels are decreased in
post-mortem brain samples of schizophrenic patients. It is unclear whether
the decrease in expression is the cause or result of the disease or whether
antipsychotic drug treatment effects the expression of synapsin II.
1
MNYLRRRLSDSTFISNLPNGYMSDLQKPDPPQPPPPGPAPVPAPASGPAPVSPRQ--ERK
MN+LRRRLSDS+FI+NLPNGYM+DLQ+P+P QPPPP P
A ++ AP +
ER+
MNFLRRRLSDSSFIANLPNGYMTDLQRPEPQQPPPPPPPGPGAASASAAPPTASPGPERR
PIQPQP---------GLGTGFFSSITNVVKQTAASAGLVEQTSVPASSKR-IKILLVIDE
P
+G+ FFSS++ VKQTAASAGLV+ +
++ R K+LLV+DE
PPPASAPAPQPAPTPSVGSSFFSSLSQAVKQTAASAGLVDAPAPAPAAARKAKVLLVVDE
58
61
108
121
PQHDWAKLFRGKKLNGDYDIKVEQAEFGDISIVAHANGSCNVAVQVLQNENKVLRSFKPD
P DWAK FRGKK+ GDYDIKVEQAEF ++++VAHA+G+ V +QVL+N KV+RSF+PD
PHADWAKCFRGKKVLGDYDIKVEQAEFSELNLVAHADGTYAVDMQVLRNGTKVVRSFRPD
168
FVLIRQHAFSMTENEDFRNLIIGLQYAGIPSINSLESVYNLCDKPWAFACLINTYKKLGQ
FVLIRQHAF M ENEDFR+LIIG+QYAG+PSINSLES+YN CDKPW FA L+ YK LG
FVLIRQHAFGMAENEDFRHLIIGMQYAGLPSINSLESIYNFCDKPWVFAQLVAIYKTLGG
228
EKFPLIEQTFYPNYKEMVTMPAFPVVVKIGHAHSGVGKVKVENHTKFQDIASVVAITQTY
EKFPLIEQT+YPN+KEM+T+P FPVVVKIGHAHSG+GKVKVENH FQDIASVVA+TQTY
EKFPLIEQTYYPNHKEMLTLPTFPVVVKIGHAHSGMGKVKVENHYDFQDIASVVALTQTY
288
STCEPFIDPKYDIRIQKIGNDYKAYMRTSVSGNWKSNTGTAMVEQVAMTDRYKLWVDTCC
+T EPFID KYDIR+QKIGN+YKAYMRTS+SGNWK+NTG+AM+EQ+AM+DRYKLWVDTC
ATAEPFIDSKYDIRVQKIGNNYKAYMRTSISGNWKTNTGSAMLEQIAMSDRYKLWVDTCS
348
EIFGGLEICAVKAINGKDGKDYITEVMGSSMPLMGEHQAQDQQLIADMVIAKMNHVMAQE+FGGL+ICAVKA++GKDGKDYI EVM SMPL+GEHQ +D+QLI ++VI+KMN ++++
EMFGGLDICAVKAVHGKDGKDYIFEVMDCSMPLIGEHQVEDRQLITELVISKMNQLLSRT
407
---NPKRP-TAIQPKQITVENNTLVEVKNAPPQKAPPQGCLQYILDCNGVAVGPKLVQAN
+P+RP T QP+ T+++
+
PPQ+ PPQGCLQYILDCNG+AVGPK VQA+
PALSPQRPLTTQQPQSGTLKDP---DSSKTPPQRPPPQGCLQYILDCNGIAVGPKQVQAS
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241
301
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421
463
478
Figure 4: Comparison of zebrafish and human Synapsin II.
Human and zebrafish SynapsinII amino acid sequences were aligned
using CLUSTALW. Dashes in sequences allow optimal alignment for
amino acid insertions/deletions. Identical amino acids are shown and
similar amino acids are highlighted by plus signs. (Identities = 314/480
(66%) ) Domains A and C are highlighted, Domain A and B contain
phosphorylation sites and Domain C stabilizes the interaction.
Table 1: Markers and Genes Syntenic with
SynII Gene in Zebrafish and Humans.
Figure 1: RT-PCR analysis of Synapsin IIa from
zebrafish embryos. The SynIIa gene was amplified
from zebrafish brain, 24 hpf, and 72 hpf. The primers
used encompassed the predicted ATG start codon and
stop codon to produce an amplicon of 1420 base pairs
(bp).
Zebrafish
Human
Gene Name
Chromosome Chromosome
SynII
rhoa
craf
plxnbl
tkt
her9
slc41a3
23
23
23
23
23
23
23
3
3
3
3
3
3
3
Conclusions
RNA Isolation
1. Synapsin IIa is expressed in the brain of adult zebrafish,
however we did not find expression in the eye, gut, muscle, or
heart.
2. The data presented here represents the first analysis of
temporal expression of the synapsin IIa gene in developing
zebrafish embryos.
PCR
Denatured at 95°C
Annealed at 46°C
Elongated at 72°C
cDNA
3. Sequence comparisons and syntenic analysis supports the
identification of a novel zebrafish synapsin II homolog.
Future Experiments
30 cycles
• Determine the spatial expression of synapsin IIa gene during different developmental
timepoints in zebrafish embryos
• Perform a whole-mount in situ hybridization on embryonic zebrafish using the cloned synapsin
IIa gene to determine its spatial and temporal expression pattern.
• Determine whether antipsychotic drug treatment alters the expression levels of synapsin IIa in
zebrafish.
Zebrafish Synapsin IIa
gene cloned
Literature Cited
•Cesca, F., Baldell, P., Valtorta, F., & Benefenat, F. (2010). The synapsin key actors of synapse function and plasticity.
Progress in Neurobiology, 91, 313-348.
•Chong, V.Z., Skoblenick, K., Morin, F., Xu, Y., Mishra, R.K., 2006. Dopamine-D1 and -D2 receptors differentially
regulate synapsin II expression in the rat brain. Neuroscience, 138, 587–599.