Transcript Kristi Tschetter

Maternal deprivation induces a
rapid decline in circulating leptin
levels and sexually dimorphic
modifications in hypothalamic
trophic factors and cell turnover
Viveros et. al.
Kristi Tschetter
2/12/10
Overview of Chiaruttini et. al.
• Coding region common to all BDNF transcipts that
contains a constitutively active dendritic targeting
signal
• Targeting signal is suppressed in transcripts containing
exons 1 or 4 these are resistricted to the soma and
proximal dendrites
• Targeting signal is mediated by translin (RNA-binding
protein implicated in RNA trafficking)
• Translin binding is disrupted by the G196A (or
Val66Met mutation)
• G196A mutation blocks dendritic targeting of BDNF
mRNA by disrupting its interaction with translin
BDNF Transcipts
• Multiple BDNF transcripts are generated by
alternative splicing on one 5’ exon with a shared
3’ exon containing the entire BDNF coding region
and either a long or a short 3’ UTR sequence
• Exon 1 and 4 transcripts are localized in the cell
soma
• Exon 2 and 6 transcripts show a somatodendritic
localization
• Splice variants appear to encode spatial
localization signals used to preferentially regulate
BDNF expression in different subcellular domains
Overall
• Translin mediates the constitutive dendritic
targeting signal located in the CDS and this
targeting mechanism is conserved in rat and
human BDNF mRNA
• Evidence that translin/trax complex mediates
dendritic targeting of BDNF mRNA
• BDNF coding region common to all BDNF slice
variants is sufficient to direct constitutive
targeting of BDNF mRNAs to the distal dendritic
compartment
Figure 6
Figure 3
Figure 4
Results
• Characterization of this signal has
demonstrated that
1) Can be overridden by the 5’ UTRs of BDNF mRNA
isoforms (exon 1 and 4) that are retained in the
cell body and proximal dendrites
2) Mediated by translin
3) Blocked by the G196A (Val66Met) mutation
Results
• G196A substitution affinity impairs dendritic
trafficking of BDNF mRNA through the disruption
of its interaction with translin
• G196A SNP blocks dendritic trafficking of BDNF
mRNA implies that phenotypic changes induced
by this mutation, such as reduced hippocampal
memory deficits complexity and volume, as well
as memory deficits and susceptibility to mood
disorders may be due to this effect
Discussion
• SNP may also impair BDNF protein sorting by
disrupting its interaction with sortilin (a
vasicular membrane protein implicated in
membrane trafficking)
• G196A SNP interferes with BDNF processing
by disrupting trafficking of both BDNF mRNA
and protein through distinct mechanisms
Sirianni et. al. Overview
• BDNF is closed linked with neuronal survival
and plasticity in psychiatric disorders
• Engineered a degradable, injectable alginate
microspheres to continuously deliver BDNF to
the dorsal hippocampus of rats for 2 days
• non-degradable implantable poly(ethylene
vinyl acetate) matrices to continuously deliver
BDNF to the dorsal hippocampus of rats for
more than 1 week
Overview
• Small dose from a single infusion or from a 2
day sustained release alginate implant
produced anti-depressant-like effects
• Prolonged delivery of BDNF resulted in a
dysregulation of plasticity-related functions:
increased dose and duration of BDNF delivery
 increased levels of TrkB, ERK, CREB, and
phosphorylated ERK, while also producing
decreased phosphorylated CREB
Overview
• Direct administration of neurotrophic factors in
animals increases sprouting and growth of
neurons
• Learning and adaptive deficiencies observed in
depressive phenotypes are the consequence of
decreased neuroplasticity due to loss of
neurotrophic growth in the brain
• Loss of BDNF may underlie major depression
• Antidepressant treatment is associated with
increased expression of BDNF
Goals
1) Examine behavioral and biochemical effects
of BDNF delivery to the hippocampus
2) Develop drug delivery devices which could be
used for future exploration of the biological
effects of compounds that do not normally
cross the blood brain barrier
Figure 1
Methods
• Stereotactic surgery
– Target the CA1 and DG regions of the dorsal
hippocampus
Behavioral Test Methods
Open Field
Forced Swim Test
Figure 2
Figure 3
Figure 4
Figure 5
Table 1
Table 2
Discussion
• EVAc and alginate materials were effective
vehicles that could be used to deliver varying
rates and doses of bioactive DNF to a local
tissue region
• Advantages of alginate implants:
– Allows for high loading of drug in polymer with
the use of an aqueous solvent
– Doses can be administered by the same infusion
protocols used for fluid infusions
Discussion
• EVAc implant advantages
– No drug is lost in the formulation steps and total
loading of drug in polymer is known exactly
– Geometry of the implants can be altered in order
to target larger tissue regions
– Can be designed to deliver sustained doses of
drug for longer periods of time than alginate
materials
Results
• One-time bilateral infusion (25µg) of BDNF
produced antidepressant-like effects in the
forced swim test
• Delivery of BDNF from alginate microspheres
was also antidepressant-like
• Delivery of BDNF from EVAc matrices was not
antidepressant-like, even when the total dose
was high (11µg)
Results
• BDNF function may be mediated via serotonergic
action
• Selectively enhanced swimming behavior is
consistent with other published reports
demonstrating enhanced serotonergic function
and the sprouting of survival of serotonergic
neurons after the direct delivery of BDNF
• Sustained exposure to BDNF may interfere with
its previously reported antidepressant-like effects
Results
• Two key features of BDNF
– Relationship between neuroplasticity-related behavior
and neuroplasticity-related biochemistry is complex
– Precise effect of BDNF individual components of
neuroplasticity-related processes depends on how it is
delivered
• Dysregulation of BDNF-associated plasticity
related pathways was observed after the
sustained delivery of BDNF in the dorsal
hippocampus of the rat
Overview of Viveros et. al.
• Circulating corticosterone levels were increased
and glucose and leptin levels decreased
throughout the study in both sexes
• Hypothalmic mRNA levels of leptin receptor
increased significantly at MD24 in both sexes,
normalizing in females at MD36, but not in males.
• Male rats insulin-like growth factor mRNA levels
decreased at MD12 and MD24, with both trophic
factors unaffected in females
Overview
• Males cell proliferation was significantly
decreased at MD36, as were the glial structural
proteins, glial fibrillary acidic protein and
vimentin
• Females nestin levels decreased significantly at
MD24
• MD differentially affects trophic factors and cellturnover in the hypothalamus of males and
females  may underlie the sex differences seen
in the endocrine and metabolic outcome
Background
• MS results in specific metabolic and hormonal
alterations and delayed corporal growth
• MD  ↑corticosterone levels  exert
changes on neurodevelopment including
neurotrophic factors (NGF and BDNF), induces
synaptic modifications, and ↑
neurodegeneration and possibly gliosis
Hypothesis
• During MD circulating and
locally produced factors
produced in the
hypothalamic
development are
modified in a sexually
dimorphic manner
differentially affecting
cell-turnover in the
developing hypothalamus
of males and females
Methods
•
•
•
•
Adult Wistar rats
Reversed 12-h light-dark cycle (lights on at 20:00)
Free access to rat chow
On day of birth PND0, litters were culled to 8 pups per
dam (4 males and 4 females) did not cross foster
• Total of 12 rats in each experimental group
• Serum from all rats run in hormone analyses (n=11-12)
• ½ hypothalami were used for protein analysis (n=6) and
½ for mRNA analysis (n=6) with 2 rats from each of the
3 litters used in each analysis
Maternal Deprivation (MD)
• On the morning of PND9 beginning at 9:00 mothers
from the deprived group were removed and placed in a
cage beside the homecage in the same room
• For baseline measures and circadian variations pups of
both sexes were sacrificed on the morning of PND9 at
9:00
• Both controls and test pups were killed 12h after the
start of MD (Ct12 n=12 and MD12 n=12) and 24h after
MD (Ct24 n=12 and MD24 n=24)
• On PND10, mothers were returned to the cage of their
respective litters and 12h later another experimental
group was sacrificed (Ct36, n=12 and MD36 n=16)
Figure 1A
Figure 1B
Figure 2A
Figure 2B
Figure 2C
Figure 3A
Figure 3B
Figure 3C and 3D
Figure 4
Figure 5A and 5B
Figure 5C and 5D
Figure 5E and 5F
Discussion
• Early MD has sexually dimorphic effects on the
developing hypothalamus
• Basal levels of tropic factors such as IGF-1 and
BDNF differ between the sexes and are
modulated in a sex-specific manner during the
separation period
• Sex differences in trophic factors are
associated with differences in cell proliferation
and cell specific markers
Discussion
• MD differentially affects cell development in the
hypothalamus of male and female rats which
could underlie the differences observed between
the sexes in the pathological effects in the adult
animal
• Both males and females have a significant
increase in circulating corticosterone and a
dramatic increase in leptin levels in response to
MD which may be involved with some of the later
endocrine outcomes
Detected Subcellular Localization
Using in situ Hybridization
Localization of these transcipts
parallels the transcripts in vivo even
through they lack the BDNF 3’UTR
KCl did not effect the localization of
any of the BDNF-GFP chimaeric
mRNAs or GFP-tubulin