Wnt2 Expression and Signaling Is Increased by Different Classes of Antidepressant Treatments

1. Wnt2 Expression and Signaling Is Increased byDifferent Classes of Antidepressant Treatments Okamoto et. al., 2010

2. Wnts • Secreted signaling proteins that orchestrate many aspects of early developmental processes • Specification of cell fate • Regulation of cell adhesion • Migration • Control of cell proliferation • Role of signaling in the brain is unclear • Localization of Wnt receptors on the postsynaptic membrane • Activation of Wnt signaling at synapses modulates synaptic plasticity • Most widely recognized Wnt receptors are Frizzled proteins (Fzd) • Fzds comprise a family of 10 different G-coupled protein receptors • Best studied is the canonical signaling pathway • Wnt proteins are involved in synapse formation • Second, many studies have implicated Wnts in synapse formation, both as pro- and antisynaptogenic molecules

3. The Synaptic Wnt Signaling HypothesisSHAO-JUN TANG • Hypothesis is that Wnts signal at mature synapses in an activity-dependent manner and that the activity-dependent synaptic Wnt signaling regulates the structural and functional plasticity of the synapse • Wnt signaling at central synapses: Synaptic activity leads to the release of Wntligands from the synapses, and released Wnts bind to Frizzled receptors on synaptic membranes to activate Wnt signaling that regulates synaptic structures, functions, and gene expression

4. The Synaptic Wnt Signaling HypothesisSHAO-JUN TANG

5. Discussion • CaMKII and calcineurin by synaptic Wnt/ Ca2+ signaling may directly affect the activity of synaptic receptors that are important for synaptic transmission • As a cell-wide mechanism, the synaptic Wnt signaling may contribute to synaptic plasticity by regulating gene expression • Involvement of the synaptic Wnt signaling in synaptic plasticity as I postulate here immediately suggests a cellular mechanism by which this signaling regulates the cognitive functions of the brain (e.g. learning and memory)

6. Wnts acting through canonical and noncanonical signaling pathways exert opposite effects on hippocampal synapse formationDavis et. al., 2008 • Used in situ hybridization to show that several Wntligands (Wnt3, Wnt5a, Wnt7a, and Wnt7b) and their receptors, Frizzled, are expressed in the developing hippocampus during the period of synapse formation in rodents • Wnt7a and Wnt7b activate canonical signaling, whereas Wnt5a activates a noncanonical pathway • Canonical pathway, either through pathway manipulations or through Wnt stimulation, increases presynaptic inputs

7. Methods • Hippocampi (from P0 Long Evans rats) were dissected, dissociated, and plated onto 8-well chamber slides • Grown for 8-12 days at 37°C with 5% CO2 • Cells were then treated for 36 hours with lithium chloride, sFRP2, recombinant Dkk-1, recombinant Wnt3a, recombinant Wnt7a or Wnt7b conditioned media

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17. Results • Wnts expressed in the hippocampus at the peak of synapse formation have been implicated in the activation of both canonical and noncanonical pathways • Wnt7a and Wnt7b specifically have been identified as canonical Wnt pathway activators • Activation of the canonical Wnt pathway promotes synapse formation based on the evidence that Wnts that activate canonical Wnt signaling increase the number of presynaptic terminals per area of dendrite

18. Results • Wnt5a decreased β-catenin levels and reduced the number of presynaptic puncta • Not yet know the pathway by which Wnt5a exerts its effect, but it is likely to be a noncanonical pathway that antagonizes the canonical pathway • Wnt5a is dominant over canonical Wnts

19. Conclusions • Wnt5a lowers β-catenin levels, even when Wnt3a is added along with Wnt5a • Pro- and anti-synaptogenic effects of Wnts on synapse formation are generally mediated by the level of activation of canonical Wnt signaling • Canonical Wnt pathway activation has the opposite effect on presynaptic inputs as noncanonical Wnt pathway activation. Previous work has shown Wnts to be both pro- and anti-synaptogenic, although these studies were done in several different systems with different Wnt proteins • Here they show, in the same system, that Wnts exert a bidirectional influence on synapse formation

20. Wnt2 Expression and Signaling Is Increased byDifferent Classes of Antidepressant TreatmentsOkamoto et. al., 2010 • Wnt2 expression and signaling is a common target of antidepressants and that increased Wnt2 is sufficient to produce antidepressant effects • Wnt signaling is crucial for normal embryonic development as well as neuroplasticity in the adult brain • Abnormal Wnt signaling has been implicated in various disorders, ranging from cancer and neurodegeneration to psychiatric illnesses • In order to identify the changes in hippocampal gene expression in response to different antidepressants, they performed microarray analysis with a focused array that included multiple classes of signaling molecules. • The expression of several genes involved in the Wnt signaling pathway was regulated by chronic antidepressant administration

21. Methods • Male Spraque-Dawley rats (160-180g for antidepressant treatment) • 250-300 g from AAV studies • 12-h light/dark cycle • Ad libitum access to food and water

22. Methods • IP injections twice daily for 5 days (subchronic treatment) or 3 weeks (chronic treatment) • Vehicle (distilled water) • Citalopram (15 mg/kg) • Fluoxetine (5 mg/kg) • Venlafaxine (15 mg/kg) • Atomoxetine (3 mg/kg) • Bilateral ECS was administered via moistened pads on spring-loaded ear clip electrodes with a pulse generator (frequency, 100 pulses/sec, .5 msec; shock duration, .5 sec; current, 55mA)

23. Methods • Microarray analysis of gene expression • Growth factor signaling, transcription factors, kinases and genes implicated in neuropsychiatric disorders and drug action • In Situ hybridization analysis • Immunoblotting • AAV • Viral production and purification • Stereotaxic Surgery and Infusions

24. Methods: Behavioral Sucrose Preference Test Learned Helplessness

25. Methods: Behavioral Forced Swim Test

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31. Conclusions • Wnt2 expression can influence LH and SPT behavior • Wnt2 signaling plays a critical role in activity-dependent formation of dendrites via intracellular β-catenin • Expression of Wnt2 significantly influenced behavior in the SPT, which is typically used as a measure of anhedonia that occurs in response to repeated stress exposure • AAV-Wnt2 into the dentate gyrus granule cell layer resulted in significant antidepressant-like responses in the LH and SPT, but no significant effects were found in NSF or FST • Possibility that Wnt2 expression alters learning

32. Discussion • Lack of effect of Wnt2 on the FST was surprising but could be explained by several possibilities • FST is a different despair paradigm relative to LH, notably being responsive to acute antidepressants, indicating that different signaling mechanisms are involved • FST/immobility could be influenced by brain regions other than or in addition to the hippocampus. • Although expression of Wnt2 in the dorsal hippocampus was sufficient to alter LH and SPT behavior, it is possible that more widespread expression or targeting of other parts of hippocampus (e.g., ventral) would influence FST behavior

33. Discussion • Regulation of multiple components of Wnt/β-catenin signaling, both canonical and noncanonical pathways (PCP and Wnt/calcium cascades), showing that a complex network of molecular changes might contribute to the efficacy of antidepressant treatment • Results demonstrate that local expression of Wnt2 in the hippocampus is sufficient to produce antidepressant-like effects in behavioral models