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Platform session 1 Translational research in child neurology (1Ц8).

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1. Identification Of A Filamin A Enhancer Gene
during Long-term Memory Formation
Francois Bolduc and Tim Tully; Cold Spring Harbor, NY
Objectives: Defects in Filamin A are responsible for periventricular nodular heterotopia (PNH). Affected patients manifest epilepsy and cognitive dysfunction. We sought to understand the molecular pathway associated with Filamin A in
the formation of long-term memory (LTM).
Methods: Our behavioral screen for genetic mutations that
affect long-term memory formation identified a Drosophila
mutant strain (Joy) with a lesion in Filamin A. We also studied gene expression in normal flies using Affymetrix DNA
microarray technology to identify genes with expression regulated during LTM formation. Statistical analysis yielded
more than 4500 candidate memory genes (CMGs). In vivo
validation was performed using a Palovian olfactory task.
Results: We identified 23 CMGs from the Filamin A pathway. Among those, we focused on lightoid, an orthologue of
Rab. An epistatic interaction approach revealed that the cognitive defects in LTM present in Filamin A mutants were
significantly enhanced by mutation of Rab, suggesting a
common physiological pathway. In addition, fly brain imaging showed a differential neuroanatomical localization for
each gene, suggesting distinct elements of a neuronal network.
Conclusions: Our study of Drosophila mutants established a
specific role for Filamin A during LTM formation. In addition, functional genomic analyses identified a target of Filamin A, Rab, to be significantly upregulated. Behavioral
study indicated that Rab and Filamin A shared a common
pathway during memory formation. Finally, our study illustrates the value of Drosophila genetics to further our understanding of the molecular bases of cognitive defects in PNH.
2. Altered Protein in Trafficking in eiF2B Mutated
Cells in Response to Stress
Adeline Vanderver, Michelle Mintz, Raphael Schiffmann,
Raphael, Yetrib Hathout; Washington, DC and
Bethesda, MD
Objective: Explain how an altered ER stress response in
eIF2B related disorders (Vanishing White Matter Disease or
Childhood onset Ataxia and Central nervous system Hypomyelination) results in cellular dysfunction.
Methods: Primary fibroblast cultures from 4 EIF2B5 mutated patients (homozygous R113H, R136H and R195H(2))
were compared to 2 fibroblast controls using stable isotope
labeling of amino acids in culture (SILAC). Control cells
were grown in 13C6,15N2-Lys and 13C6-Arg labeled medium,
while primary EIF2B mutated patient cells were grown in
standard media. All cells were treated with 1␮M thapsigargin. Cells were mixed at 1:1 ratio, then processed for ER
fractionation and proteome profiling using LC-MS/MS.
Results: Over 100 proteins were uniformly identified and
quantified in the ER fractions of patients and controls. Of
the identified proteins, a subset was found to be altered by at
least a 1.5 fold difference in patients versus controls. The
largest differences were found to affect ER chaperone resident proteins, GRP78, oxygen-regulated protein and many
other proteins involved in protein folding. Interestingly, an
additional group of proteins destined for secretion out of the
ER such as fibronectin and collagen precursors, were also
increased in the eIF2B mutated cells after stress.
Conclusion: Mutations in EIF2B, causative of VWM disease, result in abnormal protein trafficking in response to
cellular stress. This may explain why mutations of this ubiquitously expressed protein result in a primarily central nervous system phenotype with myelin destruction, since oligodendrocytes are uniquely sensitive to disturbances in
3. Major Locus for Centrotemporal Sharp Waves in
Rolandic Epilepsy Families Maps to Chromosome 11p
Lisa J Strug, Tara Clarke, Bhavna Bali, Peregrine L Murphy,
David A Greenberg, Deb K Pal; New York, NY
Introduction: Centrotemporal sharp waves (CTS) are the
EEG hallmark of rolandic epilepsy (RE). RE and its characteristic EEG trait is presumed to be genetic. Although not
yet established, the inheritance of RE appears complex while
the inheritance of CTS appears autosomal dominant. We
conducted a linkage study of CTS in RE families to (1) determine a region of the genome that contributes to the etiology of CTS in RE families; and (2) establish the mode of
inheritance of this CTS trait.
Methods: We ascertained 38 families for linkage through a
single RE proband. By definition, all RE probands have the
CTS trait. Their siblings (age 4 to 16 years) underwent
sleep-deprived EEG to determine their CTS status. Observations from individuals without an EEG, or those remaining
awake without showing CTS were treated as unknown in the
linkage analysis. We used GENEHUNTER and LIPED to
perform multipoint and two-point linkage analysis, respectively. We analyzed our families assuming dominant (gene
frequency ⫽ 0.006) and recessive (gene frequency ⫽ 0.1)
modes of inheritance, maximizing over penetrance.
Results: We observed a maximum multipoint lod score of
4.30 at marker D11S914 on chromosome 11, assuming a
dominant mode of inheritance, no heterogeneity, and 50%
penetrance. The maximum two-point lod score at D11S914
was 3.1.
Conclusion: A locus at chromosome 11p12-p13, inherited
dominantly with incomplete penetrance, plays a major role
in determining the presence of CTS in RE families. We will
conduct fine mapping studies to localize the gene responsible
for the strong linkage signal.
4. Neonatal Seizures Cause Substantial Deficits in
Frontal and Hippcampal Function
Havisha B. Karnam, Qian Zhou, Gregory Holmes;
Hanover, NH
Objective: Neonatal seizures are associated with a high risk
of cognitive dysfunction. There is a controversy regarding
the role of etiology of the seizures and the seizures per se in
cognitive impairment. The goal of this study was to determine the long-term cognitive consequences of neonatal seizures.
Methods: Rat pups were subjected to 50 (n -8) or 100 (n ⫽
8) flurothyl-induced seizures between postnatal day (PN) 1
to 15, while age-match controls ((n ⫽ 8) received sham seizures. When the animals were mature, they were tested in
the water maze, a measure of spatial learning and memory,
and the object recognition test, a measure of frontal lobe
© 2007 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Results: Flurothyl seizures were characterized by agitation,
running, myoclonus, and tonic extension of the forelimbs
and hindlimbs. When the rats were tested as adults in the
water maze there were significant differences between groups
(F ⫽ 3.730, p ⫽ 0.0121) with the rats receiving 100 seizures
having greater latencies to the escape platform than the rats
with 50 seizures (p ⬍ 0.01). Rats with neonatal seizures
demonstrated impairment both in working and reference
memory. Compared to control rats, neonatal seizures were
also associated with impaired novel recognition (t ⫽ 2.310,
p ⫽ 0.0413), demonstrating deficits in frontal lobe function.
Conclusions: Neonatal seizures are associated with longstanding impairment in both frontal lobe and hippocampal
function. The extent of cognitive impairment is directly correlated with the number of seizures. This study provides additional evidence that neonatal seizures are harmful and are
associated with both neocortical and limbic dysfunction.
5. The Chloride Transporter Inhibitor Bumetanide
Suppresses Behavioral and Electrographic Seizure in a
Rat Model of Perinatal Hypoxia
T Huynh, A Rotenberg, SN Rakhade, KJ Staley, FE Jensen;
Boston, MA
Objective: Neonatal seizures due to hypoxic encephalopathy
can be resistant to conventional anticonvulsants. We previously showed that the chloride importer NKCC1 is overexpressed in cortical neurons in the human term neonate and
immature rat. NKCC1 increases intracellular chloride, causing GABA receptors to be excitatory, and may explain the
relative resistance of neonates to GABA agonists such as barbiturates and benzodiazepines. We previously showed that
bumetanide suppressed electrographic kainate-induced seizures in immature rats. Here we tested the efficacy of bumetanide in combination with phenobarbital in suppressing
behavioral and electrographic hypoxia-induced seizures in
immature rats.
Methods: Postnatal day 10 Long Evans rats were treated i.p.
with phenobarbital (15 mg/kg), bumetanide (0.15mg/kg),
vehicle, or phenobarbital/bumetanide combination (same
doses) prior to induction of seizures during 15 min global
hypoxia. Seizure occurrence was assessed by video/EEG (Stellate Harmonie).
Results: Compared to vehicle treatment, phenobarbital alone
modestly suppressed total duration of behavioral seizures
(p⬍0.01), while bumetanide alone had no effect. However,
phenobarbital/bumetanide combination markedly suppressed
seizures (p⬍0.0001). Quantitative EEG analysis showed
combination treatment significantly reduced the cumulative
duration of spike/wave discharges (59.9⫹32.1 sec) compared
to vehicle-treated hypoxic rats (311.5⫹59.5 sec) (⫹ SE,
Conclusions: Combination treatment with bumetanide and
phenobarbital shows superior efficacy in suppressing both behavioral and electrographic seizures in a rat model of neonatal hypoxia-induced seizures. These data suggest that bumetanide may enhance the anticonvulsant effects of
phenobarbital by lowering intracellular chloride levels. Given
NKCC1 overexpression in human neonatal cortex, bumetanide may represent a potential adjuvant therapy in the clinical treatment of neonatal seizures.
Annals of Neurology
Vol 62 (suppl 11)
6. Phase 2 Study of PTC124 for Nonsense Mutation
Suppression Therapy of Duchenne Muscular Dystrophy
Brenda Wong, Carsten Bonnemann, Richard Finkel,
Kevin M. Flanigan Jacinda B. Sampson , Lee Sweeney,
Allen Reha, Gary L. Elfring, Langdon L. Miller,
Samit Hirawat; Cincinnati, OH, Philadelphia, PA,
Salt Lake City, UT, South Plainfield, NJ
Objective: PTC124 is a novel, small-molecule drug that promotes ribosomal readthrough of mRNA containing a nonsense mutation. This study is evaluating PTC124 safety,
compliance, PK, effects on full-length muscle dystrophin expression, and clinical activity in patients with nonsensemutation-mediated DMD.
Methods: Patients receive PTC124 orally for 28 days at 4, 4,
8 mg/kg (low); 10, 10, 20 mg/kg (mid); or 20, 20, 40 mg/kg
(high) dose levels, after breakfast, lunch, and dinner.
Results: 26 boys (ages: 5-13 years; stop codons: 15 UGA, 6
UAG, 5 UAA; baseline serum CK: 8,645-49,500 IU; steroid
use: 19/26) completed dosing at the low (n⫽6) or mid
(n⫽20) dose levels. All adverse events were mild-moderate.
Compliance was ⬎98% for both doses. PTC124 plasma exposures were stable over time; exposures were lower than in
PTC124-treated adult healthy volunteers and cystic fibrosis
patients. Myotube cultures from 24 evaluable patients’ pretreatment muscle biopsies showed dose-dependent increases
in dystrophin expression with in vitro PTC124 treatment.
Relative to baseline, visually appreciable post-treatment qualitative increases of in vivo dystrophin expression were noted
in 4/6 and 10/20 boys at low and mid doses, respectively.
Within the 28 days of treatment, muscle enzyme levels decreased significantly with minimal changes in muscle
strength and timed functions.
Conclusions: Preliminary evidence indicates that PTC124
safely induces full-length dystrophin expression in vitro and in
vivo and decreases serum muscle enzyme levels in boys with
nonsense-mutation-mediated DMD. Because subjects did not
achieve plasma exposures associated with maximal preclinical
activity, evaluation at the high dose level is ongoing.
Authors A.R., G.E., L.M., and S. H., are employees of PTC
Theraputics which makes PTC124. Other investigators do
not have any financial or commercial interests.
7. Methamphetamine Induces Locomotor Changes and
Striatal Synaptic Plasticity that are Dependent on D1
CA Scarlis, W Hanan, and NS Bamford, Seattle, WA
Objective: Addictive drugs such as methamphetamine release
extracellular dopamine, which modulates glutamate release
from corticostriatal terminals. Here, we determined the effect
of repeated methamphetamine on behavior and striatal function.
Methods: Mice were treated with methamphetamine (20
mg/kg/day, i.p.) for 10 days and challenged with amphetamine (2 mg/kg, i.p.) during withdrawal. Locomotor ambulations were recorded using activity-monitoring cages. Corticostriatal release (t1/2) was determined in striatal slices using
the endocytic tracer FM1-43.
Results: Repeated methamphetamine produced a persistent
depression (⬎3 months) in corticostriatal release (t1/2⫽335
sec vs. 203 sec for saline-treated controls; p⬍0.001) that was
partially reversed by drug reinstatement in withdrawal (t1/
2⫽285 sec; p⬍0.001,ANOVA). This potentiation was occluded with a D1 receptor agonist in vitro (SKF38393; 10
␮M; t1/2⫽233 sec; p⬎0.001), which had no effect on controls (t1/2⫽185 sec; p⬎0.5,ANOVA). Repeated methamphetamine enhanced locomotor ambulations to amphetamine challenges in withdrawal (p⬍0.001, repeated-measures
ANOVA). This sensitized behavioral response was also dependent on D1-receptor actions as the antagonist
SCH23390 (10 and 20 ␮g/kg s.c., 30 minutes prior to amphetamine) decreased ambulatory responses in a dose dependent manner (p⬍0.001) whereas controls were unaffected
Conclusions: Chronic methamphetamine induced a state of
persistent striatal depression that was reversed by a drug challenge. This potentiation in glutamate release from corticostriatal projections suggests that locomotor sensitization may
be determined through a new D1 receptor effect that is observed only in mice with previous exposure to psychostimulants. This psychostimulant-induced synaptic plasticity provides further insight into pharmacological alternatives for the
treatment of drug dependence, Tourette syndrome and
attention-deficit-hyperactivity disorder.
8. MeCP2 in the Cytoplasm of Neurons: Potential
Novel Function
Johana Vallejo, Shannon Snyder, Gabe Rice, Garilyn Jentarra,
Keri Ramsey, Elizabeth Solomon, Dietrich Stephan,
Vinodh Narayanan; Phoenix, AZ
Rett syndrome (RTT) is caused by mutation of the gene encoding methyl-CpG-binding protein 2 (MeCP2), primarily a
nuclear protein. Recent studies have shown that MeCP2 is
present in the post-synaptic compartment (dendrites) of neurons in human brain tissue and is in both the nucleus and
cytosol of cultured neurons. MeCP2 and other members of
the family of methyl DNA binding proteins (MBDs) are also
able to specifically bind to mRNA.
Hypothesis: MeCP2 binds to dendritic mRNAs in neurons,
and modulates translation of these mRNAs, thus affecting
synaptic function and plasticity.
The aims of this study are (1) to unequivocally demonstrate that a fraction of neuronal MeCP2 is present in dendrites, and (2) to demonstrate that MeCP2 binds to mRNAs
in vivo. A recombinant MeCP2-myc fusion protein was expressed in cultured mouse hippocampal neurons. Staining
with 9E10 (anti-myc monoclonal antibody) showed the presence of the fusion protein in neuronal dendrites. Immunostaining of cultured neurons with anti-MeCP2 (Sigma) also
detects MeCP2 in dendrites. Presence of MeCP2 in hippocampal dendrites in vivo is being confirmed by laser capture microdissection (cell bodies vs. dendrites) and Western
blotting. Finally, we immunoprecipitated MeCP2 from a
mouse brain homogenate, isolated bound mRNAs, and characterized this bound mRNA fraction by microarray hybridization. Among the MeCP2-associated RNAs were those encoding several ribosomal proteins, Na⫹/K⫹ ATPase,
neuronal glycoprotein M6a, and protein phosphatase 2.
These results support our idea, and suggest that a portion of
neuronal MeCP2 is present in dendrites, and may function
there by binding to dendritic mRNAs.
DOI: 10.1002/ana.11627
Program and Abstracts, Child Neurology Society
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