Breitling, R.

Ham, T. van

Hofstra, R.

Nollen, E.

Plasterk, R.

Thijssen, K.

University of Groningen Research Database

  
 University of Groningen
Identification of modifiers of alpha-synuclein inclusion in a C. elegans model by genome-wide
RNAi
Ham, T. van; Thijssen, K.; Breitling, R.; Hofstra, R.; Plasterk, R.; Nollen, Ellen
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2007
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Ham, T. V., Thijssen, K., Breitling, R., Hofstra, R., Plasterk, R., & Nollen, E., (2007). Identification of
modifiers of alpha-synuclein inclusion in a C. elegans model by genome-wide RNAi, 1 p.
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Poster Presentations: Alpha-synuclein and Lewy Bodies S125
2.313 Alcoholism, alpha-synuclein, and Parkinson disease:
A case-control study
L. Brighina1°, N.K. Schneider, T.G. Lesnick, M. de Andrade,
J.M. Cunningham, M.J. Farrer, S.J. Lincoln, D. Mrazek, W.A. Rocca,
D.M. Maraganore
1Monza, Italy
Objective: Variability in the allele length of a dinucleotide repeat
sequence (REP1) within the alpha-synuclein gene (SNCA) promoter is
associated with Parkinson’s disease (PD) [JAMA 2006; 296: 661–670]
and also with alcohol dependence [Hum Mol Genet 2005; 14: 967–971].
Medical diagnosis of alcoholism is inversely associated with PD [Neurology
2000; 14: 1350–1358]. The aim of this study was to determine whether
the association of REP1 genotype and PD is independent of alcoholism,
whether the association of alcoholism and PD is independent of REP1
genotype, and whether REP1 genotype and alcoholism have joint effects
on PD susceptibility.
Method: Cases were recruited prospectively from the Department of
Neurology of the Mayo Clinic in Rochester, MN, after June 1, 1996. The
controls included unaffected siblings of cases and unrelated population
controls. We assessed alcohol use using a structured telephone interview.
We screened for alcoholism (overt or covert) using the CAGE questionnaire.
Genotyping was performed using an ABI 3730 platform. Odds ratios (ORs)
and 95% conﬁdence intervals (CIs) were determined using conditional
logistic regression models.
Results: Our study included a total of 893 case-control pairs. There was
an increased risk of PD with increasing SNCA REP1 bp length (OR 1.18
for each REP1 genotype score unit, 95%CI 1.02−1.35; p = 0.02). There was
a trend of decreasing risk of PD with increasing CAGE score (p = 0.0002).
The association of REP1 genotype score with PD remained signiﬁcant after
adjusting for CAGE score, and the inverse association of CAGE score
with PD remained signiﬁcant after adjusting for REP1 genotype score.
No signiﬁcant pairwise interactions were observed for REP1 genotype and
CAGE scores.
Conclusion: Alcoholism (as measured by CAGE score) and REP1
genotype score were associated with PD susceptibility (main effects), but
they did not have multiplicative joint effects on PD susceptibility.
2.314 Identiﬁcation of modiﬁers of alpha-synuclein inclusion
in a C. elegans model by genome-wide RNAi
T. van Ham1°, K. Thijssen, R. Breitling, R. Hofstra, R. Plasterk, E. Nollen
1Groningen, Netherlands
Objective: The protein alpha-synuclein is tightly connected to devel-
opment of Parkinson’s disease: hallmark of PD is the presence of protein
inclusions in the brain containing the protein alpha-synuclein. In addition,
multiplication of the alpha-synuclein gene has been shown to be causative
for PD. We aim to identify genes involved in the formation of alpha-
synuclein inclusions to provide an understanding of the mechanism of and
genetic susceptibility to age-related sporadic PD.
Method:We created nematodes (C. elegans) transgenic for human alpha-
synuclein fused to yellow ﬂuorescent protein to visually track inclusion
formation. We used this model in a genome-wide RNAi feeding screen to
ﬁnd genetic inactivations that alter a-synuclein inclusion formation.
Results: Alpha-synuclein inclusions develop during aging in the trans-
genic C. elegans strain. The inclusions contain mostly mobile protein mate-
rial and also immobile, aggregated protein at old age. Using genome-wide
RNAi we identiﬁed genes that increase the formation of inclusions when
silenced by RNAi. Interestingly, there was a signiﬁcant overrepresentation
of the genes that function in the ER, Golgi and vesicular membranes,
when compared to random sets of genes. In addition, we found several
genes associated with aging to increase inclusion formation when silenced.
Conclusion: We developed a small animal model of alpha-synuclein
accumulation, in which age-dependent inclusion formation can be visually
tracked. Using this model we found genes that increase the number
of alpha-synuclein inclusions when silenced. The overrepresentation of
genes with a role in vesicular trafﬁcking and ER protein quality control
functioning in the endomembrane system suggests that these processes are
involved in the age-dependent formation of alpha-synuclein inclusions. The
genes found may provide an understanding of genetic susceptibility to PD
and molecular targets for therapy.
2.315 Identiﬁcation of potent small molecule inhibitors of
alpha-synuclein aggregation in cell culture and by
in vitro screening
A. Snow1°, L. Esposito, J. Cummings, T. Lake, M. Hudson, F. Cheng,
A. Ferree, S. Saha, B. Wolozin
1Kirkland, USA
Objective: A rotenone-induced cell culture model of Parkinson’s disease
(PD) was primarily utilized to test the ability of ProteoTech’s small
molecule library of compounds (representing new chemical entities) to
inhibit aggregation of alpha-synuclein, a major component of PD Lewy
bodies.
Method: In this cell culture model, A53T mutant alpha-synuclein was
overexpressed in human BEM-17 neuroblastoma cells. Cells exposed to
1 or 5uM rotenone accumulated alpha-synuclein aggregates containing a
large amount of beta-sheet secondary structure as detected by positive
Thioﬂavin S ﬂuorescence and quantitative image analysis.
Results: Treatment of rotenone-treated cells with different novel small
molecule compounds identiﬁed 4 compounds (referred to as PD-61, 86, 31
and 13) that profoundly reduce the accumulation of Thioﬂavin-S positive
alpha-synuclein aggregates by 87−91%, 73−91%, 40−84% and 57−70%,
respectively. A marked reduction of alpha-synuclein aggregation by these
compounds was conﬁrmed using Thioﬂavin T ﬂuorometry, Congo red
binding assays and circular dichroism spectroscopy. In addition to their
potent alpha-synuclein anti-aggregation properties, these compounds also
provided good protection against rotenone-induced neurotoxicity in cell
culture and in a C. elegans survival assay.
Conclusion: These studies indicate that we have identiﬁed unique small
molecule compounds that have potent alpha-synuclein anti-aggregation
and neuroprotective properties and thus may serve as promising new
therapeutics for PD and related disorders.
Funded by ProteoTech Inc. and a LEAPS Award from the Michael J. Fox
PD Research Foundation.
2.316 6-Hydroxydopamine nigrostriatal lesion modulates
alpha-synuclein expression in different rat brain regions
M. Perovic1°, A. Mladenovic, S. Ruzdijic, S. Kanazir
1Belgrade, Serbia and Montenegro
Objective: alpha-Synuclein is considered to be involved in pathogenesis
of PD and other neurodegenerative diseases. Its ﬁlamentous aggregates are
found as principal constituents of Lewy bodies and some others patholog-
ical intraneuronal deposits. Due to the localization of alpha-synuclein in
presynaptic terminals, its role in synaptic function and plasticity was pos-
tulated as well. 6-Hydroxydopamine (6-OHDA) rat model of PD is charac-
terized by acute loss of dopaminergic neurons, but speciﬁc alpha-synuclein
aggregates were not reported. Therefore, this model could reveal possible
role of alpha-synuclein in plasticity given that some compensatory sprout-
ing of dopaminergic neurons exists after period of extensive neuronal death.
Method: Adult male Mill-Hill Hooded rats (n = 24) received four-site
intrastriatal stereotaxic injection of 6-OHDA or saline (sham operated
animals) into the right lateral striatum. Five weeks later, animals were killed
by decapitation and the model was veriﬁed by tyrosine hydroxylase (TH)
imunohistochemistry. Western blot analysis was used to detemine the level
of alpha-synuclein protein and confocal microscopy for analysis of alpha-
synuclein co-localization with TH, synaptophysine, MAP 2 and GAP-43.


Identification of modifiers of alpha-synuclein inclusion in a C. elegans model by genome-wide RNAi

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Identification of modifiers of alpha-synuclein inclusion in a C. elegans model by genome-wide RNAi

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