1. BDNF Seminar Spring 2010
Maneeshi Prasad
Jan 29th 2010

2. dlPFC (dorsolateral prefrontal cortex)
dlPFC is the last area (45th) to develop (myelinate) in the human cerebrum.
dlPFC is connected to the orbitofrontal cortex, thalamus, parts of the basal ganglia (the dorsal caudate nucleus), the hippocampus, and primary and secondary association areas of neocortex, including posterior temporal, parietal, and occipital areas.

3. Jon S. Simons & Hugo J. Spiers
Nature Reviews Neuroscience 4, (August 2003)

4. dlPFC
dlPFC serves as the highest cortical area responsible for motor planning, organization, and regulation.
It plays an important role in the integration of sensory and mnemonic information and the regulation of intellectual function and action.
It is also involved in working memory.
Complex mental activities require additional cortical and subcortical circuits that are connected with dlPFC.
dlPFC development and maturation may last up to two decades in humans and may thus show differential expression of genes.

5. BDNF
Brain-derived neurotrophic factor belongs to "neurotrophin" family of growth factors
BDNF support growth and differentiation of new neurons and synapses in CNS and PNS
BDNF is active in the hippocampus, cortex, cerebellum and basal forebrain—areas vital to learning, memory, and higher thinking
BDNF is also involved in neurogenesis
BDNF binds to TrkB receptor and p75NTR receptor

6. BDNF in CNS
Alternative promoter usage provides differential mRNA stability and subcellular localization
Promoter IV of BDNF has been implicated in forming inhibitory synapses in the cortex
BDNF mRNA with long 3’UTR are localized in dendrites of cortical neurons, while short 3’UTR mRNA is restricted to soma
Loss of long 3’UTR mRNA results in denser and thinner dendritic spines of CA1 pyramidal neurons and reduced hippocampal long- term potentiation
Rat visual cortex shows expression of transcripts III-V and IV-V in cell soma, while IV-V is expressed in dendritic processes

7. BDNF in CNS
Early postnatal development shows greater increase in spine density which is reduced by ~40% in later life
Conversion of proBDNF to mBDNF promotes late-phase long-term potentiation expression in hippocampus
ProBDNF is higher during postnatal stages while mBDNF is prominent in adults

8. Activity-dependent BDNF Expression Influences Homeostatic Plasticity
Activity-dependent BDNF Expression Influences Homeostatic Plasticity. Excitatory neuronal activity increases postsynaptic BDNF levels via Ca2+ -dependent transcription factors. For example, CREB phosphorylated on serine 133 binds to CaRE3/CRE following coactivation by CREB binding protein (CBP). Postsynaptic release of BDNF subsequently promotes the formation of inhibitory GABAergic synapses. The BDNF precursor, pro-BDNF is also an actively secreted molecule that affects synaptic plasticity during development.
Yang, J. et al. (2009) Nat. Neurosci. 12:113.

9. BDNF during development
Plays a important role during cortical development and is required for formation of ocular dominance columns in visual cortex
High levels of BDNF mRNA its TrkB receptor has been seen in dlPFC of young adults which subsequently decreases in adults

10. DNA-RNA-Protein
Gene locus on chromosome 1 2 3
DNA
Transcription
mRNA 1 2 3 2 3
Translation
Pre-pro Protein
Protein
Processing by cleavage
Mature protein

11. Alternative transcripts of BDNF
West et al, 2001

12.  Brain-derived neurotrophic factor (BDNF) gene structure

13. Human BDNF gene structure
10 noncoding exons in the 5’UTR
Presence of multiple translational (ATG) start sites
The Brian

14. Table. 1. Brain cohort demographics

15. Demographic variables
Postmortem pH values:
Postmortem Interval (PMI): 4 hr-32 hr
RNA integrity (RIN) varied

16. Expression of housekeeping genes across development
Real time PCR principle:
No variation in mRNA expression of housekeeping genes with changes in pH and RIN

17. BDNF mRNA expression in dlPFC during development

18. BDNF mRNA expression in dlPFC during development
Transcript I-IX changed significantly across development
Higher during earlier stages
Peaked at infancy
Decreased after infancy
Constant level was maintained from school age till adulthood

19. BDNF mRNA expression in dlPFC during development
Transcript II-IX was low at birth, increased during 1st few years and peaked in toddlers
Lowest in neonates
Increased in infants and toddlers
Decreased during school age, similar to infants, and was maintained till adulthood

20. BDNF mRNA expression in dlPFC during development
Transcript IV-IX highest in infants and toddlers
Lowest in neonates
Increased in infants and toddlers age group
Decreased gradually from school age and stayed consistent from adolescent till adulthood

21. BDNF mRNA expression in dlPFC during development
Transcript VI-IX peaks within first years of life
Highest at infancy
Decreased subsequently from toddler age till adulthood

22. Real-Time PCR

23. v
BDNF Protein levels

24. BDNF protein expression in dlPFC during development
Both proBDNF (28 kDa) and mature BDNF (14 kDa) bands were seen at all ages
Protein expression increased from neonates to infants
Infants had highest level of BDNF expression followed by toddlers
Mature BDNF form varied across development and peaked at infancy
Increase of protein level in toddler age group might be related to increase in level of IV-IX or II-IX transcripts
Decrease in protein levels in adults matches with decrease in mRNA levels
Decrease in level of pre-proBDNF was similar to that of mature BDNF

25. BDNF distribution in dlPFC by
ISH
BDNF
transcript

26. Expression of BDNF transcripts in layer IV

27. Expression of BDNF transcripts in layer V & VI

28. BDNF distribution in dlPFC
All 4 BDNF transcripts were highest in deeper cortical layers V and VI
Layer I: no expression at any age group
Layer II: moderate expression
Layer III: robust expression was seen in neonates
Layer IV/mid cortical layer: lower expression neonates and low to moderate expression in older age group
Layer V and VI: intense staining for BDNF seen in neurons

29. Discussion
Transcripts I-IX, IV-IX and VI-IX had highest expression patters in infancy
II-IX transcript was highest at toddler age group and was delayed by 2-3 years as compared to the other 3 transcripts
Lower expression of all transcripts during school age years

30. Discussion DLPFC layer IV showed increased BDNF signal
DLPFC layer IV is enriched in inhibitory neurons: cannot express BDNF by itself
This BDNF signal may be due to the BDNF mRNA that is targeted to the apical dendrites of layer V pyramidal neurons

31. Discussion
High level of BDNF transcripts & protein during early years and overlaps with 1.5 fold increase in synaptic density
Synaptic density may decrease during adolescence and stabilize by young adulthood with cortex reaching maturity, which overlaps with the decrease in BDNF levels

32. Discussion
Current study differs from a previous study (Webster, 2002), where BDNF expression was lowest in infancy and higher in young adult group
This may be due to combining of neonatal and infant groups, and differences in cohorts

33. Conclusions
The dynamic regulation of BDNF gene in hDLPFC may be activated in a promoter-specific manner
During postnatal cortical development, neuronal morphology and synaptic density may be regulated by transcript specific BDNF expression

34. Thank You !