1. The BDNF val66met polymorphism affects activity- dependent secretion of BDNF and human memory and hippocampal function Egan et al.

2. Background Episodic memory – the ability to store and recall experiences and events – depends on the hippocampus. Molecular studies of hippocampal synapses show that memory-related modifications in synaptic transmission occur in stages.

3. The first stage, early phase long-term potentiation (E-LTP) involves rapid increases of intracellular calcium concentrations and subsequent activation of protein kinases. The second stage, late phase LTP (L-LTP), recruits the cAMP and CREB signaling pathway to direct protein synthesis-dependent changes in structure and function of hippocampal synapses.

4. BDNF Brain-derived neurotrophic factor (BDNF) appears to play an important role in both E-LTP and L-LTP. BDNF gene expression is markedly enhanced by titanic (repeated) stimulation that induces LTP and during spatial memory tasks. Increased BDNF facilitates LTP while reduction attenuates LTP.

5. BDNF enhances high frequency synaptic transmission by facilitating synaptic vesicle docking. Inhibition of BDNF signaling in rodents by gene knockout or infusion of antisense BDNF impairs spatial learning and memory.

6. BDNF's relevance in human memory and hippocampal function had not been examined directly prior to the current study.

7. Newly-synthesized BDNF protein appears to be sorted inside the cell into a regulated pathway that secretes BDNF in response to neural stimulation. Transfection experiments using BDNF- GFP (Green Fluorescent Protein) fusion constructs show that BDNF is packaged into secretory vesicles.

8. The BDNF gene, like other peptide growth factors, encodes a precursor protein, pro-BDNF, which is proteolytically cleaved to form the mature protein. Only one frequent, nonconservative polymorphism in the human BDNF gene has been identified. It is a single nucleotide polymorphism (SNP) at nucleotide 196 (G/A), which produces an amino acid substitution (valine to methionine) at codon 66 (val66met).

9. This SNP located in the 5' pro-BDNF-sequence, and thus is unlikely to alter the intrinsic biological activity of the mature protein. However, the authors hypothesized that this SNP would affect intracellular processing and secretion of BDNF, leading to impairments in hippocampal function in humans. They also hypothesized that BDNF genotype might be associated with genetic risk of schizophrenia.

10. Methods To test these hypotheses, the authors examined the effects of the BDNF val66met substitution in a cohort of normal controls, patients with schizophrenia and their unaffected siblings.

11. Three in vivo assays of hippocampal function 1. Cognitive measures of episodic memory 2. A neurophysiological measure of hippocampal activation during a memory task using function MRI (fMRI) 3. An intracellular neurochemical measure of hippocampal neuronal integrity and synapse abundance assayed with proton magnetic resonance spectroscopic imaging (1H-MRSI)

12. To examine underlying molecular mechanisms, the authors transfected rodent hippocampal neurons with val- or met-BDNF fused with GRP ad examined trafficking of val-and met- BDNF proteins under fluorescence microscopy.

13. Effects of BDNF genotype on episodic memory The authors examined the effects of BDNF genotype on measures of episodic memory in a cohort of 621 subjects, including normal controls, patients with schizophrenia and their unaffected siblings, using the Wechsler Memory Scale (WMS), a test of verbal episodic memory. Delayed recall scores on WMS reflect the amount of information from two stories with 50 total elements that subjects are able to recall following a 0.5 hour delay.

14. Patients with schizophrenia had substantially lower scores compared to controls, while siblings were intermediate between these two groups. In the entire sample, BDNF genotype had a significant effects on these memory scores (p=0.02). In the 133 controls alone, BDNF genotype also had a significant effects on these memory scores (p=0.008).

15. BDNF genotype had no significant effect in either the normal subjects or the entire cohort on a second memory test which required recall of work lists, the California Verbal Learning Test (CVLT), immediately after hearing the list (p=0.45). No effect of BDNF genotype was seen on other types of memory, such as semantic memory (p=0.98). The authors conclude that these results suggest that the val66met polymorphism most strongly affects episodic memory.

16. Effects of BDNF genotype on hippocampal activation The authors performed an in vivo assay of hippocampal physiology using the blood oxygenation level dependent (BOLD) fMRI method in subjects performing the N-back working memory task. The brain area involved in the N-back task is thought to involve primarily the neocortical regions, particularly the prefrontal cortex. However, the N-back task also deactivates the hippocampus.

17. The authors studies two independent cohorts of healthy subjects, each comprised of val/met and val/val genotype subgroups. No met/met subjects were included because of their low population frequency (less than 5%). Members of the two cohorts were selected so that N-back performance, age, and gender did no differ between the genotype subgroups.

18. val/val subjects showed the normal pattern of hippocampal deactivation (Figure 2) val/met subjects showed an abnormal pattern of increased activation of the hippocampus.

19. Val- and Met-BDNF expression in cultured hippocampal neurons The authors expressed constructs containing either valBDNF or met BDNF attached to green fluorescent protein (GFP), and examined the expression, processing, biological activity, and release of the two BDNF isoforms.

20. Confocal microscopy showed that valBDNF-GFP exhibited a punctuate fluorescence, distributed throughout the cell body and extending to processes. metBDNF-GFP neurons exhibited diffuse fluorescence pattern mainly in cell bodies. Neuronal activity-dependent secretion of valBDNF-GFP was normal, while activity- dependent secretion of metBDNF-GFP was severely reduced and sometimes not detectable.

21. valBDNF-GFP-containing particles often superimposed with punctuate staining for the synapse marker protein synaptophysin. metBDNF-GFP showed no colocalization with synaptophysin. The authors suggest that failure to localize at synapses may contribute to the in vivo phenotypes associated with the val66met polymorphism.

22. Clinical caveats The authors point out several caveats with regard to this work. 1. The magnitude of the effects of BDNF genotype on in vivo measures of hippocampal function is small. This is expected, given the likely polygenic aspects of these traits.

23. 2. Spurious results can arise when ethnic groups are mixed ("population stratification"). Because the effects are found in a single ethnic group (European Americans) and because similar effects are seen in both normal controls in patients, the authors believe that stratification is unlikely to explain their results.

24. 3. Genetic association per se cannot exclude a potential effect of another nearby SNP in linkage disequilibrium with val66met. The authors re-sequenced the BDNF coding region in 16 subjects (32 chromosomes) and scanned for polymorphisms in 66 subjects. Only one rare synonymous change was observed, suggesting that linkage disequilibrium to a nearby locus does not account for these results.

25. The authors argue that their demonstration that the met allele is both associated with relatively poorer hippocampal function in vivo and with impaired trafficking and regulated secretion in vitro indicates strongly that the effects in humans are due specifically to the met allele.